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Artificial Insemination in Dogs
Rita Payan-Carreira
1
, Sónia Miranda
2
and Wojciech Niżański
3
1
CECAV – Univ. of Trás-os-Montes and Alto Douro,
2
Escola Universitária Vasco da Gama,
3
Univ. Environmental and Life Sciences, Wrocław,
1,2
Portugal
3
Poland
1. Introduction
In Artificial Insemination (AI) the semen is collected manually from a stud male and
thereafter deposited (inseminated) in the female so that fertilization can occur in the absence
of natural mating. Artificial Insemination, one of the earliest techniques for assisted
reproduction in animals and humans, took longer to be implemented in dogs due to species-
specific particularities. In past decades, progresses in the knowledge of canine physiology
and new advances in canine semen technology allowed these services to become available
worldwide. Hence, subsequent to the increase in the artificial insemination demand among
dog breeders and owners and the broaden of the AI to preserved semen as a management
tool in canine breeding, as through international exchange of frozen semen, inbreeding
within breeds can be reduced. Therefore, with spread of canine AI dog, breeders now may
select stud dogs from all over the world to improve their kennel´ genetics, without
transport-associated stress to the animals. Also, it is possible to save semen from valuable
dogs into sperm bank to be used in next generations, after their death or the peak of
reproductive age. In addition, breeders also are aware of the sanitary benefits associated
with AI. Avoiding direct contact between the male and female, AI also prevents the spread
of sexually transmitted diseases, as those originated by Brucella canis or Herpes virus
(Farstad, 2010; Linde Forsberg, 2005a).
Although the first reports on AI in dogs subsequent to the Spallanzani experiments (in late
XVIII century) appeared by the end of the fifties, reporting the use of fresh semen, or in the
sixties, the use of frozen semen, only in the nineties this technique was introduced into dog
breeding practice, particularly in USA and Nordic countries (Foote, 2002; England & Millar,
2008). The reproductive physiology of this species and unfavourable response of the dog
sperm to freezing were the two major constraints to the initial efforts to improve the AI
technique in dogs (Linde Forsberg, 2005a). A lot of research was performed in those areas,
especially in the northern Europe, to overcome these issues, generating a large amount of
information and allowing technical development, in particular in the canine semen
technology. Nowadays, as a consequence of the demand for reproductive technologies, in
particular the AI with fresh or refrigerate semen, this is a current service offered in the small
animal veterinary practice.
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According to Linde-Forsberg (2001, 2005a), from all the AI in dogs performed by
veterinarians today in Europe, about 50-55% is done with fresh semen, collected at the clinic,
10% with chilled semen and around 35-40% with frozen semen. However, at least in
Portugal, the use of imported chilled semen is far most frequent than the use of frozen
semen when compared to other countries in Northern Europe.
Research on AI in the domestic dog, along with other reproductive technologies, proceed
worldwide, particularly on sperm survival at freezing and the identification of deleterious
components to spermatozoa or fertilization, providing important information for the
preservation of wild canidae semen that are currently threatened or endangered.
2. Indications for artificial insemination
Several main indications exist to perform AI in the dog (Linde Forsberg, 2005a; England &
Millar, 2008; Farstad, 2010). In parallel, some ethical conditions must be discussed when
facing the different interests of specific groups, namely dogs, breeders, owners and
veterinarians.
Main indications for AI in dogs include both medical and breeding-management reasons
(Table 1). As major potential advantage, AI may allow to reduce physical distances, the use
of genetically valuable stud dog semen all over the word, fighting the stress of
transportation of animals and inbreeding (Johnston et al., 2001; Linde-Forsberg, 2005a). It is
also an important technique whenever physical and behavioural abnormalities in the male
or female preventing natural mating (Table 2).
3. The ethics and role of artificial insemination in canine breeding programs
Performing canine AI may raise some ethical concerns, mostly to central institutions like the
National Kennel Clubs or Veterinarian Orders or equivalent, in particular on what concerns
the use of frozen semen and the need for intra-uterine insemination, mainly those involving
surgical procedures. In fact, several countries (such as Norway, Sweden and the United
Kingdom) refer to welfare concerns and discourage or even forbid the use of surgical
procedures to obtain intra-uterine insemination (England & Millar, 2008; Linde-Forsberg,
2005b).
Ethical issues are seldom associated with the non-surgical process of artificial insemination
per se. Most procedures used for semen deposition are neither detrimental to the bitch, nor
interfere with animal welfare, and even allow protection against certain diseases. However,
some attention may be given to the inbreeding of animals that may compromise health of
following generations (England & Millar, 2008).
Restrictions to the use of AI in animals that never matted despite all physiological
conditions met together to guarantee a successful mate, may respond to the ethical issue
that demands for ruling out clinical reasons for AI, as an underlying unaware problem
(congenital or behavioural) may exists. This concern is in fact previewed in the Fédération
Cynologique International (FCI) breeding rules (http://www.fci.be/circulaires/102-2010-
annex-fr.pdf). According to those rules, AI should not be performed in animals not having
at least one previous litter registered from natural service. Furthermore, AI to be a
recognisable breeding technique must be performed by veterinarian or a specifically
recognisable technician, which skills will avoid complications or adverse effects, as well as
stress or risks of welfare infringements towards the animals, in particular the female.
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53
Potential benefits Potential weakness
- Decrease stress, infectious deseases
transmission, travel expenses
- Semen collection without interruption of the
male activity (show or training)
- Splitting of an ejaculate to bred more females
- Reduction of the costs with maintenance of stud
dogs in a colony
- Worldwide availability of the semen of a given
dog
- Allow early castration of working dogs while
maintaining availability of their genes
- Evaluation of semen quality prior to AI
- Early detection of male reproductive
pathologies
- Semen preservation, so genetic material may be
available in the future
- Overcome problems associated with the refusal
to breed (psychological or physical reasons,
precocious ejaculation), inexperienced males
- May overcome quarantine restrictions
- Induction of physical or psychological
trauma during the AI process
- Risk of performin
g
AI for inappropriate
reasons
- Failure in careful clinical examination
of the breeding animals
- Potential risk for maintaining some
disorders in a particular genetic line
(hip dysplasia or anatomical
abnormality of the reproductive tract)
- Potential risk for introduction of
inherited diseases or abnormalities
- Potential overuse of a given male
within a programme or breed
- May allow confusion of parentage
Table 1. Main advantages and inconveniences for canine AI.
Factor Cause
Female-dependent
- Male rejection
- Aggression
- Congenital abnormalities (e.g. presence of a vaginal septum;
genital tract strictures, small vulva and vagina)
Male-dependent
- Reduced libido (due to local or s
y
stemic diseases, a
g
e, deficient
breeding management, drugs)
- Pain
- Physical deficiency (such as inability to mount or to obtain
penile erection, lumbar muscle problems, congenital
abnormalities)
Associated to both
male and female
- Inexperience
- Male to female disproportion
- Social and behavioural problems (dominant female, inversion of
the social hierarch
y
)
Table 2. Main causes for refusal of natural mating.
The competence of the operator to perform the procedures is essential to avoid all
technique-related ethical constraints to the use of AI in dogs. Before offering canine AI
services, practitioners ought to specialised themselves, acquiring profound knowledge of
the reproductive physiology and pathology of the species and the skills to collect semen and
to inseminate the female without risking animal health or welfare.
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54
4. Semen collection and evaluation
4.1 Semen collection in the dog
Semen collection in the dog is a relatively easy procedure, although requiring some training
for optimization of the technique. Semen collection and evaluation is necessary to obtain
good results in canine AI. Although practitioners are often asked to collect semen and
perform AI without detailed semen analysis, every sample of semen collected should be
evaluated (at least progressive forward motility, total sperm count and morphology) before
it is used for artificial insemination or cryopreservation. Semen evaluation prior to
insemination warrants the male potential fertility and consequently may predict the fertility
potential for the AI. In addition, when preparing semen preservation, fertility certificate
may be needed. In such cases andrological evaluation of the stud dog (breeding soundness
evaluation or BSE) has to be performed. Semen collection should be performed before the
physical exam or any stressful procedures on the stud, or can be booked to another day
(Freshman, 2002; Johnston et al, 2001).
