| Chinese water deer, Hydropotes inermis inermis is endemic to China and also a protected animal in China. Up to now, habitat loss and poaching have reduced its range and number drastically. Therefore, understanding the morphological and molecular variation of different geographic populations of Chinese water deer is important for us designing conservational strategies. At the same time, the captive breeding of Chinese water deer is becoming popular in China, but researches on reproductive behavior, genetic background of captive Chinese water deer is still lack. So, in this study, the molecular and morphological variation of different geographic populations of Chinese water deer were investigated, using mitochondrial DNA control region (mtDNA D-loop) and microsatellites as genetic markers and skull as morphological marker, and also the methods of focal animal sampling, behavior sampling and continus record were used to study quantitively the reproductive behavior of captive population mainly in Shengzhou Chinese water deer Breeding Base, Zhejiang province, from May 2003 to July 2004. The main results are as follows:1. Analysis of complete mtDNA D-loop sequences (927-928 bp) among 32 Chinese water deer individuals revealed 37 variable sites.The propotion of variable sites among the three regions of D-loop sequence, extended termination associated sequence (ETAS), central conserved region (CCR) and conserved sequence blocks(CSB) is 8.78%, 2.16% and 2.70% respectivly. Furthermore, the genetic diversity of Chinese water deer was estimated among 40 individuals, using partial D-loop sequence (403 bp). 27 variable sites were detected, which defined 18 haplotypes. No obvious phylogenetic structure among haplotypes was found forsamples of different origins. Sequence divergence within Chinese water deer ranged from 0.2% to 3.5% (average 1.7%). Overall, Chinese water deer had a relatively high genetic diversity compared to other rare cervid species with a haplotype diversity of 0.923 ± 0.025 and nucleotide diversity of 1.318% ± 0.146%. It also showed that the Zhoushan population (n = 1.061 ± 0.217%, H = 0.885 ± 0.04) had a lower diversity than that of the mainland (x = 1.551 ± 0.175%, H = 0.941 ± 0.036). Comparison between the two populations revealed an average nucleotide divergence of 1.488%. An analysis of molecular variance (AMOVA) indicated significantly differentiated between Zhoushan and mainland populations (Fst = 0.088, P < 0.001;St = 0.075, P = 0.043).2. The nuclear genetic diversity and population structure of two representative captive populations, Nanjing Hongshan Zoo population (mainland orignated;n=19) and Shengzhou population (Zhoushan orignated;n=19) with a Zhoushan wild population for control (n=25) were investigated, using 7 microsatellite loci. There was a relatively high level of genetic diversity of Chinese water deer for the total samples, comparing to the other rare cervid species. The mean number of observed alleles per locus (A) was 5.143. The average observed (Ho) and expected heterozygosity (He) values were 0.531 and 0.662, respectively. The diversity of Nanjing Hongshan Zoo populations (mainland originated;A= 4.143, Ho =0.571, He= 0.625) was higher than that of the other two (Zhoushan originated) populations, and the Shengzhou population (A=2.%S1, Ho = 0.466, He-0.561) was the lowest genetic diversity among them. Furthermore, the pairwise estimate of Fst among the three populations indicated that the Nanjing population was* significantly differentiated from the other two Zhoushan originated populations, the FSt is 0.210 (p < 10"3) and 0.211 (p < 10"3), respectively, but no significant genetic differentiation was found between Zhoushan wild and Shengzhou populations (Fst = 0.004;p > 0.05). The assignment tests also had the same result as that of pairwise Fst comparison.3. Variation in 11 measurements of skulls of Chinese water deer was studied to assess the sexual dimorphism and geographic variation, using principalcomponents analysis (PCA) methods. A total of 115 skulls of adult Chinese water deer (45 male, 70 female) was included in the analysis. Four principal components were abstracted, which explained 83.137% variations. The Chinese water deer samples were discriminated by sex over the first two principal components with 98.3% correct assignment, which are relative to the length of skull and size of the canine, respectively. Therefore, sexes were studied separately when we test the geographic variation among Chinese water deer populations. We found that despite the Chinese water deer were distributed in different habitats, variations in skull morphology among populations were not acute. The principal factor of interpopulational variability in Chinese water deer was the size of skull. The Zhoushan population had the largest skull, the Jiangxi population had the smallest one, and Zhejiang mainland populations were in-between.4. In the non-mating season (from the last ten-days of February to March), adult females behaved more stereotypes behavior than yearling females, but lower frequency of feeding than that of yearling females. There was no significant difference of behavior between adult and yearling males, and also no significant difference of behavior between female and male was found. In the mating season (from December to January in the next year), the duration of environment investigating behavior and the frequency of rumination in adult females were more than that of yearling females. No significant difference of behavior between adult and yearling male was found. Comparing to the adult female, not only some special sexual behavior, but also more