**7. Artificial insemination and oocytes collection**

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

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 (yellow-red area).

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

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

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

Wildlife Cats Reproductive Biotechnology 381

The endoscope is placed near the umbilicus for the evaluation of ovaries and observation of the follicles. Only mature follicles, larger than 2 mm, should be aspirated. The *Verres* needle is used for the follicles' size determination and for the maintenance of the ovary in an

The mature follicles are aspirated with a 22G needle attached to polypropylene tubing connected to a sterile collection tube (15mL) containing M199 culture medium and heparin, which is attached to a vacuum aspiration pump (Figure 4). After the collection, the oocytes are placed in petri dishes with culture medium and observed by stereomicroscopy at 400 X

The oocytes from carnivores are dark and contain lipidic drops. The maturation status is characterized in I, II and III according morphological aspects. Oocytes I are of excellent quality, characterized by a uniformly dark cytoplasm, nucleus with a distinct corona radiate, and an expansive cumulus cell mass. Oocytes II are of regular quality, characterized by a nonuniformly cytoplasm, nucleus with an indistinct corona radiate, and a non-expansive cumulus cell mass. Oocytes III are degenerated and characterized by an abnormal cytoplasm, nucleus without corona radiata or cumulus cell mass (Goodrowe et al., 1988; Johnston et al., 1989).

Fig. 4. Follicular aspiration using video-laparoscopy and *Verres* needle in ocelot (left).

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

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

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

Aspiration follicular system (right). Pictures: Regina Paz.

hours without maturation and changes in the fertilization potential.

medium is used for follicular aspiration and ovaries laceration.

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

adequate position, near to the abdominal wall.

magnification for their classification.

with alternating applications of Betadine scrub and alcohol. A pneumoperitoneum should be created by means of CO2 gas introduced through a *Verres* needle inserted transcutaneously into the central abdominal cavity. A 7-mm-diameter laparoscope should be inserted through a 1cm skin incision slightly cranial to the umbilicus. The ovaries could be manipulated with the *Verres* needle probe, and each ovary should be closely examined to determine the number of mature follicles ( 2 mm diameter), recently-formed corpora lutea, and corpora albicans.

To stabilize the uterine horn, where the cannula will be introduced to deposit the semen, grasping forceps is inserted laterally, 4 to 5 cm of the umbilicus. This procedure maintains the uterus close to the abdominal wall. The horn to be inseminated is the ovary that shows the corpus luteum after ovulation. The procedure should be performed in both uterine horns if both ovaries present the corpus luteum.

For the semen deposition, the uterine horn is cannulated using a 20G sterile needle catheter inserted through the abdominal cavity, near the uterine lumen. As soon as the needle pierces the uterine horn, it is removed, keeping the catheter in place. Inside the catheter, a sterile polypropylene tube must be inserted, which will be connected to a syringe containing the semen. (Figure 3).

The intrauterine artificial insemination by laparoscopy is less invasive because the semen deposition ocurrs directly in the uterine horn without laparotomy. This methodology resulted in a 46.2% increase in cheetah's pregnancy rates (Howard, 1992).

Similarly, Donoghue et al. (1993) reported the first birth of a tiger cub (*Panthera tigris altaica*) in Siberian Tiger Species Survival Plan (SSP Program) after intrauterine insemination by video-laparoscopy in females stimulated with eCG and hCG. This result demonstrates the importance in using assisted reproduction methods in the production of genetically viable population with recommended breeding by a management program.

Fig. 3. Intrauterine insemination set: 1mL syringe, polypropylene tube and 22G needle (left). Conected set (right). Pictures: Regina Paz.

The procedures for oocyte retrieval in *in vitro* fertilization are performed using the laparoscopy technique. The ovaries' visualization and the oocyte retrieval in carnivores are species-specific. Cats' ovaries are easily accessed and the follicles easily aspirated.

