生鲜牛乳体细胞检测与计量校准必要性研究

体细胞数是反映生鲜牛乳质量及奶牛健康状况的重要指标。2018年我国食品安全国家标准首次计划加入体细胞数限量值分级标准,体细胞数准确检测直接影响生乳产品正确分级。本综述基于体细胞对乳制品质量的重要性,对国内外生鲜牛乳体细胞限量标准进行了总结和比较;对现有的体细胞检测方法和原理进行了概括分析,阐明了不同原理仪器的方法性能的关键检测参数指标,指出了计量校准对于保证体细胞检测仪器结果准确可比的重要性。     

奶牛体细胞核移植胚胎产业化生产条件优化北大核心CSCD

通过优化高产奶牛体细胞克隆胚胎体外生产技术条件,制备高质量的奶牛克隆胚胎,旨在提高奶牛体细胞核移植产业化应用效率。就受体卵母细胞去核方法、不同年龄供体牛细胞来源、血清饥饿与否以及不同气相组成培养等条件对奶牛体细胞克隆胚胎生产效率的影响进行了研究和探讨。结果表明,虽然荧光染色辅助去核和盲吸法的去核率、囊胚发育率分别为100%、24.83%和92.44%、28.26%,两者之间无显著差异(P>0.05),但盲吸法操作简单、效率高;不同年龄来源供体牛的细胞系构建的克隆胚胎的囊胚发育率分别为31.43%、25.68%,两者之间没有显著差异(P>0.05);经血清饥饿和未饥饿供体细胞重构的克隆胚胎囊胚发育率分别为24%、29.9%,两者之间没有显著差(P>0.05);富氧和低氧气相培养的克隆胚胎的囊胚发育率分别为28.26%、31.55%,两者之间差异不显著(P>0.05),低氧气相组成更有利于囊胚的发育。根据上述结果,奶牛体细胞核移植胚胎(克隆胚胎)的产业化生产条件为:供体细胞无需进行同期饥饿处理,直接注入到盲吸去核后的受体卵子透明带下构建克隆胚胎,融合后的克隆胚胎在密封的混合三气(5%CO2-5%O2-90%N2)的气相组成下进行体外培养,能保持稳定的囊胚发育率。     

人-兔异种核移植构建克隆胚的实验研究

“治疗性克隆”是人类最关注的课题之一,而人体细胞核移植是治疗性克隆的基础和前提。异种核移植的方法虽已被引入人体细胞克隆胚的构建,但供体细胞的类型、培养代数及准备方法与其效率之间的关系尚有待探讨。本实验以不同培养代数和不同准备方法的人卵丘细胞、皮肤成纤维细胞和软骨细胞为供体构建了克隆胚,对其发育情况的比较表明,以卵丘细胞为供体时重构胚的体外发育率高于其余二者,差异显著（P〈0．05）;不同培养代数的成纤维细胞克隆胚和不同冷藏天数供体细胞克隆胚体外发育率无明显差异。此外,本实验还尝试用荧光原位杂交法检测所构建的异种克隆胚核遗传物质的来源,结果显示来自人体细胞。本研究表明,人一兔异种核移植构建克隆胚切实可行;体细胞的类型与核移植效率相关;供体细胞的体外培养传代对克隆胚的发育并无影响;而冷藏是一种简便有效的供体细胞准备方法;此外,用FISH方法对重构胚进行核遗传物质的鉴定切实可行。     

放线菌素D诱导植物离体细胞染色体变异

用放线菌素D处理辣椒愈伤组织,明显抑制愈伤组织细胞的离体增殖,并诱发染色体数量与结构变异。细胞核DNA荧光染色测定,表明放线菌素D干扰了DNA合成复制,是导致离体细胞染色体变异的直接原因。     

在大鼠玻璃体内移植猕猴神经前体细胞的方法探索

探索一种简单、可行的异种神经前体细胞眼内移植方法。采用机械性损伤方法造成大鼠视网膜局部受损,然后在损伤眼及对照眼玻璃体内移植绿色荧光蛋白(green fluorescence protein,GFP)标记的猕猴神经前体细胞,观察细胞能否存活。结果显示:经激光共聚焦显微镜检查发现移植细胞在损伤眼及对照眼内均可存活并整合至损伤眼视网膜。实验表明,玻璃体内异种移植GFP标记的猕猴神经前体细胞可以存活并整合,是一种可行的移植方法。     