Semen can be collected from most dogs in the absence of a teaser, in a quiet and isolated
room, where interruptions should be prevented, although the presence of a bitch would
allow better ejaculates. In reluctant males, stimulating estrus scent can be provided by the
presence of a female in estrus or by using frozen-thawed swabs or gauze sponges taken
from vaginal secretions of estrus bitches (Freshman, 2002; Kutzler, 2005; Olson & Husted,
1986). Although possible, not everyone achieves the use of a chemical pheromone (methyl p-
hydroxybenzoate, Aldrich Chemical, Milwaukee, WI) swabbed on the perineal area and tail
of an anestrus teaser (Johnston et al., 2001; Kutzler, 2005).
Collection of semen should be prepared in advance, and interval between collections or
between the natural mating and collection, should be registered, if the male is regularly used.
Ideal intervals between collections are 2 to 5 days, whilst intervals longer than 10 days may
result in an increased number of morphological abnormalities and decreased motility
(Freshman, 2002; Johnston et al., 2001). In longer periods, it is advisable to perform one
previous collection, if semen is to be chilled or frozen for shipment. If semen preservation is
planned, semen extender should be prepared before the arrival of the animal (Freshman, 2002).
The most common method for semen collection in the dog is by digital manipulation, in the
presence of a female. However, bitch presence, although desirable as it facilitates
procedures, is not essential to accomplish the collection (Farstad, 2010; Linde Forsberg,
2005a). It should be noticed that when the collection is achieved in the presence of the bitch
ejaculates present higher concentration.
The use of manual massage is the most commonly used technique (Farstad, 2010; Johnston
et al., 2001; Linde Forsberg, 2005a), although in the past semen was collected from dogs
using an artificial vagina. Nowadays, semen collection into a tube is commonly
accomplished by penile massage and the use of a cone or plastic sleeve, a funnel or a special
collecting vial (Linde Forsberg, 2005a). Briefly, the process is started with a massage of the
dog prepuce at the level of the bulbus glandis until developing partial erection, followed by
the quick retraction of the prepuce and penile expose. If the collector is right-handed, semen
must be collected from the dog’s left side, with the operator holding the dog’s penis with the
right hand and the collection container in the left hand. During pelvic thrusting, rigid vials
should be kept at a distance from the penis, to avoid trauma. When pelvic movements are
finished and the dog lifts its rear leg, a 180º backward rotation of the penis should be
obtained and the erectile penis should then be directed into the collection cone or the funnel.
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55
Some pressure may be applied with the thumb on the apex of the glans penis, at the level of
the urethral process, to stimulate ejaculation. When a crystal clear fluid (prostatic fluid)
begins to flow into the collection tube, you can gently slide the collection cone off the penis.
Watch for semen to flow in the collection tube (Farstad, 2010; Linde Forsberg, 2005a).
Canine ejaculate consists of 3 fractions, with the first and third fraction consisting of
prostatic fluid and the second being rich in spermatozoa (England et al., 1990) (Table 3). The
first fraction, the presperm portion, is emitted in 0.5 to 1 minute and is colourless, with a
volume range of 1-5 ml. It is expelled during first stage of erection, at the moment of the
presence of evident copulatory movement of male. The second fraction, the sperm-rich
portion, is also rapidly completed (1-2 minutes), and is grayish-white in colour, with a
volume of 1-3 ml. It is expelled when thrusting movement of the male ceases and full
erection is observed. The third fraction comes from the prostate and may be up to 30-40 ml;
it may take up from 5 to 30 minutes to be completed (Günzel-Apel, 1994; Johnston et al.,
2001).
Characteristics 1
st
fraction 2
nd
Fraction 3
rd
Fraction
Volume
0.1-2 mL
(average 0.33 mL)
0.1-3 mL
(average 1.17 mL)
Sometimes larger volume
1-2 to >20 mL
Quite variable dependin
g
on the animal.
Colour
clear or opaque
greyish-white, white,
milky-white
clear, transparent
Consistency
watery watery-milky, milky watery
Character
prostate
secretion with
admixture of
epithelial cells,
urine, bacteria and
sperm cells
sperm cells suspended in
seminal plasma
prostate gland secretion
pH (average) 6.37 6.10 7.20
Duration
5-90 sec.
(average 13.5 sec)
5-300 sec.
(average 52.4 sec.)
60 sec-20 min.
(average 6 min. 55 sec.)
Table 3. Main characteristics of the different fractions of the dog ejaculate.
Size of the dog Volume of the ejaculate
< 20 kg
1-22.5 mL
(average 5.38 mL)
> 20 kg
2-45 mL
(average 12.75 mL)
Table 4. Variation on the volume of the ejaculate with the size of the dog (Dubiel, 2004)
In the dog, the volume of whole ejaculate varies between breeds (Table 4) mainly with
animal size and is partially dependent on the volume of the third fraction collected, which
constitute about 95% of the volume of the ejaculate in dogs (Farstad, 2010).
In most dogs, semen can be collected twice at 30 minutes interval (Farstad, 2010), although
the second sample is usually slightly diluted.
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56
Most often, artificial insemination with freshly collected semen is performed without
fractioning the ejaculate, although for artificial insemination, only the second fraction is of
interest (Thomassen & Farstadt 2009; Root Kustritz, 2003). Furthermore, it has been
demonstrated the existence of detrimental effects on fertility when this fraction is not
separated from the second one, particularly if semen will be processed as chilled or frozen.
Consequently, ejaculate fractioning should always be accomplished, particularly separation
of the third fraction. If the ejaculate has a very small volume, it may be diluted with semen
extender, to facilitate its handling during insemination procedures.
4.2 Semen assessment
Semen assessment is an important part of the evaluation of fertility in males and it should be
performed as routine element of prebreeding examination. Furthermore, semen evaluation
ought to be completed before artificial insemination or sperm preservation. Semen should
be assessed immediately after collection and it has to be handled carefully during all the
procedures. Rapid changes of environmental temperature may be deleterious for
spermatozoal motility and structure, and may also artifactually influence the results of
examination. Any delay in semen assessment may decrease the percentage of motile sperm
and simultaneously increase the percentage of dead sperm. It is advisable to keep all
equipment necessary for semen collection and evaluation at the temperature near 37ºC
(Christiansen, 1984; Feldman & Nelson, 1996; Linde-Forsberg, 1991).
On table 5, the most frequent indications for routine semen evaluation are presented. Semen
evaluation is also frequently performed in the absence of known reproductive pathology,
upon request of the owner. In addition, it can be performed at a predetermined moment
after the diagnosis of a clinical disease that may have negative reflects on the potential
fertility of a male dog.
It should be notice that reliable in vitro estimation of the real fertilizing ability of sperm cells
is not always possible. Usually, in males with aspermic (no ejaculate), azoospermic (no
spermatozoa), or necrospermic (no motile spermatozoa) semen, the fertilizing potential may
be excluded. When the quality of semen in a dog with history of unsuccessful matings is
low, premises exist to exclude such male from the breeding programme. However, it should
always be remembered that the semen characteristics should be recheck 2-3 times at 1-2
weeks intervals, to confirm the male infertility. On the other hand, good in vitro semen
quality does not always prove the fertilizing potential of a particular dog.
