Cat reproduction is a science unfortunately characterised for too long by poor development and often old and out-of-date knowledge. Fortunately, the research developed for wild cats, some of them using the domestic cat as a model, has led to growing interest in and knowledge about cat reproduction and biotechnology during recent years. Because of the limited genetic pool in endangered species as well as in domestic breeding of very rare breeds or to facilitate exchanges between countries and increase the genetic diversity, andrology and male biotechnology of reproduction in the cat have seen a significant development over the last few years. The development of semen collection techniques and preservation protocols as well as artifical insemination (AI) procedures have permitted new clinical applications. The semen can either be used fresh after collection or be preserved for future use (chilled or frozen semen).

Semen collection and immediate AI with the collected fresh semen are used to overcome aggression between animals, behavioural incompatibility between the queen and the chosen tomcat and physical disability of the male or the female. AI may also help to prevent the spread of infections or the contamination of the female (however, it is demonstrated that the feline immunodeficiency virus (FIV) passes into the semen and can infect the queen, with the exception that frozen semen may be protective for a horizontal contamination).

When preserved, feline semen may allow transport and exchange of genetic material over a distance and thereby contribute to widening of the gene pool. Frozen semen may also be used for long-term storage for preservation of genetic biodiversity. As valuable breeding tomcats may be very rare for some breeds, chilled or frozen semen may allow AI with a better choice of individuals to diversify the lineage and to introduce some fresh blood into inbred colonies.

Semen Collection

In cats, semen can be collected by manual collection and artificial vagina (AV) (trained males!) or electroejaculation (EE). These are the most common methods in felids and enable one to obtain ejaculates of reasonably good quality. Methods of semen collection such as epididymal squeezing or slicing after neutering to recover the semen have been reported, mainly for research.

EE is probably the only option in private practice conditions and can easily be performed on all healthy animals but requires dedicated equipment (electroejaculator or EJ) including adapted size probe (approximately 1 cm diameter for the domestic cat) with three ventral electrodes to be placed into the rectum of the cat and an electroejaculator with a voltmeter ranging from 0-10 volts. This procedure requires general anaesthesia and should ideally be performed only on healthy animals. The choice of anaesthetic protocol may also influence the quality of the collected semen.

To achieve EE, after the evacuation of the faeces, a lubricated probe is inserted 6-8 cm into the rectum of the anaesthetised animal. Many different EE protocols (effect of level of voltage and method of collection on seminal characteristics) have been described with very similar results. Protocols used generally alternate cycles of increasing voltage stimulations followed by short periods of relaxation. The author's protocol involves starting with ten stimulations at 3 V separated from each other by 1 second, followed after a resting period of 1 minute by a second cycle at 4 V and a third cycle at 5 V. If ejaculation is not achieved at the end of this first cycle, a second cycle will be performed after a 1 minute resting period, but it will start with the first wave of stimulations at 4 V and then 5 and 6 volts. In general EE is obtained during the second cycle of stimulation. A third cycle is rarely needed.

The semen will be collected directly in a very small collection device (Eppendorf tube) or indirectly using a urinary catheter inserted about 5 cm into the urethra and connected to an insulin syringe without the plunger. Indeed the volume of semen in the feline species is quite small, ranging from 5-500 µl.

A common complication during collection, particularly if the bladder has not been emptied before starting the procedure, is contamination of the sample by some urine. This is most often obtained at high voltage. When observed, the semen should be cleaned as soon as possible by washing with an appropriate extender. The extender should be chosen carefully to fit the sample pH and osmolarity. Significant differences in one or both of those parameters will be sufficient to ruin the sample.

Examination of the Semen

Analysis of semen is essential in the fertility evaluation of the tomcat, but, due to the small volume of the cat ejaculate, some of the diagnostic procedures used in other species are difficult as such a small volume would be ideally entirely dedicated to the AI. In practice, sperm motility and viability, morphology and sperm concentration should be evaluated, even though determination of sperm membrane and acrosome integrity, semen chemistry and other parameters can be important for testing both fresh and frozen-thawed sperm.

The normal colour of the semen is creamy white and the intensity of the colour reflects the concentration of the spermatozoa. A yellow colour may be obtained with urinary contamination or the presence of pus, and red blood cells may also be detected.

After EE, semen volumes ranged from 0.019-0.284 ml (mean 0.076) and spermatozoa counts of ejaculates collected in the catheter ranged from 0.32-49.60 x 10⁶ (mean 11.64 x 10⁶). Some animals show retrograde ejaculation after electrical stimulation.

