Embryonic mortality in buffalo cows

Abstract

In buffalo species embryonic mortality is considered one of the major causes of fertility loss, especially in the animals that are not mated during their reproductive period. Embryonic loss in animals mated by artificial insemination (AI) is 20-40% during seasons characterized by high number of light hours. Also in buffalo naturally mated the incidence of embryonic mortality is about 20% and a higher incidence is observed between 28- 60 days of gestation in buffaloes that conceive during increasing daylight length. A reduced capacity to secrete progesterone seems to explain in part this embryonic mortality but other as yet unidentified factors contribute between 40-50% to the embryonic losses. Treatments with hCG, GnRH agonist or progesterone on Days 5 after AI not always reduce embryonic mortality in buffalo species. Embryonic mortality in buffaloes appears to occur later (Day 25-40) than in cattle and P4 treatments should perhaps be applied later in buffaloes.

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Ital.J.anIm.ScI. vol. 6, (Suppl. 2), 119-129, 200711?
Embryonic mortality in buffalo cows
G. Campanile, G. Neglia
Department of Scienze Zootecniche ed Ispezione degli Alimenti, Faculty of Veterinary Medicine,
Via F. Delpino 1, 80137 Napoli, Italy
Corresponding author: G. Campanile, Department of Scienze Zootecniche ed Ispezione degli Alimenti, Fa-
culty of Veterinary Medicine, Via F. Delpino 1, 80137 Napoli, Italy - Tel. 081 2536069 - Fax: 081 292981
- Email: giucampa@unina.it
ABSTRACT: In buffalo species embryonic mortality is considered one of the major caus-
es of fertility loss, especially in the animals that are not mated during their reproductive
period. Embryonic loss in animals mated by artificial insemination (AI) is 20-40% during
seasons characterized by high number of light hours. Also in buffalo naturally mated the in-
cidence of embryonic mortality is about 20% and a higher incidence is observed between 28-
60 days of gestation in buffaloes that conceive during increasing daylight length. A reduced
capacity to secrete progesterone seems to explain in part this embryonic mortality but other
as yet unidentified factors contribute between 40-50% to the embryonic losses. Treatments
with hCG, GnRH agonist or progesterone on Days 5 after AI not always reduce embryonic
mortality in buffalo species. Embryonic mortality in buffaloes appears to occur later (Day
25-40) than in cattle and P4 treatments should perhaps be applied later in buffaloes.
Key words: Eembryonic mortality, Bbuffalo, Progesterone, GnRH, Hcg.
BUFFALO REPRODUCTIVE CHARACTERISTICS - Buffalo is an animal species
that lives in regions found between 31°N parallel and 2°S. Currently, the distribution of
the buffalo population covers the major climatic regions of the lower latitudes (Tropical
zone) and middle latitudes (Temperate zone). This geographic origin and distribution
logically suggests that buffaloes are adapted to hot, humid macro or microclimates (Sha-
fie, 1985). Buffalo is a photoperiodic species. Like sheep, buffaloes have to be considered
a “short day” species. They have heats throughout the year but are more fertile when
daylight hours decrease. According to Zicarelli (1995), this characteristic is due to their
tropical origins; in fact, in these areas the availability of forage coincides with the period
in which dark hours increase. Therefore, it has been supposed that animals which cal-
ve in the most suitable period for survival of the offspring were selected. It seems that
they have retained this characteristic even when transferred to places where forage is
always available (Zicarelli, 1995). In countries like Italy, where market demand requires
a concentration of deliveries in the spring-summer period (not corresponding to buffalo
reproductive activity) the out-of-season technique is widely applied. As a result, buffa-
loes which are less sensitive to photoperiodic effects have been selected. When the out-of-
season technique has been applied for long periods a lower loss of fertility was observed
(15% vs. 30%) compared to the farms in which it has been adopted for shorter periods
(Campanile, 1997). These results are also due to the renewal of the herd that is very

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frequent on farms that apply the out-of-breeding-season mating technique (Campanile,
1997). Moreover, the season-dependent reproduction phenomenon is more frequent in
older buffaloes (Zicarelli et al., 1988a) that are more sensitive to the bull effect and show
seasonal acyclia more easily.
Buffalo reproduction is characterized by delayed puberty, silent oestrus, long post-partum
ovarian inactivity, and, on the whole, poor fertility (Singh, 1988; Madan et al., 1994; Sin-
gla et al., 1996). Most of these problems result from the use of the “out of breeding season
mating” technique (Zicarelli, 1997; Gasparrini B., 2002). In fact, if buffalo are bred without
modification of their natural seasonality and without controlled breeding, an inter-calving
period of less than 400 days and a culling rate of less than 12% has been observed in Italy,
Brazil, Venezuela, and Argentina (Zicarelli et al., 1993). Poor fertility has also been obser-
ved when biotechnologies are applied to reproduction.