Most frequent indications Other situations, on request
- Semen evaluation before artificial
insemination
- Semen evaluation before/after chilling or
cryopreservation
- Clinical signs of a disorder of male genital
organs
- Whenever infertility or subfertility of a
male is suspected
- New stud dog introduced to the breeding
colony
- Evaluation of young stud dog before first
mating
- In cases of serial unsuccessful matings of
particular dog
- Pathological lesions of male genital tract
observed by the owner
- Re-evaluation after the treatment of
diseases of male genital tract
Table 5. Common reasons for canine semen assessment
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57
The semen assessment performed once is not always reliable, because:
Frequent matings or semen collections may temporary result in a decreased semen
quality;
After a prolonged sexual rest dogs may ejaculate many dead, immotile spermatozoa of
abnormal morphology;
In young inexperienced males and dogs which mated earlier only naturally, without
experience on semen collection, the obtained semen sample may contain only the part
of sperm-rich fraction.
4.2.1 Conventional assessment of semen
Different approaches are available to assess the quality of the dog semen that can be
grouped in conventional and advanced techniques. The later, usually requires more
sophisticated means for the semen assessment and the support of a technical equipment,
while the former may be performed in an inhouse lab.
The conventional approaches to semen evaluation include macroscopical evaluation of the
semen (volume and colour), but also the microscopical assessment, which will give the
concentration and the number of viable cells in the ejaculate.
4.2.1.1 Macroscopic evaluation
Volume. The volume of the ejaculate may be assessed in the calibrated tubes used for semen
collection. It mainly dependends on the size of the dog, the size of the prostate gland, the
animal age, the frequency of semen collection, the level of erotisation, and the volume of 3
rd
fraction collected. A decrease of semen volume is observed in cases of benign prostatic
hyperplasia, prostatic cysts, inflammatory lesions of prostate and testicles, inflammation of
epididymis, vas deferens or urethra and at weak libido.
Colour. The colour of whole ejaculate depends on the volume of third fraction of ejaculate
collected, on the concentration of spermatozoa per mL and the potential presence of non-
germ cells in the ejaculate. When analysing the colour, one should be aware of the method
of collection, as colour varies with the fraction to be analysed and the fact that analysis may
been performed on the whole semen or on fractioned semen. The normal colour of whole
ejaculate is greyish-white. Pathological colours include: green-greyish typical for the
presence of the pus in semen; red or pink-specific for erythrocytes contamination
(haemorrhages from urethra or corpora cavernosa, prostatitis); yellow specific for urine
contamination; and brown, if in the presence of blood.
Any kind of semen contamination, such as hair or mud, exclude the specimen from further
procedures including artificial insemination or semen preservation. It is therefore important
to check the region of praeputial opening before semen collection and to clean it.
The presence of sediment consisting of sperm cells at the bottom of the tube is a normal
feature if the semen is left for several minutes.
4.2.1.2 Microscopic evaluation
Motility. One of the most important step of conventional semen assessment is the subjective
evaluation of progressively motile spermatozoa (Spz) under contrast-phase microscope. The
optimal temperature for assessment of dog sperm cell motility is 39ºC. A small drop of
about 20 µL of semen is placed on in a pre-warmed slide and cover by the coverslip. The
evaluation is performed under the objective of x20 to x40. If the highly concentrated sperm-
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58
rich fraction is collected separately, the semen should be extended with saline or Tris-buffer
to a concentration allowing the observation of particular, single sperm cells. The assessment
is based on the evaluation of the average percentage of progressively motile spermatozoa in
a few different fields of the specimen. The normal dog semen contains at least 70% of
progressively motile spermatozoa (Feldman & Nelson, 1996; Günzel-Apel, 1994).
A decrease in the percentage of motile spermatozoa may results from temperature shock,
contamination with water, urine, blood or lubricants but also from long sexual abstinence
and systemic or infectious diseases, such as brucellosis. Sperm agglutination is always
pathological and is frequently found in cases of infectious diseases.
Concentration and total sperm count. The sperm concentration in whole canine normal
ejaculate usually exceeds 80 x10
6
Spz/mL. If the second fraction of ejaculate is collected
separately, the sperm cells concentration in sperm-rich fraction varies usually between 200-
600 x 10
6
Spz/mL. It is generally assumed that the number of motile spermatozoa necessary
for successful AI should be >150 x10
6
(Linde-Forsberg, 1991). Therefore, under normal
conditions, the dog´s ejaculate contains far more sperm cells than those needed for a seminal
dose, although sometimes, especially in miniature or toy breeds, ejaculate volume and the
total number of sperm cells are relatively low (<100 x10
6
Spz/mL). The concentration of
spermatozoa in semen volume is usually assessed by cytometric method on the
haemocytometer, such as the Thoma, Thoma-Neu, Bürker or Neubauer chambers, with
semen pre-diluted at 1:200. In order to find the sperm count per mL, the number of
spermatozoa in the one or four large squares (depending of the chamber) is multiplied by
500 000. For the assessment of sperm concentration more sophisticated equipment could
also be used, such as the spectrophotometer, flow cytometer or computer assisted semen
analyser (Rijsselaere et al., 2005).
A large variety in the total number of spermatozoa per ejaculate is observed in different
breeds. It varies between 50 x10
6
up to 1575 x 10
6
Spz (Linde-Forsberg, 1991; Oettle 1993).
Small breeds do not produce as many spermatozoa as large breeds, as sperm cell production
is related to the weight of the testicular tissue. The number of spermatozoa per ejaculate also
varies according to age, testicular weight, sexual activity and the size of the dog (Amann,
1986). The total number of spermatozoa in the ejaculate may be decreased in young and
older dogs and in inbred males. Apprehension, absence of the teaser bitch, painful prostate,
spine rear limbs may also negatively influence the number of spermatozoa ejaculated.
Sperm morphology. The percentage of morphologically normal spermatozoa in canine
semen should be greater than 70% (Günzel-Apel, 1994). The morphology may be assessed
under contrast-phase microscope, but usually the evaluation is performed under light
microscope on stained slides. Smears of undiluted or diluted ejaculate are examined
microscopically for the presence of structural abnormalities of spermatozoa. The stains used
include modified Giemsa stain (DiffQuik) and Spermac® stain. The semen is smeared on a
glass slide in a similar manner to that of blood, air dried and stained. The semen may be also
stained with a nigrosin-eosin stain. A drop of this stain is gently mixed with a drop of semen
on a pre-warmed slide before being smeared, and allowed to air dry. Evaluation of sperm
morphology should be completed microscopically using oil immersion, using an objective of
x100 or x 125. A minimum number of 200 spermatozoa should be counted and evaluated for
the presence of abnormalities. The percentage of cells with particular morphological defects
and of normal cells are calculated. Traditionally sperm cells abnormalities are divided into
primary defects - originating from abnormalities of spermatogenesis and secondary defects -
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59
originating from abnormalities of semen maturation, transit through the ductal system and
specimen preparation. According to another classification sperm abnormalities may be
divided into major defects, negatively correlated with fertility, and minor defects,
unassociated with fertility (Table 6) (Oettle, 1993).
Primary spermatozoa defects Secondary spermatozoa defects
Head
Macrocephalus, microcephalus,
double, pointed, indented heads
Free, bent heads, swollen acrosomes,
detaching acrosomes
Neck
Thickened, eccentric insertion
Midpiece
Thickened, thinned, coiled,
kinked, double midpiece
Bent midpiece, extraneous material
surrounding midpiece, proximal, mid
and distal cytoplasmatic droplets
Tail
Thin, double, triple tail.
Coiled, looped, kinked, folded,
detached tail.
Table 6. Main defects of the dog spermatozoa
The acrosomal status, which is frequently assessed for the estimation of the quality of the
frozen-thawed semen, may be evaluated with the use of eosin-nigrosin, Giemsa, Trypan
blue, Bismarck brown, Rose Bengal or Spermac® stainings (Dahlbom et al. ,1997; Dott &
Foster, 1972; Watson, 1975). When a spermatozoon presents more than one abnormality, it
should be classified according to the most important abnormality or with the most prevalent
one, if they have equal significance (Oettle, 1993).
