Abstracts

Abstracts
THE PROS AND CONS OF SPA Y/NG THE S/TCH; A PRACT/CAL AND ETH/CAL
D/LEMMA
By Wenche FARSTAD
Norwegian School of Veterinary Science (NVH) , P.O. Box 8146 Dep, N-0033 Oslo,
Norway.
INTRODUCTION
Castration or spaying of a female dog implies the surgical or laparascopic removal of
gonads (ovaries) only (ovariectomy) or the more commonly used operation, the
surgical procedure termed ovariohysterectomy. An ovariohysterectomy (OHE) or spay
is the complete removal of the female reproductive tract. The ovaries, oviducts, uterine
horns, and most of the body of the uterus are removed. Alternatively, a tubai ligation
may be performed in the female. The oviducts are located during abdominal surgery,
cut and tied off with suture material. Either one of these three surgical procedures will
prevent future pregnancies if done correctly. Both have advantages and
disadvantages, but only those that involve gonadectomy, such as ovariectomy and
ovariohysterectomy; are of significance for the long-term health of the bitch. The major
disadvantage of tubai ligation is the recurrence of oestrus 1-3 times per year. Tubai
ligation to prevent nidation is rarely performed in bitches, unless specifically demanded
by the owner.
Canine reproduction possesses characteristics that distinguish female dogs from other
domestic animais, such as a monocyclic oestrus cycle, an unusually long pro-oestrus
with vaginal bleeding induced by increasing oestrogen production in follicles, a
preovulatory luteinisation of follicles in response to the LH surge, a postovulatory
maturation of the oocyte, and a non pregnant luteal phase, which is similar in duration
to the pregnant luteal phase, or even a bit longer. This luteal phase is followed by a
long resting phase or anoestrus of several months. Although not overtly seasonal,
reproduction is also influenced by moulting cycle and day length to a certain extent,
and is related to the distance from last parturition in parous animais. Many of these
characteristics make manipulation of reproduction more difficult in the dog than in many
domestic polyoestrous species. Also, the availability of the drugs used to manipulate
canine reproduction varies among countries, many of them are meant primarily for use
in the human or in large domestic animais. If the owner of a: bitch wants to prevent
unwanted pregnancies the fastest and most practical solution may be to spay the bitch.
ln the following presentation a review is given on the pros and cons of spaying and the
legislation connected to this in Europe.
LEGISLATION
ln the United States castration of dogs is allowed. Most dogs and cats are
spayed/neutered between 5 and 8 months of age. To try to control pet overpopulation,
many humane shelters have started to spay/neuter ail animais before they are
adopted. This means they are spaying/neutering animais at an even younger age, that
is, at 7-14 weeks of age. Many veterinarians in private practice have also started early
spaying/neutering. In Norway routine castration of dogs is prohibited according to the
Animal Welfare Act; so most bitches are left intact throughout their Iifetime. Castration
is only allowed for medical reasons, and in dogs in official service, such as guide dogs
for the blind. In many European countries the legal practice is between these two
extremes. Both the other Nordic countries and Germany have Animal Welfare Acts that
specifically mention the prohibition to remove animal organs or parts, but most of them
allow castration of dogs, since this not only involves modifications of the animais to
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please humans, but also because there are many perfectly good medical reasons to
remove the gonads. In most of these countries, castration is done after 6 moths of age
or after the first oestrus in females.
THE PROS AND CONS
Most arguments that are used in favour of or against spaying of bitches are either
behavioural or medical.
Sorne points held in favour of spaying
•
•
•
•
•
•
It has health benefits: Spaying a bitch before her first heat is the best way to
prevent mammary cancer. The use of contraceptives is not safe and increases
the risk of developing these tumours.
Behaviour modification: It tends to calm down the sometimes aggressive or
hyperactive bitch and increases concentration for performing tasks
Pseudopregnancy is avoided, thus, the behavioural and physical alterations of a
false pregnancy are eliminated
Castration is the treatment of choice for pyometra.
Preventing unwanted pregnancies, and thus, it is important for pet population
control
Owner related cornplaints such as, ''the inconvenience of vaginal bleeding that
typically goes on for 9 to 15 days", or ''frequently having to chase away roaming
males around the garden"
There is little controversy with regard to the negative effects of sex steroids on the
development of mammary cancer in the bitch. Oestrogen is one of the primary causes
of canine mammary cancer, the most cornmon malignant tumour in female dogs.
Animais that are spayed prior to one year of age very rarely develop this malignancy.
However, it is very cornmon in unspayed females. The chances of reaching seniority
without developing this tumour is less than 11% in sorne breeds with normal hormone
production. If the dog is spayed later in life, it may still decrease the chances for this
cancer to develop. Mammary cancer is the most common tumour altogether in female
dogs in Norway, and this represents a population of almost entirely reproductively
intact females. Data from two population-based studies in four Norwegian counties
were used to calculate the crude incidence of mammary tumours, and the age- and
breed-specific incidence of mammary tumours in female dogs of three different breeds.
The largest study comprised 14401 histologically verified tumour cases from four
counties covered by the Norwegian Canine Cancer Register. The register covers about
25% of the total Norwegian dog population. The crude incidence of malignant
mammary tumours in female dogs of any breed was 53.3%. The highest relative risk
ratio of mammary tumours was found in boxers, cocker spaniels, English springer
spaniels and dachshunds. The mean age of histologically diagnosed mammary
tumours was 7.9 years in boxers and 7.8 years in springer spaniels, compared with 8.8
years in ail other breeds (Moe, 2001).
Prevention of diseases of the reproductive tract, such as tumours and CEH/pyometra is
another non-disputable justification for spaying. Tumours of the reproductive tract of
the bitch are relatively uncommon but they do occasionally occur in the uterus and
ovaries. An OHE would eliminate any possibility of this occurring. Recent studies on
the incidence of pyometra are relatively scarce due to the fact that many countries
allow spaying routinely in non-breeding females. The incidence observed in a colony of
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beagle bitches more than 4 years old over a 12-year period in Japan, was 15.2% (n =
165), with the average age of onset 9.36 +/- 0.38 years. (Fukuda, 2001). Middle aged
and older bitches may experience problems with severe uterine disease developing
after oestrus. Pyometra may, if it remains untreated, be fatal due to extensive systemic
effects on the internai organs and the risk of septicaemia. The strain on the kidneys or
heart in sorne of these cases may be fatal or cause life long problems, even after the
uterus has been removed. Aiso increased incidence of diffuse bleeding may be seen in
older animais. Its treatment requires either the use of hormonal and intravenous fluid
therapy if the medicinal route is chosen, or more commonly, ovariohysterectomy with
the risk of systemic complications during or after surgery. A recent survey from Norway
shows that 98% of practicing veterinarians considered surgery as the treatment of
choice for closed pyometras, and 80% preferred surgery, even when the cervix was
open (Rootwelt- Andersen et al, 2003).
Another problem in sorne bitches is the occurrence of false pregnancy or Overt Canine
Pseudocyesis (OCPSC). This is the physiological syndrome characterized by c1inical
signs such as: nesting, weight gain, mammary enlargement, lactation and maternai
behaviour, which appear in non-pregnant bitches at the end of metoestrus. OCPSC is a
frequent finding in domestic dogs. Prolactin (PRL) plays a central role in the
appearance of OCPSC, but its precise aetiology is not completely understood.
Considering that OCPSC is a self-limiting physiological state, mild cases usually need
no treatment. These cases often experience no serious problems, as the behaviour
disappears
when the circulating
hormones return to their appropriate
levels.
Pseudopregnant females, however, have a greater tendency to develop pyometras.
Spaying bitches that consistently have false pregnancies prevents both the behavioural
and physical symptoms of false pregnancy. This may therefore also be considered an
acceptable medical reason to spay bitches.
However, there are now prescription
antiprolactinic hormones available in sorne countries, cabergoline (Galastop®, which
eliminates unwanted effects of this condition. Inhibition of PRL release by ergot
derivatives (bromocriptine (10-100 microg/kg per day for 10-14 days), cabergoline (5
microg/kg per day during 5-10 days), metergoline (0.2 mg/kg per day during 8-10 days)
has proved to be effective for the treatment of canine OCPSC (Gobello et al, 2001). A
colour shift in the haircoats of particular breeds has been found to occur after the use
of antiprolactins. This effect may be mediated through the inhibition of the secretion of
melanocyte-stimulating
hormone by the administration of the dopaminergic agonist
cabergoline for more than two weeks. Transient coat colour changes should be
considered a possible side effect when planning long-term treatment with dopaminergic
agonists in dogs (Gobello et al, 2003).
Sorne points he Id against spaying
•
•
•
•
•
The spayed female grows fat
Spaying involves invasive surgery
Urinary incontinence develops in spayed females
Irreversibility
Spaying and neutering is an intervention to modify the bitch for the convenience
of humans. It removes natural instincts and changes behaviour and therefore
compromises the integrity of the animal.
Spaying itself does not cause the bitch to become obese, but it may alter metabolic
rate and thus reduce calorie need. This has only been shown conclusively for the cat
(Oison et al, 2001). Every dog needs exercise and a good balanced diet. Overfeeding,
which results in obesity, is often caused by the owner who is responsible for the dog's
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food and her weight. As the bitches grow older, they tend to exercise less and need
less food to eat. This challenge of spaying is not emphasized by the producers of dog
feed. Owners often get little help to regulate their animais' weight. This issue is even
more relevant in relation to the increasing problem of human overfeeding, and
educating the owner how to manage the spayed female is becoming increasingly more
important. Furthermore, as discussed elsewhere in this paper, high bodyweight
predisposes the bitch to post spaying urinary incontinence.
Urinary incontinence is a common side effect of spaying. It is still controversial whether
a bitch should be spayed before or after the first oestrus, but it would be desirable to
spay bitches at an age that would minimize side effects. Prepubertal spaying is
recommended to prevent the occurrence of mammary tumours. On the other hand,
compared with late spaying the clinical signs of urinary incontinence were more distinct
after early spaying. Urinary incontinence after spaying was found to occur in 9.7% of
bitches. Urinary incontinence affected 12.5% of bitches that were heavy (> 20 kg body
weight) and in only 5.1 % of bitches that were lighter « 20 kg body weight) (Okkens et
al, 1997). The type of surgical procedure (ovariectomy versus ovariohysterectomy) had
no influence on the incidence, or on the period between spaying and the occurrence of
urinary incontinence. Urinary incontinence occurred on average at 2 years and 10
months after surgery and occurred every day independently of whether the bitches
were awake or asleep (Stocklin-Gautschi et al, 2001).
Furthermore, with regard to prepubertal gonadectomy or early spaying, questions have
arisen regarding the safety of an early spaying procedure and possible effects on the
animais when they become older. Younger animais may need different anaesthetics
and are more prone to hypothermia during surgery. As long as procedures are modified
to account for these differences, early spaying is probably safe. And often the recovery
after surgery is faster. Studies in which three groups of dogs were spayed or neutered
at different ages showed no significant differences in growth rate, food intake, or weight
gain (Oison et al, 2001).
The spaying of a bitch involves a general anaesthetic to open into the abdomen and
remove the ovaries and uterus. This operation involves her staying hospitalised
although for a short time, normally just one day, or, in a few cases, overnight if no
complications occur. Most bitches recover uneventfully, however, in a survey of Pollari
et al (1996) the frequency of clinically relevant postoperative complications of elective
surgedes such as, OHE, neutering and declawing in dogs and cats is substantial (19
and 12%, respectively).
The ovarian remnant syndrome is a well-recognized clinical problem in both bitches
and queens having undergone elective OHE. Of the total number of bitches (109) in a
Dutch study with complications
following ovariohysterectomy
and submitted to
examination, 55 showed gynaecological symptoms, such as discharge of the vulva
(28), attractiveness to male dogs (37), periods of heat (28) and pseudopregnancy.
When a cytological study was done in the dogs with suspected residual ovarian tissue,
an oestrus pattern was observed in 39 per cent of the cases, the progesterone level of
the peripheral blood being above the basal level in 70 per cent of the cases. Remnants
of ovarian tissue were removed during laparotomy in forty-seven dogs, on the right side
in forty-one cases and on the left in twenty-two cases. The uterocervical stump was
shortened when uterine tissue was probably or obviously present. The uterine stump
was more or less severely inflamed in nineteen cases (Okkens et al, 1981).
The spay operation cannot be reversed. The option to spay a young bitch, wh en there
is indecision as to whether to breed from her or not, can be delayed by using drugs to
124
control her seasons, in theory indefinitely. Due to the side effects, .the use of these
drugs must be discussed with the veterinary surgeon, as each case is an individual
one, and must be treated as such. In Norway this is a valid argument since more than
80% of dogs are purebred dogs registered with full breeding potential in the Norwegian
Kennel Club. These young bitches often have to await the results of mandatory health
examinations, such as eye or hip examinations before breeding is allowed. Thus, in
these cases, early spaying would eliminate the possibility to breed, and thus, limit the
potential active breeding population in small breeds, leading to reduced heterogeneity
of the population.
ln many countries castration is allowed for medical reasons and for special purposes.
Guide dogs for the blind are always spayed, but in many countries (for example, the
USA) also police dogs, and dogs used by the Armed Forces are spayed, but remain
good working dogs. Contrary to this, in Scandinavia, only the guide dogs for the blind
are routinely spayed, not the police, hunting and service dogs. These dogs seem to be
functioning weil, and it is claimed that they are better workers, more energetic and with
higher persistence than dogs that are castrated. This is contrary to the argument of
higher concentration by spayed animais because of little interest in the opposite sex
and independence of sex hormone influence. With respect to behaviour modification in
males the effect of surgical castration on aggressive behaviour is not straightforward
(Neilson et al, 1997). Likewise, there is reason to assume that female aggressive
behaviour related to humans or other dogs will not be affected by OHE. Thus spaying
and neutering may influence mainly sex steroid dependant behaviour problems, such
as urine marking and mounting in males, and pseudopregnancy- and oestrus-related
behaviour problems in females.
ln many countries, thousands of pets are destroyed annually after a longer or shorter
stay at a dog shelter or pound. Furthermore, local townships and counties may charge
an extra fee for licenses for animais that are intact (not spayed or castrated). The
bitch's chance of living longer is therefore increased because of spaying. In Norway no
such fee (dog tax) exists anymore, so this argument is not relevant for ail countries.
TO SPAY OR NOT TO SPAY, AN ETHICAL DILEMMA?
From the data presented above there are obviously good medical reasons for
castrating non-breeding females. It is difficult to argue against these justifications, but
one must bear in rnind that surgery is not necessarily complication free. Furthermore, in
areas or countries where the production of pups severely supersedes the demand for
pets, there is a very good animal welfare argument to Iimit the number of dogs that are
allowed to breed, and to leave breeding to experienced and serious breeders that offer
good facilities and a favourable environment for the rearing of puppies. In many
European countries, legislation has already been passed to limit such overproduction.
Aiso the recently passed modifications of the Animal Welfare Acts in sorne European
countries such as Norway, Sweden, Finland, Denmark and Germany do not encourage
surçlcalrernoval of organs or parts of animais for the purpose of convenience to
humans. In Norway the castration of male pigs will be completely prohibited in 2009
despite the very large economic impact this will impose upon the pig breeding industry.