conflict and anogenital sniffing behavior was expressed in male. In addition, the duration and frequency of bedding in male was significantly lower than that of adult female. Comparing to which in non-mating season, the duration of moving increased significantly both in adult females and males. Furthermore, the duration of bedding decreased significantly, while the conflict and anogenital sniffing behavior were increased significantly for the males.5. Captive female entered into estrus once in a reproductive season. The period of estrus lasted about 25 hour (n=2). In one estrus, repeated copulation was found,ejaculation mounting was 4-5 times in yearling males (n=2) and 7-8 times in adult males (n=2). The mean duration of ejaculation mounting of yearling and adult males was 9.00±0.91s (n=16) and 7.32±0.43s (n=50) , respectivly, and no significant difference was found between them.6. The multiple mating behaviors of Chinese water deer in enclosure with two males' were observed. It revealed that although the dominant male had the prior chance of copulation and expressed the behavior of driving the subordinate male away from the female, it is impossible for the dominant male to prevent subordinant male from mounting in one estrus. Furthermore, there was no chose of the rank of male for the female to mate (even for the yearling males). Therefore, mating competetion exists. The distribution of mounting for the dominant and subordinate male was interlaced.7. The result of mating competition of two males was tested, using 9 microsatellite loci for paternity identification. When the mother-young relationship was known, and only two candidate males was exist, the successful rate of paternity allocation was 100%. In five enclosures with two males, 10 females reproduced, among which 9 females with litter^2. Multiple paternities were found in the offsprings of three females, which accounting for 33.33%. Furthermore, for the whole 25 fawns, 88% paternities allocated to dominant males, while 12% paternities were assigned to the subordinate (including one yearling male).8. The gestation of captive female is 167±0.88 days (n=15). Does often select safe place for parturition. The mean duration of parturition was 302.20±15.27min (n=15). Two kinds of fetus position when dilivered were observed, one is prior leading position, accounting for 83.78%, the other is posterior leading position, accounting for 16.22%. There were no significant difference in duration from fawn on ground to suckles among the twins, triplets and quadruplets, and the average duration for the first suckle was 44.97±2.73min (n=35) . The duration from fawn on ground to fawn stands of twins and triplets was 32.25±2.49min(n=16) and 29.42±2.52min (n=12) , respectively. No significant difference was found between them, but significant difference was found between them andquadruplets (65.00±7.39min;n=6). Furthermore, comparison of birth weight of neonate from different parity revealed that the birth weight of quadruplets was lighter than that of other neonate.9. In 2004, the neonate mortality in Shengzhou Base was investigated. Results showed that the neonate mortality was up to 49.23% (n=65), and the post-natal mortality during the first 30 days was 40.43% (n=47). The neonate mortality decreased with the birth weight increasing, and among them, mortality of which birth weight lighter than 600g was 75%. The main cause for the neonate mortality including starvation (account for 34.38%), non-viability (account for 28.12%) and misfortunate (account for 18.75%).10. Timing and synchrony of parturition in captive Chinese water deer populations (including Nanjing Hongshan Zoo, Shengzhou Base and Zhoushan Baiquan Breeding Center populations) were studied between 2003 and 2004. Results should that the calving date of primiparous does was significantly late than that of multiparous does, and the level of birth synchrony of primiparous was also lower than that of multiparous in the three populations. The calving date in both primiparous and multiparous does in Nanjing population was early than that of the other two Zhoushan originated populations. The calving date of Zhoushan Baiquan and Shengzhou population in 2004 was significantly latter than that in2003. The level of birth synchrony of multiparous in Nanjing population was significantly higher than that in Zhoushan Baiquan population both in 2003 and2004, while there was fluctuation in the level of birth synchrony of multiparous between years in Shengzhou. However, no significant difference was found in the calving date and level of synchrony of parturition of primiparous and multiparous between 2002 and 2004.11. Based on the results of our studies, some conservation and management suggestion was provided as follows: (1) The Zhoushan population should be treated as one conservation unit. (2) Taking more actions on the conservation of wild Chinese water deer resources. (3) A special mainland Chinese water deer breeding center was recommended to be established. (4) The exchange individualsbetween captive populations from different origins (especially some breeding center populations) should be forbidden, and the pedigree information for the individuals in captive should be registered. (5) Mitochondrial DNA D-loop and microsatellite marker could be used as a tool to assistant the genetic management of the captive populations. (6) The enclosure design should consider more about the target animals, warrant its warfare, and easily manipulated. (7) Enhencing food nutrition of does during the pregnant period, improving the viability of fawns.
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