Early Detection of Pregnancy and Embryonic Loss in Dairy Cattle by ELISA Tests

Over a 25-month period 8118 blood samples were assayed for the presence of the serum pregnancy specific-protein B (PSPB) and progesterone (P4) concentrations on three Hungarian large-scale dairy farms. Pregnancy (n = 4085) was checked by BioPRYN assay at 30-36 days post-insemination (PI). Samples from all cows that tested not pregnant and from cows with an optical density (OD) reading in the BioPRYN test that was between 0% and 30% above the cutoff OD value were tested for serum P4 concentration. According to serum P4 concentration, cows were assigned to three categories: high (>4 ng/ml), medium (2-4 ng/ml) and low (<2 ng/ml) serum progesterone. The authors predicted a presumed (low) or possible (medium) late embryonic loss (LEL) or maintenance of the pregnancy (high). A total of 710 LELs were detected (17.4%) and 31.8% of them were predicted because of a low OD value at 30-36 days after insemination. Lower PSPB serum level significantly refers for LEL (p < 0.0001). The prediction rate for the true embryonic loss was 31.8% when OD cutoff from 0% to + 30% of cutoff was examined while it was 62.5% when the threshold was OD cutoff of 0% to 10% of cutoff. The authors conclude that BioPRYN was useful for prediction of a part of LEL in dairy cows and serum P4 concentration in these cows related to the rate of LEL.     

Pregnancy‐Associated Glycoprotein and Progesterone Concentrations during Pregnancy Failure in Bedouin Goat from the Southwest of Algeria

Thirteen female Bedouin goats living in arid land of Algeria Sahara desert were used in this study. These goats were pregnant but they sustained an abortion because of unidentified causes. None of the goats showed any signs of general disease. Plasma concentrations of caprine pregnancy‐associated glycoproteins (cPAGs) and progesterone (P4) were determined during pregnancy using radioimmunoassay. The cPAGs concentration was undetectable (<0.8 ng/ml) throughout the first 2 weeks of gestation. From week 3 after mating, cPAGs concentration was detectable with significant individual variations (p < 0.05) reaching a maximum secretion (436.1 ng/ml). Throughout gestation, cPAGs concentration remained relatively constant but decreased few days before abortion, on an average of 9.2 ± 1.2 days (n = 11), except for two females where the concentrations decreased later (1–2 days before abortion). One or two peaks of cPAGs concentrations (in 4/13 and in 9/13 females, respectively) have been measured few weeks before abortion (77–124 days after mating), when a decline of cPAGs was detected. The P4 concentration increased after mating, and was high from the first week till the end of pregnancy. The P4 concentration (9.1 ± 0.9 ng/ml) decreased rapidly (<0.5 ng/ml) after 4 ± 0.7 days (n = 6) or 9.4 ± 1.6 days (n = 7) before abortion. A positive relationship (p < 0.01) was found between P4 and cPAGs concentrations during gestation. Results indicate that cPAGs and P4 measurements can be used for monitoring gestation and for abortion prediction.     

Associations of Male Fertility Traits with Blood Groups and Blood Protein Polymorphisms in Dairy Cattle

Abstract

The relationships between 12 polymorphic systems and bull fertility were studied in the Swedish Red and White dairy cattle population. The material consisted of 1447 young bulls with non-return rates based on at least 500 inseminations each. A reduced animal model including a full additive relationship matrix was used. Multiple regressions were fitted for the estimation of the average gene substitution effects as well as the gene-frequency-independent effects of additive gene action and dominance. To evaluate the results a multivariate analysis of variance, classical segregation analyses and marker frequency studies were performed. Significant associations were found between the F, B, Tf, S and C systems, and nonreturn rates, but the contribution of the markers to the total variation of non-return rates was small.     

Dexamethasone‐Induced Eosinopenia is Associated with Lower Progesterone Production in Cattle

