Successful artificial insemination in the giant panda (Ailuropoda melanoleuca) at ueno zoo

Successful breeding of the giant panda (Ailuropoda melanoleuca) following artificial insemination was achieved at the Ueno Zoo in 2 consecutive years (1985 and 1986). The first cub, born in June 1985, unfortunately died 43 hours after birth from being crushed by the mother panda; the second cub, born in June 1986, has been growing in good health. Electroejaculation and artificial insemination procedures were performed after immobilization with diazepam (0.1 mg/kg) and ketamine HCL (4.0–5.0 mg/kg). Semen of the male panda was collected by electroejaculation using a rectal probe with a diameter of 2.0 cm and with eight rings as electrodes. Stimulation of the male was given with 3 V (30–40 mA) over a 5-sec period with 5-sec intervals. The female panda exhibited estrus between late February and early March in 1985 and also between mid-january and early February 1986. Increased excretion of urinary total estrogen showed coincidentally at maximum behavioral estrus, and a gradual rise of pregnanediol level was followed by artificial insemination. The gestational length for the first pregnancy was 110 days and that of the second 121 days.     

Semen evaluation of giant pandas (Ailuropoda melanoleuca) at the Wolong Reserve

Semen from 4 wild-caught giant pandas (Ailuropoda melanoleuca) held at the China Conservation and Research Centre for the Giant Panda was collected (22 samples during 1991–1993) by electroejaculation, and evaluated for use in artificial insemination. Semen characteristics (mean ± SD) recorded were as follows: semen volume–1.5 ± .9 ml (range—0.3–3.5); sperm density 1.5 ± 0.1 × 10⁹/ml (range—0.24–4.2); motility 79 ± 10% (range—60–95); abnormal sperm 14 ± 5% (range—10–27); and pH 7.1 ± 0.2 (range—6.7–7.5). There were significant differences from year to year (P < 0.05) in semen volume collected and in the percentage abnormal sperm in 1993 compared to other years. There were no significant differences among semen produced from the four different pandas. Data collected were similar to reports for other giant pandas, and semen from all 4 giant pandas was considered suitable for use in artificial insemination. © 1994 Wiley-Liss, Inc.     

Cloning, sequence, and function analyses of giant panda (Ailuropoda melanoleuca) CD9 gene

CD9 is a member of the tetraspanin family proteins and has recently been shown to be essential for sperm-oocyte fusion in mice. The giant panda (Ailuropoda melanoleuca) CD9 (gpCD9) cDNA was amplified for the first time by RT-PCR from ovary total RNA and cloned, sequenced and analyzed. The result revealed that the open reading frame (ORF) of gpCD9 was 681 bp, which has the same length as that of mouse. Sequence analysis and structure prediction displayed that the amino acid sequence of gpCD9 is over 80% identity to those of mammals with the conserved structures, including the four transmembrane domains (TM) and certain characteristic residues. The results of sperm-egg fusion experiments demonstrated that giant panda CD9 large extracellular loop (LEL) significantly inhibited (P < 0.05) the mouse gamete fusion when the recombinant protein was added. However, when three amino acid residues TVT (173-175) of the gpCD9 were mutated to AAA, the large extracellular loop (LELM) of mutated protein was rarely inhibiting the gamete fusion of mice. Our results may be useful in improving an insight into understanding the potential mechanism of gamete fusion and genetic characteristics of giant panda.     

Sequencing,annotation and comparative analysis of nine BACs of the giant panda(Ailuropoda melanoleuca)

A 10-fold BAC library for the giant panda was constructed and nine BACs were selected to generate finish sequences.These BACs could be used as a validation resource for the de novo assembly accuracy of the whole genome shotgun sequencing reads of the giant panda newly generated by Illumina GA sequencing technology.Complete Sanger sequencing,assembly,annotation and comparative analysis were carried out on the selected BACs of a joint length 878 kb.Homologue search and de novo prediction methods were used to ...     

Zinc‐finger intron 7: a new locus for sex identification of giant panda (Ailuropoda melanoleuca)

We developed a single‐reaction test for identifying the sex of giant panda (Ailuropoda melanoleuca) targeted to co‐amplify homologous fragments with size polymorphism that located at zinc‐finger (ZF) intron 7 by using one pair of primers. This assay produced one sex‐specific fragment in females (XX genotypes) whereas two fragments were produced in males (XY genotypes). Indels (insertion/deletion) in intron 7 of Y‐linked allele provide a significant discrimination between ZFX and ZFY, thus the amplification products can be simply distinguished by agarose gel electrophoresis, exhibiting sex‐specific banding patterns (female, 354 bp; male, 354 bp, 135 bp). The new primer set was successfully tested on known‐sex giant pandas by using template DNA extracted from both blood and fecal samples. Cross‐species test was also performed, revealing that this assay could be applied to other Ursidae species. Zoo Biol 29:526–531, 2010. © 2009 Wiley‐Liss, Inc.     

