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known dam was shown to have zero Off-Dam mismatches and, thus, was concordant with maternity. The known dam was then assigned and all resident males at the centre (regardless of age) were queried for possible paternity. Table 10.3 lists the mismatch results for the putative sire, for the next most likely male at the breeding centre and for the next most likely sire based on inspection of only those animals that were naturally mated or supplied semen for AI of the dam. In every case, the implicated dam was naturally mated to (or artificially inseminated with semen from) the putative sire.

Of 50 parentage assessments, there were only three cases with a single locus exclusion for the putative sire. For SB 495 and SB 509, both the putative sire (SB 329) and the offspring have a different single (homozygous) allele for Ame-^25, resulting in an exclusion (i.e. they inherited an allele from the dam but did not show an allele from the sire). The absence of a paternal allele can be explained by a polymorphic nucleotide site within the panda's homologous Ame-^25 primer sequence that abrogates PCR amplification due to a primer sequence mismatch. The presence of such 'null' alleles has been previously recognised (Callen et al., 1993), and we have observed them in the genotyping of a large multigenerational pedigree during construction of a microsatellite-based genetic linkage map of the domestic cat (Menotti-Raymond et al., 1999). Finally, SB 495 and SB 496 are twins resulting from the natural mating of sires SB 329 and SB 308 to the dam SB 446, who also was artificially inseminated with semen from SB 298. The putative sire of SB 495, as discussed above, is SB 329, and the putative sire of her twin SB 496 is SB 308. There is, however, one Off-Sire-Dam mismatch for SB 496 with SB 308 at locus Ame-^25. This is not consistent with the presence of a null allele because SB 496 has a unique allele not present in either parent. It is possible that the offspring allele is a new mutation or a genotyping error.

There were nine cases where more than a single male was listed as a possible sire in the blinded analysis of all males at the centre. In three cases, no genotype data existed for the dam, thereby reducing the exclusion power. In five cases, two included sires were close relatives, and the last situation involved microsatellite amplification failure. In all cases of uncertain paternity in the blind analysis, it was possible to exclude all but a single male when checking against the potential sires based on breeding records.

Insufficient genotypes to assign paternity were obtained for animals SB 388, SB 433, SB 434, SB437, SB439 and SB455 and, thus, were excluded from Tables 10.3 and 10.4. Four offspring were typed that were included in a previous microsatellite paternity study (Zhang et al., 1994). The results between the two independent laboratories agreed for offspring SB 394 and SB 399. Zhang et al. (1994) excluded both potential sires for twins SB 387 and SB 388 and suggested that one of the sire samples (SB 202) was misidentified. In contrast, our results implicated SB 202 as the sire of SB 387. Also, Zhang and colleagues proposed SB 201 as the sire of SB 386 based on exclusion of SB 343. However, breeding records at the Chengdu Research Base listed SB 343 and SB 298 as potential sires, and our data confirmed that SB 343 was the sire of SB 386.

Accessing paternity: natural mating versus artificial insemination

Fifty paternity questions were addressed with results presented in Table 10.4 and Figures 10.4 and 10.5. Of the 50 paternities, seven were not in question since offspring resulted from either AI with semen from a single sire (SB 425, SB 522, SB 523, SB 473, SB 474 and SB 512) or natural mating plus AI using a single sire (SB 516). In these confirmation cases, the presumptive sire was sustained in every case.

There were 13 cases where offspring resulted from only using AI (see Table 10.4). Six offspring were born as a result of AI with semen from a single sire and seven from multiple sires. Clearly, AI is a viable procedure, and recent evidence indicates nearly identical pregnancy/ birth success rates when using natural mating versus AI with fresh semen (Huang et al., 2001; see Chapter 20). Four offspring were produced after a single male was used for both natural mating and AI, and then a second male was used for AI (SB 490, SB 491, SB 519, SB 520; see Table 10.4). In these four cases, the male used for natural mating was the true sire but it is impossible to determine if fertilisation occurred with semen that was deposited naturally or by AI.

In 29 cases, females were mated naturally to one or more males and then artificially inseminated by one or more different males. In all 29 instances, the offspring resulted from natural mating. The predominance of natural mating success was probably due to the advantage of early semen deposition because females are virtually always allowed to mate naturally before being artificially inseminated. Thus the copulating male benefits by having his sperm at the site of fertilisation

Figure 10.4. Pedigree of giant pandas from the China Conservation and Research Centre for the Giant Panda (Wolong Nature Reserve) constructed using microsatellite data. In this study DNA was available for all animals shown except SB 230. Under each animal is its name, potential sire by natural mating (NM) or AI, and date of birth. □ male, O female, O bold - wild born, □ grey shading - multiple potential sires, * - paternity determined or confirmed with microsatellites, \ - deceased.

