HPLC法检测牛初乳中IgG含量

建立了牛初乳中IgG的高效液相色谱检测法,并测定了初乳中IgG的经时变化过程.结果显示,IgG浓度在0.2～15mg/mL的范围内峰面积与浓度呈现良好的线性关系,回收率实验平均大于98.5%.采用本法测定了六头幽门螺杆菌免疫牛初乳中IgG及两头正常牛初乳中IgG的经时变化过程.母牛分娩后24h内,初乳中免疫球蛋白IgG平均含量在40～70mg/mL之间;3d后,初乳中免疫球蛋白总量下降较快;至第7d,初乳中免疫球蛋白含量平均在1～5mg/mL之间.     

发酵法去除牛初乳中酪蛋白提高IgG含量的研究

利用乳酸菌发酵牛初乳从而降低pH达到酪蛋白等电点,通过离心去除酪蛋白,提高免疫球蛋白G(JgG)含量。结果表明,发酵法去除酪蛋白的最适pH为4.65,离心条件为1500×g,30min;随着pH的下降,酪蛋白被去除.初乳中的IgG含量上升,pH由4.70降低到4.65时,IgG含量有显著性变化(P<0.01)。2-7d原料初乳中IgG含量平均为20.09mg/g,经发酵后乳清IgG含量提高约1.5倍,平均达到52.07mg/g。     

牛初乳中免疫球蛋白的测定

以单向免疫扩散法测定了牛初乳中的IgG。结果表明,产犊后第1次挤乳,初乳gG平均为67.23mg/mL,之后随泌乳进行迅速下降,24h后降到小于5mg/mL,到第5天已降到常乳水平。     

胎次及天数对牦牛初乳活性成分的影响

采用双抗体一步夹心法酶联免疫吸附实验(ELISA),研究了五个胎次的天祝白牦牛初乳中免疫球蛋白G(IgG)、乳铁蛋白(LF)以及胰岛素样生长因子-Ⅰ(IGF-Ⅰ)在产后七天的浓度变化规律,为探讨白牦牛初乳的营养价值及其资源的合理开发和利用提供理论依据.结果表明:天祝白牦牛初乳中IgG、LF和IGF-Ⅰ在分娩后第1天且四胎浓度最高,分别为2404.41、2001.53和1206.48U/L,其后都随产后泌乳天数的增加急剧下降,5天以后下降速度趋于平缓.产后四天内牛初乳的IgG、LF和IGF-Ⅰ含量仍可达较高水平,营养价值丰富,具有一定的开发价值,因此在开发利用牦牛初乳产品时,为扩大牛初乳的利用率,可采集四天内初乳进行加工利用.     

山羊初乳成分及其免疫球蛋白构成变化的研究

动物初乳对其后代健康的作用已经得到了广泛的证实,其对人类健康的影响也受到了越来越多的重视.本研究探讨了阿尔卑斯山羊产后一周内乳成分和其中免疫球蛋白(Ig)含量的变化.结果表明:产后48h内山羊奶的蛋白质、总固形物和免疫球蛋白含量均快速下降;脂肪含量在前三次的初乳中逐渐升高,随后又缓慢下降;乳糖含量保持相对稳定于4%～5%;经产山羊前三次初乳的蛋白含量分别达到了16.5%、10.7%和6.8%,显著地高于初产山羊前三次初乳的蛋白含量(7.0%、4.8%和4.1%);经产山羊前两次初乳的IgG含量达到了59.9mg/ml和34.7mg/ml,IgM含量达到了6.14mg/ml和2.37mg/ml,显著地高于初产山羊前两次初乳的IgG含量(18.1mg/ml和13.9mg/ml)和IgM含量(1.95mg/ml和0.98mg/m1);经产和初产山羊初乳的IgA含量无显著差异.     

