孵化温度对乌龟性别及质量的影响

为评估不同孵化温度对乌龟(Chinemys reevesii)性别及质量的影响,探求更好的稚龟孵化条件,本研究将乌龟受精卵置于25、27、29、31、33℃、转温温度(33℃转29℃)条件下孵化,统计孵化率、测量稚龟形态特征以及运动性能和生理生化指标等。结果显示:27℃孵化率最高,为42.67%,且稚龟的尾长、背甲长、背甲宽、体高、腹甲长最大;27℃孵出的稚龟翻身速度最快,与31、33℃存在显著性差异;29℃孵出的稚龟游泳速度最快,与25、33℃呈现显著性差异;33℃孵化,孵化期最短和雌性比例最高,与25、27、29℃孵化差异显著,而33℃孵化的雌性比例与转温温度孵化没有显著性差异;33℃转29℃孵化温度下腹甲宽、体重最大,乌龟生理生化指标中仅总抗氧化能力存在显著性差异,33℃的总抗氧化能力最低,与29℃有显著性差异,转温温度组处于两者之间,与两者没有显著性差异。综上所述,27℃、29℃与转温温度处理能产生高质量的稚龟,而转温温度与恒定的27℃、29℃相比,其孵化周期更短,稚龟雌性比率更高,与31、33℃相比,则稚龟质量更好。综上结果表明本研究的转温温度方式可用来做为规模化扩繁全雌乌龟苗种的生产方式。

2种石斛属植物和小叶石仙桃多糖的组织化学定位

为对不同年龄的铁皮石斛(Dendrobium candidum)、铜皮石斛(D.moniliforme)及石斛伪品-小叶石仙桃(Pholidota chinensis)不同器官中的多糖进行组织化学定位,选择具有较高药用价值的石斛类药用植物和药用部位,确定铁皮石斛的最佳采收期,根据多糖的理化性质,材料经冰冻切片,用改良的高碘酸-席夫试剂(PAS)对多糖进行组织化学定位;结果表明,多糖类物质主要存在茎的薄壁细胞、根的皮层细胞、叶的叶肉细胞中。其总多糖含量有如下顺序:二年生的铁皮石斛茎>铜皮石斛茎≈>小叶石仙桃茎,铁皮石斛根≈铜皮石斛根>小叶石仙桃根,铁皮石斛叶≈小叶石仙桃叶>铜皮石斛叶。另外,在不同的器官中,铜皮石斛的多糖含量呈现“茎≈根>叶”和小叶石仙桃的多糖含量呈现“叶>茎≈>根”的规律,而二年生的铁皮石斛则为“茎>叶>根”。这表明在种间及同种植物的不同器官、不同年龄,其多糖的含量存在差异。而3种兰科植物中,以二年生铁皮石斛茎的多糖含量最高,表明其药用价值也最高。

中华草龟(♀)与中华花龟(♂)及其杂种F_1代染色体及同工酶比较

采用肾细胞体内注射法分析了中华草龟(Chinemys reevesii)(♀)、中华花龟(Ocadia sinensis)(♂)及其杂种F1代的染色体核型。三种龟染色体倍数为2 n=52,核型公式为18 m+10 st+24 t,臂数NF=80。采用聚丙烯酰胺垂直板电泳法检测了上述三种龟的肌肉、心脏、肝脏、脾脏和肾脏等5种组织的乳酸脱氢酶(LDH)、酯酶(EST)、过氧化物酶(POD)和醇脱氢酶(ADH),分析了三种龟的亲缘关系和遗传多样性。共检测50个基因座位,其中中华草龟15个,中华花龟16个,杂种龟19个,多态位点3个(Ldh-3,Pod-2和Pod-3),多态座位百分比P分别为13.3%、12.5%和15.8%,平均观测杂合度(H0)分别为0.025 6、0.041 7和0.102 3,平均预期杂合度(He)分别为0.041 6、0.040 7和0.074 6,遗传偏离指数(D)分别为-0.386 4、0.023 7和0.372 5。结果表明:杂种F1代群体遗传多样性较高,而亲本群体相对较低;在POD上,杂种F1代与母本中华草龟相近。对比发现,肝脏的POD和LDH及心脏的POD同工酶电泳图谱可以作为鉴别杂种F1代的生化遗传标记。

基于Cyt b基因的乌花杂交龟系统进化关系研究

利用PCR方法克隆乌花杂交龟线粒体DNA的细胞色素b基因(Cyt b基因),并结合GenBank中已公布的22种龟鳖类动物同源序列进行了序列变异比较和系统发生分析,采用NJ(Neighbor-joining)法和UPGMA法构建分子系统树。结果表明,乌花杂交龟与中华草龟(Chinemys reevesii)以很高的置信度聚成一支,再与中华花龟(Ocadia sinensis)聚成一组。

乌龟蛋品质分析

随机选取了乌龟(Chinemys reevesii)同一天所产蛋80枚,测定了乌龟蛋品质等相关指标和蛋白质含量。结果表明,乌龟蛋重、蛋形指数、蛋壳厚度、蛋黄颜色、蛋白高度和粗蛋白含量分别为7.47g、1.70、0.21mm、2级、4.64cm和13.95%。各项指标均达到较好的标准,且蛋白质含量高于鸡蛋和鸭蛋等禽类蛋。该结果为育种者改良乌龟品种、消费者了解乌龟蛋提供了理论依据。

Effects of Pesticide Exposure on Embryonic Development and Hatchling Traits of Turtles

Deltamethrin is a widespread environmental hormone with endocrine-disrupting properties, but its effect on embryonic development of reptiles is largely unexplored. We investigated the effects of deltamethrin on embryonic development and offspring traits in two turtle species, one with parchment-shelled eggs and the other with rigidshelled eggs. Deltamethrin exposure during egg incubation did not affect hatching success and hatchling body size in either species. However, embryonic exposure to deltamethrin resulted in reduced hatchling locomotor performance in the red-eared slider turtle（Trachemys scripta） with parchment-shelled eggs, but not in the Chinese three-keeled pond turtle（Chinemys reevesii） with rigid-shelled eggs. These results suggest that parchment-shelled eggs are likely more vulnerable to deltamethrin than rigid-shelled eggs.

