Content uploaded by Sarvpreet Singh Ghuman
Author content
All content in this area was uploaded by Sarvpreet Singh Ghuman on Oct 15, 2018
Content may be subject to copyright.
THE
INDIAN JOURNAL OF
ANIMAL REPRODUCTION
IJAR 40:1 June 2019; ISSN 0970 - 2997
THE INDIAN SOCIETY
FOR STUDY OF ANIMAL REPRODUCTION
OFFICIAL ORGAN OF
The Indian Journal of Animal Reproduction
Indexed In the Abstracting Journal of CAB International
ISSAR
View Journal Online
http://www.indianjournals.com/ijor.aspx?
target=ijor:IJAR3&type=home
OFFICIAL ORGAN OF
THE INDIAN SOCIETY
FOR STUDY OF ANIMAL REPRODUCTION
IJAR 40:1 June 2019; ISSN 0970 - 2997
THE
INDIAN JOURNAL OF
ANIMAL REPRODUCTION
The Indian Journal of Animal Reproduction
Indexed In the Abstracting Journal of CAB International
The Editor / Editorial Board and distinguished referees are in no-way responsible individually or collectively for
the views, data and technical details presented in the journal. The whole responsibility vests with authors of the
article/manuscript.
Cost of Journal (Revised From 2010)
Life members of ISSAR - Free of cost
Professional & Non-members of ISSAR - Rs. 300/- per annum
Institution & Libraries - Rs. 500/- per annum
All foreign institutions and libraries - US$100 per annum
THE INDIAN JOURNAL OF ANIMAL REPRODUCTION
Ocial Organ of the Indian Society for Study of Animal Reproduction
[Regd. No. Bom.253/78]
AIMS AND SCOPE
The Indian Journal of Animal Reproduction, an ocial organ of the Indian Society for Study of Animal
Reproduction (ISSAR) publishes basic/applied research articles, short communications and clinical articles/case
reports in Veterinary gynaecology, obstetrics, andrology, semenology, articial insemination, embryo transfer and
other assisted reproductive technologies. It is published bi-annually i.e. June and December.
Dr. D. Vecchio, DVM, Ph.D.
Researcher IZSM-National Reference Centre
on Water Bualo Farming and Productions,
Hygiene and Technologies (CreNBuf), Italy
E-mail: domenico.vecchio@izsmportici.it
Dr. S. Selvaraju, Ph.D.
Senior Scientist, NIANP, Bangalore
E-mail: selvarajuars@gmail.com
Dr. Shivprasad, Ph.D.
Professor and Head, ARGO, Pantnagar
E-mail: shivp2003@yahoo.co.uk
Dr. S. Velmurugan, Ph.D.
Scientist C, National Institute of Animal
Biotechnology, Telangana
E-mail: sathyavet@gmail.com
Dr. A.J. Dhami, Ph.D.
Professor & Head, ARGO, Anand
E-mail: ajdhami@aau.in
Dr. Inderjeet Singh, Ph.D.
Director, CIRB, Hisar
E-mail: inderjeet.dr@gmail.com
Dr. V.K. Gandotra, Ph.D.
Professor, VGO, GADVASU, Ludhiana
E-mail: vkgandotra@yahoo.com
Dr. M. Selvaraju, Ph.D.
Professor & Head, Clinics, Namakkal
E-mail: mselvaraju1969@yahoo.co.in
Dr. G.K. Dass, Ph.D.
Senior Scientist, IVRI, Izatnagar
E-mail: gkdasivri@yahoo.co.in
EDITORIAL BOARD
EDITOR
Dr. Sarvpreet Singh Ghuman, Ph.D.
Head, Department of Teaching Veterinary Clinical Complex
Professor (Gynaecology and Obstetrics)
Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana - 141 004
Email: ghuman_s@yahoo.co.in, Mobile: 08146237600
EXECUTIVE COMMITTEE
Editor
Dr. Sarvpreet Singh Ghuman
Head, Department of Teaching Veterinary Clinical Complex
Professor (Gynaecology and Obstetrics)
Guru Angad Dev Veterinary and Animal Sciences University
Ludhiana- 141 004
Email: ghuman_s@yahoo.co.in
Mobile: 08146237600
General Secretary
Dr. Shiv Prasad
Professor and Head, AGRO, CVASc,
Pantnagar, Uttarakhand - 263 145
Mobile: 09411377368
Email: shivp2003@yahoo.co.uk
Joint Secretary
Dr. K. Veerabrahmaiah
Professor, VGO, CVSc,
SVUV, Tirupati
Mobile: 9849255258
Email: bramhaiahvet@yahoo.com
Treasurer
Dr. M. Selvaraju
Professor and Head, VGO,
Veterinary College and Research Institute,
Namakkal - 637 002
Mobile: 09443008610
Email: mselvaraju1969@yahoo.co.in
Ex-General Secretary
Dr. Shiv Prasad
Professor and Head, AGRO, CVASc,
Pantnagar, Uttarakhand - 263 145
Mobile: 09411377368
President
Dr. V. Chandrasheker Murti
Professor, VGO, CVSc,
Hebbal, Bangalore
Mobile: 9341228277
E-mail: chandravet@yahoo.co.in
Vice - President
Dr. V.K. Gandotra
Professor, VGO, CVSc.,
GADVASU
Mobile: 9876051591
Email: vkgandotra@yahoo.com
Dr. P.R. Nandi
VGO, CVSc,
WBA&FSc Univ
Kolkata - 700 037
Mobile: 8420172437
Email: drprnandi@gmail.com
Ex-President
Dr. A.K. Mishra
Vice - Chancellor
MAFSU, Nagpur
Mobile: 8888373830
E-mail: prof_misra@hotmail.com
Chapter Name of the Secretary Contact Number
Andhra Pradesh Dr. Prabakar Jetti 9010475475
Assam Dr. K. Ahamed 09435548233
Bihar & Jharkhand Dr. Balraj Singh 0641-2450301
Chattisgarh Dr. R.P. Tiwari 09827168315
Goa Dr. S.B. Barbudha 09422438073
Gujarat Dr. C.T. Khasatiya 09427255039
Haryana Dr. Pawan Singh 09416035184
Himachal Pradesh Dr. Navneet Vashisht 09418010262
Karnataka Dr. M.K. Tandle 09448569337
Kerala Dr. K.N. Aravinda Ghosh 09447174765
Madhya Pradesh Dr. S.P. Shukla 09826351075
Maharashtra Dr. S.K. Sahatpure 09921929929
Orissa Dr. Srinibas Das 09861337072
Punjab Dr. H.K. Verma 09815873929
Rajasthan Dr. Sumant Vyas 09351354543
RVC Chapter Col. Dr. Devendra Kumar 09877891266
Tamilnadu Dr. C. Chandrahasan 09443089531
Uttarakhand Dr. Shiv Prasad 09411377368
Uttar Pradesh Dr. A.K. Srivasta 09839025350
West Bengal Dr. Manojit Tewari 09433139249
EXECUTIVE COMMITTEE MEMBERS
THE INDIAN JOURNAL OF ANIMAL REPRODUCTION
(Ocial Organ of the Indian Society for Study of Animal Reproduction)
IJAR 30:1 June 2019; ISSN 0970 - 2997
CONTENTS
I. REVIEW ARTICLE Page No.
1 A REVIEW OF CURRENT BOVINE OBSTETRICAL PRACTICES
S.P.S. GHUMAN AND N. SINGH
1-6
II. RESEARCH ARTICLE
1 PURIFICATION OF SPERMATOGONIAL STEM CELLS FROM RAM TESTICULAR
ISOLATE USING FICOLL DENSITY GRADIENT SEPARATION
B.K. BINSILA, S. SELVARAJU, S.K. GHOSH, J.K. PRASAD, L. RAMYA, J.P.
RAVINDRA AND R. BHATTA
7-11
2 RELATIONSHIP BETWEEN SERUM PROGESTERONE AND ESTRADIOL-17β
AND EMBRYONIC LOSS IN FRISIAN COWS UNDER EGYPTIAN CONDITION
M.A. ABU EL-HAMD, A.K. KADOOM, W.F.M FOUAD AND S.H. HASSOUNA
12-15
3 CONCEPTION RATE BASED ACCURACY OF DIFFERENT METHODS TO DIAG-
NOSE SUB-CLINICAL ENDOMETRITIS IN COWS
NEELAM, MADHUMEET SINGH AND P. KUMAR
16-18
4 EFFECT OF MELATONIN ON NUCLEAR MATURATION OF CAPRINE OOCYTES
B.S. KUSHWAH, S.D. KHARCHE, J. AGARWAL, A.K.S. SIKARWAR, J. PATHAK
AND S. AGARWAL
19-23
5 ADDITION OF CHLOROQUINE DIPHOSPHATE OR ASCORBIC ACID IN JERSEY
BULL SEMEN AND SUBSEQUENT EVALUATION WITH RESPECT TO POST-
THAW INCUBATION TIME AND CONCEPTION RATE
MADHUMEET SINGH AND A. SHARMA
24-26
6 EFFECT OF TWO DIFFERENT PERMEABLE CRYOPROTECTANTS ON FREEZ-
ABILITY OF EXOTIC STALLION AND JACK SEMEN
T.R. TALLURI, S.K. RAVI, C. SINGH, R.A. LEGHA, S.C. MEHTA, V. PRAKASH
AND B.N. TRIPATHI
27-30
7 EFFICACY OF GARLIC EXTRACT±ASHWAGANDHA FOR THE TREATMENT OF
INFECTIOUS REPEAT BREEDING IN CATTLE
S.R. LAWANGE, N.M. MARKANDEYA, B.L. KUMAWAT, A.G. SAWALE, R.S. AN-
BHULE AND P.D. KADAM
31-34
8 EFFECT OF OXYTOCIN, PGF2α AND GnRH ON UTERINE INVOLUTION AND
POSTPARTUM FERTILITY IN MURRAH BUFFALOES SUBJECTED TO FETOTO-
MY
A.A. WANI, M.B. BHAT, P.S. MAVI AND P.S. BRAR
35-37
9 EFFICACY OF NEEM SEED EXTRACT±ASHWAGANDHA FOR THE TREATMENT
OF ENDOMETRITIS IN BUFFALOES
P.D. KADAM, N.M. MARKANDEYA, B.L. KUMAWAT, A.G. SAWALE, S.R. LA-
WANGE AND R.S. ANBHULE
38-41
III. SHORT COMMUNICATION
1 HIGH BLOOD / MILK UREA NITROGEN HAD DELETERIOUS IMPACT ON FER-
TILITY PARAMETERS IN CROSSBRED COWS
I. ANAND, G.S. DHALIWAL AND P.S. CHAWLA
42-44
2 CERVICAL MUCUS FERN PATTERN IN RELATION TO FERTILITY AND MI-
CRO-MINERAL PROFILE IN ANESTRUS SURTI BUFFALOES SUBJECTED TO
OVSYNCH ALONE AND IN COMBINATION WITH PRID
H.R. SAVANI, C.T. KHASATIYA, S. SAXENA, M.D. PATEL, V.R. PATEL, L.C. MODI
AND N.F. CHAUDHARI
45-46
3 ROLE OF EFFECTIVE COMMUNICATION IN ENHANCING DOG OWNER
KNOWLEDGE ABOUT BREEDING PRACTICES IN PUNJAB
Y. ROOPA, R. KASRIJA, H.K. VERMA AND P. SINGH
47-48
4 INFLUENCE OF TYPE OF WHELPING ON NEONATAL MORTALITY IN PUPS
A. SUBRAMANI, N. ARUNMOZHI, P. SRIDEVI AND M. SHAFIUZAMA
49-50
5 SERUM METABOLIC AND MACRO-MINERALS PROFILE IN POSTPARTUM
ANESTRUS SURTI BUFFALOES TREATED WITH OVSYNCH ALONE AND IN
COMBINATION WITH PRID
H.R. SAVANI, C.T. KHASATIYA, S. SAXENA, S.B. PATEL, V.K. SINGH, S.S.
CHAUDHARY AND C.F. CHAUDHARI
51-52
6 SYNCHRONIZATION OF OVULATION USING DOUBLESYNCH PROTOCOL IN
CROSSBRED CATTLE UNDER FIELD CONDITIONS
K. NAVAMANI, K. KULASEKAR, K. KRISHNAKUMAR AND L. RADHAKRISHANAN
53-54
7 IMPROVEMENT IN CONCEPTION RATE BY GnRH OR hCG ADMINISTRATION
ON DAY 5 POST-AI IN NON-INFECTIOUS REPEAT BREEDER COWS
R.S. ANBHULE, A.G. SAWALE, N.M. MARKANDEYA, B.L. KUMAWAT, P.D. KAD-
AM AND S.R. LAWANGE
55-57
IV. CASE REPORT
1 IMPERFORATE HYMEN AND SUBSEQUENT MUCOVAGINA IN A FILLY
R.P. PANDEY, B. KUMAR, V. SCHAN, A. SAXENA AND D. YADAV
58-60
2 DYSTOCIA DUE TO BILATERAL HOCK FLEXION IN A JENNY (EQUUS ASINUS)
V. SACHAN, B. KUMAR, A. SAXENA AND M.K. CHAUDHARY
61-62
3 SURGICAL MANAGEMENT OF TESTICULAR HYPERPLASIA IN A CRYP-
TORCHID LABRADOR RETRIEVER
T.M. MOMIN, D.N. KELAWALA, V.S. SUTHAR AND D.B. PATIL
63-64
4 CERVICOTOMY FOR THE MANAGEMENT OF DYSTOCIA DUE TO PREPARTUM
VAGINO-CERVICAL PROLAPSE IN COW
S. RAJA, M. PALANISAMY, V. PRABAHARAN, R. RAJKUMAR AND P. JAYAGAN-
THAN
65-66
5 MEDICAL MANAGEMENT OF A CLOSED TYPE PYOMETRA USING MISOPROS-
TOL AND CLOPROSTENOL COMBINATION IN A PUG
R.R. PATI, J. UMAMAGESWARI AND P. SRIDEVI
67-68
6 SUCCESSFUL MANAGEMENT OF A VIOLENT ASIAN ELEPHANT IN MUSTH
UNDER XYLAZINE ANAESTHESIA
J. AHMED, N. AHMED AND S. KYARONG
69-70
7 MANAGEMENT OF DYSTOCIA DUE TO MACERATED FETUS IN A MARE
Y. SONI, J.S. MEHTA, A. KUMAR, A. TRIPATHI AND T.R. TALLURI
71-72
V.
VI.
VII.
VIII.
IX.
REPORT OF ISSAR - 2017, ISSAR AWARDS - 2017, 33RD ISSAR CONVENTION
PROCEEDINGS AND SCIENTIFIC RECOMMENDATIONS
ISSAR LIFE MEMBERSHIP APPLICATION
AUTHOR GUIDELINES
AUTHOR’S DECLARATION CERTIFICATE
AUTHOR INDEX
74-80
81
82-84
85
86
Indian Journal of Animal Reproduction 40 (1): June 2019
1
1Professor cum Head; 2Assistant Professor, Directorate Livestock
Farms; *ghuman_s@yahoo.co.in
For a veterinarian and a dairy farmer, getting a
cattle or bualo pregnant is very tough job, followed
by health care during pregnancy. Unfortunately, if
an animal suers from dicult calving at the end of
gestation period and the case is not handled properly,
then the outcome may be death of fetus or dam or
both. This leads to nancial and emotional burden on
the farmer. In case of survival of dam, if an obstetrical
case is handled carefully there will be meagre
gynaecological complications later on. In a study,
available 34% conception data revealed that 90%
bualoes treated within 36h of occurrence of uterine
torsion conceived successfully (Singh et al., 2017).
Considering the importance of bovine obstetric care,
the present paper has focused on eective strategies
that can be adopted for the judicious delivery of a calf
and save the life of dam.
Management factors associated with uterine
torsion in bualoes
Uterine torsion is the major cause of dystocia in
bualoes which warrants immediate attention. Various
existing suppositions concerning the maternal and the
fetal destabilizing factors liable for the occurrence of
uterine torsion in bualo are unrealistic, however some
of these have been justied by logical interpretations.
Nevertheless, bualo reared in open housing system
and those reared by nomads in open grazing system
rarely encounter uterine torsion. In addition, Indian
Murrah bualo imported by Brazil in 1960s and reared
in big pastures on hills, never suer from uterine torsion.
In fact, a study involving 570 bualo farmers revealed
that extensively reared bualoes were at lower risk of
uterine torsion as compared to the stall-fed bualoes
(Singh et al., 2017). Daily exercise in the form of walk/
wallowing reduced the risk of uterine torsion. Keeping
bualoes on kacha oor is associated with lower risk
of uterine torsion. However, segregation of advance
pregnant bualoes and feeding practices had no impact
on incidence of uterine torsion (Singh et al., 2017).
This suggests the possibility of poor musculature, due
to failure of exercise, in bualo suering from uterine
torsion as these bualo usually belong to farmers
who rear bualo in closed/tie housing system. Thus,
bualo farming community can be advised to expose
bualo to free movement for some period of the day so
that perineal/abdominal muscles are strong and well
A REVIEW OF CURRENT BOVINE OBSTETRICAL PRACTICES
S.P.S. GHUMAN1* AND N. SINGH2
Department of Teaching Veterinary Clinical Complex, Guru Angad Dev Veterinary and Animal Sciences
University, Ludhiana - 141 012
Received: 10.09.2018 Accepted: 20.09.2018
ABSTRACT
The dairy animal population especially bualo suers from various obstetrical problems, the solution for some
of these have been devised and are in process for the others. There is need to understand management factors
associated with uterine torsion in bualoes, the physiological and cardiovascular impact of dystocia. This will
help in timely resuscitation of dystocia-aected dam through the use of tranquilizers, glucocorticoids plus anti-
oxidants and crystalloid plus colloid based uid therapy. Huge success has been achieved in cervical massage or
intracervical treatment for dilatation of incompletely dilated cervix as well as timely and appropriate handling of an
obstetrical case by understanding the importance of time factor. The use of uterine lubricants improves success
rate. Fetotomy should be used as preferred obstetrical procedure by making the availability of an appropriate
Fetotomy kit and the knowledge of ‘Strength-Stamina-Skill’ for carrying out fetotomy procedures.
Keywords: Bualo, Doppler, Fetotomy, Obstetrics, Uterine torsion
Indian Journal of Animal Reproduction 40 (1): June 2019
2
developed.
Understanding the cardiovascular impact of
dystocia
Doppler ultrasonography-aided assessment of
uterine blood ow in relation to duration and degree
of uterine torsion was carried out in cattle (Singh et
al., 2016a). Fourteen dairy cattle with uterine torsion
were detorted and fetal delivery was completed within
30 min after detorsion. Six live calves were delivered
by cattle having torsion from lesser duration and rest
dead calves delivered by dams with prolonged uterine
torsion. Whereas the dams of majority of live (n=4/6) or
dead (n=5/8) fetus had uterine torsion ≤180° or >180°,
respectively. Doppler ultrasonography of middle uterine
artery ipsilateral (IpsiUA) and contralateral (ContUA)
to the side of torsion was carried out before uterine
detorsion for doppler indices viz. blood ow volume
(BFV), time-average peak velocity (TAP), resistive
index (RI) and pulsatility index (PI). With increase in
degree and duration of torsion, BFV in both IpsiUA and
ContUA was reduced. In long standing uterine torsion,
TAP values were found lower as compared to short
duration torsions in both ipsilateral and contralateral
uterine arteries. In ipsilateral uterine artery PI (PI-
IpsiUA) increased with an increase in duration of
torsion. The presence of Pre-diastolic notch in IpsiUA
and ContraUA validates the hindrance in blood ow
through the vessel and absence of diastole in higher
degree and/or duration uterine torsion dened the
severity of torsion which further relates to fetal viability
(Singh et al., 2016a). This suggested that assessing
the blood ow parameters in middle uterine artery in
relation to degree and duration of uterine torsion can
serve as useful prognostic indicator. The cattle having
lesser degree of uterine torsion could have more
chances of fetal survival due to lesser alterations in
blood ow.
Furthermore, the uterine blood ow during
peripartum period in bualo was associated with
shedding of placenta (Singh et al., 2016b). The values
of TAP and BFV in the uterine artery recorded at the start
of calving process invariably exhibited a decreasing
trend by 30 min post-calving which continued till 6 h
post-calving. The values of RI and PI in uterine artery
ipsilateral to side of pregnancy increased consistently
till 6 h post-calving as compared to pre-calving values.
At 6 h post-calving, both in IpsiUA as well as ContUA,
the values of TAP and BFV were higher, and RI and PI
were lower in bualo that failed to shed placenta within
6 h post-calving in comparison to their counterparts
taking >6 h after calving for shedding placenta (Singh
et al., 2016b). Thus, the reference values for doppler
indices for uterine artery during peripartal period were
generated and it was observed that the decrease
observed in blood ow in uterine artery was slow in
bualo with delayed shedding of placenta.
Understanding the physiological impact of
dystocia
The occurrence of dystocia and the extent of
obstetrical manipulations are highly stressful and are
directly reected through an increase in adrenal activity.
In dystocia-aected bualo, plasma cortisol is much
higher compared to their normal calving counterparts
(41.61±4.18 vs. 8.65±1.14 ng/ml; Ghuman et al., 1998).
Furthermore, applying uterine detorsion procedures
exhibits clear impact on plasma cortisol with excessive
adrenal stimulation due to one to two extra detorsion
rolls (Ghuman et al., 1997b). The survival rate of
bualo requiring 1-2 or 3-4 rolls for complete detorsion
of uterus was 85 and 43%, respectively (Ghuman et
al., 1997b). The type of obstetrical procedures also
impacts the physiology of the dam. The release of
plasma cortisol in bualo subjected to caesarean was
more and for longer duration than their counterparts
subjected to fetotomy, thus, leading to poor survival
rate in bualoes following delivery of calf through
caesarean section (25-95%) compared to per-vaginal
delivery (88-100%; Ghuman and Dhaliwal, 2011).
Resuscitation of dystocia-aected dam
Tranquilizers: Tranquilization of bualo before
detorsion of uterus failed to prevent the stressful impact
of uterine torsion (Ghuman et al., 1997a). Moreover,
Ghuman and Singh
Indian Journal of Animal Reproduction 40 (1): June 2019
3
during post-detorsion period, alleviation of stress was
poor in bualo detorted under chlorpromazine and
diazepam tranquilization. In fact, tranquilization with
chlorpromazine and diazepam had paralyzing action
on skeletal muscles leading to recumbency for longer
duration (Ghuman et al., 1997a). This warrants more
studies on tranquilization therapy for alleviating the
stress of dystocia.
Glucocorticoids plus anti-oxidants: Dystocia
aected bualo administered dexamethasone sodium
(40 mg, i.v.) before relieving dystocia and subsequently
once daily for 2 days postpartum lead to signicant
reduction in plasma cortisol on Day 1 postpartum, thus
suggesting that Hypothalamus-Pituitary-Adrenal axis
was responsive to dexamethasone-induced negative
feedback during the immediate postpartum period
following dystocia (Sathya et al., 2005). A decline in
plasma cortisol, erythrocytic malondialdehyde level and
superoxide dismutase activity was greater in vitamin
E and Selenium supplemented dystocia-aected
bualo (78.62±2.7%, 13.38±9.75% and 28.83±7.72%,
respectively) compared to their unsupplemented
counterparts (54.33±12.62%, 3.48±4.16% and
17.69±12.93%, respectively; Sathya et al., 2007), thus
suggesting that providing antioxidant supplementation
is benecial in reducing oxidative stress in dystocia
aected bualo.
Crystalloid plus colloid based uid therapy: The
delay in handling of dystocia leads to progression
of dam towards dehydration and toxaemia due to
decrease in plasma and blood volume. Intravenous
uid administration is considered best method for
treating dehydrated bovines. In routine dystocia
cases, a meager amount of intravenous normal saline
therapy (NSS) is used. However, a large amount of
NSS is required as the bovines suering from mild to
severe dehydration (8-12%) are recommended around
50-120 ml NSS/kg b wt, which is a huge amount
and not practicable to administer due to exuberant
cost, catheterization of vein, prolonged restraint and
periodic monitoring. The possible way out is the use
of large volume of oral uid (fresh water) with small
volumes of intravenous hypertonic saline either
with or without Dextran-40 (7.2% HSS @ 4ml/kg +
Dextran-40 @10ml/kg + Oral uid 25L). Intravenous
administration of Dextran-40 is necessary to maintain
elevated plasma and blood volume for longer durations
and thus decrease the degree of dehydration. Thus, it
was recommended that administration of fresh water
through oral route along with intravenous hypertonic
saline and Dextran-40 could be a quicker, practical,
easy and eective method for resuscitating the
dystocia aected bualoes suering from variable
degree of toxaemia and hypovolemia, and thereby
decreasing their mortality rate (Kumar et al., 2009a, b).
Cervical massage or intracervical treatment: The
challenge of achieving complete cervical dilatation in
successfully detorted uterine torsion aected bualo
carrying dead fetus can be taken care by cervical
massage with SCMC, otherwise leaving the soft or
moderately soft cervix on its own to dilate will lead
to hardening of cervical texture followed by its failure
to dilate. In a study, a procedure of manual dilatation
of cervix was developed for bualo in which cervical
massage for 15 minutes (3 times at hourly interval)
can be carried out using warm SCMC (Honparkhe et
al., 2009). Using this procedure, cervix can be dilated
in all the bualo with soft cervical texture, whereas
success rate up to 50% can be achieved in bualo
with moderately soft cervix. Out of bualo with soft
cervical texture and not being subjected to cervical
massage, only 29% achieved cervical dilatation
whereas none of the bualo with moderately soft cervix
achieved cervical dilatation. In the absence of cervical
massage, soft cervical texture was converted to hard
texture within 24 h following detorsion of uterus and
subsequently cervix failed to dilate (Honparkhe et al.,
2009). In another study, 24 bualoes with incomplete
dilation after successful detorsion were subjected to
dierent cervical dilation treatments. The complete
dilation of cervix occurred in bualoes (87.5%) treated
intracervically with hyaluronidase enzyme, whereas
Prostaglandin E1 led to dilation in 62.5% of bualoes
Bovine obstetrics
Indian Journal of Animal Reproduction 40 (1): June 2019
4
(Singh et al., 2017).
Timely and appropriate handling of an obstetrical
case
Importance of time factor: An obstetrical case
handling in the eld by quacks is a major constraint
behind the poor survivability of bovines. Depending
upon injudicious handing, survival rates of torsion
aected bovines presented in <36 h, 36-72 h and >72
h of occurrence of torsion are 52-86, 29-74 and 32-
62%, respectively (Ghuman, 2010). Cases around
lower range of the survival rate are those that are rst
handled in the eld. In fact, survival rate in torsion
aected bovine declines linearly (from 87 to 43%) with
an increase in duration of uterine torsion (Ghuman,
2010). The duration of uterine torsion and the time
taken for complete dilatation of cervix increases the
severity of uterine necrosis, fetal putrefaction, maternal
toxemia, dehydration, shock and peritonitis. The bualo
that ultimately died following detorsion and the bualo
that delayed the fetal delivery following detorsion had
prolonged elevated plasma cortisol as compared
to surviving counterparts and the early delivering
counterparts, respectively (Ghuman et al., 1998). This
warrants creating awareness among farmers and eld
practitioners’ for timely and appropriate handling of an
obstetrical case.
Uterine lubricant: The use of adequate amount
of uterine lubricant allows the correction of fetal
malpresentation and helps in carrying out procedures
like fetotomy for successful per-vaginal delivery.
In fact, for appropriate handling of dystocia, the
importance of uterine lubricant can be understood
from the fact that by the time, an obstetrical case is
presented in a referral hospital, the birth passage
becomes dry due to continuous contraction of the
uterus and the previous handling of case through
repeated vaginal examination. Moreover, the uterus
gets contracted on the fetus following the expulsion
of uterine uid. In the absence of uterine lubrication,
fetal delivery through fetal mutations by an obstetrician
becomes impossible. Thus, if the birth passage is dry,
then copious volume of a non-irritant lubricant like
sodium carboxy methylcellulose (SCMC, a commonly
available chemical from laboratory chemical suppliers)
is required for birth passage lubrication. One per cent
solution of SCMC can be prepared by boiling 200 ml of
clean water and slowly adding 10 gram SCMC powder
while stirring (Ghuman, 2015). Additional clean water
can be added while stirring to bring the total volume to
a litre. Few crystals of potassium permanganate can
be added to the gel solution. This product is extremely
slippery, and good footing is essential. A sterile
stomach tube and pump are used to gently instill the
mixture into the uterine lumen.
Fetotomy as preferred obstetrical procedure
Fetotomy is usually advantageous because dam
survival rate is high following fetotomy compared to
caesarean, future fertility is not compromised, post-
operative complications are less and excessive stress
to dam associated with forced traction is avoided
(Singh et al., 2013). Fetotomy is usually indicated
in case of emphysemated fetus to avoid the risk of
peritonitis following caesarean operation as well
as in cases of fetal malpresentation that can not be
corrected by mutation (Hip-lock, Breech presentation),
feto-pelvic disproportion (narrow pelvis, oversized
fetus) and in case of partially dilated cervix (Ghuman,
2010). However, it should be considered that fetotomy
is a time-consuming process, exhaustive for the
obstetrician and there is risk of injury to obstetrician
and dam. Moreover, fetotomy should be avoided in
case of tear in the birth canal of dam as the tear can be
aggravated with fetotomy instruments and may prove
fatal for dam.
Fetotomy kit: The non-availability of good quality
fetotomy kit is a major constraint and hence some
obstetrical cases, which can be relieved through per-
vaginal delivery, are subjected to caesarean operation.
In the kit, a good quality Thygesen’s fetotome is a
prime necessity. A poor quality fetotome usually leads
to breakage of fetotomy wire due to failure of fetotome
to allow free movement of wire. The second major kit
Ghuman and Singh
Indian Journal of Animal Reproduction 40 (1): June 2019
5
component is a strong and exible fetotomy wire (27
interwoven steel wires in a group of 3 x 3 x 3 wires)
which is required to compete the cut through soft and
bony tissues of the fetus (Ghuman, 2014). The non-
availability of this fetotomy wire in local market is also
a constraint, as the wire needs to be imported. Other
important fetotomy kit components are calving rope
carrier, guarded knife and eye hooks.
Strength-Stamina-Skill: The perfection in fetotomy
depends on technical knowledge, adequate training
and experience, correctly designed instruments and
proper lubrication. Clinician intending to carry out
fetotomy should consider the rule of 3S viz, ‘Strength-
Stamina-Skill’ for completing a fetotomy procedure and
the technical knowledge regarding where the cut(s)
should be made. If clinician is not familiar with correct
technique then the best option is caesarean section
(Ghuman, 2010). A common fault is to choose fetotomy
only after the birth canal has already been traumatized
by unproductive attempts at manual correction. Avoid
manipulating dry birth canal as this may lead to uterine
rupture and ultimately unsuccessful fetotomy. The
obstetrician should ensure that the wire threaded
around the fetal part to be amputatd is not crossed or
kinked, head of the fetotome is in the correct position,
cover fetotome head with a hand and avoid producing
sharp skeletal edges as they may damage the birth
canal. An assistant is instructed to start the cut by slow,
short, to-and-fro arm movements. Longer movements
decrease the amount of heat generated and spread the
wear on the wire. Minimize the number of cuts required
for delivery of all the fetal parts. This will shorten the
intervention time and permit a less traumatic delivery
of dead fetus.
REFERENCES
Ghuman, S.P.S. (2010). Uterine torsion in bovines: a
review. Indian J. Anim. Sci., 80: 289-305.
Ghuman, S.P.S. (2010). Fetal malpresentation - a risk
factor for perinatal mortality on a dairy farm. INTAS
Polivet, 11: 11-14.
