**5. Sperm cryopreservation**

The semen cryopreservation procedures should be initiated only after the centrifugation at 300g for 10 minutes in culture medium or HEPES HAM'SF-10. This procedure is essential for eliminating microorganisms and seminal plasma remove.

The "Double Step" cryopreservation method, using glycerol as cryoprotectant, is in general, used for the semen of most carnivores. This method use two fractions: fraction A containing nutritional constituents and antibiotics; and fraction B containing nutritional constituents, antibiotics, and the cryoprotectant. The PDV medium is used for the semen cryopreservation of the majority of wildlife cats. The fraction A contains 20% egg yolk, 11% lactose, 1000 IU penicillin/mL, 1000 mg streptomycin/mL; the fraction B contains 20% egg yolk, 11% lactose, 8% glycerol, 1000 IU penicillin/mL and 1000 mg streptomycin/mL.

*(L. tigrinus)* 1.01 182 0.110.022 78.533.82 62.15.72 - 35.66.02

**Concentr. (x106/mL)** 

28.017.01 53.817.82

79.928.11 14.25.32

12.59.41 7.24.02

10.85.71 364.0326.02

300.0233.21 66.524.42

12.01.91 13.1610.763

*(P. concolor)* 1.61 121 2.80.51 20.24.71 52.08.01 3.50.21 23.43.71

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

The semen cryopreservation procedures should be initiated only after the centrifugation at 300g for 10 minutes in culture medium or HEPES HAM'SF-10. This procedure is essential

The "Double Step" cryopreservation method, using glycerol as cryoprotectant, is in general, used for the semen of most carnivores. This method use two fractions: fraction A containing nutritional constituents and antibiotics; and fraction B containing nutritional constituents, antibiotics, and the cryoprotectant. The PDV medium is used for the semen cryopreservation of the majority of wildlife cats. The fraction A contains 20% egg yolk, 11% lactose, 1000 IU penicillin/mL, 1000 mg streptomycin/mL; the fraction B contains 20% egg yolk, 11% lactose, 8% glycerol, 1000 IU penicillin/mL and 1000 mg streptomycin/mL.

**Motility (%)** 

72.012.51 70.42.32

86.03.31 62.85.32

50.09.91 57.82.52

36.76.61 81.36.32

73.04.41 64.04.72

82.05.81 56.99.353 **Vigour (0-5)** 

4.00.51 -

4.60.21 -

3.50.41 -

2.80.21 -

4.00.31 -

4.10.31 3.020.773 **Normals (%)** 

80.80.91 58.45.82

48.56.11 39.57.72

35.414.31 25.74.62

65.923.81 56.50.52

29.011.51 46.95.02

58.211.11 65.736.73

**Volume (mL)** 

0.30.11 0.620.082

0.20.11 0.310.052

0.10.11 0.080.022

0.30.11 0.080.012

0.20.11 0.210.032

2.70.61 5.71.713

**Species Probe**

*Ocelot* 

*Tigrinus* 

*Margay* 

*Jaguarondi (H.* 

*Pampas cat* 

*Geofroy's cat* 

*Puma* 

*Jaguar (P. onca)* 

insemination.

**5. Sperm cryopreservation** 

**(cm)**

*(L. pardalis)* 1.01 <sup>51</sup>

*(L. wiedii)* 1.01 <sup>111</sup>

*yagouarundi)* 1.01 <sup>31</sup>

*(O. colocolo)* 1.01 <sup>51</sup>

*(O. geofroy)* 1.01 <sup>81</sup>

3.01 2.33

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

**Nº Ejaculates**

382

272

212

22

242

51 383

Table 2. Neotropical wildlife cats seminal characteristics.

for eliminating microorganisms and seminal plasma remove.

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 motility (scale, 0-5) before use.

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

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 material depends to the correct identification.

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, thereby facilitating the location of the material.

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 cryopreservation procedures.