Eosinophilic cells accumulate in the capillaries of the bovine Graafian follicle shortly before ovulation and in the early developing corpus luteum (CL). Suppressing the migration of these eosinophilic cells by dexamethasone allowed us to evaluate their possible function in the CL development. Brown Swiss cows (n = 10) were randomly subdivided into two groups (n = 5). Every group was used once as control group and once as experimental group with two oestrous cycles between each treatment. Eighteen hours (h) after oestrus synchronization, dexamethasone or saline was given. Ovulation was induced 24 h later with gonadotropin‐releasing hormone. Another injection of dexamethasone or saline was given 12 h later. Eosinophilic cells in the blood were counted daily until day 7 after the first dexamethasone injection. The collection of ovaries took place at days 1, 2 and 5. Gene expression, protein concentration and location of angiogenic factors, chemokines, insulin‐like growth factor 1 (IGF1) and eosinophilic cells were studied. No eosinophilic cells were found in the CL of the treatment group. Blood progesterone decreased significantly in the dexamethasone group from day 8 to 17. The protein concentration of FGF2 increased significantly in CL tissue at day 2 and VEGFA decreased. Local IGF1 gene expression in the CL was not regulated. We assume from our data that the migration of eosinophilic cells into the early CL is not an essential, but an important stimulus for angiogenesis during early CL development in cattle.     

Therapeutic Approaches to Pregnancy Loss of Non‐infectious Cause During the Late Embryonic/Early Foetal Period in Dairy Cattle. A Review

The problem of pregnancy loss during the late embryonic/early foetal period due to non‐infectious causes in dairy cattle is on the rise, especially in high milk producing cows. This review discusses recent therapeutic approaches designed to reduce the incidence of early foetal loss. These strategies are based on the fact that low plasma concentrations of progesterone are commonly related to foetal loss. Progesterone supplementation during the late embryonic/early foetal period has proven useful in curtailing losses, mainly in single pregnancies, whereas a more positive effect of treatment with GnRH than progesterone has been found in twin pregnancies. Therapeutics is not necessary in cows with an additional corpus luteum. In practical terms, suggestions include recording the number of embryos and corpora lutea on first pregnancy diagnosis and checking for normal pregnancy progression on day 60 post‐insemination.     

奶牛早孕因子重组蛋白的原核表达、纯化及多克隆抗体的制备

本研究旨在表达、纯化奶牛早孕因子重组蛋白及制备多克隆抗体.本试验构建奶牛早孕因子重组蛋白原核表达载体pET32a-EPF,转化至大肠杆菌BL21(DE3)内进行诱导表达,SDS-PAGE切胶纯化的早孕因子重组蛋白免疫BALB/c小鼠制备多克隆抗体,并用Western blotting和ELISA对重组蛋白及抗体进行检测.结果显示,成功表达并纯化了奶牛早孕因子重组蛋白,重组蛋白分子质量约37 ku,表达量约占菌体总蛋白的48.6%,纯化后目的蛋白纯度可达93%,重组蛋白可与所制备的多克隆抗体结合,ELISA测定的抗体效价为1:12 800.结果表明,本研究成功表达并纯化了奶牛早孕因子重组蛋白,为研究奶牛早孕诊断奠定基础.     

Gamma Interferon Production and Plasma Concentrations of Pregnancy‐Associated Glycoproteins 1 and 2 in Gestating Dairy Cows Naturally Infected With Neospora caninum

Gamma interferon (IFN‐γ) production and cross‐breed pregnancy have been attributed a role in protecting dairy cows infected with Neospora caninum against abortion. Plasma levels of pregnancy‐associated glycoproteins‐1 (PAG‐1) are a marker of placental/foetal well‐being and of PAG‐2 is an abortion risk indicator in chronically N. caninum‐infected animals. The present study examines, in cross‐breed pregnancies, interactions between IFN‐γ production and levels of PAG‐1 and PAG‐2 in non‐aborting naturally Neospora‐infected dairy cows. Data were obtained from 60 pregnant Holstein‐Friesian cows: 44 Neospora‐seropositive and 16 Neospora‐seronegative; 12 became pregnant using Holstein‐Friesian semen and 48 using Limousin semen. Blood samples were collected on Days 40, 90, 120, 150, 180 and 210 of gestation. Gamma interferon was only detected in the plasma of nine of the 44 Neospora‐seropositive cows, all of them became pregnant using Limousin semen. Through GLM procedures, in cows inseminated with Limousin semen and Neospora‐seropositive cows showing no IFN‐γ production, PAG‐1 concentrations were high and increased throughout gestation compared to the levels detected in cows inseminated with Holstein‐Friesian semen and Neospora‐seropositive cows producing IFN‐γ, respectively. In Neospora‐seronegative cows and in Neospora‐seropositive cows showing no IFN‐γ production, significantly increased PAG‐2 concentrations were observed on gestation Day 120. Our findings indicate that IFN‐γ production correlates negatively and the production of antibodies against N. caninum is uncorrelated with plasma PAG concentrations during gestation in Neospora‐infected dairy cows. Accordingly, IFN‐γ production could be linked to the transplacental migration of tachyzoites, which may cause a reduction in PAG levels.     