抑制素α亚基片段P33对大鼠离体培养黄体细胞凋亡的影响

我室先前的工作表明,抑制素α亚基片段Ｐ３３显著抑制离体培养大鼠黄体细胞的孕酮分泌,整体实验显示Ｐ３３促进黄体功能萎缩和细胞凋亡。本实验进一步在细胞水平探讨Ｐ３３促进黄体细胞凋亡的作用机制。应用ＤＮＡ电泳检测技术、ＤＮＡ荧光（ＡＯＥＢＰＩ）染色和流式细胞分析方法观察了Ｐ３３对ＰＭＳＧｈＣＧ假孕大鼠胶原酶ＤＮＡ酶分散的黄体细胞的自发凋亡的影响。结果三种方法一致显示,Ｐ３３（１μｇ／ｍｌ）促进黄体细胞的自发凋亡。阻断酪氨酸蛋白激酶活性（ｇｅｎｉｓｔｅｉｎ５０μｇ）则抑制Ｐ３３诱导的黄体凋亡;而阻断ＲＮＡ和蛋白质合成（Ｃｙｘ,５０μｇ／ｍｌ;ＡｃｔＤ,５０μｇ／ｍｌ）均不抑制Ｐ３３促进的黄体细胞凋亡。结果表明,Ｐ３３促进培养大鼠黄体细胞的自发凋亡,其作用机制可能与ＴＰＫ途径有关。本实验为抑制素α亚单位或其相关衍生物可能是卵巢局部调节因子之一的假说提供了又一证据。     

我国首个体细胞克隆牛胚胎移植中心成立

,中国农业大学和芦台经济技术开发区日前签署协议,中国第1个体细胞克隆牛胚胎移植中心在唐山正式成立。据了解,早在2001年,中国农业大学就和芦台经济技术开发区合作,建立了生物技术实验基地。第2年4月,中国第1头地方优质奶牛在芦台经济技术开发区诞生。此后,在这个开发区又陆续培育出5头体细胞克隆优质奶牛。今年又培育出具有世界最新药物蛋白基因的两头克隆奶牛。目前,芦台经济技术开发区的克隆牛成活率处于我国领先水平。此次成立的我国第1个体细胞克隆牛胚胎移植中心将成为华北地区克隆奶牛繁育基地和生物工程服务中心。摘自《科学时报》…     

TNF-α基因多态性及其与奶牛乳房炎的相关性分析

以417头中国荷斯坦奶牛为研究对象,根据体细胞评分(Somatic cell score,SCS)的大小将该奶牛群体划分为感染牛群(100头)和健康牛群(317头)。通过PCR-RFLP和CRS-RFLP方法检测了肿瘤坏死因子α(Tumor necrosis factor-alpha,TNF-α)基因在荷斯坦奶牛群体中的多态性,并分析这些多态位点和奶牛乳房炎的相关性。研究发现了3个单核苷酸多态位点(Single-nucleotide polymorphism,SNP):第2外显子39bp处G→A的突变;第4外显子293bp处C→T的突变;5′侧翼区(5′-flanking region,5′UTR)C→G的突变。这3个突变位点分别是DraⅠ、AfaⅠ和DdeⅠ限制性内切酶的酶切多态位点,其中DraⅠ为创造酶切位点。经过基因型分析与χ2检验表明:3个酶切多态位点在荷斯坦奶牛群中均未达到Hardy-Weinberg平衡状态。运用SPSS13.0软件,采用最小二乘拟合线性模型分析3个酶切多态位点与SCS的关系,结果表明:AA基因型个体在DraⅠ酶切位点中的SCS显著大于BB及AB基因型个体(P0.05),BB基因型表现出乳房炎抗性。AfaⅠ酶切位点中BB基因型个体的SCS显著大于AA及AB基因型个体(P0.05),AA基因型表现出乳房炎抗性。DdeⅠ酶切位点中,AB基因型个体的SCS显著低于AA基因型个体(P0.05),AB基因型为优良基因型。因此BB、AA、AB基因型分别为DraⅠ、AfaⅠ、DdeⅠ酶切位点中的优良基因型,可作为分子标记应用于奶牛乳房炎抗性筛选。     