不同年龄大熊猫肠道可培养芽孢杆菌的多样性及部分功能特性分析

【背景】大熊猫的肠道内微生物类群丰富,种群结构与宿主的年龄、生存环境、季节变化等因素有关,其中年龄是影响肠道菌群组成的重要因素之一。【目的】以不同年龄阶段的大熊猫粪便为研究对象,旨在了解不同年龄大熊猫肠道内芽孢杆菌的多样性,探究大熊猫肠道芽孢杆菌种类与年龄段之间的关系,并为优良益生菌剂的开发提供菌种资源。【方法】用稀释涂布法分离大熊猫粪便中的芽孢杆菌,对分离的菌株进行BOXA1R-PCR、16S rRNA基因系统发育及主成分分析,揭示大熊猫肠道中可培养芽孢杆菌的多样性;采用对峙生长法和药敏纸片琼脂扩散法分别检测菌株的抗菌能力和药敏性。【结果】从大熊猫粪便中共分离出90株芽孢杆菌,基于BOXA1R-PCR分析菌株的遗传多样性并从中选取41株代表菌株,经16Sr RNA基因测序分析后,结果显示归属于枯草芽孢杆菌(Bacillus subtilis)、萎缩芽孢杆菌(Bacillus atrophaeus)、贝莱斯芽孢杆菌(Bacillus velezensis)、甲基营养型芽孢杆菌(Bacillusmethylotrophicus)、解淀粉芽孢杆菌(Bacillusamyloliquefaciens)和短小芽孢杆菌(Bacillus pumilus)6个种;主成分分析结果表明大熊猫肠道芽孢杆菌种群组成与年龄存在一定的相关性。所有的供试菌株都具有纤维素降解潜力,大部分菌株对病原菌具有不同程度的抑制作用;除对青霉素具有耐药性外,供试菌株对常见的抗生素耐受性低。【结论】年龄是影响大熊猫肠道芽孢杆菌分布的重要因素,成年大熊猫肠道芽孢杆菌种类多样性最丰富,这为大熊猫益生菌制剂的开发提供了菌种资源。     

Esterified glucomannan improves aflatoxin-induced damage of sperm parameters during liquid storage of ram semen at 5 °C

The aim of the present work was to study the effects of aflatoxin (AF) on sperm parameters in rams, and to determine the protective efficiency of esterified glucomannan (EG) co-administered with AF up to 96 h of the liquid storage of ram semen at 5 °C. Thirty-two Merino rams (12–14 months old) were used. The animals were examined for their general health status. To ensure their adaptation to the environment and the new feeding regimen, a 15-day acclimatization programme was applied to the animals, prior to the start of the study. Experimental feeding was continued for ninety-two days. The experimental design consisted of four dietary treatments. The control group (C) was fed with commercial feed. The AF group was fed with commercial feed plus 250 μg/day of total AF. The EG group received commercial feed plus 2 g/day of EG. The AF + EG group was given commercial feed plus 250 μg/day of total AF and 2 g/day of EG. In the study, ejaculates were obtained from rams twice a week for 12 weeks, using an electro-ejaculator. After collected, the ejaculates were diluted with a skimmed milk extender, and stored at 5 °C. Sperm motility and rates of abnormal and nonviable spermatozoa were determined for the different treatment groups at 5 °C at 0, 24, 48, 72 and 96 h of liquid storage.     

Effect of antioxidants on preservation of motility,viability and acrosomal integrity of equine spermatozoa during storage at 5 degrees C

