**3.2 In vivo sperm collection**

The collection of semen, through digital manipulation or using an artificial vagina are indicated because they promote a natural and normal ejaculation, however, these methods require intensive animal training. These techniques are effective for wild dogs, but no routinely used in wild cats.

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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

Fig. 1. Ocelot's penis exposed before semen collection; the penis' spines can be observed

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

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

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

(left). Ocelot's electroejaculation procedure (right). Pictures: Regina Paz.

after the preparation of samples in a humidified chamber.

droplet, and proximal or distal droplet.

different species are presented in Table 2.

progression, 4 - progression, 5 - rapid progression) (Howard, 1993).

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 before start procedure.

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, seems to be related to retrograde ejaculation.

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 and gradual increase in power output.

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 species (Table 2).

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

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 should be used for resting between sets.

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