‘Live-dead’ spermatozoa. The assessment of the percentage of live and dead spermatozoa is
based on the assumption that dead spermatozoa possess disintegrated plasma membrane
allowing eosin penetration. Thus the percentage of eosin positive cells stained with nigrosin-
eosin stain is considered as percentage of dead cells. The normal dog semen consists of
maximal percentage of 30% of dead sperm cells. The evaluation of the percentage of live and
dead spermatozoa and the percentage of morphological defects may be performed on the
same nigrosin-eosin stained slides.
4.2.2 Advanced semen assessment
In the past 2 to 3 decades, several strategies were developed to escape the subjectivity in the
semen evaluation, related to the experience and skills of the observer, the method of
specimen preparation, staining technique and number of cells evaluated, and wich is
particularly important when the fertility potential of preserved sperm cells has to be
ascertain. It is well documented that variations in results of the conventional evaluation of
the same semen samples obtained by different observers and laboratories may reach 30-60%
(Coetzee et al., 1999; Davis & Katz, 1992). Moreover, implementation of such methodologies,
not routinely usable in the small to median veterinary clinics due to their costs, allows
accurate comparisons between laboratories worlwide and minimizes occurence of large
errors. Furthermore, advanced semen assessment is essential whenever the semen has to be
preserved, in particular for freezing. Advanced semen assessment techniques are sumarized
on table 7. In general, the results obtained with these methods are better correlated with the
AI outcome than the results of traditional semen evaluation.
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60
Tests Aims Procedure and Analysis References
Hypo-osmotic
swelling test.
Spz membrane
integrity
(indirect
method)
Sperm incubation with hypo-osmotic
solutions for 30 minutes at 37ºC
Spz with intact plasmalemma become
swolled and show coiled tails
England &
Plummer, 1993;
Kumi-Diaka, 1993
Computer
assisted sperm
analysis
[CASA]
Objective
evaluation of
sperm cell
motility
Determination of motility parameters
for individual spz
Characterization of Spz movement
according to the average velocity, the
trajectory, the amplitude of movement
and beat cross frequency. It allows
identification of Spz subpopulations
Verstegen et al.,
2001;
Rijsselaere et al.,
2003;
Niżański et al.,
2009
Zona
pellucida
binding assay
Assessment of
sperm
fertilizing
potential
- ZP-binding assay ( ZBA) using intact
homologous oocytes
- hemizona bindin
g
assa
y
(HZA) usin
g
bisected hemizonae
The number of spermatozoa bound to
ZP is counted with contrast-phase
microscopy. The number of bound Spz
reflects its fertilizing potential
Hermansson et
al., 2006;
Kawakami et al.,
1998;
Rijsselaere et al.,
2005;
Ström-Holst et al.,
2001
Fluorescent
probes and
flow
cytometry
Membrane
integrity and
evaluation of
live and dead
cells
Combined use of several fluorescent
dyes (i.e, propidium iodide PI and
carboxyfluorescein diacetate, SYBR-
14/PI) allow the identification of live
cells
Live cells activate fluorescence
(deacylation) which is maintained
intracellularly in intact membrane
cells. Dead Spz are stained red due to
the influx of PI through damaged
plasma membrane.
Hewitt &
England, 1998;
Peña et al., 1998;
Rijsselaere et al,
2005;
P.F. Silva &
Gadella, 2006
Capacitation
status
Fluorescent antibiotic
chlorotetracycline (CTC), when bound
to free calcium ions, is fluorescent.
Combined with Hoechst 33258 allows
also assessment of percentage of live
cells and capacitation status
Three classes of sperm cells may be
assessed: uncapacitated and acrosome
intact (F-pattern), capacitated and
acrosome intact (B-pattern) and
capacitated and acrosome reacted (AR-
pattern)
Guérin et al.,
1999;
Hewitt &
England, 1998;
Petrunkina et al.,
2004;
Rota et al. 1999b
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61
Tests Aims Procedure and Analysis References
Acrosomal
status
Lectins conjugated with fluorescein
isothiocyanate, such as Peanut
Agglutinin (FITC-PNA) or Pisum
Sativum Agglutinin (FITC-PSA). PNA
labelling is specific for the outer
acrosomal membrane whereas PSA is
labelling acrosomal matrix.
The absence of the fluorescence of the
living sperm indicates an intact
acrosome, whereas the presence of the
fluorescence is indicative for acrosome
disruption or acrosome reaction
Kawakami et al.,
2002;
Peña et al., 2001;
Sirivaidyapong et
al., 2000;
P.F. Silva &
Gadella, 2006
Mitochondria
Rhodamine 123 (R123) is a
potentiometric membrane dye used for
the selective staining of functional
mitochondria.
It fluoresces only when the proton
gradient over the inner mitochondrial
membrane (IMM) is built up
Garner et al.,
1997;
Gravance et al.,
2001
Integrity of
DNA structure
- Sperm chromatin structure assay
(SCSA) with acridine orange (AO).
- Terminal deoxynucleotidyl
transferase-mediated nick end labeling
(TUNEL)
The SCSA is a flow cytometric method
for identification of changes in the
DNA status. AO shows green
fluorescence when DNA is intact and
red fluorescence when DNA is
denaturated
Chohan et al.,
2006;
Bochenek et al.,
2001;
Garcia-Macis et
al, 2006
Table 7. Concise description of the available advanced methods for sperm quality
assessment.
5. Success rates for artificial insemination
The key-issues to obtain good results by using canine artificial insemination are:
Proper timing of the insemination
The use of adequate number of viable sperm cells per dose
Good semen preparation and handling
Adequate deposition of semen in the female reproductive tract
On next sections the major issues on timing the AI and available techniques of semen
deposition on the bitch genital tract will be discussed.
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Artificial Insemination in Farm Animals
62
5.1 Timing the moment for insemination
Obtaining successful pregnancies and adequate number of offspring per litter depends upon
the correct timing for mating, as well as for insemination, particularly because bitches are
mono-estrous, presenting usually one to two reproductive cycles per year. Although
relationship among behavioral, hormonal and physiological events for the average bitch
exists, considerable individual variation are also currently found on what concerns the
duration of the estrogenic and early luteal stages (proestrus and estrus) and of the anestrus
(Concannon et al., 1977; Concannon, 2004). The bitch usually presents a relatively long
follicular phase and considerable variability exists in the onset of estrous behavior and
acceptance of the male, making it difficult to determine occurrence of the LH surge and
onset of ovulation in this species unless specific methods for timing the ovulation and
estimating the fertile period are used (Linde-Forsberg, 1991). Furthermore, in this species,
ovulation of immature oocytes (primary oocytes, before extrusion of the first polar body)
determines the need for a maturation period in distal oviducts that may last for 96-108 hours
(Concannon, 2004, 2010; Tsuitsui, 1989; Tsutsui et al., 2009); for most bitches, the secondary
oocytes present a life span of 24-48h (Tsuitsui, 1989). Those particularities in the
reproductive physiology may explain why the major cause for infertility in the bitch is the
inappropriate breeding management (Goodman, 2001; Linde-Forsberg, 1991). Consequently,
careful planning of mating time by timing ovulation is a key step in canine artificial
insemination.
5.2 Vaginal cytology and progesterone blood levels
Determination of blood progesterone and the vaginal cell cornification on cytological
specimens are the most widely used techniques (Linde Forsberg, 2003), to which recently
has been added the vaginal endoscopy (that replaces the vaginoscopic exam) and the
ultrasonographic follow-up of the follicular development and ovulation (England &
Concannon, 2002; Hewitt & England, 2000; Fontbonne & Malandain, 2006; Levy &
Fontbonne, 2007). These evaluations should be performed in sequence and with 2-3 days
intervals for the majority of females (if the bitch has been reported to present short heat
period, of about 6 to 9 days, is possible that daily evaluations may be needed).
