Use of monoclonal antibodies for radioimmunoassay of water buffalo milk progesterone

In order to standardize a radioimmunoassay of milk progesterone as a routine method for confirmation of oestrus and diagnosis of pregnancy in water buffalo, monoclonal antibodies against progesterone were produced. Hybridomas were prepared by fusing spleen cells from a Balb/c mouse immunized with progesterone 11 alpha-hemisuccinate-bovine serum albumin conjugate with the mouse myeloma cell line NS-1. Thirty wells out of 94 secreted anti-progesterone antibodies. Of the ten independent hybridomas derived, one (AF65) was suitable for the quantification of milk progesterone by radioimmunoassay. The tracer used in the assay was progesterone-11 alpha-hemisuccinate [( 2-125I]iodohistamine). The sensitivity of the assay was 50 pg/tube. The mean progesterone concentration at oestrus was 0.8 +/- 0.2 ng/ml increasing to 8.5 +/- 0.8 ng/ml 24 d later in pregnant animals.     

Nicotine metabolism in pregnant and nonpregnant rabbits.

Smoking during pregnancy remains a major public health concern and is associated with numerous adverse effects. Recently the clearance of nicotine and cotinine was shown to be substantially increased in pregnant women compared with nonpregnant controls. The present study investigated the usefulness of the rabbit for studying the molecular basis for the observed changes in nicotine and cotinine disposition during pregnancy. Nicotine was largely metabolized to cotinine in rabbit liver microsomes (approximately 50% of total metabolism); significant amounts of nicotine-N'-oxide and nornicotine also were detected. The conversion of nicotine to cotinine also was detected in rabbit placental and fetal liver microsomes, albeit at only a fraction of the rate found in adult rabbit liver microsomes. The major products of cotinine metabolism in rabbit liver microsomes were 5'-hydroxycotinine, cotinine-N'-oxide, and norcotinine. Differences between human and rabbit liver were most apparent for cotinine. The major human metabolite, 3'-hydroxycotinine, was formed at only low levels in rabbit liver microsomes. Pregnancy had no effect on the metabolism of nicotine or on the expression of CYP2A6 immunoreactive proteins in rabbit liver microsomes. These studies provide a complete quantitative assessment of nicotine metabolism in rabbit liver microsomes and suggest that the rabbit may not be an appropriate animal model to study the effects of pregnancy on nicotine and cotinine metabolism. However, a molecular understanding of these effects is essential for prediction of the pharmacological and toxicological consequences of smoking during pregnancy.     

Hyperketonemia impairs glucose metabolism in pregnant and nonpregnant ewes

The present study was undertaken to test the hypothesis that high ketone body concentrations suppress endogenous production of glucose and in pregnant sheep facilitate development of pregnancy toxemia. Rates of endogenous glucose production [mmol.min(-1)], and rate constants of glucose turnover [min(-1)] were measured in seven 12-h fasted sheep in the presence of normo- and hyperketonemia by use of D-2-[(3)H]-glucose. The measurements were carried out in the same sheep during the nonpregnant nonlactating state, during late pregnancy (10 +/- 7 d antepartum) and during lactation (19 +/- 6 d postpartum). Hyperketonemia (5 to 7 mmol.L(-1)), similar to that present in spontaneous ovine pregnancy toxemia, was induced by continuous intravenous 4-h infusions of DL-beta-hydroxybutyrate (DL-BHB). Glucose turnover [mmol.min(-1)] in the same 7 nonpregnant nonlactating, late pregnant, and lactating sheep was significantly greater during normoketonemia (0.80, 1.16, 1.76) than during hyperketonemia (0.66, 0.92, 1.16, respectively). The rate constants of glucose turnover were not altered by elevation of the BHB concentration. The results demonstrated that high BHB concentrations significantly suppressed endogenous glucose production but showed no effect on glucose utilization. The suppressive effect of hyperketonemia on hepatic glucose production resulted in a significant reduction of plasma glucose concentration and was qualitatively the same in all three reproductive states. The results indicate that hyperketonemia, which is regularly present in late twin pregnant hypoglycemic sheep contributes significantly to the reduction of available glucose. This effect of hyperketonemia may invoke sustained hypoglycemia and may render the ewe into a vicious cycle that probably makes the animal refractory to treatment in most cases.     