酶法分离胚囊的荧光显微观察

用荧光显微技术对酶法分离的胚囊进行了三方面的研究:(1)利用与DNA结合的荧光染料Hoechst 33258对金鱼草、蚕豆、芝麻、烟草、龙葵等植物的分离胚囊??染色,可以观察胚囊中各组成细胞的核以及合子和胚乳的核。(2)用金胺O和樱草黄两种荧光染料染色,证明胚囊壁中含有耐果胶酶与纤维素酶作用的成分,可能是角质,因而虽经上述酶处理仍能保持胚囊的整体性。(3)用脱色苯胺蓝染色观察了被果胶酶分离的泡桐大孢子发生的各期材料,对这一过程中胼胝质的动态作了描述。以上各项实验表明:酶法分离技术与荧光显微技术相结合,是研究胚囊细胞生物学的一种简便有效的方法。     

家蚕幼虫咽侧体体积变化分析

【目的】本试验旨在研究不同生长发育时期家蚕幼虫咽侧体及其腺细胞的变化。【方法】选取家蚕4眠蚕中限品种作为实验材料,对其4龄、5龄等时期进行解剖处理,得到咽侧体后进行透析处理与DAPI染色,在荧光显微镜下成荧光像图,结合软件分析工具测量咽侧体横截面积直径与细胞数量来确定家蚕咽侧体及其腺细胞在不同生长时期的变化。【结果】咽侧体体积随幼虫生长发生了明显增长;咽侧体细胞数目随幼虫生长会增加,但比较缓慢。随着幼虫的生长,腺细胞体积会增大,同时咽侧体体积增长与腺细胞核体积增长有极显著相关性。【结论】家蚕咽侧体体积增长主要源于腺细胞体积的增大而稍带腺细胞数目的增加,家蚕幼虫咽侧体体积与生长发育的时间经过密切相关。     

Genetically enhanced cows resist intramammary Staphylococcus aureus infection

Mastitis, the most consequential disease in dairy cattle, costs the US dairy industry billions of dollars annually. To test the feasibility of protecting animals through genetic engineering, transgenic cows secreting lysostaphin at concentrations ranging from 0.9 to 14 micrograms/ml [corrected] in their milk were produced. In vitro assays demonstrated the milk's ability to kill Staphylococcus aureus. Intramammary infusions of S. aureus were administered to three transgenic and ten nontransgenic cows. Increases in milk somatic cells, elevated body temperatures and induced acute phase proteins, each indicative of infection, were observed in all of the nontransgenic cows but in none of the transgenic animals. Protection against S. aureus mastitis appears to be achievable with as little as 3 micrograms/ml [corrected] of lysostaphin in milk. Our results indicate that genetic engineering can provide a viable tool for enhancing resistance to disease and improve the well-being of livestock.     

云南省奶牛胚胎移植试验

实验用12头黑白花奶牛（3-9岁）,PGF[2α]作同步发情处理后,周期第10-12天,总剂量36 mg的FSH-P按递减方式每日肌肉注射2次,间隔12小时,共注射4天,在第3天加注PGF[2α]35 mg,进行超数排卵处理。12头供体牛共得胚135枚,可用胚数118枚,为总胚数的87.47%。平均每头得胚11.25枚。胚胎分别移植给20头同步发情处理的黑白花奶牛和20头黄牛受体（有3头为自然发情）,每头移植1枚胚胎。结果,奶牛受孕率为35%（受孕7头,产犊7头）;黄牛受孕率为40%（受孕8头,流产2头,产犊6头）。奶牛和黄牛总受孕率为37.5%。     

Prediction and Control of Brucellosis Transmission of Dairy Cattle in Zhejiang Province,China

