ARTIFICIAL INSEMINATION IN DOGS: A PROMISING TOOL FOR DOG BREEDERS AND OWNERS

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2 Indo-Am. J. Agric. & Vet. Sci., 2015 ISSN Pankaj Jain et al., 2015 Vol. 3, No. 2, June Meghana Publications. All Rights Reserved Research Paper ARTIFICIAL INSEMINATION IN DOGS: A PROMISING TOOL FOR DOG BREEDERS AND OWNERS Pankaj Jain 1 *, Amit Kumar 1, Priyanka Deshmukh 1 and Rahul Sharma 1 *Corresponding Author: Pankaj Jain pjain427@gmail.com Artificial Insemination (AI) is a reproductive technique wherein semen is collected manually from a fertile male and inseminated in the female so that fertilization in the absence of natural mating can be achieved. Dog AI provides convenience for owners and breeders. Today, the actual technique and methods of artificial insemination are relatively easy and done by many private individuals and most veterinary clinics. Although the field is relatively new in canine, but its prospects are very promising. AI in dogs can help avert sexually transmitted diseases besides reducing the chances of inbreeding through good management of semen records of donar dogs. Keywords: Artificial Insemination, Breeding, Fertility, Semen INTRODUCTION Among the myriads of techniques devised for the genetic improvement of animals, artificial insemination occupies an important position. The most important attributes of AI is its non employment of any cumbersome, invasive technique as encountered in other methods of genetic improvement, viz., cloning, transgenesis, etc. In bovine, it has attained a remarkable height of success, but in case of other animals it is still in its infancy particularly in dogs. In India the concept and idea of AI in dogs is pregnant with both numerous benefits and puppies from elite males, for pure bred males are not readily and easily available for mating the bitches. This adds to the woes of dog s owner both monetarily and resultant loss of a valuable breeding season. It was an Italian physiologist, Spallanzani, who in 1780 obtained Beagle puppies by employing the AI. The first reports on AI in dogs subsequent to this experiment came to the fore by the end of the fifties, reporting the use of fresh semen, or in the sixties, the use of frozen semen, only in the nineties this technique was introduced into dog breeding practice, particularly in USA and Nordic countries (England and Millar, 2008). Advances and gain in the knowledge of canine physiology and new advances in canine semen technology have made AI to become available and practicable worldwide. With the increase in demand for the AI among dog breeders and owners and the semen preservation as a management tool in canine breeding, the ill effect, i.e., inbreeding within breeds can be reduced. The breeders now have 1 College of Veterinary Science and Animal Husbandry, NDVSU South Civil Lines, Jabalpur, MP , India. 11

3 luxury of selecting pure bred dogs from all over the world to improve their kennel genetics, without being undergoing transport-associated stress. Besides, semen from precious dogs can be saved and stored into semen banks to be used in further generations in spite of their death or after the peak reproductive age. In addition, the inherent benefits associated with AI, i.e., avoiding direct contact between the male and female, prevention of sexually transmitted diseases viz. Brucellosis, Herpes virus infection, etc., can be realized (Farstad, 2010 and Linde, 2005). Research work on AI in the domestic dog, along with other reproductive technologies, have provided important information for the preservation of canine semen. AI in dogs is accomplished by involving semen collection, evaluation, preservation and insemination techniques. SEMEN COLLECTION AND EVALUATION Semen collection in the dog is an easy procedure that requires basic training for standardization and optimization to realize the goal of AI. Semen collection and evaluation are essential prerequisites to yield good results in dogs. It is of paramount importance that semen collected from the donor should be assessed before being used for AI or cryopreservation as it foretells the male potential fertility and infertility (Johnston et al., 2001 and Freshman, 2002). Semen can be collected from most dogs in the absence of a teaser, in a quiet and isolated place (Freshman, 2002 and Kutzler, 2005). Methyl p- hydroxybenzoate, a chemical pheromone, can be swabbed on the perineal area and tail of an anestrus teaser (Johnston et al., 2001 and Kutzler, 2005) to enhance the sexual urge. The ideal intervals between collections are 2 to 5 days, while intervals longer than 10 days may result in an increased number of morphological abnormalities and decreased motility (Johnston et al., 2001 and Freshman, 