Ghuman, S.P.S. (2012). Recent advances in obstetrical
procedures with special reference to fetotomy. In:
Compendium of State Symposium on ‘Recent
Advances in Reproductive Technologies and
Obstetrical procedures for Augmenting Fertility
in Farm animals’ and First joint convention of
Mahavet Satara and ISSAR Maharashtra chapter
at Panchgani, Dist. Satara, Maharashtra on
February 5, 2012. pp 19-32.
Ghuman, S.P.S. (2014). Fetotomy – A tool to resolve
dystocia and salvage reproductive future of dairy
animals. INTAS Polivet, 15: 318-323.
Ghuman, S.P.S. (2015). Gynaecological interventions
for managing the menace of infertility in dairy
animal. In: e-compendium of ‘Animal Husbandry
Ocers’ workshop’ held at Guru Angad Dev
Veterinary and Animal Sciences University,
Ludhiana on June 26-27, 2015. pp 88-96.
Ghuman, S.P.S.and Dhaliwal, G.S. (2011). Alleviating
the Stress of Dystocia in Bovines. In: Proceedings
of 27th Annual Convention of ISSAR and National
Symposium on ‘Reproductive Biotechnologies for
Augmenting Fertility and Conservation of Animal
Species with Special Reference to North Eastern
Hill Region’ at Central Agricultural University,
Aizawl, Mizoram from Sept 27-29, 2011. pp 112-
121.
Ghuman, S.P.S., Nanda, A.S., Prabhakar, S. and
Sharma, R.D. (1996). Stress related endocrine
and metabolic changes in normally calving and
torsion aected bualoes. Indian Vet. J., 73: 1142-
1146.
Ghuman, S.P.S., Nanda, A.S., Sharma, R.D. and
Prabhakar, S. (1997a). The eect of tranquilizers
on stress response in bualoes aected with
uterine torsion. Indian Vet. J., 74: 1034-1036.
Ghuman, S.P.S., Sharma, R.D., Prabhakar, S. and
Nanda, A.S. (1997b). Plasma cortisol and blood
glucose milieu as an index of stress in bualoes
with uterine torsion. Indian J. Anim. Reprod., 18:
83-84.
Bovine obstetrics
Indian Journal of Animal Reproduction 40 (1): June 2019
6
Ghuman, S.P.S., Sharma, R.D., Nanda, A.S. and
Prabhakar, S. (1998). Eect of certain clinical
procedures and stressors on plasma cortisol
milieu in dystocia aected bualoes. Bualo J.,
14: 71-79.
Honparkhe, M., Ghuman, S.P.S., Kumar, A., Sood,
N.K., Gupta, K. and Ahuja, C.S. (2009). Cervical
massage with sodium carboxy methyl cellulose
for achieving complete cervical dilatation in
successfully detorted uterine torsion aected
bualoes. Indian J. Anim. Sci., 79: 26-29.
Kumar, A., Ghuman, S.P.S., Ahuja, C.S. and
Honparkhe, M. (2009a). Eect of oral uid therapy
on endocrine and biochemical status of dystocia
aected bualoes. Indian Vet. J., 86: 1021-1023.
Kumar, A., Ghuman, S.P.S. and Honparkhe, M.
(2009b). Eect of oral uid therapy in combination
with intravenous dextran-40 and hypertonic saline
solution on plasma and blood volume in dystocia
aected bualoes. Indian J. Anim. Sci., 79: 479-
482.
Sathya, A., Prabhakar, S. and Ghuman, S.P.S. (2005).
Eects of dexamethasone administration on
cortisol concentration and biochemical prole in
bualoes suering from dystocia. Anim. Reprod.,
2: 233-239.
Sathya, A., Prabhakar, S., Sangha, S.P.S. and
Ghuman, S.P.S. (2007). Vitamin E and selenium
supplementation reduces plasma cortisol and
oxidative stress in dystocia-aected bualoes.
Vet. Res. Comm., 31: 809-818.
Singh, G., Pandey, A.K., Agnihotri, D., Chander, S.,
Chandolia, R.K. and Dutt, R. (2013). Survival
and fertility rate in bualoes following caesarean
section and mutation with/without partial fetotomy.
Indian J. Anim. Sci. 83: 251-253.
Singh, N. (2017). Doppler sonography as prognostic
indicator in uterine torsion aected bualoes.
Ph.D. Thesis. Guru Angad Dev Veterinary and
Animal Sciences University, Ludhiana (Punjab).
Singh, N., Ghuman, S.S. and Gandotra, V.K. (2016a).
Predicting the impact of degree and duration of
uterine torsion on uterine blood supply in cattle
using doppler ultrasonography. J. Anim. Res., 6:
685-689.
Singh, N., Gandotra, V.K., Ghuman, S.P.S., Dhindsa,
S.S. and Brar, P.S. (2016b). Uterine blood
ow during peripartum period in bualo and its
association with shedding of placenta. Indian J.
Anim. Reprod., 37: 33-36.
Ghuman and Singh
Indian Journal of Animal Reproduction 40 (1): June 2019
7
INTRODUCTION
Spermatogonial stem cells (SSCs) possess a vital
role in male reproduction, genetics and transgenesis
studies, however, the successful purication of SSCs
from testicular isolate is essential for culture and
transplantation (Binsila et al., 2017). The testis consists
of dierent types of cells like Sertoli cells, Leydig cells,
SSCs and dierent stages of dierentiating germ cells
with the percentage of SSCs to be <1% of testicular
cells. Attempts were made to isolate and purify SSCs
with high viability. Although, the isolation and culture
of SSCs has started in bovine, caprine, porcine and
bubaline (reviewed in Zheng et al., 2014), the isolation,
culture and transplantation of SSCs in livestock is in
a nascent stage. Considering the less population of
SSCs in the testicular cell isolate, several enrichment
procedures were tried to obtain a maximum population
of SSCs with appropriate quality which is a prerequisite
for downstream applications such as culture
experiments and or transplantation. The enrichment
procedures include magnetic-activated cell sorting
(MACS), uorescence activated cell sorting (FACS),
dierential plating, selection with extracellular matrix
(ECM), velocity sedimentation and density gradient
centrifugation (Borjigin et al., 2010; Valli et al., 2014).
In addition, the combination of enrichment techniques
may augment the purity of spermatogonia (Herrid
et al., 2009). As the initial isolate of testicular cells
contains cells of varying size and density, the density
gradient separation can be eectively used to enrich
SSCs. Recently, the Ficoll density gradient technique
provided optimum purity with high viability (Joseph et
al., 2017). Hence, the objective of present study was
to isolate and enrich SSCs from the ram testes using
Ficoll density centrifugation.
1Scientist, 2ICAR-National Fellow, 5RA-National Fellow Project, 6Acting
Head, 7Director; 3,4Principal scientist, Animal Reproduction Division,
ICAR-Indian Veterinary Research Institute, Izatnagar - 243122;
*drbinsila@gmail.com
PURIFICATION OF SPERMATOGONIAL STEM CELLS FROM RAM TESTICULAR ISOLATE
USING FICOLL DENSITY GRADIENT SEPARATION
B.K. BINSILA1*, S. SELVARAJU2, S.K. GHOSH3, J.K. PRASAD4, L. RAMYA5, J.P.
RAVINDRA6 AND R. BHATTA7
Reproductive Physiology Laboratory, Animal Physiology Division, ICAR-National Institute of Animal Nutrition
and Physiology, Adugodi, Bengaluru – 560030
Received: 16.03.2018 Accepted: 26.04.2018
ABSTRACT
Spermatogonial stem cells (SSCs) can be used to propagate male superior germ plasm and to preserve the
male of high genetic merit and endangered species. The objective was to assess the eciency of dierent Ficoll
density gradient separation in order to purify SSCs from the testicular cell isolates. The mixed testicular isolates
obtained after enzymatic digestion of testicular tissues was subjected to two dierent Ficoll density gradient
methods (method 1: Ficoll at 10 and 12% and method 2: Ficoll at 10, 12 and 16%). The percentage of SSCs
(positive for PLZF, an SSC marker) obtained from the fractions, F12 media and F12-16 Interphase (FI) yielded
(p<0.05) higher percentage of PLZF+ spermatogonia as compared to initial testicular isolate (35.1±3.8 and
22.8±4.5 vs 11.2±3.7). The viability (%) of F12 and FI enriched fraction was 55.6±4.3 and 51.2±6.5, respectively.
In brief, Ficoll purication method using F12 fraction yielded higher (p<0.05) recovery rate (4.9±1.2x106 cells/g of
testis) with improved purity (1.8±0.4x106 PLZF+ cells) when compared to FI (recovery rate: 3.28±1.2x106 cells/g of
testis and purity: 0.8±0.3x106 PLZF+ cells) and can be used either alone or in combination with other purication
methods for further enrichment of SSCs that can be used for culture.
Keywords: Ficoll density gradient, PLZF positive, Ram, Spermatogonial stem cell, Testis
Indian Journal of Animal Reproduction 40 (1): June 2019
8 Binsila et al.
MATERIALS AND METHODS
Isolation of putative SSCs: Prepubrtal ram testes
samples (n=6) procured from the slaughterhouse
were transported within 2 h in ice-cold saline to
the laboratory. After removing tunica vaginalis and
epididymis, testes tissue samples were excised
mechanically, cut into small pieces and testicular cells
were isolated immediately using enzymatic methods
(Izadyar et al., 2002; Borjigin et al., 2010) with slight
modications. In brief, testes tissue (2 g) was washed
using Dulbecco’s Modied Eagles Medium (DMEM)
and placed in DMEM. The tissues were mechanically
sliced into small pieces, ground with syringe plunger
to form homogenized tissue mass. The homogenized
samples were added with 4 ml DMEM containing
collagenase (2 mg/ml) and incubated for 1 h at 37°C.
The digested tissue was washed twice with Dulbecco’s
phosphate buered saline (DPBS) by centrifugation at
269 g for 5 min and the supernatant was removed.
The pellet containing digested tissues was added with
trypsin (0.5 mg/ml) in 5 ml DPBS and incubated at
37˚C for 5-7 min followed by inactivation of trypsin with
the addition of an equal volume of DMEM containing
5% fetal bovine serum (FBS). The digested tissues
were ltered through 70 µm strainer (352350, BD
Falcon) and the ltrate containing cells were washed
using DMEM twice by centrifugation at 269 g for 5 min
and the supernatant was discarded. The cell pellet
containing mixed testicular cells was suspended in
DMEM containing 5% fetal bovine serum (Initial cells
isolate, IC).
Enrichment using Ficoll density gradient
separation: Putative SSCs from the IC were enriched
using Ficoll density gradient centrifugation (Panta
et al., 2011) with modications. Briey, two dierent
gradients were carried out viz. Ficoll at 10 and 12%
and Ficoll at 10, 12 and 16%. The isolated cells from
each gram testes were mixed in 2 ml of 10% Ficoll
in DMEM and slowly layered on to the top of 2 ml of
12% Ficoll in DMEM in a 15 ml centrifuge tube. The
tubes were centrifuged (5810R, Eppendorf, Germany)
at 800 g for 30 min at 18˚C. From 10 and 12% gradient
(method 1), 12% Ficoll fraction (F12: pellet and 12%
media) and from 10, 12 and 16% gradients (method
2), the interphase of 12 and 16% fraction (FI) were
collected. These fractions were enriched in SSCs
based on our preliminary study. F12 and FI fractions
were centrifuged at 1680 g for 5 min. Supernatants
were discarded and the pellet was washed in the
media (DMEM added with 5% FBS) by centrifugation
at 269 g for 5 min. The supernatant was removed and
pellet was re-suspended in the media. Ficoll enriched
cells were analyzed for the stem cell property using
marker Promyelocytic leukemia zinc nger protein
(PLZF), viability and concentration (Figure 1).
Figure 1: Steps followed for the enrichment of ram
SSCs using Ficoll density gradient separation. The
initial testicular isolate was subjected to Ficoll
density gradient (10 and 12%; 10, 12 and 16%)
separation. The SSCs were enriched in 12% Ficoll
fraction (F12: Pellet and 12% media; FI: interphase
of 12 and 16%).
Localization of dierent SSC markers: The purity of
putative SSCs in dierent fractions of Ficoll separation
was assessed using PLZF (SSC marker) localization in
the initial isolate and enriched fractions (Somashekar
et al., 2017) with slight modications. Briey, the
smears were xed with 4% paraformaldehyde for 10
min and rinsed with TBST (Tris-buered saline with
Indian Journal of Animal Reproduction 40 (1): June 2019
9Spermatogonial stem cell isolation from ram
0.05% Tween 20). Then the slides were incubated in
0.01% Triton-X in TBS for 5 min and washed with TBS.
The slides were incubated in TBS containing 0.6%
H2O2 for 20 min. The nonantigenic sites were blocked
by adding a solution of 3% BSA in TBST onto the
cells and incubating at room temperature for 30 min.
The smear was added with primary antibody (rabbit
polyclonal IgG: PLZF, 1:100 dilution, sc-22839, Santa
Cruz Biotechnology, Santa Cruz, USA) and incubated
overnight at 4ᵒC. After incubation, the slides were
washed three times using TBST for 5 min each and
incubated with secondary antibody (105499, Goat
anti-rabbit IgG-HRP, GeNei, India) for 30 min at room
temperature. Then the slides were washed three times
with TBST for 5 min each and incubated with DAB
substrate for 10 min for the development of brown color.
For negative controls, the smears were incubated in
the buer (TBST) without primary antibody. The cells
were counterstained with hematoxylin. The cells were
observed under 400X magnication using a phase
contrast microscope (Nikon 80i, Nikon, Japan) and
the percentage of cells positive for PLZF activity was
calculated.
Viability of isolated and enriched cells: The viability
of the isolated and enriched cells was assessed by
trypan blue (0.5%) staining. Equal volume (5 µl) of
trypan blue and cells suspension in medium were
mixed and incubated for 2 min and observed at 400X
magnication in a phase contrast microscope (Nikon
80i, Nikon, Japan). The cells with blue color were
considered as dead and those did not take up the
stain were considered as viable and the percentages
of viability were calculated.
Cell concentration: The number of cells obtained
following isolation and enrichments was calculated
using hemocytometer and the total number of
cells /g of testis were estimated. Based on the
immunocytochemistry (ICC) results, the number of
SSCs yield /g of testis was calculated in the initial
isolate and enriched fraction.
Cell morphology documentation: The cell
morphology was estimated in the initial isolate and
enriched fractions using a phase contrast microscope
(Nikon 80i, Nikon, Japan).
RESULTS AND DISCUSSION
The present study was carried out to enrich ram
SSCs from initial testicular isolate using Ficoll density
gradient separation. In the method 1, SSCs (PLZF+
cells) were enriched in 12% Ficoll fraction (F12: pellet
and 12% media; 35.1±3.8%) and using the method
2, Ficoll at 10, 12 and 16% gradients, the SSCs were
enriched in the interphase of 12 and 16% fraction
(FInterphase; 22.8±4.5%; Table1). The percentage
of PLZF+ cells in F12 fraction was higher (p<0.05)
than the initial isolate, however, did not dier (p>0.05)
with FI fractions. The morphologically enriched cells
were round in shape with a homogenous size (Figure
2a). In the present study, 3.6 (FI) and 4.1 (F12) Fold
enrichment obtained through Ficoll gradient separation
Figure 2: (a) Morphological appearance of pre-
enriched and enriched (round in shape with a
homogenous size) SSC fractions, (b) The cells
stained with trypan blue are dead and others are
live, and (c) The putative SSCs (brown colour)
were identied using PLZF marker. 400x
Indian Journal of Animal Reproduction 40 (1): June 2019
10
is in agreement with the 2.9 fold in bovine (Herrid et
al., 2009) and 3.6 fold in sheep (Rodriguez-Sosa et
al., 2006) through percoll enrichment. Further, the
inclusion of density gradient separation along with
dierential plating method improved the purity of SSCs
(42.70 to 64.39% GFRα+ cells) in cats (Tiptanavattana
et al., 2015).
The viability of puried fraction following Ficoll
density gradient though reduced (p<0.05) as compared
to initial isolate, did not adversely aect the culture
procedure (unpublished data; Table 1, Figure 2b).
Similarly, an earlier study reported reduced viability
of enriched bovine SSCs obtained through Percoll
density gradient centrifugation (de Barros et al., 2012).
The average yield of number of mixed testicular cells
from each gram of testicular tissue was 17.67x106 and
the puried fractions, F12 and FI yielded 4.85x106
and 3.28x106 cells from prepubertal ram, respectively
(Table 1). The total number of PLZF+ cells in F12
and FI fractions were 1.8±0.4 and 0.8±0.3 million,
respectively (Figure 3).
The study revealed that even though there was
no signicant dierence in PLZF+ cells between the
two enriched fractions (F12 and FI), the F12 fraction
was better based on higher recovery rate. Hence, F12
fraction of 10 and 12% Ficoll density gradient may be
used for the culture of SSCs. Through PLZF localization
study, it was revealed that SSCs can be enriched by
Ficoll density gradient separation and the procedure
is suitable for eliminating the other testicular cells like
dierentiating spermatogonial population and Sertoli
cells as these cells will be separated out in dierent
layers based on the density (Figure 2c). A similar
study for the enrichment of SSCs using Ficoll density
gradient was reported in rodent (Jeong et al., 2003)
and sh (Panda et al., 2011). Percoll density gradient
though chemically dierent, but separates cells based
on density and has been used for SSCs isolation in
dierent studies (Izadyar et al., 2002; Tiptanavattana
et al., 2015). As the initial isolate of the testicular cells
contains cells of varying size and density, density
gradient separation might be a suitable option to
segregate the subsets of homogenous cells. The
present study, though provide the optimum number
of PLZF positive cells for culture, the SSCs enriched
fraction from density gradient separation can be
combined with other protocols like dierential plating
(Izadyar et al., 2002; Tiptanavattana et al., 2015) or
Figure 3: Total number of PLZF+ cells (x106) in
12% Ficoll fraction (F12: pellet and 12% media,
Method 1) and in the interphase of 12% and 16%
fraction (FI, Method 2) of ram testes isolates
(n=6). *p<0.05.
Table 1: Enrichment of putative spermatogonial stem cells (SSCs) from the initial ram testes isolates (n=6)
using Ficoll density gradient centrifugation. Method 1: 12% Ficoll fraction (F12: pellet and 12% media),
Method 2: Interphase of 12% and 16% fraction (FI). PLZF, Promyelocytic leukemia zinc nger protein.
Parameters Initial isolate Ficoll enrichment
Method 1 Method 2
Total no. of cells (x106) /g of testis 17.7±2.8 4.9±1.2 3.3±1.2
Viability, % 72.0±4.1c51.2±6.5ab 55.6±4.3a
PLZF+ve, % 11.2±3.7a35.1±3.8bc 22.8±4.5ab
a,b,cp<0.05
Binsila et al.
Indian Journal of Animal Reproduction 40 (1): June 2019
11Spermatogonial stem cell isolation from ram
magnetic activated cell sorting (MACS) (Panda et al.,
2011) for improving enrichment eciency. Another
concern from the experiment is the reduced viability of
isolated cells. The viability of the cells may be probably
improved by reducing the duration of experimental
procedures.
ACKNOWLEDGEMENTS
We gratefully acknowledge The Director, ICAR-
NIANP, Bengaluru for the facilities and support
provided.
REFERENCES
Binsila, B.K., Selvaraju, S., Somashekar, l., Archana,
S., Arangasamy, A., Ravindra, J.P. and Bhatta,
R. (2017). Molecular advances in semen quality
assessment and improving fertility in bulls-a
review. Indian J. Anim. Reprod., 39(1)
Borjigin, U., Davey, R., Hutton, K. and Herrid, M.
(2010). Expression of promyelocytic leukaemia
zinc-nger in ovine testis and its application in
evaluating the enrichment eciency of dierential
plating. Reprod. Fertil.Dev., 22: 733–742.
de Barros, F.R.O., Worst, R.A., Saurin, G.C.P.,
Mendes, C.M., Assumpção, M.E.O.A. and Visintin,
J.A. (2012). α‐6 Integrin Expression in Bovine
Spermatogonial Cells Puried by Discontinuous
Percoll Density Gradient. Reprod. Domest.
Anim., 47: 887-890.
Herrid, M., Davey, R.J., Hutton, K., Colditz, I.G.
and Hill, J.R. (2009). A comparison of methods
for preparing enriched populations of bovine
spermatogonia. Reprod. Fertil. Dev., 21: 393-399.
Izadyar, F., Spierenberg, G.T., Creemers, L.B., Den
Ouden, K. and De Rooij, D.G. (2002). Isolation
and purication of type A spermatogonia from the
bovine testis. Reproduction, 124: 85-94.
Jeong, D., McLean, D.J. and Griswold, M.D. (2003).
Long‐Term Culture and Transplantation of Murine
Testicular Germ Cells. J. Androl., 24: 661-669.
Joseph, J.G., Liu, S.X., Cheung, M.K., Haey, P.J.,
Kurabayashi, K. and Fu, J. (2017). Centrifugal
microuidics for sorting immune cells from whole
blood. Sens Actuators B Chem., 245: 1050-1061.
Panda, R.P., Barman, H.K. and Mohapatra, C.
(2011). Isolation of enriched carp spermatogonial
stem cells from Labeo rohita testis for in vitro
propagation. Theriogenology, 76: 241-251.
Rodriguez-Sosa, J.R., Dobson, H., and Hahnel,
A. (2006). Isolation and transplantation of
spermatogonia in sheep. Theriogenology, 66:
2091– 2103.
Somashekar, L., Selvaraju, S., Parthipan, S., Patil, S.K.,
Binsila, B.K., Venkataswamy, M.M., Karthik Bhat,
S. and Ravindra, J.P. (2017). Comparative sperm
protein proling in bulls diering in fertility and
identication of phosphatidylethanolamine‐binding
protein 4, a potential fertility marker. Andrology, 5:
1032-1051.
Tiptanavattana, N., Techakumphu, M. and Tharasanit,
T. (2015). Simplied isolation and enrichment
of spermatogonial stem-like cells from pubertal
domestic cats (Felis catus). J Vet Med Sci., 77:
1347-1353.
Valli, H., Sukhwani, M., Dovey, S. L., Peters, K.A.,
Donohue, J., Castro, C.A., Chu, T., Marshall,
G.R. and Orwig, K.E. (2014). Fluorescence-
and magnetic-activated cell sorting strategies to
isolate and enrich human spermatogonial stem
cells. Fertil. Steril., 102: 566-580.
Zheng, Y., Zhang, Y., Qu, R., He, Y., Tian, X. and
Zeng, W. (2014). Spermatogonial stem cells
from domestic animals: progress and prospects.
Reproduction, 147: R65-R74.
Indian Journal of Animal Reproduction 40 (1): June 2019
12
INTRODUCTION
There is no practical way to reduce the
early pregnancy loss in dairy cattle, however the
management strategies may be helpful in increasing
the calving rate. Prior to the development of
ultasonography for pregnancy diagnosis, it was dicult
to determine the viability of fetuses at an early stage of
gestation (Chaudhary and Purohit, 2012). Moreover,
an estimation of early pregnancy loss by progesterone
(milk or blood) can not be detected with certainty. The
aim of present study was to assess the embryonic loss
with the help of transrectal ultrasonography and serum
progesterone (P4) and estradiol (E2) determination in
Frisian cows under Egyptian condition.
MATERALS AND METHODS
Friesian cows (n=105; age, 30-96 mo.; b. wt.,
440-660 kg) were used in this study which was carried
out between Oct, 2012 to July, 2013. The tagged
experimental animals were fed on concentrate feed
mixture, maize silage and rice straw in summer. While,
in winter, animals were fed concentrate feed mixture,
fresh berseem and rice straw according to their body
weight and milk production. All heifers were free
from any disease with healthy appearance and were
housed in separated two groups under semi-open
sheds, partially roofed by asbestos.
Estrus was visually detected two times daily
between 6 a.m. and 7 p.m. using a teaser bull. Cattle
detected in estrus in morning were recorded and
articially inseminated (AI) in the afternoon of same
day and cattle detected in estrus in the afternoon were
inseminated next morning. Pregnancy was diagnosed
by serum progesterone on d4, 7, 11, 13 and 22 post
AI, as well as using ultrasonography starting at d22
and d45 post AI.
Blood samples were collected for the determination
of progesterone (P4) and Estradiol-17β (E2) in blood
serum. Within an hour after collection, samples
were centrifuged for 15 min at 3000 rpm for serum
separation. Serum samples were stored at -20 ºC till
the hormonal assay.
A direct radioimmunoassay technique was
performed for determination of serum P4 using
RELATIONSHIP BETWEEN SERUM PROGESTERONE AND ESTRADIOL-17β
AND EMBRYONIC LOSS IN FRISIAN COWS UNDER EGYPTIAN CONDITION
M.A. ABU EL-HAMD, A.K. KADOOM*, W.F.M FOUAD AND S.H. HASSOUNA
Animal Production Research Institute, Agricultural Research Center, Egypt
Received: 20.11.2017 Accepted: 06.01.2018
ABSTRACT
Association between circulating progesterone and oestradiol-17β concentrations and embryonic loss in Frisian
cows (n=105) under Egyptian condition was investigated. Total embryonic losses in present study were 26.4%.
On d25, progesterone (P4) concentrations and sonography revealed 79.3% cows were continuing gestation
and 20.7% lost their pregnancies (early embryonic loss, EEL). Furthermore, 7.4% cows with late embryonic
loss (LEL) were detected between d25-42. An increase in P4 levels started from day 4 to 7 in cows diagnosed
pregnant compared to low P4 in cows that lost pregnancy (p<0.05). Cows with LEL had decrease in P4 from d28.
Estradiol (E2) concentrations increased (p<0.05) in cows with EEL starting from d4 to 21 after AI compared to
cows diagnosed pregnant. Starting from d4 to 40 after AI, P4/E2 ratio was higher (p<0.05) in cows diagnosed
pregnant compared to cows exhibiting EEL loss, while cows with LEL had highest P4/E2 ratio till d25 post-AI and
declined from d28 to 40.
Keywords: Embryonic loss, Friesian cows, Estradiol-17β, Pregnancy, Progesterone
*abkadoom@yahoo.com
Indian Journal of Animal Reproduction 40 (1): June 2019
13
ready antibody coated tubes kit (Diagnosis Systems
Laboratories, Texas, USA). The standard curve of P4
ranged from 0.0 to 40.0 ng/ml. The sensitivity value
was reported as 0.12 ng/ml. The intra and inter-assay
coecients of variation were 8 and 13.1%. Serum E2
was determined by radioimmunoassay procedure
(Sirois and Fortune, 1990). Intra-assay coecient
of variation for E2 was 4%. Inter-assay coecient of
variation was 10.9%. The detection limit of assay was
2.9 pmol/l.
For diagnosing pregnancy and embryo loss of
mated animals, ultrasonographic examinations were
performed using a B-mode ultrasound scanner (Falco,
Easote/Piemedical, Maastricht, 6-8 MH2 Linear array
transducers, Alliance Medical Int.) on day 25 and 42
post AI. The animals were considered pregnant on the
basis of presence of anechoic uid with visualization
of embryo and heart beat in either of uterine horns.
Early pregnancy loss was conrmed by non-visibility
of embryo/fetal heart beat or non-visibility of embryo
or the absence of positive signs of pregnancy in an
animal previously diagnosed pregnant or the presence
of signs of embryo/ fetal degeneration.
The results were statistically analyzed using
completely randomized design for the data of
diagnostic and therapeutic studies. The signicant
dierences among treatment groups were tested using
Duncan’s Multiple Range Test.
RESULTS AND DISCUSSION
From 105 Friesian cows, 70.8% cows were
pregnant on day 25 by both serum P4 and sonography.
Out of these, 79.3% continued their gestation and
20.7% lost their pregnancies by d25 (Table 1). Late
embryonic loss was detected in 7.4% from d25-42,
thus the total embryonic losses were 26.4% (Table
1). This estimate of early embryonic loss was in close
agreement with an earlier study exhibiting 22.8%
(Chaudhary and Purohit, 2012).
An increase in serum P4 started from d4 to d7 in
cows diagnosed pregnant (p<0.05) compared to low
P4 in cows that lost pregnancy (Table 2). In addition,
serum P4 was higher (p<0.05) from d4 to d40 post-
AI in pregnant cows compared to cows that lost
their embryos. A reduced capacity P4 secretion can
explain around 5% embryonic mortalities in bualo
synchronized and mated by AI during a period of low
reproductive activity (Campanile et al., 2005). Cows
with late embryonic loss exhibited increasing serum
P4 till d25, thereafter, P4 started to decrease from d28
onward (Table 2). A signicant negative correlation
was reported between serum P4 and late embryonic
loss as P4 is responsible for maintaing the pregnancy
(Gabor et al., 2008).
Serum E2 increased (p<0.05) in cows with
early embryonic loss starting from d4 to 21 post-AI
compared to cows diagnosed pregnant (Table 3).
Estrogen secretion from a large follicle from d14 to 17
of pregnancy may negatively aect embryo survival
(Inskeep, 2004). In addition, cows pregnant at day
28 had lower E2 (p<0.05) compared to cows with late
embryonic loss (Table 2).
The present results showed that P4/E2 ratio was
Table 1: Early (EEL) and late (LEL) embryonic losses in Friesian cows
Parameter Total Pregnant Pregnancy loss
N N % N %
Conception rate, % 105 87 82.8 - -
EEL, till d 25 87 69 79.3 18 20.7a
LEL, d 25-42 69 64 92.7 57.4b
Total 87 64 73.6 23 26.4
a,bp<0.05
Steroid hormones and embryonic loss
Indian Journal of Animal Reproduction 40 (1): June 2019
14
higher (p<0.05) in cows diagnosed pregnant starting
from d4 to 40 post-AI compared to cows that lost their
embryos between d4 to 25 post-AI (Table 3). The cows
with late embryonic loss had highest P4/E2 ratio till
d25 post-AI and declined on d28 to 40 (Table 3). The
high ratio occurs when P4 is high relative to E2 and this
describes the classic situation of P4 dominance. An
embryo must secrete sucient amounts of interferon-
tau by d16 to prevent the regression of corpus luteum
(Vasconcelos et al., 1997). It was also noticed that
P4/E2 ratio was high on d0 (day of estrus and AI) in
cows with early embryonic loss than cows diagnosed
Table 3: P4/E2 ratio during post-mating period in Friesian cows. EEL, Early embryonic loss; LEL, Late
embryonic loss
Day Pregnant EEL LEL
00.03±0.02 0.04±0.003 0.03±0.03
41.87±0.15a0.57±0.23b2.07±0.20a
72.44±0.32a0.59±0.22b2.34±0.51a
11 2.92±1.35a0.41±0.24b3.17±0.52a
12 3.19±1.21a0.51±0.34b3.12±0.45a
13 3.55±0.98a0.79±0.28b3.26±0.36a
21 3.42±0.57a0.28±0.11b1.88±0.28a
28 3.13±0.57a- 0.63±0.54b
40 4.46±2.02a- 0.49±0.31b
a,bp<0.05, within a column
Table 2: Serum progesterone (P4) and Estradiol-17β (E2) concentrations (ng/ml) during post-mating period
in Friesian cows. EEL, Early embryonic loss; LEL, Late embryonic loss
Day Pregnant EEL LEL
P4E2P4E2P4E2
0 0.43±0.05b16.36±1.0 0.60±0.04a15.79±0.69 0.49±0.06ab 15.65±1.02
48.03±0.75a4.28±1.57b4.40±1.17b7.67±1.10a7.57±0.65a3.66±1.62b
7 8.98±0.99a3.68±0.68b4.91±1.17b8.23±1.52a8.37±0.87a3.57±0.84b
11 9.23±1.23a3.17±0.58b4.56±1.53b11.12±1.81a9.06±1.11a2.87±1.06b
12 10.11±0.79a3.16±0.70b4.61±1.33b11.36±1.96a9.27±0.86a2.96±0.95b
13 11.20±0.87a3.15±0.53b6.85±1.55b8.67±1.35a9.96±0.92a3.06±1.27b
21 9.61±0.76a2.81±0.47b3.77±0.80b 13.17±2.80a8.57±0.78a4.56±1.35b
28 9.09±0.61a2.83±0.54b- - 5.15±1.12b8.14±1.21a
40 9.56±0.84a2.14±0.43b- - 2.95±0.46b5.91±0.96a
a,bp<0.05, within a column
pregnant or cows with late embryonic loss (d25-40,
Table 3).
