**2. General reproductive characteristics in wildlife cats**

The large number of felines and their wide geographical distribution determine many of the species' particularities, mainly in the reproductive aspects. The reproductive seasonality is one of the most variable aspect between species.

In domestic cats (*Felis catus*), the reproductive seasonality is related to photoperiod (Johnston et al., 1996; Shille et al., 1979; Tsuitsui & Stabenfeldt, 1996), whereas in wild animals, it is also related to high or low food supply during the seasons (Ewer, 1975).

The ovulation mechanism is another variable aspect among cat species. Studies evaluating serum hormone levels confirmed that some wild cats like tiger (*Panthera tigris*) (Seal et al., 1985), snow leopard (*Panthera uncia*) (Schmidt et al., 1993), jaguars (*Panthera onca*) (Wildt et al., 1979) and cougars (*Puma concolor*) (Bonney et al., 1981), have induced reflex ovulation, similar to the domestic cat (Johnston et al., 1996; Shille et al., 1979; Tsuitsui & Stabenfeldt, 1996).

However, female leopards (*Panthera pardus*) presented two ovulation mechanisms in two different situations. When kept isolated, they showed typical hormonal profile for ovulation reflex mechanism; but, when housed in pairs with another female, the ovulation was probably stimulated by physical contact (Schmidt et al., 1988).

Lionesses (*Panthera leo*), when isolated from the males, showed an ovulation pattern distinct from other cats. Schmidt et al. (1979), using serum hormone levels and corpus luteum

Artificial insemination has been conducted in different species of carnivores such as cougar (*Felis concolor*), leopard (*Panthera pardus saxicolor*), cheetah (*Acinonyx jubatus*), tiger (*Panthera tigris altaica*), ocelot (*Leopardus pardalis*) tigrina (*Leopardus tigrinus*), and jaguar (*Panthera onca*) (Dresser et al., 1982; Donoghue et al.; 1993, Howard et al., 1992a; Jimenez et al., 1999; Moore

*In vitro* fertilization (IVF) has also been performed in captive wild cats such as tiger (*Panthera tigris altaica*), jaguar (*Panthera onca*), ocelot (*Leopardus pardalis*) and tigrina (*Leopardus tigrinus*)

The application of artificial reproductive methods in wild animals has not being successful, showing low reproductive rates. Some of the several reasons for these low reproductive rates are lack of knowledgement on the species' physiology, poor sperm or oocytes quality,

Considering the zoos' limitation to maintain genetically viable populations of threatened species, the establishment of genetic banks containing semen, oocytes, embryos and cells emerge as a strategy to ensure the genetic diversity of populations (Lasley et al., 1994). The potential of assisted reproduction for endangered species should be emphasized by the possibility of semen and embryos cryopreservation, which are genetically valuable for the

Although there are some records related to the female reproductive physiology (Table 1) and semen characteristics for various Neotropical felids species (Table 2), there is a little knowledge about the fertilization ability using artificial methods (Table 3) in these animals. Studies about the application of assisted reproduction techniques in Neotropical felids showed limitations because of the lack of basic knowledge on the physiology of the species.

The large number of felines and their wide geographical distribution determine many of the species' particularities, mainly in the reproductive aspects. The reproductive seasonality is

In domestic cats (*Felis catus*), the reproductive seasonality is related to photoperiod (Johnston et al., 1996; Shille et al., 1979; Tsuitsui & Stabenfeldt, 1996), whereas in wild

The ovulation mechanism is another variable aspect among cat species. Studies evaluating serum hormone levels confirmed that some wild cats like tiger (*Panthera tigris*) (Seal et al., 1985), snow leopard (*Panthera uncia*) (Schmidt et al., 1993), jaguars (*Panthera onca*) (Wildt et al., 1979) and cougars (*Puma concolor*) (Bonney et al., 1981), have induced reflex ovulation, similar to the domestic cat (Johnston et al., 1996; Shille et al., 1979; Tsuitsui & Stabenfeldt, 1996).

However, female leopards (*Panthera pardus*) presented two ovulation mechanisms in two different situations. When kept isolated, they showed typical hormonal profile for ovulation reflex mechanism; but, when housed in pairs with another female, the ovulation was

Lionesses (*Panthera leo*), when isolated from the males, showed an ovulation pattern distinct from other cats. Schmidt et al. (1979), using serum hormone levels and corpus luteum

animals, it is also related to high or low food supply during the seasons (Ewer, 1975).

and difficulties in adapting from the methodologies used in experimental models.

et al., 1981; Moraes et al., 1997; Silva et al., 2000; Swanson et al., 1996a).

(Donoghue et al., 1990; Morato et al., 2000; Swanson & Brown, 2004).

**2. General reproductive characteristics in wildlife cats** 

probably stimulated by physical contact (Schmidt et al., 1988).

one of the most variable aspect between species.

future populations.

visualization, demonstrated that this species presents spontaneous ovulation in a higher frequency compared to what is described for other felines.

The genus Leopardus is polyestral and can cycle all year round (Morais et al., 1996; Moreira et al., 2001); the margay (*Leopardus wiedii*) is the only species in this genus presenting spontaneous ovulation (Moreira et al., 2001).

According to Tebet (1999), the estrous cycle in ocelots is characterized by the presence of serum estradiol peaks associated with relatively low levels of serum progesterone (<2.61 ng/mL). This demonstrates the polyestral characteristic of this species, similar to previously observed for other species such as cats (Shille al., 1979; Tsuitsui & Stabenfeldt, 1996; Verhage et al., 1976), tigers (Seal et al., 1985), and cheetahs (Brown et al., 1996).

Ocelot females that ovulated and were not fertilized showed a period of increased progesterone serum concentration and estrus inhibition (Tebet, 1999), called pseudopregancy or diestrus, similar to domestic cats (Feldman & Nelson, 1996) and leopards (Schmidt et al., 1988).

Under an evolutionary analysis and among other factors, the process of spontaneous or induced ovulation may be related to the sociability of the species. Thus, solitary cats would require a longer estrus period, extended viability of oocytes, and extended time for the ovulation to occur, after the couple meet in the wild (Ewer, 1975).

The detection of fecal estrogens and progestins, throught the analyses of fecal metabolites in domestic and wild cats such as the leopard cat (*Felis bengalensis*), cheetah (*Acinonyx jubatus*), clouded leopard (*Neofelis nebulosa*), and snow leopard (*Panthera uncia*) was successfully performed by Brown et al. (1994). Likewise, this methodology has been widely used to monitor ovarian function in Neotropical felines such as the ocelot and margay (Morais et al, 1996; Moreira et al., 2001).

In this context, noninvasive methods such as the quantification of fecal hormonal metabolites are increasingly being used in wildlife animals.

The estrus cycle, gestational time, and number of pups observed in different species of Neotropical wild cats are presented in Table 1.


Wildlife Cats Reproductive Biotechnology 373

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

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

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

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,

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 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).

The collection of semen, through digital manipulation or using an artificial vagina are indicated because they promote a natural and normal ejaculation, however, these methods require intensive animal training. These techniques are effective for wild dogs, but no

presence and quality in epidydimis, at the collection time (Maksudov et al., 2008).

death is worse than that of animals that died accidentally (Maksudov et al., 2008).

freezing and thawing may be different.

1999).

the same species.

**3.2 In vivo sperm collection** 

routinely used in wild cats.


Mellen 19891; Morais et al., 19962; Morato & Paz, 20013; Moreira et al., 20014; Oliveira, 19945; Oliveira & Cassaro, 19976.

Table 1. Neotropical wildlife cats reproductive characteristics.