水牛奶乳制品深加工的研究进展

简单介绍了国内外奶水牛业的发展,详细展望了水牛奶深加工的产品,对发展奶水牛业和提升水牛奶的附加值具有一定的指导作用。     

水牛乳蛋白质的组成

分析了摩拉水牛（M）、尼里-拉菲水牛（N）、一代杂交水牛（F1）、二代杂交水牛（F2）和高代杂交水牛（Fh）5个品代水牛的乳蛋白主要组分的相对百分比含量．同时分析了总氨基酸组成及钙、磷含量。结果表明，水牛乳蛋白的主要组分有：α-乳清蛋白（α-LA）、β-乳球蛋白（β-LG）、免疫球蛋白轻链（IgG—L）和重链（IgG—H）、αs1-酪蛋白（αs1-CN）、αs2-酪蛋白（αs2-CN），β-酪蛋白（β-CN）、κ-酪蛋白（κ—CN）、血清白蛋白（SA）和乳铁蛋白（LF）等；CN在水牛乳蛋白中占优势，与荷斯坦牛乳相比，水牛乳中CN的质量分数稍低，而且各品代水牛乳中的CN有显著性差异（P〈0．05）；乳清蛋白中β-LG含量最高；杂交水牛乳蛋白高于纯种摩拉水牛和尼里一拉菲水牛，差异显著（P〈0．05）：各品代水牛乳的氨基酸比例比较接近；不同品代水牛乳中钙、磷含量没有显著性差异。     

广西水牛奶与荷斯坦牛奶工艺学特性比较

为了探索、比较广西水牛奶与荷斯坦牛奶的工艺学特性,对二者的缓冲容量、表面张力、粒度分布、稳定系数进行了比较分析。结果表明,广西水牛奶的缓冲容量和粒径分别是荷斯坦牛奶的1.1倍和2倍。在研究范围内,广西水牛奶在pH值为6.8时缓冲容量最大,温度50℃时表面张力最小,pH值为4.7时稳定系数最小;而荷斯坦牛奶在pH值为6.6时缓冲容量最大,温度为50℃时表面张力最小,pH值为4.6时稳定系数最小。广西水牛奶的粒径分布在91.28~458.7 nm之间,荷斯坦牛奶的粒径主要分布为91.28~190.1 nm之间,因此,认为两种牛奶的工艺学参数存在多方面的差异。     

Relationship between cellular and whey components in buffalo milk

High somatic cell count (SCC) affects milk quality and cheesemaking, resulting in a reduction in cheese yield and quality. In dairy cows, quarter milk samples with > 200,000 cells/ml are considered to have subclinical mastitis, while there is much uncertainty on the corresponding levels of SCC in buffalo milk. In this study 30 lactating water buffaloes were selected and SCC, differential somatic cell counts and several whey components were tested in quarter milk samples to assess the relationship between inflammation markers and milk quality. Overall 236 quarter milk samples were considered. To evaluate the relationship between cellular markers (SCC, polymorphonuclear leucocytes, PMN, and N-Acetyl-beta-glucosaminidase, NAGase) and other milk components, three classes were defined (low, medium and high). Analysis of milk yield showed a significant reduction in the high class of each of the three markers chosen. Overall, the highest class was characterized by significant changes in milk composition and a lower milk quality. The presence of an inflammatory status of the udder was frequent after the first trimester of lactation and in buffaloes with two or more parturitions. This study showed that significant changes in milk components can be observed when SCC are > 400,000 cells/ml, PMN are > 50% and NAGase is > 100 units. These thresholds could be suggested as levels to define udder health status in buffalo cows.     