The prohibition of docking and ear cropping in dogs in an increasing number of
European countries signais a stricter rather than a more lenient policy. Public opinion
may turn against using invasive procedures unnecessarily in veterinary medicine. Many
Europeans find elective surgeries to be an intervention only to modify the bitch for the
convenience of humans. It removes natural instincts and changes behaviour and
therefore compromises the integrity of the animal. This is indeed an ethical dilemma for
many of us, considering that spaying of bitches and neutering of dogs do have clear
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medical indications, since castration prevents some health problems related to
reproduction.
Do we have any alternatives to surgical intervention to prevent unwanted puppies? ln
the USA The Alliance for Contraception in Cats and Oogs is a collaborative initiative to
develop and test non-surgical technologies for the humane control of cat and dog
overpopulation. This Alliance wishes to increase public awareness of the
overpopulation problem, as weil as educate communities about the time, moneyand
research required to find humane, workable solutions. Their strategy is to support
researchers who are working to develop and test a number of products, including
immunocontraceptive vaccines. The immunocontraceptive vaccines can be delivered
by injection and in the case of ferai cats and dogs or wild animais, vaccine forms
suitable for delivery in baited foods are being explored. On the European market a
progestagen-based contraceptive drug, proligestone, Covinan® Intervet, has been
marketed for some time. The manufacturers claim that this gestagen has less affinity to
the progesterone receptors in the mammary gland, and thus, the risk for developing
mammary cancers should be diminished compared with the first generation gestagens,
such as medroxyprogesterone acetate. Faced with the controversies connected to
invasive surgical procedures, there seems to be an increased demand for further
research into medical alternatives to spaying of bitches, as weil as neutering of male
dogs. Furthermore, the practical aspects of pet overpopulation must be addressed
educationally and legally.
REFERENCES
Fukuda S. Incidence of pyometra in colony-raised beagle dogs. Experimental Animais 2001; 50(4): 325-9.
Gobello C, de la Sota RL, Goya RG. A review of canine pseudocyesis. Reproduction in Domestic Animais, 2001; 36(6):
283-8
Gobello C, Castex G, Broglia G, Corrada Y. Coat colour changes associated with cabergoline administration in bitches.
Journal of Small Animal Practice 2003; 44(8): 352-4.
Moe L. Population-based
incidence of mammary tumours in some dog breeds. Journal of Reproduction and Fertility;
2001, Suppl. 57:439-43.
Neilson J, Eckstein R, Hart B. Effects of castration on problem behaviours in male dogs with reference to age and
duration of behaviour. Journal of the American Veterinary Association, 1997; 211 :180-82.
Okkens AC, Dieleman SJ, v d Gaag 1. Gynaecological complications following ovariohysterectomy
in dogs, due to: (1)
Partial removal of the ovaries. (2) Inflammation of the uterocervical stump (in Dutch). Tijdschritt Diergeneeskunde
1981;106 (22): 1142-58.
Okkens AC, Kooistra HS, Nickel RF. Comparison of long-term effects of ovariectomy versus ovariohysterectomy
in
bitches. Journal of Reproduction and Fertility, 1997; Suppl.51 :227-31.
Oison PN, Kustritz MV, Johnston SD. Early-age neutering of dogs and cats in the United States (a review). Journal
Reproduction and Fertility 2001 ; Suppl 57:223-32.
Pollari FL, Bonnett BN, Bamsey SC, Meek AH, Allen DG. Postoperative complications of elective surgeries in dogs and
cats determined by examining electronic and paper medical records. Journal of the American Veterinary Medical
Association, 1996; 208: 1882-6.
Rootwelt-Andersen,
Heiene, R, Farstad, W. Treatment of pyometra in dogs in Norway: Two case studies and a survey
among practicing veterinarians. In: Proceedings of The III Annual Meeting of the European Veterinary Society for Small
Animal Reproduction, Dublin, 2003, pp. 87-91.
Stocklin-Gautschi
NM, Hassig M, Reichler lM, Hubler M, Arnold S. The relationship of urinary incontinence to early
spaying in bitches. Journal of Reproduction and Fertility,2001 ;SuppI.57:233-6.
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Abstracts
NON-SURGICAL
INTRAUTERINE
ARTIFICIAL
INSEMINA TION IN THE BITCH
By Wenche FARSTAD
Norwegian School of Veterinary Science (NVH) P.O. Box 8146 Dep, N-0033 Oslo,
Norway.
INTRODUCTION
Artificial insemination (AI) in dogs, although performed by Lazzaro Spallanzani as early
as in 1780, is still performed in various ways and with variable success. Vaginal
deposition of fresh semen, as was done by dr. Spallanzini more than 200 years ago, is
still today the common AI method for breeders and most small animal veterinary
practitioners. In cattle a standardized method is used for semen deposition into the
uterus, i.e., the transcervical
catheterisation
method using a stainless steel
insemination gun covered by a disposable plastic sheath and transrectal fixation of the
cervix. Even non-surgical flushing of embryos and embryo transfer is routinely
performed via the cervical route in cows. In cattle frozen semen is used almost
exclusively, since sire and dam are usually separated either in distance, by legal trade
restrictions, by sanitary regulations, or in time, if the sire is already slaughtered. The
demands for transport of canine semen and for using cryopreserved material are
increasing for the same reasons as in cattle. There are obviously major differences
between cows and bitches, yet common elements to consider that may lead to a higher
degree of standardization of procedures with AI in dogs. In the following, the different
aspects of sperm deposition in relation to type of sperm (fresh or frozen) will be
presented.
THE SIGNIFICANCE OF INTRAUTERINE DEPOSITION
First of ail, one must consider the difference between the deposition of semen in cattle
and dogs during natural service. The bull produces a high-density ejaculate of a small
volume, deposited in the cranial vagina, and the coitus is very short. Sperm transport is
delayed through the cervix, although uterine contractions transport dead as weil as live
sperrn equally weil in the female oviduct. Sperm storage during natural mating is in the
cervix, the uterotubal junction from which sperm release is synchronized to fit
favourably with ovulation time, and finally in the isthmus of the oviduct. In cattle, due to
the cervical delay there is a significant gain by artificially bypassing the cervix, or when
depositing the semen deeply into the uterine horns. The delay bypass obtained by
cervical catheterisation enables a large reduction in insemination dose (number of
spermatozoa from billion/ml by natural service to million/ml by AI) in this species.
Dogs ejaculate less concentrated, somewhat larger volumes of semen, and the semen
is indirectly transported into the uterus during coitus. This is due to the swelling of the
penile bulb, which is believed to create pressure gradients between the uterus and the
vaginal compartments, leading to rapid passive transport of the ejaculate into the
uterus. In bitches it is assumed that no delay of sperm transport occurs during oestrus,
when the cervical folds are relaxed and the cervical canal is open, and the
spermatozoa are passively transported into the uterus during coitus (England and
Pacey, 1998.) The majority of sperm are first stored in the uterine glands, and freshly
ejaculated canine sperm survive there for several days (Doak et al, 1967).
Synchronisation between sperm deposition time, as weil as sperm mobilisation from
the uterine storage site, and ovulation, or more precisely fertilization time, may be less
optimal in dogs than in cattle. This is compensated for by the longevity of the canine
spermatozoa. Wh en sperm are frozen however, longevity is considerably reduced, due
to capacitation and acrosome reaction-like alterations in the sperm membranes and the
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acrosome after thawing. Therefore, timing of AI as weil as site of deposition may be
crucial. One important aspect of this is that the cervical canal closes late in oestrus,
despite the fact that oocytes still may be fertile (Allen and France, 1985; Silva et al,
1995; Silva, 1995).
Thus, in the dog there is less to gain with regard to reducing insemination dose than in
cattle. Transport of spermatozoa from the vagina occurs during oestrus when the cervix
is open. Vaginal deposition of semen in early or mid oestrus results in rapid transport
to the first storage site inn the uterus or to the tip of the horns. Sperm may be mobilised
from their storage site when the oocytes are ready to be fertilized, provided they
survive long enough. Indeed, Nôtling et al (1995) observed a very high whelping rate
(88%) after vaginal deposition of frozenlthawed semen, however, using an average
number of inseminations per bitch during oestrus of 5.6. A high number of sperm per
insemination dose or frequent Ais seem necessary to attain whelping rates similar to
intrauterine AI.
INTRAUTERINE
INSEMINATION
TECHNIQUES
The Norwegian Intrauterine Method (NIU)
ln the early 1970s a transcervical intrauterine AI technique was developed for dogs,
and with this procedure a litter of mongrel puppies was born after AI with frozen semen
in 1972 (Andersen, 1975). This was the first reported litter from frozen semen in
Scandinavia, the second Iitter in ail of Europe (van Gemert, 1972) and the third litter in
the world (Seager, 1969). The intrauterine insemination equipment consisted of a
simple stainless steel catheter and a plastic guiding tube (Andersen 1975). The guiding
tube is used to protect and steady the catheter and to stretch the vagina and protect
the vaginal mucosa against damage during insertion of the catheter. The cervix is fixed
through abdominal palpation, and the semen is deposited after insertion of the catheter
through the cervix and into the uterus. Control of correct intrauterine deposition is done
by feeling the tip of the catheter cranially to the cervix inside the uterine body.
The Norwegian Kennel Club (NKK) registered the first purebred litter obtained by
frozen semen in Norway in 1975. Our insemination team have inseminated over 1000
bitches using this procedure over a period of 31 years 1975-2003. Before 1994 the
recording procedure for the frozen semen AI's was only based on owners' report, so
data were not complete. From 1994, however, The Norwegian Kennel Club's semen
bank was organized, and the co-operation with the Norwegian Kennel Club's official
pedigree pup register was initiated to obtain accurate AI data. The second report of our
results was published 11 years after the first, and here a whelping rate of 67% and a
mean litter size of 5.6 pups were reported for. 30 bitches inseminated with frozen
semen into the uterus in a controlled trial (Farstad, 1984). Later, records of 150 bitches
inseminated with frozen semen during 1980-1989 showed the same whelping rate of
67% and 6.4 pups per litter (Farstad and Andersen Berg, 1989). Since the technique
was now considered standard procedure, no further reports were published from our
group until 2001, when our team of inseminating veterinarians had increased from
three to five. During the period 1994 to 1998 we reported an overail whelping rate in
312 bitches of 70% and a mean litter size of 5.3 pups (Thomassen et al, 2001).
Furthermore, a total of 381 bitches of 90 breeds were inseminated with frozen-thawed
semen from 250 different dogs over the 5-year period from 1999 to 2003. A total of
281/371 bitches (76%) gave birth to a mean litter size of 6.0 ±0.19 (SEM) pups. In 258
bitches the semen was inseminated into the uterus, and in 13 bitches the semen was
deposited deeply into the vagina due to failure to pass through the cervical canal. The
whelping rate was 79 and 0%, respectively. Bitches inseminated once into the uterus
130
had the same whelping rate as bitches inseminated twice (79%), but they had a smaller
litter size, 5.4 ±O.3 (SEM) versus 6.4 ±O.25 (SEM) pups.
Thus, the overall fertility results improved by nearly 10% from 1984 to 2003 (67%
versus 76%, respectively and with a small increase in Iitter size (5.5. and 6.0 pups,
respectively).
The improvement may be due to several factors, probably increased skill of individual
operators, improved freezing procedures both for domestic and imported frozen
semen, and more accurate oestrus detection procedures (improved accuracy of
quantitative progesterone analyses). The frozen semen fertility results by the NIU in
large numbers of bitches without selection for semen quality after freezing and thawing,
freezing method, storage vial (pellets or straws) breed (ie genetic litter size), operator
or timing of AI have probably reached their maximum in the range of 72-76% (whelping
rate). However, with selection for optimal timing and very good semen quality the
whelping rates increase to over 80% with this method (Thomassen et al, 2001).
The endoscope method (ElU)
Transcervical intrauterine insemination of the bitch with the aid of an endoscope and a
urinary or angioscopic catheter was first reported in 1993. The whelping rate presented
for the bitches inseminated by frozen semen in this material (32/40, 80%), was equal to
that first reported by Andersen using the NIU (Wilson, 1993). This author has since
published another review of the results (Wilson, 2001). Using a similar technique, but
with an angiography catheter to pass the cervix Battista et al (1988) obtained only 25%
whelping rate. Endoscopic insemination allows visualization of the insertion of the
catheter, but this method also requires sorne training to manipulate the cervix. The
team of Onclin et al (2003) claims to succeed weil with this technique, a number of
brochures from private semen banks and internet based information also refer to
whelping results in the proximity of 80%, but very few peer reviewed publications have
yet appeared with accurate fertility data from endoscopy aided intrauterine
insemination.
The surgical method (SIU)
Surgical AI, such as laparoscopic AI, is yet another procedure by which to secure that
the semen is properly deposited into the uterus. This method seems to be the method
of choice in veterinary clinics of North America and sorne practices in Europe. This
usually involves semen deposition only once. When done correctly by an experienced
surgeon, this method should be safe with relatively smail risk of post-surgical
complications. It does, however, necessitate general anaesthesia and thus poses a risk
of unwanted side effects of anaesthesia in older females or sensitive animais.
DISCUSSION AND CONCLUSION
ln many countries, in dogs as weil as in cattle, intrauterine AI is considered an invasive
technique only to be pertormed by veterinarians, trained AI technicians, or occasionally
by trained owners. Unauthorised use of "assisted insemination" is weil known among
breeders of certain breeds of dog. Thus, for sorne time already veterinarians have
competed with the experienced and skilled breeder, who knows how to collect an
ejaculate and insert semen into the vagina. A reasonable conception rate and litter size
may result from such inseminations if the bitch is inseminated within the time limit of
oestrus. There are no official records of the success rate of these owner pertormed
inseminations, but breeders claim that it is working very weil, while others say that it is
131
only a 50/50 chance of having puppies, which is probably close to the real rate or
somewhat higher (Farstad et al, 1984). The success rate also depends on the skill of
the operator, the breed and timing of the AI.
ln a study by Linde- Forsberg et al (1999) the insemination results with the Norwegian
lU catheter (NIU), lntrauterine AI by fiberoptic endoscope (ElU) or vaginal AI (VAG)
were compared with significantly higher results for the Norwegian lU catheter method
(84.4%, 57.9 and 58.9%, respectively for NIU, ElU and VAG). Assisted intrauterine
insemination irrespective of method has the advantage over vaginal AI that it is
possible to deposit the semen into the uterus also when the cervix is undergoing
closure. Thus it allows frozen semen, which has shorter longevity than freshly
ejaculated semen, to be used late in oestrus when the oocytes are ready to be
fertilized. The number of Ais may therefore be reduced to one or two. With intrauterine
AI conception rates and litter size increase even for fresh semen. This needs to be
communicated to the breeders in such a way that they understand the benefit of using
skilled veterinarians as opposed to inseminating the females themselves. It is
imperative that the veterinarians obtain better pregnancy results than the skilled
breeder, if not, using veterinarians for this procedure and having to bring the breeding
animais to a c1inic,may seem too impractical and expensive for the breeders.
The Norwegian intrauterine insemination technique enables the use of frozen semen
with an overall pregnancy rate that is as good as or better than the results from
intrauterine AI in cattle (75% whelping rate versus 45-50% calving rate in cattle). The
scarce reports in the literature from private canine practices and commercial semen
banks makes it difficult to assess the real whelping rate under field conditions for
endoscopie and surgical intrauterine inseminations, but it is assumed that the results
may be in the range of 70-90% whelping rate depending on the procedure. Since the
NIU and the ElU method need training, however, it is reasonable to believe that fertility
results will vary among different operators, and also results may vary between
laboratories and practices.