Figure 10.4. Pedigree of giant pandas from the China Conservation and Research Centre for the Giant Panda (Wolong Nature Reserve) constructed using microsatellite data. In this study DNA was available for all animals shown except SB 230. Under each animal is its name, potential sire by natural mating (NM) or AI, and date of birth. □ male, O female, O bold - wild born, □ grey shading - multiple potential sires, * - paternity determined or confirmed with microsatellites, \ - deceased.

OuiiCwt ZhuZhu Al 343 AI 343 b. 19« h- I»1 A 2001

Chcnggong AI3M b. 2000

Chcngji,

AI3B6 b 2000

Al 342 AI 298 b-1993

Eryatou Xiwyalau

AI 298 AI298

b 1093 hi. 199S

Qi 7-licii

Da Schuuig X«o Schilfig LiingliHig Yilaodi YaJaoct fiu Hu Bingxiii Bingdiw

NM2*7 XM2B7 N"M287 NM 2*7 NM 287 AI3fli NM 287 NM 2S7

AI 298 AI298 AI 3B6 AI3Bd AI3S6 Ailffltl Zoo AI 287 AI2S7

AI 231 AI 231 b. 2000 h. 1999 b. 1999 U 1997 AD86 A1386

OuiiCwt ZhuZhu Al 343 AI 343 b. 19« h- I»1 A 2001

Chcnggong AI3M b. 2000

Chcngji,

AI3B6 b 2000

Al 342 AI 298 b-1993

Eryatou Xiwyalau

AI 298 AI298

b 1093 hi. 199S

Qi 7-licii

Da Schuuig X«o Schilfig LiingliHig Yilaodi YaJaoct fiu Hu Bingxiii Bingdiw

NM2*7 XM2B7 N"M287 NM 2*7 NM 287 AI3fli NM 287 NM 2S7

AI 298 AI298 AI 3B6 AI3Bd AI3S6 Ailffltl Zoo AI 287 AI2S7

AI 231 AI 231 b. 2000 h. 1999 b. 1999 U 1997 AD86 A1386

Figure 10.5. Pedigree of giant pandas from the Chengdu Research Base facility constructed using microsatellite data. DNA was available from all displayed pandas except SB 152, SB 243, SB 373, SB 393, SB 452 and SB 461. Under each animal is its name, potential sire by natural mating (NM) or AI, and date of birth and death. □ male, O female, O bold - wild born, □ grey shading - multiple potential sires, * - paternity determined or confirmed with microsatellites, \ - deceased.

(oviduct) during early oestrus and at least a few hours (if not a day or more) before the competing AI donor.

Data analysis for twins and the issue of genetic over-representation

Paternity information for 15 sets of twins is presented in Table 10.5. Of 13 cases where multiple paternity was possible, we observed only a single case (SB 495/496) where offspring were sired by different males, through natural mating. All other pairs were sired by singleton males via copulation or AI. There were four sets of twins born by AI only. Of the 15 pairs, all were dizygotic, ten of mixed sex, suggesting that monozygotic twins are rare if they occur at all (Fang et al., 1997c).

At the Wolong facility, three males were capable of natural mating (SB 329 and SB 308, which were wild caught, and SB 394, which is a son of SB 308). Twenty-one of 29 offspring at the Wolong facility (72.4%) - shown in Table 10.4 - are offspring of SB 308 or SB 394. The genetic consequences of this type of over-representation are discussed in more detail by Ballou et al. (in Chapter 21).

There is a broader representation of founder individuals in the Chengdu pedigree (see Fig. 10.5) where AI in the absence of natural mating has been more common. However, the dilemma here is that paternity remains unsolved for 17 living offspring and grand-offspring of key dams SB 278, SB 297 and SB 314 (see Fig. 10.5). Breeding records indicate SB 174, SB 201 and SB 202 as potential sires, but SB 202 can be excluded as a potential sire in all but three cases. DNA samples were not available for SB 174 and SB 201 for our study so these key paternities remain unresolved.

An important presumption in designing a maximally outbred ex situ breeding programme is that the founder individuals are unrelated or at least not first-order relatives. However, for rare species where wild populations often are under 30 or 40 individuals, the likelihood of recent inbreeding and consanguinity among captive founders may be appreciable. It is possible, however, to assess roughly the degree of relatedness among members of a captive population by comparing their composite microsatellite genotypes and examining the overall distribution (Queller & Goodnight, 1989; Vigilant et al., 2001; Uphyrkina et al., 2002).

We computed the genetic 'relatedness' of all pairs of individual genotypes at the Wolong and Chengdu facilities and plotted their

Table 10.5. Paternity information for 15 pairs of gia nt pa nda twins

Potential sires in order of mating/

Twins' Number of Current Date insemination (sire determined studbook numbers Names genotypes Sexes locations Sire 1/Sire 2 Dam of birth in bold)

362/363

Ya Ya/Jun Jun

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