T. Rijsselaere, A. Van Soom

Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, B-9820 Merelbeke, Belgium

tom.rijsselaere@ugent.be

INTRODUCTION

Up till recently, the reproduction in cats has hardly been studied, despite the fact that the reproductive cycle of the cat is unique in a number of aspects, such as the induced ovulation and the sequence of events in the course of natural mating. In recent decades how- ever, a considerable amount of new information has been published, mainly due to the increased interest in cat reproduction in daily practice and due to the use of the cat as a research model for endangered wild felids and for several human pathologies (Axner and Linde- Forsberg, 2002). The first successful artificial insemi- nation (AI) in cats was described 40 years ago by Sojka et al. (1970). Subsequently, several research groups worldwide published new information al- though AI in cats is at present not as commonly per- formed as AI in dogs in daily veterinary practice. The main reasons to perform an insemination in the cat are

(1) if natural mating is impossible due to anatomical, physiological or behavioral problems, (2) the preser- vation (chilled or frozen) and subsequent transport of genetic material of valuable breeding animals and (3) the research and use of information obtained in the do- mestic cat for the conservation of endangered wild fe- lids.

SPERM COLLECTION

Various techniques have been described for the sperm collection in cats: artificial vagina, electroeja- culation, urethral catheterization after sedation with medetomidine and retrieval of epididymal sperm.

Artificial vagina

A trained tomcat mounts a queen in heat and grasps her in the neck to fix her (Figure 1). Subsequently, sperm is collected by using a small artificial vagina consisting of a 2 ml rubber bulb-pipette and a small test tube which is placed in a water bottle at 37°C (Sojka et al., 1970). However, not all tomcats allow this procedure and if they allow it, they have mostly been trained for this procedure since puberty. Conse- quently, this method is largely restricted to universi- ties or research facilities with large breeding colonies of queens and trained tomcats but it is difficult to apply with tomcats that are presented only occasionally in daily practice.

Electroejaculation

The tomcat is anesthetized by using medetomidine (80-100 µg/kg IM) and ketamine (5 mg/kg IM) (Platz and Seager, 1978). After the removal of feces a probe with 3 electrodes is inserted 6 to 8 cm into the rectum (Figure 2A and B). Subsequently, a series of electrical stimuli is applied (e.g. 80 stimuli of 2, 3 and 4V in 3 successive series) which makes the tomcat to ejacu- late. By using this method sperm can be collected without any previous training or the presence of a queen in heat. Moreover, this procedure can be app- lied in every male which can be safely anaesthetized but is ethically questionable and is therefore prohibi- ted in several countries. Moreover, the required equip- ment is expensive and many cat owners and breeders are reluctant to apply this method.

Urethral catheterization

This recently developed technique requires the se- dation of the tomcat using medetomidine (100-150 μg/kg; Zambelli et al., 2007, 2008; Filliers et al., 2010), which stimulates the α2-adrenergic receptors and allows the release of a small volume of highly con- centrated sperm from the cauda epididymis in the ure- thra. Subsequently, the sperm is collected by using a urinary catheter (Buster® Cat Catheter, 1.0 mm x 13.0 cm), with its tip cut to get a shorter, open-ended ca-

Figure 1. Sperm collection in a tomcat using an artificial vagina. The trained male mounts the queen in heat, fixing her in the neck while an artificial vagina is inser- ted over the penis to collect the sperm (see arrow).

Figure 2. A. Electro-ejaculator with a rectal probe cotaining 3 electrodes; B: Technique of electro-ejaculation in the cat (the probe is inserted in the rectum while an eppendorf tube is placed over the top of the penis to collect the semen).

theter which is inserted approximately 9 cm into the urethra, taking care not to reach the bladder. Subse- quently, the catheter is removed from the urethra and the semen sample is collected. Immediately after col- lection, the sperm sample is placed in a pre-warmed Eppendorf tube with a diluent. Currently, this method is probably the most practical and the least invasive procedure for the sperm collection in tomcats in daily practice.

Epididymal sperm

After castration the testicles and epididymi are col- lected and placed in a petridish. The epididymi are se- parated from the testicles, incised repeatedly using a scalpel after which the spermatozoa are allowed to dis- perse during 10 minutes in the surrounding medium (Hepes TALP or physiological saline solution; Filliers et al., 2008). Finally, the medium with the spermato- zoa is collected and purified by density gradient cen- trifugation (such as Percoll® or Isolate®). This procedure is rather easy to perform and is routinely performed in laboratories for in vitro fertilization ex- periments (Filliers et al., 2008). Additionally, it can be used for genetically valuable tomcats that unexpec- tedly died.

SPERM EVALUATION

After the collection, the quality of the semen sam- ple is evaluated by assessing the main sperm parame- ters, i.e. the concentration, motility, morphology and the membrane integrity. The concentration can be mea- sured by means of a counting chamber (Bürker or Thoma), the motility by using subjective assessment of the percentage motile and progressively motile sper- matozoa on a pre-warmed glass slide and the morpho- logy and membrane integrity on e.g. eosin/nigrosin or diff-quick stained slides. Recently, several new tech- niques for sperm assessment have been described in cats,such as computer assisted sperm analysis and fluorescent stainings, which allow a more detailed sperm assessment (Filliers et al., 2008, 2010). One of the most important disadvantages for the sperm eva-

Table 1. Sperm quality parameters in the tomcat (Axner and Linde-Forsberg, 2002).