双抗体夹心酶联免疫法测定水牛初乳中的IgG

为快速、准确地测定水牛初乳中IgG活性质量浓度,建立了双抗体夹心酶联免疫分析法(ELISA)。结果表明,IgG活性质量浓度的对数值与吸光值存在很好的线性关系,其标准曲线方程式为γ=-0.2907x2+2.0819x+0.8689,相关系数R2=0.9937,线性范围在7.8～1000ng/mL,平均回收率在84.40%～96.71%之间,灵敏度高、重现性好,可用于水牛初乳及其制品中IgG活性质量浓度的检测。     

高静水压处理对牛初乳中IgG的影响

采取不同的高静水压条件（处理压强、施压温度、保压时间）处理产犊后48 h内的牛初乳,研究高静水压处理牛初乳对免疫球蛋白G（immunoglobulin G,IgG）活性的影响。将牛初乳离心去除酪蛋白、乳脂肪等,取上清液进行高静水压处理,采用高效液相色谱法和十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法（sodium dodecyl sulfatepolyacrylamidegel electrophoresis,SDS-PAGE）对处理后的样品进行检测。结果表明：高静水压处理后的样品,经高效液相色谱仪检测,其与亲和色谱柱发生特异性吸附的能力下降,IgG检出质量浓度降低;经SDS-PAGE检测和凝胶电泳成像仪扫描处理,不同处理条件下IgG的轻链和重链的质量变化不显著。因此,当牛初乳在200 MPa、30 ℃和20 min的高静水压处理条件下,IgG的活性最好,检出质量浓度为16.843 9 mg/mL。     

一定工艺下牛初乳生产投料IgG含量与对应产品IgG含量关系的探究

牛初乳中含有大量的免疫球蛋白(Ig),其中IgG的含量最高。作为食用功效成分,准确控制牛初乳粉中的IgG含量是牛初乳生产的核心,而投料生鲜牛初乳IgG在生产过程中的保留比率不稳定,经常出现投料IgG含量较高,得到对应产品并不理想的问题。因此,在一定工艺下了解牛初乳生产投料IgG含量与产品牛初乳粉IgG含量之间的关系,有利于提升产品质量的一致性,以及内控标准的符合性。     

水牛初乳中免疫因子和生长因子质量浓度的研究

研究水牛初乳中免疫球蛋白IgG,IgA和类胰岛素样生长因子(IGF-I)质量浓度的变化特点。用ELISA方法检测水牛分娩后7d内初乳样品的IgG,IgA和IGF-I质量浓度,同时检测3种成分在水牛常乳中的质量浓度。结果表明:分娩后3h的水牛初乳IgG,IgA和IGF-I质量浓度最高,分别为78.22g/L,8.83g/L和1099.78μg/L,常乳中的质量浓度分别为1.87g/L,0.71g/L和358.30μg/L。IgG在泌乳的第1天内下降91.18%,IGF-I下降57.02%,IgA在头2d内下降85.84%,之后缓慢下降接近常乳。因此,水牛泌乳最初的1~2d内初乳含有丰富的IgG,IgA和IGF-I,具有很高的开发利用价值。     

牛初乳中免疫球蛋白G双抗夹心ELISA检测方法的建立

将牛免疫球蛋白G（immunoglobulin G,IgG）作为免疫原免疫BALB/c小鼠,通过细胞融合、筛选获得分泌抗牛IgG单克隆抗体的细胞株。制备小鼠腹水抗体,进一步纯化获得抗牛IgG单克隆抗体。建立双抗夹心酶联免疫吸附法检测牛初乳中IgG质量浓度,该方法在7.8～1 000 ng/mL范围内有良好的线性关系,最低检出限为7.06 ng/mL,批内变异系数为4.52%,批间变异系数为4.94%,回收率为91.85%～102.45%。此法操作简便、准确度高、稳定性好,可用于实际牛初乳样品的快速检测。     