On the vaginal cytology, epithelial cells of the vagina change their form in response to
estrogen impregnation, and passes from small round cells with a clearly visible cytoplasm in
non-estrogenic stages, to larger, cornified, angular shaped-cells with small pyknotic nucleus,
almost to the point of disappearing, under the influence of estrogens (Figure 1). At
beginning of estrus, vaginal cytology presents its maximum cornification index (>70%). By
that time, serial blood sampling for progesterone determination should start to detect the
initial progesterone rise (2-3 ng/mL) which correlates with LH surge, which in turn triggers
ovulation within 2 days. On the day of ovulation (day “0” of the cycle) progesterone
concentrations may vary between 4 and 10 ng/mL. The sudden increase in the number of
round-shaped cells and of neutrophils reflects the onset of diestrus (Fontbonne &
Malandain, 2006).
Progesterone semi-quantitative immunoenzymatic assays are available for clinical routines,
but although rapids, these test lack accuracy. They give progesterone concentration
according to a colorimetric scale for values corresponding to basal progesterone levels (0-1
ng/mL), intermediate levels corresponding to the LH surge (around 1-2.5ng/mL) and the
ovulation periods (2.5 – 8ng/mL), and high progesterone levels (more than 8 or 10,
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Artificial Insemination in Dogs
63
depending on the kit). A recent study showed that, in dogs, semi-quantitative methods for
progesterone determination are less accurate than the quantitative methods, in particular at
intermediate plasma progesterone concentrations (Moxon et al., 2010). According to this
study, the tested semi-quantitative assay estimated higher progesterone concentration than
RIA (radioimmunoassay), which could suggest that the fertilization period had commenced
earlier than it was actually the case. In addition to those assays, quantitative radio or
chemiluminescent assays can also be used, even if not always available in the house lab,
since cross-reactivity exist to the molecule between different species, for example with
human progesterone.
Fig. 1. Schematic representation of the major morphological changes of the predominant
epithelial cell in the vaginal cytology during the dog estrous cycle. On the bottom, images of
the vaginal péri-estrus cytological preparations stained with Harris-Shorr.
5.2.1 Ultrasound examination
Although ovarian ultrasound examination is a reliable and accurate method to determine
ovulation in most domestic females, in the bitch fat accumulation in ovarian bursa that
encloses the ovaries may difficult the value of the technique. In addition, several studies
demonstrated that ultrasound images of the ovaries around ovulation are more difficult to
analyze, due to the fact that ovarian follicles do not differ much in the immediate pre- and
post-ovulatory period (England & Concannon, 2002), as not all dog follicles collapse at
ovulation (Yeager & Concannon, 1996) and also because non-ovulated follicles frequently
remain in the ovary (Wallace et al. 1992).
Consequently, follicular dynamics evaluation through ultrasonography (US) in dogs is still
experimental and must follow a very precise protocol, which accuracy increase with the use
of frequent examinations. In a recent study, Fontbonne (2008) reported that US was accurate
enough to detect the occurrence of ovulation and obtain comparable numbers of ovarian
structures between US examinations and macroscopic visual count on the surface of the
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Artificial Insemination in Farm Animals
64
ovaries after surgical removal, even if only one daily examination was performed. However,
that author accepts that features of ovulation may be difficult to visualize in large breeds
and in overweight animals. Pre-ovulatory follicles may present different aspects at US.
Usually they appear as round to slightly triangular anechoic structures, sometimes slightly
flattened, giving a honeycomb aspect to the ovary (Figure 2). At ovulation, different degrees
of follicular collapse can be found in the US images, and usually a clear change of the
ovarian echogenicity has been detected in a large number of bitches, giving the ovary a
more homogeneous aspect (Fontbonne, 2008; Fontbonne & Malandain, 2006). Persistence of
non-ovulatory follicular structures was perceived in US images after ovulation. Also, in the
immediate post-ovulation period, until 24 hours after US changes of the ovaries at
ovulation, hypoechoic structures were observed in most cases (Figure 2). These structures
were very similar to the pre-ovulatory follicles, although slightly smaller, and tending to
increase in echogenicity (from the border to the interior of the structure) with time
(Fontbonne, 2008; Fontbonne & Malandain, 2006).
Fig. 2. Ultrasonographic scans of canine ovaries before and after LH surge and ovulation. US
are compared with images of longitudinal sections of canine ovaries of similar stages of
follicle developement.
5.2.2 Vaginal endoscopy
It is possible to use vaginal endoscopy to determine the fertile period although it does not
allow accurate timing of ovulation. However, this method requires expensive equipment.
Nevertheless, it may give a huge contribution to the vaginal evaluation and detection of
anatomical abnormalities that may impair proper reproductive performance.
The fluctuation of estrogen and progesterone concentrations in the blood at consecutive
stages of estrous cycle in the bitch results in specific morphologic changes of the vaginal
mucosa. Analysis of these changes allows for exact assessment of the stage of the estrous
cycle and for determination of the optimal insemination time (Goodman, 2001; Jeffcoate &
Lindsay, 1989; Lindsay 1983). The observation of the cranial part of the vagina is performed
for this purpose. The deep introduction of the tip of endoscope into the narrow part of the
vagina close to the cervix (dorsal median postcervical fold) or paracervix, is of less
diagnostic value (Pineda et al., 1973).
Vaginoscopic examination is performed using a rigid endoscope 3-4 mm in diameter, with
diagnostic sheath and a length of 30-33 cm or longer. The examination should be done on
the standing animal. Usually there is no need of administration of sedatives. The tip of the
endosope is introduced at the beginning at angle of 45-60º cranially and dorsally. When the
tip of the optics reaches the vagina it should be repositioned at horizontal axis.
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Artificial Insemination in Dogs
65
During proestrus the increase of the estrogen concentration results in the oedema of the
vaginal mucosa. Vaginoscopy reveals rounded folds in the vagina. The mucosa of the folds
is turgid, pink in colour and with a smooth surface. Normally the bloody discharge is also
visible in the vagina. Sometimes, periodic blood outflow from the cervix, through the
paracervix may be observed. The lumen of the vagina is narrow, which can be appreciated
when the endoscope is advanced cranially. At the last days of proestrus and at beginning of
estrus, the decrease of estrogen concentration and increase of progesterone (P
4
) level is
noted. It results in the collapse of vaginal folds. Formerly turgid and smooth, the mucosa,
becomes wrinkled and shrunked. Vaginal folds become smaller. Maximal intensity of
shrinkage of vaginal mucosa is observed between 3 and 7-8 days of estrous cycle. This time
the loss of fluid from the tissue of vaginal mucosa and submucosa is great and the shape of
vaginal folds become angulated with sharp angles at the top of folds. As the result, the
lumen of the vagina is wider in comparison to proestrus. During diestrus vaginal folds
become flat and round. The mucosa is red and small petechia may be visible at places
touched by the tip of the endoscope. This is due to the fact that epithelium of the vagina is
thin and consists of only 2-3 cell layers in diestrus and anestrus. An opaque, thick mucus is
sometimes visible on the surface of epithelium (Figure 3).
5.2.3 Proposed alternative methods
Other methods has been proposed to monitor the bitch oestrous cycle, such as serial reading
of the electrical resistance of the vaginal mucus around the time of ovulation, using probes
inserted into the vagina during the heat period (Fontbonne, 2008), or the crystallization
patterns in anterior vaginal fluids (England & Allen, 1989) or in saliva (Pardo-Carmona et
al., 2010), which have been found up not to present an acceptable reliabily in the
identification of the canine ovulation.
Fig. 3. Vaginal endoscopy of the bitch. [From left to right] Aspect of the vaginal folds at early
proestrus, proestrus, estrus and diestrus.
5.3 The moment for insemination
In the bitch, when timing the day of ovulation as accurately as possible is essential to
guarantee adequate fertility in natural mating systems, it becomes even more important to
determine precisely when to inseminate bitches according to the sort of semen to be used
(fresh, chilled or frozen semen), as usually semen longevity and sperm cells survival
decreases with time. In addition, in frozen/thawed semen sperm cell capacitation is shorter
due to secondary effects of the frozen procedure.
