水牛牛巴贝斯虫体外培养的研究：Ⅱ.冷冻血清的应用和筛选

应用冷冻血清对１株采自自然感染的水牛牛巴贝斯虫进行了长达７２ｄ的体外连续培养，共继代２０次，培养７２ｈ红细胞染虫率最高达１４．１％，平均为８％～１０％。培养２０ｄ和３０ｄ的牛巴贝斯虫经液氮保存复苏后，接种于去脾水牛犊均引发了严重的牛巴贝斯虫病，从而说明已建立了水牛牛巴贝斯虫的体外连续培养，且经培养后的牛巴贝斯虫致病力没有改变。本试验利用６头份的冷冻健康水牛血清同时进行培养，结果发现，并非所有的健康水牛血清均适合于体外培养牛巴贝斯虫。这一发现对建立水牛牛巴贝斯虫的体外培养和研究水牛牛巴贝斯虫病均具有重要意义     

水牛附睾尾精子的体外受精

采用肝素诱导获能 ,比较了 TAL P液和 BO液处理水牛附睾尾精子进行体外受精和细管冷冻精液体外受精的效果。结果表明 ,用 BO液和 TAL P液分别处理水牛附睾尾精子 ,受精后的卵裂率分别为 4 6 .6 7%和 5 3.73% ,发育率分别为 2 1.6 7%和 2 6 .87% ,其受精效果差异不显著。综合 2种方法 ,水牛附睾尾精子的受精率为 5 7.14 % ,受精后的卵裂率为 5 0 .39% ;发育率相对于培养卵为 2 4 .4 1% ,相对于卵裂卵为 4 8.4 4 % ;与细管冷冻精液 (5 6 .0 0 % ,5 4 .31% ,2 6 .72 % ,4 9.2 1% )相比 ,差异不显著。形态学观察还表明 ,用保温干储的方法可获得活率好、存活时间长的附睾尾精子。试验结果说明水牛附睾尾精子用于体外受精可以得到与细管冷冻精液相当的效果。     

水牛精子在胞质内注射后的早期形态变化

应用胞质内精子注射 (intracytoplasmic sperm injection,ICSI)技术探讨了水牛卵母细胞胞质与注射精子间的相互作用以及 ICSI精子的形态变化。ICSI后 13h,5 9.4 %的精子头部已膨大 ,且有 18.8%的精子进入解聚状态。雌雄原核发育不同步 ,雄原核在 ICSI后 16 h和 19h的形成率分别为 3.2 %和 4 0 .0 % ;雌原核在 ICSI后 13h已达到 71.9% ,16 h时提高到 90 .3%。经离子霉素与 6 - DMAP联合激活 ,ICSI后 19h产生的胚胎核型以 2 PN PB1 为主 ,其雌原核形成 (激活 )率为 91.3% ,雄原核形成率为 4 0 .0 % ,5 1.3%为孤雌胚。用 5 mm ol/ L 的 DTT预处理精子 1h,可提高精子的解聚率 (30 .9%比 12 .7% ,P<0 .0 5 ) ,但对雄原核的形成率无显著影响 (33.3%比 32 .7% ,P>0 .0 5 )。结果表明 ,水牛精子 ICSI后的雄原核形成时间晚于卵子雌原核形成时间 ,且其比率低于卵子 ,DTT处理精子能提高其解聚率 ,但对雄原核形成无影响     

中国水牛瘤胃纤毛虫分布与挥发性脂肪酸的研究

对中国广西西林水牛和摩拉水牛瘤胃纤毛虫种类分布进行了鉴定检测,并测定了其瘤胃液挥发性脂肪酸浓度,同时进行了比较研究。全部检出17属63种25型瘤胃纤毛虫,比以前进行的8种反刍动物瘤胃纤毛虫分布研究中出现的总数和每个宿主个体中出现的平均纤毛虫种数多,种类构成最复杂,其纤毛虫构成特点类似于东南亚地区水牛瘤胃纤毛虫构成特点。其中广西西林水牛中检出了Entodinium biconcavumsp n,是至今从未鉴定记载过的认为是新种的特殊种类。广西西林水牛检出13属54种20型、摩拉水牛检出16属45种11型瘤胃纤毛虫。广西西林水牛瘤胃纤毛虫平均密度为2.13×105/mL,摩拉水牛瘤胃纤毛虫密度为3.43×105/mL。中国水牛瘤胃纤毛虫属别构成中Entodinium属出现率最高,但两个水牛品种间存在明显差别,其他属别构成也不尽一致。这与不同品种原来的地理环境分布与饲养管理条件有关。广西西林水牛瘤胃总VFA浓度为75.60 mmol/mL,摩拉水牛的为58.74 mmol/mL。瘤胃总VFA含量中均为乙酸含量最高,属于典型的粗饲料发酵类型。     

Improved milk production through PG–PL system by provision of in‐house shelter management in lactating Murrah buffaloes during winter season