REFERENCES
Campanile, G., Neglia, G., Gasparrini, B., Galiero, G.,
Prandi, A., Di Palo, R., D’occhio, M.J., and Zicarelli,
L. (2005). Embryonic mortality in Embryonic
mortality in bualoes synchronized and mated
by AI during the seasonal decline in reproductive
function. Theriogenology, 63: 2334-2340.
Chaudhary, A.K. and Purohit, G.N. (2012).
Ultrasonographic detection of early pregnancy
Abu El-Hamd et al.
Indian Journal of Animal Reproduction 40 (1): June 2019
15
loss in dairy cows. J. Anim. Sci. Adv., 2: 706-710.
Gabor, G., Toth, F., Ozsvari, L., Abonyi-Toth, Z.,
and Sasser, R.G. (2008). Factors inuencing
pregnancy rate and late embryonic loss in dairy
cattle. Reprod. Dom. Anim., 43: 53-58.
Inskeep, E.K. (2004). Preovulatory, postovulatory, and
post-maternal recognition eects of concentrations
of progesterone on embryonic survival in the cow.
J. Anim. Sci., 82 (Suppl E): E24-39.
Sirois, J. and Fortune, J.E. (1990). Lengthening the
bovine estrous cycle with low levels of exogenous
progesterone: a model for studying ovarian
follicular dominance. Endocrinology, 127: 916-
925.
Vasconcelos, J.L.M., Silcox, R.W., Lacerda, J.A.,
Pursley, J.R., and Wiltbank, M.C. (1997).
Pregnancy rate, pregnancy loss and response
to heat stress after AI at two dierent times from
ovulation in dairy cows. Biol. Reprod., 56 (Suppl.
1): 140.
Steroid hormones and embryonic loss
Indian Journal of Animal Reproduction 40 (1): June 2019
16
INTRODUCTION
In repeat breeder cows, the high prevalence
of sub-clinical endometritis (SCE) is hypothesized
as the main etiological factor as most of the cases
go undiagnosed (Parkinson, 2009). Due to the
presence of polymorphonuclear (PMN) cells in the
endometrial lumen, the SCE was rst described as
cytological endometritis (Gilbert et al., 1998), and was
subsequently standardized based on the negative
eects on reproductive performance (Madoz et al.,
2014). The most reliable method for diagnosing SCE is
cytobrush cytology (Barlund et al., 2008). Despite the
recognized negative eects of SCE on reproduction,
the standardization of SCE diagnosis is not fully
established (Pascottini et al., 2015). The lack of on-
farm method to diagnose SCE leads to inability of
farmers to routinely monitor SCE in commercial herds.
In this study, attempts were made to record prevalence
of SCE by using dierent diagnostic methods and the
eect of SCE on bovine reproductive performance.
MATERIALS AND METHODS
For this study, 140 clinically normal cows were
selected based on the absence of abnormal discharge
on external inspection. Apparently clear cervico-vaginal
discharge samples were collected in a sterile screw
capped vial containing sterile swabs. Subsequently,
the cows were inseminated with frozen thawed semen
(0.25 ml) and their reproductive performance was
recorded.
For bacteriological culture analysis, the collected
discharge samples (n=140) were inoculated in nutrient
broth and incubated for 24-48 h and turbidity was
observed. Whiteside test was conducted in 114 cows.
One ml uterine discharge was mixed with one ml 5%
sodium hydroxide solution in a test tube, heated upto
boiling point and subsequently cooled in running tap
water. The appearance of yellow color was positive
indication of infection. Depending upon the intensity
of color development, the degree of infection was
classied; no color (absence of infection), mild yellow
color change (mild infection), and intense yellow color
(severe infection; Anilkumar and Devanathan, 1996).
Endometrial samples were collected by cytobrush
in 15 cows. Sample must contain epithelial cells to
CONCEPTION RATE BASED ACCURACY OF DIFFERENT METHODS TO
DIAGNOSE SUB-CLINICAL ENDOMETRITIS IN COWS
NEELAM1, MADHUMEET SINGH2* AND P. KUMAR3
Department of Veterinary Gynaecology and Obstetrics, CSK Himachal Pradesh Krishi Vishvavidyalaya,
Palampur – 176 062
Received: 26.02.2018 Accepted: 16.04.2018
ABSTRACT
The status of genitalia (through rectal palpation) and cervico-vaginal discharge at spontaneous estrus was
examined in 140 clinically normal cows (Jersey and Jersey crossbred). Endometrial cytology (n=15), bacteriological
culture analysis (n=140) and Whiteside test (n=114) used for the diagnosis of sub-clinical endometritis (SCE) in
cows recorded the prevalence as 40.0, 58.8 and 55.0%, respectively. With respective methods, the conception
rate in SCE positive cows was 40.0, 32.4 and 16.7% and in SCE negative cows was 48.6, 55.6 and 77.8%. Thus,
based on conception rate, the endometrial cytology was the most eective test for the diagnosis of SCE.
Keywords: Bacteriological culture, Conception rate, Endometrial cytology, Endometritis, Whiteside test
1MVSc. Scholar, 2Professor cum Head, 3Associate Professor;
Department of Veterinary Gynaecology and Obstetrics, CSK
Himachal Pradesh Krishi Vishvavidyalaya, Palampur - 176062;
*madhumeet2004@gmail.com
Indian Journal of Animal Reproduction 40 (1): June 2019
17
conrm the correct site of collection. If no epithelial
cells are seen, there is no assurance that the sample
is taken from uterus. Slides for cytological examination
were prepared by rolling the cytobrush on to a clean
glass microscope slide, air dried and xed in methanol
for 15-20 min and stained with Giemsa stain for 45
min. The stained smears were examined at 100x by
counting 100 inammatory and other cell types. Cows
with >5% PMN in the smear were regarded positive for
SCE (Gilbert et al., 2005).
RESULTS AND DISCUSSION
In the present study, the inoculation of clear
cervical mucus in nutrient broth indicated SCE in 55%
cows, whereas, it was 58.8% on the basis of Whiteside
test and 40% according to endometrial cytology. In
a previous study, bacteriological culture of cervical
mucus showed growth of aerobic organisms in 57.14%
and SCE was diagnosed in 72% cows based on
Whiteside test (Raja et al., 2012). Using the threshold
of ≥5% PMN in endometrial cytology, the prevalence of
SCE was higher in present study than a previous study
with 13.8% prevalence (Pothmann et al., 2013).
In this study, following the collection of uterine
discharge for the analysis of SCE, the cows were
inseminated. Based on microbial growth, SCE positive
cows exhibited 40% conception rate, whereas based
on Whiteside test and endometrial cytology ndings,
32.4% and 16.7% infected cows conceived (Table
1). The conception rate in non-SCE cows based on
bacteriological, Whiteside and cytology test was
48.6, 55.6 and 77.8%, respectively (Table 1). Others
recorded 5% and 47% CR after sampling cows with or
without SCE (Salasel et al., 2010).
Thus, the comparison of diagnostic techniques
indicated endometrial cytology as the best method to
diagnose SCE because the CR was lowest in infected
animals. In fact, neutrophils constitute the rst defensive
barrier against invading pathogenic organisms,
resulting in an increase in the PMN cell population
within the uterine lumen (Herath et al., 2009). Once
a pathogen comes into contact with endometrium, the
endometrium is stimulated to produce cytokines and
chemokines that attract immune cells, in particular
PMN cells, into the uterus (Galvao et al., 2011).
In conclusion, uterine cytology was the most
accurate method as compared to Whiteside test
and bacteriological culture to diagnose sub-clinical
endometritis in cows.
REFERENCES
Anilkumar, R. and Devanathan, T.G. (1996). Correlation
between white side test for quality of cervical
mucus and sperm penetration test. Indian Vet. J.,
73: 1099-1100.
Barlund, C.S., Carruthers, T.D., Waldner, C.L. and
Palmer, C.W. (2008). A comparison of diagnostic
techniques for postpartum endometritis in dairy
cattle. Theriogenology, 69: 714-723.
Galvao, K.N., Santos, N.R., Galvao, J.S. and Gilbert,
R.O. (2011). Association between endometritis and
Table 1: Reproductive performance of non-infected and sub-clinical endometritic (SCE) cows based on
dierent diagnostic methods.
Methods SCE Inseminated Conception rate, %
Bacteriological culture, n=77 + 40 40.0
- 37 48.6
Whiteside test, n=55 + 37 32.4
- 18 55.6
Endometrial cytology, n=15 + 6 16.7
- 9 77.8
Diagnosis of sub-clinical endometritis in cows
Indian Journal of Animal Reproduction 40 (1): June 2019
18
endometrial cytokine expression in postpartum
Holstein cows. Theriogenology, 76: 290-299.
Gilbert, R.O., Shin, S.T., Guard, C.L. and Erb, H.N.
(1998). Incidence of endometritis and eects
on reproductive performance of dairy cows.
Theriogenology, 49: 251.
Gilbert, R.O., Shin, S.T., Guard, C.L., Erb, H.N. and
Frajblat, M. (2005). Prevalence of endometritis
and its eects on reproductive performance of
dairy cows. Theriogenology, 64: 1879-1888.
Herath, S., Lilly, S.T., Fischer, D.P., Williams, E.J.,
Dobson, H., Bryant, C.E. and Sheldon, I,M.
(2009). Bacterial Lipopolysaccharide Induces
an Endocrine Switch from Prostaglandin F2α
to Prostaglandin E2 in Bovine Endometrium.
Endocrinology 150: 1912-1920.
Parkinson, T. (2009). The repeat breeder syndrome.
In: Noakes, D.E., Parkinson, T.J., and England,
G.C.W. (eds) Veterinary reproduction and
obstetrics. 9th ed., Saunders Elsevier, Edinbourgh,
463-466.
Pascottini, O.B., Dini, P., Hostens, M., Ducatelle,
R. and Opsomer, G. (2015). A novel cytologic
sampling technique to diagnose subclinical
endometritis and comparison of staining methods
for endometrial cytology samples in dairy cows.
Theriogenology, 84: 1438-1446.
Pothmann, H., Prunner, I., Wagener, K., Jaureguiberry,
M., de la Sota, R.L., Erber, R., Aurich, C., Ehling-
Schulz, M. and Drillich, M. (2013). The prevalence
of subclinical endometritis and intrauterine
infections in repeat breeder cows. Theriogenology,
83: 1249-1253.
Raja, S., Devanathan, T.G., Kulasekar, K., Pazhanivel,
N. and Balachandran, C. (2012). White side
test and endometrial biopsy for diagnosis of
endometritis in repeat breeding cows. J. Anim.
Reprod., 33: 56-58.
Salasel, B., Mokhtari, A. and Taktaz, T. (2010).
Prevalence risk factors for and impact of
subclinical endometritis in repeat breeder dairy
cows. Theriogenology, 74: 1271-1278.
Neelam et al.
Indian Journal of Animal Reproduction 40 (1): June 2019
19
INTRODUCTION
The manipulation of gametes and embryo increases
the risk of exposure of these cells to high levels of
reactive oxygen species (ROS) (Agrawal et al., 2006).
Increased oxidative stress damages mitochondria and
consequently impairs ATP production, and hampers
meiotic and mitotic spindles formation in growing
oocyte. It also destroys oocyte cell membrane lipids
and DNA and progresses apoptosis quickly to inhibit
fertilization (Kowaltowski et al., 1999).
Oxidative stress can be decreased by the
presence of an antioxidant or radical scavenger in
in vitro culture medium. Melatonin (MT; N-acetyl-
5-methoxytryptamine) was successfully tested for
promoting in vitro embryo development in many
species including bovine (Manjunatha et al., 2009)
and porcine (Kang et al., 2008). However, the eect of
melatonin in maturation media for in vitro maturation
of caprine oocytes has not been evaluated. Therefore,
the present study was undertaken to investigate the
optimum concentration of melatonin required for in
vitro maturation of caprine oocytes.
MATERIALS AND METHODS
During spring season (February - April), goat
ovaries (n=184) were collected from a local abattoir
and were transported within 4h to laboratory in warm
saline (35-370C), containing100 IU penicillin-G and
100 µg streptomycin sulfate per ml. Oocytes were
retrieved by slicing goat ovaries and the recovered
oocytes were graded (Kharche et al., 2008).
Selected cumulus oocyte complexes (COCs) were
washed two or three times in Oocyte Holding Medium
(OHM) (TCM-199 medium, EGS 10%, Sodium
Pyruvate 0.25 mM, Gentamicin 50 µg/ml, L-glutamine
100 µg/ml, BSA 3 mg/ml) and subsequently 8 to 10
times in 50-100 µl drops of oocyte maturation media
supplemented with TCM-199 with 10% FBS, 10%
follicular uid, sodium pyruvate 0.25 mM, L-glutamine
100 µg/ml, LH 10µg/ml, FSH 5 µg/ml, estradiol-17β 1
EFFECT OF MELATONIN ON NUCLEAR MATURATION OF CAPRINE
OOCYTES
B.S. KUSHWAH1, S.D. KHARCHE*, J. AGARWAL2, A.K.S. SIKARWAR, J. PATHAK1 AND S.
AGARWAL1
ICAR-Central Institute for Research on Goats, Makhdoom, Mathura
Received: 15.07.2018 Accepted: 08.09.2018
ABSTRACT
Melatonin at the concentrations of 0, 5, 10, 20, 30, 40, and 50 ng/ml was used in maturation medium to
assess the impact on in vitro maturation of caprine oocytes. The oocytes from slaughter house derived ovaries
were subjected to in vitro maturation at 38.5°C with 5%CO2. After culture for 27h, over 90% COCs had full
cumulus cell expansion. The degree of cumulus cell expansion remained similar (p>0.05) on increasing melatonin
concentration, however, oocytes incubated in 30 and 50 ng/ml melatonin containing maturation media for 27 h,
result in 80% and 18.9% nuclear maturation rate, respectively, which were dierent (p<0.05) from control (45.3%).
The nuclear maturation rate decreased (p<0.05) when melatonin concentration was increased from 30 to 40
and 50 ng/ml (80% vs. 28.3% and 18.9%, respectively). In conclusion, melatonin improved nuclear maturation
of caprine oocytes at 30 ng/ml, whereas a high concentration of melatonin may aect caprine oocytes meiotic
maturation at metaphase-II stage and can be toxic for caprine oocytes.
Keywords: Caprine, in-vitro maturation, Melatonin, Metaphase II, Oocytes.
1Ganeshi Lal Agarwal University, Mathura, 2Uttar Pradesh Pandit Deen
Dayal Upadhyaya Pashu Chikitsa Vigyan Vishwavidyalaya Evam Go
Anusandhan Sansthan; *kharche62@gmail.com
Indian Journal of Animal Reproduction 40 (1): June 2019
20
µg/ml, EGF 10ng/ml, BSA 3 mg/ml and gentamicin 50
µg/ml and allowed for maturation in 50 µl droplets of
maturation media covered with sterile mineral oil for
27h in humidied atmosphere of 5% CO2 at 38.50C in
CO2 incubator.
Matured oocytes (Fig. 1) were randomly divided
into dierent treatment groups of maturation media on
the basis of concentrations of melatonin added viz.
group 1 (control, n=172), group 2 (5 ng/ml, n=120),
group 3 (10 ng/ml, n=105), group 4 (20 ng/ml, n=125),
group 5 (30 ng/ml, n=105), group 6 (40 ng/ml, n=113)
and group 7 (50 ng/ml, n=111).
After 27 h maturation, oocytes were stripped o
their cumulus cells by gentle pipetting for 1 min in
0.1% hyaluronidase enzyme. Denuded oocytes were
selected and washed in PBS (1X) followed by xation
with Para-formaldehyde for 10 min. Oocytes were
stained with Hoechst33342 dye (1µL/mL dissolved in
DMSO was stored at 2–6° C, protected from light) for
30 min in dark. Therafter, oocytes were washed with 1X
PBS and evaluated under an Inverted phase-contrast
microscope. Nuclear stages were distinguished by the
morphology of chromatin material (Hewitt et al., 1998;
Yadav et al., 2013). Oocytes with second metaphase
plate (two chromatin spot) and rst polar body were
classied as mature phase of second meiotic cell
division (Fig. 2).
The maturation rates between dierent treatment
groups were compared using the Chi-square test. The
level of signicance was recorded at the 5% level of
condence.
RESULTS AND DISCUSSION
From slaughtered goat ovaries, by slicing
technique, the oocyte recovery rate was 3.94. The
nuclear stages of matured oocytes were distinguished
by the morphology of chromatin material (Hewitt et al.,
1998 and Yadav et al., 2013). Oocytes with rst polar
body or two chromatin spots were classied as mature
phase of second meiotic cell division (MII).
Oocytes that show polar body or two chromatin
spots were considered as matured oocytes (Fig.
1). The rate of oocytes maturation to MII stage was
higher (p<0.05) with 30 ng/ml MT (80%), 20 ng/ml
MT (68%), and 10 ng/ml MT (55.2%) as compared to
control (45.3%, Table 1), possibly due to antioxidant
role of melatonin. However, the oocyte maturation
rate lowered in 40 ng/ml MT (28.3%) and 50 ng/ml
MT (18.9%, Table 1). This could be due to toxic eect
of melatonin at higher concentration, thus leading to
lowering maturation rate and resulting in degeneration
of oocytes (Tamura et al., 2009).
Melatonin (N-acetyl-5-methoxytryptamine) directly
destroys free radicals, indirectly stimulates antioxidant
enzymes and inhibits peroxidation enzymes such as
nitric oxide synthetase (Galano et al., 2011). Melatonin
accelerates the action of maturation-inducing hormone
on maturation-promoting factor and germinal vesicle
breakdown of oocytes (Chattoraj et al., 2005). The
Table 1: Nuclear maturation rate of caprine oocytes at dierent Melatonin concentrations
Melatonin, ng/ml Oocytes, n Matured oocytes, n Maturation, %
0 172 78a,c 45.4±0.4
5120 56a,c 46.5±0.2
10 105 58a,b 55.1±0.3
20 125 86a,b 68.6±0.1
30 105 84b80.2±0.1
40 113 32c28.2±0.3
50 111 22c19.5±0.1
a,bp<0.05
Kushwah et al.
Indian Journal of Animal Reproduction 40 (1): June 2019
21
present study demonstrates that melatonin has a
powerful antioxidative eect during IVM of bovine
oocytes. In addition, the melatonin receptors were
identied in granulosa cells, which suggest another
possibility by which melatonin may take part in oocyte
maturation.
The eect of dierent doses of melatonin on degree
of nuclear maturation of oocytes investigated the
optimal concentration for IVM of oocytes. The nuclear
stages were identied as germinal vesicle stage (GV),
germinal vesicle breakdown stage (GVBD), metaphase
I stage (M I) and metaphase II stage (M II) with
extruded polar body oocytes and Metaphase II plates
were counted as mature (Fig. 2). The chromosomes in
polar bodies with intact plasma membranes uoresced
blue. The observations from present study revealed
Fig. 1: Morphological maturation of caprine oocytes. A, B - Immature oocytes with compact cumulus cells
(cc); C, D - Oocytes (O) with expanded cumulus cells (ECC) after in vitro maturation; E - Denuded oocytes
(DO) after in vitro maturation; F - Matured oocytes with extruded polar body (PB) and peri-vitelline space
(PS)
Melatonin and caprine oocytes
Indian Journal of Animal Reproduction 40 (1): June 2019
22
Fig. 2: Nuclear maturation of caprine oocytes. A - Oocyte showing germinal vesicle (GV) stage. Nuclear
membrane is visible; B - Oocyte showing germinal vesicle break down stage (GVBD); C - Oocyte after
GVBD with condensed chromatin mass (C); D - Oocyte with chromosomes arranged in equatorial plane
at metaphase I (M I Stage); E, F - Oocytes showing metaphase II (M II stage) and extruded polar body (PB)
under Hoechst staining (40X)
that all polar bodies had a sharply dened, smooth
membrane and clear cytoplasm with chromosomes as
scattered, stretched, or adherent to each other (Fig. 2).
In fact, the high concentrations of melatonin
in follicular uid (Brzezinski et al., 1987), and the
presence of receptors in granulose cells (Na et al.,
2005), suggest that melatonin might be important to
ovarian functions. The addition of melatonin to culture
medium may increase the cumulus expansion and
in vitro maturation of oocytes. The cumulus cells are
known to play a crucial role during oocyte maturation.
For acquiring developmental competence by oocyte in
vitro, the cumulus cells during maturation are essential
(Gordon, 2003). It was reported that 1 μM melatonin
Kushwah et al.
Indian Journal of Animal Reproduction 40 (1): June 2019
23
reduces cumulus cells apoptosis by activating its
receptors on cumulus cells (Na et al., 2005).
Thus, melatonin improved the nuclear maturation
of caprine oocytes at 30 ng/ml, whereas a high
concentration of aected caprine oocytes meiotic
maturation at metaphase-II stage and can be toxic for
caprine oocytes.
ACKNOWLEDGEMENTS
The authors wish to thanks to Assistant Director
General, NFBSFARA, New Delhi for providing funding
and Director, C.I.R.G., Makhdoom, Farah, Mathura for
providing the facilities.
REFERENCES
Agrawal, A., Said, T.M., Bedaiwy, M.A., Banerjee,
J. and Alvarez, J.G. (2006). Oxidative stress in
assisted reproductive techniques setting. Fertil.
Steril., 86: 503-512.
Brzezinski, A., Seibel, M., Lynch, H., Deng, M.
and Wurtman, R. (1987). Melatonin in human
preovulatory follicular uid. J. Clin. Endocrinol.
Metab., 64: 865-867.
Chattoraj, A., Bhattacharyya, S., Basu, D.,
Bhattacharya, S., Bhattacharya, S. and Maitra,
S.K., (2005). Melatonin accelerates maturation
inducing hormone (MIH): induced oocyte
maturation in carps. Gen. Comp. Endocrinol., 140:
145-155.
Galano, A., Tan, D. and Reiter, R. (2011). Melatonin
as a natural ally against oxidative stress: a
physicochemical examination. J. Pineal Res., 51:
1-16.
Gordon, I. (2003). Laboratory production of cattle
embryos. 2nd edn., CABI International publishing,
Wallingford. pp. 157.
Hewitt, D.A., Watson, P.F. and England, G.C. (1998).
Nuclear staining and culture requirements for
in vitro maturation of domestic bitch oocytes.
Theriogenology, 49: 1083-1101.
Kang, J.T., Koo, O.J., Kwon, D.K., Park, H.J., Jang, G.
and Kang, S.K. (2009). Eects of melatonin on in
vitro maturation of porcine oocyte and expression
of melatonin receptor RNA in cumulus and
granulosa cells. J. Pineal Res., 46: 22-28.
Kharche, S.D., Goel, A.K., Jindal, S.K., Sinha, N.K.
and Yadav, P. (2008). Eect of somatic cells co-
cuture on cleavage and development of in-vitro
fertilized caprine embryos. Indian J. Anim. Sci.,
78: 686-692.
Kowaltowski, A.J. and Vercesi, A.E. (1999).
Mitochondrial damage induced by conditions of
oxidative stress. Free Radical Bio. Med., 26: 463.
Manjunatha, B.M., Devaraj, M., Gupta, P.S.P.,
Ravindra, J.P. and Nandi, S. (2009). Eect of
taurine and melatonin in the culture medium on
bualo in vitro embryo development. Reprod.
Domest. Anim., 44: 12-16.
Na, K., Kim, J., Lee, J., Yoon, T., Cha, K. and Lee, D.
(2005). Eect of melatonin on the maturation of
mouse GV oocytes and apoptosis of cumulus cells
in vitro. Fertil Steril., 84: 103.
Tamura, H., Nakamura, Y., Korkmaz, A., Manchester,
L.C., Tan, D.X. and Sugino, N. (2009). Melatonin
and the ovary: physiological and pathophysiological
implications. Fertil. Steril., 92: 328-343.
Yadav, P., Kharche, S.D., Goel, A.K., Jindal, S.K.
and Goel, P. (2013). Assessment of on nuclear
maturation of caprine oocytes with dierent
supplementations in maturation medium. Indian J.
Anim. Sci., 83: 1048-1052.
Melatonin and caprine oocytes
Indian Journal of Animal Reproduction 40 (1): June 2019
24
INTRODUCTION
Mammalian spermatozoa are extremely sensitive
to oxidative damage, in-vivo as well as in-vitro. The
process of peroxidation induces structural alterations,
particularly in the acrosomal region, a fast and
irreversible loss of motility, a deep change in metabolism
and a high rate of intracellular components release
(Cecil and Baskt, 1993). Membrane stabilizers and
antioxidants have benecial eect on the membrane
integrity and biofunctional activity of spermatozoa.
Ascorbic acid, a biologically active reducing agent,
restores fertility possibly by the reduction of anti-
agglutination factor on sperm membrane from inactive
form to active form (Lindahl, 1966). Chloroquine
diphosphate as membrane stabilizer was used earlier
in preservation of bualo semen (Kumar, 1992). The
success of cryopreservation depends largely on the
specic susceptibility of sperm cells to low temperature.
The overall impact is seen in fertility in terms of lowered
conception than with the fresh semen.
The present study investigated the impact of semen
additives (Chloroquine diphosphate and Ascorbic acid)
on routine semen evaluation parameters with respect
to post-incubation time and conception rate.
MATERIALS AND METHODS
The study was conducted on 36 ejaculates (6
from each bull) collected twice a week from each of
six apparently healthy purebred Jersey breeding
bulls (age, 2.5-8.0 yr), maintained at Sperm Station,
Palampur, Himachal Pradesh, India (32.6ON, 76.3OE,
altitude 1290.8 m). After initial evaluation, the neat
semen extended in TRIS-based extender was divided
into three aliquots (10 ml diluted semen) viz. control
(G1), Chloroquine diphosphate @ 10-5M concentration
(G2) or Ascorbic acid @ 0.02% concentration (G3).
All the semen dilution, extension and modied
extension procedures were carried out at 37oC with
80M spermatozoa/ml of diluted semen. Tested semen
samples were lled in 0.25 ml French mini plastic straws
and these were frozen as per the standard procedures.
Semen straws from all the groups were thawed at 37oC
for 30 second and were evaluated at 0, 1 and 2 h post-
thaw for percent viability, progressive motility, reaction
to 150 mOsmol hypo-osmotic solution, and acrosomal
integrity. For fertility trials, 55 cows were inseminated
ADDITION OF CHLOROQUINE DIPHOSPHATE OR ASCORBIC ACID IN
JERSEY BULL SEMEN AND SUBSEQUENT EVALUATION WITH RESPECT
TO POST-THAW INCUBATION TIME AND CONCEPTION RATE
MADHUMEET SINGH1* AND A. SHARMA2
Department of Veterinary Gynaecology and Obstetrics, College of Veterinary and Animal Sciences, Himachal
Pradesh Agricultural University, Palampur-176 062
Received: 26.02.2018 Accepted: 10.04.2018
ABSTRACT
Total 36 ejaculates (6 from each Jersey bull) were used in the study and aliquots were added with Chloroquine
diphosphate or Ascorbic acid or served as control. Semen straws from all the groups were analyzed at 0, 1
and 2 h post-thaw incubation at 37°C for percent viability, progressive motility, reaction to hypo-osmotic solution
and acrosomal integrity. Following post-thaw incubation, an improvement (p<0.05) in all the evaluated semen
parameters was recored in groups with semen additives compared to control. The conception rate was better
(p<0.05) using semen fortied with ascorbic acid.
Keywords: Acrosomal integrity, Ascorbic acid, Chloroquine diphosphate, Conception rate, HOST
1Professor cum Head, 2Associate Professor; *madhumeet2004@gmail.
com
Indian Journal of Animal Reproduction 40 (1): June 2019
25
with straws of each group. The data were analyzed by
SPSS® 20 level version for windows.
RESULTS AND DISCUSSION
All the semen quality assessment traits viz.
live sperms, progressive motility, HOST reactive
spermatozoa and acrosomal integrity were negatively
and signicantly (p<0.05) correlated with the post-
thaw incubation time (Table 1), as observed earlier
(Sharma et al., 2012; Rastegarnia et al., 2013).
Furthermore, the post-thaw deterioration of semen
quality was comparatively less in semen with additives
in comparison to unfortied one (Table 1). Out of 165
cows inseminated (n=55 in each group), the conception
rate was 45.5, 56.4 and 65.5% in cows of control,
Chloroquine diphosphate and Ascorbic acid group,
respectively. Thus, ascorbic acid as an anti-oxidant
improved (p<0.05) the post-thaw quality of frozen
semen of Jersey bulls. Higher conception rates with no
statistical dierence were also observed in the semen
preserved with Chloroquine diphosphate in bualoes
(Singh et al., 2000). The decline in progressive motility
observed in the present study after storage may be
associated with decrease in live-dead sperm ratio and
gradual exhaustion of some vital endogenous reserves
in a highly dierentiated cell which has lost its capacity
for protein synthesis (Kumar, 2007).
In brief, it can be concluded that semen parameters
had negative correlation with post-thaw incubation
time, however, post-thaw deterioration of the semen
quality was comparatively less in semen with additives
and the conception rate was better following the use of
fortied semen.
REFERENCES
Cecil, H.C. and Baskt, M.R. (1993). In vitro lipid
peroxidation of turkey spermatozoa. Poultry Sci.,
72: 1370-1378.
Table 1: Relationship between post-thaw incubation time with semen evaluation parameters following the
addition of semen additives. G1, Control; G2, Chloroquine Diphosphate; G3, Ascorbic acid
Group Correlation
Coecient
Regression
Estimate
Regression Equation
Live Sperms, %
G1-0.72** -6.34±0.81 y = 53.85±1.04–6.34x±0.81
G2-0.73** -6.67±0.81 y = 57.28±1.11–6.67x±0.81
G3-0.75** -6.62±0.17 y = 57.98±0.93–6.62x±0.17
Progressive Motility, %
G1-0.80** -9.65±0.71 y = 47.84±0.91–9.65x±0.71
G2-0.76** -9.06±0.72 y = 50.10±0.93–9.06x±0.72
G3-0.83** -9.31±0.61 y = 51.24±0.80-9.31x±0.61
HOST, %
G1-0.78** -7.71±0.59 y =40.74±0.77–7.71x±0.59
G2-0.81** -7.38±0.53 y =46.18±0.63–7.38x±0.53
G3-0.75** -7.53±0.65 y =45.53±0.84–7.53x±0.65
Acrosomal Integrity, %
G1-0.78** -7.82±0.61 y =67.78±0.79–7.82x±0.61
G2-0.69** -5.96±0.59 y =68.25±0.76–5.96x ±0.59
G3-0.79** -6.63±0.49 y =70.01±0.64–6.63x±0.49
**p<0.01
Improving bull semen quality
Indian Journal of Animal Reproduction 40 (1): June 2019
26
Kumar, S. (1992). Eect of dierent thawing
temperatures on the leakage of bualo
spermatozoon enzymes. M.V.Sc. Thesis, Punjab
Agricultural University, Ludhiana, India.
Kumar, S. (2007). Eect of certain additives in the
diluter on post-thaw recovery rate of bovine
semen. Indian J. Vet. Res., 16: 29-42.
Lindahl, P.E. (1966). Sperm agglutinating and anti-
agglutinating factors in normal follicular uid from
cattle. Inter. J. Fert., 11: 297
Rastegarnia, A., Shahverdi, A., Topraggaleh, T.R.,
Ebrahimi, B. and Shapour, V. (2013). Eects
of dierent thawing rates on post-thaw viability,
kinematic parameters and chromatin structure of
bualo (Bubalus bubalis) spermatozoa. Cell J., 14:
201.
Sharma, M., Singh, M., Kapoor, S. and Jasial, S.