General anesthesia is needed to perform this procedure; isoflurane inhalation anesthesia is generally used. After anesthetized, the animals are placed in the supine position (45 degrees) and pneumoperitoneum is created with the *Verres* needle, which can be coupled to a CO2 gas automatic insulflator or a manual pump.

with alternating applications of Betadine scrub and alcohol. A pneumoperitoneum should be created by means of CO2 gas introduced through a *Verres* needle inserted transcutaneously into the central abdominal cavity. A 7-mm-diameter laparoscope should be inserted through a 1cm skin incision slightly cranial to the umbilicus. The ovaries could be manipulated with the *Verres* needle probe, and each ovary should be closely examined to determine the number of mature follicles ( 2 mm diameter), recently-formed corpora lutea, and corpora albicans.

To stabilize the uterine horn, where the cannula will be introduced to deposit the semen, grasping forceps is inserted laterally, 4 to 5 cm of the umbilicus. This procedure maintains the uterus close to the abdominal wall. The horn to be inseminated is the ovary that shows the corpus luteum after ovulation. The procedure should be performed in both uterine

For the semen deposition, the uterine horn is cannulated using a 20G sterile needle catheter inserted through the abdominal cavity, near the uterine lumen. As soon as the needle pierces the uterine horn, it is removed, keeping the catheter in place. Inside the catheter, a sterile polypropylene tube must be inserted, which will be connected to a syringe containing

The intrauterine artificial insemination by laparoscopy is less invasive because the semen deposition ocurrs directly in the uterine horn without laparotomy. This methodology

Similarly, Donoghue et al. (1993) reported the first birth of a tiger cub (*Panthera tigris altaica*) in Siberian Tiger Species Survival Plan (SSP Program) after intrauterine insemination by video-laparoscopy in females stimulated with eCG and hCG. This result demonstrates the importance in using assisted reproduction methods in the production of genetically viable

Fig. 3. Intrauterine insemination set: 1mL syringe, polypropylene tube and 22G needle (left).

The procedures for oocyte retrieval in *in vitro* fertilization are performed using the laparoscopy technique. The ovaries' visualization and the oocyte retrieval in carnivores are

General anesthesia is needed to perform this procedure; isoflurane inhalation anesthesia is generally used. After anesthetized, the animals are placed in the supine position (45 degrees) and pneumoperitoneum is created with the *Verres* needle, which can be coupled to

species-specific. Cats' ovaries are easily accessed and the follicles easily aspirated.

resulted in a 46.2% increase in cheetah's pregnancy rates (Howard, 1992).

population with recommended breeding by a management program.

horns if both ovaries present the corpus luteum.

Conected set (right). Pictures: Regina Paz.

a CO2 gas automatic insulflator or a manual pump.

the semen. (Figure 3).

The endoscope is placed near the umbilicus for the evaluation of ovaries and observation of the follicles. Only mature follicles, larger than 2 mm, should be aspirated. The *Verres* needle is used for the follicles' size determination and for the maintenance of the ovary in an adequate position, near to the abdominal wall.

The mature follicles are aspirated with a 22G needle attached to polypropylene tubing connected to a sterile collection tube (15mL) containing M199 culture medium and heparin, which is attached to a vacuum aspiration pump (Figure 4). After the collection, the oocytes are placed in petri dishes with culture medium and observed by stereomicroscopy at 400 X magnification for their classification.

The oocytes from carnivores are dark and contain lipidic drops. The maturation status is characterized in I, II and III according morphological aspects. Oocytes I are of excellent quality, characterized by a uniformly dark cytoplasm, nucleus with a distinct corona radiate, and an expansive cumulus cell mass. Oocytes II are of regular quality, characterized by a nonuniformly cytoplasm, nucleus with an indistinct corona radiate, and a non-expansive cumulus cell mass. Oocytes III are degenerated and characterized by an abnormal cytoplasm, nucleus without corona radiata or cumulus cell mass (Goodrowe et al., 1988; Johnston et al., 1989).

Fig. 4. Follicular aspiration using video-laparoscopy and *Verres* needle in ocelot (left). Aspiration follicular system (right). Pictures: Regina Paz.
