**6. Ovarian activity induction and superovulation**

The currently used ovarian stimulation and superovulation protocols require injections of exogenous gonadotropins, which consist of large complexes of glycoproteins. Equine Chorionic Gonadotropin (eCG) and Human Chorionic Gonadotrophin (hCG) are frequently used due to their long half-life in circulation (24-48h) and good ovarian response with a single application. Other hormones used are the porcine Follicle Stimulating Hormone (pFSH) and porcine Luteinizing Hormone (pLH), these hormones are characterized as short half-life (~ 2h) gonadotropins, therefore, they present the disadvantage of requiring multiple applications to produce a good ovarian response (Crichton et al., 2003; Dresser et al., 1988; Pope, 2000; Wildt et al., 1981).

Studies on wild cats report the use of eCG/hCG in combination, mainly to avoid the stress associated with multiple injections of FSH (Roth et al., 1997). However, the use of porcine FSH/LH determined equivalent number of oocytes compared to the established protocol for eCG/hCG used in tigers (*Panthera tigris*) (Crichton et al., 2000), ocelot (*Leoparuds pardalis*) and tigrinas (*Leopardus tigrinus*) (Paz et al., 2005, 2006), demonstrating that the stress caused by daily injections did not influence the ovarian response.

Wildlife Cats Reproductive Biotechnology 379

they have different sensitivities to exogenous gonadotropins (Roth et al., 1997; Swanson et al., 1996b). The nutritional status of the animal also influences the fertilization success rate. Swanson et al. (2002a), studying ocelots and tigrinas in Brazil, observed better quality of oocytes and increase fertilization rates after supplementing the diet with vitamins and

The rate of artificial insemination success in carnivores is influenced by the localization of the semen deposition. Non-surgical methods of semen deposition in the vagina has shown inferior results compared with the surgical method, with semen deposition directly into the uterus. This can be explained by the chemical restraint need in wild animals, with the anesthesia compromising the sperm transportation in non-surgically insemination (Howard, 1993).

The artificial insemination success with semen deposition in the uterine horn is described in several species of wild cats such as puma (*Puma concolor*) (Moore et al., 1981); leopard (*Panthera pardus saxicolor*) (Dresser et al., 1982); cheetah (*Acinonyx jubatus*) (Howard, 1992); tiger (*Panthera tigris altaica*) (Donoghue et al., 1993); ocelot (*Leopardus pardalis*) (Swanson,

Artificial insemination using video-laparoscopy technique has been developed for the semen deposition directly into the uterine horn, close to the oviduct where fertilization occurs, in addition to being a less invasive method. In this procedure, the ovaries and uterine horns can be accessed and evaluated for thickness, consistency and color in all species. In cats, the ovaries are easily observed, facilitating the counting and characterization of pre-ovulatory follicles (brighter small elevated areas) and post-ovulatory corpus luteum

According to ovarian stimulation protocols, the animals should be inseminated within 24 to 48 hours after hCG or pLH administration, or after the ovulation process. Inhalatory

Fig. 2. Anesthesia with isoflurane gas mask (left) and intubation (right). Pictures: Regina

The anesthetized cats should be secured in dorsal recumbency with the use of leg ties on a tilting surgical table, and the abdominal region of each female should be clipped and prepped

**7. Artificial insemination and oocytes collection** 

1996a) and tigrina (*Leopardus tigrinus)* (Moraes et al., 1997*)*.

anesthesia is necessary to perform this procedure (Figure 2).

minerals.

(yellow-red area).

Paz.

The eCG/hCG combination has been used successfully in tigers (*Panthera tigris*), cheetahs (*Acinonyx jubatus*), clouded lepards (*Neofelis nebulosa*), pumas (*Puma concolor*), ocelots (*Leopardus pardalis*) and tigrinas (*Leopardus tigrinus*) (Barone et al., 1994; Donoghue, 1993; Donoghue et al., 1990; Howard, 1992b; Moore et al., 1981; Moraes et al., 1997; Morato et al., 2000; Swanson, 1996a). The FSH and hCG combination was used successfully in the Indian desert leopard (*Felis sylvestris ornata*) (Pope et al., 1989), and the pFSH and pLH combination in tigers (Crichton et al., 2000, 2003) jaguars (*Panthera onca*) (Morato et al., 2000), ocelots (*Leopardus pardalis*) and tigrinas (*Leopardus tigrinus*) (Paz et al., 2005, 2006).

Swanson et al. (1995, 1996a) suggest that the repeated administration of exogenous gonadotropins, within short time intervals is a problem because it causes a reduction in the ovarian stimulation which is immunologically mediated. The repeated administration of exogenous gonadotropins has been associated with the production of neutralizing immunoglobulin, which prevents the ovarian response to superovulation protocols.