(2012). Inter relationship between some routine
semen evaluation parameters in Jersey X local hill
cattle crossbred bulls. Open Vet. J., 2: 26-31.
Singh, M., Pant, H.C. and Patial, K.K. (2000). Eect of
post-thaw incubation procedures on semen quality
of Jersey bulls. Him. Vet. J., 4: 15-18.
Singh and Sharma
Indian Journal of Animal Reproduction 40 (1): June 2019
27
INTRODUCTION
Equine semen is a one of the most dicult in
industry to cryopreserve eciently without causing
damage to membrane or apoptosis. The successful
use of cryopreserved sperm largely depends on
cryosurvival rates, which show large variation
among species and individuals of the same species
(Vidament et al., 2009; Wu et al., 2015). In fact, only
20% of fertile stallions produce sperms that survive
the freezing and thawing processes (Tischner, 1979).
Glycerol (Gly) is a preliminary cryoprotectant that has
not only benecial cryoprotective eects (Homan
et al., 2011), but also toxic eects on spermatozoa
(Alvarenga et al., 2005), despite with contraceptive
eects in mare (Vidament et al., 2009). Glycerol
lowered the fertility of equine semen when included
in extenders for fresh, cooled, and frozen semen
(Wu et al., 2015). Other cryopritectant like Dimethyl
formamide (DMF) were less toxic and yielded similar
or superior results as compared to glycerol (Alvarenga
et al., 2005). Therefore, the present study aimed to
assess the eects of two permeable cryoprotectants
viz. Glycerol (GLY) and Dimethyl Formamide (DMF) on
the freezability of exotic stallion and jack semen.
MATERIALS AND METHODS
Fiive healthy exotic (Thoroughbred) stallions
and ve exotic Jacks (Poitou breed), aged 6-10 yr,
maintained in well-ventilated boxes on a standard
diet (5 kg concentrate with mineral mixture, salt and
15 kg fodder - green: dry in 3:1 ratio) and ad lib fresh
drinking water were used in the present study during
the breeding season.
The semen from all the animals were collected
using articial vagina (Colorado model) equipped with
a disposable liner as per the standard method (Talluri et
al., 2017). Semen collection, evaluation and processing
for freezing were done according to described methods
(Talluri et al., 2017). Immediately after semen collection,
seminal parameters like appearance, volume, colour,
consistency, pH were recorded by visual observation.
The gel portion of fresh semen was sieved through a
sterilised gauze lter and volume of total and gel free
EFFECT OF TWO DIFFERENT PERMEABLE CRYOPROTECTANTS ON
FREEZABILITY OF EXOTIC STALLION AND JACK SEMEN
T.R. TALLURI1*, S.K. RAVI1, C. SINGH2, R.A. LEGHA3, S.C. MEHTA3, V. PRAKASH4 AND
B.N. TRIPATHI5
Equine Production Campus, ICAR-National Research Centre on Equines, Bikaner
Received: 30.08.2018 Accepted: 12.09.2018
ABSTRACT
Semen was collected from the exotic stallions and exotic jacks (n=5 each) using an articial vagina. The
fresh semen was treated with primary extender and centrifuged to remove seminal plasma. Sperm pellet
obtained after centrifugation was equally divided to receive secondary extender containing same amount (5%)
of dierent permeable cryoprotectants, either glycerol (Gly) or dimethyl formamide (DMF) before submitting for
cryopreservation. Various sperm parameters were assessed at pre-freeze and post-thaw stage which revealed
dierences (p<0.05) in motility, liveability, plasma membrane integrity and acrosome integrity of stallion as well as
Jack semen cryopreserved with DMF or Gly. A variation (p<0.05) existed between individual stallions and jacks
extended either with DMF or Gly. In brief, cryoprotectant DMF for stallion and Gly for jack semen were suitable for
retaining the plasma and acrosome integrity of spermatozoa.
Keywords: Cryopreservation, Di-Methyl Formamide, Glycerol, Jack, Semen, Stallion
1Scientist, 3Principal Scientist, 5Director; 2Major, Equine Breeding Stud,
Hisar; 4Scientist, ICAR-NRC on Camel, Bikaner; *raotalluri79@gmail.
com
Indian Journal of Animal Reproduction 40 (1): June 2019
28
semen was calculated visually through graduated
sterile semen collection bottle. For calculating total gel
volume, the gel free semen volume was deducted from
fresh semen collected before sieving. After performing
macroscopic and microscopic evaluation of fresh
semen, the semen samples were processed, extended
and frozen according to the described methods (Talluri
et al., 2012a).
Semen samples having progressive motility
>60% were processed for cryopreservation. Gel free
semen was mixed with modied Citrate-EDTA primary
extender in the ratio of 1:1 and centrifuged at 550 g for
3 min. The supernatant was discarded and sperm pellet
was extended with Glucose-EDTA-lactose secondary
extender having cryoprotectant agents either 5% GLY
or 5% DMF. The diluted semen (100×106 sperm cells/
ml) was kept in semen cooling cabinet at 4ºC for 2 h
as equilibration period. Semen samples were again
assessed for pre-freeze seminal characteristics.
Equilibrated semen was manually lled in 0.5 ml
straws using vacuum pump. The straws were sealed
using PVC powder and were cooled for 30 min at 4ºC.
The straws were spread over freezing racks, 4 cm
above liquid nitrogen (LN2) in a traditional styrofoam
box for exposure to LN2 vapours for 10-12 min and
then plunged directly into LN2. Later, the semen straws
were transferred to LN2 cryocans. The microscopic
evaluation of frozen thawed sperm was done at least
24 h after storage. The straws were thawed in 37ºC
water bath for 30 sec. Each frozen thawed semen
sample was evaluated for determining the post-thaw
motility, live and dead percentage, plasma membrane
integrity through hypo-osmotic swelling (HOS) test and
acrosome integrity as per the standard procedures.
As repeated collection of semen was done on
same stallion/jack at dierent time intervals, a repeated
measure ANOVA was done to partition the variability
attributable to dierences between treatments and
individual variation among stallion/jack/subjects in
treatment groups. Statistical analyses were performed
using the SPSS 20.0 statistical software package. One-
way ANOVA was used to test statistical dierences
between dierent treatment groups. Pair wise
comparisons (or post hoc test) were performed using
the T-method (Tukey’s honestly signicant dierence
method).
RESULTS AND DISCUSSION
The fresh semen collected from exotic stallions
and jacks was white to creamy white and consistency
was variably thick and viscous as observed earlier
(Talluri et al., 2018). The fresh semen volume collected
from stallions ranged from 25-125 ml and in jacks
from 45-83 ml (Table 1). The total volume of stallion
semen varies between 30-250 ml (Ricketts, 1993).
In the current study, average total semen volume,
gel free semen and gel in semen was recorded less
than reported for indigenous stallions (Talluri et al.,
2012a and 2012b). The pH of stallion semen ranged
from 6.92-7.21 and of jacks was 6.52-7.10 (Table 1).
The pH range was in correlation with earlier reports
for stallions (Talluri et al., 2018) and Jacks (Rabindra
Kumar et al., 2018).
The sperm concentration in fresh semen was
185.7-294.7×106 ml-1 in stallions and 281.7-341.1×
106 ml-1 in jacks. with a mean of 318.29±16.29× 106
ml-1 (Table 1). The variation in sperm concentration
between individual stallions and jacks was simular
(p<0.05), however, between stallions and jacks was
dierent (p<0.05). The sperm concentration for exotic
stallions and jacks observed in this study was higher
compared to earlier reports for Stallions (Talluri et al.,
20018) and jacks (Rabindra Kumar et al., 2018). Total
sperm motility in gel fractioned semen was 79.3-88.2%
in stallions and was 80.9 to 91.1% for jacks (Table 1),
which was higher than observed for Marwari stallions
(Pal et al., 2009) and Poitou jacks (Rabindra Kumar et
al., 2018). The progressive sperm motility in gel free
semen was 70.2-79.9% for stallions and 76.3-84.7%
for jacks (Table 1). The stallion semen exhibiting
>60% progressive sperm motility can be considered
appropriate for cryopreservation.
Talluri et al.
Indian Journal of Animal Reproduction 40 (1): June 2019
29
Table 1: Fresh seminal characteristics of exotic
Stallion and Poitou Jack (n=40 each).
Seminal attribute Stallion Jack
Total Volume, ml 45±6.7 53.8±8.3
Gel Volume, ml 9±1.18 9.6±0.81
Final volume, ml 36±5.7 45.2±8.3
pH 7.03±0.01 6.82±0.06
Sperm Concentration, x10
6
205.4±12.6 389.0±15.5
Total Initial Motility, ml 81±5.3 85±2.7
Progressive Motility, ml 74.7±1.70 80±2.73
A major dierence was observed in pre-freeze
and post-thaw motility of stallion spermatozoa
cryopreserved with either DMF or Gly (Fig. 1). The
stallion semen extended with DMF had better (p<0.05)
progressive motility compared to Gly both at pre-freeze
and post-thaw stages. Furthermore, at pre-freeze
and post-thaw stages, a dierence (p<0.05) existed
in sperm motility between individual stallions treated
with DMF or Gly (Fig. 1). In case of jacks, the semen
cryopreserved with Gly had better (p<0.05) motility
both at pre-freeze and post-thaw stages. The jack
semen treated with Gly exhibited superior (p<0.05)
motility over DMF or Gly treated stallion semen with at
both stages (Fig. 1).
At post-thaw stage, the liveability was better
(p<0.05) for stallion and jack semen cryopreserved
with DMF but not for semen extended with Gly (Fig.
1). The functional integrity of plasma membrane as
determined by HOS test was dierent (p<0.05) in
spermatozoa extended with DMF or Gly at pre-freeze
stage but not the post-thawed stallion semen. The jack
semen extended with Gly had good plasma membrane
integrity than semen extended with DMF and the
same was vice versa for the stallion semen (Fig. 1). A
highly signicant dierence (p<0.05) was observed in
acrosome integrity of stallion spermatozoa extended
with DMF or Gly at pre-freeze stages only.
Fig. 1: Pre-freeze and post-thaw seminal characteristics of exotic Stallion and Jacks (n= 40 each group).
A,Bp<0.05 - within a group (between pre-freeze and post thaw). a-gp<0.05 - across the groups.
Freezability of stallion and jack semen
Indian Journal of Animal Reproduction 40 (1): June 2019
30
The stallion semen treated with DMF had higher
(p<0.05) percentage of sperms with acrosome
integrity than that of Gly at both pre-freeze and post-
thaw stages. Furthermore, the dierence (p<0.05) in
acrosome integrity was observed between individual
stallion semen treated with DMF or Gly at post-thaw
stage only (Fig. 1). In jacks, the semen extended with
either DMF or Gly had dierence in acrosome integrity
at post-thaw stage (p<0.05), with the semen extended
with Gly had higher acrosome integrity than DMF
(Fig. 1). The freezability of semen from exotic horses
and exotic jacks was 90% (20 ejaculates out of total
22 ejaculates) and 95% (21 ejaculates out of total 22
ejaculates), respectively.
In brief, the stallion and jack spermatozoa may be
damaged by glycerol, but the toxic eect of glycerol is
more obvious for the stallions. Thus, DMF can be used
as an alternative to Gly as cryoprotectants for stallions
and Gly can be used for jack semen to obtain better
post-thaw motility and integrity of sperm membranes.
ACKNOWLEDGEMENTS
The authors thank Dr Iqbal Hyder and Dr
Guruvishnu, Assistant Professor, SVVU, Tirupati,
Andhra Pradesh for their immense contribution in
critical analysis of the data.
REFERENCES
Alvarenga, M.A., Papa, F.O., Landim-Alvarenga,
F.C. and Medeiros, A.S.L. (2005). Amides as
cryoprotectants for freezing stallion semen: A
review. Anim. Reprod. Sci., 89:105-113.
Homann, N., Oldenhof, H., Morandini, C., Rohn, K.
and Sieme, H. (2011). Optimal concentrations of
cryoprotective agents for semen from stallions that
are classied ‘good’ or ‘poor’ for freezing. Anim.
Reprod. Sci., 125: 112-118.
Pal, Y., Legha, R.A. and Tandon, S.A. (2009).
Comparative assessment of seminal
characteristics of horse and donkey stallions. Ind.
J. Anim. Sci., 79: 1028-1029.
Rickets, S.W. (1993). Evaluation of stallion semen. Eq.
Vet. Edu., 5: 232-237.
Talluri. T.R., Arangasamy, A., Ravi, S.K., Pal, Y., Gupta,
A.K. and Singh, R.K. (2012a). Characteristics of
fresh and frozen semen of zanskari stallion. Ind
Vet. J., 89: 62-64.
Talluri, T.R., Arangasamy, A., Ravi, S.K. and Pal, Y.
(2012b). Hypo-osmotic swelling test (HOST) for
quality evaluation of fresh and frozen semen in
stallions. Ind. Vet. J., 89: 68-70.
Talluri, T.R., Ravi, S.K., Legha, R.A., Yash Pal.,
Thomas K.T., Kashyap, S.S. and Tripathi, B.N.
(2017). Seminal traits and suitability of semen for
cryopreservation in exotic stallion semen reared
in a government farm. Ind. J. Anim. Reprod., 39:
44-45.
Tischner, M.(1979). Evaluations of deep frozen semen
in stallions. J. Reprod. Fert., 27: 53-59.
Vidament, M., Vincent, P., Martin, F.X., Magistrini, M.
and Blesbois, E. (2009). Dierences in ability of
jennies and mares to conceive with cooled and
frozen semen containing glycerol or not. Anim.
Reprod. Sci., 112: 22-35
Wu, Z., Zheng, X., Luo, Y., Huo, F., Dong, H., Zhang,
G., Yu, W., Tian, F., He, L., and Chena, J. (2015).
Cryopreservation of stallion spermatozoa using
dierent cryoprotectants and combinations of
cryoprotectants. Anim. Reprod. Sci., 163: 75-81.
Talluri et al.
Indian Journal of Animal Reproduction 40 (1): June 2019
31
INTRODUCTION
The success of dairy farming lies in ensuring
the optimal reproductive eciency as reproductive
failure results in progressive economic losses. Among
various reproductive disorders, repeat breeding is
one of the major gynaecological problems (Kumar
et al., 2017). Uterine infection implies adherence of
pathogenic organisms to uterine mucosa and followed
by colonization, penetration of the endometrial
epithelium and release of bacterial toxins setting up
uterine inammation. Incidence of clinical and sub-
clinical endometritis in cows is 12 % and 29.7%,
respectively (Kumar et al., 2018). Several approaches
including antibiotics, antiseptics and hormonal
therapies were employed to treat repeat breeding
cows with endometritis. However, there are certain
limitations in the use of antibiotics like drug resistance,
inhibition of normal uterine defense mechanisms and
residual eect of antibiotics and hormones in the milk
and meat (Rahi et al., 2013). Various herbs possess
antibacterial, antiviral, antifungal, antioxidant and
immunomodulatory properties ensuring prevention
and cure of several diseases and disorders without
side eects. Hence, dierent herbs were used as a
source of valuable medicines globally (Gebreyohannes
et al., 2013). Therefore, in the present study, herbal
Garlic and Ashwagandha preparations were used for
the treatment of repeat breeding due to infectious
endometritis in cows.
EFFICACY OF GARLIC EXTRACT±ASHWAGANDHA FOR THE TREATMENT
OF INFECTIOUS REPEAT BREEDING IN CATTLE
S.R. LAWANGE1, N.M. MARKANDEYA2, B.L. KUMAWAT3*, A.G. SAWALE4, R.S. ANBHULE5
AND P.D. KADAM6
Department of Animal Reproduction, Gynaecology and Obstetrics, College of Veterinary and Animal
Sciences, Parbhani - 431 402
Received: 23.08.2018 Accepted: 12.09.2018
ABSTRACT
The present research work was conducted to evaluate the ecacy of Garlic extract intrauterine (IU) versus
Garlic extract (IU) plus Ashwagandha powder (oral) for the treatment of fresh (repeated ≤7 times) and chronic
(repeated >7 times) infectious repeat breeding in cattle, respectively. Uterine infection was diagnosed by pH
estimation, PMN cell count and White Side Test of cervico-vaginal mucus in both fresh and chronic cases while
Immunoglobulin occulation test with serum in chronic cases only. This indicated 92.8% cows having alkaline pH,
PMN cell count >7% and 100% positive for White Side Test and increased immunoglobulin level in blood. Total
36 cows aected with endometritis were divided into three groups to undertake treatment on day of estrus viz.
Group-I consisting of 12 fresh cases were treated with Garlic extract @10 µg in 30 ml PBS by intrauterine route
once; Group-II consisting of 12 chronic cases were treated with Garlic extract as per Group-I plus Ashwagandha
powder @ 15 gm with concentrates per oral for 5 days; and Group-III consisting of 12 cows as control were treated
with placebo @ 30 ml PBS intrauterine. The clinical recovery of cows was conrmed by normal pH, negative white
site test and PMN cell count <5% in cervico-vaginal mucus at subsequent estrus. Immunoglobulin occulation
test revealed raised immunoglobulin level in recovered cases of chronic endometritis. The recovered cows were
inseminated with frozen-thawed semen at mid stage of estrus and pregnancy was conrmed at 50-60 day post
insemination. The results of present investigation revealed highest recovery rate (83.3%) in group-I, whereas the
highest conception rate (66.7%) and pregnancy rate (55.6%) was observed in group-II, thus suggesting the use
of Garlic extract alomg with Ashwagandha for infectious repeat breeder cattle.
Keywords: Ashwagandha, Cows, Garlic extract, Infectious, Repeat breeder
1,5,6M.V.Sc. Scholar, 2Professor and Head, 3SMS, 4Assistant Professor;
*drblkivri@gmail.com
Indian Journal of Animal Reproduction 40 (1): June 2019
32
MATERIALS AND METHODS
The study was carried out in 36 infectious repeat
breeder cows under eld and farms. Infectious repeat
breeding cows were selected on the basis of history
and breeding records (repeated ≥3 times), results
from laboratory investigation of cervico-vaginal
mucus (CVM) viz. alkaline pH, positive white site test,
presence of white akes (Popov, 1969) and increased
PMN cell count (>7%) in endometrial cytology smear
and immunoglobuline occulation test of serum
(Deshpande et al., 1991). Selected 36 endometritic
cows were equally divided into 3 groups. Group-I
repeat breeder cows (n=12) repeated seven times or
less were administered with single intra uterine infusion
of 10 mcg Methanolic Garlic extract dissolved in 30 ml
PBS during mid-estrus stage. Group-II repeat breeder
cows (n=12) repeated >7 times were administered
with single intrauterine infusion of 10 mcg Methanolic
Garlic extract as per group-I and 15 gm Ashwagandha
powder in concentrate was fed additionally once daily
for 5 days and Group-III repeat breeder cows (n=12)
were administered placebo treatment @ 30 ml PBS
intrauterine during mid estrus and were inseminated
at next estrus only. The CVM and blood samples
were collected from test animals at estrus before and
after treatment to evaluate the treatment ecacy. The
serum was separated and stored at 200C till laboratory
investigation.
Methanolic Garlic extract (20% W/V) was
prepared in advance by adding 200 gm Garlic seeds
pest in 100% methanol to make nal volume 1000
ml in a conical ask. The solution was kept at room
temperature for 48 h with constant shaking followed
by sieving with muslin cloth and then ltration through
ordinary lter paper. The ltrate was poured in wide
stainless-steel plate and allowed to evaporate under
room temperature till the formation of semi-solid pest
which took around one month. From the prepared
Garlic pest, each dose was prepared by dissolving
10 mcg pests in 5 ml PBS as stock solution. Thus,
the prepared stock doses were stored in refrigeration
temperature (40C). Transportation of doses to eld
was attempted in proper thermos ask on ice packs.
Each dose was reconstituted with addition of 25 ml
PBS for administration through intrauterine route.
Ashwagandha powder available in market was used
for therapeutic purpose and the powder was fed in
concentrate @ 15 gm/day to the chronic cases for 5
days as an immuno-booster.
The clinical recovery of cows was conrmed
by normal pH, negative white site test and PMN cell
count <5% at subsequent oestrus. The recovered
cows were inseminated with frozen-thawed semen at
mid stage of estrus. Conception was ensured by non-
return of estrus in subsequent cycle. Pregnancy was
conrmed by trans-rectal palpation after 50-60 days of
insemination. The data was statistically analyzed using
analysis of variance (ANOVA) to nd out the signicant
dierences of mean values (Snedecor and Cochran,
1989).
RESULTS AND DISCUSSION
The present study was conducted to evaluate the
antimicrobial property of Garlic and immunomodulatory
eect of Ashwagandha herbs. Uterine infection in
repeat breeder cows was diagnosed on the basis of
pH estimation and White Side Test of cervico-vaginal
mucus, uterine cytology of endometrial contents and
Immunoglobulin Flocculation Test in serum. The
diagnosis was made on the basis of cumulative results
of all tests and no single test result was considered.
The pH observations in infectious repeat breeder
cows were recorded as mildly acidic in 2.8%, normal
in 13.9%, moderately alkaline in 61.1% and highly
alkaline in 22.2% cases. A highly signicant (p<0.05)
number of cases were carrying pH from 8.1 to 8.5.
White Side Test showed variable grade of severity of
endometritis viz. 27.8% mild, 36.1% moderate and
19.5% severe. Endometrial cytology revealed PMN
cell count as >5% in all selected cases. The PMN
cell count ranged between 05-18% with an average
of 9.8±0.3% and, thus diagnosed as subclinical stage
Sawale et al.
Indian Journal of Animal Reproduction 40 (1): June 2019
33Herbal treatment of infectious repeat breeding
of endometritis (Sheldon et al., 2009; Bajaj et al.,
2017). The average immunoglobulin concentration in
endometritic cows was 6.9-7.0 mg/ml in 24 cases.
On treatment of Garlic extract by intrauterine
route in infectious repeat breeding cows from Group-I,
10 cows (83.3%) responded to treatment against nil
response in control group. Ecacy of Garlic extract
was studied and reported previously with variable
recovery rate from 50-100% in cases of infectious
repeat breeder cows (Sarkar et al., 2006; Rahi et al.,
2014; Yildiz, 2016; Kumar et al., 2018; Sharma et
al., 2018). However, the current ndings regarding
recovery rate as 83.3% by garlic extract corroborate
with others (Kumar et al., 2018; Sharma et al., 2018).
Mean pH value before treatment was 8.3±0.1
which changed to 7.3±0.1 in recovered cows after
treatment; however, the same remained almost similar
in non-recovered (8.4±0.0). The pH value of CVM was
7.8±0.2 before treatment which slightly increased to
8.4±0.1 after treatments in control cases which may
be due to progression of uterine infection in untreated
animals. Negative White Side Test was conrmed
in all responded cases, but it remained positive in
non-recovered and control group. Average PMN cell
count was recorded as 9.4±0.5% before treatment,
which reduced (p<0.05) to 2.9±0.3 in recovered cows,
whereas, the same decreased (p>0.05) to 8.0±0.0 in
non-recovered cows after treatment.
Conception rate was recorded as 60.0% in
inseminated (only recovered) cows after Garlic extract
treatment in the present study, which was slightly
higher than earlier ndings (Sarkar et al., 2006; Kumar
et al., 2011; Rahi et al., 2014; Yildiz, 2016; Sharma
et al., 2018; Kumar et al., 2018) who reported same
(range: 50.0-52.4%). Further, the pregnancy was
conrmed by trans-rectal palpation after 50-60 days of
insemination and it was recorded as 50.0% as against
nil pregnancy in control group.
Endometritis is basically caused by non-specic
bacterial agents and moreover the infection is mostly
undulant. Mixed infection evident in endometritis
indicates body stress and lowered immune status. Garlic
is natural protection even against physiological threats
like oxidative stress, cardiovascular complexities and
immune dysfunction. Garlic was recommended for
immune-modulatory eect as it increases T-lymphocyte
blastogenesis and phagocytosis with modulation of
cytokine production (Mukherjee et al., 2014).
Combined treatment of Garlic extract with
Ashwagandha in infectious repeat breeder cows
from Group-II showed 75% recovery rate as against
nil recovery in control group. Conception rate was
recorded as 66.7% in inseminated cows. Subsequently,
pregnancy rate was recorded as 55.6% on per-rectal
palpation as against nil pregnancy in control group. It
was observed that similar treatment was employed
earlier (Rahi et al., 2014), who reported 75% recovery
rate and 62.5% conception rate in endometritis cows
treated with Garlic extract IU plus Ashwagandha
powder orally. However, they found higher recovery
(87.5%) and conception (75.0%) rate when both Garlic
extract and Ashwagandha were administered through
intrauterine route.
Table 1: Mean pH and PMN cell count before and after treatment in infectious repeat breeder cows.
Group Before treatment After treatment
Recovered Non-recovered
pH PMN, % pH PMN, % pH PMN, %
Garlic Extract 8.3±0.1a9.4±0.5c7.3±0.1b2.9±0.3d8.4±0.0 8.0±0.0
Garlic + Ashwagandha 8.5±.01a10.0±0.3c7.4±0.1b4.7±0.3d8.2±0.0 8.5±0.0
Control 7.8±0.2 9.9±0.4 - - 8.4±0.1 9.7±0.5
a,b,c,dp<0.05
Indian Journal of Animal Reproduction 40 (1): June 2019
34
The mean pH value of CVM reduced from 8.5±0.1
to 7.4±0.1 in recovered cows whereas, remained same
(8.2±0.0) in non-recovered cows after treatments. WST
became negative in all recovered cases while remained
positive in non-recovered cases on subsequent estrus
after treatment. Average PMN cell count was 10±0.3%
before treatment in this group, which reduced to
4.7±0.3% (p<0.05) in recovered cases, whereas,
reduced (8.5±0.0%; p>0.05) in non-recovered cows.
The mean immunoglobulin level (mg/ml) in group-II
increased from 6.8±0.4 to 12.3±0.9 after treatment. No
report citing immunoglobuline occulation test results
could be traced from the literature for comparison of
the current ndings in infectious repeat breeding.
In conclusion, the study showed that although bare
Garlic extract yielded higher recovery rate (83.3%),
however the nal pregnancy rate on treated basis
was similar for both treatment protocols irrespective of
chronic or fresh repeat breeding. Statistically, recovery
(83.3 vs. 75.0%), conception (60.0 vs. 66.7%) and
pregnancy rate (50.0 vs. 55.6%) in Garlic extract
therapy vs. combination therapy diered (p>0.05) in
endometritis cows.
REFERENCES
Bajaj, N.K., Jain, S.K., Swamy, M. and Shrivastava,
O. (2016). Diagnosis subclinical endometritis in
post-partum murrah bualoes using cytobrush
technique. J. Anim. Res., 6: 1065-1068.
Deshpande, A.R., Gujar, M.B. and Bannalikar, A.S.
(1991). Passive transfer of some immunological
activities in new born calves from levamisole
treated dams. Indian Vet. J., 68: 614-620.
Gebreyohannes, G. and Gebreyohannes, M. (2013).
Medicinal values of garlic: A review. International
J. Med. Medical Sci., 5: 401-408.
Kumar, A., Srivastava, S., Kumar, S., Kumar, A.,
Yadav, S., and Chaudhary, V. (2017). Physico-
Chemical Characterstics of Cervical Discharge
in Endometritic Repeat Breeder Cow. Int. J. Pure
App. Biosci., 5: 821-831.
Kumar, R., Sinha, M.P., Kumar, A., Kurmi, D.J., and
Kumar, P. (2018). Management of Endometritic
Repeat Breeding Cross Bred Cow with Herbal
Extract to Improve the Conception Rate. Int. J.
Curr. Microbiol. App. Sci., 7: 4621-4626.
Kumar, S., Gupta, H.P., Prasad, S. and Singh, B.
(2011). Immunomodulatory eects of garlic and
tulsi in repeat breeding crossbred cows. Indian J.
Anim. Reprod., 32 (1).
Mukharjee, P.K., Nema, N.K., Bhandra, S., Mukherjee,
D., Braga, F.C. and Matsabisa M.G. (2014).
Immunomodulatory lead from medicinal plants.
Indian J. Traditinal Knowledge, 13: 235-256.
Popov, Y.N. (1969). Diagnosis of occult endometritis
in cows (using white site test on cervical mucus).
Vet. Moscow, 4: 85-87.
Rahi, S., Gupta, H.P., Prasad, S., and Baithalu,
R.K. (2014). Phytotherapy for endometritis and
subsequent conception rate in repeat breeding
crossbred cows. Indian J. Anim. Reprod., 34 (1).
Sarkar, P., Kumar, H., Rawat, M., Varshney, V.P.,
Goswami, T.K., Yadav, M. C. and Srivastava, S.K.
(2006). Eect of administration of Garlic Extract
and PGF2α on a hormonal changes and recovery
endometritis cows. Asian-Aust. J. Anim. Sci., 19:
964-969.
Sharma, P., Srivastava, S., Kumar, R., and Singh,
V.B. (2018). Phytotherapy: an alternative low cost
therapeutic management of endometritis in dairy
animals. Int. J. Curr. Microbiol. App. Sci., 7: 4581-
4591.
Sheldon, I.M., Price, S.B., Cronin, J., Gilbert, R.O.
and Gadsby, J.E. (2009). Mechanisms of infertility
associated with clinical and subclinical endometritis
in high producing dairy cattle. Reprod. Dom. Anim.,
44 (Suppl. 3): 1-9.
Yildiz, A. (2016). Eect of intrauterine Allium sativum
extract on recovery and conception rate in dairy
cows with subclinical endometritis. Indian J. Anim.
Sci., 86: 154-157.
Sawale et al.
Indian Journal of Animal Reproduction 40 (1): June 2019
35
INTRODUCTION
During postpartum period, the important
events include uterine involution, regeneration of
endometrium, elimination of bacterial contamination of
uterus and the return of ovarian cyclical activity (Peter
et al., 1987). Dystocia leads to uterine contamination
and aects involution and subsequently fertility. Thus,
dystocia aected bualoes subjected to fetotomy were
administered uterine ecbolics like oxytocin and PGF2α
and GnRH postpartum to augment the process of
involution and revival of ovarian cyclicity.
MATERIALS AND METHODS
In 40 Murrah bualoes, the dystocia was corrected
by fetotomy and the animals were divided into two
groups. Group I bualo (n=20) were a) Oxytocin (50IU
I/M) immediately and 4h after fetotomy, b) Prostaglandin
F2α (500µg I/M on d7 and d21 postpartum), and c)
GnRH (buserlin acetate @ 20µg I/M) on day 28
postpartum. Group II bualo (n=20) were injected only
with routine antibiotics, analgesics and calcium. Group
III comprised of 10 normally calved bualoes and were
not given any medication.
All the bualoes subjected to fetotomy were
sampled for blood and uterine uid immediately after
obstetrical treatment and subsequently on d7, 14, 21
and 28 postpartum. An autoclaved one-way stainless-
steel catheter 15 inches length and 3 mm diameter
was used for collection of uterine uid with the help
of a sterile syringe. Five ml of the aspirated uid was
transferred to a sterile plastic tube, placed in ice, and
transported to laboratory within 4 h of collection for
further analysis. A drop of the uterine uid collected
was put on a clean grease free slide and a smear
was prepared immediately. The smears were air
dried, wrapped in a tissue paper and transported to
laboratory. The smear was stained with Leishman
stain for 10 min and allowed to dry on slide warming
stand. Under a microscope (100x), 100 cells per slide
were calculated for total percentage of PMN cells and
lymphocytes.