微滤-超滤联用技术优化牛初乳乳清中IgG富集工艺

为了高效富集IgG的同时减轻牛初乳的浪费问题,提高产品价值,本文采用微滤-超滤联用技术对牛初乳乳清中IgG进行富集。首先探究了微滤技术在牛初乳乳清除菌中的应用,并对其操作工艺进行优化,其次,利用超滤技术对微滤除菌后的牛初乳乳清进行富集,在单因素实验基础上,采用响应面对超滤工艺进行优化,并对富集后的牛初乳乳清进行品质分析。结果表明:牛初乳乳清微滤除菌的最佳工艺参数为:微滤压力为0.2 MPa、温度为30 ℃,超滤富集的最佳工艺参数为:超滤压力为0.15 MPa、温度为35 ℃、浓缩倍数为6倍、稀释次数为4次,按此条件进行牛初乳乳清的微滤-超滤操作,此时的IgG浓缩率为58.19%,膜通量为204.46 L/m2·h。富集后的牛初乳乳清品质分析表明:IgG含量为22760 μg/mL,IgG活性为718.31 IU/L,蛋白质含量为7.86%,脂肪含量为0.035%,菌落总数为2.4 lg CFU/mL。本研究为牛初乳乳清中IgG的进一步开发与综合利用提供了一定的参考依据。     

Heat treatment of bovine colostrum. I: effects of temperature on viscosity and immunoglobulin G level

The objective of this study was to identify the critical temperature, at or below which heat-treatment of bovine colostrum would produce no significant changes in viscosity, IgG concentration, or Ig activity. Results of preliminary work, using a Rapid Visco Analyzer (RVA) to heat 50-mL aliquots from 6 unique batches of bovine colostrum at 59, 60, 61, 62, and 63°C, suggested that colostrum could be heated to 60°C for up to 120 min without changing viscosity or IgG concentration. This finding was confirmed by heating 50-mL aliquots from 30 unique batches of colostrum in an RVA for 120 min at 60 and 63°C. Heating colostrum to 63°C resulted in an estimated 34% decrease in IgG concentration and 33% increase in viscosity. However, there was no difference in IgG concentration between preheat-treated (73.4 ± 26.5 mg/mL) and post-heat-treated (74.5 ± 24.3 mg/mL) samples after heating colostrum to 60°C in an RVA for 120 min. Similarly, viscosity was unaffected after heating colostrum to 60°C in an RVA for 120 min. High quality colostrum (≥73.0 mg/mL) suffered greater losses of IgG and greater viscosity changes when heated to 63°C than did moderate quality colostrum (<73.0 mg/mL). However, the effects of colostrum quality were minor if high quality colostrum was only heated to 60°C. The results of a bovine viral diarrhea serum neutralization assay suggested that antibody activity was unchanged after heating colostrum to either 60 or 63°C. However, these results were interpreted as being inconclusive due to a high proportion of missing results because of the congealing of many samples after heat treatment. The results of this study indicate that 50-mL volumes of bovine colostrum can be heat treated at 60°C for up to 120 min in an RVA without affecting IgG concentration or viscosity.     

Evaluation of 2 different treatment procedures after calving to improve harvesting of high-quantity and high-quality colostrum