The laparoscopie procedure may provide less of a challenge to the skilled surgeon.
The surgical method may be preferred if the operator has no training in non-surgical lU
methods, the semen is of very poor quality, or there is very little semen available from a
particularly valuable stud dog. In some European countries, such as the Nordic
countries, it is considered unethical to perform surgical inseminations if there is an
alternative, non-surgical route. This seems not to be the case in the USA and in sorne
European countries where the surgical approach is the common method for AI (Brittain
et al, 1995; Silva et al, 1995). The endoscopie AI technique is also a safe and reliable
method, but also needs training. The operator is able to control the passage of the AI
catheter through visual inspection, an option preferred by many veterinarians. The
Norwegian transcervical method is based on palpation of the insemination deposition
site. Its greatest advantage over the endoscopie method is the cost of the equipment,
since it avoids the use of very expensive endoscopes. The catheters can be easily
sterilized by boiling in water and transported. Thus, AI can be carried out even in
private homes. From an animal welfare point of view insemination by the NIU or ElU
methods should be preferred since they eliminate surgical intervention and
consequently relieve the female from the stress caused by surgical trauma and
anaesthesia.
REFERENCES
Allen, WE and France C. A contrast radiography study of the vagina and uterus of the bitch. Journal of Small Animal
Practice 26, 153-166.
Andersen K. Insemination with frozen dog sem en based on a new insemination technique. Zuchthygiene 1975, 10,1-4.
132
Battista M Parks J Concannon PW . Canine sperm post-thaw survival following freezing in straws or pellets using
PIPES, lactose, TRIS or TEST extenders. Proceedings 11th International Congress on Animal Reproduction (ICAR)
Dublin, Ireland, 1988, 3, pp 229·231.
Brittain D, Concannon PW, Flanders JA, Flahive WJ, Lewis BL, Meyers-Wallen V, Moise NS. 1995. Use of surgical
intrauterine Insemination to manage infertility in a colony of research German shepherd dogs. Laboratory Animal
Science 45,404-407.
Doak RL, Allen H and Dale HE. Longevity of spermatozoa in the reproductive tract of the bitch. Journal of Reproduction
and Fertility 13, 51-58.
England GCW and Pacey AA. Transportation and interaction of dog spermatozoa within the reproductive tract of the
bitch: comparative aspects. Advances in Canine Reproduction, Centre for Reproductive Biology Report 3, Swedish
University of Agricultural Sciences, Uppsala, Sweden, 1998, pp 57-92.
Farstad W. Bitch Fertility after natural mating and after artificial Insemination with fresh or frozen semen. Journal of
Small Animal Practice 1984, 25, 561-565.
Farstad W, Andersen Berg K. Factors influencing the success rate of artifical Insemination with frozen semen in the dog.
Joumal of Reproduction and Fertility 1993, Suppl 39, 289-292.
Farstad W, Thomassen R, Krogenœs AK, Fougner JA. Artificial Insemination using the Norwegian transcervical catheter
for intrauterine semen deposition. Proceedings, the III EVSSAR Annual Meeting, Dublin, 2003, pp 42-47.
Nôtling JO, Gerstenberg C, Volkmann DH. Success with intravaginal Insemination of frozen-thawed semen- a
retrospective study. Journal of the South African Veterinary Medical Association 1995, 66, 49-55.
Rota A, Iguer-Ouada M, Verstegen J, Linde-Forsberg C. Fertility after vaginal or uterine deposition of dog semen
frozen in a Tris extender with or without Equex STM paste.Theriogenology 1999, 51, 1045-1058.
Silva LDM. Artifical vaginal and intrauterine Insemination after the cervical closurew in the Beagle bitch. PhD thesis,
University of Liege, Belgium, 1995, pp 11-120.
Silva LDM, Onclin K, Snaps F, Verstegen J. Laparoscopie intrauterine Insemination in the bitch. Theriogenology 1995a
43,615-23.
Silva LDM; Onclin K and Verstegen JP. Cervical opening in rlation to progesterone and oestradiol during heat in beagle
bitches. Joumal of Reproduction and Fertiltiy 1995b 104, 85-90.
Thomassen R, Farstad W, Krogenœs A, Fougner JA and Andersen Berg K. Artificial insemination with frozen semen in
dogs: a retrospective study. Journal of Reproduction and Fertility Supplement 2001,57,341-346.
van Gemert W. Diepvries-pups. Tijdschrift Diergeeneskunde 1970, 95, 697-699.
Wilson MS. Non-surgical intrauterine artificial Insemination in bitches using frozen semen. Journal of Reproduction and
Fertility Suppl. 47, 307-311.
Wilson MS . 1993. Non Surgical Intrauterine artificial Insemination in bitches using frozen semen. J ReprodFertii
(Suppl), 47, 307-311.
Wilson MS. Transcervical Insemination techniques in the bitch. Veterinary Clinics of North America Smail Animal
Practice, 2001, March 31, 291-304.
133
Abstracts
Chemotherapy in canine and feline mammary tumours.
A review of the existing and future protocols
Sonia FERNANDEZ PEREZ
The mammary neoplasms in the bitch are the second most common cancer, after skin
tumors. In the queen, mammary tumors are the third most common cancer followed by
skin and Iymphoid tissue tumors, which are more frequently reported in this specie 1•
Considering the existing differences among both canine and feline mammary cancer
(higher % of malignancy and higher incidence of metastasizing (50-90%)2 of feline
tumors compared with canine, different incidence of tumor type, different cancer
behaviour, ...), the treatment for both species is quite similar: surgery remains the
primary mode of therapy.
Chemotherapy is the second step, whether used alone or combined with surgery.
Radiation is infrequently used in this type of cancer. The use of chemotherapy is widely
extended among veterinarians for managing cancer, and many studies support its use
and efficacy, especially for Iymphoma and skin tumors. In the case of mammary
tumors, there is an important lack of literature to demonstrate the efficacy of
chemotherapy as a treatment for canine or feline mammary tumors. Even though, there
are many similarities between canine and human breast cancer such as age of onset,
histological type, metastasis, presence of estrogen receptors, the use of human
antibodies to detect canine proteins, ...especially with spontaneous canine mammary
tumor. This can be used as an excellent model to test new anticancer drugs for both
human and canine mammary cancer.
SAFE HANDLING OF CHEMOTHERAPEUTIC AGENTS
•
•
•
•
•
•
•
•
•
•
4,5
Always wear PVC or rubber gloves, even when handling oral medication (tablets,
pills, ...). It is also advisable to use gown with long sleeves.
NEVER break or crush capsules or tablets, especially cyclophosphamide, except if
allowed by rnanutacturer.
Always use surgical mask (to avoid inhalation) and protective visor or glasses (to
protect eyes in case of accidentai spilling).
Never work with mechanical ventilation, air conditioning or draughts. Setter to work
in a cabinet.
It is advisable to wash hands after handling any chemotherapeutic agent, and to
keep a water source near in case of spilling.
When spelling air from a filled syringe, exhaust into a pad, not into atmosphere.
Always use a gauze or pad to open vials or ampoules.
Always inform your staff and pet owners about medication handling and via of
excretion (urine, saliva, ...).
Always keep the animal well-confined and if necessary, sedate.
Use special containers for medications, used vials, syringes, needles, ...
135
COMMON CHEMOTHERAPY
Aoent
Doxorrubicin
Cyc/ophosphamide
Fluorouracil (5-FU)
USED FOR MAMMARY NEOPLASMS
Dose
Indication
30 mg/m~ IV every 2-3 weeks for 5 *Feline mammary carcinoma ti
tts.
* FAC protocol, for canine
mammarv carcinoma 7
100 mglm~ daily for 4 days ti
* Combined with doxorrubicin
for feline mammary carcinoma
200-250 mg/m2 q 3 weeks 8,7
or rnetastatic"
* FAC protocol, for canine
50 mg/m2 PO 9
mammary carcinoma 7
* VAC protocol, for canine
mammarv carcinoma 9
150 mg/m~ IV weekly
* FAC protocol, for canine
mammarv carcinoma 7
Mitoxantrone
Cisplatin/Carboplati
n
Vincristine
* VAC protocol, for canine
mammary carcinoma 9
NEW CHEMOTHERAPEUTIC
Agent
CCNU (Lomustine)
AGENTS
Dose
90 mglm2 PO every 21
days, for 4-5 cycles,
th en q 6-8 wk (doq)"
50-60 mg/m2 PO q
6wk.(cat)6,10
Tamoxifen (Nolvadex)/
Raloxifen
Paclitaxel
-
Doxorrub inhal
-
Doxil
-
-
REFERENCES
1.
2.
3.
4.
5.
Hahn KA, Adams W.H.: Feline Mammary Neoplasia. Feline Practice. Vol 5 N° 2. 1997
Johnston s.D., Root Kustritz M.V., Oison P.N.s.: Canine and Feline Theriogeno/ogy. 474-483. 2001
Dhaliwal RS : Canine Mammary Neoplasia. ACVIM 2003
Dobson J.M, Gorman N.: Cancer Chemotherapy in Small Animal Practice. BSAVA, 1993
FernàndezS. : Protoco/os de tratamiento y manejo de pacientes oncol6gicos. Quimioterapia: Tendencias actuales
y perspectivaS'. Jornadas de Trabajo de Oncoloçia y Reproducci6n. GERPAC. Fac Veterinaria Madrid, 1999.
6. Ogilvie G.K., Moore A.S. : Feline Onco/ogy. A comprehensive guide to compassionate care. VLS, 2001
7. Kitchell B.E., Dhaliwal RS.: Update anticancer drugs and protocols using traditional drugs. Kirk's Current
Veterinary Therapy XIII. W.B. Saunders , 2000.
8. Selting KA: Chemotherapy: Principles and Practice. 2002 SAVMA Symposium.
9.
Hahn KA, Richardson RC. CancerChemotherapy.
A Veterinary Handbook. Williams&Wilkins, 1995
10. Rassnick K.M. et al. Phase 1Evaluation of CCNU in tumor-bearing cats. JVIM, May-June 2001.
136
Abstracts
CANINE MALE INFERTILlTY:
CLiNICAL APPROACH
Alain FONTBONNE, DVM, MSc, Senior Lecturer, Dip. ECAR
Alfort Veterinary College, Paris, France
afontbonne @vet-alfort.fr
For many breeders, reproductive expectations are high for a dog which has won
several exhibitions or competition shows or has been genetically selected after
several generations. These persons are prepared to conduct several c1inical
investigations. Therefore, veterinarians are more and more often asked to diagnose
and solve fertility problems in the male dog.
Nowadays, very little is still known about male infertility in the dog. Regarding
human male infertility, the cause remains unknown in around 70% of cases. In the
area of human medicine, when the semen is of poor quality, most of the time
assisted reproduction techniques are utilized such as in vitro Fertilization (IVF) or
Intra-Cytoplasmic
Sperm Injection (ICSI). These techniques are not routinely
available to the dog. Only 10% of dogs presented with infertility may fertility be
restored after diagnosis and appropriate treatment.
Veterinarians willing to solve a fertility problem in the male dog must first conduct a
very detailed history of the general health and reproductive history of the dog. A
complete general and genital clinical examination must then be conducted.
Complementary
examinations
may, depending on the case, include sperm
collection and analysis, hormonal assays, urinalysis, ultrasonography, radiography
with or without contrast compounds, serological or bacteriological tests, testicular
biopsy, PCR. Of course, complementary evaluation should be designed to progress
from relatively easy and· inexpensive to more difficult and time consuming
diagnostic tests.
The main causes of male dog infertility include:
anatomical abnormalities: congenital or acquired;
low quality semen;
- oligospermia: lack of volume of the ejaculate
- aspermia: no ejaculate in a dog which has a normal libido and signs of
ejaculation
- oligozoospermia: lack of quantity of sperm cells in a normal volume
ejaculate
- teratozoospermia: presence of too many abnormal sperm cells in a
normal volume ejaculate
- asthenozoospermia: lack of moti/ity of sperm cells
- OAT
(term
often
used
in human
medicine):
Oligo-AsthenoTereiozoospermie;
prostate problems;
epididymal or testicular problems;
urinary problems (cystitis or uretritis);
hormonal problems, including central causes, hypothyroidism
or testicular
tumors;
infectious causes;
drugs;
139
genetic problems;
abnormal sexual behavior;
miscellaneous causes.
The following text provides a general pathway towards diagnosis. Our lecture will
concentrate on sorne c1inicalcases iIIustrating sorne major clinical situations.
1. LACK Of VOLUME Of SEMEN (ASPERMIA OR OLiGOSPERMIA)
The veterinarian may suspect a problem related to:
an incomplete collection of semen (Iack of libido or interest of the dog).
Normally, a male should begin to ejaculate within 20 to 30 seconds after
manually induced erection;
lack of sexual maturity;
the existence of pain during ejaculation;
a prostate problem;
a retrograde ejaculation;
other causes like neuropathy, spinal cord injury or diabetes mellitus.
We first recommend trying to collect the dog a second time, maybe under better
conditions (for example, using a teaser bitch in heat). Sorne experienced stud dogs
will not ejaculate during manual semen collection unless presented with an estrous
teaser bitch. Sometimes, they even refuse to give semen if they are not allowed to
mount the bitch.
If the result is still a lack of volume of semen, a general c1inicalexamination of the
dog must be done in addition to a urine collection by cystocentesis after semen
collection (to verify the presence of many spermatozoa in the bladder) and
ultrasonography of the prostate gland. If temporary aspermia is suspected,
veterinarians can perform a vaginal smear in the few minutes following a natural
mating with a bitch to see if any spermatozoa can be viewed. For treatment of
retrograde ejaculation: see lecture from Pro Stefano Romagnoli.
2. LACK Of SPERM MOTILITY (ASTHENOZOOSPERMIA)
Normally, dog semen samples should have more than 70% of the spermatozoa
exhibiting vigorous forward motility. In infertile men, asthenozoospermia occurs
when less than 25% of spermatozoa have normal motility. In dog, this terminology
may be used when less than 50% of spermatozoa have a normal forward motility.
This problem couId be due to the following:
the collection material that has not been correctly rinsed and may harbor
spermicidal substances like detergents or toxics: recollect the dog a few hours
later after having checked everything;
an inflammation of the urinary or genital organs, such as prostatitis, cystitis,
uretritis. It is useful to control the pH of semen and to perform a urinalysis,
ultrasonography of the genital tract including the prostate gland, examination of
the cytology of prostate fluid, bacteriologic examination of seminal fluid, etc...
the start of another genital problem, including testicular tumors.
140
3. AZOOSPERMIA
(OR VERY SEVERE OLiGOZOOSPERMIA)
Veterinarians should be aware that in many cases of true azoospermia the size of
testes remain unchanged.
The first problem is to determine if the case represents a true azoospermia or if the
collection of semen was incomplete, providing only the pre-sperm fraction without
spermatozoa. The semen always should be collected several times as artifactual
azoospermia may occur in dogs that are apprehensive at the time of collection and
may ejaculate only the first (pre-sperm) fraction. For similar reasons, the existence
of a prior vasectomy must be ruled out. It is first recommended to assay the alkaline
phosphates concentration in seminal fluid. This concentration is much higher in the
epididymis than in the testes or prostate. A normal intact dog with an incomplete
ejaculation usually shows a concentration of this enzyme in seminal fluid at less
than 5000 units/L, because little epididymal fluid has been ejaculated.