Sperm motility is assessed using an optic or phase-contrast microscope on a warming plate at 38°C. At least, 10 µl are evaluated on a prewarmed slide. Fresh cat semen, undiluted and stored at 38°C, maintains motility for around 60 minutes; nothing is known about the time they can survive in the female genital tract during natural mating. However, it is possible to extend that period to more than 2 hours if the temperature is reduced to 23°C and even more if the semen is diluted with an appropriate extender. Normal sperm motility ranges from 60-90%. Progressive motility may also be evaluated, ranging from 0 to 5 (steady, rapid, forward progression) and a sperm motility index can then be calculated. Computer-assisted semen analysis (CASA) can also be used to measure varied kinematic parameters, such as curvilinear velocity, linearity, straight-line velocity and amplitude of lateral head displacement. A hyperactivated status of the spermatozoa may be detected this way, suggesting their capacitated condition. CASA systems are expensive but in reproductive centres where semen analyses are commonly performed, they are really valuable. They allow for objective evaluation and comparison of results, objective evaluation of treatments (parameters before and after process) but require good calibration and validation.

Semen morphology is evaluated by counting the abnormalities in 10 µl of semen stained with eosin-nigrosin, Spermac, Diff-Quick or other common staining methods. Abnormalities may be classified in different ways. One classification distinguishes between primary and secondary abnormalities. The primary abnormalities include all defects occurring during spermatogenesis in the testis (coiled flagellum, microcephalic, macrocephalic) while the secondary abnormalities consider abnormalities of maturation, occurring while the spermatozoa are transiting in the epididymis (bent mid-piece, bent flagellum, protoplasmic droplets and acrosomal defects). Primary defects are considered as major drawbacks to fertility.

In cats, abnormalities are frequent, to the point that we talk about teratospermia. The most often encountered morphology defects are bent mid-piece with or without a cytoplasmic droplet, bent flagellum or tightly coiled flagellum.

As acrosomes are highly vulnerable and often altered with the processes of semen cooling or freezing, checking their integrity may be very useful. The fluorescent chlortetracycline stain allows discrimination between uncapacitated and capacitated acrosome-intact spermatozoa and appears to be a suitable method to evaluate functional integrity of cat spermatozoa. Feline ejaculates may show high proportions of acrosomal defects such as large vacuoles, protrusion of acrosomal matrix and folding of the acrosome back on to itself.

The frequency of semen collection has thus far not been shown to affect sperm morphology in felids. In a study comparing sperm morphology and motility of two ejaculates collected from the same cat within a short interval, better sperm morphology and motility was demonstrated in the second ejaculate.

Semen Freezing

Frozen-thawed spermatozoa, either electro-ejaculated or recovered from the epididymis have already been successfully used in artificial insemination in the domestic cat, proving to be a valuable resource for the reproduction of wild felid species, which are threatened by extinction

The effects of freezing and thawing on domestic cat semen collected by electroejaculation (EE) and from the epididymes and vasa deferentia have been compared, spermatozoa being frozen-thawed following a single classical protocol. Sperm motility, sperm progressive status (0-5), plasma membrane integrity and morphology (light and transmission electron microscope) showed no significant differences between the electroejaculated and epididymal fresh or frozen-thawed spermatozoa. However, the incidence of acrosome defects after freezing and thawing increased by 19% based on light microscopy, whereas ultrastructural images revealed acrosome damages in most sperm cells. Since these acrosome changes are known to affect sperm fertilising capacity, further studies are needed to optimise cryopreservation techniques for epididymal as well as electroejaculated domestic cat spermatozoa. Recently, a study confirmed the importance of acrosome damage by demonstrating a reduced ability of cat frozen-thawed semen to penetrate in vitro maturated oocytes.

Artificial Insemination

AI in cats represents an important technique for increasing the contribution of genetically valuable individuals in specific populations, whether they are pedigree purebred cats, medically important laboratory cats or endangered non-domestic cats.

Sperm samples can be used for AI immediately after collection, after temporary storage above 0°C or after cryopreservation.

There have been three and five reports on intravaginal and intrauterine insemination, respectively, and one report on tubal insemination with fresh semen.

In studies using fresh semen, it was reported that conception rates of 50% or higher were obtained by intravaginal insemination with 10-50 x 10⁶ spermatozoa, while, in another report, the conception rate was 78% after AI with 80 x 10⁶ spermatozoa. After intrauterine insemination, conception rates following deposition of 6.2 x 10⁶ and 8 x 10⁶ spermatozoa were reported to be 50 and 80%, respectively.

With tubal insemination, the conception rate was 43% when 4 x 10⁶ spermatozoa were used, showing that the number of spermatozoa required to obtain a satisfactory conception rate was similar to that of cats inseminated directly into the uterus.

When frozen semen was used for intravaginal insemination the conception rate was rather low, but intrauterine insemination with 50 x 10⁶ frozen--thawed spermatozoa resulted in a conception rate of 57% in one study.

Overall, there have been few reports on artificial insemination in cats. The results obtained to date show considerable variation, both within and among laboratories, depending upon the type and number of spermatozoa used and the site of sperm deposition. Undoubtedly, future studies will identify the major factors required to consistently obtain reliable conception rates, so that AI can become a practical technique for enhancing the production of desirable genotypes, both for veterinary practice and laboratory and conservation purposes.

Acknowledgement

BSAVA thanks John Verstegen DVM MSc PhD DipECAR for his assistance with these notes.