2002). The most common method for semen collection in the dog is by digital manipulation although in the past semen was collected from dogs using an artificial vagina (Johnston et al., 2001; Linde, 2005 and Farstad, 2010). Collection of semen into a tube is commonly achieved by penile massage and the use of a cone or plastic sleeve or a special collecting vial (Linde, 2005). Briefly, the process is started with a massage of the dog prepuce at the level of the bulbus glandis until developing partial erection, followed by the quick retraction of the prepuce and penile expose. When a crystal clear fluid (prostatic fluid) begins to flow into the collection tube, collector can gently slide the collection cone off the penis (Linde, 2005 and Farstad, 2010). Canine ejaculate consists of 3 fractions, with the first and third fraction consisting of prostatic fluid and the second being rich in spermatozoa. The first fraction, the presperm portion, with a volume range of 1-5 ml while the second fraction, the sperm-rich portion is grayish-white in colour with a volume of 1-3 ml. However, third fraction which also comes from the prostate and may be up to ml in volume (Johnston et al., 2001). In the dog, the volume of whole ejaculate varies between breeds mainly with animal size (Dubiel, 2004 and Farstad, 2010). Most often, artificial insemination with freshly collected semen is performed without fractioning the ejaculate, although for artificial insemination, only the second fraction is of interest (Thomassen and Farstadt, 2009). Semen assessment is an integral part of the evaluation of fertility in males and should be performed as routine element of pre-breeding examination. Furthermore, semen evaluation ought to be completed before artificial insemination or preservation. Semen should be assessed immediately after collection and needs to be handled carefully during all the procedures. It is advisable to keep all equipment necessary for semen collection and evaluation at the temperature near 37ºC (Christiansen, 1984 and 12

4 Feldman and Nelson, 1996). It should always be remembered that the semen characteristics should be recheck 2-3 times at 1-2 weeks intervals, to confirm the male infertility. On the other hand, good in vitro semen quality does not always prove the fertilizing potential of a particular dog. Different approaches are available to assess the quality of the dog semen that can be grouped in conventional and advanced techniques. The later, usually requires more sophisticated means for the semen assessment and the support of technical equipment, while the former may be performed in an in laboratory. The conventional approaches to semen evaluation include macroscopical evaluation of the semen i.e. volume and colour and also the microscopical assessment, which gives the concentration and the number of viable cells in the ejaculate. The parameters and their respective characteristics are listed in Table 1. The number of spermatozoa per ejaculate also varies according to age, testicular weight, sexual activity and the size of the dog (Amann, 1986). In the past 2 to 3 decades, several strategies were developed to escape the subjectivity in the semen evaluation, related to the experience and skills of the observer, the method of specimen preparation, staining technique and number of cells evaluated, and which is particularly important when the fertility potential of preserved sperm cells has to be ascertain. It is well documented that variations in results of the conventional evaluation of the same semen samples obtained by different observers and laboratories may reach 30-60% (Coetzee et al., 1999). CASA is a sophisticated technique employing a computerized system that tracks multiple motility parameters. The computer takes video images of the sperm and stores them for analysis. The system recognizes motile from non-motile sperm and other organic debris by comparing luminosity (gray-scale intensity) and size of the object. There are also preset userdefined thresholds for size and luminosity that help prevent mistaking other cells and debris for non-motile sperm. While some studies have shown that CASA systems are more accurate than subjective assessment of sperm motility, other studies have found that subjective analysis progressively motility is well correlated with computer-based analysis SEMEN PRESERVATION Fresh Semen The use of fresh semen insemination is useful in a number of different circumstances including when the bitch is unwilling to stand to be mated, when mating is difficult owinng to Table 1: Characteristics of Different Parameters for Dog Semen Evaluation Sl.No. Parameters Characteristics 1. Color Opalescent to milky white with a clear prostatic supernatant or homogeneous greyish white. 2. Volume Pre sperm fraction: 0.1 to 3 ml, Sperm_rich fraction: 0.1 to 6 ml Prostatic fraction: one to 50 ml Totalvolume: one to 60 ml 3. Progressively Motile Sperm 60 to 90% 4. Number of Sperm per Ejaculate 60 to 600 X Morphologically Normal Sperm: 70 to 90% 6. Bacteria Many; usually more than10,000/ ml. However, only the presence of many white blood cells is an indication for bacterial culture of the semen. 13