4. LOW QUALITY SEMEN (OAT)
The diagnosis procedure of an infertile dog suffering from OAT will be the same as
in the previous case: good clinical examination, ultrasonography, hormonal
assessment, bacteriological culture of seminal fluid, etc. Of course, Brucella canis
serological testing should always be performed, especially if sperm agglutination is
observed. Everything must be done quite quickly, because most of the time OAT is
a step towards complete azoospermia, and it may be reversible only during a short
time period.
Treatment of OAT in dogs depends upon etiology. With any treatment, no
improvement will occur for at least 62 to 70 days after the start of treatment, that is,
the duration of a normal spermatogenetic cycle.
Sorne authors recommend to do nothing in the first run: they let the dog rest
sexually and re-evaluate after 2 months, hoping that the OAT will have been
transient. Unilateral castration of dogs with unilateral testicular tumors may allow
spermatogenesis to improve in the contra-Iateral testicle. In the case of
inflammation, the return to normal fertility is unlikely to happen if fibrosis or
degenerative changes have occurred. Medical therapy for idiopathie OAT may be
tried but is very often very disappointing. Based upon our own experience,
repeated intrauterine inseminations of bitches could be successful. In case of
genital or urinary infection, specifie antibacterial compounds, depending upon the
antibiogram, may be used for a long period (at least 3 weeks to one month). Finally
a dog with frequent sexual usage and low quality semen may regain fertility when
used sparingly to allow spermatozoa to accumulate in the epididymis.
5. PRESENCE OF PUS IN THE EJACULATE (PYOSPERMIA)
Normal dogs should normally have less than 2000 white blood cells per microliter in the
first and second fraction. But the correlation between the number of leukocytes and
infection is not clear. Still, if many leukocytes are present in the sperm after
centrifugation, bacterial culture of the ejaculated seminal fluid may be done. Do not
forget to ask for mycoplasma and ureaplasma identification which usually necessitate
specifie transport and culture media.
141
Cultures must be interpreted cautiously. Bacteria that are isolated may represent a
primary infection causing infertility, but they may be contaminants.
6. BLOOO IN THE EJACULATE (HEMATOSPERMIA)
If no lesion of the penis is observed, a thorough clinical examination of the prostate,
including ultrasonography, must be conducted because a prostate problem is often the
initial cause of hematospermia. The specific treatment will depend upon the nature of
the prostate disease.
Sometimes, the coagulation parameters should be checked if nothing is viewed either
in the prostate or on the penis.
7. FAILURE TO ACHIEVE ERECTION
Oiagnosis of the cause of failure to achieve erection requires the careful observation of
copulation and/or semen collection. The veterinarian will notice if the dog experiences
pain. The localization of the pain will direct further diagnostics. If no semen can be
obtained, a blood sample should be taken to assay androgens and thyroxin levels. In
sorne cases, electroejaculation under general anesthesia could be considered as a
mean of obtaining semen from very recalcitrant dogs.
8. FAILURE TO ACHIEVE COPULATION
Non steroidal anti-inflammatory drugs in dogs with painful orthopedic conditions may
allow limited use of such dogs. Oogs with idiopathic poor libido could be given GnRH or
hCG injections prior to semen collection or breeding attempt. If they admit semen
collection, artificial insemination is often the best solution.
REFERENCES:
ENGLAND GCW, VERSTEGEN
Record, 2001, 148,20-22.
JP and HEWITT DA, Pregnancy
following in vitro fertilization
FRESHMAN JL, AMANN RP et al. Clinical evaluation of infertility in dogs. Compend.
10(43),433-460.
FULTON RM, KESKINTEPE
Theriogenology
JOHNSTON
Philadelphia
L et al. Intracytoplasmic
S.D., ROOT-KUSTRITZ
2001, 592 p.
of canine oocytes, Vet.
Contin. Educ. Pract. Vet. 1998,
Sperm Injection (ICSI) for the treatment of canine infertility.
M.V. and OLSON P.N.S.: Canine and Feline Theriogenology,
WB Saunders
KEENAN L.R.J. "The infertile Male" in England and Harvey, BSAVA Manual of Small Animal Reproduction
Neonatalogy, BSAVA Ed. 1998, 83-93.
MEYERS-WALLEN
V.N. "Clinical approach
Small. An. Pract. 1991, 21 (3), 609 - 633
for evaluating dogs with azoospermla
Ed.
and
or aspermia" Vet. CI. North Am.
OLSON P. "Clinical approach to infertile male with sperm in the ejaculate" Vet. CI. North Am. Small. An. Pract. 1991,
21(3),531-608
142
Abstracts
CANINE SEM EN BAN KING AND SHIPMENT FROM A EUROPEAN
PERSPECTIVE
Catharina LINDE FORSBERG DVM, PhD, Dipl ECAR
(Lectured by Alain Fontbonne)
Department of Obstetrics and Gynaecology, P.O. Box 7039, Faculty of Veterinary
Medicine, Swedish University of Agricultural Sciences, S-750 07 Uppsala,
Sweden. Tel.: + 46 18 6721 63. Fax.: + 46 18673545, email: Catharina.LindeForsberg@og.slu.se.
The interest in international shipment of dog semen is steadily increasing and the
number of storage facilities for frozen canine semen is also increasing, although at a
somewhat slower pace. Dog semen can be shipped as extended and chilled, or frozen. What
is most convenient will depend on a number of factors, such as shipping distance, and
whether the shipped sernen is intended for artificial insemination (AI) of one bitch or of
several bitches. Sometimes the regulations in the importing country preclude the possibility of
using chilied semen. In sorne countries there are different rules for semen coming from
different countries and sorne (eg the UK and the USA) have rules that also regulate the
export of semen. Kennel Clubs may request prior application for permission to use imported
semen, and usually also request proper identification by microchip or ID-tattoo, or a DNA
test. The regulations may change at any time. Before undertaking to export dog semen it is
therefore strongly adviced that the bitch owner contacts the Ministry of Agriculture and the
Kennel Club of the importing country. These contacts should be made weil in advance of the
planned semen export, so that ail the necessary health certificates and blood tests can be
made in accordance with the requirements. Sorne countries have no regulations, whereas
others request an import permit, a health certificate at the time of semen collection, and/or at
a stipulated time interval before or after semen collection, including a serological test for
brucella canis and leptospirosis. Sorne countries request that the dog from which the semen
is collected must not have been outside its home country for, for instance, 6 months before
the semen collection, or used for natural breeding from the time of the blood test until semen
collection, and that the inseminated bitch must not be bred by another dog during the same
cycle and that in case she aborts the fetuses should be examined. Minimum documentation
to accompany the dog semen shipment should consist of a health -certificate, and a certificate
of the quality of thesemen including its identifying markings and a recommendation of the
amount of semen to be used per AI. Those certificates should be enclosed with the
shipment, with a full set of copies inside the outer casing in case the originals are lost
underway, or removed by mistake for instance by customs in a transit country.
CHILLED EXTENDED DOG SEMEN
Chilled dog semen can retain its motility and membrane integrity for 1 - 3 weeks,
although it still remains to be tested for how long it also retains itsfertilizing capacity.
The chilled semen is usually sent in an ordinary thermos flask, or a styrofoam box with
ice-packs. It is important that the temperature stays above zero so that the semen
doesn't freeze. The thermos f1askand the styrofoam box weigh little and usually need
not be returned, thus keeping the shipping costs low.
Mark the semen vial with breed, name, registration number, date of collection and
place (see below). Whenever this is practically possible an official seal should be
applied on the thermos flask or the styrofoam box. This is required in sorne countries,
and it prevents unauthorized persons from opening the inner package containing the
semen. The identification of the seal should be stated in the veterinary certificate.
145
Frozen dog semen
Frozen dog semen can be stored practically indefinitely, probably for 1- 2000 years or
more. Dog semen can be frozen in 0.5 or 0.25 ml French straws or in pellets. Frozen
semenis shipped in a liquid nitrogen container that keeps the temperature at -197°C.
Today most semen agencies use so-called dry shippers, which absorb the Iiquid
nitrogen into a porous matèrial in their walls.These need not to be shipped as
dangerous goods but should always be sent as fragile goods, because they are easily
broken by rough handling. The rules pertaining to air transportation of dry shippers can
be found in the IATA packing instruction 202, Note.
It should be made a routine to check that the Iiquid nitrogen dewar is not broken.
Therefore, always fill the dry shipper with liquid nitrogen at least 24 hours before the
shipment is due. The tank needs to be filled over a period of time, and left to saturate
between fillings. Some liquid nitrogen seen to remain at the bottom of the tank is the
sign that it is full. It can also be weighed before and after filling to check that it contains
the proper amount of liquid nitrogen. The tank should be left over-night and its weight
checked again before it is shipped. The most commonly used dry shippers take from 2
to 4 kg of LN2, and have a holding time of 10-20 days. The containers are expensive
and are usually on loan from the semen agency and should be returned as soon as
possible. When they are full and in their outer protective casing their weight is usually
between 7 and 15 kg.
General recommendations
for minimum documentation
• It is recommended that each international shipment of canine semen should
be accompanied bya general veterinary health certificate, including a
statement that the dog has normal testicular status.
• It should also be stated in the certificate that the identity of the dog was
controlled and how this was done. Remember always to use the dogs
registered name, not its pet name, for ail official documents, including the
blood test report.
• Always enclose a set of copies of the certificates attached on the outside of
the box, or inside it in case the freight bill with the original certificates is lost on
the way.
• There should always be an accompanying semen quality assessment and
thawing instructions, together with a recommendation of how many straws, or
vials of pelletted semen, that should be used for each AI. (See the Appendix.)
• Straws (or pellet vials) should be packaged so that they are easy to move from
the dry-shipper to the storage tank, i.e. put in canes, goblets, or plastic sheats
which are properly marked (see below). If pellets are sent, don't forget to
enclose the plastic bags used for the thawinq (Whirl-pak, Nasco, USA) as in
many countries these are not available.
• Always seal the thermos flask or LN2 dewar, also when this is not a requirement. This prevents unauthorized persons from opening it and minimizes the
risks of damage. Do not put the seal on the outer case, but directly on the f1ask
dewar. The LN2 dewars usually have a ring for seals.
The semen straws (or vials) should always be marked with:
• the breed (which may be abbreviated),
• the dogs name as registered with the Kennel Club (which may be
146
abbreviated) ,
• its registration number in the Kennel Club (or corresponding body),
• the date of semen collection (absolutely necessary in the cases
where blood tests and veterinary certificates are required), and
• where the semen was processed.
Example:
!Afghan hd B.S.5andstorm
5-06689-99
12-3-2004
SLul
The identification markings of the straws should also appear in the certificates.
How much semen to send.
It is still generally recommended to use at least 150-200 million motile, morphologic-ally
normal spermatozoa per AI, and to inseminate twice. If the ejaculate contains abnormal
spermatozoa or the post-thaw motility is low the total number of sperma-tozoa per breeding
unit should be increased accordingly in an attempt to compen-sate for this. If the semen is of
poor quality, however, it should not be shipped unless the bitch owner is informed about the
situation and has given his/her consent. Surgical AI, which is still done in sorne countries,
requires less spermatozoa and is usually only done once, while for vaginal Ais 10 times more
spermatozoa are needed than for intrauterine Ais. Considering the high costs of shipping
frozen semen it might be a good idea to send enough semen for a repeat breeding in case
the bitch doesn't get pregnant on the first occasion. If the importer has paid for the freezing, it
may also be reasonable to send ail the resulting semen doses, unless other agreements
have been made.
Freight companies
It can be quite time consuming to organize shipments, and it almost always involves
customs clearance. Freight charges appear to be quite negotiable why it may be better to
leave this ail to the dog owners to organize. They will need to know that the code for Iiquid
nitrogen is "UN1977", and for the dry-shippers the "IATA Packing instruction 202, Note"
which clarifies that this kind of tank can be shipped as non-dangerous goods.There should
also be an accomanying statement for customs that the tank is on loan and that it will be
returned as an empty packaging, to avoid VAT or a customs fee.
Canine frozen semen banking
The rules and regulations for the canine semen banks vary considerably between
countries. Again, both the Ministry of Agriculture (or the corresponding authority) and
the Kennel Clubs and the Breed Clubs may have regulations pertaining to canine
semen banks and use of frozen semen. The major concern of the Ministry is the
prevention of spread of disease, the correct identification of semen doses, and animal
welfare. The main interest of the Kennel Clubs and Breed Clubs usually is to ascertain
that the semen donors are correctly identified, and the frozen semen straws properly
marked with the identity of the dog. A number of countries, as of yet, have no
regulations whatsoever, whereas in sorne the use of artificial insemination in dogs is
not allowed.
Who may run a canine semen bank?
It may be necessary to apply for permission to store canine semen both from the
National Ministry of Agriculture and the Kennel Club or Breed Club. In many countries
this right is restricted to the veterinary profession. The Kennel Clubs and Breed Clubs
may only accept to register Iitters from frozen semen if it has been stored in one of the
acknowledged semen banks. The American Kennel Club (AKC) acknowledges quite a
large number of semen freezing and storing localities in the USA, The French Kennel
Club (LOF) three, at the veterinary colleges in Maisons Alfort, Lyon and Nantes, and for
instance the Swedish Kennel Club (SKK) at the moment only one, the semen bank at
the Swedish University of Agricultural Sciences (SLU).
147
From which dogs may semen be frozen?
There may or may not be rules for from which dogs semen may be frozen and stored.
The most common requirements are that the dog should have two normal testicles, be
permanently identified byan ID-tattoo or a micro-chip, and in sorne breeds they must
be declared to be free from various hereditary diseases Le. hip dysplasia or various
eye or kidney diseases. Another requirement may be that they have shown that they
can mate normally and/or have proved that they are fertile.
What records should be kept by the semen bank?
Sorne basic data should always be supplied for each dog who's semen is frozen, in
order that the semen can be eligible for use in the future. This is:
• breed,
• the dog's registered name,
• registration number,
• date of birth,
• a health certificate including testicular status,
• how the correct identity of the dog was assessed,
• name etc. of the owner of the dog
• name etc. of the owner of the stored semen
• date of the semen collections and freezings,
• semen quality
• the identitying markings on the semen straws (see above),
• number of straws in storage and how many breeding units they
constitute.
Additional requirements.
If ail or sorne of the semen is likely to be exported to other countries blood tests,
usually for brucella canis and for leptospira ichterohaemorrhagica and canicola may be
required, taken either before (usually within 15 to 45 days) or after (between 20 and 30
days, or from 3-6 weeks) the semen collection. The regulations pertaining to each
country should always be checked weil in advance of the freezings to avoid problems
and the risk of having to discard the semen or the frustration and extra costs of
returning it to its country of origin.
Who is the owner of the stored semen?
The canine semen bank should have a contract with the person(s) using its storage
facilities. The owner of the stored semen in most cases is the dog owner, but just as
likely and especially when the semen has been imported, it may belong to the
importer/bitch owner, who has payed for the freezing and ail the other costs for the
importation. In sorne countries it is the bank itself that owns the semen. A declaration
from the owner of the stud dog that the semen may be used for a particular bitch and
that the stud fee has been payed may or may not be required for the registration of
the resulting litter(s). From the storage contract between the bank and the owner of
the semen it should also be c1earwho has the right to use the semen stored in the
bank. If more than one person is registered as owner of the semen, either ail of them
may have to be present when semen is used, or a written agreement between the
owners should be presented to the bank, stating how many breeding units may be
taken out. The contract should also state what should be done with the semen in
case of the death of the registered owner, the options usually being that the
ownership is transferred to someone else, returned to the owner of the stud dog, or
that the semen be destroyed. Another aspect that should be discussed is what should
be done with the semen should the semen bank for sorne reason close down.