Brucellosis is a bacterial disease caused by brucella; mainly spread by direct contact transmission through the brucella carriers, or indirect contact transmission by the environment containing large quantities of bacteria discharged by the infected individuals. At the beginning of 21st century, the epidemic among dairy cows in Zhejiang province, began to come back and has become a localized prevalent epidemic. Combining the pathology of brucellosis, the reported positive data characteristics, and the feeding method in Zhejiang province, this paper establishes an dynamic model to excavate the internal transmission dynamics, fit the real disease situation, predict brucellosis tendency and assess control measures in dairy cows. By careful analysis, we give some quantitative results as follows. (1) The external input of dairy cows from northern areas may lead to high fluctuation of the number of the infectious cows in Zhejiang province that can reach several hundreds. In this case, the disease cannot be controlled and the infection situation cannot easily be predicted. Thus, this paper encourages cows farms to insist on self-supplying production of the dairy cows. (2) The effect of transmission rate of brucella in environment to dairy cattle on brucellosis spreading is greater than transmission rate of the infectious dairy cattle to susceptible cattle. The prevalence of the epidemic is mainly aroused by environment transmission. (3) Under certain circumstances, the epidemic will become a periodic phenomenon. (4) For Zhejiang province, besides measures that have already been adopted, sterilization times of the infected regions is suggested as twice a week, and should be combined with management of the birth rate of dairy cows to control brucellosis spread.     

Animal board invited review: Specialising and intensifying cattle production for better efficiency and less global warming: contrasting results for milk and meat co-production at different scales

Cattle are the world’s largest consumers of plant biomass. Digestion of this biomass by ruminants generates high methane emissions that affect global warming. In the last decades, the specialisation of cattle breeds and livestock systems towards either milk or meat has increased the milk production of dairy cows and the carcass weight of slaughtered cattle. At the animal level and farm level, improved animal performance decreases feed use and greenhouse gas emissions per kg of milk or carcass weight, mainly through a dilution of maintenance requirements per unit of product. However, increasing milk production per dairy cow reduces meat production from the dairy sector, as there are fewer dairy cows. More beef cows are then required if one wants to maintain the same meat production level at country scale. Meat produced from the dairy herd has a better feed efficiency (less feed required per kg of carcass weight) and emits less methane than the meat produced by the cow-calf systems, because the intake of lactating cows is largely for milk production and marginally for meat, whereas the intake of beef cows is entirely for meat. Consequently, the benefits of breed specialisation assessed at the animal level and farm level may not hold when milk and meat productions are considered together. Any change in the milk-to-meat production ratio at the country level affects the numbers of beef cows required to produce meat. At the world scale, a broad diversity in feed efficiencies of cattle products is observed. Where both productions of milk per dairy cow and meat per head of cattle are low, the relationship between milk and meat efficiencies is positive. Improved management practices (feed, reproduction, health) increase the feed efficiency of both products. Where milk and meat productivities are high, a trade-off between feed efficiencies of milk and meat can be observed in relation to the share of meat produced in either the dairy sector or the beef sector. As a result, in developing countries, increasing productivities of both dairy and beef cattle herds will increase milk and meat efficiencies, reduce land use and decrease methane emissions. In other regions of the world, increasing meat production from young animals produced by dairy cows is probably a better option to reduce feed use for an unchanged milk-to-meat production ratio.     

Polymorphisms of the ATP1A1 gene associated with mastitis in dairy cattle

Mastitis affects the concentrations of potassium and sodium in milk. Since sodium-potassium adenosine triphosphatase (Na(+), K(+)-ATPase) is critical for maintaining the homeostasis of these two ions, and is involved in cell apoptosis and pathogenesis, we presumed that polymorphism of the ATP1A1 gene, which encodes the bovine Na(+), K(+)-ATPase α1 subunit could be associated with mastitis. The ATP1A1 gene was analyzed in 320 Holstein cows using PCR low ionic strength single-strand conformation polymorphism (PCR-LIS-SSCP) and DNA sequencing methods. A C/A SNP was identified at nucleotide position -15,739 in exon 17 of the ATP1A1 gene, but it did not induce any change in amino acids. We examined a possible association of polymorphism of the ATP1A1 gene with somatic cell score and 305-day milk yields. Individuals with genotype CC in ATP1A1 had significantly lower somatic cell scores and 305-day milk yields than those with genotype CA. We also examined changes in Na(+), K(+)-ATPase activity of red cell membranes. The Na(+), K(+)-ATPase activity was significantly higher in dairy cows with genotype CC compared to the other two genotypes, and the Na(+), K(+)-ATPase activity of the resistant group was significantly higher than that of the susceptible group in dairy cows. We conclude that this polymorphism has potential as a marker for mastitis resistance in dairy cattle.     