PCR detection of cows’ milk in water buffalo milk and mozzarella cheese

Polymerase chain reaction (PCR) has been applied for the specific detection of cows’ DNA in water buffalo milk and mozzarella cheese by using primers targeting the mitochondrial 12S ribosomal RNA gene. The use of specific primers for cow yielded a 346 bp fragment from cows’ milk DNA, whereas no amplification signal was obtained in sheep's, goats’ and water buffalo's milk DNA. Analysis of both buffalo milk and buffalo mozzarella cheese mixtures containing different percentages of cows’ milk or bovine mozzarella cheese, enabled the specific detection of cow's DNA with a sensitivity threshold of 0.1%. The proposed PCR assay represents a rapid and straightforward method for the detection of adulterations in water buffalo milk and mozzarella cheese.     

Follicular dynamics and corpus luteum growth and function in pregnant versus nonpregnant cows

The effects of pregnancy on follicular dynamics and corpus luteum growth and function are somewhat contentious. In an effort to learn more about the effects of pregnancy, the reproductive tracts of 16 bred dairy cows were monitored using an ultrasound device with a rectal probe through the first 60 d of pregnancy or upon a return to estrus. Additionally, blood samples were collected for progesterone determination. Eleven of 16 cows were diagnosed pregnant by 25 d postbreeding. There were two embryonic mortalities between 28 and 32 d. A two-wave pattern of follicular growth and atresia, each wave resulting in a large dominant follicle, was seen in both pregnant and nonpregnant cows during the first 24 d postbreeding. Pregnant cows had more follicles than nonpregnant cows; however, there was no difference in the size of the largest follicle. A wave-like pattern of growth of large follicles continued throughout the study period in pregnant cows. Progesterone profiles, corpus luteum growth rate, and maximum size of the corpus luteum were similar in both pregnant and nonpregnant cows. It is concluded that growth of dominant follicles and of the corpus luteum is unaffected during the first 60 d of pregnancy.     

Regulation of the prostaglandin enzymatic system by estradiol and progesterone in nonpregnant sheep cervix

In the present study, we examined the in vivo effects of estradiol (E(2)) and progesterone on cyclooxygenase (COX) 2, prostaglandin F synthase (PTGFS, also known as PGFS), and membrane-associated prostaglandin E synthase 1 (mPTGES1) expression at both mRNA and protein levels using a nonpregnant ovariectomized (OVX) sheep model. Sixteen ewes were OVX shortly after ovulation. After 40 days, ewes were treated with saline (Cont, n=5), or E(2) infused intravenously for 2 days (50 microg/day, n=5) or intravaginal progesterone (P) sponges for 10 days (0.3 g P, n=6). Cervical COX2, PTGFS, and mPTGES1 mRNA and protein were quantified by northern and western blot analyses respectively. In situ hybridization and/or immunocytochemistry were used to localize the cellular distribution of COX2, PTGFS, and mPTGES1 mRNAs and proteins. COX2 mRNA abundance increased significantly in the cervix after E(2) treatment (P<0.05). However, progesterone was a more potent stimulator than E(2) of COX2 mRNA and protein abundance in the cervix (P<0.01). In contrast, PTGFS and mPTGES1 mRNA and protein concentrations did not change after E(2) or progesterone treatment (P>0.05). COX2, PTGFS, and mPTGES1 mRNA and protein were only localized in cervical glandular epithelial cells. This study shows that increased cervical COX2 mRNA and protein, but not PTGFS and mPTGES1 mRNA and protein, were associated with E(2) and progesterone treatment in nonpregnant sheep. More strikingly, progesterone was a more potent stimulator of cervical COX2 expression than E(2). The expression of COX2, PTGFS, and mPTGES1 mRNA and/or protein was confined in the cervical glandular epithelial cells of nonpregnant sheep.     