Immediately after parturition buffaloes show several physiological modifications, which
are fundamental to sustain the new pregnancy. The first step is the resumption of ovarian
cycle. This is blocked during pregnancy by progesterone, which avoids other ovulations
and maintains hypotonic the uterus. In buffalo species, the resumption of ovarian acti-
vity is affected by the calving season. In fact, buffaloes that delivered during the spring
period, showed an intercalving period on average longer by 30 or 70 days, respectively if
they are pluriparous or primiparous (Zicarelli, 1994). This phenomenon is observed until
the delivery happens in July-August period. Usually, after 90 days of lactation during the
spring period, 44% of pluriparous and 80% of primiparous are acyclic (Zicarelli, 1994). The
reproductive activity of Italian buffalo cows is also influenced by climatic variation. As
regards spontaneous heats, temperatures lower than 8°C and continuous light for more
than 11 hours cause a delay in ovulation, starting from the end of heats. This is proba-
bly a delay in the pituitary response to ovarian steroid secretion (Zicarelli et al., 1988a).
Thermal excursions higher than 7°C and 9°C appreciably increase the incidence of double
ovulation in both spontaneous oestrus and prostaglandin-induced oestrus (Zicarelli et
al., 1988b). Such conditions limit the adoption of A.I. during specific periods of the year.
Moreover, Sastry and Georgie (1978) found a correlation between conceptions and tem-
perature, relative humidity or rainfall. Specifically, a lower temperature and increased
rainfall improve the conception rate. Obviously, rainfall during the three months previous
to the conceptions improves the availability of herbage, meeting productive requirements.
This represents an indirect effect of climate on buffalo reproduction; a hot climate, in fact,
affects living and reproductive behaviour directly by its effect on their systemic functions
and indirectly by governing the availability of nutrients.
EMBRYO DEVELOPMENT - A fundamental condition for the maintenance of a new
gestation, is the presence of a suitable uterine environment, which is able to guarantee an
adequate embryo development (Campanile, 2006). In bovine the first cleavage division is
at around 30 hours after insemination and the second at around 48 hours. During the fol-
lowing 4-5 days, each blastomere undergoes subsequent divisions in the oviduct, yielding
4, 8, 16, 32 cells, tight morula and, finally, blastocyst (Senger, 2003). On day 6.5-7 the new
embryo reaches the uterus and during the second week of gestation it begins to elongate
and to send signals to the mother, in order to prevent luteolysis and maintain adequate
progesterone circulating levels. Progesterone is important for allowing uterine secretions
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and reducing myometrial tone, favouring embryo development and attachment. In buffalo
species Karaivanov et al. (1987) observed that from the flushing of oviducts and uterine
horns of slaughtered superovulated donors between 74 and 100 h, eggs were recovered only
from the oviducts, while flushing conducted between 102 and 108 yielded eggs from both the
oviducts and uterine horns. This observation suggests that embryo development is faster in
buffalo than in bovine, as confirmed by several trials carried out in vitro, during which tight
morulae and blastocysts were observed already on day 5-6 (Neglia, et al., 2001). Therefore,
the maintenance of pregnancy is due to either the embryo capacity of signalling its presence
and the mother capacity of recognizing these signals and maintaining an adequate uterine
environment.
EMBRYONIC MORTALITY - Embryonic loss is increased when physiological regula-
tion of oviductal and uterine function is inadequate or when the mother is exposed to one
or more of the many stresses that can compromise embryonic survival (Hansen, 2002).
Embryonic mortality usually happens during the first phases of gestation in various spe-
cies: in cattle, for example, it is evident within 40 days of pregnancy. In particular, 30-40%
of embryonic losses in bovine occurs between 7 and 17 days post fertilization and, in some
cases, it can take place before embryo becomes foetus (Thatcher et al., 1995). Vasconcelos et
al. (1997) recorded that during the subsequent phases of pregnancy (after 42 days), embry-
onic loss is a remote eventuality (around 10%).
In buffalo species embryonic mortality is considered one of the major causes of fertility
loss, especially in the animals that are not mated during their reproductive period. In
Italy, in fact, the application of the out of breeding season mating technique guarantees
milk production in accordance with market requirements, but it forces the breeders to
mate buffaloes during the less favourable periods. It was observed that embryonic loss in
animals mated by artificial insemination (AI) is 20-40% during seasons characterized by
high number of light hours (Campanile et al., 2005; Campanile et al., 2007a; Campanile
et al., 2007b), whereas values of around 7% were recorded in Brazil during decreasing
light days (Baruselli et al., 1997). In contrast to the previous work, an embryonic morta-
lity rate of 20% was reported for buffaloes close to the equator (Vale et al.,1989). In any
case, embryo mortality in buffalo occurs later than in bovine, usually between 25 and
40 days from AI (Campanile et al., 2005). In buffaloes naturally mated (Vecchio et al.,
2007), independently from the conception period, 8.8% and 13.4% showed respectively
embryonic mortality between 28-45 days (embryonic mortality-EM) days and between
46-90 days (foetal mortality – FM) of pregnancy. In this work no differences were found
between the incidence of EM in relation to the conception period (Table 1), while a high
incidence (P<0.01) of FM was found (Table 1) during a period of increasing daylight length
(transitional period: December-March) compared to the April-July period. It is hypothesi-
zed that this condition is due to the presence, in the transitional period, of subjects that
become pregnant, even if they have a lower function of the corpus luteum because they
are going into anoestrus. In the subsequent months (April-July) an increased incidence
of acyclic buffaloes is observed and, hence, only the subjects that are not sensitive to the
photoperiod are cyclic and become pregnant. In fact, the incidence of FM is similar to that
observed in the decreasing daylight length period (August-November), that is the favou-
rable period for reproductive activity.