The objective of this study was to evaluate 2 different treatment procedures at the first milking after calving to increase colostrum quantity and to improve colostrum quality in dairy cows. We hypothesized that either exogenous treatment with oxytocin or the presence of the calf at first milking would lead to higher colostrum quantity and higher IgG concentration. The study was conducted from October to December 2017 on a commercial dairy farm in Germany. A total of 567 cows at the time of calving were enrolled, but for the final analyses only 521 animals were considered. The cows were randomly assigned on a daily basis into 1 of 3 groups: (1) control group (n = 177), (2) application of 20 IU of oxytocin i.m. (OXY; n = 163), and (3) presence of the calf (CA; n = 181) before and during milking. Cows in the control and oxytocin group had no contact with their calves after calving and were milked in a separate milking parlor. Cows in the oxytocin group were injected with 20 IU of oxytocin i.m. 3 min before manual stimulation. For cows in the third group, the calf was placed into a calf cart and located in front of the cow 3 min before manipulation of the cow. Colostrum quantity was determined by a digital hanging scale. The colostrum quality was assessed with digital Brix refractometry and ELISA. To evaluate the effect of 2 different treatment procedures, a generalized linear mixed model was constructed using SPSS (SPSS Inc., IBM, Ehningen, Germany). The mean (±SE) colostrum quantity was 4.17 ± 0.30 kg. The treatment procedures and the harvesting time after calving had no effect on colostrum quantity. Parity, calf birth weight, and calving time affected colostrum quantity. Cows in second parity had the lowest quantity of colostrum (3.74 ± 0.37 kg) compared with cows in parity 1 (4.75 ± 0.34 kg) and cows in parity 3 or greater (4.75 ± 0.38 kg). Cows calving during the night (2200 until 0600 h; 4.93 ± 0.37 kg) had the highest quantity of colostrum compared with cows calving in the morning (0600 until 1400 h; 4.17 ± 0.38 kg) or afternoon (1400 until 2200 h; 4.14 ± 0.34 kg). Regarding colostrum quality, 48% of the colostrum samples contained ≥50 mg of IgG/mL. The mean IgG concentration was 54.6 ± 2.80 mg of IgG/mL. Colostrum quality was affected by the treatment procedures, colostrum quantity, parity, calving time, harvesting time after calving, and the calving day during the week. Both treatment procedures (i.e., OXY with mean IgG concentration results of 57.0 mg of IgG/mL and CA with 56.0 mg of IgG/mL) resulted in higher IgG concentrations in colostrum compared with the control group (50.7 mg of IgG/mL). With increasing colostrum quantity, the colostrum quality decreased in primiparous and multiparous cows. A longer time lag between calving and milking negatively affected the colostrum quality. Concentration of IgG was higher for cows in parity 3 or greater (64.6 ± 2.59 mg of IgG/mL) compared with cows in parity 1 (48.5 ± 2.86 mg of IgG/mL) and cows in parity 2 (50.7 ± 2.89 mg of IgG/mL). Cows calving during the night had greater IgG concentrations (60.4 ± 2.92 mg of IgG/mL) compared with cows calving in the morning (51.9 ± 2.98 mg of IgG/mL) or afternoon (51.3 ± 2.71 mg of IgG/mL). Harvesting colostrum on quieter days, such as Sundays, resulted in higher IgG concentrations (61.4 ± 3.70 mg of IgG/mL). The assessment by Brix refractometry resulted in a mean result of 26.0 ± 0.20% Brix. Treatment procedures and the harvesting time after calving had no effect on colostrum quality. A negative association was observed between colostrum quantity and quality in primiparous and multiparous cows determined by Brix refractometry. Brix readings were greater for cows in parity 3 or higher (27.7 ± 0.26% Brix) compared with cows in parity 1 (25.3 ± 0.30% Brix) and cows in parity 2 (25.0 ± 0.32% Brix). In conclusion, the treatment procedure for the first milking is irrelevant to improve the quantity of colostrum. Both treatment procedures, however, increased IgG concentrations as determined by ELISA.     

Effect of feeding single-dam or pooled colostrum on maternally derived immunity in dairy calves