利用雅致放射毛霉制备水牛乳豆乳混合干酪工艺参数的优化

为提高水牛乳豆乳混合干酪品质,分别利用单因素试验及正交试验对以雅致放射毛霉为表面发酵剂制备水牛乳豆乳混合发酵干酪的工艺进行了优化。通过优化确定了水牛乳豆乳混合干酪的最佳发酵条件为豆乳添加量15%,程序升温至41℃,霉菌孢子喷雾浓度1×10^6 C FU /m L,成熟时间为30 d。在此优化的工艺条件下,能得到较好品质的霉菌干酪产品。     

Genetic parameters for buffalo milk yield and milk quality traits using Bayesian inference

The availability of accurate genetic parameters for important economic traits in milking buffaloes is critical for implementation of a genetic evaluation program. In the present study, heritabilities and genetic correlations for fat (FY305), protein (PY305), and milk (MY305) yields, milk fat (%F) and protein (%P) percentages, and SCS were estimated using Bayesian methodology. A total of 4,907 lactations from 1,985 cows were used. The (co)variance components were estimated using multiple-trait analysis by Bayesian inference method, applying an animal model, through Gibbs sampling. The model included the fixed effects of contemporary groups (herd-year and calving season), number of milking (2 levels), and age of cow at calving as (co)variable (quadratic and linear effect). The additive genetic, permanent environmental, and residual effects were included as random effects in the model. The posterior means of heritability distributions for MY305, FY305, PY305, %F, P%, and SCS were 0.22, 0.21, 0.23, 0.33, 0.39, and 0.26, respectively. The genetic correlation estimates ranged from −0.13 (between %P and SCS) to 0.94 (between MY305 and PY305). The permanent environmental correlation estimates ranged from −0.38 (between MY305 and %P) to 0.97 (between MY305 and PY305). Residual and phenotypic correlation estimates ranged from −0.26 (between PY305 and SCS) to 0.97 (between MY305 and PY305) and from −0.26 (between MY305 and SCS) to 0.97 (between MY305 and PY305), respectively. Milk yield, milk components, and milk somatic cells counts have enough genetic variation for selection purposes. The genetic correlation estimates suggest that milk components and milk somatic cell counts would be only slightly affected if increasing milk yield were the selection goal. Selecting to increase FY305 or PY305 will also increase MY305, %P, and %F.     

广西水牛原料乳微生物和理化指标分析

按不同种群从广西取80个水牛原料乳样品，通过理化和微生物指标检测，对水牛乳营养价值和安全性进行了分析比较．主要理化检测结果如下：脂肪7．32％，蛋白质4．71％，乳糖4．67％。固形物18．42％，灰分0．84％（均为质量分数），pH值为652，密度1.030g／mL。纯种河流型水牛摩拉和尼里-拉菲的脂肪、蛋白质、固形物、灰分舍量显著低于（P〈0.05）河流-沼泽型杂交水牛。微生物检测结果如下（对数值）：细菌总数5．69mL^-1，茅孢菌3．03mL^-1，乳酸菌4．79mL^-1，真菌1．80mL^-1，大肠菌群2．48mL^-1，大肠杆菌1．83mL^-1，金黄色葡萄球菌1.80mL^-1，所有样品中李斯特氏苗均未检出。地区间微生物指标差异较大。微生物含量均在可接受范围之内，但是致病菌的出现说明需要建立相关的卫生质量标准.     