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These data are in accordance with Baruselli (personal communication), that found an em-
bryonic mortality (after 30 days from AI) of 13.2% and 7.0% respectively in decreasing and
increasing daylight length period. In 1994, Zicarelli (1994) found an embryonic mortality of
21.8% in buffaloes naturally mated. The phenomenon was not correlated with the breeding
season (spring vs. summer), but with the farm and the ovarian resumption after calving. In
another trial (Zicarelli, unpublished data) performed on 3000 conceptions, a higher inciden-
ce of embryonic mortality was reported between 30 and 90 days in buffaloes that conceived
during increasing daylight length (Figure 1). However, also in this case the phenomenon
was affected by farm, management and environment. The incidence of embryonic mortality
in Farm A was 5% vs. 14% observed in Farm B, but either these values were lower than
those recorded in 1994.
The incidence of embryonic mortality found in Italy was higher between 28-60 days of
gestation and lower after 71 days (Figure 2). This result is different from that reported in
cattle (Silke et al., 2002), in which the embryonic loss from 28-87 days of gestation was
similar.
Table 1. Incidence of embryo (EM) and fetal (FM) mortality during the periods.
EM %
?.1
7.0
10.8
8.8
FM %
16.6 a
10.? b
11.2 ab
13.4
December-March
April-July
August-November
Total
a vs. b = P<0.05
Figure 1. Incidence of embryonic mortality in relation to mounth of conception and
daylight lenght in two farms.
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Embryonic mortality, in buffalo species, was not affected by age, parity or lactation stage
and infectious agents explained only about 2-8% of the cases (Campanile et al., 2005; Cam-
panile et al., 2007a). Campanile et al. (2005) found a higher P4 plasma levels in pregnant
buffaloes than in buffaloes which showed embryonic mortality since day 10 after AI, whilst
P4 in non-pregnant buffaloes was intermediate. Pregnant buffaloes had also higher pla-
sma P4 on day 20 than both non-pregnant buffaloes and buffaloes that showed embryonic
mortality. P4 plasma concentration significantly decreased only in non-pregnant buffaloes
between day 10 and 20. In a further trial, it was observed that pregnant buffaloes showed
higher concentrations of P4 milk whey than both animals showing embryonic mortality and
non-pregnant buffaloes on day 20 and day 25 but only than non-pregnant buffaloes on Day
10 (Campanile et al., 2007b).
It may be hypothesised that embryonic mortality in buffalo species is primarily due to a
reduced secretion of P4 by corpus luteum. This conclusion would be consistent with several
findings in cattle and sheep, where early embryonic mortality was associated with reduced
circulating concentrations of P4 (Garret et al., 1988; Mann and Lamming, 1999; Mann and
Lamming, 2001). During a trial carried out in Italy in a period of increasing daylight length
it was proposed that the relatively high incidence of buffaloes with low circulating concen-
trations of P4 after oestrus synchronisation was reflective of a reduced activity of the repro-
ductive endocrine system. As previously specified, buffaloes are seasonal animals ,showing
increased reproductive activity in response to decreasing daylight length (Zicarelli, 1997).
Impaired P4 secretion has been linked with a reduced capacity of the developing embryo to
secrete interferon-tau (IFNτ) at threshold amounts necessary to prevent luteolysis (Wathes
et al., 1998). In fact, as above mentioned, the maintenance of pregnancy is due either to the
maternal recognition of pregnancy and to the embryo capability of blocking luteolysis since
day 16 post-AI (Mann and Lamming, 1999). This process occurs by the production of bovine
trophoblastic protein-1 (bTP-1), also called IFNτ (Roberts et al., 1992). This protein is able
to avoid corpus luteum regression by two mechanisms: i) by inhibiting oxytocin receptors
(OTR) development on endometrium (Robinson et al., 1997); ii) by activating a prostaglan-
din inhibitor (Thatcher et al., 1995).
Figure 2. Embryonic loss rate during stage of gestation.
