**4. Sperm evaluation**

The appearance is the first evaluation of the semen: changes in color may be associated with diseases in the accessory organs and testes. The ejaculate's volume is the second aspect to be evaluated and must be determined immediately after collection. The volume provides information about the semen production in different species. The pH determination is important because it may indicate urine (acidic pH) or bacteria contamination (basic pH).

For sperm evaluation, an aliquot of the ejaculate is placed on a microscope slide warmed at 37°C, covered with a warmed glass coverslip, and examined at 400 X magnification. The

The electroejaculation is the most used method in wild cats because it can be performed in anesthetized animals. However, one of the disadvantages of using this method in cats is the urine contamination of the semen. The urine contamination occurs when the voltage exceeds the minimum necessary level for ejaculation or when the electrode is positioned cranially. One alternative to minimize this problem would be the catheterization or cystocentesis

The Tiletamine-Zolazepam combination has been the most used anesthetic protocol for this procedure because it produces insignificant changes in the ejaculate. The ketamine hydrochloride and xylazine association in the same syringe has been used for semen collection in small cats, but according to Dooley et al. (1991), these drugs, in combination,

The electroejaculator should indicate both the voltage and amperage. The voltage should reach up to 12V, controlled by the command button, which should provide smooth control

The rectal bipolar electrode used for electroejaculation should have three longitudinal strips of copper. The copper strips must have a 0.4 cm apart and protruding approximately 0.2 cm. The electrode, previously lubricated with mineral oil, should be introduced into the rectum with the longitudinal strips ventrally positioned, applying light pressure to increase contact with the pelvic plexus region. The diameter of the electrode should be specific to each

The electrical series follows a specific protocol with 80 stimuli divided into three series: 30 (series 1: 10 stimuli at 2, 3 and 4V), 30 (Series 2: 10 stimulations in 3, 4 and 5V) and 20 (series 3: 10 stimulations in 4 and 5V) (Howard 1993). In jaguars, the last series of stimuli can reach

The stimulation cycle starts at 1 second from 0 voltage to the desired voltage, 2 to 3 seconds at the desired voltage, and 3 seconds returning to 0 voltage. An interval of 10 minutes

Before the start of the series, the penis must be exposed, examined, and washed with saline solution and gauze. The semen collection should be performed in plastic tubes that are maintained warm at 37° C water bath. For each series, the tubes should be replaced in order to avoid urine contamination. All ejaculates should be used; the total volume of semen is the

The appearance is the first evaluation of the semen: changes in color may be associated with diseases in the accessory organs and testes. The ejaculate's volume is the second aspect to be evaluated and must be determined immediately after collection. The volume provides information about the semen production in different species. The pH determination is important because it may indicate urine (acidic pH) or bacteria contamination (basic pH).

For sperm evaluation, an aliquot of the ejaculate is placed on a microscope slide warmed at 37°C, covered with a warmed glass coverslip, and examined at 400 X magnification. The

before start procedure.

species (Table 2).

seems to be related to retrograde ejaculation.

up to 6V to achieve ejaculation (Paz et al., 2000).

should be used for resting between sets.

sum of each ejaculate's volume (Figure 1).

**4. Sperm evaluation** 

and gradual increase in power output.

semen should be evaluated for motility and progressive sperm motility. Motility is expressed in percentages, with 0% being the value for immobile spermatozoa and 100% for maximum spermatozoa performance. The sperm type of movement is evaluated by the progressive sperm motility in scale from 0 to 5 (0 - no motility, 1 - poor lateral movement with some progression, 2 - moderate lateral movement with occasional progression, 3 - slow progression, 4 - progression, 5 - rapid progression) (Howard, 1993).

Fig. 1. Ocelot's penis exposed before semen collection; the penis' spines can be observed (left). Ocelot's electroejaculation procedure (right). Pictures: Regina Paz.

The sperm morphology and concentration can be evaluated by fixing an aliquot of semen (1:3 dilution) in a 10% formaldehyde saline solution or in a 2.5% glutaraldehyde solution after the preparation of samples in a humidified chamber.

For the determination of the sperm morphology, 200 cells per slide were counted at 1000 X magnification under light microscopy; the abnormalities were classified as primary or secondary defects expressed as percentages. According to the primary defects presented in the sample, the sperm can be classified as macrocephalic, microcephalic, bicephalic, pyriform head, rounded head, abnormal acrosome, abnormal midpiece, no midpiece, tightly coiled tail and biflagelat. According to the secondary defects presented in the sample, the sperm can be classified as bent midpiece with or without droplet, bent tail with or without droplet, and proximal or distal droplet.

The concentration can be evaluated using a Neubauer chamber at 400 X magnification under ligth microscopy. The volume, concentration, motility, vigor and abnormal sperm data in different species are presented in Table 2.

Wildlife Cats Reproductive Biotechnology 377

After being removed from liquid nitrogen, the semen straws should be immediately thawed for 1 min in waterbath at 37°C, evaluated for total motility (%) and progressive sperm

The first step in the process of freezing semen is the removal of the supernatant after centrifugation of the semen collected in HAM'S F-10 or HEPES culture media, and the subsequent ressuspension of the pellet in PDV fraction A at 37 °C. This mixture should be kept in the refrigerator for 2 hours followed by a subsequent slow addition of the PDV fraction B. The material is them transferred to cooled 0.25 mL straws and kept in the refrigerator for 30 minutes. Afterwards, each straw is placed in liquid nitrogen vapor for 20 min, immersed in liquid nitrogen, transferred to the racks, and loaded into the canisters for

The straws, racks and canister identification are extremely important, being the determining factor for the germplasm bank establishment and successful operation. The material collected might be extremely valuable for populations in the future and the safety use of this

The straws identification must contain the animal species (scientific name), tattoo or microchip number, the institution to which the animal belongs, and the date. For free-living animals, the straws must contain the species, the location where the animal was captured, and the date. The racks may be identified by numbers or if applicable, by species. A registry, which can be computerized, is essential to record all of the straws, racks, and canisters,

The reasons for the poor quality of wildlife semen after thawing are still unknown and involve a range of information and specific characteristics for each species, which are also not yet clearly understood. New tests with different protocols and different cryoprotectors for each species of interest are required in order to maximize the spermatozoa viability after

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;

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

motility (scale, 0-5) before use.

long-term liquid nitrogen storage at -196°C.

material depends to the correct identification.

thereby facilitating the location of the material.

**6. Ovarian activity induction and superovulation** 

by daily injections did not influence the ovarian response.

cryopreservation procedures.

Pope, 2000; Wildt et al., 1981).


Howard, 19931; Morais, 20012; Paz et al., 20003;

Table 2. Neotropical wildlife cats seminal characteristics.

The poor semen quality in carnivores may be related to the nutritional status of the animals. Rodrigues da Paz et al. (2006), studying the reproduction of jaguars, observed a positive correlation between the improvement in the semen quality and the decrease of primary defects, after diet supplementation with vitamins and minerals. Ocelots, tigrinus and margays showed an increase in the number of ejaculates and 20-30% improvement related to sperm defects after receiving vitamin and mineral supplementation (Morais, 2001).

The seminal plasma constituents compromise the sperm viability in some species. Thus, washing the ejaculates in culture media (HEPES, HAM'SF-10) by centrifugation is efficient in removing the seminal plasma, which could contain bacteria and other undesirable microorganisms, especially when the semen will be used for intrauterine artificial insemination.