Ex vivo digestion of proteins and fat in buffalo milk

Ex vivo digestion of proteins and fat in whole buffalo milk (WBM) and skimmed buffalo milk (SBM) was studied. Proteolysis, generation of peptides, lipolysis and generation of fatty acids were monitored. In both types of milk, β- and κ-caseins were hydrolysed almost completely after 20 min of gastric digestion. Residual α_S-caseins were digested completely on 5 min of duodenal digestion. Of the main whey proteins, α-lactalbumin was only partially hydrolysed and β-lactoglobulin seemed to be resistant during 40 min of gastric digestion. However, by 5 min of duodenal digestion, both α-lactalbumin and β-lactoglobulin were fully hydrolysed in both WBM and SBM. Only small variations between WBM and SBM were found for the release of peptides. In the WBM, 30% lipolysis was observed after 30 min of duodenal digestion. Different extents of lipolysis of fatty acids among/within different groups, based on carbon number and double bond(s), were observed.     

Detection of buffalo milk adulteration with cow milk by capillary electrophoresis analysis

The addition of cow milk during the production of buffalo mozzarella is a common fraud in dairy industries because of the lower price and greater availability of cow milk throughout the year. The aim of this study was to develop a new, rapid, and robust capillary electrophoresis method for detecting and quantifying cow milk in buffalo milk by exploiting cow α-lactalbumin as a marker of adulteration. In particular, a linear calibration curve was generated, using a training set of calibrators consisting of 7 series of 17 buffalo/bovine whey mixtures, obtained after casein precipitation, with increasing percentages of cow whey. The capillary electrophoresis method showed high linearity (R² = 0.968), repeatability [relative standard deviation (RSD) = 2.11, 3.02, 4.38, and 1.18%, respectively for 5, 10, 20, and 50% of buffalo/bovine whey mixtures], and intermediate precision (RSD = 2.18, 2.49, 5.09, and 3.19%, respectively, for 5, 10, 20, and 50% buffalo/bovine whey mixtures). Moreover, the minimum amount of detectable fraudulent cow milk was 1%, and the limit of quantification was 3.1%.     

Concentrations of progesterone in milk of cows administered an intravaginal progesterone insert

Milk from pregnant cows contains concentrations of progesterone (P4) considered safe for human consumption. The objective of this study was to determine if concentrations of P4 in milk during administration of an intravaginal progesterone insert (CIDR insert) are less than concentrations of P4 in milk associated with pregnancy. Results have implications for human use of milk from cows receiving CIDR inserts. Holstein cows (N = 64; > 40 and < 150 d after calving) were administered 25 mg of PGF2alpha i.m. (study d 0) and 20 cows detected in estrus from 2 to 4 d later were assigned randomly to either control (N = 10; no further treatment) or CIDR insert (N = 10; 1.38 g of P4) inserted on study d 17 (14 +/- 1 d after estrus) and removed 7 d later. Composite milk samples were collected contemporaneously from each of the 20 estrous cycling cows and from 10 pregnant cows (> or = 60 and < or = 220 d of gestation) twice daily from study d 17 to 27. Concentrations of P4 in defatted milk samples were quantified using a validated radioimmunoassay. Mean logs of areas under the curve of concentrations of P4 from the afternoon on study d 17 through the afternoon on study d 27 were 3.05 ng day/ml for control, 3.33 ng day/ml for CIDR insert, and 3.81 ng day/ml for pregnant cows. Therefore, increased P4 due to pregnancy was 0.76 ng day/ml (3.81-3.05), whereas the increase in P4 due to CIDR insert was only 0.28 ng day/ml (3.33-3.05). Applying a 95% confidence interval to 0.28 ng day/ml provided an upper value of 0.70 ng day/ml, lower than the increase due to pregnancy. Because milk from pregnant cows is considered safe for human consumption, it follows that milk from cows administered CIDR inserts should also be considered safe, based on concentrations of P4.     

量化筛选中国水牛奶生产Mozzarella干酪所需发酵剂

为了获得水牛奶Mozzarella干酪生产发酵剂所需菌种及其配比和用量的最佳方案,以自行筛选的保加利亚乳杆菌、嗜热链球菌、乳酸乳球菌、干酪乳杆菌为素材进行研究.通过检测所得水牛奶Mozzarella干酪样品的10项质量参数,对参数统计处理分析得到量化筛选结果,发现同时选用4个菌种、以相同的比例和3.0％的总添加量作为发酵剂制作的中国水牛奶Mozzarella干酪品质较优.     