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It has been supposed that oestradiol is another factor involved in the luteolytic process, ei-
ther by promoting OTR development and by stimulating prostaglandin secretion (Wathes et
al., 1998). In fact, it has been demonstrated in ovine that the number of oestradiol receptors
on endometrium is significantly lower in pregnant vs. not pregnant animals (Lamming et
al., 1995; Spencer et al., 1995). However, in buffalo species oestradiol plasma levels do not
differ between pregnant, not pregnant and buffaloes undergone embryonic mortality on day
0, 10, 20 and 25 after A.I. (Spagnuolo et al., 2007).
Gametes quality is one of the main factors involved in the phenomenon of embryonic morta-
lity in domestic animals. Oocyte quality is able to affect embryo development and interfere
with the following gestation. In buffalo species this phenomenon may be more frequent
during the seasonal anoestrus, which coincides with day length increase (Campanile et al.,
2005) and, consequently, with the resumption of sexual promiscuity in the farms in which
the out of breeding season mating technique is applied. Campanile et al. (2005) demonstra-
ted that 51% of buffaloes which showed embryonic mortality had P4 concentrations on days
10 and 20 similar to those of animals which maintained pregnancy. Therefore, it is possible
that other factors, rather than reduced circulating P4 concentrations, also contributed to
embryonic mortality. With this regard, it was reported that oocyte quality, judged as the
capacity to result in embryonic development and pregnancy, is worse in buffaloes during the
anoestrous period (Abdoon et al., 2001), occurring when daylight length increases (Zicarelli,
1997). Furthermore, the incidence of embryonic mortality between 40th and 60th day post
AI is three times higher in buffaloes that are acyclic 70 days post partum (Zicarelli, 1994),
compared to those that are cyclic. It is known that in buffalo species high incidence of atre-
sia is present and the mean recovery of good quality oocytes per ovary is low? (Gasparrini,
2002). The maturation and the quality of oocyte depend on the function of the granulosa
cells that are sensitive to oxidative stress (Dharmarajan et al., 1999). It is worth mentioning
that the antioxidant defence system plays a key role in preventing apoptosis and atresia,
thus preserving steroidogenic function of granulosa cells (Cassano et al., 1999). Spagnuolo
et al. (2007) found no significant differences in redox status between pregnant, not pregnant
and cows with embryonic mortality.
TREATMENTS FOR PREVENTING EMBRYONIC MORTALITY IN BUFFALO
SPECIES - The importance of progesterone (P4) concentration during the first weeks of
pregnancy for reducing embryonic mortality has been demonstrated in cattle (Mann and
Lamming; 1999 and 2001). According to some reports the presence of an early P4 peak
(within 5 days after mating or AI) facilitates the elongation of the conceptus and, conse-
quently, the secretion of adequate interferon-tau (Starbuck et al., 1999; Mann, 2002). In
cattle, interferon-tau extends the lifespan of the corpus luteum (Plante et al., 1989) by
suppressing estradiol receptor and oxytocin receptor genes (Spencer and Bazer; 1996) and
by attenuating the endometrial secretion of PGF2α (Helmer et al., 1989a). It has also been
shown that interferon-tau reduces PGF2α secretion by bovine endometrial explants (Hel-
mer et al., 1989b) and endometrial epithelial cells (Danet-Desnoyers et al., 1994). Several
approaches have been used to increase P4concentration in blood in order to reduce the
occurrence of embryonic mortality. Increased plasma P4 concentrations were achieved ei-
ther by inducing increased endogenous secretion or by administering exogenous P4 (Mann
and Lamming; 1999). Studies have shown that administration of natural sequence GnRH,
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GnRH agonists or hCG after AI can stimulate corpus luteum function, induce accessory
corpus luteum formation, increase P4, and reduce estradiol production, with a consequent
positive effect on embryonic survival (Kerbler et al., 1997; Thatcher et al., 2003; Bartolome
et al., 2005). In buffalo species there are some controversial results, regarding the best
moment for hormonal treatment. Campanile et al. (2007a) reported that treatment with
exogenous P4 (PRID®, Vetem) on day 5 after A.I gave the lowest pregnancy rate and highest
incidence of embryonic mortality, suggesting that exogenous P4 can have had a detrimental
effect on conception. It is possible that exogenous P4 may contribute to the regulation of
LH and reduce the capacity of the preformed corpus luteum to increase P4 synthesis and
release. After removal of the exogenous source of P4 the corpus luteum may not be able to
secrete P4 in the amount required to maintain pregnancy. Furthermore, the injection of 12.6
µg GnRH agonist (buserelin) or 1500 I.U. of hCG on Day 5 after A.I. increased P4 concentra-
tions without reducing the incidence of embryonic mortality. It should be noted, however,
that P4 in buffaloes treated with buserelin and hCG was significantly different to control
buffaloes only on Day 15 after AI. It is therefore possible that P4 was not elevated for a
sufficient time in the period after AI to have a major effect on uterine function and embryo-
maternal interactions (Campanile et al., 2007a). The present findings are in contrast with
Kumar et al. (2003) who reported an increase in conception rate in buffaloes treated with
125 mg of 17-α hydroxyprogesterone caproate s.c. on Day 4 after AI. It is possible that the
type and mode of exogenous P4 treatment may influence the response in buffaloes. With
this regard, 341 mg of 17-α hydroxyprogesterone caproate administered i.m. 3 times, at 4-
day intervals, starting on Day 25 after AI, reduced the incidence of embryonic mortality in
a buffalo herd characterised by a high incidence of embryonic mortality (Campanile et al.,
2007b). Treatment with buserelin or hCG on Day 25 after A.I. in pregnant buffaloes also
reduced the incidence of embryonic mortality in buffaloes bred in a farm characterized by
high incidence of embryonic mortality (Campanile et al. 2007b).