148
Who may perform A.I.?
Both national legislation and the Kennel Clubs also may have regulations regarding the
right to perform AI in dogs. In severai countries only veterinarians may perform AI in
dogs, sometimes they need to have passed a special course for AI in dogs. In other
countries the breeders themselves may inseminate their own dogs. When it cornes to
frozen semen, however, this is unlikely to occur, because of the special qualifications
needed for the handling of the semen and the generally poor results obtained with
vaginal AI especially of frozen-thawed semen.
149
Appendix.
CERTIFICATE
Name of dog:
Breed:...
ID-tattoo no:
OF COLLECTION
VETERINARY
CERTIFICATE
OF DOG SEMEN
KCReg.no:
Date of birth:
Photograph:
Nose print:
.
.
.
1hereby certify that the dog described above was presented for semen collection
on
(date)
From the collection resulted
or
The semen can be identified
a)
medium straws
b)
ml of fresh semen
as follows
[breed,
(0.5 ml)
name (may be abbreviated),
reg.no., date and place of collection]:
KC
.
Colour of straws/plug:
'"
.
Type of extender/cryoprotective:
.
Recommended thawing procedure:
.
Recommended
number of straws per artificial insemination:
Semen gualitv
(if frozen. state post-thaw guality):
Total number of spermatozoa:
.
Motility:
Number of spermatozoa/straw:
%
Abnormal sperm
%
At the time of semen collection 1examined the semen donor and found him free
from clinical signs of disease. The sem en donor has two normal testicles, fully
descended in the scrotum.
Signature of veterinary surgeon:
.
Printed name:
.
Address:
Phone:...
.
..
Fax:
.
Declaration by stud owner
1hereby certify that the above dog from which semen has been collected is the alleged dog,
which righttully belongs to me.
150
Signature of owner:
,
,
.
Printed name:
.
Address:
Phone:
.
Fax:
.
151
Abstracts
OVULATION
DIAGNOSIS ATTEMPT
USING OVARIAN UL TRASONOGRAPHY
Alain FONTBONNE, Delphine RAUL T, Elise MALANDAIN, Stéphanie BOSCHIERO, Nicolas
TRUELLE, Marie DUMASY and Dominique BEGON.
Alfort Veterinary College, Paris, France
afontbonne @vet-alfort.fr
For many years, ovarian ultrasonography of non-pathological ovaries was supposed to be
difficult, or even impossible to perform in the bitch, due to the presence of a fatty ovarian
bursa hiding the good imaging of intra-ovarian follicles. Nowadays, the new generations of
ultrasound machines in veterinary medicine are of much better quality and offer a valuable
method to observe the ovarian changes during the oestrous cycle.
ln the queen, following of the reproductive cycle has been empirical for a long time, but the
development of new techniques of assisted reproduction, like artificial insemination, has lead
to a more rational use of ovarian ultrasonography.
We have been trying to use ovarian ultrasonography in bitches and queens at the Alfort
Veterinary College (Paris, France) for more than 2 years. Our lecture will emphasize on the
practical points of view.
1. IN THE BITCH.
1.1. INDICATIONS:
There are numerous cases in which performing ovarian ultrasonography
may be valuable:
in bitches in heat
- Very precise timing of ovulation;
- improving the results obtained after natural mating or A.I., especially when using fresh
chilled or frozen semen;
- ln situ visualisation of non-ovulating follicles or ovulation problems, helping to make a more
rational use of hormonal treatments and to check the result of such treatments,
- counting the number of follicles and thus evaluating the fertility potential of the bitch
(especially for ail bitches);
- Diagnosis of ovarian cysts or tumours.
1.2."
METHOD:
Bitches are often put on a dorsal or dorso-Iateral position. Most of the time, it is not
necessary to sedate them. Hair may be shaved on a limited area, in order to improve the
contact between the probe and the skin. It is recommended to c1ean the skin using alcohol;
then, a chlorhexidine solution (Hibitane ~ may be applied.
It is necessary to use high frequency linear probes (7,5 à 12 MHz). It takes from 5 to 15
minutes to make a good ultrasonographic survey of the two ovaries, provided that the bitch
does not move too much.
1.3.
ULTRASOUND
ASPECTS Of THE OVARIES:
Although there are sorne individual specificities, the ultrasonographic
during the estrous cycle follow the same general pattern:
appearance of ovaries
153
PRE-OVULATION PERIOD:
The ovaries appear like ovoid structures, slightly hypoechoic, with a regular smooth shape.
The growing follicles appear to be round anechoic structures. Their size increases from 0.3
cm at the beginning of prooestrus up to 0.6 to 0.9 cm at the beginning of oestrus. At the
same time, the thickness of the follicular wall increases in thedays just prior to ovulation
(pre-ovulatory luteinisation of follicles). Their number decreases when comparing the
beginning of prooestrus and the pre-ovulation period. During oestrus, the ovaries appear
slightly bigger and easier to identify by ultrasound.
OVULATION PERIOD:
During a short period (half a day or maximum one day), the ovaries become difficult to find
by ultrasound. This change of the ovarian aspect is easy to identify when repeated
ultrasound examinations have been made in the preceeding days. Most of the time, the
ovaries do not appear smooth and homogeneous. Intra-ovarian structures are still observed,
but they appear smaller and not so round than before. Quite often, sorne « non-ovulated
follicles », showing no collapsus, may persist in the middle of collapsed follicles, which can
be confusing (in such a case, it is very useful to perform also progesterone assays). The
shape of the ovaries appears often much more irregular than at the beginning of the heat
period.
POST-OVULATION PERIOD:
The ovaries remain with an irregular shape. Corpora lutea appear soon after ovulation: at
first they look like round anechoic structures with a thick wall. It very much looks like preovulatory follicles. Thus, ovaries look more or less the same just before and just after
ovulation. Therefore, it is important to perform daily ovarian examinations in order not to miss
the ovulation period and to get confused.
1.4.
UTRASONOGRAPHY
OTHER METHOOS.
OF THE OVARIES:
INTEREST IN COMPARISON
WITH
ln most bitches, we have observed a good synchronisation of ovulation between the two
ovaries. In an unpublished study, estimated time of ovulation using ultrasound was found to
occur 3 days after the LH peak in 4 bitches / 7. Slood progesterone level at the day of
ovulation was always close to 5 or 6 ng/ml, with a chemi-Iuminescence assay (Elecsys®.
Roche Diagnostics. Germany). Still, ovarian ultrasonography seems to offer a better timing
and precision, and is the easiest method to monitor ovulation problems in the bitch.
2. IN THE QUEEN:
ln this species, it is essential to use high frequency probes. Queens are not always easy to
manipulate, therefore the ultrasound survey of both ovaries takes longer. A very quiet
environment is absolutely required !
Very often, the ovarian imagings, and the observation of ovarian follicles, are of better quality
than what can be seen in the bitch, due the absence of fat around the ovaries. It is therefore
easier to count the number of follicles. In a preliminary study (Ma/andain et al. 2002), the
maximal diameter of follicles was found to be between 0.25 to 0.35 cm. This maximal size
was observed at different intervals from the beginning of oestrous behaviour. In the queen,
ultrasonography of the ovaries offers a much better precision than vaginal smears; it could
154
also prove to be a good method to decide when to induce ovulation. Jt is also a mean to
check if inducing ovulation has succeeded or not.
REFERENCES
England G.C.w.
1989.
and Allen W.E. : Ultrasonographic
and histological
appearance
of the canine ovary. Vet. Rec., 125,555-556,
Hase M, Hori T, Kawakami E. and Tsutsui T.: Plasma LH and progesterone levels before and after ovulation
ovarian follicles by ultrasonographic diagnosis system in dogs. J.Vet. Med.Sci. 62 (3): 243-248, 2000.
Hayer P., Günzel-Appel A.R., Luerssen D. and Hoppen H.a.: Ultrasonographic
and the earty luteal phase in the bitch. J. Reprod. Fert. ,Suppl. 47, 93-100,1993.
monitoring
of follicular
and observation
development,
of
ovulation
Malandain E., Rault D., Froment E., Ficheux C., Pichard J.P., Fontbonne A., Begon D. and Chastant-Maillard
S.: Follow-up of
d
follicular growth during non-ovulatory estral phases in queen by ultrasonography. 3r EVSSAR congress. Liège, May1 0-12 2002.
155
EMERGENCY SURGERY IN REPRODUCTION
Félix GARCIA, Anna ANDALUZ. Departament of Animal Medicine and 8urgery .
Veterinary Faculty of Barcelona (UAB).
Felix.Garcia @uab.es
VAGINAL PROLAPSEI HIPERPLASIA
Vaginal prolapse/hiperplasia is most common in large breed-dogs and it seems to have a
familial predisposition. The most important finding is the protrusion of a mass from the vulva.
Normally the protrusion affects ail the circumference of the vulva. Nevertheless the partial
protrusion may occur.
This pathology appears during the oestrus or proestrus and it is more common during one f
the first three cycles. During the oestrus the mucosa becomes hyperaemic, oedematous and
keratinized. Prolapse or hyperplasia occurs because of the eversion of the mucosa. The
amount of eversion is variable and it may involve ail the circumference of the vulva.
Nevertheless the origin of the mass is usually small (1 cm) and located on the vaginal floor
cranial to the urethral orifice.
The oedema increases after the eversion of the tissues. The abrasion with the floor and
licking traumatizes the tissues resulting in ulceration and bleeding. Hematuria and disuria
may appear if the oedema affects the urinary orifice. After the oestrus the oedema
disappears. Nevertheless the ulcerated tissues recommend the surgical treatment. The
problem may recur in each succeeding oestrus cycle.
For the surgical approach is necessary to lavage the protruded tissues. Reduce the
prolapsed tissue by digital manipulation and place two or three horizontal mattress sutures
between the vulvar Iips. The tension of the suture may be diminished using buttons or rubber
tubs. Ovariohystectomy is recommended to prevent recurrence and injury to the everted
mucosa.
UTERINE TORSION
Uterine Torsion is infrequent in dogs. One or both uterine homs can twist along the long axis
or around the opposite homo Normally 1 see torsion of gravid uterus. They are not racial
predisposition but in Pomeranian is more common.
The ethioloqy include jumping in pregnancy, fetal movement, partial abortion and
abnormalities in length and mobility of the proper ovarian ligament.
The first symptom is abdominal pain. The queen begins the movements of parturition, but
they are not discharge. The abdomen can be tense and distended,radiographic examination
shows a large air or f1uid-filledtubular structure.
The treatment of choice is ovariohisterectomy. Because massive sequestration of fluid into
the uterus and vascular compression are possible preoperative supportive therapy may be
necessary. If they are a viable hom, and the queen have an special reproductive interest, we
can practice partial ovariohisterectomy of the affected hom with histeropexia of the another
homo
157
TESTICULAR TORSION
Testicular torsion is infrequent in dogs, only in cryptorchid dogs is possible. The testicular
torsion happens because the testicle is not protected and fixed by the vaginal tunica so it can
move freely in the abdominal cavity. The physical examination shows acute abdominal pain.
Ultrasonography is useful to perform the diagnosis. The surgical treatment includes a
laparotomy and orchiectomy.
PENIS NECROSIS
Penis necrosis is the last state of paraphimosis. When the penis is unable to retract it is
easily traumatized, the circulation is impaired and it becomes edematous. The increased size
and the inability to retract the penis into the prepuce induce the necrosis. Vascular
engorgement may progress to trombosis of the corpus spongiosum and necrosis.
The process requires the penis amputation and scrotai urethrostomy. Make an elliptical
incision around the prepuce, and penis preserving adequate skin for closure. Dissect the
penis from the body wall from cranial to caudal, ligate the preputial vessels, locate and Iigate
the dorsal penile vessels. Place a circunferential ligature in the penis with reabsorbible
material (Monosyn~ caudal to the place of amputation. Now we perform the urethrostomy,
we begin with the ablation of the scrotum preserving adequate skin for the suture,
orchydectomy with open technique and lateralitation of retractor penis muscle to open the
urethra. Appose urethral mucosa to skin at the urethrostomy site with simple continous
suture (6/0 reabsorbible material) take cavernous tissue to prevent the postoperative
hemorrage.
158
Abstracts
long term release GnRH agonists and anti-estrogens
in canine reproduction
CRISTINA GOBELLO, DR MED VET, DIPL ECAR
Faculty of Veterinary Medicine, National University of La Plata, Argentina
New pharmacological presentations and compounds are constantly being tested in
canine reproduction, being most of them, not authorized for their use in this species yet.
Long term release gonadotropin releasing hormone (GnRH) agonists and anti-estrogens
have been used in human medicine for years. Although, not many studies have been carried
out with these drugs in dogs. Further clinical trials are necessary to establish their safety and
eventual indications before they could be widely recommended. The aim of this presentation
is to summarize the main pharmacological properties and published results with long term
release GnRH agonists and antiestrogens in dogs. Possible indications of these compounds
on canine reproduction are also briefly quoted.
LONG TERM RELEASE GnRH AGONISTS
Synthetic GnRH agonists like nafarelin, leuprolide, deslorelin, buserelin and goserelin
stimulate production and release of gonadotrophins from the pituitary with different
potencies. Conversely, GnRH agonists, when used at sustained doses, reversibly inhibit
the gonadal axis after a short period of stimulation. Inhibition is produced by
downregulation of anterior pituitary GnRH receptors (16). Continuous administration or
long term release formulations of GnRH agonists have shown to reversibly suppress
reproductive function in male and female dogs for periods that exceeded one year in
sorne studies (4, 5, 8, 16, 21, 22).
The previous disadvantage with this compounds was the need for frequent subcutaneous
injections or pumps over prolonged periods of time. A significant advance in this field has
been the development of slow-release formulations of these agonists that can be easily
implanted subcutaneously every month/s or even year depending on the compound.
Thus, in a first report in which a single subcutaneous injection of leuprolide acetate at 1mg/kg
administered to 5 male dogs caused decreased ejaculatory volume and appearance of
morphological abnormal spermatozoa. An initial rise in luteinizing hormone (LH) and
testosterone (T2) serum concentrations followed by a marked decline to below the normal
concentrations for 6 weeks was also described in this study. Twenty weeks after treatment, a
return to normal spermatogenesis was observed (8).
ln a more recent study, 30 mature dogs received subcutaneous implants with doses ranging
from 0.08-0.79 mg/kg of deslorelin (6-D-tryptophan-9-[N-Ethyl-L Prolinamide]-10desglycinamide) acetate and 11 were re-irnplanted either before or after the suppression
period. Their T2 concentrations decreased to <1 ng/ ml within a mean of 17 days after the
implantation and remained at this level from 3 months to 2.7 years. The length of the
inhibitory effect seemed to be dose related and restoration to normalcy was evidenced in
scrotal circumference, T2 concentrations, semen quality and fertility (21). In another report,
0.5 -1mg/kg of the same agonist implanted in 5 dogs significantly decreased serum T2 and
prostatic size for 32 and 48 weeks, respectively (18). Recently, in seven dogs which were
injected 6.6 mg of buserelin, T2 and estradiol decreased to basal concentrations within 15
days of implantation and remained so for a mean of 233 days. Testicular and prostatic size
reversible reduced and no semen could be collected 21 days after injection (19).