When fresh or chilled semen is used, insemination should be performed on the day of
ovulation, and a second insemination must be schedule for 2 days later. On the contrary,
when frozen/thawed semen is used, and considering the need of canine oocytes to mature
in the oviducts, insemination should be performed 2 days after ovulation, and the second
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Artificial Insemination in Farm Animals
66
insemination 48h later (Figure 4). However, scheduling for the artificial inseminations may
be slightly adjusted according to the experience of the operator, the place for semen
deposition and the limitation on the number of inseminations. Consequently, regimes for
canine AI may vary with authors (Root Kustritz, 2003). Table 8 condenses the available
information on the AI schedules for fresh, chilled and frozen semen.
Fig. 4. Graphic representation of the fertile period and the ideal moment for canine AI
according to the type of semen.
Semen Dosis
Expected
spz
survival
Insemination schedule
Expected
fertility
Fresh
150-200x10
6
spz/mL
(extended)
4-6 days
- Every other day, when P
4
rise above 4ng/mL, up to
3 times.
- Day 1 to 4 post-ovulation
- P
4
levels between 8 and
15ng/mL
- 80-90%
(either with
transcervical
or vaginal
deposition)
Chilled
150 - 200x10
6
spz/mL
(extended)
24-72hrs
- Breeding once or twice 2-
4 days post ovulation
(P
4
= 4 -10ng/mL).
- Day 2 to 4 post-ovulation
- P
4
levels between 8 and
15ng/mL
- 80-90%
(either with
transcervical
or vaginal
deposition)
Frozen
50 - 300x10
6
spz/mL
(extended)
12-24hrs.
- Twice, at P
4
levels above
8ng/mL and estrus
vaginal cytology
- Day 5 to 7 post-ovulation
- P
4
levels between 18 and
28 ng/mL
- 45% if vaginal
deposition
- 67 -84% if
transcervical
or intrauterine
Table 8. Artificial insemination schedules for dogs, according to the type of semen used.
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Artificial Insemination in Dogs
67
If in the AI with fresh semen the success of the procedure is strongly related to the quality of
semen used and the moment for AI (Table 9), when using chilled semen both the quality of
semen and the site of semen deposition are important factors for success, whilst in the AI
with frozen semen, the intra-uterine semen deposition is critical (Table 10).
2AI, 48h apart Multiple AI
2AI, day 3 and 5
post LH surge
Day 9-13 after blood
y
vaginal discharge
Eosinophilic
index >80%
Eosinophilic index
>80%
Progesterone
measurements
n % n % n % n %
Pregnancy
rates
29 55.8
a
33 66.0
ab
37 78.7
b
37 80.4
b
Whelping
rates
27 51.9
a
31 62.0
a
36 76.6
b
36 78.3
b
Length of
pregnancy
63.71.4
a
64.52.6
b
64.42.4
b
63.11.3
c
Number
per group
52 50 47 46
a,b,c – different superscript
mean significant differences (P<0.05) between groups
Table 9. Success of canine AI with fresh semen according to the method used to timing AI
(n=136) (Adapted from Niżański et al. 2005)
Semen
Sperm deposition
Intra-vaginal (n) Intra-uterine (n)
Fresh
47.7 (1212) 62 (121)
Chilled
45.4 (348) 65.0 (40)
Frozen
36.7 (30) 55.5 (290)
Table 10. Comparison of whelping rates (%) in vaginal and intrauterine inseminations
according to the type of dog semen (n=2041) (Adapted from Linde Forsberg, 2002a).
5.4 Insemination techniques
In dogs, during natural mating, occurs the projection of a considerable portion of the
ejaculate into the uterus, through the cervical canal, during the coital tie (England et al.,
2006; Thomassen & Farstad, 2009). When performing AI we should be aware that vaginal
deposition per se will negatively influence sperm cell survival and their transport in the
female genital tract, and therefore impairs ability to achieve normal whelping rates and/or
litter sizes. However, deep vaginal insemination shows acceptable results when using fresh
semen, and also to some extent for chilled semen. However, to obtain satisfactory success
rates when using frozen/thawed semen, intra-uterine insemination is necessary.
As in other species, in dogs sperm cell number in the uterine lumen may be influenced by
many factors, such as the moment of estrus, the type of breeding (natural mating or
insemination) the method of insemination (intravaginal or intrauterine), the type of semen
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Artificial Insemination in Farm Animals
68
(fresh, chilled or frozen) and sperm quality (total and progressive motility and sperm
speed), besides some individual variations (Rijsselaere et al., 2004). However, despite the
influence of the intrauterine vs. vaginal insemination on the success of the procedure, once
intrauterine insemination is achieve, the exact place of semen deposition is not of the main
importance for the sperm distribution within the uterus (England et al., 2006; Rijsselaere et
al., 2004). Consequently, no potential differences or advantages exist between the vaginal
endoscopic approach and laparoscopy when the intra-uterine insemination is intended, as
no differences were found in the deposition of the semen in the uterine body or the cranial
tip of the uterine horns (Fukushima et al., 2010). Nevertheless, abdominal laparoscopy or
surgery is strongly discouraged on the basis of animal welfare issues, as non-healthy related
invading procedure that should be avoided.
Based on the last two years inseminations performed at the clinic (with a global success rate
of around 75%), using a comercial kit for progesterone determination, fresh semen and
intra-vaginal deposition (2 AI, 48h apart), it was found that when inseminations were
performed with progesterone levels above 8 ng/mL a higher success rate and a closer to
predicted whelping dates were achieved (Table 11), comparing to inseminations at lower
levels of progesterone (2.5-8 ng/mL). No diferences were found in the litter size between
these groups, which were very similar in age and parity of the bitches.
Semen
Number of
animals
Range of
age (mean)
Progesterone
at AI (ng/ml)
Days from
last IA to
whelping
Success rate
(nº females
whelped
/inseminated)
Liter size
(± SD)
Fresh 39
18
1-6 years
(2.94 years)
2.5 to 8
ng/mL
63 66.6 6.33± 2.84
12
1-6 years
(3.23 years)
> 8 ng/mL 61 80.95 6.29 ± 2.26
Chilled 42
1-6 years
(3.6 years)
>8 ng/mL 61 73.8 6.1 ±2.51
Table 11. Results for the AI procedures with fresh and chilled semen and 2 AI per animal,
48h apart.
Independently of the place for semen deposition, repeating the AI at 24-48 hrs intervals
results in a significantly higher fertility: for fresh semen both the pregnancy rate and litter
size present a significantly increase when multiple AI are performed (Linde-Forsberg &
Forsberg, 1993), whilst for frozen semen the differences on the pregnancy rates are not
significant, although litter size tended to increase with the number of inseminations (Linde-
Forsberg, 2000, 2002a).
5.4.1 Deep vaginal insemination
Deep vaginal insemination is probably the widestly used method for insemination with fresh
semen when the technique is performed by the breeder or in small budget clinics. For vaginal
AI a simple plastic catheter of proper length may be used, to which a plastic disposable
syringe containing the semen is attached. Or a commercial catheter in flexible latex tube
presenting an inflatable balloon at the tip, like the Osiris gun, may be used; when inflated, this
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Artificial Insemination in Dogs
69
kind of device has the advantage of increasing the probability for intrauterine transport of the
semen and of preventing semen backflow (Farstadt, 2010; Linde Forsberg, 2005a).
Before AI procedures start, cleaning of the perineal area, in particular the peri-vulvar area, is
needed. As transabdominal palpation is usually used to guide or ascertain the vaginal
catheter position, the owner of the female should be instructed to bring the animal with an
empty stomach, which facilitates the procedure (Linde Forsberg, 2005a).
