Methods

Semen processing for assisted breeding

Semen was collected by electroejaculation from nonbreeding males and assessed for ejaculate volume, sperm concentration, percentage sperm motility and sperm forward progression (on a scale of 0 to 5; 5 is best) using protocols described earlier (Huang et al., 2000b; see also Chapter 7). For AI, ejaculates were diluted in either SFS (prepared fresh at the giant panda breeding facilities in China; described in Chapter 7) or TEST (Irvine Scientific, Santa Ana, CA) egg-yolk diluent containing 0% or 4 to 5% glycerol. These mixtures were then used for AI either while still fresh or after cold storage at 4°C (for up to 48 hours) (Olson et al., 2003a,b). For future AI, some aliquots were cryopreserved using the pellet or straw (0.25 ml) method of freezing (Huang et al., 2000a,b; see also Chapter 7).

Artificial insemination

Female giant pandas were monitored beginning mid January through May for natural oestrus. The time of AI was based on behavioural signs of oestrus (Kleiman et al., 1979; Bonney et al., 1982; Zeng et al., 1984) and urinary hormones (increased oestrogen) assessed by enzyme immunoassay (see Chapter 8). Early indications of oestrus included increased vocalisation (particularly bleating), scent marking, restlessness and decreased appetite. These changes were followed by more overt behaviours including backwards walking and lordosis in the female (tail lifted and arched back) and strong interest in males. Vaginal cytology (increased cornified superficial cells) was assessed in selected females either during anaesthesia scheduled for AI or throughout the breeding season (Zeng et al., 1984; Durrant et al., 2002, 2003; see also Chapter 9). Longitudinal vaginal cytology profiles were mostly generated in USA institutions because pandas in China were not trained to accept vaginal swabs.

For AI, anaesthesia was induced in each female using injectable ketamine hydrochloride (Ketaset; Fort Dodge Laboratories, Inc., Fort Dodge, IA; ~5 mg kg-1 BW). The anaesthetised female was placed in a supine position, and a lubricated vaginal speculum (12 cm in length and 2 cm in diameter; Fig. 20.1) was inserted to visualise the cervix (Fig. 20.2). The insemination consisted of inserting a plastic or stainless steel catheter into the external cervical os and advancing the catheter approximately 18 cm from the vulva. A total of 1 to 2 ml of fresh, cooled or frozen-thawed spermatozoa typically was used for each insemination, although a larger volume (3 to 6 ml) was used in some females. Females were monitored for pregnancy and birth of cubs (Fig. 20.3).

Historical data on AI success outside China were summarised for giant panda cubs born in Spain in 1982, in Japan on three occasions (1985, 1986 and 1988) and most recently in the USA in 1999 and 2005. Numbers were sufficient at the Wolong Breeding Centre to allow comparing the efficiency of AI only with the combined use of natural

Figure 20.1. (a) A glass vaginal speculum and (b) plastic catheter used for transcervical artificial insemination in the giant panda. The speculum is used to expose the cervix, and then the insemination catheter is inserted into the external cervical os approximately 18 cm from the vulva.

Figure 20.1. (a) A glass vaginal speculum and (b) plastic catheter used for transcervical artificial insemination in the giant panda. The speculum is used to expose the cervix, and then the insemination catheter is inserted into the external cervical os approximately 18 cm from the vulva.

Figure 20.2. The external cervical os in a giant panda as viewed through a 12-cm long glass speculum.

breeding and AI (for breeding seasons 1998, 1999 and 2000). The reproductive competency of frozen-thawed spermatozoa was evaluated during the 2000, 2001, 2002 and 2003 breeding seasons at the Chengdu Research Base of Giant Panda Breeding.

Figure 20.3. A giant panda neonate is approximately the size of the palm of a person's hand (an estimated mother-to-cub body size ratio of approximately 900 to 1).
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