5 differences in size, or when the male is unable to mate normally because of age, debility or non inherited disease. Fresh semen may be inseminated immediately after collection, or can be stored for three or four hours by allowing it to cool to room temperature. For longer periods, semen should be diluted and cooled. In most cases vaginal insemination is suitable but if the semen quality is poor it may be necessary to perform uterine insemination. The success rate with fresh semen insemination depends upon its quality and the fertility of the bitch. Assuming that both are normal and insemination occurs at the optimal time, pregnancy rates of approximately 85% can be expected. CHILLED SEMEN Semen can be chilled and stored for up to 48 h and in some cases up to 72 h. Chilled semen is increasingly used for transportation between countries and in general, fertility is greater than with frozen thawed semen. Semen is diluted in an extender to protect the sperm during the cooling and re warming process. The extender is changed during storage. Several extenders are used commonly with the choice of extender being based on semen storage tests conducted with each dog. When the semen is to be inseminated, it is warmed slowly to body temperature and its quality is reassessed. The semen can then be inseminated. Cervical or uterine insemination is used depending upon the length of time stored and the original quality of the sample. For samples inseminated within two days of collection the pregnancy rates may be approximately 65% for fertile bitches that are inseminated at the optimal time. FROZEN THAWED SEMEN Semen that is required for long term storage must be frozen. Although frozen semen can be thawed as required, fertility is almost always lower, and associated costs are higher (Romagnoli, 2002). For practical freezing, straws are placed in a wire basket four cm over the level of liquid nitrogen. They are held in the vapor for five minutes then lowered into the liquid nitrogen and after a few minutes, transferred with forceps to the canes used at storage units in liquid nitrogen containers. One should standardize ones freezing procedure and document all steps accurately making gradual changes as required (Concannon and Battista, 1989). In the absence of specific thawing suggestions, the semen should be thawed in water at body temperature for at least 20 s. It should then be used as soon as possible. The freeze thaw process causes a considerable decrease in sperm morphology, motility and longevity. For this reason fertility rates are highest when uterine insemination is performed. NUMBER OF SPERMS TO BE INSEMINATED In general, when fresh semen is used, the whole ejaculate is collected and inseminated. The situation is similar for chilled semen except that the semen has been diluted at a ratio of 1:1 to 1:2 (semen: extender) and consequently the volume increases. When frozen semen is used, it is most sensible to thaw one straw after freezing so that the semen quality can be reassessed and the minimum number of straws can be established for successful insemination. Usually, a total of 150 to 200 million motile spermatozoa is inseminated on two to three occasions. TECHNIQUES OF ARTIFICIAL INSEMINATION In dogs, during natural mating deposition of a considerable portion of the ejaculate into the uterus occurs through the cervical canal (England et al., 2006 and Thomassen and Farstad, 2009). While performing AI one must be aware that vaginal deposition per se will negatively influence sperm cell survival and their transport 14

6 in the female genital tract, which in turn impairs ability to achieve normal whelping rates and/or litter sizes. However, to obtain satisfactory success rates when using frozen/ thawed semen, intra-uterine insemination is necessary. As in other species, in dogs sperm cell number in the uterine lumen may be influenced by many factors, such as the moment of estrus, the type of breeding (natural mating or insemination), the method of insemination (intravaginal or intrauterine) and individual variations (Rijsselaere et al., 2004). Nevertheless, abdominal laparoscopy or surgery is strongly discouraged on the basis of animal welfare issues and should be avoided and be undertaken in extraordinary circumstances only. DEEP VAGINAL INSEMINATION Deep vaginal insemination