The bitch is placed in a standing position on an examination table or on the floor (according
to the size of the female). To avoid catheterization of the urethra (the urethral opening in the
bitch is located at the pelvic brim), particular attention should be paid not to unintentionally
introduce into the urinary bladder. The insemination catheter is carefully introduced in the
vagina of the bitch, first steeply upwards until the pelvic brim has been passed, and then in
a horizontal angle, when it is carefully pushed further ahead (Farstad, 2010). In alternative,
the vulva may be elevated to just below the anus (as the bitch does when stimulated by the
male dog) (Linde Forsberg, 2005a). At this point, the position of the AI catheter must be
learn by palpation, and orientated. If the catheter is in the urinary bladder, the cranial part
of the vagina and the cervix may be palpable above the catheter and also the tip of the
catheter stands out more clearly, due to the thinner walls of the urinary bladder in
comparison to those of the vagina (Linde Forsberg, 2005a). After certification that the
catheter is correctly placed, it is moved onward through the cranial portion of the vagina
delimited by the dorsal medial folds. In smaller or primiparous bitches this point can be
difficult to overcome, and may not be possible to pass the catheter into the cervical fornix.
Except for those females, the AI catheter should be further introduced until it reaches the
paracervical area, which can be palpated as a 1- to 2-cm-long, firm structure that ends at the
cervix (a firm, rounded to ovoid structure, freely movable). The semen is deposited once the
catheter has been located in the paracervical area, close to the external cervical os.
During AI the bitch is held with the hindquarters up and head down, in an angle of 45-60º.
This position facilitates transabdominal palpation of the cervix and ensures that the semen
will not be expelled through backflow. According to earlier reports, the bitch should be
maintained in the same position up to period of time varying from 5 to 20 min after AI.
However, reducing the interval of elevated hindquarters to 1 min seems not affect fertility
(Pinto et al., 1998). Also, feathering or stroking of the vulvar or perineal region is reported
by several authors as form of stimulating the semen transport into the uterus, in an attempt
to mimic the vaginal stimulation by the thrusting movements of the dog during natural
mount. However, the contribution of such procedures to the exit of the technique has not
been proven yet.
5.4.2 Intrauterine insemination
Intrauterine insemination may be performed by using non-surgical transcervical
catheterisation (Linde-Forsberg, 1991; Linde-Forsberg and Forsberg, 1989, 1993; Linde-
Forsberg et al., 1999) or by surgical semen deposition by laparotomy (Brittain et al., 1995;
Günzel-Apel & Thiet, 1990) or laparoscopy (L.D.M Silva et al., 1995, 1996). The majority of
European centers working on small animal reproduction prefer transcervical intrauterine
insemination (TCI) due to reasons associated with animal welfare. However, catheterisation
of uterine cervix in the bitch is a difficult procedure and demand skill and experience. The
semen of lower quality, such as frozen-thawed or that collected from subfertile dogs have to
be deposited intrauterine to assure satisfactory results of artificial insemination (Linde-
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Artificial Insemination in Farm Animals
70
Forsberg et al., 1999; Thomassen et al., 2006). The conception rates after intravaginal
insemination with frozen-thawed semen are significantly lower when compared with the
results of intrauterine insemination.
5.4.2.1 The Norwegian or Scandinavian technique
The method of non-surgical transcervical intrauterine insemination was first time described
in 1975 (Andersen, 1975). The technique has been adapted from the artificial insemination
performed in foxes. Two catheters are used in this method - the outer plastic catheter and
inner metal thin catheter. There are 3 sizes of the catheters, for small, medium and large
breeds. The catheterisation should be made on standing animal. Sometimes there is no need
for administration of sedatives, but usually a small dose of alpha-mimetic, such as
medetomidine, is advisable for abdominal muscles relaxation. The outer plastic catheter
should be introduced into the vagina. It should be advanced as far as is possible. In many
bitches, especially those of larger breeds, the tip of the catheter passes into the cranial
narrow part of vagina. However, in some smaller bitches the introduction of the outer
catheter through the paracervix is difficult. It is necessary to palpate the end of the catheter
and the cervix through the abdominal wall. The cervix is palpable at estrus as solid, ovoid
structure. It is advisable to move the tip of the catheter ventrally towards the ventral region
of abdominal wall. This procedure is helpful in palpation of the cervix. The inner metal
catheter should be introduced through the plastic catheter. The cervix should be fixed
between the thumb and other fingers and tilted to horizontal axis. The metal catheter is
introduced into the cervical canal under the control of the position of the cervix by palpation
through the abdominal wall (Andersen, 1975; Linde-Forsberg, 1991). This technique
demands skill and experience. It is harder to perform uterine catheterisation in obese or
nervous bitches and in giant breeds.
The scandinavian method of uterine catheterization is recommended for routine
insemination of bitches (Ferguson et al., 1989; Günzel-Apel 1994; Linde-Forsberg, 1991,
1995). The use of this technique of insemination is especially advisable in cases when using
of semen of lower quality due to male subfertility or sperm cryopreservation. Linde-
Forsberg and Forsberg (1989) obtained 83.9% and 69.3% of pregnant bitches (data corrected
for the stage of estrus) after insemination with fresh and frozen-thawed semen, respectively.
The litter sizes were lower by 23.3%, when frozen-thawed semen was used in comparison to
fresh semen. Rota et al. (1999a) reported 25% higher pregnancy rate after intrauterine semen
deposition when using scandinavian technique than after vaginal semen deposition. On the
basis of analysis of 327 inseminations Linde-Forsberg et al. (1999) concluded that success
rate of scandinavian method and vaginal insemination with frozen-thawed semen was
84.4% and 58.9, respectively. Niżański (2006) proved that results of vaginal insemination
with frozen-thawed semen are significantly lower in comparison with fresh semen, in spite
of the use of modification of the technique of vaginal semen deposition, plasma addition
and adjustment of the number of spermatozoa.
5.4.2.2 Endoscope-assisted vaginoscopic method (New Zealand method)
Intrauterine insemination of the bitch under the visual control of endoscopic equipment was
first time described by Wilson (Wilson, 1993, 2001), using a rigid endoscope – cysto-
uretroscope of the length 29 cm with diagnostic external sheath. The procedure is performed
on the standing animal. Uterine catheterisation is made with the use of flexible catheter
introduced into the working channel of the endoscope. The endoscope is introduced into the
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Artificial Insemination in Dogs
71
cranial narrow part of the vagina, while a flexible catheter is introduced cranio-dorsal into
the external orifice of the cervical canal under the visual control performed through the
endoscope. Usually it is not necessary to administer any sedatives.
The results for the intrauterine deposition of frozen-thawed semen when using this
technique are quite satisfactory (Table 12). Wilson (1993), with the use of frozen semen,
refers a pregnancy rate and litter size 83.3% and 7.5 puppies per litter, respectively. Niżański
(2005) obtained whelping rates of 68.7% and 27.8%, when frozen-thawed semen was
deposited by intrauterine vaginoscopic method and by vaginal insemination, respectively.
Results obtained by Linde-Forsberg et al. (1999) were poorer in comparison with the
Scandinavian method. However, vaginoscopic intrauterine insemination is currently
considered as the practical, modern and useful tool in assisted reproductive techniques in
dogs which may become in the future the routine method of insemination.
Insemination
technique
n
Whelping rate
(nº females
whelped/inseminated)
Litter size
at birth
(range)
Litter size
at weaning
(range)
Percentage of
male
pups/litter
Intravaginal,
using an
infusion pipete
18
27.8
a
(5/18)
3.0 ±1.2
a
(2-5)
2.6 ±0.9
a
(2-4)
51.5 ±25.9
a
Intrauterine,
using the
endoscope
16
68.7
b
(11/16)
4.9 ±1.7
b
(3-8)
4.6 ±1.7
b
(2-8)
54.7 ±18.7
a
Different superscripts in the same column indicate significant difference (p<0.05)
Table 12. Whelping rate and mean litter size in bitches intravaginally or intrauterine
vaginoscopically inseminated with frozen-thawed semen. AI were performed at 4
th
and 6
th
days of oestrous cycle.