Alternating gonadotropins regimens in sequential treatments are indicated because of variable immunoglobulin affinities to different exogenous gonadotropins (Maurer et al., 1968; Swanson et al., 1995).

Ocelots and tigrinas treated four to six times, at 4-month intervals, with alternating exogenous gonadotropin regimens (eCG/hCG and pFSH/pLH) did not show a reduction in ovarian response (total follicles and Corpora Lutea), oocyte maturation or exogenous gonadotropins antibodies production over time (Paz et al., 2005, 2006). The findings suggest that, these endangered cat species may be managed intensively with the use of alternating exogenous gonadotropin regimens in assisted reproduction procedures without compromising ovarian responsiveness to these hormones.


Barone et al., 19941 ;Jimenez et al., 19992 ;Morato et al., 20003 ; Paz et al., 20054 ; Paz et al., 20065 ; Swanson et al., 1996a6 ; Swanson (Personal Communication)7 .

Table 3. Ovarian stimulation with exogenous gonadotropins used in wildlife cats reproduction (AI= Artificial Insemination and IVF= *In vitro* fertilization).

The exogenous gonadotropins dosage used for ovarian stimulation is another important factor to the fertilization rate and subsequent embryonic development (Donoghue et al., 1993). Species with similar size and weight may require varying dosages, possibly because

The eCG/hCG combination has been used successfully in tigers (*Panthera tigris*), cheetahs (*Acinonyx jubatus*), clouded lepards (*Neofelis nebulosa*), pumas (*Puma concolor*), ocelots (*Leopardus pardalis*) and tigrinas (*Leopardus tigrinus*) (Barone et al., 1994; Donoghue, 1993; Donoghue et al., 1990; Howard, 1992b; Moore et al., 1981; Moraes et al., 1997; Morato et al., 2000; Swanson, 1996a). The FSH and hCG combination was used successfully in the Indian desert leopard (*Felis sylvestris ornata*) (Pope et al., 1989), and the pFSH and pLH combination in tigers (Crichton et al., 2000, 2003) jaguars (*Panthera onca*) (Morato et al., 2000), ocelots

Swanson et al. (1995, 1996a) suggest that the repeated administration of exogenous gonadotropins, within short time intervals is a problem because it causes a reduction in the ovarian stimulation which is immunologically mediated. The repeated administration of exogenous gonadotropins has been associated with the production of neutralizing

Alternating gonadotropins regimens in sequential treatments are indicated because of variable immunoglobulin affinities to different exogenous gonadotropins (Maurer et al.,

Ocelots and tigrinas treated four to six times, at 4-month intervals, with alternating exogenous gonadotropin regimens (eCG/hCG and pFSH/pLH) did not show a reduction in ovarian response (total follicles and Corpora Lutea), oocyte maturation or exogenous gonadotropins antibodies production over time (Paz et al., 2005, 2006). The findings suggest that, these endangered cat species may be managed intensively with the use of alternating exogenous gonadotropin regimens in assisted reproduction procedures without

**Specie Procedure Treatment 1 Treatment 2** 

4006 5004,5

754,5 2006

1007 2007

2001 -

2002 -

Table 3. Ovarian stimulation with exogenous gonadotropins used in wildlife cats

reproduction (AI= Artificial Insemination and IVF= *In vitro* fertilization).

Barone et al., 19941 ;Jimenez et al., 19992 ;Morato et al., 20003 ; Paz et al., 20054 ; Paz et al., 20065 ; Swanson

The exogenous gonadotropins dosage used for ovarian stimulation is another important factor to the fertilization rate and subsequent embryonic development (Donoghue et al., 1993). Species with similar size and weight may require varying dosages, possibly because

**ECG (UI) hCG (UI) pFSH (UI) pLH (UI)** 


304,5 -

> - -

> - -



104,5 -

> - -

> - -


2006 2254,5

1004,5 1506

> 757 1507

1001 -

1502 -

immunoglobulin, which prevents the ovarian response to superovulation protocols.

(*Leopardus pardalis*) and tigrinas (*Leopardus tigrinus*) (Paz et al., 2005, 2006).

compromising ovarian responsiveness to these hormones.

AI6 IVF4,5

AI4,5 IVF6

AI7 IVF7

AI1 IVF

AI2 IVF3

et al., 1996a6 ; Swanson (Personal Communication)7 .

1968; Swanson et al., 1995).

Ocelot *(Leopardus pardalis)*

> Tigrinus (*Leopardus tigrinus)*

Gato mourisco *(H. yagouarundi)*

Puma *(Puma concolor)*

Jaguar *(Panthera onca)* they have different sensitivities to exogenous gonadotropins (Roth et al., 1997; Swanson et al., 1996b). The nutritional status of the animal also influences the fertilization success rate. Swanson et al. (2002a), studying ocelots and tigrinas in Brazil, observed better quality of oocytes and increase fertilization rates after supplementing the diet with vitamins and minerals.