The role of colostrum management in providing adequate immunological protection to neonatal calves has been widely investigated, and thresholds for colostrum quality, as well as optimum volume and timing for colostrum feeding have been established. However, limited information is available on the effect of colostrum source (single dam or pooled) on passive immunity, as well as subsequent antibody survival in the calf. This study aimed to assess the effect of feeding single-dam colostrum (own and other dam) or pooled colostrum on transfer of passive immunity, and also investigate the rate of depletion of disease-specific antibodies among dairy calves. In total, 320 cows and 119 dairy heifer calves were enrolled in the study. Calves were blood-sampled immediately after birth and received either own-dam, other-dam, or pooled colostrum. Calves were blood-sampled at 24 h to assess serum IgG concentrations and at monthly intervals thereafter to document disease-specific antibody survival. Mean colostrum IgG concentration was higher for other-dam treatment group, whereas own-dam and pooled treatments were similar. For all treatment groups, the mean IgG concentration was >80 mg/mL, exceeding the quality threshold of 50 mg/mL. Mean calf serum IgG concentration was lower for calves fed pooled colostrum compared with those that received colostrum from a single cow. There was a negative association with 24-h serum IgG and calf birth bodyweight; calves <30 kg at birth had the highest 24-h serum IgG concentration. Survival of antibodies to bovine viral diarrhea, Salmonella infection, leptospirosis, bovine parainfluenza 3 virus, bovine respiratory syncytical virus, rotavirus, and coronavirus was not associated with colostrum source; however, antibodies to infectious bovine rhinotracheitis had a greater period of survival among calves fed own-dam colostrum. We found that feeding single-dam colostrum can thus improve calf immunity through increased serum IgG levels and antibody survival rates. Furthermore, we hypothesize that immune exclusion may occur with pooled colostrum; therefore, providing pooled colostrum may still be a good practice as long as it can be ensured that enough antibodies are absorbed into the blood stream to deal with pathogens calves may encounter because different dams may have antibodies against different strains of viruses and bacteria, yielding cross protection.     

Technical note: Serum total protein and immunoglobulin G concentrations in neonatal dairy calves over the first 10 days of age

Efficacy of passive transfer of immunity in young calves is commonly assessed using total serum protein (STP) or serum immunoglobulin G (IgG) concentration tested within the first few days of life. To our knowledge, no research has measured changes in these concentrations over this period to establish an appropriate age range for testing. The aim of this study was to monitor changes in STP and serum IgG concentrations from birth until 10 d of age to provide a basis for recommendations for when passive transfer of immunity in dairy calves can be measured. Concentrations of STP and IgG of 12 calves were measured at 11 time points: at approximately 30 min before colostrum feeding, at 24 h after colostrum feeding, and daily from d 2 to 10 of age. Mean (± standard deviation) STP and IgG concentrations were 4.61 ± 0.3 g/dL and 0.6 ± 0.6 mg/mL at birth, 5.83 ± 0.73 g/dL and 22.2 ± 9.6 mg/mL at 24 h after colostrum feeding, and 5.78 ± 0.52 g/dL and 16.1 ± 7.3 mg/mL at d 10 of age, respectively. The IgG concentration declined over subsequent days relative to IgG measured at 24 h at a rate of approximately 0.69 mg/mL per day, declining by 27.6 ± 6.2% (mean ± SD) on d 10. The concentration of STP did not decrease over time. Concentrations of IgG at 24 h after colostrum feeding were highly correlated with each of the measures of IgG over the 10-d period (r ≥0.97). These correlations were supported by the Bland-Altman plots of agreement between the 24-h sample and subsequent samples. Compared with the reference value at 24 h, STP concentrations were highly correlated on d 2 and 3 (r ≥0.98), highly correlated but variable from d 4 to 9 (r ≥0.88), and lower at d 10 (r = 0.76). These results indicate that calves may be reliably tested for passive transfer of immunity using IgG or STP concentrations up to 9 d of age.     

Immunoglobulin G content and colostrum composition of different goat and sheep breeds in Switzerland and Germany