Molecular Cloning and Tissue-specific Expression of Cu/Zn and Mn-superoxide Dismutase in the Three-keeled Pond Turtle, Chinemys reevesii

Both copper/zinc superoxide dismutase （SOD; Cu/Zn-SOD, SOD1） cDNA and manganese SOD （Mn-SOD, SOD2） cDNA were cloned for the first time from the three-keeled pond turtle, Chinemys reevesii, using RT-PCR and RACE methods in this work. The SOD1 cDNA was 749 bp long and consisted of a 32-bp 5＇-untranslated region （UTR）, a 249-bp 3＇-UTR, and a 468-bp open reading frame （ORF） encoding a 155-amino-acid protein with 16.0 kDa predicted molecular mass and 5.95 theoretical isoelectric point （p/）. The SOD2 cDNA was 1687 bp long and comprised 94-bp of 5＇-UTR, 912-bp 3＇-UTR and 681-bp ORF encoding a 226-amino-acid protein with 25.0 kDa predicted molecular mass and 8.83 pI. The deduced amino acid sequence of SOD1 showed relatively high similarity （77.4%-87.1%） and identity （65.4%-74.4%） with the published sequences of SOD1 from other vertebrate species, whereas SOD2 protein shared slightly higher similarity （83.6%-95.6%） and identity （76.1%-88.9%） with other reported vertebrates SOD2s. Phylogenetic analysis revealed that the C. reevesii SOD1 and SOD2 were separately clustered together, and were highly conserved during evolution. Both SOD mRNA expression was detected widely in the brain, liver, muscle, kidney, gut, spleen, lung and heart at variable levels. The highest expression of the two SODs was observed in muscle, and followed in brain, liver, kidney, gut and heart, whereas low transcriptional levels were found in spleen and lung. Meanwhile, high activity of SOD 1 was kept in brain, liver, muscle, kidney and heart, and followed in gut, spleen and lung. The activities of SOD2 in brain, liver, muscle, kidney, gut and heart were significantly higher than those in spleen and lung.

Differentiating Trachemys scripta elegans Shell Glue from Chinemys reevesii Shell Glue by UPLC-QTOF/MS Coupled with Binary Compare Tool of UNIFI

Objective This study was conducted to develop an method for identification of Trachemys scripta elegans(Ba Xi Gui,巴西龟)shell glue from Chinemys reevesii(Zhong Hua Cao Gui,中华草龟)shell glue.Methods In this research,an ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry(UPLC-QTOF/MS)method coupled with Binary Compare tool of UNIFI software was validated to differentiate Trachemys scripta elegans(Ba Xi Gui,巴西龟)shell glue from Chinemys reevesii(Zhong Hua Cao Gui,中华草龟)shell glue.The gelatins were digested enzymatically into peptides using trypsin.The UPLC-QTOF/MS data of the trypsin digested samples was subjected to Binary Compare of UNIFI software in order to screen out the unique peptides.PEAKS■Studio software was utilized to identify the sequence of the marker peptides.Results It was found that m/z 641.3 was the marker peptide in samples of Trachemys scripta elegans(Ba Xi Gui,巴西龟)shell glue,and m/z 649.3 was the marker peptide in samples of Chinemys reevesii(Zhong Hua Cao Gui,中华草龟)shell glue,with possible amino acid sequence of GEAGPSGPAGPTGAR and GESGPSGPAGPTGAR respectively.Conclusions The results will be helpful for the differentiating between these 2 gelatins,and it can be also used for quality control of Tortoise shell glue(Gui Jia Jiao,龟甲胶).

Effect of Dietary Vitamin C on the Antioxidant Defense System of Hibernating Juvenile Three-keeled Pond Turtles (Chinemys reevesii)

Juvenile three-keeled pond turtles(Chinemys reevesii) were fed diets supplemented with vitamin C(Vc) at doses of 0(basal diet, Vc0), 100(Vc100), 200(Vc200), 500(Vc500) and 2500(Vc2500) mg/kg diets at 28°C for 4 weeks, respectively. Then, the water temperature was gradually reduced to 10°C, and the turtles were induced into hibernation. Liver tissue samples were collected at three time points: start of hibernation(T1), 4 and 6 weeks’ hibernation(T2 and T3). A control group fed with the basal diet was set to parallel the whole treatment process, but reared at 28°C constantly. The results showed that hibernation mildly affected the antioxidant system and the influence varied with hibernating time. Hepatic malondialdehyde content of the Vc100 group was significantly lower than that of the other groups at T1. At T2, hepatic MDA in the groups of Vc500 and Vc2500 decreased significantly, while no clear differences were found among all groups at T3. The activities of antioxidant enzymes showed a positive correlation with dietary Vc dose before hibernation. After hibernation, total antioxidant capability was not affected by Vc. Superoxide dismutase activity became similar in different groups at T2, but decreased in higher Vc groups(≥ 200 mg/kg) at T3. Glutathione peroxidase and glutathione-S-transferase activities decreased significantly with dietary Vc supplementation(≥ 100 mg/kg) at T2, but recovered at T3. The result indicates that under normal rearing condition, low dietary Vc supplementation(< 100 mg/kg) might be beneficial to the antioxidant defense system. The effect of dietary Vc on the antioxidant defense system differed during hibernation.