161
Deslorelin acetate is available in the veterinary market of New Zealand and Australia in two
sizes (5 and 10 mg) for fertility control, treatment of T2 related behavior and benign prostatic
hyperplasia in male dogs with an expected duration of at least 6 and 12 months, respectively.
The GnRH agonist [D-Trp6, des-Gly-NH210] GnRH ethylamide subcutaneously administered
daily to prepubertal male and female dogs for 23 months decreased steroid hormones and
reversible delay puberty with normal fertility after a period of recovery (14)
ln 3 female dogs implanted subcutaneously with osmotic pumps of nafarelin acetate (18
f.lg/kg/day) during proestrus, estrous cycles were suppressed for 18 months. They
recovered cyclicing 3 to 18 weeks after cessation of treatment. When the same drug was
administered to anestrous bitches an infertile ovulatory heat was induced 1-2 weeks after the
start of treatment. Finally, postponement of puberty during 18 months of treatment was
obtained in 3 bitches in the same study (16).
ln a more recent report, pregnant, diestrous and anestrous bitches were treated with doses
ranging from 0.1-2.4 mg/ kg of deslorelin acetate as subcutaneous implants. Treatment
prolonged interestrous intervals up to 27 months independently of the stage of the cycle at
which it was initiated. When serum progesterone (P4) was below 5 ng/ml an induced estrous
cycle was provoked 4-8 days after implantation. Six of 9 bitches that were mated after
recovery of the treatment became pregnant (21). In a study in which azagly- nafarelin was
subcutaneously administered to 6 bitches no estrous behavior and low P4 and follicle
stimulating hormone was found for one year of treatment (20).
ln a first trial, the expected initial estrous response induced in anestrous bitches after 6 mg
deslorelin implantation was suppressed by a 21 or 14 day oral treatment of 2.2 mg/kg
megestrol acetate began 2 or 1 week/s, respectively before implantation (24). A shorter, 8
day protocol of the same dose of megestrol acetate began one day before implantation of
10 mg/ bitch of the same agonist prevented estrous response in 4 of 8 treated bitches (5).
Further refinement of these combined protocols is still necessary to assure practicality, safety
and effectiveness of contraceptive use of GnRH agonists in anestrous bitches.
Goserelin was used successfully every 21 days for 12 months in the treatment of canine
mammary gland tumors (15). Systematic re-implantation of long term release of GnRH
agonists couId offer an essentially permanent non-surgical option for dogs with high
anesthesia risk suffering hormone dependent diseases.
Conversely, taking advantage of the initial stimulatory effect of long term release GnRH
agonists, they have been used for estrous induction in pospubertal and prepubertal bitches.
A single subcutaneous injection of a sustained release formulation of the potent GnRH
agonist, leuprolide acetate ([D-Leu6 ,Pro9 Net]-GnRH; 100 f.lg/kg), followed by fertirelin on the
first day of induced estrus provoked behavioral estrus and pregnancy in 100, and 50 to
100%, respectively of the treated bitches (9). Removal of the 2.1 deslorelin implant at the
time of the preovulatory LH peak to prevent premature luteal failure by excessive long
release of the compound induced 100 % estrus and 50 % pregnancy in 6 treated bitches in a
recent report (12). Only one of 8 bitches treated with a same protocol but without removal of
the implants led pregnancy to term in a previous report by same authors (13).
ANTIESTROGENS
Several pharmacologie groups of compounds that inhibit or modify estrogens are classified
as antiestrogens. Sorne of them are the GnRH agonists and aromatase inhibitors which
inhibit estrogen synthesis. Another specific antiestrogenic group are the receptors blockers
antiestrogens (23). Clomiphene and tamoxifen citrate are synthetic non steroideal Type 1
162
antiestrogenic compounds which competitively block estrogen receptors with a mixed
antagonist-agonist effect. Type 1estrogen antagonists partially inhibit the action of agonists,
but due to their own agonistic properties, they also induce to sorne extent estrogenic
response. The manifestation of these different actions depends on each species, organ,
tissue and cell type considered (7). For example, in women, tamoxifen exerts antiestrogenic
activity on the mammary gland and agonistic effect on the uterus (10). The exact
mechanisms of this duality is not completely understood but it may depend on the expression
of specific cell estrogen receptor variants (7). In humans, receptors blocker antiestrogens
have been indicated in the treatment of male and female infertility and breast cancer (1,10,
23).
Little is known about the effect of receptors blockers antiestrogens in dogs, and most of it
has been carried out using tamoxifen, which seems to act rather like an agonist than
antagonist (2, 3, 6, 11,17), severely Iimiting its use in female dogs.
Thus, signs of proestrus were induced in 2 of 4 bitches after a 10 day, 12.5- 25 mg
clomiphene citrate treatment given orally (6). Although, tamoxifen showed to be efficacious
in preventing or terminating canine pregnancy when administered during proestrus, estrus or
diestrus it was associated with estrogen like side effects. Endometris, pyometra and ovarian
cysts developed in 9 out of 20 treated bitches with the oral dose of 1mg/kg twice daily (2).
ln one study in which the same drug was administered in dosages raging from 2.5 to 10 mg
orally twice daily to 7 bitches with inoperable or metastatic mammary carcinoma, tamoxifen
reduced tumor burden in 5 animais. Clinical toxicities were vulvar swelling, vaginal discharge,
incontinence, and pyometra (11). In another report, 18 bitches were treated with tamoxifen
along with mastectomy and ovariectomy. Ten bitches showed estrogen like signs and no
antitumor activity could be documented (17). Additional studies are needed to establish the
potential benefit of antiestrogen therapy for canine mammary tumors, particularly when
coupled with hormone receptors assays. Caution should be exercised in prescribing
tamoxifen because of the high percentage of estrogen like side effects on the female genital
tract.
ln a first study in which 7 Beagle male dogs were daily administered 2.5 mg tamoxifen for 28
days, it showed to negatively affect testicular size and libido. It also decreased prostatic
volume and T2 concentrations during treatment. Semen quality deteriorated to nadir results
one' spermatic cycle after treatment. Then, it returned to pre-treatment values on the second
cycle after treatment, except in one oligoazoospermic dog in which sperm count was higher
at that time. No clinical nor haematological side effects were observed and fertility was
conserved at the end of the study (3).
REFERENCES
Ain Melk Y, Belisle S, Carmel M, Tetreault J P 1987 Tamoxifen citrate in male infertility Fertil Steril 48: 113-117
Bowen RA, Oison PN, Young S, Withhrow SJ. 1988 Efficacy and toxicity of tamoxifen for prevention and termination of
pregnancy in bitches. Am J Vet Res 49:27-31.
Corrada Y, Arias D, Rodriguez R, Spaini , Fava F, Gobello C. Effect of tamoxifen citrate on reproductive parameters of male
dogs. Theriogenology
(in press)
Dube D, Assaf A, Pelletier G, Labrie F 1987 Morphological study of the effects of an GnRH agonist on the canine testis after 4
months treatment and recovery. Acta Endocrinol 116: 413-417
Gobello C, Corrada Y, Trigg T. 2003 Reversible prolongation of anestrus in the bitch with deslorelin acetate. Revista Brasileira
de Reproduçâo Animal. 27 (3) : 518-519
Gobello C, dei Amo A. 1993. Estrus induction with clomiphene citrate in bitches. First International Congress FCV-UNLP and VII
International Veterinary Meeting, La Plata. Argentina. 4-6
Hoffmann B, Schuler G 2000. Receptors blockers- general aspects with respect to their use in domestic animal reproduction.
Anim. Reprod. Sei. 60-61: 295-312
Inaba T, Umerhara T, Mori J, Torri R, Tani H, Gonda M, Nakagawa A, Ohnura M, Tamada H, Sawada T. 1996 Reversible
Suppression of Pituitary testicular function by a sustained release formulation of GnRH agonist (Ieuprolide acetate) in dogs.
Theriogenology. 46: 671-677
Inaba T, Tani H, Gonda M, Nakagawa A, Ohmura M, Mori J, Torii R, Tamada T 1998 Induction of fertile estrus using a sustained
release formulation of a GnRH agonists ( leuprolide acetate) Theriogenology 49: 975-982
Jordan VC 1992 The role of tamoxifen in the treatment and prevention of breast cancer. Curr Probl Cancer 16: 129-176
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Kutzler MA, Wheeler, R Lamb SV, Volkmann OH 2002 Deslorelin implant administration beneath the vulvar mucosa for the
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treatment to prepubertal male and female dogs with the GnRH agonist [D-Trp6, des-Gly-NH21~ GnRH ethylamide. J Androl10:
456- 465
Lombardi P, Florio S, Pagnini U, Crispido A, Avallone L 1999 Ovarian function suppression with a GnRH analogue goserelin in
hormone dependent canine mammary gland cancer J Vet Pharmacol Therap 22: 56-61
Mc Rae GI, Roberts BB, Worden AC, Bajka A, Vickery BH. 1985 Long term reversible. suppression of estrus in bitches with
nafarelin acetate, a potent LHRH agonist. J Reprod Fertil. 74: 389-387
Morris JS, Dobson JM, Bostock DE 1993 Use of tamoxifen in the control of canine mammary neoplasia. Gynecol Oncol 39: 82-
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Trigg TE. 2002 The effect the GnRH agonist deslorelin on prostatic volume in
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dogs. 3 EVSSAR Meeting. 150
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dog. De Praktische Tierarzt. 83: 512- 520
Rubion S, Guerin C, Crivieri- Godet E, Horspool L, Rutten F, Drlancourt MA 2003 Treatment with a subcutaneous GnRH agonist
containing devise suppresses FSH and ovarian function in bitches. Reprod Dom Anim. Abst 79. 355
Trigg TE, Wright PJ Armour AF, Williamson PE, Junaidi A, Martin GB, Doyle AG, Walsh J 2001. Use of a GnRH analogue
implant to produce reversible, long term suppression of reproductive function of male and female domestic dogs. J. Reprod.
Fertil 57: 255-261
Vickery BH, Mc Rae GI, Goodpasture, JC, Sanders LM 1989. Use of potent LHRH analogues for chronic contraception and
pregnancytermination.
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Willams CL, Stancel GM 1996 Estr6genos y progestâgenos.
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Wright PJ, Verstegen JP, Onclin K, Jochle WJ, Armour AF, Martin GB, Trigg T.E. 2001. The suppression by progestin of oestrus
responses of the bitch to the GnRH analogue deslorelin. J. Reprod. Fertil 57: 263-268.
164
Abstracts
Inferti/itv in the bitch: Brucellosis and other etiologies
CRISTINA GOBELLO.
DR MED VET. DIPL ECAR
Faculty of Veterinary Medicine, National University of La Plata, Argentina
INTRODUCTION
Infertility is a reproductive clinical sign that, is defined in the bitch, as the failure to produce
quantitatively and/or qualitatively, normal litters. There are also different grades of infertility,
thus, the term sub fertility is used for intermediate grades of this problem. Independently of
the grade of its presentation, infertility should be quickly recognized. Only a systematic
approach to the primary cause of this reproductive problem will lead to an aetiologic
diagnosis and an appropriate treatment. This stepwise approach includes a detailed
signalment, history, physical examination and corresponding diagnostic tests. Therapy
should only be initiated if a definitive diagnosis has been established. Indiscriminate use of
hormones, antibiotics or vitamins is potentially dangerous and frequently confusing. Infertility
is routinely c1assifiedas congenital or acquired, depending on if it appears early in sexual life
or after having been fertile, respectively. From the practical point of view, there are three
common breeders complaints concerning bitch infertility Le. failure to allow mating, abnormal
oestrus cycles and failure to produce litters after normal matings.
IT IS, THEREFORE, THE AIM OF THIS PRESENTA TlON TO GUIDE THE VETERINARIAN
THROUGH A SYSTEMATIC APPROACH FROM THE OWNER'S COMPLAINT, TO THE
DEFINITIVE DIAGNOSIS OF THIS REPRODUCTIVE
PROBLEM OF THE BITCH. A
SPECIAL EMPHASIS ON CANINE BRUCELLOSIS HAS BEEN DONE
SIGNALMENT, HIS TORY AND PHYSICAL EXAMINA TION
A COMPLETE SIGNALMENT
OF THE AFFECTED
BITCH ASSURES
THA T THE
CORRECT INTERPRETATION OF THE PRIMARY COMPLAINT WITHIN THE FRAME OF
THE STUD BITCHES BREED AND AGE CERTAIN BREEDS, EG. BEAGLES, ARE
PRONE TO AUTOIMMUNE GENITAL DISORDERS WHICH MA y ALSO BE ASSOCIATED
WITH AUTOIMMUNE HYPOTHYROIDISM.
FEA TURES SUCH AS AGE OF PUBERTY,
INTER-OESTRUS
INTERVALS, HEA T DURA TION, PROLIFICACY, SHOULD ALSO BE
INTERPRETED WHEN TAKING INTO ACCOUNT EACH PARTICULAR BREED. FINALL Y,
ADVANCING AGE HAS A NEGA TIVE INFLUENCE ON FERTILlTY.
CLINICIANS HA VE TO ASK THE OWNERS ABOUT ANY CHANGE IN THE BITCHES
MANAGEMENT,
HOUSING, MEDICA TlONS, SUPPLEMENTS,
AND DIET. GENERAL
HEAL TH HISTORY AND PREVIOUS LABORA TORY TESTS SHOULD BE RECORDED.
SPECIAL EMPHASIS SHOULD BE GIVEN TO REPRODUCTIVE
HISTORY (SILENT
HEA TS, FAILURE TO CONCEIVE, INFECTIONS, ABORTIONS) AND PERFORMANCE
(PA RI TV, PROLIFICACY,
MATERNAL BEHAVIOUR)
OF THE BITCH. A DETAILED
DESCRIPTION OF BREEDING MANAGEMENT
USED IS ESSENTIAL TO RULE OUT
MANAGEMENT AETIOLOGY OF INFER TILl TY. QUESTIONS ABOUT OTHER FEMALES
IN THE KENNEL COULD ALSO HELP WHEN THERE ARE INFECTIOUS OR GENE TIC
PROBLEMS.
A complete physical examination of the genital tract should be performed, including the
vulvar size, consistency and discharges, abdominal palpation of the uterus and/or cervix.
Special attention to the skin and hair coat is necessary as they may indicate endocrine
diseases that are potentially related to reproductive problems. Size and eventual milk
secretion of mammary glands frequently help to confirm ovulation and normal biphasic
cycles.
167
OWNERS' COMPLAINTS
Owners' complaints can be summarized in: failure to cycle normally, failure to allow mating
and failure to produce a litter after apparently a normal oestrus cycle and mating. The third is
the most frequent complaint.
a) Failure to cycle normally
Common complaints about failure to cycle normally include, cases of primary or secondary
anoestrus, short inter-oestrus intervals and prolonged follicular phase. Primary anoestrus is
the lack of estrous activity in a bitch after 24 months of age. lt may be caused by husbandry
problems, hermaphroditism, endocrine disorders and systemic disease or confused with
silent heats. Secondary anoestrus is diagnosed wh en the inter-oestrus interval exceeds 12
months. It can be due to acquired disorders that have been described for primary anestrous
as weil as by non secretory ovarian cysts or tumours. A common non sexual endocrine
aetiology of anoestrus may be hypothyroidism. Hypothyroidism can be suspected by low total
serum thyroxin concentrations, and generally confirmed by low thyrotrophic stimulating
hormone (TSH) and a non responsive TSH stimulation test. Treatment consists in exogenous
administration of thyroxin.