Parameter                           Value

Volume                                 0.03-0.3 ml

Concentration                       100-5000 x 106/ml

Sperm/ejaculate                    3-160 x 106

Motility                                56-85%

Normal morphology             38-95%

luation in the tomcat however is the very low volume of the ejaculate, which limits the number of sperm eva- luations dramatically. Moreover, up till now, the ‘cut- off values’ for sperm quality in cats have hardly been studied and defined, and the relation between sperm quality and in vivo fertility is not completely clear (Table 1). Finally, the sperm quality is influenced by the season (Axner and Linde-Forsberg, 2002).

TIMING OF AI AND INDUCTION OF OESTRUS

The insemination in queens can be performed du- ring natural or induced heat with a subsequent induc- tion of the ovulation by using e.g. hCG (100-250 IU) on the second or third day of the estrus. AI during na- tural heat is preferable because the gonadotropins used for the induction of heat may cause an inappropriate maternal response. The ovulation in cats generally oc- curs 24-48 hours after coitus (Shille et al., 1983). After the determination of the optimal moment by means of behavioral changes and/or vaginal cytology, AI can be performed on the second day of the estrus with a se- cond AI 2 days later (Chatdarong et al., 2002).

ARTIFICIAL INSEMINATION

After the collection and evaluation, mostly fresh spermatozoa are inseminated. However, cat semen can be conserved either chilled at 4-5°C for several days or cryopreserved at -196°C for a long period, which makes (inter)national transport and use of the genetic material of valuable breeding animals possible (Zam- belli et al., 2008).

Table 2. Pregnancy results obtained after AI in the cat with fresh and frozen-thawed semen.

To perform AI successfully in cats, the basic know- ledge of the female genital tract is required. During the estrus, the vestibulum, vagina and cervix measure 20.2

± 3.3, 26.8 ± 3.9 and 1.2 ± 0.1 mm, respectively (Zam- belli and Cunto, 2005). The insemination can be per- formed in the vagina or directly into the uterus. General anesthesia or sedation is required in both cases. Intravaginal AI is performed by inserting a needle (9 cm long – 20 G) with a bulb on the tip (Sojka et al., 1970) or by a nylon probe (9 cm long – 1.5 mm diameter) and a stainless probe (9 cm long – 2 mm dia- meter) into the anterior part of the narrow and non-dis-

Figure 3. Several methods for intra-uterine AI in cats using a transcervical catheter of 1 to 2.7 mm (a) and a speculum (b) described by (A) Hurlbut et al. 1988, (B) Swanson et al., 1994, (C) Chatdarong et al., 2001, (D) Zambelli et al., 2005.

(Picture Reprinted from Transcervical artificial insemi- nation in the cat. Zambelli D., Cunto M. (2005). Therio- genology 64, 698-705. Copyright (2005), with permission from Elsevier).

tensible vagina (Tanaka et al., 2000). The animals are in dorsal recumbency (up to 20 minutes after AI) with the hind quarters elevated to improve the transport of the spermatozoa into the uterus.

Intrauterine AI can be obtained by performing a la- parotomy and direct injection of the spermatozoa into the uterus of an anesthesized queen by means of a sy- ringe and a small needle (25G) (Tsutsui et al., 2000). Although this procedure is easy to perform, it is con- sidered invasive and unethical and is therefore prohi- bited in several countries. Alternatively, several techniques have been described using specific types of transcervical catheters and a speculum (Figure 3), which requires practice to master. Zambelli and Cunto (2005) successfully applied a transcervical intra-ute- rine technique with the aid of transrectal digital mani- pulation (Figure 3D). The latter technique takes only several minutes to perform but requires an extensive training period.

PREGANCY RESULTS

The results that can be obtained by AI in the cat are variable and depend on many factors, such as the type of semen used (fresh, chilled or frozen), the site of in- semination (intravaginal or intrauterine), the number of inseminated spermatozoa, the quality of the sper- matozoa. Moreover, studies published on the results obtained after AI in the cat are scarce and frequently include only a small number of animals. In Table 2, the results that can be obtained by AI in cats are sum- marized. In general, the results obtained after AI with fresh semen are acceptable, whereas the results after AI with frozen semen are at present poor. The number of spermatozoa necessary to obtain acceptable preg- nancy rates (75-80%) is lower when the sperm is in- seminated directly into the uterus than in the case of intravaginal insemination. Intrauterine AI is therefore recommended for cryopreserved and poor quality semen.

REFERENCES

Axnér E., Forsberg L. (2002). Semen collection and assess- ment, and artificial insemination in the cat. In: Concan- non et al. (Editors). Recent Advances in Small Animal Reproduction. International Veterinary Information Ser- vice (www.ivis.org), Ithaca, New York, p.1-9.