Colostrum represents the sole source to acquire humoral immunity and is an important energy source for newborn lambs and goat kids. However, colostrum composition (i.e., the contents of IgG, fat, protein, and lactose) is affected by various factors such as parity and litter size and, potentially, by breed. In the present study, we examined the colostrum composition of different goat and sheep breeds raised for milk and meat production in Switzerland and Germany. Ten goat breeds (Anglo-Nubian, Appenzell, Boer, Bunte Deutsche Edelziege, Chamois-colored, Grisons Striped, Peacock, Saanen, Toggenburg, and Valais Blackneck) and 10 sheep breeds (Brown-Headed Meat, East Friesian Milk, German Blackheaded Mutton, Gray Horned Heath, Lacaune Dairy, Merino Land, Swiss Black-Brown Mountain, Swiss Charollais, Swiss White Alpine, and Valais Blacknose) were involved in this study. First colostrum samples were obtained from ewes (n = 100) and goats (n = 116) between 10 and 390 min after parturition and analyzed for total IgG, fat, protein, and lactose contents. Colostral IgG concentrations varied between 4.8 and 75.0 mg/mL in goats, and between 6.2 and 65.4 mg/mL in ewes, and the time interval between milking and parturition did not affect colostral IgG concentrations. In goats, the highest IgG concentrations were found in Boer (meat-type; 61.0 ± 10.3 mg/mL; mean ± SD) and the lowest concentrations were observed in Bunte Deutsche Edelziege (milk-type; 26.5 ± 12.5 mg/mL). In sheep, East Friesian Milk and Lacaune Dairy showed the lowest colostral IgG concentrations (17.9 ± 7.3 and 20.2 ± 8.0 mg/mL, respectively), and the highest values were observed in the Merino Land breed (44.2 ± 15.7 mg/mL). The lowest fat and protein concentrations and concomitantly highest lactose concentrations were observed in colostrum of East Friesian Milk and Lacaune Dairy sheep. Parity number did not affect colostrum composition in sheep or goats. In contrast, colostral fat content was higher in ewes bearing twins and triplets than in those carrying singletons. Increasing litter size tended to be associated with higher protein and lower lactose concentrations in ovine (i.e., singletons vs. twins vs. triplets) and caprine colostrum (i.e., singletons vs. twins), whereas colostral IgG concentrations were not affected by litter size. In conclusion, IgG and concentrations of other colostrum constituents showed a wide range in goats and ewes and were mainly affected by the type of breed.     

Effects of feeding heat-treated colostrum on passive transfer of immune and nutritional parameters in neonatal dairy calves

The first objective of this study was to describe the effect of on-farm heat treatment of colostrum on colostral bacteria counts and IgG concentrations. The second objective was to describe the effect of feeding heat-treated (vs. raw) colostrum on passive transfer of colostral immune and nutritional parameters in neonatal calves. Pooled batches of colostrum were mixed and divided equally: one half was fed raw whereas the other half was fed after heat treatment at 60°C for 60 min using a commercial on-farm batch pasteurizer. Colostrum samples were cultured for total bacteria count and total coliform count and analyzed for total IgG concentration. Forty-nine Holstein calves were fed either raw colostrum (n = 24) or heat-treated colostrums (n = 25) within 1 to 2 h after birth. Serum samples collected from calves at 0 h (precolostrum) and 24 h (postcolostrum) were assayed for serum total protein; IgG, IgA, and IgM concentrations; peripheral total leukocyte counts; neutrophil counts; lymphocyte counts; lymphocyte phenotypes; vitamin A, vitamin E, cholesterol, and β-carotene concentrations. Serum samples collected from 2- to 5-d-old calves were tested for immunoglobulin function via a bovine viral diarrhea virus type I serum neutralization titer and for neutrophil bacterial opsonization activity. On-farm batch heat treatment of colostrum at 60°C for 60 min resulted in lower colostrum bacteria concentrations while maintaining colostral IgG concentration. Calves fed heat-treated colostrum had significantly greater serum total protein and IgG concentrations at 24 h, plus greater apparent efficiency of IgG absorption (total protein = 6.3 mg/dL; IgG = 22.3 mg/mL; apparent efficiency of absorption = 35.6%) compared with calves fed raw colostrum (TP = 5.9 mg/dL; IgG = 18.1 mg/mL; apparent efficiency of absorption = 26.1%). There was no effect of treatment on serum concentrations of IgA, IgM, vitamin A, vitamin E, cholesterol, β-carotene or vitamin E:cholesterol ratio, or on serum bovine viral diarrhea virus type I serum neutralization titers. There was no difference between treatment groups when examining calf plasma total leukocyte counts, neutrophil counts, lymphocyte counts, or neutrophil opsonization activity. However, the latter results were considered inconclusive.