The uterine dynamics were obtained by palpation
1Veterinary Assistant Surgeon, Animal Husbandry Department Jammu
and Kashmir; 2Assistant Professor, Department of Public Health and
Epidemiology, Sher-e-Kashmir University of Agricultural Sciences
and Technology, Jammu - 180 009; 3Professor (Retd.), Department
of Teaching Veterinary Clinical Complex; 4Dean, College of Veterinary
Science; *aejaz748@gmail.com
EFFECT OF OXYTOCIN, PGF2α AND GnRH ON UTERINE INVOLUTION
AND POSTPARTUM FERTILITY IN MURRAH BUFFALOES SUBJECTED TO
FETOTOMY
A.A. WANI1*, M.B. BHAT2, P.S. MAVI3 AND P.S. BRAR4
Department of Veterinary Gynaecology and Obstetrics, Guru Angad Dev Veterinary and Animal Science
University, Ludhiana - 141 004
Received: 19.02.2018 Accepted: 09.03.2018
ABSTRACT
Out of dystocia aected Murrah bualoes subjected to fetotomy operation (n=40), one group of 20 bualoes
was administered a) Oxytocin (50IU I/M) immediately and 4h after fetotomy, b) Prostaglandin F2α (500µg I/M on
d7 and d21 postpartum), and c) GnRH (buserlin acetate @ 20µg I/M) on day 28 postpartum. The second group
of 20 animals subjected to fetotomy were not administered any test medication. The third group consisting of
10 normally calving bualo served as control. The bualoes of frist group had improved uterine health, ovarian
rebound and became pregnant in lesser time than others. In brief, administration of ecbolics and GnRH in early
postpartum period hastens uterine involution and improves reproductive eciency of dystocia aected bualoes.
Key words: Bualo, Ecbolics, Fetotomy, GnRH, Involution, PGF2α, Postpartum
Indian Journal of Animal Reproduction 40 (1): June 2019
36 Wani et al.
per rectum during postpartum period. The complete
involution was judged on rectal palpation by return
of uterus to its normal location in the pelvic cavity,
normal and approximately equal size of uterine horns,
and attainment of normal uterine texture, tone and
consistency. On each per rectal examination, the
observations regarding location and size of uterine
horns and cervix, consistency and tonicity of uterine
musculature, degree of reduction in size of gravid and
nongravid uterine horns, exhibition of ovarian activity
were recorded. In addition, exhibition of behavioural
estrus signs and service period postpartum were also
recorded.
The data was analysed by SPSS software
programme and one-way analysis of variance. The
signicant interactions were tested using Duncan’s
multiple range test.
RESULTS AND DISCUSSION
On d7 postpartum, in all the bualoes, uterus
and cervix were hanging below pelvic symphysis and
located in the abdominal cavity. By d14, 80% and 100%
bualo of group I and III had their genitilia returned to
pelvic brim and pelvic cavity, respectively, whereas,
the group II bualo had their genitilia still in abdominal
cavity. On d28 postpartum, all the animals in group I
and III had their genitilia in pelvic cavity, whereas in
group II only 60% had their genitilia in pelvic cavity. The
diameter of cervix, non-gravid and gravid uterine horn
were 4.70±1.00, 4.20±0.74, >5.0 cm and 7.00±2.91,
5.50±1.22, >5.0 cm in group I and II, respectively as
compared to 3.50±0.70, 3.16±3.36 and 3.20±2.83 cm,
respectively in control group on d28 postpartum.
Polymorphonuclear (PMN) cells in the uterine uid
of bualoes of group I and II were higher (p<0.05) than
group III during postpartum period (Table 1). However,
PMN cells in group I bualoes on d28 postpartum were
lower (p<0.05) than group II bualoes (Table 1), which
indicated better uterine environment in group I.
Table 1: Polymorphonuclear (PMN) cells in uterine uid as well as postpartum (pp) reproductive behavior
of fetotomy operated and control bualoes
Parameter Day Ecbolic+GnRH, n=20 No Ecbolic/ GnRH, n=20 Control, n=10
PMNs 7 68.10±2.16C2 83.10±1.56C3 60.80±3.00D1
14 66.50±4.22BC2 75.10±2.95B3 50.20±2.57C1
21 60.80±1.27B2 72.30±4.09B3 46.00±4.33B1
28 56.85±5.90A2 63.10±2.39A3 41.60±0.74A1
First postpartum behavioural estrus
Days 110.2±6.2D127.4±6.4E65.00±3.53F
Range, days 80-128286-13523 55-751
Estrus in d<120 pp 95%330%2100%1
Estrus in d>120 pp 5%170%2-
Postpartum fertility
Service period, days 111.20±6.15D127.60±6.38E88.60±4.80F
Range, days 105-1482116-156374-1221
d75-120 pp 30%2- 90%1
d≥120 pp 70%130%210%3
Conception rate
d120 pp 70% 30% 80%
A,B,Cp<0.05, within the groups; D,E,F,1,2,3p<0.05, between the groups
Indian Journal of Animal Reproduction 40 (1): June 2019
37Uterine involution in dystocia aected bualo
During postpartum period, the number of days
for the occurrence of rst behavioural estrus, animals
showing estrus within d120 postpartum, overall
service period, animals subjected to AI within study
period as well as conception rate were better in group
I (ecbolic+GnRH) compared to group II (no ecbolic or
GnRH; Table 1).
The bacterial presence in uterus is usual in >90%
of cows in rst 10-14 days postpartum, regardless
of disease signs (Sheldon and Dobson, 2004). In
the present study, the average bacterial count (CFU/
ml) in bualoes subjected to fetotomy was higher
(p<0.05) than the eutocic bualoes on all days of
sampling, which increased from d7 postpartum to d14
postpartum, followed by a decline in all the groups.
On d28 postpartum, average colony counts were
240.00±45 CFU/ml, 354±66 CFU/ml and 82.00±9
CFU/ml in group I, II and III, respectively.
Thus, the administration of PGF2α in early
puerperum had a positive chronotropic eect on
the uterine musculature, which facilitates quick
expulsion of lochia and induction of estrus. The latter
is responsible for physical expulsion of bacterial
contaminants and inammatory products as well as a
possible improvement in uterine defences under low
progesterone (Kasimanickam et al., 2004).
REFERENCES
Peter, A.T., Narshima, K.S., John, D. and Patabiraman,
S.R. (1987). Studies on involution of uterus in
postpartum Murrah bualoes. Indian J. Anim.
Reprod. 8: 1-3.
Sheldon, I.M. and Dobson, H. (2004). Postpartum
uterine health in cattle. Anim. Reprod. Sci. 82:
295-306.
Kasimanickam, R., Dueld, T.F., Foster, R.A., Gartley,
C.J., Leslie, K.E., Walton, J.S. and Johnson, W.H.
(2004). Endometrial cytology and ultrasonography
for the detection of subclinical endometritis in
postpartum dairy cows. Theriogenology, 62: 9-23.
Indian Journal of Animal Reproduction 40 (1): June 2019
38
INTRODUCTION
Antimicrobial drugs hamper the uterine defense
mechanism by inhibiting the phagocytic activity of
polymorphonuclear leucocytes (PMNs). Besides,
antimicrobial drugs have many disadvantages
like high cost of treatment, reduced milk yield,
development of resistance and varying success rate
(Shukla and Pandit, 1989). Burgeoning literature cite
the antimicrobial potential of many herbs like Neem,
Garlic, Ginger, Tulsi, Turmeric and Aloe vera. Neem
(Azadirachta indica) was extensively used in India
as traditional Ayurvedic medicine for the treatment
of various diseases. It was demonstrated that Neem
has immuno-modulatory, anti-inammatory, antifungal,
antibacterial, antiviral, and antioxidant properties
(Kumar, 2014). Recent studies have proved the ecacy
of aqueous Neem seed extract to cure endometritis in
both cows and bualoes (Thombre, 2017).
MATERIALS AND METHODS
Repeat breeder bualoes were examined
transrectally for the conrmation of normal genitalia.
The bualoes having poor body condition score and
pathology of genitalia like cervicitis, kinked cervix and
ovaro-bursal adhesions were excluded. The bualoes
with alkaline pH (>7.5) of CVM, positive white side test
and increased PMN cell count (>4%) in endometrial
cytology were considered as positive for endometritis.
Total 36 bualoes were selected for present research
trial. On the basis of insemination record from
owners, the cases were classied into fresh and
chronic endometritic. The cases with history of repeat
subsequent inseminations at seven times or less were
considered as fresh endometritic cases and more than
seven times were considered as chronic endometritic
cases.
The CVM samples were collected in petridish with
help of 20 ml syringe and AI sheath on the day of oestrus
(pre and post treatment) for pH estimation by digital pH
EFFICACY OF NEEM SEED EXTRACT±ASHWAGANDHA FOR THE
TREATMENT OF ENDOMETRITIS IN BUFFALOES
P.D. KADAM1, N.M. MARKANDEYA2, B.L. KUMAWAT3*, A.G. SAWALE4, S.R. LAWANGE5
AND R.S. ANBHULE6
Department of Animal Reproduction, Gynaecology and Obstetrics, College of Veterinary and Animal
Sciences, Parbhani - 431 402
Received: 28.08.2018 Accepted: 20.09.2018
ABSTRACT
Repeat breeder bualoes (n=36) with subclinical uterine infection tested positive by pH evaluation and White
side test of cervico-vaginal mucus (CVM) and endometrial cytology were divided equally into three groups. Group-I
containing fresh endometritic bualoes (repeated ≤7 times) was administered once with Methanolic Neem seed
extract (100 mcg/ml) @ 30 ml by intrauterine (IU) route, Group-II consisting of chronic endometritic bualoes
(repeated >7 times) was administered with Methanolic Neem seed extract (100 mcg/ml) @ 30 ml IU once only
plus 15 gm Ashwagandha powder by oral route for 5 days and Group-III as a control group was administered with
placebo as 30 ml PBS through IU. In both treated groups, pH of CVM deceased (p<0.05) after treatment and white
side test turned negative in all recovered bualoes. Polymorphonuclear cell count was reduced (p<0.05) post-
treatment in recovered cases. Immunoglobulin level in serum of chronic endometritic bualoes increased (p<0.05)
post-treatment. A higher recovery rate was observed in Group-II as 75% against 66.7% in Group-I and nil recovery
in control group. Pregnancy rates were recorded as 62.5%, 55.5% and nil in group-I, II and III, respectively.
Keywords: Ashwagandha, Bualoes, Ecacy, Endometritis, Methanolic Neem seed extract
1,5,6M.V.Sc. Scholar, 2Professor and Head, 3SMS and 4Assistant
Professor; *drblkivri@gmail.com
Indian Journal of Animal Reproduction 40 (1): June 2019
39
meter and White side test. The positive and negative
CVM samples by White side test were scored as ‘1’
and ‘0’, respectively. Blood samples were collected
and serum was separated for estimation of general
serum immunoglobulin level. General immunoglobulin
levels were estimated using immuno-occulation test
(Deshpande et al., 1991) which is quantitative and
rapid test. Endometrial smear prepared by cytobrush
technique and smear was prepared and stained with
Geimsa stain.
The selected bualoes with endometritis were
equally divided into three groups viz. Group-I
consisting of fresh endometritic bualoes (n=12) were
treated with100 mcg/ml Methanolic Neem seed extract
@ 30 ml by IU route once on the day of estrus; Group-
II comprising of chronic endometritic bualoes (n=12)
were at par with Group-I plus oral Ashwagandha
powder 15 gm in feed for 5 days. Group-III bualoes as
control were administered with placebo treatment as
30 ml PBS solution by IU route on the day of oestrus.
Data pertaining to pH, PMN cell count, recovery
and pregnancy rate was analyzed statistically by using
complete randomized design and ANNOVA.
RESULTS AND DISCUSSION
Bualoes were diagnosed for endometritis on the
basis of alkaline pH of CVM, positive White side test
and PMN cell count >4% (Gilbert et al., 2005; Barlund
et al., 2007; Singh et al., 2016) and selected for the
experiment to evaluate comparative ecacy of two
herbal protocols. White side test was ecitively used
previously also for the diagnosis of endometritis in
bualoes (Puro, 2016; Thombre, 2017).
Mean pH of CVM in all the selected bualoes
was 8.5±0.1 before treatment (Table 1). The current
observation regarding pH of CVM in repeat breeder
bualoes corroborates with earlier ndings (Kumar
et al., 2004). After treatment, average pH, white
side test and PMN cell count diered (p<0.05) in
recovered animals from both treatment groups.
However, the same in control group as well as non-
recovered bualoes was similar (p>0.05, Table 1).
The immunoglobulin occulation test in serum yielded
mean immunoglobulin level as 10.3 mg/ml in group-II
and control bualoes before treatment and that same
increased (p<0.05) in Group-II bualoes.
The bualoes with fresh endometritis recovered
after intrauterine infusion of methanolic Neem seed
extract with a recovery rate of 66.7% in Group-I.
Higher recovery rates as 75-80% were reported in
previous studies for the treatment of endometritis
in bualoes using Neem seed oil through IU route
(Kumar et al., 2009, 2013c). However, 87.5% and
75% recovery rates were documented earlier after
treatment with Methanolic and acetonic Neem bark
extract, respectively, in endometritic cows (Kumar et
al., 2013b). Further, in the current study, the mean
pH value reduced (p<0.05) from 8.6±0.1 to 7.5±0.1
in recovered animals while, the same was similar
(p>0.05) in non-recovered and control bualoes (Table
1). The mean PMN cell count in endometrial cytology
reduced (p<0.05) from 14.8±1.9% to 2.5±0.6% in
Group-I, however, it remained almost similar in non-
recovered and control cases (p>0.05, Table 1).
The combined herbal therapy in Group-II bualoes
with chronic endometritis yielded 75% recovery rate
as against nil recovery in control group. No report
of similar herbal combination for the treatment of
endometritic bualoes and even cows could be traced
from published literature for the comparison of current
ndings. However, the ecacy of herbal blends likes
oral Ashwagandha powder with IU Garlic extract
(Rahi et al., 2013) and IU Ashwagandha extract with
oral Gilloy extract (Kumar et al., 2017) in cows was
studied with 75% recovery rate. The mean pH value in
this group declined (p<0.05) from 8.5±0.1 to 7.5±0.01
after treatment and the same was similar (p>0.05)
in non-recovered and control bualoes. Further,
the average PMN cell count reduced (p<0.05) from
13.7±2.1% to 2.3±0.6% after treatment in recovered
cases, whereas it remained almost unchanged in
non-recovered and control cases (Table 1). The mean
Herbal treatment of endometritis in bualo
Indian Journal of Animal Reproduction 40 (1): June 2019
40
serum immunoglobulin level increased (p<0.05) in
recovered cases from 10.5±0.9 to 15.3±0.7 mg/ml
after treatment, whereas, it declined slightly (p>0.05)
in non-recovered cases (9.1±0.1 mg/ml) and in control
group (9.5±.07 mg/ml).
Pregnancy rate in Group-I and II was 62.5 and
55.5%, respectively as against nil pregnancy in control
group. The pregnancy rate in present study was higher
than earlier studies who recorded only 25% pregnancy
rate in endometritic repeat breeder cows treated with
Methanolic Neem seed extract (Kumar et al., 2013a),
whereas, the same was reported by as 44.4% in
endometritic bualoes treated with aqueous Neem
seed extract (Thombre, 2017).
Thus, the combination therapy (Group-II) yielded
higher recovery rate (75%) as against with bare Neem
seed extract (Goup-I, 66.7% recovery). Conversely,
the pregnancy rate was recorded higher in Group-I
(62.5%) as against Group-II (55.5%).
REFERENCES
Bajaj, N.K., Jain, S.K., Swamy, M. and Shrivastava,
O. (2017). Diagnosis subclinical endometritis in
post-partum murrah bualoes using cytobrush
technique. J. Anim. Res., 6: 1065-1068.
Barlund, C.S., Carruthers, T.D., Waldner, C.L. and
Palmer, C.W. (2008). A comparison of diagnostic
techniques for postpartum endometritis in dairy
cattle. Theriogenology. 69: 714-723.
Deshpande, A.R., Gujar, M.B. and Bannalikar, A.S.
(1991). Passive transfer of some immunological
activities in new born calves from levamisole
treated dams. Indian Vet. J., 68: 614-620.
Gilbert, R.O., Shin, S.T., Guard, C.L., Erb, H. Frajblat,
N.M. (2005). Prevalence of endometritis and its
eect on reproductive performance of dairy cows.
Theriogenology 64: 1879-1888.
Kumar, P., Shrivastava, S.K., Rawat, M., Yadav,
M.C. and Kumar, H. (2004). Eect of certain
immunomodulators on uterine infection and
fertility in postpartum bualoes. M.V.Sc thesis
submitted to Indian Veterinary Research Institute,
Izatnager-243122, Barelly (UP), India.
Kumar, H., Bhooshan, N., Barman, P. and Yadav, M.C.
(2009). Administration of herbal antimicrobials
recovers the endometritis in bualoes. Ind. J.
Anim. Sci., 79: 679-680.
Kumar, H., Singh, B., Goswami, T.K., Rawat, M.
(2013a). Use of Neem preparations for the
treatment of Endometritis in Cows. Ind. J. Anim.
Reprod., 34: 194-196.
Kumar, H., Booshan, N. Dass, R.S. and Garg, A.K.
(2013c). Eect of treatment of
immunomodulators on recovery and conception
rate in endometritic bualoes. Ind. J. Anim. Sci.,
83: 129-132.
Kumar, A., Gupta, H.P. and Shivprasad (2013b).
Studies on the immunomodulatory and therapeutic
ecacy of Neem (AZADIRACHTA INDICA) on
endometritis in repeat breeder cow. Ind. J. Anim.
Reprod., 34: 1-4.
Table 1: Mean pH and PMN cell count before and after treatment in endometritic bualoes
Treatment Before treatment After treatment
Recovered Non-recovered
pH PMN, % pH PMN, % pH PMN, %
Neem extract 8.6±0.1a14.8±1.9a7.5±0.1b2.5±0.6b8.5±0.1 14.0±2.9
Neem + Ashwagandha 8.5±0.1a13.7±2.1a7.5±0.1b2.3±0.6b8.4±0.3 12.0±3.1
Control 8.1±0.1 14.5±1.5 - - 8.3±0.1 13.7±1.6
a, bp<0.05
Kadam et al.
Indian Journal of Animal Reproduction 40 (1): June 2019
41
Kumar, A., Srivastava, S., Yadav, S.K., Yadav, A.K.,
Saurabh and Chaudhary,
V. (2017). Physico-chemical Characteristics of
cervical discharge in endometritic repeat breeder
cow. Int. J. Pure App. Biosci., 5: 821-83.
Kumar, A. (2014) Evaluation of immunomoduatory and
therapeutic ecacy of turmeric, neem and garlic on
endometritis in repeat breeding in crossbred cows.
M.V.Sc. thesis submitted to G.B. Pant University of
Agriculture and Technology, Pantnagar.
Puro, N.A. (2016). Ecacy of E. coli LPS and lugol’s
iodine in endometritis treatment of dairy animals.
M.V.Sc. thesis submitted to College of Veterinary
and Animal Sciences, Parbhani.
Rahi, S., Gupta, H.P., Shiv Prasad and Baithalu,
R.K. (2013). Phytotherapy for endometritis and
subsequent conception rate in repeat breeding
crossbred cows. Ind. J. Anim. Reprod., 34: 9-12.
Singh, J., Honparkhe, M., Chandra, A., Kumar,
A., Ghuman, S.P.S., and Dhindsa, S. (2016).
Diagnostic ecacy of uterine cytobrush technique
for subclinical endometritis in crossbreed dairy
cattle. Ind. Vet. J., 93: 11-13.
Shukla, P.S. and Pandit, R.K. (1989). Incidence of
repeat breeding and its remedial measures in Gir
cow and their crosses. Ind. Vet. J., 66: 626-630.
Thombre, G.A. (2016). Ecacy of Neem extract,
Lugol’s iodine and E. coli LPS for the treatment of
endometritis in cows and bualoes. M.V.Sc thesis
submitted to College of Veterinary and Animal
Sciences, Parbhani.
Herbal treatment of endometritis in bualo
Indian Journal of Animal Reproduction 40 (1): June 2019
42
An improvement in milk production in a crossbred
cow warrants feeding strategies like feeding of high
protein diet, however, this leads to an increase in blood
(BUN) or milk (MUN) urea nitrogen. The monitoring
of BUN or MUN is a reliable technique that can be
used for measuring protein status of cattle. However,
it is not known how the levels of urea in circulation,
consequent upon feeding of protein rich diet would
aect the reproductive eciency of crossbred cows.
Recently calved apparently healthy crossbred
cows (n=82) were selected from seven organized
private dairy farms. Each animal was subjected to
four blood and milk samplings on a particular date of
each month starting from rst month of their calving
(rst sample taken within 5-30 day after calving).
Both blood and milk samples were analyzed for
Blood Urea Nitrogen (BUN) and Milk Urea Nitrogen
(MUN), respectively using commercial kits (BUN -
Ortho-Clinical Diagnostics India Pvt. Ltd., Thane;
MUN - Bayer Diagnostics, India) under recommended
protocols. To calculate BUN and MUN of individual
postpartum animals; Firstly, mean BUN/MUN values of
all 4 months was calculated for each individual animal
and then their overall means were calculated for all
animals. The animals with values above and equal
to mean BUN (15.03 mg/dl) and MUN (13.07 mg/dl)
were considered under high BUN/MUN groups and
the others with values below mean BUN/MUN were
considered under low BUN/MUN groups.
The fertility parameters of each cow like calving
to rst estrus interval, calving to rst service interval,
calving to conception interval, service per conception
and percent animal conceived were recorded from
the respective farms. The data were analyzed
statistically using SAS 9.2 software. Signicance level
of percentage animals conceived between the groups
was calculated using CHI Square Test. Signicance
level of fertility parameters was calculated using
Unpaired T Test.
The cows with high BUN or MUN had similar
number of days to exhibit estrus and to rst service
post calving as their counterparts with low BUN or
HIGH BLOOD / MILK UREA NITROGEN HAD DELETERIOUS IMPACT ON
FERTILITY PARAMETERS IN CROSSBRED COWS
I. ANAND1*, G.S. DHALIWAL2 AND P.S. CHAWLA3
Department of Veterinary Gynaecology and Obstetrics, Guru Angad Dev Veterinary and Animal Sciences
University, Ludhiana-141004
Received: 14.09.2018 Accepted: 25.09.2018
ABSTRACT
Eighty-two healthy and recently calved crossbred cows from seven dairy farms were subjected to monthly
blood and milk sampling for a four-month period for blood and milk urea nitrogen (BUN/MUN) assessment. The
fertility parameters like calving to rst estrus interval, calving to rst service interval, calving to conception interval,
services per pregnancy and percent animal conceived were recorded. The cows with high BUN or MUN had
similar number of days to exhibit estrus and to rst service compared to their counterparts (p>0.05). The cows with
low BUN or MUN had lesser number of days to conceive than those with high BUN (p<0.05) or MUN (p>0.05). The
percent animals conceived followed a similar trend in both BUN and MUN groups; low level groups had higher
(p<0.05) conception rates. The number of services per conception was less among cows having low BUN or
MUN. In brief, high BUN/MUN have deleterious eects on fertility of lactating crossbred cows.
Keywords: BUN, Crossbred cows, Fertility, MUN, Protein
1M.V.Sc. Scholar; 2Professor, Department of Teaching Veterinary
Clinical Complex; 3Professor, Department of Veterinary and Animal
Husbandry Extension Education; *drishantvmo@gmail.com
Indian Journal of Animal Reproduction 40 (1): June 2019
43
MUN (p>0.05, Table 1). Similarly, high BUN (Harris,
1992) or MUN (Larson et al., 1997) had no impact on
days to rst estrus and days to rst service intervals.
Others observed that cows with low MUN (<10 mg/
dl) presented longer intervals between calving to rst
estrous and calving to rst service with justication that
animals fed with low protein resulted in delayed post-
partum ovarian activity (Carlsson and Pehson, 1993).
Furthermore, cows with high MUN also exhibited
signicant extension in calving to rst estrous interval
(Veena et al., 2016).
The cows with low BUN or MUN took lesser number
of days to conceive than those with high BUN (p<0.05)
or MUN (p>0.05, Table 1) as reprted earlier (Rajala-
Schultz et al., 2001). The percent cows conceived
followed a similar trend in both BUN and MUN groups,
and low-level groups had higher (p<0.05) conception
rates (Table 1), as reported earlier (Tshuma et al.,
2014).
The logic for lower fertility in cows having high BUN
has been attributed to uterine pH, which decreased
approximately by 0.1 pH units for each 5 mg/100 ml
increase in BUN. These changes might have aected
the uterine environment, which in turn retarded the
embryo development and hence, inuenced conception
adversely. Alternatively, the reason behind negative
association between high BUN and reproductive
performance could be due to the concurrent energy
decit in such animals (Gulinski et al., 2016). Most of
studies pointed out that BUN/MUN at >19 mg/dl and
<7 mg/dl could be detrimental to fertility. Since the
cut o level in the present study was 15.5 mg/dl for
BUN and 13.07 mg/dl for MUN, therefore, it could be
inferred that fertility was not aected to that extent as
at the cut-o level of 19 mg/dl.
To conclude, high BUN/MUN could have
deleterious eects on fertility of lactating crossbred
cows. Therefore, the periodic screening for BUN/
MUN during postpartum period should be encouraged
that would provide early insights regarding proactive
dietary protein adjustments for achieving optimum
reproductive eciency.
REFERENCES
Carlsson, J. and Pehrson, B. (1993). The Relationships
between seasonal variations in the concentration
of urea in bulk milk and the production and fertility
of dairy herds. J. Vet. Med. Series A., 40: 205-212.
Gulinski, P., Salamonczyk, E. and Mlynek, K. (2016).
Improving nitrogen use eciency of dairy cows in
relation to urea in milk. Anim. Sci. Papers Reports,
34: 5-24.
Harris, B. (1992). Protein intake and dairy cow fertility.
University of Florida Cooperative Extension
Service, Institute of Food and Agriculture Sciences,
EDIS.
Urea and fertility in crossbred cows
Table 1: The comparison of fertility parameters between High BUN / MUN or Low BUN / MUN exhibiting
crossbred cows.
Parameter(s) Blood urea nitrogen (BUN) Milk urea nitrogen (MUN)
High BUN,
>15.3 mg/dl,
n=46
Low BUN, <15.3
mg/dl, n=36
High MUN,
>13.1 mg/dl,
n=37
Low MUN,
<13.1 mg/dl,
n=45
Calving-1st estrous interval, d 89.5±1.9 84.4±2.4 89.1±2.4 85.3±1.9
Calving-1st service interval, d 93.9±2.2 90.7±2.6 93.2±2.8 91.8±2.0
Calving-conception interval, d 121.6±5.6 104.0±4.7* 113.0±4.2 108.6±8.4
% conceived 45.6 75.0* 32.4 80*
Services/conception 2.1±0.1 1.7±0.1 2.1±0.1 1.8±0.1
*p<0.05; between groups for BUN or MUN
Indian Journal of Animal Reproduction 40 (1): June 2019
44 Anand et al.
Larson, S.F., Butler, W.R. and Currie, W.B. (1997).
Reduced fertility associated with low progesterone
postbreeding and increased milk urea nitrogen in
lactating cows. J. Dairy Sci., 80: 1288-1295.
Rajala-Schultz, P.J, Saville, W.J.A., Frazer, G.S, and
Wittum, T.E. (2001). Association between milk
urea nitrogen and fertility in Ohio dairy cows. J.
Dairy Sci., 84: 482-489.
Tshuma, T., Holm, D.E., Fosgate, G.T. and Lourens,
D.C. (2014). Pre-breeding blood urea nitrogen
concentration and reproductive performance of
Bonsmara heifers within dierent management
systems. Trop. Anim. Health Prod., 46: 1023-1030.
Veena, M.P., Gowrakkal, M., Kumar, V.G., Gupta,
P.S.P. and Narayanaswamy, M. (2016). Relation
of milk parameters with postpartum reproductive
eciency in cattle. International J. Biomed. Res.,
7: 405-409.
Indian Journal of Animal Reproduction 40 (1): June 2019
45
Low reproductive eciency in bualoes remains
a major economic problem globally. Various hormonal
preparations were used in anestrus and subestrus
bualoes with variable success (Rathore et al., 2006;
Singh et al., 2006). The present study investigated
the impact of cervical mucus fern pattern and serum
micro-mineral prole on conception rate in anestrus
Surti bualo subjected to estrus synchronization.
Eighteen Surti bualoes between 45 and 120 days
postpartum were included in the present study. Estrus
was detected daily with the help of teaser bull parading
during morning and evening hours. The animals not
exhibiting overt signs of estrus were segregated and
subjected to rectal palpation. The animals with smooth
ovaries on rectal palpation at eleven-day interval were
divided at random into three groups of six animals
each. The rst group was subjected to standard
ovsynch protocol followed by xed time inseminations
(FTAI) twice, morning and evening, on day 10 of
protocol. The second group bualoes were inserted
PRID (0.9 g of progesterone) intra-vaginally which
was kept in-situ from day 0 to 7 of ovsynch protocol.
Rest of the ovsynch protocol and FTAI was same as in
group-1. The third group control (no hormone therapy)
bualoes were inseminated at spontaneous estrus.
The pregnancy was conrmed per rectally 60 days of
last AI.
The procedures for arborization or fern pattern
of cervico-vaginal mucus collected befrore AI were
carried out as per the known standard pattern using
a microslide. Blood samples (5-6 ml) were collected
from all animals on day 0 (prior to treatment), day 4
(during treatment), day 8 (after cloprostenol inj.), and
day of estrus / FTAI and on day 28 (18th day post-AI) by
jugular vein puncture in serum clotting vaccutainers,
and serum separated was stored at –20°C until
analysis. Serum was subjected to estimation of trace
elements (copper, zinc, iron and manganese) on an
Atomic Absorption Spectrophotometer.
The test of signicance among and within the
groups for micro elements prole was made by analysis
of variance and the mean dierences between and
within the groups were tested using Duncan’s multiple
range test at 5% level of signicance.
In the present study, the overall conception rate,
irrespective of estrus synchronization group, was
higher (77.8 vs. 42.8%, p>0.05) in bualoes with
CERVICAL MUCUS FERN PATTERN IN RELATION TO FERTILITY AND
MICRO-MINERAL PROFILE IN ANESTRUS SURTI BUFFALOES SUBJECTED
TO OVSYNCH ALONE AND IN COMBINATION WITH PRID
H.R. SAVANI, C.T. KHASATIYA*, S. SAXENA, M.D. PATEL, V.R. PATEL, L.C. MODI AND N.F.
CHAUDHARI
Department of Veterinary Gynaecology and Obstetrics, College of Veterinary Science and Animal Husbandry,
Navsari Agricultural University, Navsari, Gujarat
Received: 10.04.2018 Accepted: 15.05.2018
ABSTRACT
Eighteen anestrus Surti bualoes were subjected to Ovsynch or Ovsynch plus PRID or inseminated at
spontaneous estrus (n=6 in each group). Overall conception rate was 77.8% in bualoes with typical fern pattern
as compared to 42.8% with atypical fern pattern. Serum copper, zinc, iron and manganese concentrations were
similar (p>0.05) between animals during the study period.
Key words: Anestrus, Bualo, Cervical mucus, Fern pattern, Ovsynch, Surti
*drctkhasatiya@yahoo.in
Indian Journal of Animal Reproduction 40 (1): June 2019
46
typical compared to atypical fern pattern. However,
the serum copper, zinc, iron and manganese and in
acyclic Surti bualoes was similar (p>0.05) within as
well as between all the treatment and control groups
including overall mean values at dierent days.
In brief, similar concentrations of various serum
micro-minerals values between treated and control
groups at dierent days or between groups suggested
optimum nutritional supplementation and healthcare
strategies adopted in the organized farm of Surti
bualoes. Nevertheless, the typical fern pattern was
associated with better chances of conception in Surti
bualo.
REFERENCES
Rathore, K.K., Pandit, R.K., Agrawal, R.G. and
Shrivastava, O.P. (2006). Evaluation of
comparative eciency of certain drugs for
treatment of anestrous, assessment of cervical
mucus, quality and fertility in Murrah bualoes.
Indian. J. Anim. Prod. Mgmt., 20: 63-65.
Singh, J., Verma, H.K., Singh, K.B. and Singh, N.
(2006). Incidence of reproductive disorders in
dairy animals in dierent regions in Punjab. J.