Evaluation of an Electrochemical Point‐of‐Care Meter for Measuring Glucose Concentration in Blood from Periparturient Dairy Cattle

Background

The Precision Xtra^® meter is a promising low cost electrochemical point‐of‐care unit for measuring blood glucose concentration ([gluc]) in cattle blood. The meter uses an algorithm that assumes the intra‐erythrocyte [gluc] equals the plasma [gluc] on a molal basis, and that the hematocrit is similar in humans and cattle.

Objectives

The primary objective was to determine the accuracy of the meter for measuring plasma [gluc] in dairy cattle. Secondary objectives were to characterize the influence of hematocrit and sample temperature on the measured value for [gluc].

Animals

A total of 106 periparturient Holstein‐Friesian cattle.

Methods

Blood and plasma samples (1,109) were obtained and Deming regression and Bland–Altman plots were used to determine the accuracy of the meter against the reference method (plasma hexokinase assay). Multivariable regression and linear regression were used to determine the effect of hematocrit and sample temperature on the plasma [gluc] measured by the meter.

Results

Intra‐erythrocyte [gluc] was 18% of plasma [gluc] on a molar basis. Sample temperature had a significant linear effect on plasma [gluc] as measured by the meter for 3/5 plasma samples when measured [gluc] > 160 mg/dL.

Conclusions and clinical importance

The meter utilizes an algorithm that is optimized for human blood and is inaccurate when applied to bovine blood. Until a cattle‐specific algorithm is developed, we recommend using plasma as the analyte instead of blood and calculating plasma [gluc] using the equation: [gluc] = 0.66 × [gluc]_p‐meter + 15, where [gluc]_p‐meter is the value reported by the meter. If blood is measured, then we recommend using the equation: [gluc] = 0.90 × [gluc]_b‐meter + 15.     

奶牛乳房性状与产奶量及排乳性能的关系

选择胎次相同,泌乳月份相近的经产荷斯坦牛197头,分别测定其后乳房宽度,后乳房深度,后乳房高度,乳房纵沟深,乳头直径,乳头长度等6个高遗传力性状,并与排乳性能及产奶量的相关性进行研究.结果表明:奶牛乳房纵沟深,乳头直径,乳头长度等6个高遗传力性状,并与排乳性能及产奶量的相关性进行研究.结果表明:奶牛乳房纵沟深、后乳房宽度、乳头长、乳头直径与排乳速度有极显著正相关(P＜0.01),后乳房高度与排乳速度呈现中等正相关(P＜0.05),平均排乳时间与后乳房宽度呈现显著正相关(P＜0.05);日产乳量与乳头长度呈中等负相关(P＜0.05),与乳房纵沟深、乳头直径呈显著正相关(P＜0.05),305 d产乳量与乳房纵沟深、后乳房宽度呈极显著强正相关(P＜0.01),与乳头直径呈显著正相关(P＞0.05),与乳头长度有极显著负相关(P＜0.01).     

奶牛双胎的发生及其对奶业生产的影响

奶牛在一个发情周期内如果同时排出两个成熟卵子，经过受精和发育，就可能出现双胎现象。奶牛双胎的发生与遗传、产奶量、胎次、受孕年份和季节等因素有关。由于奶牛双胎出现了异性孪生母犊不育、胎衣不下率升高、胎间距延长、流产率和难产率升高、胎儿成活率降低等负面影响。因此，奶牛双胎对奶业生产是不利的。如果能结合性别控制等先进技术，奶牛双胎将有很大的发展前景。     

奶牛乳房性状与产奶量及排乳性能的关系

选择胎次相同,泌乳月份相近的经产荷斯坦牛197头,分别测定其后乳房宽度、后乳房深度、后乳房高度、乳房纵沟深、乳头直径、乳头长度等6个高遗传力性状,并对其与排乳性能及产奶量的相关性进行研究.结果表明：奶牛乳房纵沟深、后乳房宽度、乳头长度、乳头直径与排乳速度有极显著正相关（P＜0.01）,后乳房高度与排乳速度呈中等正相关（P＜0.05）,平均排乳时间与后乳房宽度呈显著正相关（P＜0.05）;日产奶量与乳头长度呈中等负相关（P＜0.05）,与乳房纵沟深、乳头直径呈显著性正相关（P＜0.05）,305d产奶量与乳房纵沟深、后乳房宽度呈强正相关（P＜0.01）,与乳头直径呈显著正相关（P＞0.05）,与乳头长度有极显著负相关（P＜0.01）.     

