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。     

奶水牛牛分枝杆菌的分离与鉴定

从患疑似结核病的广西奶水牛群中进行牛分枝杆菌的分离和鉴定。共收集了238份临床样品(鼻黏液及牛奶),病料经1.25 mol/mL NaOH溶液预处理后,接种罗氏培养基进行细菌分离。分离菌经进一步纯化后进行抗酸性染色并镜检,镜检后判为阳性的细菌再接种到鉴别培养基上进行鉴别培养,同时,应用PCR方法对镜检为阳性的细菌进行进一步的鉴别检验。结果收集的所有临床样品,经37℃培养后共获得细菌12株。这12株菌经抗酸性染色后镜检都为分枝杆菌阳性,进一步经鉴别培养基鉴定12株菌中有2株为牛分枝杆菌,其余均为非典型分枝杆菌。2株牛分枝杆菌经PCR方法鉴定得以确诊。     

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...     

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.     

Effect of Weaning Stress on Blood Physiology and Biochemistry Indexes and Immune Function in Dairy Buffalo Calves

To study the effect of weaning stress on dairy buffalo calves,5 healthy calves were chosen and weaned,and the blood samples were collected at 1 day before weaning,and 1,14 and 21 days after weaning to analysis blood physiology and biochemistry indexes and immune function.The results showed that the count of white blood cell,lymphocyte,erythrocyte,monocytes and hematocrit,aspartate aminotransferase activity,blood glucose,cortisol and immunoglobulin G contents of dairy buffalo calves were no significant difference between before and after weaning treatments (P > 0.05).The count of neutrophils at 21 d after weaning were significantly lower than that of 1 d before weaning of dairy buffalo calves (P < 0.05).However,lactate dehydrogenase and alanine aminotransferase activity in weaned treatments were significantly higher than those of animals in the treatment before weaning (P < 0.05).The serum urea nitrogen of dairy buffalo calves at 21 d after weaning was significantly higher than that of 1 d before weaning (P < 0.05) and hemoglobin content at 21 d after weaning was significantly lower than that at 1 day before weaning (P < 0.05).It indicated that the physiological and immunity function of dairy buffalo calves were challenged under weaning stress.     

Comprehensive transcriptional profiling and functional study of lymphangiogenic growth factor in placentome of early-stage pregnant water buffalo

The aim of the present study was to evaluate the expression and localization of lymphangiogenic factors (VEGF-C and VEGF-D), their receptor (VEGFR3) and lymphatic endothelial marker (LYVE1) in buffalo placenta during early pregnancy [EP], and to investigate the functional role of lymphangiogenic growth factors in placental lymphangiogenesis. The mRNA and protein expression of VEGF-C, VEGF-D, their receptor VEGFR3 and LYVE1 showed significant expression in EP1 (29–42 days) and EP2 stages (51–82 days) both in caruncle (maternal part) and cotyledon (foetal part) of the buffalo placenta. Immunoreactivity of VEGF-C, VEGF-D and LYVE1 was observed around the endometrial gland, in lymphatics and trophoblast cells, whereas VEGFR3 mainly localized in lymphatics of the caruncle and cotyledons. Cultured trophoblast cells were treated with VEGF-C/VEGF-D (50, 100 and 150 ng/ml) and combined doses of VEGF-C and VEGF-D (150 ng/ml) each for different time durations (24, 48 and 72 h). The mRNA expression of LYVE1 and PCNA was significantly (p < .001) upregulated with VEGF-C and VEGF-D and combined treatment (@150 ng/ml), as well as significantly downregulating Caspase-3 at 48 and 72 h. Thus, the present study provides evidence that lymphangiogenic factors are expressed in buffalo placental compartments and they may play a significant role in the regulation of placental function in water buffaloes.     

尕海湿地区沼泽草甸退化对土壤氮转化酶活性的影响

为探讨高寒湿地退化对土壤氮转化酶活性的影响,以青藏高原东缘尕海湿地未退化（ND）、轻度退化（LD）、中度退化（MD）和重度退化（HD）4种不同退化程度0~40 cm土层沼泽草甸为研究对象,研究不同退化与土层中土壤氮转化酶（蛋白酶、脲酶、硝酸还原酶和亚硝酸还原酶）活性的变化特征及其与土壤理化性质之间的关系。结果表明：1）随沼泽草甸退化程度加剧,土壤含水量、全氮、铵态氮和微生物生物量氮含量均显著降低,土壤温度与硝态氮含量却显著增加。2）随退化程度加剧,各土层土壤脲酶活性增加、蛋白酶活性降低,且仅在20~40 cm土层存在显著差异;硝酸还原酶活性增加、亚硝酸还原酶活性降低,在0~20 cm土层存在显著差异。3）各退化程度中,土壤脲酶、蛋白酶、亚硝酸还原酶活性均随土层深度的增加而显著下降,硝酸还原酶活性仅在HD显著下降。4）退化程度和土层对4种土壤氮转化酶活性均存在显著影响,且对土壤硝酸酶和亚硝酸还原酶活性存在显著交互作用。5）冗余分析表明土壤含水量对土壤氮转化酶活性变化的贡献率高达67.1%,其是驱动尕海沼泽草甸退化演替过程中土壤氮转化酶活性变化的主导因素。研究结果可为高寒湿地生态系统退化中的土壤酶活性变化规律提供理论依据。     

德宏水牛及其杂交品种体尺测定

试验对36～39月龄全放牧饲养的15头德宏、摩本、尼本水牛进行了体尺测定研究，此项试验根据水牛品种不同分为三个处理组。结果显示，杂交水牛尼本的鬐甲高、十字部高、坐骨节宽比德宏水牛分别提高5.5%、7.4%、9%，差异显著。杂交水牛摩本的鬐甲高、十字部高、坐骨节宽比德宏水牛提高5.9%、8.0%、11.9%，差异显著。摩本的肢长指数优于尼本，差异显著。此项试验结果表明，用尼里、摩拉与德宏水牛杂交，提高了德宏水牛的体尺与体况。