Preservation of liquid semen at 5 degrees C is an important technique in the breeding management of horses. Oxidative damage to spermatozoa during storage is a potential cause of the decline in motility and fertility during hypothermic storage of liquid semen. The objective of this study was to evaluate the use of water-soluble and lipid-soluble antioxidants to improve the maintenance of motility of equine spermatozoa at 5 degrees C during storage for 72 to 96 h. In Experiment 1, the effect of addition of catalase on the maintenance of motility, viability and acrosomal integrity was determined. Semen was collected, and these treatments were applied: catalase (0, 100 or 200 U/mL) in nonfat, dried skim milk extender (NFDSM; with or without seminal plasma) or 10% seminal plasma + NFDSM. Motility was determined by computerized semen analysis (CASA) at 0, 24, 48 and 72 h. Viability and acrosomal integrity were determined at 72 h of storage. There was no significant treatment effect on the maintenance of sperm motility during 72 h storage. In Experiment 2, the effect of adding lipid-soluble antioxidants on maintenance of motility was evaluated. Semen was diluted to a final concentration of 25 x 10(6) sperm/mL in NFDSM containing butylated hydroxytoluene (BHT; 2.0, 1.0, or 0.5 mM), Vitamin E (4.0, 2.0, 1.0 mM), or Tempo (2.0, 1.0, or 0.5 mM). Although the addition of BHT significantly reduced (P < 0.05) progressive motility during storage compared to the control, there were no positive treatment effects of either Vitamin E or Tempo on maintenance of motility. In Experiment 3, the effect of adding water-soluble antioxidants on maintenance of motility was evaluated. Semen was diluted in NFDSM containing these treatments: Trolox (2.0 mM), Tempo (1.0 mM), Vitamin C (0.45 mg/mL), BSA (3% w/v), combinations of these antioxidants, or control. Adding these water-soluble antioxidants did not significantly improve the maintenance of motility during cooled storage at 5 degrees C. In conclusion, adding the enzyme scavenger, catalase, or a variety of lipid- and water-soluble antioxidants did not significantly improve the maintenance of motility during liquid semen storage at 5 degrees C.     

Characteristics and in vitro fertilizing ability of giant panda (Ailuropoda melanoleuca) frozen‐thawed epididymal spermatozoa obtained 4 hours postmortem: A case report

When Chu‐Lin, a male giant panda (studbook #249), died at Madrid Zoo, his reproductive tract was removed 4 hr postmortem and the epididymal spermatozoa were collected. Extended sperm were kept at 5°C for 4 hr, loaded into straws, and frozen for 7 min in liquid nitrogen vapor before the straws were plunged into liquid nitrogen. Two straws were thawed and evaluated. Sperm motility was assessed in fresh, refrigerated, and thawed spermatozoa (75%, 60%, 35%, respectively). Sperm viability and acrosome status were estimated using a triple‐stain technique (TST). The results showed 33% live sperm with intact acrosomes after thawing. A hypoosmotic swelling (HOS) test demonstrated the retention of membrane integrity in 72% of thawed sperm. To evaluate the in vitro fertilizing ability of thawed sperm, a sperm penetration assay (SPA) was performed. The values obtained for the percentage of penetration and the penetration index were 62% and 1.78 sperm/oocyte, respectively. The results obtained demonstrate that epididymal sperm recovered from a giant panda postmortem can be successfully cryopreserved. The sperm fertilizing ability demonstrated in vitro after thawing may provide a final opportunity for this male to contribute to the currently small germplasm reserves of this endangered species, and to reproduce in the future through assisted reproductive technology. Zoo Biol 23:279–285, 2004. © 2004 Wiley‐Liss, Inc.     

The effects of drone age, semen storage and contamination on semen quality in the honey bee (Apis mellifera)

Abstract. The effect of semen storage time, drone age and semen contamination on honey bee semen quality was investigated using assays for motility and viability of semen in vitro. Four age groups (1, 2, 4 and 6 weeks) and five storage times (0, 1, 2, 4 and 6 weeks) were examined. As storage time increased, sperm viability and motility significantly decreased. However, motility patterns of unstored semen samples were significantly lower than those samples that were stored up to 2 weeks. Sperm viability decreased significantly with increasing drone age, but motility patterns did not change. Those semen samples that were found to be contaminated with foreign particles or microorganisms had a significantly lower mean viability than uncontaminated samples.     

Growth and development of infant giant pandas (Ailuropoda melanoleuca) at the Wolong Reserve,China

From 1991 to 1993 inclusive, seven infant giant pandas (Ailuropoda melanoleuca) were born at the China Conservation and Research Center for the Giant Panda. Average daily weight gain in the first 6 months for mother-reared infants (n = 5) was 71.3 g/day; for one partially mother-reared and partially hand-reared infant, 41.5 g/day; and for one completely hand-reared infant, 50.3 g/day. There was a significant difference in growth rates across the first 6 months in all methods of rearing. In addition, a comparison of growth rates across the three rearing methods showed significant differences in the first, second, third, fifth, and sixth months. Average daily body length increase for mother-reared infants was 4.1 mm/day; for partially mother-reared/partially hand-reared infants, 4.0 mm/day; and for the completely hand-reared infant, 2.8 mm/day. In mother-reared infants, body length increase during the first month was significantly greater than during the following months, and was slowest during the sixth month. At birth, infants were all pink in color with a light white coat of lanugo. Black pigmentation was first noted at 7–10 days of age, which was also the time that initial hair coat growth was seen. Eyes opened at 35–48 days of age. Ears opened at 31–50 days of age. Deciduous dentition was first seen at 82–121 days of age, while permanent dentition began to erupt at 350 days of age. © 1996 Wiley-Liss, Inc.     