平阳县奶牛生产的特点及存在问题调查

平阳县地处浙东南沿海,水陆交通便捷.全县陆地面积1052 km2,其中耕地33.15万亩(1亩=667 m2)、山林702.7万亩、草地11.02万亩、可利用滩涂54.6万亩,蔬菜、牧草及农副产品等资源丰富,适合奶牛等食草动物养殖.尤其是麻步镇地处中部平原与山区结合部,经济作物及蔬菜生产是全镇的主导产业,为发展奶牛生产提供了良好的发展基础和前景.为加快平阳县的奶业生产发展,稳步扩大优质奶牛后备资源,为此有关部门组织有关人员深入奶牛养殖场户开展调查,在掌握情况的基础上,提出对策措施,推进奶牛生产方式转变,全面提升奶业的综合生产能力,以实现奶牛业优质高产.     

Interrelationships among Production and Clinical Disease in Dairy Cattle: A Review

Epidemiological evidence is presented in order to answer two questions. The first question is: “Does high milk production put a cow at increased risk of disease?” The answer to this question seems to be “maybe” for milk fever, but “no” for most other common diseases (veterinary-assisted dystocia, retained placenta, metritis, cystic ovary, ketosis, left displaced abomasum, and mastitis). The second question is: “Is low milk production a consequence of disease?” For most diseases the answer is a cautious “yes”.     

奶牛血液保存条件和DNA提取方法的优化

本文研究中国荷斯坦牛血液在不同温度、时间保存的条件下（4℃保存0d为A组；-70℃保存30d为B组；4℃保存7d为C组）对DNA提取效果的影响和对比试剂盒法（E）、试剂盒改良法（F）、酚-氯仿法（G）提取中国荷斯坦牛血液基因组DNA的效果。试验结果表明：A组与B组差异不显著（P〉0．05）；A组、B组与C组差异显著（P〈0．05）。4℃保存0d对DNA提取效果影响最小。在A组中，E与F差异不显著（P〉0．05）；E、F与G差异显著（P〈0．05）。综合各方面因素。试剂盒改良法最好。     

甘肃马鹿血液蛋白多态性及其与产茸量关系的研究

采用垂直板聚丙烯酰胺凝胶电泳技术,分析了66头甘肃马鹿的8个血液蛋白位点的基因频率、基因型频率和群体遗传变异指标,建立了产茸量与多态位点之间的线性相关模型.结果表明:甘肃马鹿各蛋白位点中,Tf、Hb、Alb、Pa、Prt1、Prt2、血清ES分别有3、1、1、2、2、2和3种基因型.未检测到红细胞ES;Tf和Prt2的基因型频率偏离Hardy-Wein-berg平衡;Tf的变异程度最大(杂合度为0.586 3),杂合度的大小顺序为Tf>Prt2>ES>Pa>Prt1>Hb=Alb.群体的平均杂合度为0.350 5;各多态住点中仅有Prt1与产茸量之间存在显著正相关(P<0.05),而且Prt1的AA型产茸量明显高于其他各位点基因型.由此可见,Prt1位点的AA型可用来标记甘肃马鹿的品种特征.     

Formation of Corpora Lutea and Central Luteal Cavities and Their Relationship with Plasma Progesterone Levels and Other Metabolic Parameters in Dairy Cattle

The corpus luteum (CL) may be looked upon as a compact or cavitary structure. A number of papers have addressed the relationship between CL type and parameters such as fertility or progesterone levels. The present study assessed the morphological and functional sequence observed in cows with different CL types, comparing pre-ovulatory follicle size, progesterone levels, luteal tissue formation and some blood biochemical parameters (calcium, albumin, inorganic phosphorus, glucose, magnesium, copper and zinc), oestrus cycle length and oestrus expression, as a function of CL type. Twenty-eight lactating dairy cows from two commercial dairy farms in southern Spain were studied. Oestrus detection was performed by monitoring daily oestrus behaviour, and artificial insemination (AI) was perfomed using the AM/PM rule. Ovaries and uterus were sonographically examined and blood samples were collected to measure progesterone and various biochemical parameters. There was a slight tendency towards the appearance of luteal cavities when pre-ovulatory follicles were larger (1.9 ± 0.2 vs 1.7 ± 03; p = 0.074). Fertility was not affected by cavity presence (cavity = 42.9% and compact = 57.1%; n.s.). Luteal tissue and function were not modified as a function of CL type. Cows with luteal cavities displayed significantly higher levels of albumin, suggesting a possible metabolic influence on the formation of these structures, although specific research is required to confirm this observation.     

