Methods

Animals

Adult male giant pandas were evaluated in February and March (during the early breeding season) in 1998, 1999 and 2000. Of the 24 males in the Biomedical Survey, 17 were of an age that allowed a detailed fertility assessment. They were distributed among four facilities: Chengdu Zoo (n = 1 male); the Chengdu Research Base of Giant Panda Breeding (n = 3); Beijing Zoo (n = 4); and the China Conservation and Research Centre for the Giant Panda (n = 9) (within the Wolong Nature Reserve). Animals were 5.5 to 16.5 years of age: 5.5 years (n = 4 males), 6.5 (n = 2), 7.5 (n = 1), 8.5 (n = 1), 10.5 (n = 3), 12.5 (n = 1), 13.5 (n = 2), 14.5 (n = 2) and 16.5 (n = 1). All males were maintained in individual enclosures with indoor/outdoor access. Diets consisted of freshly cut bamboo and a concentrate in the form of gruel or steamed bread, all of which was fed several times a day. Historical data were collected on each male that included origin (wild-born or captive-born), date ofbirth and reproductive history, including past opportunities to breed (see Chapter 5).

Physical examination, morphometric traits and testicular development

Each male was induced into a surgical plane of anaesthesia using injectable ketamine HCl (Ketaset, Fort Dodge Laboratories, Inc., Fort Dodge, IA) with maintenance often sustained with isoflurane gas (for details, see Chapter 4). Body weight and numerous morphometric measurements, including neck girth, chest girth and body length, were obtained (see Chapter 4). Testes were examined for location (i.e. scrotal sac versus inguinal) and palpated for tone and consistency. Each testis was measured for length and width, which permitted calculating testis volume using the formula for a prolate spheroid: volume = (4/3)pab2 where a is 1/2 length and b is 1/2 width) (Beyer, 1987). For quick reference, the formula was recalculated (volume = 0.524 x L x W2 where L is length and W is width). Total testicular volume per male was determined by adding the right and left testis volumes.

Semen collection and evaluation

Semen was collected by an already described electroejaculation technique for the giant panda (Platz et al., 1983; Howard, 1993, 1999) which relied on a 2.6-cm diameter rectal probe with three longitudinal, stainless-steel electrodes and a 60-Hz sine wave stimulator (P. T. Electronics, Boring, OR) (Fig. 7.1). Each male was placed in a dorsal recumbent position for electroejaculation. The probe was inserted into the rectum with the middle electrode positioned ventrally. A standardised set of low-voltage stimulations (2-8 V) over three series of 30 stimuli each was adequate to elicit ejaculation. The electrical stimuli consisted of a slow steady rise from 0 V to initial peak voltage over a three second time period. The current was held at this peak for two to three seconds and then returned rapidly to zero. Voltage was increased after administering ten stimuli at the same voltage. The entire semen collection interval generally required less than 20 minutes. A predetermined amount of semen (0.5 or 1.0 ml) from each male was allocated to these characterisation assessments and comparative sperm cryopreservation studies. The remaining semen was used for AI or cryopreserved for long-term storage.

Semen assessments were conducted on 16 of the 17 males. The remaining male (8.5 years) was in poor health with Stunted Development Syndrome (see Chapter 4) and bilaterally retained inguinal testes, so only morphometric traits were measured. Freshly obtained semen from the other males was evaluated immediately for ejaculate volume, pH (via test strips; EM Science, Gibbstown, NJ) and sperm concentration (via a standard haemocytometer method; Howard, 1993). Subjective estimates of sperm motility and forward progression (i.e. the type of forward movement of sperm from a rapid, straight direction to

Electroejaculator Machines
Figure 7.1. (a) Electroejaculator and rectal probe used for semen collection in the giant panda, and (b) close-up of the glans penis and semen collection vial.

quivering with no progression on a scale of 0 to 5; 5 being best) was determined at x400 using a microscope with stage warmer (Howard, 1993). Spermatozoa were maintained in a plastic vial (Vangard International, Inc., Neptune, NJ) at 37°C in a water bath or dry-bath incubator.

Sperm morphology and acrosomal membranes

Sperm morphology was determined from fixed aliquots of raw semen as previously described (Howard, 1993) (Fig. 7.2). A 5 ml aliquot of the fresh electroejaculate was added to 100 ml of fixative (0.3% glutaraldehyde in saline) for morphological examination of spermatozoa by phase-contrast microscopy (x 1000) (Howard, 1993). Sperm were categorised as normal or as having one of the following anomalies: an abnormal head

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