Conversely, short inter-oestrus intervals of less than 4 months can be caused byovulation
failure or an abnormal luteal phase Le. hypoluteoidism.
Prolonged follicular phase is
manifested by persistent oestrus and may be caused by secretory ovarian cysts or tumors as
weil as by exogenous administration of oestrogens.
b) Failure to allow mating
Actual failure to accept mating is often confused with an inappropriate breeding management
and presentation of the male dog, either too early or to late in the oestrus cycle. Improper
timing of breeding is the most common cause of apparent infertility in dogs. A complete
workup of the bitch including vaginal cytology, vaginoscopy and serum progesterone (P4)
and/or luteinizing hormone (LH) determinations throughout the oestrus cycle can rule out
breeding management problems.
Conversely, mating refusai can be due to physical congenital or acquired diseases of the
genital or locomotor system e.g.: different causes of vulvar or vaginal stenoses or infections,
hip dysplasia or lumbar pain. Finally, behavioural disorders such as incomplete socialization,
excessive
aggressiveness
or simply male preference
can impede normal sexual
performance. These problems are best circumvented by artificial insemination.
c) Failure to produce a lifter after apparently normal oestrus cycle and mating
Failure to produce a litter after being bred can be caused by abnormalities at different levels
Le. ovulation, fertilization, implantation or maintenance of gestation. Infertility in either the
female or male should be distinguished from pregnancy loss. The lack of early pregnancy
markers in this species is an important limiting factor. The most frequent cause of
spontaneous pregnancy termination in Central and South America is Brucella canis. If a
bitch aborts in these countries, Brucella canis infection is assumed until it is proved
otherwise.
168
CANINE BRUCELLOSIS IS CHARACTERIZED, IN 75% OF THE CASES, BY ABORTION
AFTER 45 - 55 DAYS OF GESTATION IN AN OTHERWISE HEALTHY BITCH. ABORTION
IS FOLLOWED BY A 1 TO 6 WEEK, BROWN TO GREY- GREEN VAGINAL DISCHARGE.
ABORTED PUPS ARE OFTEN PARTIALL Y AUTOL YZED. ABORTING BITCHES MA Y
LOSE 2 OR 3 LlTTERS IN SUCCESSION. EARL Y EMBRYONIC DEA TH AND
RESORPTION 10- 20 DA YS AFTER MA TING MA Y ALSO OCCUR, THIS MA Y GO
UNNOTICED AND IS FREQUENTL Y DIAGNOSED AS FAILURE TO CONCEIVE.
CONVERSELY, A BITCH MA Y CARRY PUPS TO TERM AND DELIVER BOTH LIVING
AND DEAD PUPPIES. LIVING PUPPIES DIE WITHIN HOURS TO DA YS. NO AGE OR
BREED PREDISPOSITION HAS BEEN REPORTED FOR THIS INFECTIOUS DISEASE.
BITCHES WHO ABORT SHOULD BE KEPT ISOLATED AND THE PREMISES MUST BE
DISINFECTED AS INFECTION MAINL Y OCCURS BY INGESTION OF CONTAMINATED
ABORTED MATERIALS AND VAGINAL DISCHARGES. SHEDDING OF THE ORGANISM
INTO VAGINAL DISCHARGE IS CONTINUOUS UP TO 6 WEEKS AFTER ABORTION.
BRUCELLA CANIS IS SHORT LlVED OUTSIDE THE HOST AND IS INACTIVATED BY
COMMON DISINFECTANTS. ELIMINATION OF INFECTED DOGS IS THE PRINCIPAL
CONTROL STRA TEGY IN THE KENNELS. THUS, ALL DOGS OF THE KENNEL SHOULD
BE TESTED MONTHLY FOR 3 MONTHS UNTIL THE COLONY IS NEGA TIVE ON 2
SUCCESSIVE TESTS AND THEN TESTING CARRIED OUT EVERY 6 MONTHS.
ADDITIONALL y, TWO NEGA TlVE TESTS DONE AT 4-6 WEEK INTERVALS SHOULD BE
REQUIRED FOR ALL DOGS TO BE INTRODUCED INTO BREEDING COLONIES.
Canine brucellosis diagnosis requires laboratory confirmation. Serologie tests are rapid, easy
to perform, widely available, either for use in the hospital or in diagnostic laboratories, but
are imprecise. Moreover they cou Id be negative during the first 3 to 4 weeks after infection,
despite bacteraemia. Recrudescence of bacteraemia and elevation of antibodies titers occur
during proestrus, oestrus, pregnancy and abortion, making these the most reliable times to
test. Antibiotics may suppress bacteraemia and associated serologie response and therefore
should be avoided before screening is complete.
Serologie tests include the rapid si ide and tube agglutination tests which are sensitive but not
specifie and false positives are frequent. Thus, dog's positive on these tests should not be
considered infected until additional serological studies are done. Moreover, serologie
negative results may occur in recent infections and therefore testing should always be
repeated in 30 days. More specificity is obtained by agar gel irnrnunodlffuslon: therefore, it
may be used as a confirmatory test for dogs that are positive to the agglutination tests. Ali
serological tests should always be confirmed by bacteria isolation from aborted material or
blood culture. More than 50% of infected dogs have a bacteraemia lasting 1 year or longer.
Treatment of individually owned dogs is expensive and cures are difficult to achieve,
although greater success can be expected in early infections. Repeated blood cultures and
serologie monitoring is required for at least 6 months after treatment before a dog can be
declared negative. Treatment consists of neutering and specifie antibiotic therapy and it is
not recommended for breeding bitches, or where follow-up is unlikely. Owners should also be
informed that human infection by Brucella canis is possible.
Tissue persistence of the organism has been demonstrated after ovariohysterectomy
but
shedding of Brucella is less likely. Neutered dogs should receive a course of antibiotics. The
intracellular localization of Brucella canis organisms makes achievement
of effective
antibiotic levels difficult. Medical treatment consists of a combination of a tetracycline drug,
e.g. tetracycline hydrochloride (25 mg/kg PO tid for 4 weeks) or preferable minocycline or
doxycycline (25 mg/kg PO bid for 2 weeks) and streptomycin (10 mg/kg lM bid the1st and 4th
week). If the first course of treatment failed, a second one may be necessary.
169
Other infectious aetiologies of abnormal pregnancy termination are canine herpes virus,
aerobic bacteria and mycoplasma. Naïve bitches are likely to show clinical evidence of
canine herpes virus during the last 3 weeks of gestation by the production of stillbirths,
mummified or macerated pups ail born in one litter. Affected bitches usually lose only one
litter. Isolation of females from 3 weeks before to 3 after whelping is indicated for prevention.
Sub clinical endometritis due to ubiquitous vaginal bacteria or mycoplasma may occur.
Culture from the anterior vagina using a guarded swab in early proestrus shows a moderate
to heavy growth of a single organism.
Furthermore, vaginal cytology evidence an
inflammatory smear. Appropriate 2 to 4 week antibiotic therapy based on culture and
sensitivity assures cure.
Non-infectious causes of spontaneous pregnancy loss include: genetic abnormalities of the
puppies, inbreeding, metabolic or endocrine diseases of the bitch, trauma, cystic endometrial
hyperplasia, exposure to drugs, chemicals or, toxins and nutritional deficiencies.
Hypoluteoidism is a sexual endocrine cause of pregnancy termination. It is defined as
abnormally low serum P4 concentrations « 5 nglml) in the presence of viable normal foetus
in the uterus of a pregnant bitch. Treatment of primary hypoluteoidism consists of P4 or
progestin supplementation when serum concentration of this hormone falls to less than 5
nglml before day 58 to 60 from ovulation.
Adverse drug effects may be embryo toxic. The conceptus is extremely sensitive to chemical
insult early in gestation (Le. 6 to 20 days after LH surge) when bathed in uterine fluid. If any
pharmaceutical drug has to be administered during the critical period or even during the
remaining pregnancy the safety, of this drug should be evaluated.
DISCUSSION AND CONCLUSIONS
Infertility is one of the most puzzling aspects of small animal reproduction, representing a
challenge for practitioners. The inaccessibility of much of the female reproductive tract, the
long inter-oestrus intervals of this species and the multiple causes that can provoke infertility
make aetiologic diagnosis of bitch infertility not an easy task. Inappropriate breeding
management is frequently confused with infertility, infectious diseases are the first cause of
"real" infertility in the female dog. In Central and South America Brucella canis is the main
primary infectious disease of canine genital tract. Thus, a correct breeding management and
a complete Brucella testing should precede any other more sophisticated ancillary test.
It is concluded that a systematic approach to this disappointing reproductive problem is
necessary to correctly arrive to the primary aetiology and to establish preventive strategies at
subsequent oestrus cycles.
REFERENCES
Carmichael LE, Greene CE. Canine Brucellosis. 1990 ln: Greene CE (Ed) Infectious Diseases of the Dog and Cat. WB
Saunders, Philadelphia. Pp 573-584.
Feldman EC, Nelson RW. 2004 Canine and Feline Endocrinology and Reproduction, 3rd. WB Saunders, St Louis. p 1045.
Gobello C. 1992. Infertilidad en la perra: guia para el diagn6stico. Veterinaria Argentina. 11 (83): 195-199
Gobello C. 2004 Infertilidad en la perra ln: Gobello C, Stanchi N (eds) Signos clinicos dei perro: aproximaci6n diagn6stica.
Graficar, La Plata. Pp 7-15
Gobello C, Corrada Y. 2002. Non infectious Spontaneous Pregnancy Loss in Bitches. Compend Contin Educ Pract Vet. 24 (10):
778-783
Johnson CA 1986. Infertility in the bitch. In: Morrow, DA
Philadelphia.Pp 466-469
(Ed). Current Veterinary Theriogenology 2nd ad. W.B. Saunders,
Johnson CA, Walker RD. 1992 Clinical signs and diagnosis of Brucella canis infection. Compend Cont Educ Pract Vet. 14: 763772.
170
Johnston SD, Root Kustritz MV, Oison PN. 2001 Clinicalapproach
Theriogenology. WB Saunders, Philadelphia. Pp 257-274.
to infertility in the bitch. In: Canine and Feline
Shin SJ, Carmichael, and LE.1999 Canine Brucellosis Caused by Brucella canis
Canine Infectious Diseases. International Veterinary Information. www.ivis.org
ln: Carmichael L (Ed) Recent Advances in
Wright PJ, Watts JR. 1998 Female Infertility. In: Simpson GM, England GCW, Harvey M (Eds) Manual of small animal
reproduction and neonatology. BSAVA Cheltenham. Pp 23-46.
171
Abstracts
PASSERIFORMES REPRODUCTION MANAGEMENT
Jordi Grffols Ronda, DVM MS
Hospital Zoologie Badalona SL. Conquista, 74. 08912 Badalona (Barcelona).
zbv@hzb.es
HISTORY
The first written reports on canary (Serinus canaria) domestication and captivity date
1402, when Canary Island's monks started its breeding. It was after a French
expedition to the Canary Islands that it was brought to France and Italy, and from
these Countries to the rest of Europe. The first book treating about canaries dates
1709, it was written by Hervieux de Chanteloup.
TAXONOMY
Order Passeriformes
Family fringillidae (148 species)
Species:
Serinus canaria
Origin:
Canary Islands, Madeira and Azores
Songster canaries: Harzer, roller or flute (German),
Malinois (Belgian), Timbrado (Spanish) ...
Colour canaries: Melanie group: black, brown, agate,
isabelline ... Lopochromic group: red, yellow, white ...
Stance canaries: border, crested, hunched ...
Family strildidae (124 species)
Little Afrîcan, Aslatlc and Australian exotic finches ...
Family emberizidae (552 species)
New World exotic flnches, cardinals ...
The species most commonly kept in captivity in our Country is the canary. The following text describes
the main reproductive aspects to be taken into account of the canaries; aspects common to the rest of
Passeriformes.
CANARY BIOLOGICAL DATA
Sexual maturit
Breedin season
Mating
Signs of heat
8 ears u to 20 ears
12-30 .
Monomorphic species. During the breeding
season the male sings and its chloacal region
is bulky due to the final portion of the ductus
deferens develo ment.
6-10 months.
Janua
to Jul .
Monogamy (one male with one female).
Polygamy: in harem or one male passing
through various cages with one female each,
durin 8-10 da s.
Male: more intense singing, restlessness,
courtship (swinging, stretched legs, fallen
wings while singing).
Female: restlessness, opens and closes wings,
nest building; before the male she opens
wings, crouches with head backwards and
chee s until she is mated.
173
Clutches
Incubation
Nestin
Chicks emanci
Feathering
CAPTIVITY KEEPING
Environmental conditions:
Canaries are perfectlv adapted to the Iberian Peninsula climate. Extreme
temperatures and air draughts should be avoided.
Minimal temperature required to start the reproductive season is 1SoC. It can then
fluctuate in environmental conditions similar to those in origin. Appropriate ventilation
is needed.
Humidity during breeding season should be between 60% and 80%. With greater humidity values,
more infectious diseases are present. On the other hand, very low environ mental humidity values lead
to a lesser percentage of eggs hatching.
Premises:
Indoor premises:
For a breeding couple, the cage should measure at least SOx40x40cm, with two
perches. It should be located in a lighting place, with no air draughts, avoiding
noisy places and smoke. Perches should allow the bird to embrace them
completely with its fingers; they can be made of plastic, word or branches. The
more advisable cages are those with an extracting floor and a metallic grill.
During the breeding season they should be provided by nests (made of plastic,
esparto grass or rope), goat hair (as nesting material) and small feeders (to
contain breeding paste).
Outdoor premises or Aviaries:
The aviaries are used to accommodate young fledging birds and adults outside
the breeding season. In case we keep young fledglings, it is advisable to
introduce an adult in period of rearing, so it will brood the youngest demanding
food.
Social behaviour:
Canaries are nervous and easily stressable birds. Aggressivity problems can arise in
overcrowded premises, an adequate number of perches, feeders and water
containers is advised in order to decrease stress and aggressivity.
Feeding:
Canaries are mainly seed-eaters. They ingest more than 30% of their body weight
each day. Its basal metabolic rate is 6S% greater than other non Passeriformes
bird's.
Most of the commercial seed mixes are nutritionally deficient. Seeds lack of vitamins
A, D3, E, K, lysine, methionin and their Ca:P ratio is very unbalanced.
ln captivity their diet can be based on a seed mix (birdseed, millet, oat, colza and
linseed) and vegetables (chicory, sprouts, broccoli, spinach, wild greens chickweed, dandelion- ...) in a 1:1 ratio.
174
They can also be bed with canary-formulated dry food supplemented by vegetables.
Some colour variety need an exogenous source of carotenes to maintain
pigmentation.
During the breeding and growing phase we need to use breeding paste to reach the greater protein
and minerai demand. This paste is made ot boiled egg (protein and phospholipids source), grated
toasted bread, dry milk and a minerai supplement; it can also be marketed already tormulated. This
paste is supplied trom January until the young t1edglings are separated trom their parents and housed
in aviaries with other young birds.
Water needs:
Canaries need 200 to 300 ml/kg body weight water each day, this is 3,5 ml water per
canary daily (with a medium weight of 20 grams).