Res., 43: 224-227.
Savani et al.
Indian Journal of Animal Reproduction 40 (1): June 2019
47
Breeding, an essential component in canine
rearing, is an art as well as science as it needs skill
and knowledge. A responsible breeder should take
care of dog before pregnancy, during pregnancy and
after whelping (Ivanova and Georgiev, 2018). Based
on preventive measures, proper managemental
practices and knowledge level of dog owner, the
outcome of pregnancy depends. The extension
machinery serves as a useful media for the
dissemination of latest technological knowledge to
dog owners via various communication methods. The
present study was carried out to assess the knowledge
level of dog owners about breeding practices and
correlation of these breeding knowledge levels with
communication proles like extension contact, social
participation and mass media exposure.
The present study was conducted in Teaching
Veterinary Clinical Complex (TVCC) of the University
and in dierent districts of Punjab state. The randomly
selected dog owners (n=200) were categorized in
Group I (Urban dog owners) and Group II (Rural dog
owners). The data was collected through pretested
interview schedule by personally interviewing the
dog owners. For breeding practice, dog owners were
categorised into 3 categories based on the knowledge
scores as low, medium and high knowledge with scores
0-≤8, >8-16, and >16 respectively. The knowledge
index was calculated by dividing obtained knowledge
score by maximum possible score and multiplying the
result with 100.
The extension contacts of dog owners with
veterinary ocers/ university/ breeders were
measured on two-point continuum either as ‘No’ (score
0) or ‘Yes’ (score 1). Hence, the maximum score for
extension contact could be 6. The social participations
of dog owners in dog shows/ Livestock championship/
Pashu Palan or Kisan Melas and Animal welfare
camps were measured on two-point continuum
either as ‘No’ (score 0) or ‘Yes’ (score 1). Hence, the
maximum score for social participation could be 8. The
mass media exposure of dog owners with television/
radio/ newspaper/ dog magazines/ internet/ mobile
phone/ books were measured on two-point continuum
either as ‘No’ (score 0) or ‘Yes’ (score 1). Hence, the
maximum score for mass media exposure could be 14.
The collected date was tabulated and analysed with
the help of SAS 9.3 system Carry N C, USA.
ROLE OF EFFECTIVE COMMUNICATION IN ENHANCING DOG OWNER
KNOWLEDGE ABOUT BREEDING PRACTICES IN PUNJAB
Y. ROOPA1, R. KASRIJA2*, H.K. VERMA3 AND P. SINGH4
Department of Veterinary and Animal Husbandry Extension Education, Guru Angad Dev Veterinary and
Animal Sciences University, Ludhiana - 141 012
Received: 05.07.2018 Accepted: 25.08.2018
ABSTRACT
At Teaching Veterinary University Hospital and various districts of Punjab, the dog owners (100 each from
urban and rural area) were randomly selected and personally interviewed using pretested interview schedule.
Breeding knowledge score of both groups was dierent (p<0.05), however, the knowledge level of both groups
was under low category. A positive correlation (p<0.05) existed between breeding practice knowledge index and
their communication proles. The present study underlines the importance of communication prole in improving
the knowledge level of dog owners about correct breeding practices.
Key words: Breeding practice, Communication, Dog, Knowledge, Punjab
1M.V.Sc. Scholar, 2Assistant Professor; 3Director Extension Education;
4Professor cum Head, Department of Veterinary Gynaecology and
Obstetrics; *dr.rajesh.kasrija@gmail.com
Indian Journal of Animal Reproduction 40 (1): June 2019
48
According to communication prole, 50% of urban
dog owners and 70-76% of rural dog owners had low
extension contact and social participation. Overall,
a very few dog owners had high extension contact
(10%), social participation (6%) and mass media
exposure (22.5%).
Regarding dog breeding practices, 26.4% urban
and 14.6% rural dog owners had awareness. In
fact, 70% urban and 95% rural dog owners were not
aware about breed specic characters of dogs. Only
5% rural dog owners have knowledge about number
of times a male dog used for service in a week and
knowledge about the day of mating. Very few dog
owners had knowledge about vaginal cytology, care
during pregnancy, complications during pregnancy and
whelping, pseudo pregnancy, sexually transmissible
diseases, venereal granuloma, causes of pregnancy
loss and new borne pup care. Others suggested that
owner knowledge about length of gestation, methods
for pregnancy diagnosis, weight management,
parturition signs and timings are more important
(Fontaine et al., 2007).
The breeding knowledge score was high
(6.34±0.37) in urban compared to rural (3.51±0.47,
p<0.05) dog owners, however both groups had
low knowledge score. Furthermore, there was a
positive (p<0.05) correlation of breeding knowledge
index of dog owners with extension contact, social
participation and mass media exposure (Table 1).
Others reported that extension contact, exposure to
extension mass-media, management orientation and
innovation proneness among dog owners of 3 urban
cities of Gujarat state had signicant relationship with
knowledge of dog owners (Raval et al., 2015). This
shows that communication prole plays an important
role for improving knowledge level of dog owners
relating to dog breeding practices.
In brief, the present study highlights the dierence
in knowledge level of urban and rural dog owners
abut breeding practices, thus, suggesting the need of
organising extension programmes for enhancing their
knowledge. Also, the communication prole of dog
owner was correlated (p<0.05) with knowledge level.
This indicates that participation of owner in extension
drive can enrich their knowledge level.
REFERENCES
Fontaine, E., Million, C., Levy, X., Grellet, A.,
Fontbonne, A. (2007). Risk factors aecting
parturition and neonatal mortality: a retrospective
study on 1615 bitches. In: Proceedings of 5th
Annual European Veterinary Symposium on Small
Animal Reproduction (EVSSAR), Estoril, Portugal.
Ivanova, C., Georgiev, P. (2018). Pregnancy in the
bitch - a physiological condition requiring specic
care - review. Tradition Modernity Vet. Med. 3: 77-
82.
Raval, H.S., Nayak, J.B., Patel, B.M. and Bhadesiya,
C.M. (2015). Zoonotic importance of canine
scabies and dermatophytosis in relation to
knowledge level of dog owners. Vet. World, 8:
765-766.
Table 1: Spearman Correlation coecient of breeding knowledge index of all dog owners with
communication prole.
Spearman correlation
coecient, n=200
Breeding practice
knowledge index
(BPKI)
Extension
contact (EC)
Social
participation (SP)
Mass media
exposure (MME)
BPKI 10.65748* 0.54317* 0.66914*
EC 0.65748* 10.56284* 0.31778*
SP 0.54317* 0.56284* 10.42269*
MME 0.66914* 0.31778* 0.42269* 1
*p<0.05
Roopa et al.
Indian Journal of Animal Reproduction 40 (1): June 2019
49
The newborn puppy is an immature animal,
dependent on its dam for survival in the rst three
weeks and as a consequence the etiology of neonatal
death is frequently complex and often undetermined
(Blunden, 1998). The majority of pup losses are
stillbirths and deaths within the rst week of life known
as perinatal mortality. The mortality can occur in utero,
during expulsion, after birth, in rst weeks of life or after
weaning. The causes for high pup mortality in perinatal
period can relate to several factors concerning bitch
(mismothering, lack of milk, trauma), birth process
(prolonged labor, dystocia, obstetrics), puppy (low
birth weight, congenital malformations, starvation),
environment and presence of infectious agents
(Munnich, 2008).
The puppies born to bitches (age, 2-6 yr) of
dierent breeds with the history of progressive
whelping and / or dystocia were used in the present
analysis. A total of 100 puppies born to bitches that
underwent Spontaneous Whelping (SW), Assisted
Whelping (AW) and Caesarean section (CS) were
selected. The puppies born to ve bitches without any
medical, manual or surgical assistance either to dam
or puppies were kept in group I (SW, n=30).
The puppies born to ten bitches that delivered
through either manual or medical assistance were kept
in group II (AW). Manual assistance was attempted to
puppies which were partly expelled from the vagina
and/or the puppies whose parts were within reach on
vaginal examination but not progressed further. The
bitches were allowed in standing position or restrained
on their lateral recumbency and the vaginal passage
was well lubricated with liquid paran. The body of
puppies were grasped gently and pulled steadily away
from the bitch caudo-ventrally. While applying traction,
care was taken to ensure that force was not applied on
the limbs of puppies. Induction protocol was initiated
with slow intravenous injection of oxytocin @ 1.1-
2.2 IU/kg IM or SC with a dose range between 5-20
IU every 30 min and concurrent administration of
10% calcium gluconate @ 0.5-1.5 ml/kg to augment
the eect of oxytocin on myometrial contraction and
intravenous uids to correct hydration, electrolyte, and
blood glucose abnormalities. In between, whenever
the bitches were straining, manual assistance was
given to deliver the puppies.
The puppies born to nine bitches that underwent
caesarean section were in group III (CS). Under
general anaesthesia using propofol @ 3 mg/kg and
diazepam @ 0.5mg/kg for induction and 2% isourane
for maintenance, caesarean section was performed
INFLUENCE OF TYPE OF WHELPING ON NEONATAL MORTALITY IN PUPS
A. SUBRAMANI1, N. ARUNMOZHI2*, P. SRIDEVI3 AND M. SHAFIUZAMA4
Department of Veterinary Gynaecology and Obstetrics, Madras Veterinary College, Tamil Nadu University of
Veterinary and Animal Sciences, Chennai - 600 007
Received: 08.08.2018 Accepted: 10.10.2018
ABSTRACT
The puppies (n=100) born to bitches that underwent Spontaneous Whelping (SW), Assisted Whelping (AW)
and Caesarean section (CS) were divided into groups as SW (n=30), AW (n=35) and CS (n=35). Out of 100
puppies, 25% were born dead (2% SW, 7% AW and 16% CS). Among viable pups at birth, the early neonatal
mortality till 24 h was 18% (0% SW, 5.7% AW and 45.7% CS). In brief, the stillbirth and early neonatal mortality
was high in pups delivered through caesarean compared to spontaneous or assisted whelping.
Key words: Assisted whelping, Caesarean section, Neonatal mortality, Pups, Spontaneous whelping
1M.V.Sc. Scholar, 2*Assistant Professor; 3Professor, Department of
Clinics; 4Professor, Department of Surgery and Radiology
Indian Journal of Animal Reproduction 40 (1): June 2019
50
through mid-ventral approach by adopting standard
surgical procedures.
Out of 100 pups evaluated, 25% were born dead
(6.7% SW, 20% AW, 16% CS), 8% died within 30 min
(0% SW, 5.7% AW, 17.1% CS), 6% died within 2h (0%
SW and AW, 17.1% CS), 4% died in 2-24h (0% SW
and AW, 11.4% CS), and 57% survived beyond 24h
(93.3% SW, 74.3% AW, 8.6% CS).
The incidence of born dead or stillbirth in the
present study (25%) is higher than earlier studies
reporting between 4.3-11.5% (Indrebo et al., 2007;
Tonnessen et al., 2012), that could be due to higher
stillbirths in CS group which were delayed cases.
Further, the eect of fetal distress arising from dystocia
or the fetus retained for long time in the uterus or birth
canal or exposed to eects of oxytocin prior to resorting
emergency CS may be responsible for increased
incidence of stillbirths in present study (Gropetti et
al., 2010; Jayakumar et al., 2015). Hence, most of
the still born puppies may be saved if timely adequate
veterinary assistance is given.
Among viable pups at birth, the early neonatal
mortality till 24 h was 18% (0% SW, 5.7% AW and
45.7% CS) in the present study, which was higher when
compared to an earlier report (Potkay and Bacher,
1977). The high incidence of neonatal mortality was
mainly in pups delivered through CS which might be
due to hypoxia, hypoglycemia and hypothermia due to
delayed whelping (Münnich and Küchenmeister, 2014)
followed by the eects of anaesthetic agents on the
cardiac, respiratory and nervous systems during CS.
Hence, the number of early neonatal mortalities may
be reduced if CS is performed at the earliest.
In brief, an early and adequate veterinary
assistance can reduce the incidence of stillbirth and
neonatal mortality in pups.
REFERENCES
Blunden, A.S. (1998). The neonate: Congenital defects
and fading puppies. In: Manual of Small Animal
Reproduction and Neonatology, Simpson, G.M.,
England, G.C.W. and Harvey, M. (eds.). Bri. Small.
Anim. Vet. Assoc., 143-152.
Groppetti. D., Pecile, A., Del Carro, A.P., Copley, K.,
Minero, M. and Cremonesi, F. (2010). Evaluation
of newborn canine viability by means of umbilical
vein lactate measurement, Apgar score and
uterine tocodynamometry: Theriogenology, 74:
1187-1196.
Indrebo, A., Trangerud, C. and Moe, L. (2007). Canine
neonatal mortality in four large breeds. Acta. Vet.
Scand. Suppl, 49(S2).
Jayakumar, C., Krishnaswamy, A., Sudha, G. and
Honnappa, T.G. (2015). Assessment of canine
neonate by apgar scoring system and its value as
an index of short term neonatal survival. Indian J.
Anim. Reprod., 36: 14-20.
Munnich, A. and Kuchenmeister, U. (2009). Dystocia
in numbers: evidence-based parameters for
intervention in dogs: causes for dystocia and
treatment recommendations. Reprod. Domest.
Anim. 44: 141-147.
Munnich, A., (2008). The pathological newborn in small
animals: the neonate is not a small adult. Vet. Res.
Commun., 32: S81-85.
Potkay, S. and Bacher, J.D. (1977). Morbidity and
mortality in a closed foxhound breeding colony.
Lab. Anim. Sci., 27: 78-84.
Tonnessen, R., Sverdrup Borge, K., Nodtvedt, A. and
Indrebo, A. (2012). Canine perinatal mortality: A
cohort study of 224 breeds. Theriogenology, 77:
1788-1801.
Subramani et al.
Indian Journal of Animal Reproduction 40 (1): June 2019
51
True anestrus is a result of suppression of
reproductive hormone release through the eect of
lactation, nutrition and systemic diseases. This study
assessed the metabolic and macro-mineral prole
in postpartum Surti bualoes subjected to estrus
synchronization protocol.
Eighteen Surti bualoes between 45 and 120 days
postpartum were included in the present study. Estrus
was detected daily with the help of teaser bull parading
during morning and evening hours. The animals not
exhibiting overt signs of estrus were segregated and
subjected to rectal palpation. The animals with smooth
ovaries on rectal palpation at eleven-day interval were
divided at random into three groups of six animals
each. The rst group was subjected to standard
ovsynch protocol followed by xed time inseminations
(FTAI) twice, morning and evening, on day 10 of
protocol. The second group bualoes were inserted
PRID (0.9 g of progesterone) intra-vaginally which
was kept in-situ from day 0 to 7 of ovsynch protocol.
Rest of the ovsynch protocol and FTAI was same as in
group-1. The third group control (no hormone therapy)
bualoes were inseminated at spontaneous estrus.
The pregnancy was conrmed per rectally 60 days of
last AI.
Blood samples (5-6 ml) were collected from all
animals on day 0 (prior to treatment), day 4 (during
treatment), day 8 (after cloprostenol inj.), and day
of estrus / FTAI and on day 28 (18th day post-AI) by
jugular vein puncture in serum clotting vaccutainers,
and serum separated was stored at –20°C until
analysis. Serum was subjected to estimation of serum
metabolic prole (glucose, protein, cholesterol) as
per standard procedures, as well as macro-mineral
prole (calcium, phosphorus and magnesium) using
an Atomic Absorption Spectrophotometer.
The test of signicance among and within the
groups for micro elements prole was made by analysis
of variance and the mean dierences between and
within the groups were tested using Duncan’s multiple
range test at 5% level of signicance.
Serum glucose in acyclic Surti bualoes was
similar (p>0.05) between days within and between
treatment and control groups. (Table 1). However,
serum total protein and cholesterol was higher in
SERUM METABOLIC AND MACRO-MINERALS PROFILE IN POSTPARTUM
ANESTRUS SURTI BUFFALOES TREATED WITH OVSYNCH ALONE AND IN
COMBINATION WITH PRID
H.R. SAVANI, C.T. KHASATIYA*, S. SAXENA, S.B. PATEL, V.K. SINGH, S.S. CHAUDHARY
AND C.F. CHAUDHARI
Department of Veterinary Gynaecology and Obstetrics, College of Veterinary Science and Animal Husbandry,
Navsari Agricultural University, Navsari, Gujarat
Received: 19.04.2018 Accepted: 14.05.2018
ABSTRACT
Eighteen anestrus Surti bualoes were subjected to Ovsynch or Ovsynch plus PRID or inseminated at
spontaneous estrus (n=6 in each group). Serum protein and cholesterol was higher (p<0.05) in synchronized
bualoes compared to their control counterparts. Serum glucose, calcium, phosphorus and magnesium
concentrations were similar (p>0.05) between animals during the study period.
Key words: Anestrus, Bualo, Metabolic, Mineral, Ovsynch
*drctkhasatiya@yahoo.in
Indian Journal of Animal Reproduction 40 (1): June 2019
52
synchronized bualo than control group. Similar values
were obtained earlier in anestrus bualoes (Kumar
et al., 2010; Parmar et al., 2015). Moreover, higher
serum cholesterol concentration observed in Ovsynch
plus PRID group compared to Ovsynch group was in
agreement with earlier ndings (Buhecha et al., 2016).
Serum macro-mineral prole (calcium, phosphorus
and magnesium) of acyclic Surti bualoes neither
diered (p>0.05) within group nor between treatment
and control groups at any of the intervals.
REFERENCES
Buhecha, K.V., Dhami, A.J., Theodore, V.K., Thakor,
R. and Parmar, S.C. (2016). Eect of various
ovulation synchronization protocols on estrus
response, conception rate and blood biochemical
prole in anoestrus bualoes. Intl. J. Adv. Vet. Sci.
Technol., 5: 232-238.
Kumar, H., Bhooshan, N., Patra, M.K. and Yadav, M.C.
(2010). Treatment with progestagen and PMSG to
prevent prolonged anestrus in bualoes. Indian J.
Anim. Sci., 80: 623-625.
Parmar, S.C., Khasatiya, C.T., Chaudhary, J.K.,
Patel, R.V. and Dhamsaniya, H.B. (2015). Serum
metabolic and minerals prole in Norgestomet
primed postpartum anestrous surti bualoes. Vet.
World, 8: 625-630.
Savani et al.
Indian Journal of Animal Reproduction 40 (1): June 2019
53
Low fertility in cow has multifactorial etiology
involving genetic improvement, inadequate nutrition
and poor reproductive management. The partitioning
of the relative impact of the various factors on infertility
is not well understood. Doublesynch protocol has
the potential to increase the pregnancy rates in
primiparous dairy cows (Oztruk and Baran, 2009).
Thus, the present study was conducted to assess the
ecacy of doublesynch protocol in improving infertility
in crossbred cattle under eld conditions.
Healthy pluriparous crossbred cows and pubertal
heifers free from any palpable abnormalities of the
reproductive tract were randomly divided in two groups
and provided with area specic mineral mixture for
45 days @ 30 gm/day. Group I (n=10) animals were
regular cyclic with no failure of conception and these
were inseminated at observed estrus, twice at 24h
interval, without any treatment. The group II animals
(n=100) consisted of repeat breeders (n=46) with no
palpable abnormality of reproductive tract, anestrous
animals (n=35) without corpus luteum in the ovary, and
pubertal heifers (n=19) of >4 yr age with no reproductive
tract abnormalities but failed to conceive. Group II was
subjected to Doublesynch protocol (PGF2α analogue
@500 µg on day -2, followed by ovsynch protocol from
day 0 onward) and subsequent FTAI as per standard
procedures.
Body Condition Score (BCS) was assessed for all
the animals as per the standard technique (Wildman et
al., 1982) and was recorded to be in the range of 2.4
to 3.4. Blood was collected at the time of screening,
plasma was separated and stored at -20°C for the
estimation of BHBA by spectrophotometer. In all the
groups, the intensity of estrus and duration of estrus
were recorded and pregnancy was conrmed by rectal
palpation on day 60 post AI. The accumulated data
were analysed statistically.
Conception rate in group I and group II cows was
40% and 54%, respectively. Others also reported
an increase pregnancy rate in cattle subjected to
doublesynch protocol (Oztruk and Baran, 2009).
Plasma BHBA levels in cattle that conceived or failed
to conceive in group I was 0.45 mmol/l, whereas in
group II, their values were 0.54 and 1.00 mmol/l,
SYNCHRONIZATION OF OVULATION USING DOUBLESYNCH PROTOCOL
IN CROSSBRED CATTLE UNDER FIELD CONDITIONS
K. NAVAMANI1, K. KULASEKAR2*, K. KRISHNAKUMAR3 AND L. RADHAKRISHANAN4
Department of Veterinary Gynaecology and Obstetrics, Madras Veterinary College, Tamilnadu Veterinary and
Animal sciences University, Vepery, Chennai - 6000 007
Received: 19.07.2017 Accepted: 10.09.2017
ABSTRACT
The crossbred cattle (n=110) were randomly divided to receive either no treatment and were regular cycling
(group 1, n=10), and the remaining (group II, n=100) were pubertal heifers, repeat breeders and anestrous. The
animals were fed area specic mineral mixture @ 30 gm/d/animal for 45d and group II animals were subjected
to doublesynch protocol followed by FTAI. Body condition scores (BCS) was accessed and ranged 2.4-3.4 in all
the animals. Blood samples was collected for the estimation of plasma Beta hydroxy butyric acid (BHBA) and
the animals that failed to conceive to doublesynch had higher plasma BHBA. The conception rate was 54% in
synchronized compared to 40% in control group. Thus, doublesynch is a useful protocol to improve the conception
rate in infertile cattle under eld condition.
Keywords: BHBA, Conception, Crossbred cattle, Doublesynch, Hormone
1Veterinary Assistant Surgeon, Government of Tamilnadu; 2Professor;
3Professor, 4Professor and Head, Post Graduate Research Institute
and Animal Sciences, Kattupakkam; *kannankulasekar@gmail.com
Indian Journal of Animal Reproduction 40 (1): June 2019
54
respectively. In fact, BHBA values >0.7 mmol/l indicate
severe negative energy balance (Adewuyi et al.,
2004). In this study, even though doublesynch protocol
was initiated to improve the conception rate, 46% of
repeat breeders failed to conceive because of negative
energy balance.
In present study, the conception rate in doublesynch
treated repeat breeders as well as anestrous cattle
was similar (56.5 vs. 54.3%, respectively), where as
pleuriparous animals showed better conception rate
than heifer (55 vs. 45%, respectively).
The intensity of estrus ranged from mild to intense,
with animals showing moderate estrus in both the
control (2/10) and treatment group (55/100) when
compared with mild and intense estrus. The mean
duration of estrum was 19.05, 18.75, 22.02 and 20.39
h in animals that conceived or failed to conceive in
group I and group II, respectively.
It can be concluded from the present study that
doublesynch protocol can be a useful tool to improve
reproductive eciency in crossbred cattle under eld
conditions.
REFERENCES
Adewuyi, A.A., Gruys, E. and Van Eerdenburg, F.J.C.M.
(2005). Non-esteried fatty acids (NEFA) in dairy
cattle. A review. Vet. Quarterly, 27: 117-126.
Ozturk, C. and Baran, A. (2010) Is doublesynch protocol
a new alternative for timed articial Insemination in
anestrous dairy cows? Theriogenology, 73: 568-
576.
Wildman, E.E., Jones, G.M., Wagner, P.E., Boman,
R.L., Troutt Jr, H.F. and Lesch, T.N. (1982). A
dairy cow body condition scoring system and its
relationship to select production characteristics. J.
Dairy Sci., 65: 495-502.
Navamani et al.
Indian Journal of Animal Reproduction 40 (1): June 2019
55
About 32-35% cases of repeat breeding in dairy
animals are due to non-infectious causes and out of
these early embryonic mortality is one of the most
important cause (Diskin et al., 2008). Inadequate
functioning of corpus luteum (CL) is responsible for
early embryonic mortality in cattle (Kimura et al.,
1987). Delayed leutinization may lead to inadequate
release of progesterone during early embryonic
period, thereby reducing the chances for embryonic
survival. Several measures were adapted to correct
luteal dysfunction including use of GnRH (Lewis et al.,
1990) or hCG (Santos et al., 2001) after insemination
which increases progesterone secretion and renders
high conception rate. Considering the post-fertilization
reproductive events and endocrine changes, it is
proposed to attempt GnRH and hCG therapies in non-
infectious repeat breeder cows for improvement of
conception rate.
Thorough clinical investigations were conducted
to select the specic cases of repeat breeding due to
luteal insuciency. The cases were evaluated during
estrus stage by per-rectal palpation as well as through
laboratory investigations to rule out other etiological
factors causing repeat breeding like improper timing
of insemination or embryonic mortality due to uterine
infection. The cervico-vaginal mucus (CVM) was
collected in a sterilized petridish on the day of estrus
simultaneously with per-rectal palpation. Fern patterns
in cervico-vaginal mucus smear were evaluated under
low power (10 X) and were classied as Typical type
(fern pattern with primary, secondary and tertiary
branches), Atypical type (fern pattern with primary
and secondary branches) and Nil type having no fern
pattern. Cervical discharges were also evaluated with
digital pH meter. The observation of physical properties
like color, consistency and odour of CVM reected
the presence of uterine infection and the same was
conrmed with white side test (Bhat et al., 2014).
Uterine mucus samples were aseptically collected
by using cytobrush and smears were prepared and
stained with Geimsa stain for PMN cell count under
oil immersion using 1000X magnication. The positive
cases for uterine infection as conrmed by white side
IMPROVEMENT IN CONCEPTION RATE BY GnRH OR hCG
ADMINISTRATION ON DAY 5 POST-AI IN NON-INFECTIOUS REPEAT
BREEDER COWS
R.S. ANBHULE1, A.G. SAWALE2, N.M. MARKANDEYA3, B.L. KUMAWAT4*, P.D. KADAM5
AND S.R. LAWANGE6
Department of Animal Reproduction, Gynaecology and Obstetrics, College of Veterinary and Animal
Sciences, Parbhani - 431 402
Received: 23.08.2018 Accepted: 08.09.2018
ABSTRACT
Thirty-six non-infectious repeat breeder cows were selected on the basis of typical fern pattern, negative white
side test, normal pH of cervical mucus and <4% polymorphonuclear (PMN) cell count in endometrial content.
Selected animals were randomly distributed in three groups containing twelve animals each. Group-I and Group-
II were treated with inj. GnRH @ 10 mcg and inj. hCG @ 2000 IU by intramuscular route, respectively on day 5
post-AI, whereas group-III served as controls. Conception rates in group I, II and III was recorded as 58.3, 66.7
and 25.0%, respectively. Thus, hCG treatment on day 5 post-AI improved conception rate in non-infectious repeat
breeder cows.
Key words: Conception rate, GnRH, hCG, Non-infectious, Repeat breeder
1,5,6M.V.Sc. Scholar, 2Assistant Professor, 3Professor and Head, 4SMS;
*drblkivri@gmail.com
Indian Journal of Animal Reproduction 40 (1): June 2019
56
test (yellow color), uterine cytology (PMN cells >4%)
and pH (>7.5) were excluded and only non-infectious
repeat breeder cows were selected for the study.
Articial insemination was performed only in the cows
showing typical fern pattern. Thus, total 36 cases of
repeat breeding cows were taken for the experiment
in three groups viz. Group-I (n=12) cows were treated
with inj. GnRH @ 10 mcg by intramuscular route and
Group-II (n=12) cows were assigned with inj. hCG @
2000 IU intramuscularly on day 5 post-AI whereas,
Group-III (n=12) cows were kept untreated post-AI as
a control. The eect of treatment in dierent groups
was evaluated in terms of rst service conception rate
as conrmed by trans-rectal examination after 60 days
of AI.
Typical type of fern pattern with tertiary branching
in cervical mucus is indicative of ovulatory heat,
whereas, atypical fern pattern is observed in silent or
weak estrus (Galhotra et al., 1971). In present study,
pH values of mucus sample in cows from group-I, II
and III were recorded between 7.0-7.5. Samples of
endometrial content were carrying <4% PMN cells with
average number as 3.04±0.36% in group I, II and III. A
positive correlation was reported between PMN cells
and bacterial infection in uterus (Dutt et al., 2017). If
PMN cell count is >10%, the chances of subclinical
endometritis are highly evident.
First service conception rate of group I, II and III
was 58.3, 66.7 and 25.0%, whereas the number of
services per conception was recorded as 1.7, 1.5 and
4.0, in the respective groups. The result of conception
rate in group-I (58.7%) corroborated with an earlier
study in GnRH treated non-infectious repeat breeder
cows (62.5%; More et al., 2012). In another study,
higher conception rate (75%) was recorded (Pandey
et al., 2016) compared to current ndings (66.7%) in
repeat breeder crossbreed cows treated with hCG.
Administration of hCG or GnRH in early luteal phase
between d4-7 of estrus cycle induced pre-ovulatory
luteinization of the dominant follicle of rst wave and
thereby formation of accessory CL. This may further
aid in progesterone concentration with a positive
impact on embryo survival (Mehni et al., 2012). Thus,
the present study reects that hCG treatment on day 5
post-AI was eective to achieve higher conception rate
in non-infectious repeat breeder cows.
REFERENCES
Bhat, F.A., Bhattacharya, H.K. and Hussain, S.A.
(2014). White side test: A simple and rapid test
for evaluation of nonspecic bacterial genital
infections of repeat breeding cattle. Vet. Res.
Forum, 1-5.
Diskin, M.G., and Morris, D.G. (2008). Embryonic and
early fetal losses in cattle and other ruminants.
Reprod. Domest. Anim., 43: 260-267.
Dutt, R., Singh, G., Singh, M., Sharma, M., Dalal,
J. and Chandolia, R.K. (2017). Diagnosis of
subclinical endometritis in Murrah bualoes
through cytobrush Technique. Int. J. Curr. Microbil.
App. Sci., 6: 494-499.
Galhotra, A.P., Tyagi, R.P.R. and Baherjee, A.K.
(1971). Diagnostic signicance of arborization of
cervical mucus in bualoes and heifers. Haryana
Agri. Uni. J. Res. 1: 97-104.
Kimura, M., Nakao, T., Moriyoshi, M. and Kawata,
K. (1987). Luteal phase deciency as a possible
cause of repeat breeding dairy cows. British Vet.
J., 143: 560-566.
Lewis, G.S., Caldwell, D.W., Jr. Rexorad, C.E.,
Dowlen, H.H. and Owen, J.R. (1990). Eect of
gonadotropin releasing hormone and human
chorionic gonadotropin on pregnancy rate in dairy
cattle. J. Dairy Sci., 73: 66-72.
Mehni, S.B., Shabankaeh, H.K., Kazemi-Bonchenari,
M. and Eghbali, M. (2012). The comparison of
treating Holstein dairy cows with progesterone,
CIDR and GnRH after insemination on serum
progesterone and pregnancy rates. Reprod.
Domest. Anim., 47: 131-134.
Anbhule et al.
Indian Journal of Animal Reproduction 40 (1): June 2019
57
More, R.M., Patil, A.D., Kumbhar, U.B. and Mungle,
R.R. (2012). Fertility improvement by hormonal
therapies in repeat breeding cows. Indian J. Anim.
Reprod., 33: 61-63.
Pandey, N.K.J., Gupta Shiv Prasad, H.P. and
Sheetal, S.K. (2016). Plasma progesterone
prole and conception rate following exogenous
supplementation of gonadotropin-releasing
hormone, human chorionic gonadotropin and
progesterone releasing intra-vaginal device in
repeat breeder crossbred cows. Vet. World,
EISSN: 2231-0916.
Santos, J.E.P., Thatcher, W.W., Pool L. and
Overton, M.W. (2001). Eect of human chorionic
gonadotropin on luteal function and reproductive
performance of high-producing lactating Holstein
dairy cows. J. Anim. Sci., 79: 2881-2894.