Improvement of Prediction Ability for Genomic Selection of Dairy Cattle by Including Dominance Effects

Dominance may be an important source of non-additive genetic variance for many traits of dairy cattle. However, nearly all prediction models for dairy cattle have included only additive effects because of the limited number of cows with both genotypes and phenotypes. The role of dominance in the Holstein and Jersey breeds was investigated for eight traits: milk, fat, and protein yields; productive life; daughter pregnancy rate; somatic cell score; fat percent and protein percent. Additive and dominance variance components were estimated and then used to estimate additive and dominance effects of single nucleotide polymorphisms (SNPs). The predictive abilities of three models with both additive and dominance effects and a model with additive effects only were assessed using ten-fold cross-validation. One procedure estimated dominance values, and another estimated dominance deviations; calculation of the dominance relationship matrix was different for the two methods. The third approach enlarged the dataset by including cows with genotype probabilities derived using genotyped ancestors. For yield traits, dominance variance accounted for 5 and 7% of total variance for Holsteins and Jerseys, respectively; using dominance deviations resulted in smaller dominance and larger additive variance estimates. For non-yield traits, dominance variances were very small for both breeds. For yield traits, including additive and dominance effects fit the data better than including only additive effects; average correlations between estimated genetic effects and phenotypes showed that prediction accuracy increased when both effects rather than just additive effects were included. No corresponding gains in prediction ability were found for non-yield traits. Including cows with derived genotype probabilities from genotyped ancestors did not improve prediction accuracy. The largest additive effects were located on chromosome 14 near DGAT1 for yield traits for both breeds; those SNPs also showed the largest dominance effects for fat yield (both breeds) as well as for Holstein milk yield.     

Mycoflora of cervicovaginal fluids in dairy cows with or without reproductive disorders

Fungal infection of reproduction system of dairy cattle have not been received much more attention. The aim of this study was to determine the fungal infection of Holstein dairy cows with reproductive disorders or healthy. Fungal isolates of cervicovaginal fluids of 176 Holstein dairy cows were collected by using the double rod swabs for cervix and the sterilized cotton swabs for discharges of vagina. They were evaluated for fungal infections. The treatment group included 70 dairy cows with reproductive diseases, such as abortion, repeat breeder, endometritis, metritis, retention of fetal membrane, dystocia, cervicitis, and vaginitis. The control groups were included 42 healthy non-pregnant cows and 64 pregnant cows. Isolates of fungi were obtained from cervix and vagina of 27.1% and 28.6% of treatment group, 26.7% and 31.2% of pregnant cows, and 33.3% and 21.4% of healthy non-pregnant cows, respectively, indicating no significant differences. It is showed that the cervix and vagina of the treatment group have been infected by six different mycotic isolates. However, the cervix and vagina of pregnant and non-pregnant healthy cows in control group were infected with 5, 6 and 5, 4 different fungal agents, respectively. Penicillium and yeast were the most common isolated agents. Regarding to the result of this study, it is concluded that fungal infections can occur in cervicovaginal cavity of Holstein dairy cows with or without reproductive diseases.     

Seroprevalence of Neospora caninum infection in dairy cattle of Southern China

A seroepidemiological survey of Neospora caninum infection in dairy cattle was carried out in China's southern Guangdong Province between July 2009 and March 2010. A total of 370 serum samples of dairy cattle was collected from 5 farms and examined for antibodies to N. caninum by enzyme-linked immunosorbent assay. The overall seroprevalence of N. caninum in dairy cattle was 18.9% (70/370). The seroprevalence of N. caninum in aborting cows (22.7%) was higher than that in nonaborting cows (16.3%), but the difference was not statistically significant (P > 0.05). Five-yr-old dairy cattle had the highest seroprevalence (27.8%), followed by those that were 6-yr-old (20.4%). Dairy cattle with 4 pregnancies had the highest seroprevalence (29.2%). There was no apparent association of N. caninum seropositivity with age or number of pregnancies (P > 0.05). The results of the present survey indicated that the infection with N. caninum is prevalent in dairy cattle of all ages in southern China, which may be one of the causes of bovine abortion. This is the first report of seroprevalence of N. caninum in dairy cattle in southern China.