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 121 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 122 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 123 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 125 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. 126 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 129 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 Fernà ndezS. : 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: [email protected]. 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: 129-176 163 Kitchell BE, Fidel JL 1992 Tamoxifen as a potential therapy for canine mammary carcinoma. Proc Vet Can Soc 91 Kutzler MA, Wheeler, R Lamb SV, Volkmann OH 2002 Deslorelin implant administration beneath the vulvar mucosa for the induction of synchronous estrus in bitches. 3rd EVSSAR Meeting, Liege. 96 Kutzler MA, Wheeler R, Volkmann OH 2001 Deslorelin implants for induction of estrus in bitches. 2nd EVSSAR Meeting, Milan. 160-161 Lacoste D, Dube D, Trudel C, Belanger A, Belanger A, Labrie F 1989 Normal gonadal functions and fertility after 23 months of 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- 84 Ponglowhapan S, Lohachit C, Swangchanuthai, Trigg TE. 2002 The effect the GnRH agonist deslorelin on prostatic volume in rd dogs. 3 EVSSAR Meeting. 150 Riesenbeck A, Klein R, Hoffmann B 2002 Down regulation, a new and reversible approach to eliminate testicular function in the 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. J. Reprod. Fertil, 39: 175-187 Willams CL, Stancel GM 1996 Estr6genos y progestâgenos. In: Herdman JJ, Limbird LE Goodman Gilman A. Goodman& Gilman's Bases Farmacol6gicas de la terapéutica. Mc Graw- Hill Comp. Inc. Interamericana, Mexico. 1489- 1529 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). [email protected] 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 Cà RDOBA 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: • • • • • • 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. 179 As far as the chemical composition, the semen contains: • • • • • • 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: • • • 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: • • • • • Low molecular weight sugars (glucose, fructose, etc) Protein and lipoproteins (egg yolk) Antimicrobial substances (antibiotics) Ions and buffers (Tris, sodium citrate) Water 180 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: 181 • • • • • • • 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). • • ~:,.~ • • • • • • • 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 REFERENCES Fontbonne, M. Lenoz, P. Mimouni, Poitiers 1999. c. Dumon, s. Prigent, B. Silliart, F. Fienni. Cours avancé en reproduction du GERES. A. Prats. Reproducci6n de la perra y la gata. Canis et Fells n° 21993. 182 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.. Harcourt 2002 (www.harcourt.com). Academie press dictionary of science and technology. C. Dumon. Inseminaci6n en la especie canina e inseminaci6n artificial XVIII Congreso anual AMVAC Madrid 2001 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 G.C.W. England, M. Harvey BSAVA manual of small animal reproduction and neonatology UK GL515TO.1998. L.D.M. Silva, J.P. Verstegen .Comparisons betwen three different extenders for canine intrauterine Insemination with frozenthawed spermatozoa. Theriogenology 44. 1995 Ponglowhapan, S. Influence of glucose and fructose in the extender on long-term storage of chilled canine semen. I.M. of Science Programme. F.V.M. Swedish University of Agricultural Sciences 183
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