Post-AI treatment for fertility improvement
Indian Journal of Animal Reproduction 40 (1): June 2019
58
INTRODUCTION
In mare, congenital vaginal obstruction is most
frequently associated to an imperforate hymen, an
incomplete persistent hymen or a vaginal hypoplasia
(Hughes, 1992; Freeman and England, 1997; Raggio
et al., 2005). Generally, a variable degree of persistency
of hymen is observed in maiden mare but in rare cases,
an imperforate hymen is noted leading to accumulation
of uid within the vagina and uterus. Mostly, hymen
is swept by ngers and hand before breeding a
maiden mare but sometimes the hymen is so tough
that it can only be ruptured using a guarded scalpel
blade or scissors. The present case in a lly explains
imperforate hymen with subsequent development of
mucovagina and its surgical management.
CASE HISTORY AND OBSERVATIONS
A lly (age, 18 month) of upgraded Kathiawari
breed had the history of straining while urination and
defecation along with the frequent protrusion of a
large part like a uid lled balloon coming out of vulva
(Fig. 1) when she lay down and also during canter.
Filly was attended by local veterinary practitioner and
suspected for vaginal prolapse and given treatment
accordingly. But there was no relief over a week-long
treatment thereafter case was referred to university
hospital. Per rectal examination revealed a uid
lled uctuating swelling in the vagina behind cervix,
however, the uterine horns were normal on palpation.
Transrectal ultrasonography revealed granular free-
oating uid distending the posterior vagina, however,
a tough membrane was obstructing the hand to palpate
cervix per vaginally. The vaginoscopic examination
conrmed an imperforate membrane with similar
appearance to the adjacent tissue but slightly less
reddish and obstructing the visualisation of cervix (Fig.
2). The lly was diagnosed with imperforate hymen
and subsequent development of mucovagina due to
obstruction to natural drainage.
TREATMENT AND DISCUSSION
The lly was restrained properly in standing
position under trevis and was administered epidural
anaesthesia (2% lignocaine HCl; 8 ml in rst coccygeal
interspace). The tough hymenal membrane was taken
out with the help of soft tissue holding forceps and
an incision was made dorsal to ventral direction (Fig.
3) and around 2 L mucoid uid without any o smell
was drained o. (Fig. 4). The endoscopic examination
revealed normal vagina and a tightly closed cervix
(Fig. 5). The lubricated gloved hand was passed
gently through vagina to tear hymenal membrane
1Professor and Director, Teaching Veterinary Clinical Complex;
3Assistant Professor, 4Professor, 5M.V.Sc Scholar; 2Scientist, Animal
Reproduction, ICAR-RC for NEH Region Sikkim Centre, Tadong - 737
102; *drbrijeshvet02@gmail.com
IMPERFORATE HYMEN AND SUBSEQUENT MUCOVAGINA IN A FILLY
R.P. PANDEY1, B. KUMAR2*, V. SCHAN3, A. SAXENA4 AND D. YADAV5
Department of Veterinary Obstetrics and Gynaecology, Pt. Deen Dayal Upadyaya Pashu-chikitsa Vigyan
Vishwidayalya Evam Go-Anusandhan Sansthan, Mathura - 281 001
Received: 25.05.2018 Accepted: 10.06.2018
ABSTRACT
An eighteen-month-old lly was presented with imperforate hymen and subsequent development of
mucovagina. The hymen was incised and accumulated secretion was drained. The linear incision of hymenal
membrane was bluntly extended through digital manipulation till smooth passage of hand in vaginal canal without
any resistance and cervix was palpated. Animal recovered uneventfully, bred after seven months and diagnosed
positive for pregnancy at three months of gestation.
Key words: Imperforate hymen, Mucovagina, Filly, Vaginoscopy
Indian Journal of Animal Reproduction 40 (1): June 2019
59
to its maximum and to ensure smooth passage of
hand. The vagina was ushed with normal saline
2-3 times and Xylocain jelly (4% lignocaine HCl) and
Neosporin powder was smeared throughout vagina.
The postoperative treatment of dam comprised mainly
of injecting combination of antibiotic for ve days.
During the subsequent period, the lly had no vaginal
discharge or associated systemic abnormalities. The
tenesmus ceased and normal posture to urination
and defecation was noted with no further protrusion
of membrane from vulva. The lly was successfully
breed after 7 months and three months after breeding,
a positive pregnancy diagnosis was made by per rectal
examination.
The hymen is formed from epithelial lining of
paramesonephric ducts and urogenital sinus at the
vestibulovaginal junction. The canalization of hymen is
usually complete at birth and leads to communication
between the lumen of caudal vagina and vestibule
(Roberts, 1986). The most frequent developmental
anomaly concerning the caudal reproductive tract
in the mare is imperforate hymen or persistence of
variable degree of hymen (Mc Entee, 1990; Hughes,
1992). In mare, few reports exist on developmental
anomalies of cervix and cranial vagina besides those
associated with pseudohermaphroditism and testicular
feminization syndrome (Kieer, 1976; Crabbe et al.,
1992).
Hydrometra is a common sequale of vaginal
obstruction in cyclic females as the normal outow
of the uterine secretions is prevented leading to
accumulation of uid with an increase in age and cyclic
ovarian activity of the female (Troiano and McCarthy,
2004). Iin present case, lly was not reported for any
cyclic symptoms though there was accumulation of
vaginal secretion and epithelial debris. In this case, the
cervix was closed as visualised by vaginoscope, thus,
no chance of entry of vaginal secretion into uterus. The
latter was conrmed by ultrasonography as uterine
horns had no accumulation of secretions.
In cattle, the most common developmental
aberration, due to a sex-linked recessive gene,
of female reproductive tract is variable degree of
hymen persistence with white shorthorn breed being
most aected (Parkinson, 2001). The accumulation
of secretions associated with complete hymen
obstruction can be relieved by trocar and cannula. In
present case, the obstruction was relieved by incision
of hymenal membrane with scalpel and blade, thus
leading to drainage of mucoid uid. Nevertheless, the
surgical intervention to enable successful breeding
is not advisable due to hereditary origin (Parkinson,
2001). In horses and other ruminant species, persistent
hymen is reported, but heritability is unknown.
Fig. 1: Protrusion of
Imperforate hymen
Fig. 2: Vaginoscopic
appearance of
persistent hymen
Fig. 3:
Incision of hymen
Fig. 4:
Flow of mucous after
incision
Imperforate hymen in lly
Indian Journal of Animal Reproduction 40 (1): June 2019
60
REFERENCES
Crabbe, B.G., Freeman, D.A., Grant, B.D., Kennedy,
P., Whitlatch, L. and MacRae, K. (1992). Testicular
feminization syndrome in a mare. J. Am. Vet. Med.
Assoc., 200: 1689-1691.
Freeman, S.L. and England, G.C. (1997). Vestibulo-
vaginal hypoplasia in a mare. Vet. Rec., 141: 521-
522.
Hughes, J.P. (1992). Development anomalies of the
female reproductive tract. In: Mckinnon, A.O.,
Voss, J.L. (Eds.), Equine Reproduction. Lea &
Febiger, Philadelphia, pp. 408-414.
Kieer, N.M., (1976). Male pseudohermaphroditism of
the testicular feminizing type in a horse. Equine
Vet. J., 8: 38-41.
McEntee, K. (1990). Cervix, vagina and vulva-
congenital anomalies. In: Reproductive Pathology
of Domestic Mammals. Academic Press, San
Diego, pp. 194-201.
Pandey et al.
Parkinson, T.J. (2001). Infertility in the cow: Structural
and functional abnormalities, management and
non-specic infection. In: Noakes D.E, Parkinson
T.J, England GCW, editors. Arthur’s Veterinary
Reproduction and Obstetrics. 8. Philadelphia:
Saunders; pp. 383-472.
Raggio, I., Lefebvre, R. and Vaillancourt, D. (2005).
Theriogenology question of the month. J. Am. Vet.
Med. Assoc., 226: 205-207.
Roberts S.J. (1986). Veterinary Obstetrics and Genital
Diseases. New York: David & Charles, Female
gential anatomy and embryology, pp. 3-13.
Troiano, R.N. and McCarthy, S.M. (2004). Mullerian
duct anomalies: imaging and clinical issues.
Radiology, 233: 19-34.
Indian Journal of Animal Reproduction 40 (1): June 2019
61
INTRODUCTION
The process of foaling in equines is a rapid (30
minutes) and violent process with very low incidence
of dystocia (Bhoi et al., 2010). Nevertheless, the
majority of dystocia cases were of fetal origin with
postural disposition being very common due to long
foal extremities (Arthur et al., 1989). The present case
report places on record the successful management of
dystocia due to bilateral hock exion in jenny through
percutaneous fetotomy.
CASE HISTORY AND OBSERVATIONS
A four-year-old primiparous jenny had the history
of unsuccessful labor since 24 h and the water bags
had ruptured 18 h earlier. The case was handled by
a local practitioner but failed to relieve the dystocia.
The animal was dull, depressed and lying down with
labored breathing and intermittent straining. The per
vaginal examination revealed fully dilated and dry birth
canal with variable degree of inammation. Fetal tail
was coming out from the vulva (Fig. 1), The fetus, whch
was not emphysemated, was diagnosed in posterior
presentation with bilateral hock exion and fetal tail
presented out of vulva (Fig. 1).
TREATMENT AND DISCUSSION
The birth canal was thoroughly lubricated with
liquid paran, however, the attempts made to correct
the hock exion were unsuccessful. Hence, both the
hind limbs were disarticulated at the level of hock using
a double barrel Thygesen fetotome. After thorough
lubrication of breech area of fetus and birth canal,
initially one-point traction followed by simultaneous
two points traction was applied just above the hock
to ensure that both the stie joints enter the pelvic
cavity (Fig. 2). While applying traction, the precaution
was taken to protect birth canal from being damaged
by amputated stumps and a dead male fetus was
extracted out. The jenny was administered with routine
antibiotics and supportive therapy for four days along
with local dressing of vaginal laceration. The animal
recovered uneventfully as informed by owner.
Equine dystocia is a true emergency and threatens
the survival of dam and fetus both (Freeman et al.,
1999). Long extremities of foal tend to predispose a
Jenny to dystocia (Chauhan et al., 2013), whereas,
dystocia due to malformations like schistosomus
reexus and ankylosis of joints is also reported (Dubbin
et al., 1990). In the present case, partial fetotomy using
double barrel Thygesen fetotome proved benecial to
deliver the malpostured foal per-vaginum.
1Assistant Professor, 3Professor, 4M.V.Sc Scholar; 2Scientist, Animal
Reproduction, ICAR-RC for NEH Region Sikkim Centre, Tadong – 737
102; *drbrijeshvet02@gmail.com
DYSTOCIA DUE TO BILATERAL HOCK FLEXION IN A JENNY (EQUUS
ASINUS)
V. SACHAN1, B. KUMAR2*, A. SAXENA3 AND M.K. CHAUDHARY4
Department of Veterinary Obstetrics and Gynaecology, Pt. Deen Dayal Upadyaya Pashu-chikitsa Vigyan
Vishwidayalya Evam Go-Anusandhan Sansthan, Mathura - 281001
Received: 25.05.2018 Accepted: 10.06.2018
ABSTRACT
A case of fetal dystocia due to bilateral hock exion in a jenny and the successful per-vaginum delivery of
fetus is reported through partial fetotomy using double barrel Thygesen fetotome.
Key words: Dystocia, Fetotomy, Hock exion, Jenny
Indian Journal of Animal Reproduction 40 (1): June 2019
62
REFERENCES
Arthur, G. H., Noakes, D. E. and Pearson, H. (1989).
Veterinary Reproduction and Obstetrics. 6th edn.
ELBS, BailliereTindall, London, UK. pp 175.
Bhoi, D.B., Suthar, D.N., Parmar, J.J. and Patel, J.B.
(2010). Dystocia in Mare due to fetal Postural
defect. Vet. World, 3: 332.
Chauhan, P.M., Sindhi, S.H. and Thakor, K.B. (2013).
Fetal dystocia due to dorso-pubic position and
postural defects in a Jenny: A case report. Vet
World, 6: 116-117.
Fig. 2: Two-point traction on amputated hind limbFig. 1: Fetal tail presented at vulva
Dubbin, E.S., Welker, F.H., Veit, H.P., Modransky,
P.D. and Nemovet Talley, M.R. (1990). Dystocia
attributable to a fetal monster resembling
schistosomus reexus in a donkey. J. Am. Vet.
Med. Assoc., 197: 605-607.
Freeman, D.E., Hungerford, L.L. and Schaeer, D.
(1999) Caesarean section and other methods for
assisted delivery: comparison of eects on mare
mortality and complications. Equine Vet. J., 31:
203-207.
Indian Journal of Animal Reproduction 40 (1): June 2019
63
INTRODUCTION
Cryptorchidism occurs most commonly in
stallion, boar and some breed of dogs like Boxers,
Pomeranians, Dachshunds, Sealyhams, Cairn Terriers
(Noakes, 2009). Among cryptorchid dogs, unilateral
cryptorchidism is more common with 1-7% incidence
(Noakes et al., 2009 and Sridevi, 2015). This condition
followed by development of neoplasia is common in
stallions and dogs. Other complication in response
to raised intra-testicular temperature and endocrine
disturbance is testicular degeneration (Noakes et al.,
2009). In dogs, the etiology of canine cryptorchidism
may be heritable and linked to sex-limited autosomal
recessive trait. Although, non-genetic factors such as
relative size of testis and inguinal canal may also be
involved (Sridevi, 2015).
CASE HISTORY AND OBSERVATIONS
A 6-year-old Labrador retriever was presented with
subcutaneous mass increasing in size in the past 15-
20 days. On scrotal palpation, the dog was bilaterally
cryptorchid and ultrasonography (Mylab Delta, Esaote
pvt ltd., India) revealed an enlarged testicle as large
mixed echogenic and cavitated within subcutaneous
tissue, while the other could not be visualized. (Fig.
1). The decision was taken to remove the enlarged
testicle surgically.
TREATMENT AND DISCUSSION
The dog was fasted overnight and pre-medicated
with Atropine sulphate @ 0.03 mg/kg b. wt. and Xylazine
@ 1.0 mg/kg b. wt. intramuscularly. Following aseptic
precautions and under intravenous anaesthesia
(Ketamine @ 5mg/kg b. wt. and Midazolam @ 0.2 mg/
kg b. wt.), the enlarged testicular mass was removed
following standard surgical procedure (Kudnig and
Seguin, 2012). A 4-4.5 cm incision was made over
the mass at the right ventral abdominal region. The
narrow end of the mass was directed towards the
incision to facilitate dislodgement (Fig. 2). Blood
vessels in thickened spermatic cord were ligated at
the base of testicular mass using Vicryl 2-0 and the
mass was resected and checked for any bleeding. The
absorbable gelatin sponge was placed to minimize the
existing dead space. Incision closure involved simple
continuous suture for subcutaneous tissue using Vicryl
2-0, followed by simple interrupted sutures of skin
using Ethilon 2-0. Ceftriaxone @ 25 mg/kg b. wt. for
5 days and Meloxicam @0.3 mg/kg b. wt. for 3 days
were given. The recovery was uneventful and sutures
were removed after 15 days.
1Senior Research Associate, 2Assistant Professor, 3Director of Research
and PG studies, *vsuthar28@gmail.com
SURGICAL MANAGEMENT OF TESTICULAR HYPERPLASIA IN A
CRYPTORCHID LABRADOR RETRIEVER
T.M. MOMIN1, D.N. KELAWALA2, V.S. SUTHAR2* AND D.B. PATIL3
Department of Animal Reproduction Gynaecology and Obstetrics, PG Institute of Veterinary Education and
Research, Kamdhenu University, Gandhinagar, Gujarat - 382010
Received: 26.04.2018 Accepted: 10.05.2018
ABSTRACT
A 6-year-old Labrador retriever was presented with subcutaneous inguinal mass increasing in size. On scrotal
palpation, the dog was bilaterally cryptorchid. Ultrasonography revealed mixed echogenic enlarged testicle, while
the other could not be visualized. The surgical removal of subcutaneous testicular mass was done with uneventful
recovery.
Key words: Cryptorchid, Dog, Surgical management
Indian Journal of Animal Reproduction 40 (1): June 2019
64
REFERENCES
Kudnig, S.T. and Seguin, B. (2012). Veterinary Surgical
Oncology. 1st Ed., Wiley & Blackwell; pp 341-364.
Noakes, D.E., Parkinson, T.J and England, G.C.W.
(2009). Veterinary Reproduction and Obstetrics,
9th Ed., W.B. Saunders. pp 739.
Fig. 1: Large mixed echogenic and cavitated
testicle
Fig. 2: Surgically removed intra-abdominal
testicular mass
Sridevi, P. (2015). Canine Reproduction: The theory
and practice. 1st Ed., Active Ads & Printers Pvt.
Ltd. pp 110.
Momin et al.
Indian Journal of Animal Reproduction 40 (1): June 2019
65
1Assistant Professor, 2Professor and Head; *argoraja@gmail.com
INTRODUCTION
Ischemia of cervical region due to prepartum
prolapse in a cow may be responsible for imperfect
cervical dilatation, thus leading to diculty in normal
expulsion of fetus even with normal presentation,
position and posture of fetus. In such cases,
cervicotomy is the simple procedure for alleviating the
condition compared to cesarean section (Noakes et al.,
2009). The present report describes a case of dystocia
due to vagino-cervical prolapse coupled with imperfect
cervical dilatation and its successful management by
cervicotomy in a HF crossbred cow.
CASE HISTORY AND OBSERVATIONS
A HF crossbred cow (age, 4.6 yr; b. wt., 300
kg) was presented with the history of straining and
was unable to deliver the full-term calf for past 3 h.
The animal was in lateral recumbency and clinical
examination of the cow revealed edematous vulva with
congested vaginal mucus membrane. A torn snare
tied to fetal extremities present in vaginal passage
revealed an earlier handling of the case. Further
vaginal examination explored a dry vaginal passage
with stenosed and partially dilated (3 ngers) cervical
rim. The fetal limbs were in external os of cervix and
remaining fetal parts were not palpable.
TREATMENT AND DISCUSSION
Treatment was initiated with intravenous
administration of 3.5L 5% dextrose normal saline.
Carboxymethly cellulose gel was infused through
available space in cervix along with fanning of cervical
os. After 40 min of fanning, there was no progress
in cervical relaxation. The cow was subjected to low
caudal epidural anaesthesia (1ml 2% lignocaine HCl/
50kg b. wt.). Thereafter, a snare was applied to fetal
extremities followed by mild traction, but this lead to
prolapse of entire cervix. Hence, it was decided to
relieve the fetus by cervicotomy.
For cervicotomy, mild traction force was applied
to place the cervix in a position so that the incision
can be made at 10’O clock position involving only the
circular muscles. Consequently, the cervical dilatation
was sucient enough to deliver the live male calf by
slight traction. The cervical incision was sutured by
continuous interlock pattern with absorbable catgut
(size - 2) and was replaced in position (Fig. 1).
Thereafter, the cervix was lavaged with warm saline
to remove the debris and cetrimide cream was applied
over cervix and vaginal region. The prolapsed mass
was reduced and simple vulval tape retention suture
was applied as preventive measure for recurrence.
The dam was treated with routine parenteral antibiotic
plus supportive therapy for the next ve days. The
CERVICOTOMY FOR THE MANAGEMENT OF DYSTOCIA DUE TO
PREPARTUM VAGINO-CERVICAL PROLAPSE IN COW
S. RAJA1*, M. PALANISAMY2, V. PRABAHARAN1, R. RAJKUMAR1 AND P. JAYAGANTHAN1
Department of Veterinary Gynaecology and Obstetrics, Veterinary College and Research Institute, Tamil Nadu
University of Veterinary and Animal Sciences, Orathanadu - 614 625
Received: 24.04.2018 Accepted: 08.05.2018
ABSTRACT
A case of dystocia due to peripartum vagino-cervical prolapse and imperfect cervical dilatation in a HF
crossbred cow and it’s per vaginal delivery by cervicotomy is reported.
Key words: Cow, Cervicotomy, Cervico-vaginal prolapse, Dystocia
Indian Journal of Animal Reproduction 40 (1): June 2019
66
examination of animal after a week revealed an
uneventful recovery.
In cows, vaginal prolapse is usually a chronic,
hereditary, recurrent, pre or peripartum condition and
most commonly occurs in heavy dairy cattle before
calving, usually in third trimester of pregnancy (Noakes
et al., 2009). Cervicotomy as a management technique
in a fresh case of imperfect cervical dilatation was
reported earlier in cow (Sathiamoorthy et al., 2011).
In brief, cervicotomy is a simple and eective method
for approaching a case of imperfect cervical dilatation
in cow.
Fig. 1: Cervicotomy followed by cervical suture in a cow
Raja et al.
REFERENCES
Noakes, D.E., Parkinson, T.J and England, G.C.W.
(2009). Veterinary Reproduction and Obstetrics,
9th Ed., W.B. Saunders. pp 239.
Sathiamoorthy, T., Balasubramanian, S., Rangasamy,
S., Raja, S. and Asokan, S.A. (2011). Cervicotomy
approach for dystocia due to imperfect cervical
dilatation (ICD) in a cow. J. Ind. Vet. Assoc., 9: 46-
46.
Indian Journal of Animal Reproduction 40 (1): June 2019
67
INTRODUCTION
Pyometra is a hormonally mediated reproductive
disorder that aects sexually intact mature bitches
before they reach the age of 10 year (Baithalu et
al., 2010). This disease generally occurs during
or immediately following a period of progesterone
dominance (Blendinger et al.,1997). The elevated
progesterone in diestrus period leads to suppression
of uterine contractions, an increase in endometrial
gland secretions, closure of cervix, and suppression
of immune response, thus creating a favourable
environment for bacterial growth. It can be acute or
chronic systemic disorder with presence (open cervix
pyometra) or absence (closed cervix pyometra) of
vaginal discharge; of which, the later being a medical
emergency requiring rapid intervention to prevent
subsequent sepsis and potential patient death
(Baithalu et al., 2010).
CASE HISTORY AND CLINICAL OBSERVATIONS
A 11-month-old female pug had the history of
dullness, depression, inappetance for one week and
was crossed 20 days back in her rst heat. The clinical
examination revealed body temperature (101.6◦F)
and pulse rate (110/min) to be within a normal range.
The vaginal mucus membrane was mildly congested
and dry without any vaginal discharge. On ultrasound
examination, multiple anechoic sacculations were
noticed and blood picture revealed leucocytosis
(neutrophilia). On the basis of history, clinical
examination and haematological report, the case was
diagnosed as closed cervix pyometra.
TREATMENT AND DISCUSSION
Ovariohysteroctomy is the choice of treatment
for pyometra in older and non-breeding bitches. If
the condition is not life threatening and the animal is
young, then valuable restoration of fertility may be
done with medical management using prostaglandin.
However, the use of prostaglandins in closed pyometra
is contraindicated. In the present case, as the dog
was young and the owner was interested in future
breeding, medical management with Misoprostol
(prostaglandin E2) @ 400µg total dose pervaginum
was initiated in an attempt to relax the cervix prior to
PGF2α administration and the treatment was repeated
48h later. A foul smelling purulent vaginal discharge
was reported 48h later and thereafter, the dog was
treated with Inj. PGF2α (Cloprostenol) @ 2.5µg/kg b.
¹PG Scholar, Department of Veterinary Gynaecology and Obstetrics;
²Assistant Professor, ³Professor; *drjumamageswari@gmail.com
MEDICAL MANAGEMENT OF A CLOSED TYPE PYOMETRA USING
MISOPROSTOL AND CLOPROSTENOL COMBINATION IN A PUG
R.R. PATI¹, J. UMAMAGESWARI²* AND P. SRIDEVI³
Department of Clinics, Madras Veterinary College, Tamil Nadu University of Veterinary and Animal Sciences,
Chennai - 600 007
Received: 26.06.2018 Accepted: 14.07.2018
ABSTRACT
A female pug had the history of dullness, depression, inappetance for one week and was crossed 20 day
earlier in her rst heat. The clinical examination revealed mild congested and dry vaginal mucus membrane
without any vaginal discharge. Ultrasound examination revealed multiple anechoic sacculations. The case was
diagnosed as closed cervix pyometra and was treated with Misoprostol and PGF2α combination. The female pug
showed an uneventful recovery subsequent to treatment.
Key words: Bitch, Closed cervix pyometra, Misoprostol, Pug, PGF2α
Indian Journal of Animal Reproduction 40 (1): June 2019
68
wt., S/C, once daily for 7 days. In order to minimize
the side eects of PGF2α, atropine sulphate (0.02 mg/
kg b. wt.) was administered S/C prior to PGF2α inj. In
addition, Tab. Amoxicillin and Clavulanic acid @ 20
mg/kg b. wt. PO was administered twice daily for 14
days. Ultrasound examination on day 7 revealed mild
anechoic sacculations but the dog was active and
feeding habits had improved. The review on day 14 post
treatment revealed no uterine involvement suggesting
complete response to PGF2α administration.
In bitches, the incidence of pyometra was reported
as 9.0-15.2% (Pretzer, 2008) with higher incidence
noticed in smaller breeds (Shiju Simon et al., 2011).
The exogenous or endogenous concentration of
circulating steroid hormone particularly estrogen and
progesterone inuence the distribution of steroid
receptors within the uterus. The regulation of estrogen
and progesterone receptors in endometrial glands plays
an important role in pathogenesis of pyometra complex
in bitch (Baithalu et al., 2010). The administration of
prostaglandin E2 pervaginally leads to remodelling of
cervical extracellular matrix (Ledger et al., 1983) and
relaxation of cervix while prostaglandin F2 increases
myometrial contractility, regression of corpus luteum
and, thereby causing expulsion of uterine contents.
In conclusion, the combination of Misoprostol and
PGF2α can be used to manage closed pyometra in
young and clinically active bitches in order to restore
future fertility of bitches.
REFERENCES
Baithalu, R.K., Maharana, B.R., Mishra, C., Sarangi,
L. and Samal, L. (2010). Canine Pyometra. Vet.
World, 3: 340-342.
Blendinger, K., Bostedt, H. and Homann, B. (1997).
Hormonal eects of the use of an antiprogestin
in the bitches with pyometra. J. Reprod. Fertil.
Suppl., 51: 317-325.
Ledger, W.L., Ellwood, D.L. and Taylor, M.J. (1983).
Cervical softening in late pregnant sheep by
infusion of prostaglandin E2 into a cervical artery.
J. Reprod. Fertil., 69: 511-515.
Pretzer, S.D. (2008). Clinical presentation of
canine pyometraand mucometra: A review,
Theriogenology, 70: 359-363.
Shiju Simon, M., Ganesh, R., Arunprasad, A. and
Suresh Kumar, R. (2011). Incidence of pyometra
in bitches - a survey of 278 cases, Tamilnadu J.
Vet. Anim. Sci., 7: 252-253.
Pati et al.
Indian Journal of Animal Reproduction 40 (1): June 2019
69
INTRODUCTION
Musth is similar to rutting behaviour in ungulates
and is associated with periodic increases of testosterone
secretion. Most matings occur in this period (Poole,
1989). During musth, the animal becomes more
restless, energetic, aggressive and generally irritable
and oversensitive to sound and movements. Musth
is generally a normal phenomenon but it becomes
dangerous in case of man-elephant interactions
(Rajaraman, 2006). During musth, the elephants are
uncontrollable and become a risk to human life and
property. Therefore, this condition needs immediate
veterinary interventions for its better management.
CASE HISTORY AND OBSERVATIONS
A male Asian elephant of (Age, 40 yr; b. wt. 4000
kg) was reported as violent and destructive in Tekang
village, East Siang district of Arunachal Pradesh. The
owner reported that elephant injured his mahout and
was on the run of 21 days destroying and damaging
houses in villages. Also, the elephant charged to
everyone as soon as he got the smell of any nearby
human. The owner approached the local forest
department for veterinary interventions. The elephant
was detected to be in musth on the basis of clinical
signs. It was nally decided to chemically restrain the
elephant and to tie his legs with chains to restrict the
movements until the musth period is over.
A total volume of 7 ml or 700 mg Xylazine HCl
was loaded in a metal dart and projected through Dist-
inject N 60 rie to the rump region. As the elephant
had the tendency to charge everyone, we had to re
a gun shot in air simultaneously with ring the dart so
as to safeguard ourselves and to deter the attention
of elephant from charging. After the dart injection, the
visual contact was lost as the site was in deep forest.
The elephant was found with the signs of anaesthesia
after 15 minutes of dart injection about 200 meter away
from the site of darting. The legs of elephant were tied
with metal chains and tethered him in a nearer tree to
restrict his movement until the musth period is over.
The elephant was in constant watch till recovered from
the standing anaesthesia.
TREATMENT AND DISCUSSION
Xylazine HCl is extensively used in elephants as
a popular sedative because of its high therapeutic
index, smooth induction and recoveries (Sarma and
Pathak, 2001).The use of Xylazine @ 0.1-0.2 mg/kg b.
SUCCESSFUL MANAGEMENT OF A VIOLENT ASIAN ELEPHANT IN MUSTH
UNDER XYLAZINE ANAESTHESIA
J. AHMED1*, N. AHMED2 AND S. KYARONG3
Centre for Bear Rehabilitation and Conservation, Wildlife Trust of India, Seijosa, Pakke Tiger Reserve,
Arunachal Pradesh
Received: 07.02.2018 Accepted: 15.02.2018
ABSTRACT
Successful management of a violent male Asian elephant (Elephas maximus) in musth was carried out using
700 mg Xylazine HCl to obtain desired standing anaesthesia. The legs of elephant were tied with chains and
tethered by using special jute rope to prevent his movement until the musth period was over. In brief, better
awareness of people on physiological behavoiur of elephants for detection of musth as well as to adopt appropriate
management procedures is referred.
Keywords: Anaesthesia, Elephant, Management, Musth, Xylazine
1M.V.Sc. Scholar, College of Veterinary Science, Khanapara, Assam;
2Veterinary Ocer, ARDD, Unakoti, Tripura; 3Joint Director; *jahan.
ahmed6@gmail.com
Indian Journal of Animal Reproduction 40 (1): June 2019
70
wt. is recommended for sedation in Asian elephants.
However, in extreme aggressiveness, musth, painful
conditions and ambient disturbances may necessitate
higher doses (Cheeran et al., 2002). After 15 minutes
of the dart injection, the elephant manifested with signs
of standing anaesthesia. After 20 minutes, complete
standing anaesthesia was achieved and the signs of
anaesthesia were recorded as diminished trunk, tail,
restricted ear movement and relaxation of penis with
dribbling of urine and loud breathing sound.
Once the musth elephant was completely sedated,
all the legs were tied with chains and tethered with
special jute rope to a nearer big tree for restricting his
movements as reported earlier (Nigam et al., 2006).
The anaesthesia lasted up to 3.5 h which was similar
with the ndings of others (Ahmed et al., 2017). The
mahout was advised to keep constant watch on the
elephant and not to remove the chains until the musth
period is over. He was also instructed to provide
reduced feed, sucient clean water and to observe
for any behavioral change. After 12 days, the elephant
became uneventfully normal and with veterinarian
instruction the chains were removed.
Musth appears to be similar to rutting behaviour
which is associated with periodic increase secretion of
testosterone. As it a normal physiological phenomenon
among physically healthy bulls, it does not need
any treatment. Musth is an emergency condition
and might be the important cause for man-elephant
conicts as reported in the present case. Therefore,
it needs special management which included chaining
the animal away from contact with people and other
elephants and withdrawal of feed to reduce the musth
period by deteriorating physical health.
In the present case, chemical restraining and
securing of musth elephant is adopted as the elephant
became a threat to human life. This paper elaborates
a successful chemical restraining of a violent elephant
with Xylazine HCl anaesthesia in musth. We refer
better awareness of people on physiological behavior
of elephants for detection of musth as well as to adopt
appropriate management procedures.
REFERENCES
Ahmed, J. and Ahmed, N. (2017). Successful chemical
restraining and management of an Asian elephant
(Elephas maximus) during musth. J. Ent. Zool.