Comprehensive information on the role of β‐casein and plasminogen–plasmin (PG–PL) system in milk secretion of Murrah buffaloes during winter season is lacking, although effects of cold stress can be ameliorated to an extent by altering microclimate at farm level. Hence, this study was aimed to determine the changes in productivity along with PG–PL system of milk, plasma hormones and metabolites of buffaloes during winter (December–January) season under two different management systems. Average minimum temperature and wind chill index during this season were 7.02 and 12.74 °C respectively. Buffaloes were divided in two groups of six animals each: control and treatment, where treatment group animals were placed in‐house with floor bedding of paddy straw and the control group animals in loose housing system without straw bedding. Physiological responses were recorded, and milk and blood samples were collected at weekly intervals for six‐week experimental period. Under in‐house management system, buffaloes experienced better comfort by alleviating environmental stress as their physiological responses such as respiration rate and pulse rate were significantly reduced (p < 0.01) as compared to the control, which subsequently resulted higher milk yield by 9.92% (p < 0.05). Analysis of milk samples revealed higher concentration of plasminogen (10.6 vs. 8.05 μg/ml; p < 0.01) and β‐casein (p < 0.05), and lower plasmin level (0.299 vs. 0.321 μg/ml; p < 0.05) in buffaloes under treatment group. It was also found that plasma cortisol, glucose and non‐esterified fatty acids levels were higher (p < 0.01) in control group as compared to the treatment animals by 13.6%, 8.14% and 12.6% respectively. However, milk composition, growth hormone, epinephrine and norepinephrine level in plasma were similar in both the groups. Hence, it may be concluded that provision of in‐house shelter management with floor bedding of paddy straw during winter was effective to minimize environmental stress and improved milk production through manipulation of PG–PL system in buffaloes.     

Cloning of Buffalo BMP1 Gene and its Expression Pattern Investigation in Different Tissues

Buffalo BMP1 gene was cloned in the present study, the BMP1 sequence was systemically analysed by bioinformatics techniques, and the expression level of BMP1 gene in different tissues were also assayed with Real-time fluorescence quantitative PCR (QRT-PCR).The results showed that with RT-PCR a 3 195 bp buffalo BMP1 gene was cloned and sequenced, including the whole ORF of 2 967 bp (coding 988 amino acid).The sequence multialigned results showed that buffalo BMP1 gene shared 99%, 96%, 96%, 96% and 95% of similar amino acid sequence with Bos taurus, Sus scrofa, Equus, Homa sapiens and Mus musclus, respectively.It was predicted that buffalo BMP1 protein contained a signal peptide domain, a preregion, a metalloproteinases domain, five complement-Uegf-BMP-1 domain (CUB domain) and two epitheloid growth factor-like domain (EGF-like domain).In addition, we also analyzed the expression level in different tissues through QRT-PCR, the results showed that BMP1 gene mRNA existed in all nine tissues with the most abundant expression in heart, followed by testis, ovary, genital ridge, while with lower amount of bone and other tissues, the minimal expression in liver was observed.     

Effect Comparison of Adenovirus Vector Transfer into Different Buffalo Cells

Buffalo mammary epithelial cell,cumulus cell and fibroblasts were transfected by adenovirus vectors and compared their transfection efficiency.293 cells were transfected with pBHGloxdelE13cre and pDC316-eGFP by liposome,the virus was collected and titer was detected.Buffalo mammary epithelial cell,cumulus cell and fibroblasts were exposed to different multiplicity of infection (MOI) of adenovirus vectors.After 72 h,the cells were observed with inverted fluorescence microscope,and transfection efficiency was calculated.When the MOI was 25,50,100,200 and 400,the transfection efficiency of fibroblasts were 0.7%,7.0%,9.0%,12.5% and 34.0%,the transfection efficiency of cumulus cells were 42.5%,55.3%,57.4%,76.0% and 80.0%,the transfection efficiency of mammary epithelial cells were 88.7%,100%,100%,100% and 100%.The results showed that the transfection efficiency of mammary epithelial cell was the best,followed by cumulus cell,and fibroblast was poor.     

Cloning and Sequence Analysis of Buffalo Keap1 Gene and Investigation of Its Expression Pattern in Different Tissues

In this study,the CDS sequence of buffalo Keap1 gene was cloned and analyzed,then its expression pattern in different tissues was also investigated.A pair of primers of buffalo Keap1 gene was designed based on the nucleotide sequence of Bos taurus Keap1 gene from GenBank,and then the buffalo Keap1 gene was amplified.Using the bioinformation techniques,the gene sequence and the protein structure were analyzed.The expression level of Keap1 gene in different tissues were detected with Real-time quantitative PCR.The results showed that the length of buffalo Keap1 gene coding sequence was 1 875 bp and encoded 624 amino acids.The multiple sequence alignment results showed that buffalo Keap1 gene shared 99%,96%,92% and 90% of similar nucleotide sequence with that of Bos taurus,Ovis aries,Sus scrofa and Homo sapiens,respectively.And the phyogenetic tree also showed the conservatism between several different species.The second structure of buffalo Keap1 protein was predicted as 24 alpha regions,40 beta regions,38 turn regions and 27 coil regions.In addition,the results of Real-time quantitative PCR showed that Keap1 mRNA exists in all seven tissues,but the most abundant expression was in heart and the minimal expression was in liver and spleen.The results provided an foundation for further study of Keap1-Nrf2-ARE signal pathway,for enhancing the ability of antioxidant of buffalo embryo in vitro culture.     

水牛卵泡颗粒细胞对卵母细胞体外成熟的影响

为了系统研究颗粒细胞对水牛卵母细胞体外成熟的影响,使用颗粒细胞条件液处理或单层颗粒细胞和卵母细胞共培养的方法,探讨颗粒细胞共培养对水牛卵母细胞体外成熟和早期胚胎发育的影响。结果显示,添加颗粒细胞传代接种第2天收集的20%颗粒细胞条件液到水牛卵母细胞成熟液中能显著提高水牛卵母细胞体外成熟率和囊胚发育率(P<0.05);然而单层颗粒细胞却显著抑制水牛卵母细胞体外成熟和早期胚胎发育(P<0.05)。结果表明,与单层颗粒细胞共培养相比,与颗粒细胞条件液共培养更有利于水牛卵母细胞体外成熟和早期胚胎发育。本研究为水牛卵母细胞体外成熟培养体系的完善提供一定理论依据。