Currently a large variety of rigid endoscopes of different brands is available. The most
suitable are those of the length 30-35 cm with 30º or 0º view angle and the diameter of 3 mm
or similar. Some companies market the equipment dedicated specifically for artificial
insemination in bitches. The optics of endoscope should be used with outer sheath equipped
with working channel allowing for introduction of flexible catheter of the diameter of 5-7
French gauge. The mechanism of Albaran is not useful in the endoscope for artificial
insemination of bitches, as it is not practical and the diameter of the outer sheath becomes
too large when it is attached.
The catheterization may be problematic in both toy and giant breeds. In the first case, the
diameter of the equipment may be too large for introduction of the tip of optics near the
uterine cervix, impairing visualization of external cervical orifice. In contrast, in giant breeds
the length of the vagina may exclude the possibility to access the region of the cervix. One
additional problem may be associated with the presence of the blood or mucus within the
vagina. Sometimes the fluid present in the genital tract may cover the terminal lens of the
endoscope making the observation impossible. Withdrawal the optics and cleaning of the
lens is necessary in such cases. For better visualization of the vagina, especially within the
cranial narrow part, insuflation with CO
2
made with insuflator or even with rubber bulb is
advisable. Therefore the view of tissues is focused, sharper and clearer, which makes the
procedure easier. The time necessary for catheterization is variable. It depends mainly on
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72
the skill of the operator and on the anatomical features of the vagina, and varies usually
between 0.5-3 minutes, but it may be longer. In nervous bitches the administration of small
doses of sedatives, such medetomidine, is advisable, although in not too high doses, as the
catheterisation should be done on standing bitch.
Currently the vaginoscopic method of intrauterine insemination appears to be advantageous
and useful technique of semen deposition in the uterine lumen in bitches. The technique
demands skill but it is practical and quick to perform for experienced operators. The visual
control of introduction of the catheter into the uterus is the important advantage of the
technique. The observation of the moments of semen deposition and control if there is no
semen backflow is therefore possible. Moreover, it allows also uterine sampling when a
female is suspected of infertility due to uterine disease (Thomassen & Farstad, 2009). For
these reasons this method of intrauterine insemination is becoming more popular.
5.4.3 Surgical technique
Surgical insemination technique have been proposed once for frozen semen or when the
bitch presents an anatomical obstruction that prevents the insertion of the catheter or
endoscope. Both the laparoscopic approach and the laparotomy requires anaesthesia and
good surgical skills. The semen is introduced into the uterus by puncture of uterine wall or
incision with a scapel and passage of a tom cat catheter (Farstad, 2010; Thomassen &
Farstad, 2009). However, in such methods, semen deposition is performed only once.
Some restrictions may exists to application of this method in different countries, that may
compromise the registration of litters obtained without fulfilment of the legal requirements,
such as previous evaluation of the situation or previous authorization of the local Kennel
Club for the procedure. Furthermore, some ethical constraints have been raised regarding
the use of surgical techniques for AI in dogs. Surgery is an invasive procedure, so it is
unlikely to carry it out in the best interest of the animal, and the possibility of transmission
of an undesirable trait in a particular animal genetic line should be kept in mind.
6. Rules and regulations concerning the import or export of sperm
Before implementing canine artificial insemination the owner and the clinician should be
aware of the national or international regulations on semen import, if applicable, and of the
local Kennel Club requirements respecting the use of canine AI and litter registration. In
addition, procedures may differ between the use of national or imported semen.
Consequently, attention should be paid well in advance to this matter.
In the absence of a specific national regulation, most Kennel Clubs follows FCI (Fédération
Cynologique Internacionale) determinations for AI, transposed to the FCI International
Regulation for Breeding (http://www.fci.be/circulaires/102-2010-annex-fr.pdf). To ensure
that ethical issues are minimised, FCI recommends that AI should only be done in healthy
dogs with proven fertility (article number 13). In addition, in the introductory section of this
regulation, FCI specifically limit the use of dogs presenting diseases possible to be
transmitted to following generations and those presenting major, eliminatory defects in
regard to the breed standard. Furthermore, it presumes that the AI is performed by a
Veterinarian, which should certify the quality of the dog semen (either for the fresh and the
processed semen, the later being certified in a standard document to be released upon
semen collection and preservation) and also to attest the Kennel club to the occurrence of an
AI for a specific female. In both documents, correct identification of the animals (either the
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73
male or female) is mandatory, and can be obtained through the use of a tattoo or a
microchip. The time to submit the AI certificate may differ between national Kennel Clubs.
In some countries the Club must be informed of the AI procedure within the first 2 weeks,
whilst in others, only when the litter is to be registered.
Besides regulations on performing AI to a bitch, attention must be paid to issues concerning
the semen collection and use. Besides the use of a recognisable male, with a certified
pedigree, particular requirements may exist from national Kennel Clubs or the Official
Agriculture entities, which may vary for chilled and frozen semen. In some situations the
permit to import dog semen is required, which may or may not need to be accompanied by
a DNA sample, and a health certificate that may include blood testing against the most
important infectious or congenital canine diseases. Awareness of the latest official
requirements is essential when considering semen international shipment. Additional
information on the shipment regulations may be obtained throught the references Linde
Forsberg, 2001, 2005b.
7. Conclusion
Demands for canine artificial insemination is growing worldwile together with an increase
request for semen preservation in sperm banks. Furthermore, a tendency exists to increase
the demands for the use of frozen/thawed semen over fresh semen AI, as part of breeding
tools for genetic improvement. Nowadays is possible to achieve adequate whelping rates
and litter sizes regardless of the type of semen used, as long as proper timing of AI and
proper semen deposition are used. Client education and technical councelling must
complete the AI services to be offered by specialized practicioners, in particular when
breeding a problematic bitch.
8. Acknowledgments
The authors are greatly indebted to Dr Malgorzata Ochota for her valuable cooperation
and thoughtful comments during language revision of this manuscript.
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  • [Show abstract] [Hide abstract] ABSTRACT: The aim of this study was to define a protocol to store dog sperm before and after sorting to obtain an insemination dose sufficient to allow the conception by artificial insemination. Experiment 1 and 2 were performed to evaluate the more appropriate extender for preserving at room temperature dog sperm before and after sorting. Four extenders were tested: (1) Tris-fructose-citrate (TFC), (2) Tris-glucose-citrate (TGC), (3) modified Tyrode's albumin lactate pyruvate medium (mTALP), and (4) third fraction of the ejaculate (after centrifugation at 5000× g for 10 minutes; III FRAC). Experiment 3 and 4 were performed to evaluate the ability of dog semen to withstand sex sorting and freezing/thawing. Modified Tyrode's albumin lactate pyruvate medium was the best extender for canine sperm storage at room temperature (20 °C-25 °C) before (total motility: TFC, 8.3 ± 1.7; TGC, 50.0 ± 11.5; mTALP, 70.0 ± 0.1; III FRAC, 25.0 ± 1 0.4; P < 0.05) and after sorting (total motility: TFC, 7.3 ± 1.5; TGC, 10.3 ± 1.5; mTALP, 33.3 ± 6.7; III FRAC, 8.7 ± 5.8; P < 0.05), even if at 24-hour sorted sperm quality was impaired in all extenders tested herein. Sperm quality decreased after sorting (total motility: control, 92.5 ± 0.9; sorted, 52.9 ± 6.0; P < 0.05) and, especially, after freezing/thawing (total motility: frozen control, 25.7 ± 4.1; frozen sorted, 2.4 ± 1.2; P < 0.05). In conclusion, mTALP is an appropriate medium for canine sperm storage before and soon after sorting (hours), but a long storage period of sexed sperm at room temperature is not adequate. Cryopreservation greatly impaired sperm quality, and further studies are needed to optimize the freezing protocol for sexed dog sperm. Copyright © 2015 Elsevier Inc. All rights reserved.
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