Stud., 5: 2072-2073.
Cheeran, J.V., Chandrasekharan, K. and
Radhakrishnan, K. (2002). Tranquilization and
translocation of elephants. J. Indian Vet. Assoc.
Kerala, 7: 42-46.
Nagam, P., Sinha, S., Malik, P. and Chowdhary, S.
(2006). Management of elephant in musth: A case
report. Zoos’ Print J., 21: 2265-2266.
Poole, J.H. (1989). Mate guarding, reproductive
success and female choice in African elephants.
Anim. Behaviour, 37: 842-849.
Rajaram, A. (2006). Musth in elephants. Resonance,
11: 18-27.
Sarma, K.K. and Pathak, S.C. (2001). Cardio vascular
response to Xylazine and Hellabrunn mixture with
Yohimbine as reversal agent in Asian elephants.
Indian Vet. J., 78: 400-402.
Ahmed et al.
Indian Journal of Animal Reproduction 40 (1): June 2019
71
INTRODUCTION
Any fetal disposition other than anterior
presentation, dorsal position and normal posture is
likely to result in dystocia (Sane et al., 1994). The
gestation period in the mare is 340 days, but foals
with delayed development may be born normally up
to 385 days and some mares may foal prior to 315
days (Davis Morel et al., 2002). Foaling in the mare
is a violent and short process with the birth of foal
completing within 30-70 minutes following the rupture
of chorioallantoic membrane. Occurrence of dystocia
in mares is comparatively less common but due
foals having long neck and legs may assume many
postures, which could cause problem during foaling
(Narale et al., 2007).
CASE HISTORY AND OBSERVATIONS
A seven-year-old Marwari mare was presented
with prior history of vaginal discharges 7 days prior
to presentation at the clinic and general treatment
given by local veterinarian. The mare was at 335
days of gestation and was intermittently straining,
showing standing and sitting postures. There was
discharge of blood tinged uid from the vagina. The
1Teaching Associate, 3Assistant Professor; 2Director, Teaching
Veterinary Clinical Complex; 4Scientist (SS), Equine Production
Campus, ICAR-NRC on Equines,
rectal temperature was 101.3°F, pulse rate 42 beats/
min and respiration rate was 32 breaths/min. Mucous
membrane was pink in color and hydration status was
normal. On rectal examination, fetus was in anterior
position and forelimbs were exed from carpal joint
and the neck was deviated laterally. Fetal membranes
were putreed and maceration of fetus had initiated.
TREATMENT AND DISCUSSION
The mare was placed in a dust free quiet
area. Animal was kept in standing position with
epidural anesthesia (8 ml 2% lidocaine) and general
anaesthesia (10 ml Xylazine IV). The perineal area
was washed with non-irritant antiseptic solution and
250 ml of liquid paran was pumped into the uterus
with a sterile exible plastic tube. The carpal exion of
both limb was corrected by mutation and ropes were
applied at fetlock joint. A repulsion was attempted
at shoulder joint to facilitate an approach to neck,
which was corrected manually. Again, the lubrication
was done with liquid paran, thereafter fetus was
removed just by traction applied through rope snares.
Uterine lavage with look warm normal saline and 2%
betadine was performed, and 25 IU oxytocin in normal
saline was also administered. Antibiotic therapy, anti-
inammatory drugs and uid therapy was appropriately
administered for 5 days.
MANAGEMENT OF DYSTOCIA DUE TO MACERATED FETUS IN A MARE
Y. SONI1, J.S. MEHTA2, A. KUMAR3, A. TRIPATHI1 AND T.R. TALLURI4*
Department of Veterinary Gynecology and Obstetrics, College of Veterinary and Animal Sciences
Bikaner - 334 001
Received: 25.08.2018 Accepted: 10.09.2018
ABSTRACT
The present case report puts on record successful management of a mare suering from dystocia due to
initial stages of maceration of fetus. A full-term pregnant seven-year-old mare was presented with the history
of vaginal discharges followed by mild straining without any progress. The vaginal examination revealed a fully
dilated cervix with the presence of a fetus with carpal exion. The exion was corrected and fetus was retracted
successfully. The mare was administered uid, analgesic and antibiotic parentally apart from intra-uterine drug
treatment and the mare has resumed normal health.
Keywords: Carpal exion, Dystocia, Fetus, Maceration, Mare
Indian Journal of Animal Reproduction 40 (1): June 2019
72 Soni et al.
The malposture of long fetal extremities is
major cause of dystocia in mare although positional
and presentational abnormalities occur to a lesser
degree. In the present case, the cause of dystocia
was fetal postural abnormality (exion of carpal joint).
Classically, the methods of dystocia correction were
divided into assisted vaginal delivery, controlled
vaginal delivery, fetotomy and caesarean section
(Embertson et al., 1995). In assisted vaginal delivery,
the mare is aware and assisted to a small or large
degree for vaginal delivery of an intact foal within
10-15 minutes (Embertson et al., 1995), Controlled
vaginal delivery employs general anesthesia, if the foal
cannot be delivered within 15 minutes a fetotomy (if the
foal is dead) or caesarean section (if foal is live) was
performed. In the present case, the forelimbs of fetus
were exed from carpal joint and neck was deviated
laterally, therefore, normal and assisted delivery was
not possible. Due to severe straining shown by mare
during handling, the controlled vaginal delivery under
general anesthesia with xylazine was performed.
REFERENCES
Davis Morel, M.C., New, J.R. and Holland, S.J.
(2002). Factors aecting gestation length in the
thoroughbred mare. Anim. Reprod. Sci., 74: 175-
185.
Embertson, R.M., Bernard. W.V., Hance, S.R. and
Smith, S. (1995). Hospital approach to dystocis in
the mare. Am. Ass. Equine. Practnrs. 41: 13-14.
Narale, N.L., Amle, M.B., Shelar, R.R. and Parthiban,
M. (2007). Studies on parturition traits in
Thoroughbred mares. J. Bom. Vet. Col., 15 (1, 2):
125-26.
Sane, C.R., Deshpande, B.R., Kaikini, A.S., Velhankar,
D.P., Kodagali, S.B., Luktuke, S.N. and Threlfall,
W.R. (2007). Parturition and dystocia. In:
Youngquist, R.S., Threlfall, W.R. eds. Current
Therapy in Large Animal Theriogenology, 2nd
edition, Missourri Saunders Elsevier, pp 118-130.
Fig. 1: Macerated Foal
Indian Journal of Animal Reproduction 40 (1): June 2019
73
Dr. R.D. Sharma, Former Additional Director of Research (Veterinary and Animal Sciences) and Professor-
cum-Head, Department of Veterinary Gynaecology and Obstetrics, Punjab Agricultural University born on 28
August, 1933 in a small town, Bhawanigarh, Punjab, left for his heavenly abode on 6 December, 2017. Dr. Sharma
graduated (Bachelor of Veterinary Science & Animal Husbandry) from Punjab University in 1956, did his Masters
from OHIO State University, Columbus, Ohio, USA in 1968 and Ph.D. (Veterinary Gynaecology and Obstetrics) from
Punjab Agricultural University, Ludhiana in 1976. He initially worked as Veterinary Assistant Surgeon, Technical
Assistant at Govt. Progeny Testing Farm, Hisar and later as Research Assistant (A.I.) at Department of Animal
Husbandry, PAU, Hisar. He joined as Assistant Professor of Veterinary Gynaecology and Obstetrics at College
of Veterinary Science, PAU, Ludhiana in 1969. Dr. Sharma remained Professor-cum-Head of the Department of
Veterinary Gynaecology and Obstetrics for 14 years before taking over as Additional Director Research in 1991
till his superannuation in 1993. He also worked as Professor Emeritus for ve years thereafter in the Department.
He guided 12 M.V.Sc. and 09 PhD students as Major advisor and had more than 150 publications to his credit.
Many laboratories for Obstetrics, Gynaecology, Andrology, Radioimmunoassay, Postgraduate Research and Cold
Room for preserving fetuses for obstetrical training were established under his guidance because of which the
department, later on, was recognized as Centre of Advance Faculty Training.
Dr. Sharma was a dedicated teacher and a clinician par excellence who had great love for animals. Owing to
his seless service to the animals and the farmers, he earned a lot of respect from the community. He was famous
for his softspoken nature, ever helping attitude and dedication towards profession. Dr. Sharma had a passion
for Obstetrics and could not bear the suering of the animals. He therefore, established a 24 h clinical service
in the Veterinary Hospital where the cases were attended even at odd hours. Keeping in view the contributions
of Dr. R.D. Sharma towards society and science, Indian Societies for Study of Animal Reproduction started a
prestigious award in his name for best scientic presentation in Obstetrics during the National Conventions. Many
of his students are presently working on many eminent positions in India and Abroad. Sudden demise of Dr. R.D.
Sharma has left a void which will not be fullled so easily.
Renowned Veterinarian Dr. R.D. Sharma Passes Away
Indian Journal of Animal Reproduction 40 (1): June 2019
74
REPORT OF INDIAN SOCIETY FOR STUDY OF ANIMAL REPRODUCTION
(ISSAR) 2017
Indian Society for Study of Animal Reproduction (ISSAR) has organized 33rd Annual Convention of ISSAR and
National Symposium on “Use of reproductive technologies and production improvement in livestock species aiming
to socioeconomic development of rural mass” during February 9-11, 2018 at City of Joy, Kolkata. The symposium
began with colourful inaugural session on February 9, 2018 which was inaugurated by Hon’ble Purnendu Basu,
Minister of Technical Education, Training and Skill Development, Govt. of West Bengal in the presence of Hon’ble
Vice–Chancellor of West Bengal University of Animal and Fishery Sciences, Prof. Purnendu Biswas, Director,
Animal Husbandry and Veterinary Sciences, Govt. of West Bengal, Dr. (Capt.) A.G. Bandopadhyay, Chairman,
West Bengal cooperative milk producer federation Ltd., Sri Paras Dutta and other dignitaries. A total of 409
delegates from dierent states of country participated. The entire symposium was spread over 8 technical
sessions. A total of 12 invited lead speakers gave exhaustive presentations on various aspects of reproductive
technologies covering most important livestock species. This year eight young scientists were selected for young
scientist award on the basis of their performance during research paper presentations. Total 415 abstracts were
included in this symposium. There were two special sessions; Dr. S.N. Luktuke Extempore presentation for young
scientist (≤35 yr) and another for eld veterinarian award i.e. clinical case presentation.
Glimpses of ISSAR 2017 Inaugural Session
Inaugurated by Hon’ble Purnendu Basu, Minister
of Technical Education, Training and Skill
Development, Govt. of West Bengal
Dr. Madhusudan Sarkar, Dr. J.L. Chakraborty with
CEC, ISSAR members
Address by Hon’ble Purnendu Basu Participation of ISSAR convention by delegates
from dierent parts of the country
Indian Journal of Animal Reproduction 40 (1): June 2019
75
ISSAR activity report by general Secretary, ISSAR
Dr. Shiv Prasad
Release of Souvenir of 33rd ISSAR convention
during inaugural session
C.R. Sane oration delivered by Dr. D. KathiresanAddress by Hon’ble president, ISSAR Dr. V.
Chadrashermurty
Poster session presentation by scientist during
convention
Dierent scientic sessions held during convention
Indian Journal of Animal Reproduction 40 (1): June 2019
76
ISSAR AWARDS 2017 - AWARDED DURING 33RD ANNUAL CONVENTION AT
KOLKATA
Life Time Achievement Award: Dr. D. Kathiresan, Ex-Dean, College of Veterinary and Animal Sciences,
Aizawl, Mizoram was honoured with Life Time Achievement Award-2017 of the society for his contribution
to the eld of Animal Reproduction for more than 3 decades
ISSAR Fellow - 2017: Dr. J.B. Phogat, Dean, Post graduate Studies, Lal Lajpat Rai University of Veterinary
and Animal Sciences, Hissar, Haryana was awarded with Fellow, Indian Society for Study of Animal
Reproduction
ISSAR Fellow - 2017: Dr. S.P.S. Ghuman, Professor &Head, TVCC, GADVASU, Ludhiana, Punjab was
awarded with Fellow, Indian Society for Study of Animal Reproduction
Indian Journal of Animal Reproduction 40 (1): June 2019
77
Nil’s Lagerlof Memorial Award; This award is given
to young scientist who has published best paper in
the eld of Animal Reproduction in preceding year.
This year it goes to Dr. M.K. Patel and co-authors
Drs. R.S. Cheema, A.K. Bansal and V.K. Gandotra
Dr. G.B. Singh Memorial Award: This award is
given to the young scientist who has published
best paper in the eld of Animal Reproduction
in preceding year. This year it goes to Dr. N.K.J.
Pandey and co-authors Drs. H.P. Gupta, Shiv
Prasad and S.K. Sheetal.
Dr. N.C. Sharma award: This award is given to
the scientist who has published best research
paper in Indian Journal of Animal Reproduction
in preceding year. This year it goes to Dr. Bilawal
Singh and co-authors Drs. S.P.S. Ghuman, R.S.
Cheema and A.K. Bansal
Dr. R.D. Sharma Award: This award is given to the
scientist who has published best research paper
in the eld of Veterinary Obstetrics. This year it
goes to Dr. N. Singh and co-authors were Dr. V.K.
Gandotra, S.P.S. Ghuman, S.S. Dhindsa and P.S.
Brar
Indian Journal of Animal Reproduction 40 (1): June 2019
78
Best Field Veterinarian Award: This award is given
to the best eld veterinarian on the basis of his
presentation on clinical cases handled under
eld conditions. This year it goes to a very senior
veterinarian Dr. K. Ganesan.
Young scientist winner of dierent scientic
sessions during convention
S.N. Luktuke Extempore Award: This award is
based on extempore presentation by young
scientist below the age of 35 years on a specied
topic during convention. This year it was won by
Dr. Shilpa V.S. from Tamil Nadu.
INTAS quiz competition won by Dr. Meenakshi
Rawat, C.V.A.Sc., GBPUA&T., Pantnagar,
Uttarakhand.
Indian Journal of Animal Reproduction 40 (1): June 2019
79
PROCEEDINGS OF 33RD ANNUAL CONVENTION OF ISSAR HELD AT
KOLKATA, FEBRUARY 9-11, 2018
General body meeting of ISSAR was held on February 10, 2018 in the evening and the following decisions
were taken.
1. It was approved by GBM that Rs 10,000 (Rs. Ten thousand) per issue as demanded by www.indianjournal.
com for uploading of our journal (IJAR) is viable solution as it will be free of cost for all the members for
downloading of their papers or other materials.
2. GBM approved a cash award of Rs. 2,100 per winner scientist (8) instead of travel grant.
3. Financial status and expenditure presented by treasurer, ISSAR was approved by GBM.
4. CEC of ISSAR has expressed great concern about poor reporting of the state chapters about their
nancial status, executive body elections and technical activities conducted in the state and any signicant
achievement of the members at national or international level. State secretaries were impressed upon to
collect, compile the information and report to General Secretary, ISSAR for wider dissemination.
5. The dates to apply for various awards of ISSAR remain unchanged i.e. 31st March of every year.
6. It was decided in GBM that mid convention meeting of CEC is compulsory for shaping the activities and
making it more meaningful. CEC should meet preferably at the venue of next proposed convention.
7. From the next year R.D. Sharma Award shall be known as R.D.Sharma Memorial Award.
8. The next convention of ISSAR which was decided in GBM for Rewa (M.P.) has been changed to College of
Veterinary and Animal Sciences, Anand (Gujarat) by CEC after consultation looking into various aspects.
9. For reviewing of ISSAR by-laws, a committee under the chairmanship of Dr. K. Kulasheker was constituted
with Dr. Shiv Prasad, Dr. M. Selvaraju, Dr. F.A. Ahmad, Dr. M.K. Tandley and Dr. D.K. Nandi. They will
complete the work within 3 months.
Indian Journal of Animal Reproduction 40 (1): June 2019
80
Scientic recommendations of 32nd Annual Convention of ISSAR and National Symposium on “Use of
reproductive technologies and production improvement in livestock species aiming to socioeconomic
development of rural mass” held at West Bengal University of Veterinary and Fishery Sciences, Kolkata
w.e.f. 9-11 February, 2018.
1. Use of ultrasonography should be encouraged under eld conditions for diagnosis of various reproductive
ailments and also for pregnancy diagnosis.
2. Development of diagnostic tools for various reproductive disorders also needs fresh attention.
3. Molecular markers for selection of breeding bulls at early stage should be established for indigenous cattle
and bualoes.
4. Infrastructural boosting is required to reach up to end users. Elite bull semen should only be allowed to be
used for breeding.
5. Frozen semen from elite indigenous breeds should also be given good attention.
6. Regular training on obstetrical techniques should be conducted to acquire skill in the eld of Obstetrics by
using various models.
7. Research on sexing of spermatozoa for skewing of sex ratio needs to be given priority.
8. Research work on embryo biotechnology and stem cell technology should be enhanced and it should
continue.
9. Research work on equine reproduction should also nd place in veterinary colleges of the country as in
domestic animals.
Indian Journal of Animal Reproduction 40 (1): June 2019
81
APPLICATION FOR LIFE MEMBERSHIP IN THE INDIAN SOCIETY FOR
STUDY OF ANIMAL REPRODUCTION (REG. NO. F 5156/MUMBAI)
Name :
Address :
Professional qualication :
Field of specialization :
Address to which all
correspondence should be made
(with Pin Code)
:
Telephone No.
Home
Oce
:
:
Mobile No. :
e-mail address :
Whether Membership is direct or
through State Chapter
:
Details of remittance for life
membership fee (Cash / DD /
NEFT / RTGS)
:
Place :
Date :
Signature of the Applicant
Note:
• Life Membership fee - Direct application Rs.1510 (Rs One thousand ve hundred and ten only)
• Life Membership fee - Through state chapter Rs. 1135 (Rs One thousand one hundred and thirty ve only)
• The fees may be drawn as the cheque / demand draft in favour of The Treasurer, ISSAR, payable at Namakkal
or fees may be sent through NEFT / RTGS for the following account. Name: Treasurer, ISSAR, Name of
the Bank: Union Bank of India, Branch Name: Namakkal, Account number: 548802010014809, IFSC code:
UBIN0554880, MICR code: 637026002.
Indian Journal of Animal Reproduction 40 (1): June 2019
82
AUTHOR GUIDELINES
General
The Indian Journal of Animal Reproduction (IJAR) is the ocial organ of the Indian Society for the Study
of Animal Reproduction (ISSAR) and publishes basic/applied research articles, short communication and case
reports in the eld of Veterinary Gynaecology, Obstetrics, Andrology, Semenology, Articial Insemination, Embryo
Transfer and other Assisted Reproductive Technologies. The journal is published biannually i.e. June and
December. The manuscripts are subjected to a thorough peer-review by referees and journal publishes only well-
written manuscripts contributing towards scientic and practical advancement in the eld of reproduction. The
publication language of journal is English. The manuscripts are accepted for publication with the understanding that
they have not been published, submitted or accepted for publication elsewhere in any language. The manuscript
will be accepted after editorial revision based on comments. The manuscript found unsuitable for publication in the
IJAR will be communicated to the author and no correspondence will be entertained in this regard. The decision of
the editorial board shall be nal. The responsibility with regard to the data and technical details of the article would
be with the authors. The copyright of manuscripts, accepted for publication, rests with ISSAR.
Manuscript submission
The manuscript submission and review process is handled online. To submit an article to The Indian Journal
of Animal Reproduction, please go to http://www.indianjournals.com/ijor.aspx?target=ijor:IJAR3&type=home and
please ensure that the corresponding author’s valid email is present in the manuscript. Authors submitting a
manuscript need to upload online Author’s Declaration Certicate along with the manuscript as a supplementary
le. The research article, short communication or case report should be strictly formatted as mentioned below.
Review article: The length including gures, tables and references, should not exceed 4,000 words. The
standard format of review article shall include abstract, keywords, review and references. Other guidelines as
mentioned in original research article shall be followed. The main focus of review should be the research carried
out by the authors in India.
Research article: The length including gures, tables and references, should not exceed 2,000 words. The
standard format of original research article must be as per the example given in the box below. The manuscript
must be computer typed (Arial, font 12) with single spacing using standard software (Microsoft Word). Margins
should be 2.54 cm on Top and Bottom and 3.17 cm on Left and Right. Title should be concise, informative and
containing keywords necessary to enable retrieval by search engines. Abstract should limit to maximum 150
words. The abstract should not just contain repeated/rewritten results. Eorts should be made to give principal
signicant ndings only and concluding the ndings keeping in view the importance of work done. The abstract
should be self-explanatory to generate interest in the reader for reading/citing the article. Keywords should be
ve and alphabetically arranged. Introduction should be related to parameters done in the study and justifying
the importance of work carried out. Materials and Methods should be described in sucient detail (animal
management, methods, statistics etc.) to allow for repetition of the experiments. Results and Discussion should
focus on the interpretation of ndings and repetition of data in text, already presented in table should be avoided.
Figure and Table number should be kept to a minimum. Tables should be elegantly made, in a vertical format and
within the page margins. Photographs or drawings must be sharp, of high contrast and to be submitted in JPEG
format. All the gures and tables should be referred in the text.
Indian Journal of Animal Reproduction 40 (1): June 2019
83
References: Citations in the text should be given by placing in parenthesis the name(s) of author(s) and the
year of publication, e.g. (Rao, 1996), (Mann and Lamming, 2001), (Binelli et al., 2001), (Rao, 1996; Binelli et al.,
2001 and Mann and Lamming, 2001). Avoid writing citations in text as Rao (1996) reported……, Binelli et al.
(2001) found……. All publications cited in the text should be presented in the form of list arranged alphabetically
according to author’s surnames. The number of references should be restricted to maximum 15. The examples
are given for citing a journal article and a book (chapter). For conference proceedings, include the name(s) of the
editor(s) of the proceedings, the publisher and the place of publication. References of thesis and dissertations
more than three year old should be avoided.
Rao, A.R. (1996). Pioneers in Animal Reproduction. Indian J. Anim. Reprod., 17(2): 1546-1547.
Mann, G.E. and Lamming, G.E. (2001). Relationship between maternal endocrine environment, early embryo
development and inhibition of luteolytic mechanism in cows. Reproduction, 121(1): 175-180.
EARLY LUTEAL PHASE HORMONAL TREATMENT FOR
INCREASING CONCEPTION RATE DURING SUMMER SEASON IN
BUFFALO
A. KUMAR1*, S. SINGH2 AND J. SINGH3
Department of Veterinary Gynaecology and Obstetrics
Guru Angad Dev Veterinary and Animal Sciences University,
Ludhiana - 141 004
1Veterinary ocer, Punjab Government, Barnala - 141 003;
2Asssistant Professor, 3Professor; *aba_aba@yahoo.co.in
Received: 02.04.2016 Accepted: 04.06.2016
ABSTRACT
Keywords:
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
ACKNOWLEDGEMENTS (if any)
REFERENCES
Table: (if any)
Figure: (if any)
Indian Journal of Animal Reproduction 40 (1): June 2019
84
Binelli, M., Thatcher, W.E., Mattos, R. and Baruselli, P.S. (2001). Antiluteolytic strategies to improve fertility in
cattle. Theriogenology, 56: 1451-1463.
Niemann, H., and Elsaesser, F. (1983). Steroid hormones in early pig embryo development. In: Bavister, B.D.
(ed.), The Mammalian preimplantation Embryo. 1st ed., Plenum Press, New York. pp. 117-132.
Units and abbreviations: Any abbreviations of chemical, biological, veterinary, or other terms should only be
employed when it is certain that they are internationally known. The full name must be stated in brackets when
the abbreviation is rst used in the manuscript. Metric system should be followed in the manuscript. Authors are
requested to use the following abbreviations. Body weight - b wt, Kilogram - kg, International Units - IU, Centimeter
- cm, Kilo calories - kcal, At the rate of - @, Count per minute - cpm, Milligram - mg, et al - et al, Cubic centimeter
- cm3, Microgram - μg, Inch - in, Square centimeter - cm2, Millilitre - ml, Intramuscular - im, Degree centigrade - ºC,
Microlitre - μl, Intravenous - iv, Degree Fahrenheit - ºF, Picogram - pg, Subcutananeous - sc, Decilitre - dl, Parts
per million - ppm, Once a day - od, Gram - g, Hour(s) - h, Twice a day - bid, Litre - l, Minute(s) - min, Thrice a day
- tid, Metre - m, Second(s) - sec, Revolution per min - rpm, Per cent - %, Year(s) - yr, Articial Insemination - AI.
Short communication: In the same general format as full-length research manuscripts, but should not
exceed 1,200 words including tables and illustrations. The subheadings, except for ABSTRACT, Keywords,
ACKNOWLEDGEMENTS and REFERENCES, should not be written in the manuscript. The manuscript under
this head should be clearly marked “Short Communication” at the right hand corner, on the top of the rst page of
manuscript. Photos and tables (Vertical format) should be restricted to one.
Case reports: Case reports of interesting and rare nature sent for publication should not exceed 1,000 words
including references and illustrations. The manuscript should contain ABSTRACT, Keywords, INTRODUCTION,
CASE HISTORY AND OBSERVATIONS, TREATMENT AND DISCUSSION and REFERENCES. Photos, if any,
should be restricted to one.
Publication charges
For publication in the IJAR, presently the journal levies Rs. 500/- (Five hundred only) per article as processing
and publication charges. If authors and co-authors are not life member of the ISSAR then each one has to pay Rs.
300 in addition to the publication charges. The details of payment through electronic mode will be communicated
following acceptance of the manuscript.
Indian Journal of Animal Reproduction 40 (1): June 2019
85
INDIAN SOCIETY FOR THE STUDY OF ANIMAL REPRODUCTION
(Regd. No. Bom - 253 / 78)
Certicate required for sending article for publication to
INDIAN JOURNAL OF ANIMAL REPRODUCTION
AUTHOR’S DECLARATION CERTIFICATE
Article entitled:
Author’s:
1. The article has been seen by all the authors (signatures given below who are satised with its form and
content).
2. Due credit of authorship has been given to every scientist who has made a notable contribution to the paper
and we are statised with the sequence in which the authors names appear in the by-line (authors’ names).
3. The by-line of the article does not include any name of a scientist who has not made a notable contribution to
the paper.
4. The name of the Institute appearing below the by-line is that of the Institute where the research was conducted
and not of the Institute where the rst author (or the author who has sent the paper) is currently employed.
5. Two copies of the article and illustrations have been sent
6. The results reported in the article have not been sent simultaneously for publication in any other journal.
7. The article has not been published elsewhere either in a Journal or Souvenir or Research / Extension Bulletin.
8. The article has not been rejected for publication in any other Journal. Rejection elsewhere does not necessarily
disqualify the paper for publication in the Indian Journal of Animal Reproduction
9. If published, the copyright of this article would vest with the Indian Society for the Study of Animal Reproduction
(ISSAR) which has the right to enter into any agreement with any organization in India or abroad engaged in
reprography, photo-copying, storing and dissemination of information contained in it, and neither we nor our
legal heirs will have any claims on any royalty.
Signatures of all authors, their Designations and present correspondence addresses:
Sr. No. Author Life membership No. Present Address Signature
1.
2.
3.
Indian Journal of Animal Reproduction 40 (1): June 2019
86
AUTHOR INDEX
ABU EL-HAMD M.A. 12
AGARWAL J. 19
AGARWAL S. 19
AHMED J. 69
AHMED N. 69
ANAND I. 42
ANBHULE R.S. 31
ANBHULE R.S. 38
ANBHULE R.S. 55
ARUNMOZHI N. 49
BHAT M.B. 35
BHATTA R. 7
BINSILA B.K. 7
BRAR P.S. 35
CHAUDHARI C.F. 51
CHAUDHARI N.F. 45
CHAUDHARY M.K. 61
CHAUDHARY S.S. 51
CHAWLA P.S. 42
DHALIWAL G.S. 42
FOUAD W.F.M 12
GHOSH S.K. 7
GHUMAN S.P.S. 1
JAYAGANTHAN P. 65
KADAM P.D. 31
KADAM P.D. 38
KADAM P.D. 55
KADOOM A.K. 12
KASRIJA R. 47
KELAWALA D.N. 63
KHARCHE S.D. 19
KHASATIYA C.T. 45
KHASATIYA C.T. 51
KRISHNAKUMAR K. 53
KULASEKAR K. 53
KUMAR A. 71
KUMAR B. 58
KUMAR B. 61
KUMAR P. 16
KUMAWAT B.L. 31
KUMAWAT B.L. 38
KUMAWAT B.L. 55
KUSHWAH B.S. 19
KYARONG S. 69
LAWANGE S.R. 31
LAWANGE S.R. 38
LAWANGE S.R. 55
LEGHA R.A. 27
MARKANDEYA N.M. 31
MARKANDEYA N.M. 38
MARKANDEYA N.M. 55
MAVI P.S. 35
MEHTA J.S. 71
MEHTA S.C. 27
MODI L.C. 45
MOMIN T.M. 63
NAVAMANI K. 53
NEELAM 16
PALANISAMY M. 65
PANDEY R.P. 58
PATEL M.D. 45
PATEL S.B. 51
PATEL V.R. 45
PATHAK J. 19
PATIL D.B. 63
PATI R.R. 67
PRABAHARAN V. 65
PRAKASH V. 27
PRASAD J.K. 7
RADHAKRISHANAN L. 53
RAJA S. 65
RAJKUMAR R. 65
RAMYA L. 7
RAVINDRA J.P. 7
RAVI S.K. 27
ROOPA Y. 47
S.H. HASSOUNA 12
SACHAN V. 61
SAVANI H.R. 45
SAVANI H.R. 51
SAWALE A.G. 31
SAWALE A.G. 38
SAWALE A.G. 55
SAXENA A. 58
SAXENA A. 61
SAXENA S. 45
SAXENA S. 51
SCHAN V. 58
SELVARAJU S. 7
SHAFIUZAMA M. 49
SHARMA A. 24
SIKARWAR A.K.S. 19
SINGH C. 27
SINGH MADHUMEET 16
SINGH MADHUMEET 24
SINGH N. 1
SINGH P. 47
SINGH V.K. 51
SONI Y. 71
SRIDEVI P. 49
SRIDEVI P. 67
SUBRAMANI A. 49
SUTHAR V.S. 63
TALLURI T.R. 27
TALLURI T.R. 71
TRIPATHI A. 71
TRIPATHI B.N. 27
UMAMAGESWARI J. 67
VERMA H.K. 47
WANI A.A. 35
YADAV D. 58
RNI - Registration No. 4924 (Registered under press and registration of Books Act 1867
By the Registrar of newspaper for India, New Delhi)
Edited and Published by Dr. Sarvpreet Singh Ghuman, 376-G, Bhai Randhir Singh Nagar,
Ludhiana - 141 012.
Cell: 081462-37600, Email: ghuman_s@yahoo.co.in
M/s. FOIL Printers, 2051, Gobind Nagar, Ludhiana-141 001. Ph.: 0161-2404093
STATEMENT ABOUT PUBLISHER, EDITOR AND PRINTER OF
THE INDIAN JOURNAL OF ANIMAL REPRODUCTION
Sd/
Prof. Sarvpreet Singh Ghuman
Editor
1. Title of the Journal : The Indian Journal of Animal Reproduction
2. Language : English
3. Periodicity of its Publication : Half yearly
4. Editor/Publisher's Name : Prof. Sarvpreet Singh Ghuman
5. Nationality and address : Indian, 376-G, Bhai Randhir Singh Nagar,
Ludhiana - 141 012. Published on behalf of The
Indian Society for Study of Animal Reproduction
(ISSAR)
6. Place of Publication : 376-G, Bhai Randhir Singh Nagar, Ludhiana -
141 012. Published on behalf of The Indian
Society for Study of Animal Reproduction (ISSAR)
7. Printer : M/s. FOIL Printers
2051, Gobind Nagar, Ludhiana-141 001
I, Prof. Sarvpreet Singh Ghuman hereby declare that the particulars given
above are true to the best of my knowledge and belief.
