a All females demonstrated behavioural signs of oestrus and bred naturally. In all females (except SB 230), females bred naturally for one, two or three days before AI with fresh, cold-stored (4°C) or frozen-thawed semen. In SB 230, AI was conducted prior to natural breeding. Duration of gestation was calculated as first day of natural breeding = Day 0. h Sire of cub(s) was confirmed by molecular genetic analyses (see Chapter 10). All cubs were sired by males that mated females on the first day of oestrus.

Figure 20.4. Female giant panda cub SB 473 born after two transcervical artificial inseminations with fresh (AI number 1) and cold-stored (AI number 2) spermatozoa. (See also Plate XXXIX.)

pregnancy also demonstrated the reproductive competency of a 4.5-year-old female, while also revealing important information on the timing of AI since inseminations were performed 48 and 72 hours after the peak in urinary oestrogens (see Table 20.3). After combined natural mating and AI, females in 12 of 18 (66.7%) trials became pregnant and produced a total of 20 cubs (see Table 20.4). Interestingly, all the cubs were sired by the male that bred on the first day (as confirmed by molecular analyses; see Chapter 10), even when multiple males bred on two consecutive days (e.g. SB 397 in 1999 and 2000) (see Table 20.4). Furthermore, one set of twins with different sires was produced by female SB 446 after breeding with two different males on the first day of oestrus (see Table 20.4).

Overall, these results indicated that AI without natural mating was as effective for propagating giant pandas as natural mating combined with AI. Data also revealed that cold storage of semen at 4°C was a viable method for maintaining high-quality semen for AI over consecutive days.

Efficiency of artificial insemination with cryopreserved sperm at the Chengdu Research Base

The availability of a GRB and the efficient use of AI with frozen-thawed spermatozoa would facilitate genetic management of the ex situ giant panda population. Basic scholarly knowledge in gamete cryobiology is essential for allowing the consistent use of AI with frozen-thawed semen. There have been years of scientific research in China and the USA with the most recent efforts occurring during the Giant Panda Biomedical Survey. For details, see Chapter 7, which summarises the significant amount of new information generated about post-thaw sperm function, metabolism, membrane integrity, capacitation and the ability to undergo the acrosome reaction and decondensation in the presence of oocyte cytoplasm.

However, there have also been efforts to test the feasibility of producing giant panda offspring using AI with thawed spermatozoa. The Chengdu Research Base of Giant Panda Breeding, established in 1987, has had a long interest in sperm cryobiology beginning with its milestone cub birth in 1980 using thawed semen. From 2000 to 2003, and exploiting new information learned from the Biomedical Survey on sperm freezing, seven females (4.5 to 17.5 years of age) were serially anaesthetised in 14 insemination trials for a total of 55 transcervical inseminations without mating (Table 20.5). In each case, these females were demonstrating weak behavioural signs of oestrus and would not breed naturally. In 2000, two females (SB 297 and 425) were inseminated with a combination of cold-stored (4°C) and frozen-thawed sperm on different days of oestrus. Only frozen-thawed sperm were used in the remaining 12 trials.

For AI in all 14 trials, mean inseminate traits were: spermic volume inseminated 1.2 ± 0.1 ml; sperm motility 59.6 ± 1.2%; sperm forward progression 3.1 ± 0.1; normal sperm 62.9 ± 3.2%; and total motile sperm inseminated per AI 73.7 ± 10.1 x 106. Overall, seven of the 14 (50.0%) insemination trials using cold-stored and/or frozen sperm resulted in a pregnancy, one of which resulted in abortion (see Table 20.5). Of the pregnancies going to term, the mean gestation and litter size were 165.7 ± 12.3 days and 1.2 ± 0.2 cubs per litter, respectively. For females receiving only thawed spermatozoa, six of the 12 (50.0%) became pregnant and produced a total of six cubs (including the aborted fetus) (see Table 20.5; Fig. 20.5; Plate XXXX). Together, these

Table 20.5. Results of artificial inseminations (AI) using cold-stored (4°C for 24 or 48 hours) or frozen-thawed semen in nonbreeding giant pandas at the Chengdu Research Base of Giant Panda Breeding (14 AI trials; seven femalesf

Motile Pregnant

Female Age Male Volume Sperm Sperm sperm (gestation) No. of cubs

SB no. (years) AI no. Date of AI SB no. Semen type Diluent-glycerol (ml) motility (%) progression (xlO6) (birth date) (sex SB no.)

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