In cattle, treatment with hCG on Day 5 or Day 7 after AI increases P4 concentrations by
enhancing secretion from the existing corpus luteum and also by inducing ovulation and
formation of an accessory corpus luteum (Kerbler et al., 1997; Schmitt et al., 1996; Santos et
al., 2001). In buffalo species, P4 increased on Day 10 after injection of 1.500 I.U. of hCG. It is
speculated that in buffaloes hCG may not increase the P4 secreting capacity of the existing
corpus luteum, but can induce ovulation and formation of an accessory corpus luteum which
leads to increased P4 some time later. It is known that hCG administered at Day 25 after
AI induces ovulation in around 57% of buffaloes (Campanile et al., 2007c) and that there
is a similar response to GnRH agonists, using which ovulation rates of 62% (Campanile et
al., 2007d) and 68.6% (Campanile et al., 2007c) are observed, respectively after administra-
tion on day 5 or 25 post AI. The mean follicular diameter which resulted sensitive to the
hormonal treatment was about 8.9 mm in both treatments, varying between 4.2 and 13.0
mm (Campanile et al., 2007c; Campanile et al., 2007d). It is worth pointing out that the di-
mensions of the follicles recorded in buffaloes responsive to the treatments were similar to
those of buffaloes in which ovulation did not occur. These data are in accordance with those
reported in bibliography in cattle (Martinez et al., 1999), regarding the incidence of subjects
responsive to the treatment with GnRH and the dimensions of responsive follicles. Buffa-
loes that ovulated in response to the treatment with a GnRH agonist showed a progressive
increase in milk whey progesterone concentrations on Days 10, 15 and 20, while progester-
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one levels remained relatively constant for buffaloes that did not ovulate. The injection of a
GnRH agonist on Day 5 after AI increased milk whey progesterone concentrations in 97% of
buffaloes subsequently pregnant on Day 40, compared to 68% in the non-pregnant buffaloes
(P<0.01). A greater (P<0.05) proportion of the buffaloes that ovulated (96.7%), compared to
buffalo that did not ovulate (68.4%) recorded a gestational chamber on Day 40 after AI and
were judged to be pregnant (Campanile et al., 2007d). Ovulation also increased milk whey
progesterone levels and reduced embryonic mortality in buffalo cows treated with 1500 I.U.
of hCG or 12.6 µg of GnRH agonist on Day 25 after A.I. (Campanile et al. 2007c).
CONCLUSIONS - In buffalo species embryonic mortality is one of the major causes of
fertility loss, above all in those animals that are not mated during their reproductive period.
Buffaloes that undergo oestrus synchronisation and AI during a period of increasing day
length have a relatively high occurrence of embryonic mortality. In naturally mated buf-
falo embryonic loss is about 20% and a higher incidence is reported between 28-60 days of
gestation in buffaloes that conceive during increasing daylight length. This phenomenon
is probably affected by farm, management and environment. A reduced capacity to secrete
progesterone seems to explain in part this embryonic mortality but other as yet unidenti-
fied factors contribute between 40-50% to the embryonic losses. It will be important to
further elucidate the factors that can contribute to embryonic mortality in buffaloes so that
strategies can be developed to optimise fertility after synchronisation and AI during periods
of reduced reproductive activity.
The treatments to increase P4 early in pregnancy, that have proven successful in increasing
pregnancy rates in cattle, are not necessarily applicable to buffaloes. It is however acknowl-
edged that whilst P4 tended to be elevated on Day 10 in buffaloes treated with buserelin
and hCG, concentrations were significantly different to controls only on Day 15. As noted
above, this finding was interpreted to suggest that hCG and buserelin did not stimulate
increased P4 secretion from the existing corpus luteum but rather induced ovulation and
formation of an accessory corpus luteum, at least in a proportion of buffaloes. Embryonic
mortality in buffaloes mated by AI in mid-winter appears to occur later than in cattle and
hence P4 treatments applied on day 25 after A.I. reduce embryo mortality in farms in which
embryo loss is highly present.
ACKNOWLEDGMENTS - The authors gratefully acknowledge Prof. Luigi Zicarelli, for
his support and critical reading throughout manuscript preparation.