凉山山系大熊猫和黑熊适宜生境预测及重叠分析

研究同域物种的分布格局及重叠状况对物种的区域整合保护管理及区域生物多样性保护具有重要实践价值。本研究基于全国第四次大熊猫调查及长期野外调查数据, 利用MaxEnt模型预测了凉山山系两种同域分布的熊科动物——大熊猫(Ailuropoda melanoleuca)和黑熊(Ursus thibetanus)的适宜生境, 基于适宜生境预测结果, 分析了两个物种的生境需求因子、生境破碎化现状及重叠状况。结果显示: (1)大熊猫和黑熊的适宜生境分布格局相似, 主要分布在凉山山系的山脊地带, 适宜生境面积分别为1,383.84 km²和2,411.49 km²; (2)两个物种的适宜生境都较为破碎, 且存在一些隔离分布区, 相较而言, 黑熊适宜生境的连通性要优于大熊猫; (3)两个物种生态位重叠度较高(D = 0.654, I = 0.901), 适宜生境重叠面积为958.29 km², 分别占大熊猫和黑熊适宜生境总面积的69.25%和39.74%; (4)两个物种对环境因子的选择和响应表现出了相似性和差异性。相似性在于对两个物种生境分布影响最大的两个因子均为距居民点距离和海拔; 差异性在于对大熊猫生境分布影响次之的因子是植被类型和最冷季均温, 而黑熊的是年最大EVI指数和距道路距离。为了更有效地保护两个物种, 应加强对人类干扰的控制和植被的恢复, 对栖息地实行连通管理, 并建立多物种保护规划。     

Developmental competence of domestic cat oocytes from ovaries stored at various durations at 4 °C temperature

Temporal storage of ovaries can provide opportunity to rescue oocytes from ovaries of endangered felids. The objective of the study was to examine the effect of different storage periods (2, 24 and 48 h) of ovaries at 4 °C for maturation of cat oocytes in vitro. Ovaries were collected from 25 domestic cats at various stages of the estrous cycle by routine ovariohysterectomy following anesthesia at different local veterinary clinics, and maintained in physiological saline at 4 °C for 2, 24 or 48 h until oocytes recovery. Selected COCs were maturated at 38 °C for 48 h in four-well petri dishes, which included 500 μL modified synthetic oviduct fluid (mSOF) medium under mineral oil in a humidified 5% CO₂, 5% O₂, and 90% N₂ atmosphere incubator. After the in vitro maturation period, there were no differences between the rate of oocytes matured at MII stages in 2 and 24 h storage groups (50.7% and 48.2% respectively, p > 0.05). However, the same result for the 48 h group was significantly lower than the 2 and 24 h groups (28.0%, p < 0.001).Our results suggest that while 2 or 24 h storage of ovaries at 4 °C does not affect the meiotic competence of oocytes in vitro, 48 h storage of ovaries decrease the results dramatically.     

Liquid storage of Asian elephant (Elephas maximus) sperm at 4 degrees C

The Asian elephant (Elephas maximus) population in the wild has been in decline for several decades and breeding in captivity has not been self-sustaining. The use of artificial insemination (AI) can help overcome many of the difficulties associated with breeding elephants in captivity; however, the ability to store semen for extended periods of time is critical to the successful application of AI to elephants. The objective of the present study was to assess the effects of four different semen extenders and the presence of egg yolk on the viability and motility of Asian elephant semen stored at 4 °C. High quality ejaculates (n=4) were collected from two Asian elephant bulls by rectal massage. Aliquots of each ejaculate were extended in four different diluents (Beltsville thawing solution (BTS); Tris–citric acid (TCA)/fructose-based; Beltsville F5 (BF5); dextrose-supplemented phosphate-buffered saline (PBS)) with or without egg yolk then cooled and stored at 4 °C. The percentages of viable (viability) and motile (motility) sperm were evaluated at 8, 24 and 48 h following collection. The addition of egg yolk significantly reduced the percentage loss in viability from initial collection to 48 h compared to extenders without egg yolk (17.0±8.2 versus 32.6±8.9 decline in percent viable sperm in the population, respectively; P<0.05). Extender and egg yolk affected (P<0.005) total motility and percent progressively motile sperm at all evaluation times during incubation. TCA + egg yolk maintained higher (P<0.05) levels of progressive motility compared to other extenders supplemented with egg yolk. These results indicate that Asian elephant semen extended in TCA diluent supplemented with egg yolk can maintain at least 50% viability and motility when stored at 4 °C for 48 h.     