水牛奶乳清蛋白的热稳定性

以水牛奶为材料,利用native-PAGE和SDS-PAGE,通过不同温度和加热条件下总乳清蛋白和单体蛋白质量浓度的变化,研究水牛奶乳清蛋白的热稳定性。结果表明:水牛奶乳清蛋白在native-PAGE条件下只有β-LG一条条带,在SDS-PAGE条件下得到4条单体乳清蛋白条带;随着提取温度和时间的增加,水牛奶单体乳清蛋白和总乳清蛋白质量浓度均呈下降趋势,说明水牛奶乳清蛋白热稳定性较差;4种乳清单体蛋白中,α-LA的热稳定性最好,IG的热稳定性最差,热稳定性顺序为:α-LAβ-LGBSAIG。     

Effect of parity, days in milk, and milk yield on detailed milk protein composition in Mediterranean water buffalo

The effects of some nongenetic factors on milk protein fraction contents and relative proportions were estimated in 606 individual milk samples of Mediterranean water buffalo. Content of α(S1)-casein (CN), α(S2)-CN, β-CN, γ-CN, κκ-CN, glycosylated κ-CN (glyco-κ-CN), α-lactalbumin, and β-lactoglobulin was measured by reversed-phase HPLC. Relative contents of α(S1)-CN%, α(S2)-CN%, β-CN%, and κ-CN% were, respectively, 32.1, 17.1, 34.5, and 15.7%, whereas γ-CN% accounted for 0.6% of total casein content. Increasing total casein content in milk would result in a greater proportion of β-CN% at the expense of all of the other major casein fractions, especially of κ-CN%. Values of α(S2)-CN%, β-CN%, and γ-CN% tended to decrease with parity, although their variations were not significant, whereas α(S1)-CN% and glyco-κ-CN% showed the opposite trend. Contents of most protein fractions showed the typical trends observed for milk components as lactation progressed, with high contents in early lactation, a minimum in midlactation, followed by a gradual increase toward the latter part of lactation. Values of α(S1)-CN% increased during lactation, whereas α(S2)-CN% decreased. The proportion of β-CN% had its maximum value between 60 and 160 d of lactation, followed by a decrease, whereas κ-CN% had its minimum value in early lactation (<60 d) and remained relatively constant in the period of mid and late lactation. Glyco-κ-CN% and β-lactoglobulin% decreased in the first part of lactation, to reach their minimum values in midlactation, followed by an increase. Milk of top-producing buffaloes, compared with that of low-producing ones, had a significantly greater value of β-CN% and glyco-κ-CN%, and lower proportion of α(S1)-CN%. The possible effect exerted by protein genetic variants in affecting variation of milk protein fraction contents and relative proportions should be further considered to better get insight into buffalo milk protein composition.     

广西水牛奶和荷斯坦牛奶中蛋白质和氨基酸的含量及组成分析

目的研究广西区内生水牛奶和荷斯坦生牛奶中蛋白质、氨基酸含量及其组成特性,分析水牛奶和荷斯坦牛奶的差异。方法采用盐酸水解法处理牛奶样品,通过氨基酸自动分析仪测定氨基酸含量,分析水牛奶和荷斯坦牛奶氨基酸的含量和组成;通过凯氏定氮法测定牛奶中蛋白质含量。结果 18份牛奶样品中蛋白质、氨基酸的测定结果表明,生水牛奶的蛋白质和氨基酸含量均高于荷斯坦生牛奶,而水牛奶和荷斯坦牛奶中各氨基酸含量占氨基酸总量的比值相近。结论该方法为广西生水牛奶和荷斯坦生牛奶的鉴别提供参考。