REFERENCES - Abdoon, A.S.S., Kandil, O.M., 2001. Factors affecting number of sur-
face ovarian follicles and oocytes yield and quality in Egyptian buffaloes. Reprod Nutr Dev
41: 71-77. Bartolome, J.A., Melendez, P., Kelbert, D., Swift, K., McHale, J., Hernandez, J.,
Silvestre, F., Risco, C.A., Arteche, A.C.M., Thatcher, W.W., Archibald, L.F., 2005. Strategic
use of gonadotrophin-releasing hormone (GnRH) to increase pregnancy rate and reduce
pregnancy loss in lactating dairy cows subjected to synchronization of ovulation and timed
insemination. Theriogenology. 63: 1026–1037. Baruselli, P.S., Visintin, J.A., Barnabe, V.H.,
Barnabe, R.C., Amaral, R., Souza, A.C., 1997. Early pregnancy ultrsonography and embry-
onic mortality occurence in buffalo. Proc. V World Buffalo Congress, 776-778. Campanile,
G. , Vecchio, D., Rendina, M., Grassi, C., Balestrieri, A, Di Palo, R., 2007c. Ovary response
Ital.J.anIm.ScI. vol. 6, (Suppl. 2), 119-129, 2007126
vIII World Buffalo congreSS

Page 9

and embryonic mortality in buffaloes treated with GnRH or hCG. Proc. VIII Buffalo Con-
gress. In press. Campanile, G., Vecchio, D., Neglia, G., Di Palo, R., Prandi, A., D’Occhio,
M.J., 2007d. Progesterone and pregnancy status in buffaloes treated with a GnRH agonist.
Livestock Science. In press. Campanile, G., 1997. Relationship between nutrition and re-
production. Third course on biotechnology of reproduction in buffaloes. Suppl. Bubalus bu-
balis. II/98: 217-235. Campanile, G., 2006. Gestione riproduttiva e redditività dell’azienda
bufalina. Proc. 8° Congresso Nazionale Multisala SIVAR: 25. Campanile, G., Di Palo, R.,
Neglia, G., Vecchio, D., Gasparrini, B., Prandi, A., Galiero, G., D’Occhio, M..J., 2007a. Corpus
luteum function and embryonic mortality in buffaloes treated with a GnRH agonist, hCG
and progesterone. Theriogenology. 67: 1393-1398. Campanile, G., Neglia, G., Gasparrini,
B., Galiero, G., Prandi, A., Di Palo, R., D’Occhio, M.J., Zicarelli, L., 2005. Embryonic mortal-
ity in buffaloes synchronized and mated by AI during the seasonal decline in reproductive
function. Theriogenology. 63: 2334-2340. Campanile, G., Vecchio, D., Zicarelli, L., Neglia,
G., Di Palo, R., Balestrieri, A., D’Occhio, M.J., 2007b. Strategies to reduce embryonic mortal-
ity in buffalo cows. Proc. VIII Buffalo Congress. In press. Cassano, E., Tosto, L. Balestrieri,
M., Zicarelli, L., Abrescia, P., 1999. Antioxidant defense in the follicular fluid of water buf-
falo. Cellular Physiology and Biochemistry. 9: 106-116. Danet-Desnoyers, G., Wetzels, C.,
Thatcher, W.W., 1994. Natural and recombinant bovine interferon 7 regulate basal and oxy-
tocin-induced secretion of prostaglandins F h and & by epithelial cells and stromal cells in
the endometrium. Reprod. Fertil. Dev. 6:193-202. Dharmarajan, A.M., Hisheh, S., Singh,
B., Parkinson, S., Tilly, K.I., Tilly, J.L., 1999. Antioxidants mimic the ability of chorionic go-
nadotropin to suppress apoptosis in the rabbit corpus luteum in vitro: a novel role of super-
oxide dismutase in regulating bax expression. Endocrinology. 140: 2555-2560. Garrett, J.
E., Geisert, R.D., Zavy, M.T., Morgan, G.L., 1988. Evidence for maternal regulation of early
conceptus growth and development in beef cattle. J Reprod. Fertil. 84: 437-46. Gasparrini,
B., 2002. In vitro embryo production in buffalo species: state of the art. Theriogenology 57:
237-256. Hansen, P.J., 2002. Embryonic mortality in cattle from the embryo’s perspective.
J. Anim. Sci., 80: E33-E44. Helmer, S.D., Hansen, P.J., Thatcher, W.W., Johnson, J.W., Ba-
zer, F.W., 1989a Intrauterine infusion of highly enriched bovine trophoblast protein-1 com-
plex exerts an antiluteolytic effect to extend corpus luteum lifespan in cyclic cattle. J. Re-
prod. Fertil. 87:89-101. Helmer, S.D., Gross, T.S., Newton, G.R., Hansen, P.J., Thatcher,
W.W., 1989b. Bovine trophoblast protein-I complex alters endometrial protein and prosta-
glandin secretion and induces an intracellular inhibitor of prostaglandin synthesis in vitro.