Assessment of different sperm functional tests in golden‐headed lion tamarins (Leontopithecus chrysomelas)

The golden‐headed lion tamarin (Leontopithecus chrysomelas) is an endangered species endemic to Brazil's Atlantic Forest, a shrinking biodiversity hotspot. As in other Neotropical primates, its semen characteristics and freezability are poorly studied. Hence, reproductive technologies for callitrichids would greatly benefit from reliable methods of semen analysis. In a bid to promote reproductive research in tamarins, we validated simple and inexpensive sperm function tests that can be used to monitor sperm‐egg binding, plasma membrane and acrosome integrity, mitochondrial activity, and DNA fragmentation. Ejaculates from adult males were individually diluted and divided into control and damage‐induced aliquots, and then samples comprising assorted amounts of damaged spermatozoa were examined by organelle‐specific tests. Our findings showed that sperm‐binding in chicken egg perivitelline membrane (EPM) positively correlated with the number of spermatozoa injured by snap‐freezing. Eosin‐nigrosin (EN) and propidium iodide readings were correlated with each other, and both provided robust measurements of plasma membrane integrity. A high correlation between expected and measured amounts of acrosome‐intact spermatozoa was found using Fast Green‐Rose Bengal (FG‐RB), Coomassie Blue (CB), and FITC‐PSA stains, and all three methods exhibited comparable results. Likewise, different percentages of UV‐irradiated spermatozoa were accurately assessed for DNA integrity by Toluidine Blue (TB) and sperm chromatin dispersion (SCD) tests. Comparisons between 3,3′‐diaminobenzidine (DAB) and JC‐1 stains also indicated the reliability of the former assay to ascertain gradual increases in spermatozoa with greater mitochondrial function. These data confirmed that different parts of the tamarin spermatozoa can be simply and consistently evaluated by EPM, EN, FG‐RB, CB, TB, and DAB protocols.     

No short‐term change in avian assemblage following removal of Yellow‐throated Miner (Manorina flavigula) colonies

The overabundance of Yellow‐throated Miner (Manorina flavigula) has been shown to negatively affect the abundance and richness of small birds in areas they occupy, leading to homogenization of the avifauna across the fragmented landscape. In this study, we took advantage of a planned management cull to ask the question, does the removal of Yellow‐throated Miner colonies cause an immediate change in avian species richness and abundance? This cull was undertaken around the Bronzewing Flora and Fauna Reserve (north‐western Victoria, Australia) in order to protect a resident population of endangered Black‐eared Miner (M. melanotis) from hybridization. We conducted avian surveys along roadsides surrounding the reserve at Yellow‐throated Miner colonies (n = 6), control sites with no miners (n = 7), and where colonies were removed (n = 3). We found that the cull was followed by only a very modest increase in the species richness and abundance of small birds, with no significant effects on avian assemblage overall. This result contrasts with far more dramatic increases following culls of other species of miner. Sites where miners were removed were not depauperate of other species prior to the cull, which could have been due to a combination of proximity to refuge for small birds in a neighbouring reserve or the low numbers of miners that made up each culled colony. This study highlights that assumed effects of a management action may be highly dependent upon spatial and temporal context.     

Sequencing,annotation and comparative analysis of nine BACs of giant panda (<Emphasis Type="Italic">Ailuropoda melanoleuca</Emphasis>)

A 10-fold BAC library for giant panda was constructed and nine BACs were selected to generate finish sequences. These BACs could be used as a validation resource for the de novo assembly accuracy of the whole genome shotgun sequencing reads of giant panda newly generated by the Illumina GA sequencing technology. Complete sanger sequencing, assembly, annotation and comparative analysis were carried out on the selected BACs of a joint length 878 kb. Homologue search and de novo prediction methods were used to annotate genes and repeats. Twelve protein coding genes were predicted, seven of which could be functionally annotated. The seven genes have an average gene size of about 41 kb, an average coding size of about 1.2 kb and an average exon number of 6 per gene. Besides, seven tRNA genes were found. About 27 percent of the BAC sequence is composed of repeats. A phylogenetic tree was constructed using neighbor-join algorithm across five species, including giant panda, human, dog, cat and mouse, which reconfirms dog as the most related species to giant panda. Our results provide detailed sequence and structure information for new genes and repeats of giant panda, which will be helpful for further studies on the giant panda.