REPRODUCTION IN THE CANARY
Physiology:
Canaries need the following conditions to start the breeding phase:
• Sexual maturitv (10 months of age) and good health.
• Compatible partner.
• Minimal photoperiod (14-15h light/day).
• Nest and nesting material.
• Food and water supplv.
• Minimal temperature (>14°C).
• Photoperiod stimulation (natural or artificial).
Other factors involved in breeding stimulation are:
• Effect of direct sunlight exposition of the skull bones.
• Singing of a male in heat.
These last factors stimula te testicular growth (up to 5 times the normal
size) and fertile sperm production in males; in females, thev stimulate
ovarian activitv and follicular growth. Males need a longer adapting
period to the increasing dailv light hours than females (changes in
photoperiod should start 15 davs ear/ier than in fema/es).
Importance of photoperiod:
Light ~ Retinal Receptors ~ Optic Nerve ~ Hypothalamus ~ Activating Factors
Release ~ Adenohypophysis ~ Gonadotrophins Production ~ Gonads Activation ~
Sexual Hormones Production.
Photoperiod length and light cycle are determinant factors in starting and maintaining
breeding. When these two factors suffer fluctuations, a negative hormonal feed-back
activates leading to a start in feathering and a stop in breeding.
We can manipulate photoperiod in two ways to ensure breeding:
- Graduai increase in day length: two months before the breeding season daylight
hours increase from 8-10h lightlday to 15h lightlday gradually 30 minutes per week.
Longer th an 17h lightlday lead to worse annual breeding results than 15h lightlday.
- Immediate increase in day length: day length increases from 10h to 15h lightlday.
suddenly. Birds last 3-4 weeks reaching optimal breeding conditions. This method
leads to a smaller percentage of fertile eggs in the first clutch and a greater female
mortality index.
175
Technical aspects related ta Iight:
-a
tical radiation s ectrum:
UV-C
UV-B
UV-A Visible IR-A IRIR-C
---li ht B
UV lieht stimulates skin production of vitamin Dl which allows calcium
intestinal absorption needed for maintenance of calcemia. skeleton
calcification and egg shell calcification. When direct sunlight exposure is not
possible. broad spectrum light lamps should be used to assure vitamin Dl
production.
• Light intensity:
At least 500 to 1.000 lux are reguired (sunlight gives 100.000 lux in a
normal day). Lux = lumen / m2. Lumen show the totallight produced bv a
light source per second. We should assure an adeguate light intensity.
• Light freguency reguired (Hz):
Fluorescent lights don't produce continuous light: they oscillate in a
stroboscopie effect (they switch on and off 50 times per second (50 Hz)).
Different light sources should be used in combination to diminish the
stroboscopie effect. Another possibility is to use high freguency lamps to
assure up to 28.000 Hz freguencies that suppress the bird's perception
of the on/off effect of light.
• "Colour' temperature:
The terro is used to differentiate a hot or cold light source. It expresses in
Kelvin units.
Incandescent lamps
2.700K
Hot white lamps
3.000K
Cold white lamps
4.000K
Daylight
5.000K
Cold daylight
6.500K
At higher colour temperature, the perceived light is bluer and more UV
light Hill be produced.
• Environmental temperature:
Some lamps (fluorescents) produce optimal light at a temperature of
20°C.
• Advisable aspects on Iight:
Use a combination of difterent types of lamps to achieve the technical
aspects required.
o Simulate dawn and dusk (graduai increase and decrease of Iight along
the day).
o Provide a smalllight source (7W) at night.
o
BIBLIOGRAPHY
•
Alvarez-Borda B, Nottebohm F. Gonads and singing play separate, additive roles in new neuron
recruitment in adult canary brain. J Neurosci 2002;22:8684-8690.
176
•
•
•
•
•
•
•
•
•
•
Aschoff J, von Goetz G. Masking of circadian activity rhythms in canaries by light and dark. J Biol
Rhythms 1989;4:29-38.
Bentley GE, Audage NG, Hanspal EK, et al. Photoperiodic response of the hypothalamo-pituitary-gonad
axis in male and female canaries, Serinus canaria. J Exp Zoolog Part A Gomp Exp Biol 2003;296:143151.
Gostantini V, Ginone F, Lacalandra GM. Early oviposition in Serinus canaria after endonasal
administration of Gn-RH. (Preliminary research). Boil Soc Ital Biol Sper 1985;61:633-638.
Goutteel, P. Ganary and finch medicine. European association of avian veterinarians, 2001:1-16.
Goutteel, P. The importance of manipulating the daily photoperiod in canary breeding. Proc. 3rd conf.
EAAV, Jerusalem, 1995 pp166-170.
Del Negro G, Edeline JM. Differences in auditory and physiological properties of HVc neurons between
reproductively active male and female canaries (Serinus canaria). Eur J Neurosci 2001; 14:1377-1389.
Del Negro G, Kreutzer M, Gahr M. Sexually stimulating signais of canary (Serinus canaria) songs:
evidence for a female-specific auditory representation in the HVc nucleus during the breeding season.
Behav Neurosci 2000;114:526-542.
Gil D, Leboucher G, Lacroix A, et al. Female canaries produce eggs with greater amounts of
testosterone when exposed to preferred male song. Horm Behav 2004;45:64-70.
Leboucher G, Beguin N, Mauget R, et al. Effects of fadrozole on sexual displays and reproductive
activity in the female canary. Physiol Behav 1998;65:233-240.
Schwabl H. Environment modifies the testosterone levels of a female bird and its eggs.
J Exp ZooI1996;276:157-163.
177
Abstracts
SEMEN EXTENDERS
Ricard CàRDOBA 1VIVES
CLiNICAN - Cassà de la Selva - Spain
(Lectured by Xiomara Lucas)
ABSTRACT
The interest in viably conserving the semen of certain canine specimens for as long as
possible in order to use it at an appropriate moment in the future has led researchers in the
field to develop substances that enable this biological material to be conserved for a long
time without significantly affecting its function and qualities; on the contrary, they protect it
against external agents, giving it energy and considerably lengthening its life.
These substances are semen extenders. The paper we present to you here aims to describe
what an extender is, its composition, its effects on spermatozoids, the types most commonly
used and the results obtained in comparative studies in the use of several of these extenders
by different veterinary schools.
Semen is a complex substance with one majority component, which is water. If we want to
use the extender as a preservative at low temperatures, we must take into account the
behaviour of water along with the types of cryoprotectors that are to make up the extender
(penetrative or non-penetrative). In addition, we must also think about the nutrition of cells in
the latent state, pH changes in the medium, protection of the membranes, damage to the
acrosome that may release enzymes, and so on.
ln the same way that the world's finest chefs try out their new ideas in the kitchen, many
researchers are currently testing different 'recipes' to lengthen the lifespan of spermatozoids
without altering their characteristics and their fertilising power, achieving more and more
spectacular results.
INTRODUCTION
To dilute, is to add a solvent, reliable of characteristics similar to a solution or a certain
substance, to obtain a greater volume and a minor concentration without altering to its
properties.
UTILITY OF EXTENDERS
The extenders were used to:
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Increase the ejaculate volume allowing obtaining a large number of insemination
doses.
Improve sperm viability and longevity during preservation.
Protect spermatozoa against external agents.
Allow the transport of semen doses to large distances.
Preserve characteristics of genetically important individuals.
Have the possibility of choosing a male that more interest to us in a certain moment.
SEMEN: CHARACTERISTIC AND COMPOSITION
A healthy mature dog of 25 kg of weight produces (McDonald, 1969) around 7 ml of semen,
with 200 x 106 spzlml, a pH range between 6.5-6.8, density of 1.011 and a electrical
conductivity of 129-138 x 10-4Mho.
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As far as the chemical composition, the semen contains:
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water
Total nitrogened substances
total lipids
fructose
sorbitol
citric acid
97.86%
480 mg/100
182 mg/100
0.6 mg/100
0.1 mg/100
traces
ml
ml
ml
ml
EXTENDERS
Optimal diluents should provide a suitable environment for spermatozoa,
having an
osmolarity similar to that of semen, maintaining pH, providing substrate for metabolism
together with protection from cold shock and bacterial growth.
TYPES Of EXTENDERS
We can divide extenders in 3 great groups:
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Ejaculate fractions (seminal plasma homologues)
Lactic derivates
Laboratory extenders
PLASMA SEMINAL HOMOLOGUES
After collection ail the ejaculate (both sperm rich fraction and seminal plasma), semen can be
centrifuged between 300-1 000 9 and the supernatant keep frozen for a few days for a later
use. Supernatant allow to increase the volume gives protection, food and allows to facilitate
spermatozoa transport at the moment of artificial insemination. Seminal plasma does not
serve us to maintain semen cooled or frozen. Moreover this fluid can be contaminated by
bacteria, virus or blood coming from the prostate reason why it must be used if we are safe
of the health of the donor.
LACTIC DERIVA TES
These extenders are useful to preserve fresh chi lied semen during a few days. Sorne of the
most used are those (Rota et al, 1995).
EXTENDER2
EXTENDER 1
Pasteurized milk O. % fat
Egg yolk
Benzyl penicillin
Dihydrostreptomycin
80%
20%
1 mgr/ml
1 mgr/ml
High pasteurized cream
egg yolk
Benzyl penicillin
Dihydrostreptomycin
80%
20%
1 mg/ml
1 mg/ml
The disadvantages of this kind of diluents are that it is difficult to observe the spermatozoa at
the Iight microscope due to the refraction which produce the globules of milk fat.
LABORATORYEXTENDER
Almost ail these diluents contain:
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Low molecular weight sugars (glucose, fructose, etc)
Protein and lipoproteins (egg yolk)
Antimicrobial substances (antibiotics)
Ions and buffers (Tris, sodium citrate)
Water
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LABORA TORY EXTENDERS COMPOSICTION
AND FUNCTION
TRIS (HYDROXYMETHYL) AMINOMETHANE
It's a buffer substance. It's a white, crystal powder highly used in biologic systems for
containing low concentration of metals, being good at maintaining pH nearly 7.5 and
stabilizing NADH and NADPH coenzymes.
CITRICACID
It is an acidic substance that allows to fit the pH of a solution to the wished one. Normal
semen pH ranged between 6.5-6.8, and diluent pH is neutral, so it lets a slight decreasing in
the pH solution. Citric acid is a source of biosynthetic precursors in the Cycle of Krebs of
cells essential for the synthesis of ATP in mitochondrias.
WATER
Its main function is being solvent of other substances, allowing the entry of those inside of
spermatozoa. rehydrating them and contributing with nutritious substances. During freezing
and thawing, the main stresses to spermatozoa are induced by osmotic changes,
dehydration and ice crystal formation, events occurring at temperatures closed to 0° degrees.
At -0.S2°C an overfusion effect is produced, and spermatozoa can be damaged by exposure
to a too high concentration of solutes, also if water freezes inside the cells is a potential
source of damage.
EGG YOLK
The plama membrane structure is a fluid bilayer of amphipathic lipids containing integral and
associated peripheral membrane proteins which acts as a selective barrier. The major lipid
classes found are phospholipids, glycolipids and sterols.When the plasma membrane is
cooled between 20°C to SoC,· its components can become reorganized through the
aggregation of constituent non-bilayer lipids and a protein redistribution., lipid phase
transition effects cause responses in sorne enzymes, including sorne of the membrane
ATPases, potassium channels and sodium potassium pump.
The low-density lipoprotein fraction of egg yolk, specifically the phospholipids within that
fraction, has been identified as the effective component in protecting sperm. Loss of
membrane phospholipids from sperm is known to occur during cold shock and it is generally
assumed that addition of egg yolk prevents these losses or modulates their detrimental
effects; the egg yolk interacts with the sperm plasma membrane making it more fluid and
elastic.
CRYOPROTECTANTS
Cryoprotectant compounds can be classified into 2 groups, with differing modes of action,
permeate into the cellular cytoplasm and not permeate. Glycerol, together with substances
such as ethylene glycol, propanediol, acetamide, belong to a group which permeate into the
cellular cytoplasm. The protective effects. of these were mediated by depression of freezing
point and the consequent lowering of electrolyte concentrations in the unfrozen fraction,
lowering the lost of water and stabilizing the membrane. Non-permeate cryoprotectants are
not able to pass into the cellular cytoplasm so are less effective.
CURRENTSEXTENDERS
The most commonly canine extender used is that provided by the Alfort method, or modification
of this extender. The composition is the following:
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TRIS
Citric acid
fructose
distilled water
egg yolk
benzyl penicillin
dihydroestreptomycin
3,025 g
1,7 g
1,25 g
to 100 ml
20% (add at the time of use)
Img/ml
sulphate
1mg/ml
As an example of the last few extenders tested with aditives to improve sperm viability and
longevity, we can show one used by I.K.Kong (2001).
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~:,.~
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TRIS
Citric acid
Glucose
Dihydrostreptomycin
Penicillin
egg yolk
Glicerol
Equex stm paste
Royal Jelly
2'4g
1'4g
0'8g
O'lmglml
100UIImi
20ml/l00ml
7ml/l00ml
Iml/l00ml
Iml/l00ml
DIFFERENTSPAPER ABOUT SEMINAL EXTENDER
Fertility after vaginal or uterine deposition of dog semen frozen in a tris extender with
or without Equex STM paste Rota,Ouada,VerstegenLinde-Forsberg,1998
Artificial insemination with fresh semen in beagle bitches Tsutsui et al. Theriogenology
1988;50
Storage of domestic cat spermatozoa for extended periods at 4°C. Harris et al.
Theriogenology, Proceedings of the annuai conference international embryo transfer
societry, Omaha, Nebraska, USA 13-16January 2001 Vol 55 N°. 1
Effect of addition of royal jelly in tris-buffer extender on the post thaw viability of
canine semene I.K. Kong et al. Sunchon National University of S. Korea. Theriogenology ,
Proceedings of the annual conference international embryo transfer societry, Omaha,
Nebraska, USA 13-16January 1 2001 Vol 55 NO. 1
Efficacy of the anticaspase agent z-vad fmk on post thaw viability
spermatozoa. Peter et al. Theriogenology,2003; 59
of canine
Effects of Equex from different sources on post-thaw survival, longevity and
intracellular Ca2+ concentration of dog spermatozoa. Pefia et al. Theriogenology 2003;
59: 1725-1739
Cryopreservation of canine semen using a coconut water extender whith egg yolk and
three different glycerol concentrations.
Cardoso et al. Theriogenology2003; 59:743-751
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c. Dumon, s. Prigent,
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A. Rota, B. Strôm, C. Linde-Forsberg.
Theriogenology n° 44 1995
Effects of seminal plasma and three extenders on canine semen stored at 4°C.
.
A. Rota, B. Strôm, C. Linde-Forsberg, H. Rodriguez. Effect of EOUEX STM PASTE on viability of frozen-thawed dog
spermatozoa during in vitro incubation at 38°C.
A.Rota, M. Iguer Ouada, J.P. Verstegen, C. Linde-Forsberg Fertility after vaginal or uterine deposition of dog semen frozen
in a tris-extender with or without Equex STM paste..
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C.R.F. Pinto et al. The effect of reducing hindquarter elevation time after artificial Insemination in bitches. Theriogenology 50.
1998
C.R.F. Pinto et al. Fertility in bitches artifiacial inseminated with extended, chilled semen. Theriogenology 52 , 1999
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