is the most widely used method for insemination with fresh semen when the technique is performed by the dog breeder or in small clinics. For vaginal AI a simple plastic catheter of proper length is used, to which a plastic disposable syringe containing the semen is attached. A commercial catheter in flexible latex tube having an inflatable balloon at the tip may also be used. This kind of device has the advantage of increasing the probability for intrauterine transport of the semen and of preventing semen backflow (Linde, 2005 and Farstadt, 2010). The owner of the female should be instructed to bring the animal with an empty stomach, which facilitates the procedure (Linde, 2005). Before proceeding to the AI procedures in female the cleaning of the perineal area, in particular the perivulvar area, is to be done and tansabdominal palpation is usually used to guide or ascertain the vaginal catheter position. The bitch is placed in a standing position on an examination table or on the floor (according to the size of the female). The insemination catheter is carefully introduced in the vagina of the bitch, first steeply upwards until the pelvic brim has been passed, and then in a horizontal angle, when it is carefully pushed further ahead (Farstad, 2010). Alternatively, the vulva may be elevated to just below the anus (Linde, 2005). At this point, the position of the AI catheter must be felt by palpation, and oriented. On being confirmed that the catheter is correctly placed, it is moved onward through the cranial portion of the vagina delimited by the dorsal medial folds. AI catheter should be further introduced until it reaches the paracervical area, which then can be palpated as a 1- to 2-cm-long, firm structure that ends at the cervix. The semen is deposited once the catheter has been located in the paracervical area, close to the external cervical os. During AI the bitch is held with the hindquarters up and head down, in an angle of 45-60º. This position facilitates transabdominal palpation of the cervix and ensures that the semen will not be expelled through backflow. Earlier reports suggest that the bitch should be maintained in the same position up to period of time varying from 5 to 20 min after AI. However, reducing the interval of elevated hindquarters to 1 min seems not to be affecting the fertility (Pinto et al., 1998). INTRAUTERINE INSEMINATION Intrauterine insemination is done by using nonsurgical transcervical catheterisation or by surgical semen deposition by laparotomy or laparoscopy (Silva et al., 1996). However, catheterization of uterine cervix in the bitch is a difficult procedure and warrants skill and experience. The semen of lower quality, such as frozen-thawed or that collected from subfertile dogs should be deposited intrauterine to assure satisfactory results of artificial insemination (Thomassen et al., 2006). The conception rates after intravaginal insemination with frozenthawed semen are significantly lower when compared with the results of intrauterine insemination. 15

7 THE NORWEGIAN OR SCANDINAVIAN TECHNIQUE This method of non-surgical transcervical intrauterine insemination was first time described in 1975 (Andersen, 1975). In this technique two catheters are used in this method the outer plastic catheter and inner metal thin catheter. There are three sizes of the catheters, for small, medium and large breeds. The catheterisation should be made on standing animal. There is no need for administration of sedatives, but usually a small dose of alpha-2 agonist, such as xylazine, is advisable for abdominal muscles relaxation. In many bitches, especially those of larger breeds, the tip of the catheter passes into the cranial narrow part of vagina. However, in some smaller bitches the introduction of the outer catheter through the paracervix is difficult. The metal catheter is introduced into the cervical canal under the control of the position of the cervix by palpation through the abdominal wall (Andersen, 1975). This technique demands skill and experience. It is harder to perform uterine catheterisation in obese or nervous bitches and in giant breeds. The use of this technique of insemination is especially advisable in cases when using of semen of lower quality due to male subfertility or sperm cryopreservation. ENDOSCOPE ASSISTED VAGINOSCOPIC METHOD (NEW ZEALAND METHOD) Intrauterine insemination of the bitch under the visual control of endoscopic equipment was first described by Wilson (1993) using a rigid endoscope cystouretroscope. The procedure is performed on the standing animal. Usually it is not necessary to administer any sedatives. The results for the intrauterine deposition of frozenthawed semen when using this technique are quite satisfactory. Wilson (1993) with the use of frozen semen, refers a pregnancy rate and litter size 83.3% and 7.5 puppies per litter, respectively. Nizanski (2005) obtained whelping rates of 68.7% and 27.8%, when frozen-thawed semen was deposited by intrauterine vaginoscopic method and by vaginal insemination, respectively. SURGICAL TECHNIQUE Surgical insemination techniques have been proposed for frozen semen or when the bitch presents an anatomical obstruction that prevents the insertion of the catheter or endoscope. Both the laparoscopic approach and the laparotomy require anaesthesia and good surgical skills. The semen is introduced into the uterus by puncture of uterine wall or incision with a scalpel and passage of a tom cat catheter (Thomassen and Farstad, 2009 and Farstad, 2010). However, in such methods, semen deposition is performed only once. Furthermore, some ethical constraints have been raised regarding the use of surgical techniques for AI in dogs. Surgery is an invasive procedure, so it is unlikely to carry it out in the best interest of the animal, and the possibility of transmission of an undesirable trait in a particular animal genetic line should be kept in mind. REFERENCES 1. Amann R P (1986), Current therapy in theriogenology, Saunders, Philledelphia. 2. Andersen K (1975), Insemination with frozen dog semen based on a new insemination technique, Zuchthygiene, Vol. 10, pp Christiansen I J (1984), Reproduction in the Dog & Cat, Bailliere Tindall, London. 4. Coetzee K, Kruger T F, Lombard C J, Shaughnessy D, Oehninger S, Ozgur K, Pomeroy K O and Muller C H (1999), Assessment of interlaboratory and intralaboratory sperm morphology readings with the use of a Hamilton Thorne 16

8 Research integrated visual optical system semen analyzer, Fertil. Steri, Vol. 71, pp Concannon P W and Battista M (1989) Canine semen freezing and artificial insemination. Current Veterinary Therapy, Small Animal Practice, p Dubiel A (2004), Andrological examination of the stud dog, In: Rozrod psow and A Dubiel (Eds.) Wydawnictwo AR, Wroc³aw, pp England G C and Millar K M (2008), The ethics and role of AI with fresh and frozen semen in dogs, Reprod Domest Anim, Vol. 43, pp England G C, Burgess C M, Freeman S L, Smith S C and Pacey A A (2006), Relationship between the fertile period and sperm transport in the bitch, Theriogenology, Vol. 66, pp Farstad W K (2010), Artificial Insemination in Dogs. In: BSAVA Manual of Canine and Feline Reproduction and Neonatology, England G and von Heimendahl A (Eds.), 2 nd Edn, British Small Animal Veterinary Association Gloucester, UK. 10. Feldman E C and Nelson R W (1996), Canine and Feline Endocrinology and Reproduction, W.B. Saunders Comp., Philadelphia. 11. Freshman J L (2002), Semen collection and evaluation, Clinical Techniques in Small Animal Practice, Vol. 17, pp Johnston S D, Root K M V and Olson P N S (2001), Canine and Feline Theriogenology, W.B. Saunders Comp., Philadelphia. 13. Kutzler M A (2005) Semen collection in the dog, Theriogenology, Vol. 64, pp Linde Forsberg C (2005), Artificial Insemination, In ESAVS-EVSSAR Course Reproduction in companion, exotic and laboratory animal, Nantes 12th-17th September 2005 online ( net/course_notes/reproduction1_05/ artificial_) insemination.pdf) 15. Nizanski W and Klimowicz M (2005), Success of artificial insemination with fresh semen with the use of different methods for determination of optimal insemination time in bitches, Medycyna Wet, Vol. 61, pp Pinto C R, Eilts B E and Paccamonti D L (1998), The effect of reducing hindquarter elevation time after artificial insemination in bitches, Theriogenology, Vol. 50, pp Rijsselaere T, Van Soom A, Van C S, Coryn M, Gortz K, Maes D and Kruif A (2004) Sperm distribution in the genital tract of the bitch following artificial insemination in relation to the time of ovulation, Reproduction, Vol. 128, pp Romagnoli S (2002), Canine artificial insemination with fresh, refrigerated and frozen semen -proceedings of the veterinary sciences congress, Animais de Companhia, Oct. pp Silva L D M, Onclin K, Lejeune B and Verstegen J P (1996), Comparisons of intravaginal and intrauterine insemination of bitches with fresh or frozen semen, Vet. Rec, Vol. 17, pp Thomassen R and Farstad W (2009), Artificial insemination in canids: a useful tool in breeding and conservation, Theriogenology, Vol. 71, pp Thomassen R, Sanson G, Krogenæs A, Fougner J A, Andersen B K and Farstad W (2006), Artificial insemination with frozen semen in dogs: A retrospective study of 10 years using a non-surgical approach, Theriogenology, Vol. 66, pp Wilson M S (1993), Non-surgical intrauterine artificial insemination in bitches using frozen semen, J. Reprod. Fert, Vol. 47, pp

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