**3. Sperm collection**

Two methods are used for the collection of semen from wild animals: the first is the epididymis' semen collection from dead animals or after castration or vasectomy. The second is through an artificial vagina, electroejaculation, or digital manipulation.

#### **3.1 Post mortem sperm collection**

In the post mortem sperm collection, immediately after death or castration, the testes should be kept cold at 5°C (Howard, 1993). Generally, postmortem epididymidal spermatozoa remain viable for several days after the animal's death. However, this period depends on the species, the storage conditions of the testes, and methods used for sperm collection. Several techniques could be used including: totally cutting (homogenization) the cauda epididymis; washing out or aspiration (flushing) through sperm duct; making cuts (mincing) in the cauda epididymis and squeezing of content; or just squeezing (pressing). The first two techniques are the most frequently used (Maksudov et al., 2008).

For cats, the epididymis is washed and homogenized in HEPES medium plus Ham's F10, and the sperm is recovered after centrifugation. Live sperm can be recovered within the first twelve hours of the epididymis isolation using this technique (Howard, 1993). In vasectomized animals, the semen is collected by aspiration from the epididymis' tail using syringe and needle. The syringe should contain HEPES medium plus HAM'S F10 and the sperm is recovered after centrifugation.

Sperm recovered from the epididymis of cats are known to be motile, viable, and capable of penetrating oocytes (Goodrowe & Hay, 1993; Hay & Goodrowe, 1993). However, epididymal sperm have naturally more cytoplasmic droplets, which normally are lost during transport through the duct (Briz et al., 1995).

According to Tebet et al. (2006) there were no significant differences between the fresh or frozen-thawed domestic cat spermatozoa for the variables: sperm motility, plasma membrane integrity and morphology of electroejaculated and epididymal spermatozoa analyzed immediately after collection, and after freezing and thawing. Jewgenow (Jewgenow et al., 1997) reported motility of frozen-thawed epididymal sperm of lions (*Panthera leo*), tigers (*Panthera tigris*), leopards (*Panthera pardus*), pumas (*Felis concolor*) and jaguar (*Panthera onca*).

Pallas cat *Oncifelis colocolo* - - 80-856 1-36

Puma *Puma concolor* 8.05 23.05 84-986 1-66 Jaguar *Panthera onca* 12.01.03 47.25.43 90-1116 1-26 Mellen 19891; Morais et al., 19962; Morato & Paz, 20013; Moreira et al., 20014; Oliveira, 19945; Oliveira &

Two methods are used for the collection of semen from wild animals: the first is the epididymis' semen collection from dead animals or after castration or vasectomy. The

In the post mortem sperm collection, immediately after death or castration, the testes should be kept cold at 5°C (Howard, 1993). Generally, postmortem epididymidal spermatozoa remain viable for several days after the animal's death. However, this period depends on the species, the storage conditions of the testes, and methods used for sperm collection. Several techniques could be used including: totally cutting (homogenization) the cauda epididymis; washing out or aspiration (flushing) through sperm duct; making cuts (mincing) in the cauda epididymis and squeezing of content; or just squeezing (pressing).

For cats, the epididymis is washed and homogenized in HEPES medium plus Ham's F10, and the sperm is recovered after centrifugation. Live sperm can be recovered within the first twelve hours of the epididymis isolation using this technique (Howard, 1993). In vasectomized animals, the semen is collected by aspiration from the epididymis' tail using syringe and needle. The syringe should contain HEPES medium plus HAM'S F10 and the

Sperm recovered from the epididymis of cats are known to be motile, viable, and capable of penetrating oocytes (Goodrowe & Hay, 1993; Hay & Goodrowe, 1993). However, epididymal sperm have naturally more cytoplasmic droplets, which normally are lost

According to Tebet et al. (2006) there were no significant differences between the fresh or frozen-thawed domestic cat spermatozoa for the variables: sperm motility, plasma membrane integrity and morphology of electroejaculated and epididymal spermatozoa analyzed immediately after collection, and after freezing and thawing. Jewgenow (Jewgenow et al., 1997) reported motility of frozen-thawed epididymal sperm of lions (*Panthera leo*), tigers (*Panthera tigris*), leopards (*Panthera pardus*), pumas (*Felis concolor*) and

second is through an artificial vagina, electroejaculation, or digital manipulation.

The first two techniques are the most frequently used (Maksudov et al., 2008).

**Estrous Cycle (days)** 

*yaguarundi* 3.170.751 53.632.411 72-756 1-46

**Gestation (days)** 

**Pups (nº)** 

**Estrus (days)** 

**Common Name** 

Cassaro, 19976.

**3. Sperm collection** 

**3.1 Post mortem sperm collection** 

sperm is recovered after centrifugation.

jaguar (*Panthera onca*).

during transport through the duct (Briz et al., 1995).

**Scientific Name** 

Table 1. Neotropical wildlife cats reproductive characteristics.

Jaguarondi *Herpaelurus* 

Epididymal spermatozoa were collected from immobilized adult male lion by caudal epididymectomy, cryopreserved, and used for IVF of *in vitro* matured lionesses' ova. The post-thawed motility ranged from 55-65%, and the percentages of fertilized ova were 12.7% and 11.5% for 30 and 36 hours of *in vitro* maturation, respectively (Bartels et al., 2000).

It has been shown, for several species, that the methods developed for ejaculated sperm are effective for freezing epididymidal spermatozoa. However, the physiology of ejaculated and epididymidal spermatozoa are different thus, it can be assumed that optimum methods of freezing and thawing may be different.

Postmortem material that can be retrieved in zoos usually belonged to the aged animals with different diseases or that died because of accidents (stress, fights). Moreover, some species display seasonal breeding. All these factors can influence on the spermatozoa's presence and quality in epidydimis, at the collection time (Maksudov et al., 2008).

Analysis of different factors showed that the concurrency of death with breeding season has the strongest affect on the spermatozoa content in testicles of dead animals. The spermatozoa content and quality were almost equal in males that died of sickness (cancer, chronic cardiovascular or excretory system disorders) and in males that died in accidents (stress, fights). However, the quality of postmortem semen of animals that died of natural death is worse than that of animals that died accidentally (Maksudov et al., 2008).

The energy invested by a male with copulation is minimal compared to that of the female, for whom the costs continue throughout pregnancy and lactation. Therefore, it is a significant genetic advantage for cats to be reproductively active throughout the year, or at least to remain active well outside of the usual female breeding season (Spindler & Wildt, 1999).

This strategy is apparent in wild felids. The female Siberian tiger exhibits more estrual activity in January-June than at any other time of the year (Seal et al., 1985), however, the sperm quality is fairly consistent throughout the year (Byers et al, 1990). The quantity and quality of the sperm from some felids can vary when a distinctive female reproductive seasonality is know, as in the snow leopard (Johnston et al., 1994) and Pallas' cat (Swanson et al., 1996), the males remains reproductively active for longer periods than the females of the same species.

The collection of the postmortem sperm of recently dead animals belonging to endangered species can be of substantial importance, and therefore, the method of choice for the preservation of reproductive cells, in the wild, at zoos, and in national parks. The preservation and utilization of postmortem represent the last chance to obtain offspring from the dead males in cases of unexpected loss of valuable animals (Maksudov et al., 2008).
