**8.** *In vitro* **fertilização and embryo transfer**

The *in vitro* fertilization technique has been applied in wild animals after follicular aspiration using laparoscopy tecnhique, oocyte retrieval post-mortem, or after ovariohysterectomy. According Swanson (1998), oocytes collected from ovaries can be refrigerated at 5°C for 24 hours without maturation and changes in the fertilization potential.

Oocytes recovery from refrigerated ovaries can be achieved using the follicular aspiration technique with a syringe and needle, or through ovary laceration and oocyte harvest using stereomicroscopy. The second technique is used in small animals, which present ovaries with small diameters because the follicle aspiration would be difficult. The M199 culture medium is used for follicular aspiration and ovaries laceration.

However, laparoscopic follicular aspiration is the most used oocyte retrieval technique for IVF, which should be preceded by hormonal treatment. According Howard (1999), the

Wildlife Cats Reproductive Biotechnology 383

The methods for processing semen without centrifugation and removal of the seminal plasma were responsible for the development of pyometra in 40% of inseminated domestic cats, regardless of the semen being diluted in culture medium containing antibiotics penicillin and streptomycin (Howard, 1993). It is believed that the donors might carry

In a study conducted by Paz et al. (1999), aiming at determining preputial microbiota in nine adult male jaguars (*Panthera onca*), the most frequently observed microorganism were *Staphylococcus sp* (40%), followed by *Streptococcus sp* (30%), *Escherichia coli* (20%) and

The preputial microbiota in the genus Leopardus was assessed by Guido et al. (2000), and the results were *Escherichia coli* (40%), *Proteus rettgeri* (40%) and *Yersinia psedotuberculosis* (20%) in tigrina (*Leopardus tigrinus*) (n = 5), *Staphylococcus sp* (42.9%), *Escherichia coli* (28.5%), *Streptococcus sp* (14.3%), *Staphylococcus sp* + *Streptococcus sp* (14.3%) in ocelots (*Leopardus* 

The feline immunodeficiency virus is present in the semen of domestic cats and can be transmitted to females by AI (Jordan et al., 1995, 1996). This aspect should be taken into

In addition, a reproductive evaluation and clinical examination should be performed in wild cats before the animal inclusion in the management programs using assisted reproduction.

Bartels, P.; Lubbe, K.; Killian, L.; Friedmann, Y.; Van Dyk, G. & Mortimer, D. (2000). In vitro

Barone, M. A.; Wildt, D. E.; Byers, A. P.; Roelke, M. E.; Glass, C. M. & Howard, J. G. (1994).

Bonney, R. C.; Moore, H. D. M. & Jones, D. M. (1981). Plasma concentrations of oestradiol-

Briz, M. D.; Bonet, S.; Pinart, B.; Egozcue, J. & Campos, R. (1995). Comparative study of boar

Brown, J. L.; Wasser, S. K.; Wildt, D.E. & Graham, L. H. (1994). Comparative aspects of

Brown, J. L.; Terio, K. A. & Graham, L. H. (1996). Fecal androgen metabolite analysis for non

Byers, A. P.; Hunter, A. G.; Seal, E. S.; Graham, E.F. & Tilson, R.L. (1990). Effect of season on

noninvasively in feces*. Biology of Reproduction*, Vol.51, pp. 766-86.

*tigris*). *Journal of Reproduction and Fertility,* Vol. 90, pp. 119-125.

maturation and fertilization of lion (*Panthera onca*) oocytes using frozen-thawed epididymal spermatozoa recovered by cauda epididymectomy of an immobilized

Gonadotrophin dose and timing of anaesthesia for laparoscopic artificial insemination in the puma (*Felis concolor*). *Journal of Reproduction and Fertility*,

17 and progesterone, and laparoscopic observations of the ovary in the puma (*Felis concolor*) during oestrus, pseudopregnancy and pregnancy. *Journal of* 

sperm coming from the caput, corpus, and cauda regions of the epididymis*. Journal* 

steroid hormone metabolism and ovarian activity in felids, measured

invasive monitoring of testicular steroidogenic activity in felids. *Zoo Biology*, Vol.15,

seminal traits and serum hormone concentration in male Siberian tiger (*Panthera* 

bacteria in their normal flora, eg *E. coli*, which would cause infection in the females.

*pardalis*) (n = 6) and only *Staphylococcus sp* in margay (*Leopardus wieddi*) (n = 1).

consideration during assisted reproduction procedures performed in wild cats.

lion. *Theriogenology*, Vol. 53, pp. 325.

*of Andrology*, Vol. 16, pp. 175-188.

*Reproduction and Fertility*, Vol.63, pp. 523-532.

Vol.101, pp. 103-108.

pp. 425-434.

*Corynebacterium sp* (10%).

**10. References** 

treatment with exogenous gonadotropins and laparoscopy are the basic requirements for ovarian stimulation and oocyte retrieval for IVF procedures.

The immature oocytes collected ( 60%) in cats become mature in 24-32 hours in culture media. These about 70% are fertilized, however, only a small percentage, from 20 to 30% develops in blastocysts (Johnston et al., 1989).

Donoghue et al. (1990) reported the birth of the first wild cat from IVF after an embryo transfer. Tiger cubs (*Panthera tigris*) were produced *in vitro* using excellent quality embryos, containing two to four cells, and surgically transferred to the oviducts of two females. Pregancy was successful in one of these females and three kittens were born after 107 days.

The first embryos produced by IVF in Brazil were jaguar (*Panthera onca*) embryos. The ovarian stimulation with pFSH/LH produced 25 follicles/female ( 80%), however, despite the recovery of high quality oocytes, the fertilization percentages were low ( 25%) (Morato et al., 2000).

In a Project involving the São Paulo University/Brazil (USP), the Mata Ciliar Association/Brazil (AMC) and the Cincinnati Zoo/USA (CREW), 128 ocelot (*Leopardus pardalis*) and tigrina (*Leopardus tigrinus*) embryos were produced. The animals were treated with eCG/hCG and produced 10 follicles/female. Follicles were aspirated by laparoscopy and 7-9 of excellent quality oocytes/female were recovered. Of these, 60% were fertilized *in vitro*, resulting in 76 ocelot and 52 tigrina embryos (Swanson & Brown, 2004). Two ocelots became pregnant with normal pregnancy development and birth, one from the Cincinnati Zoo/USA and another from the Sao Bernardo do Campo Zoo/Brazil. However, only the offspring born to the American female survived after birth (Swanson, 2002b).

Among all the Neotropical cats, the ocelot is the only species that produced offspring after the transfer of frozen embryos (Swanson, 2001, 2002b). These births were the result of a cooperative effort between Brazil and the USA for the development of adequate management programs for this species in captivity, and for the establishment of genetically viable populations between the Brazilian and American populations.

Based on the percentage of successful embryonic cleavage after thawing domestic cat embryos, which is less than 70% (Pope, 2000), the difficulties to develop feline embryos used post-freezing is recognized. The difficulties may be related to inappropriate timing for the embryo implantation or fetal survivel (Swanson & Brown, 2004) and not only related to the quality of the embryos. Thus, it is necessary to detect the female natural receptor estrus in order to perform the transfer of thawed embryos. This determination is achieved through measurement of fecal steroid levels according to the enzyme-immunoassay (EIA) technique for the fecal estrogens metabolites.

After the determination of the natural estrus, the ovulation should be induced with GnRH (Gonadotropin Releasing Hormone) and the embryos should be transferred by videolaparoscopy directly in the ostium of the oviduct.