J. Reprod. Fertil. 87: 421-430. Karaivanov, C., Vlahov, K., Petrov, M., Kacheva, D., Sto-
janova, M., Alexiev, A., Polihronov, O., Danev, A., 1978. Studies on preimplantation develop-
ment of buffalo embryos. Theriogenology. 28: 747 - 753. Kerbler, T.L., Buhr, M.M., Jordan,
L.T., Leslie, K.E., Walton, J.S., 1997. Relationship between maternal plasma progesterone
concentration and interferon-tau synthesis by the conceptus in cattle. Theriogenology. 47:
703-714. Kumar, R., Phogat, J.B., Singh, I., Kumar, V., Singh, U., Sethi, R.K., 2003. Efficacy
of post-insemination progesterone supplementation for enhancement of fertility in buffa-
loes. Bubalus bubalis. 3: 76-82. Lamming, G.E., Wathes, D.C., Flint, A.P.F., Payne, J.H.,
Stevenson, K.R., Vallet, J.L., 1995. Local action of trophoblast interferons in suppression of
the development of oxytocin and oestradiol receptors in ovine endometrium. J Reprod. Fer-
til. 105: 165-175. Madan, M.L., Chauhan, M.S., Singla, S.K., Manik, R.S., 1994. Pregnan-
cies established from water buffaloes (Bubalus bubalis) blastocysts derived from in vitro
Ital.J.anIm.ScI. vol. 6, (Suppl. 2), 119-129, 2007127
vIII World Buffalo congreSS

Page 10

matured, in vitro fertilized oocytes and co-cultured with cumulus and oviductal cells. The-
riogenology. 42: 591-600. Mann, G.E. 2002. Corpus luteum function and early embryonic
death in the bovine. XXII World Buiatrics Congress, Hannover, Germany, August 18-23:
300-306. Mann, G.E., Lamming, G.E. 1999. The influence of progesterone during early
pregnancy in cattle. Reprod Dom Anim. 34: 269–274. Mann, G.E., Lamming, G.E. 2001.
Relationship between maternal endocrine environment, early embryo development and in-
hibition of the luteolytic mechanism in cows. Reproduction. 121: 175–180. Martinez, M.F.,
Adams, G.P., Bergfelt, C., Kastelic, J.P., Mapletoft, R.J. 1999. Effect of LH or GnRH on the
dominant follicle of first follicular wave in beef heifers. Anim. Reprod. Sci. 57: 23-33. Neg-
lia, G., Gasparrini, B., Caracciolo di Brienza, V., Presicce, G.A., Zicarelli, L. 2001. Buffalo
and Bovine in vitro embryo production from ovum pick up and abattoir derived oocytes.
Proceedings of the A.S.P.A. XIV Congress: 624-626. Plante, C., Hansen, P.J., Martinod, S.,
Siegenthaler, B., Thatcher, W.W., Pollard, J.W., Leslie, M.V. 1989. Effect of intrauterine and
intramuscular administration of recombinant bovine interferon- alpha 1 on luteal lifespan
in cattle. J. Dairy Sci. 72: 1859-1865. Roberts, R.M., Cross, J.C., Leaman, D.W. 1992. Inter-
ferons as hormones of pregnancy. Endocrine Reviews. 13: 432-452. Robinson, R.S., Mann,
G.E., Wathes, D.C., Lamming, G.E. 1997. The effect of pregnancy on oxytocin and oestrogen
receptor expression in the bovine uterus. J. Reprod. Fertil. Abstr. Ser. 19: 122. Santos,
J.E.P., Thatcher, W.W., Pool, L., Overton, M.W. 2001. Effect of human chorionic gonadotropin
on luteal function and reproductive performance of high producing lactating Holstein dairy
cows. J. Anim. Sci. 79: 2881-2894. Sastry, N.S.R. ,Georgie, G.C. 1978. An appraisal of factors
contributing to a seasonal breeding tendency in Indian water buffaloes. Proc. Indo-Soviet
Symp. Buff. Breed: 137-142. Schmitt, E.J.P., Drost, M., Diaz, T., Roomes, C., Thatcher, W.W.