西北地区奶牛养殖生产特点及发展策略

本文通过分析西北地区奶业的发展历史、现状、优势以及存在问题等特点,根据本地区实际情况,提出未来奶业发展策略,从而充分发挥西北地区的发展潜力和优势,实现西北地区的奶业产业化。     

Development of Y‐chromosome‐Specific SCAR Markers Conserved in Taurine,Zebu and Bubaline Cattle

Sex pre‐selection of bovine offsprings has commercial relevance for cattle breeders and several methods have been used for embryo sex determination. Polymerase chain reaction (PCR) has proven to be a reliable procedure for accomplishing embryo sexing. To date, most of the PCR‐specific primers are derived from the few single‐copy Y‐chromosome‐specific gene sequences already identified in bovines. Their detection demands higher amounts of embryonic genomic material or a nested amplification reaction. In order to circumvent this, limitation we searched for new male‐specific sequences potentially useful in embryo sexing using random amplified polymorphic DNA (RAPD) analysis. Random amplified polymorphic DNA (RAPD) assay reproducibility problems can be overcome by its conversion into Sequence Characterized Amplified Region (SCAR) markers. In this work, we describe the identification of two bovine male‐specific markers (OPC16₃₂₃ and OPF10₁₁₆₈) by means of RAPD. These markers were successfully converted into SCARs (OPC16₇₂₆ and OPF10₉₈₄) using two pairs of specific primers.Furthermore, inverse PCR (iPCR) methodology was successfully applied to elongate OPC16₃₂₃ marker in 159% (from 323 to 837 bp). Both markers are shown to be highly conserved (similarity ≥95%) among bovine zebu and taurine cattle; OPC16₃₂₃ is also highly similar to a bubaline Y‐chromosome‐specific sequence. The primers derived from the two Y‐chromosome‐specific conserved sequences described in this article showed 100% accuracy when used for identifying male and female bovine genomic DNA, thereby proving their potential usefulness for bovine embryo sexing.     

Timing of Early Foetal Loss for Single and Twin Pregnancies in Dairy Cattle

The incidence of early foetal loss is increasing under intensive management systems for dairy cattle. The aims of the present study were to determine whether there is any peak period of pregnancy loss during the early foetal period and to evaluate possible differences between single and twin pregnancies. The study population consisted of 1442 pregnant cattle from a single herd. Pregnancy was diagnosed by transrectal ultrasonography between 36 and 42 days after insemination, and then weekly until day 90 of gestation or until pregnancy loss. A total of 1310 cows (90.8%) bore single embryos and 132 (9.2%) carried twins. Pregnancy loss was registered in 139 (9.6%) cows before day 90 of pregnancy: 101 (7.7%) in single and 38 (28.8%) in twin pregnancies. The average time of pregnancy loss for all animals was 58.4 ± 12.6 days and ranged from 45 to 90 days. Seventy‐five per cent of the pregnancy losses were registered between 45 and 60 days of gestation. The average time of pregnancy loss for cows with singletons was 52.1 ± 4.1 days and ranged from 45 to 61 days and that for those with twins was 75.1 ± 12.4 days and ranged from 46 to 90 days. Seventy‐five per cent of the twin pregnancy losses were registered between 68 and 90 days of gestation. Our data show that the foetal loss in singleton pregnancies occurs earlier than in twin pregnancies. Assessment of normal development of gestation on days 60 and 90 after insemination is suggested.     

供体牛外周血AMH浓度对荷斯坦青年奶牛体内胚胎生产效率的影响

旨在研究供体牛超数排卵(简称超排)过程中不同时间外周血AMH浓度与荷斯坦青年奶牛体内胚胎生产效率的相关性,优化供体牛筛选标准,提高荷斯坦青年奶牛体内胚胎生产效率.本试验共选用96头荷斯坦青年奶牛进行超排处理,分别在孕酮阴道栓(CIDR)埋置、人工授精和胚胎回收当天通过尾根采集外周血检测抗缪勒管激素(anti-Mülle...