1996. Effect of a gonadotropin-releasing hormone agonist on follicle recruitment and preg-
nancy rate in cattle. J. Anim. Sci. 74: 154-161. Senger, P.L. 2003. Chapter 13. Early Em-
bryogenesis and Maternal Recognition of Pregnancy. In: Pathways to pregnancy and partu-
rition: 284- 303. Shafie, M.M. 1985. Physiological responses and adaptation of water
buffalo. In: Mohamed K. Yousef (Ed.). Stress Physiology in livestock, vol. II, Ungulates, CRC,
Florida: 67-80. Silke, V., Diskin, M.G., Kenny, D.A., Boland, M.P., Dillon, P., Meec, J.F.,
Sreenan, J.M. 2002. Extent, pattern and factors associated with late embryonic loss in dairy
cows. Animal Reproduction Science. 71: 1–12. Singh, G. 1988 Seasonal trend of calving and
subsequent service-period in rural buffaloes in Punjab (India). Acta Vet. Stand. Suppl 83:
80-84. Singla, SK, Manik, RS, Madan, ML. 1996. Embryo biotechnologies in buffaloes: A
review. Bubalus bubalis. 1: 53-63. Spagnuolo, M.S., Vecchio, D., De Rosa, R., Polimero, F.,
Balestrieri, A., Zicarelli, G., Ferrara, L., Campanile, G. 2007. Effect of different housing con-
ditions on several indices of blood redox status and on reproductive performance in buffalo
cows. Proc. VIII Buffalo Congress. In press. Spencer, T.E., Ing, N.H., Ott, T.L., Mayes, J.S.,
Becker, W.C., Watson, G.H., Mirando, M.A., Bazer, F.W. 1995. Intra-uterine injection of inter-
feron-tau alters oestrogen receptor and oxytocin receptor expression in the endometrium of
cyclic ewes. J. Mol. Endocrinol. 15: 203-220. Spencer, TE, Bazer, FW.1996.Ovine interferon
tau suppresses transcription of the estrogen receptor and oxytocin receptor genes in the
ovine endometrium. Endocrinology. 137: 1144-1147. Starbuck, G.R., Darwash, A.O., Mann,
G.E., Lamming, G.E. 1999. The detection and treatment of post insemination progesterone
insufficiency in dairy cows. In Diskin MG (editor), Fertility in the High-Producing Dairy
Cow. Occasional Publication Nº26, British Society of Animal Science. 2: 447-450. Thatcher,
Ital.J.anIm.ScI. vol. 6, (Suppl. 2), 119-129, 2007128
vIII World Buffalo congreSS

Page 11

W.W., Guzeloglu, A., Meikle, A., Kamimura, S., Bilby, T., Kowalski, A.A. 2003. Regulation of
embryo survival in cattle. Reproduction. 61: 253–266. Thatcher, W.W., Meyer, M.D., Danet-
Desnoyers, G. 1995. Maternal recognition of pregnancy. J. Reprod. Fertil. Suppl. 49: 15-28.
Vale, W.G., Ohashi, O.M., Sousa, J.S., Ribeiro, H.F.L., Silva, A.O.A., Nanba, S.Y. 1989. Morte
embrionária e fetal em bufalos, Bubalus bubalis Lin. Revista Brasileira de Reprodução Ani-
mal. 13: 157–165. Vasconcelos, J.L.M., Silcox, R.L., Lacerda, J.A., Pursley, J.R., Wiltbank,
M.C. 1997. Pregnancy rate, pregnancy loss and response to heat stress after AI at 2 differ-
ent times from ovulation in dairy cows. Biol. Reprod. 56: 140. Vecchio, D., Di Palo, R., Zi-
carelli, L., Grassi, C., Cammarano, A., D’Occhio, M.J., Campanile, G. 2007. Embyonic mor-
tality in buffalo naturally mated. Proc. VIII Buffalo Congress. In press. Wathes, D.C.,
Robinson, R.S., Mann, G.E., Lamming, G.E. 1998. The establishment of early pregnancy in
cows. Reprod Dom Anim. 33: 279–284. Zicarelli, L. 1994. Anaestro e induzione dell’estro in
bufale acicliche. Agricoltura Ricerca. 153: 25-40. Zicarelli, L. 1995. Management in differ-
ent environmental conditions. Buffalo J. 2: 17-38. Zicarelli, L. 1997. Reproductive season-
ality in buffalo. Proc. Third Course on Biotechnology of Reproduction in Buffaloes (Issue I):
29–52. Zicarelli, L., Campanile, G., Infascelli, F., Esposito, L., Ferrari, G. 1988b. Incidence
and fertility of heats with double ovulations in the Mediterranean buffalo cows of Italy.
Proc. II World Buffalo Congress: 56-62. Zicarelli, L., Di Palo, R., Palladino, M., Campanile,
G., Esposito, L. 1993. Embryo transfer in Mediterranean bubalus bubalis. Proc Int Symp
Prospects of Buffalo Production in the Mediterranean and the Middle East, Cairo, Egypt,
9-12 Nov: 73-75. Zicarelli, L., Infascelli, F., Esposito, L., Consalvo, F., de Franciscis, G.
1988a. Influence of climate on spontaneous and alfaprostol-induced heats in Mediterranean
buffalo cows bred in Italy.. Proc. II World Buffalo Congress: 47-56.
Ital.J.anIm.ScI. vol. 6, (Suppl. 2), 119-129, 200712?
vIII World Buffalo congreSS