Development and validation of a sensitive enzyme immunoassay (EIA) for blood plasma cortisol in female cattle,buffaloes, and goats

A highly sensitive enzyme immunoassay (EIA) that used the second antibody coating technique and the cortisol-horseradish peroxidase conjugate as a label for determination of free and total cortisol in blood plasma of dairy animals (cows, buffaloes, and goats) was developed. For biological validation of the EIA, blood samples were collected from the animals at 48 and 24 h before and 0, 12, 24, 36, 48, 60, 72, 84, 96, 108, 120, and 132 h after dexamethasone administration. The EIA was performed directly with 20 μL of fresh plasma (for free cortisol) and also with 20 μL of heat-treated plasma (for total cortisol) after 1:5 dilutions with PBS. Cortisol standards ranging from 0.39 to 200 pg/well/20 μL were used, and the sensitivity of the EIA procedure was found to be 0.39 pg/well/20 μL, which corresponded to 0.02 ng/mL. In comparison with RIA the EIA was at least 4 times more sensitive and required 5 times less cortisol antiserum. In female cattle, buffaloes, and goats, the total, free, and bound plasma cortisol before dexamethasone administration was significantly (P < 0.05) higher than the total, free, and bound cortisol after dexamethasone administration. It can be concluded from these studies that the direct, sensitive EIA validated for estimating the free and total cortisol concentrations was sufficiently reliable and quick for studying the dynamics of cortisol distribution in blood plasma of dairy animals.     

Comparison of 2 enzyme immunoassays and a radioimmunoassay for measurement of progesterone concentrations in bovine plasma, skim milk, and whole milk

The objective of this study was to compare 2 enzyme immunoassays (EIAs) with a radioimmunoassay (RIA) as to sensitivity and accuracy in the measurement of the progesterone (P4) concentration in bovine plasma, skim milk, and whole milk. The 72 samples from 24 lactating dairy cows expected to have either a high P4 concentration (cows in diestrus or pregnant) or a low P4 concentration (cows in estrus or anestrus) were analyzed by RIA, solid-phase EIA (SPEIA), which included a solvent extraction step, or direct EIA (DEIA) without solvent extraction. The overall mean concentrations of P4 did not differ (P < 0.4) among the assays. However, for the cows that were in diestrus or pregnant, the mean P4 concentrations (and standard error) were higher (P < 0.03), regardless of sample type, with RIA than with SPEIA, at 7.3 (0.7) and 6.1 (0.6) ng/mL, respectively. When only the high-P4 samples analyzed by RIA were compared, the mean P4 concentration was higher (P < 0.001) in whole milk than in skim milk, at 9.8 (1.0) and 4.1 (0.7) ng/mL, respectively. Although the mean P4 concentrations in the low-P4 samples did not differ (P < 0.80) among assays, the proportions of cows with a P4 concentration > or = 1 ng/mL were 3%, 14%, and 44% for RIA, SPEIA, and DEIA, respectively (P < 0.01; DEIA > SPEIA > RIA).     

Accuracy of pregnancy/non pregnancy diagnosis in zebu and crossbred cattle and Murrah buffaloes by milk progesterone determination post insemination

Tropical Animal Health and Production - Milk samples collected on days 0, 6, 20, 22 and 24 post insemination from Sahiwal and Tharparkar (zebu) cows, Karan Swiss (Sahiwal × Brown Swiss) and...     

Validation of a direct time-resolved fluoroimmunoassay for progesterone in milk from dairy and beef cows

The aim of this study was to validate a direct time-resolved fluoroimmunoassay (TR-FIA) for quantifying progesterone concentrations in milk during the bovine oestrous cycle. Holstein-Friesian and suckled and non-suckled Japanese Black cows were used to demonstrate the relationship between milk and plasma progesterone concentrations and to monitor progesterone profiles in milk and plasma during the oestrous cycle. The minimum detection level of the assay was 1.53ng/mL. Progesterone concentrations in milk and plasma changed in a similar manner throughout the oestrous cycle in dairy and beef cows, and milk and plasma progesterone profiles were significantly correlated (P<0.001). The study confirmed that a direct TR-FIA can be used to monitor the oestrous cycle in cattle and to quantify progesterone concentrations in whole milk.     

Direct measurement of progesterone in plasma and milk by a simple solid-phase radioimmunoassay

A rapid method for determination of progesterone in bovine, goat and porcine plasma as well as in bovine milk was evaluated. The method employed was a solid-phase (125)I radioimmunoassay equipped with progesterone standards in human serum and called the Coat-A-Count procedure. The dilution curves of bovine plasma samples with high progesterone content were parallel with the standard curve based on human serum. The relation between measurements of progesterone levels in bovine plasma using the reference extraction method and the direct Coat-A-Count procedure was highly significant, resulting in the linear regression equation Y = 1.06x-0.04. In case of goat and porcine plasma, the direct method yielded higher results than the reference extraction method (Y = 1.37x + 1.38 and Y = 1.69x - 6.47, respectively). Progesterone concentration in bovine whole milk was much higher when measured by the Coat-A-Count procedure than by the reference Farmos kit (Y = 1.59x + 1.51). However, when the same samples were assayed by a modified Coat-A- Count procedure, i.e. progesterone standards from Farmos kit, the values were more or less identical (Y = 0.88x - 0.21).     

牛奶中孕酮直接竞争ELISA检测方法的建立

采用直接竞争ELISA方法检测牛奶中孕酮含量.采用碳化二亚胺法,应用辣根过氧化物酶(HRP)标记11-α羟基孕酮琥珀酸酯(11α-OH-P4-HS),制备孕酮酶标抗原;酶标抗原与牛奶样品中的孕酮共同竞争结合固相包被的孕酮单克隆抗体,建立直接竞争ELISA反应体系.试验结果表明,最佳抗体包被浓度为1∶2 000,最佳酶标抗原工作浓度为1∶16 000,建立的标准曲线为y=-2.4598x+0.999(R2=0.996),牛奶中孕酮检测范围0.12～40 ng/mL,最低检测量为0.12 ng/mL,批内和批间变异系数分别为1.63%和2.49%.成功建立了可用于牛乳中孕酮快速检测的直接竞争ELISA方法.     

Validation of a human progesterone enzyme immunoassay (EIA) kit for use on serum of cattle in Cameroon

A study aimed at validating a human progesterone enzyme immunoassay kit was carried out on cattle at Bambui, Cameroon. Progesterone ELISA Kits (EH-511) were obtained from Clinpro International. Forty-one cows were selected, of which 19 were pregnant and 22 within 14 days post partum. Blood samples were analysed and progesterone levels were deduced from a curve obtained from standard absorbance values (A ₄₅₀). Results show that 95.5% of postpartum cows had progesterone levels below 1 ng/ml, with the highest level being 0.75 ng/ml. The mean level was 0.5 ± 0.26 ng/ml. The cows in the ‘pregnant group’ had progesterone levels ranging from 3.5 to 17.5 ng/ml. This kit can be used for measuring progesterone levels in cattle. Levels of 1 ng/ml for two consecutive samples or one sample at or above 3 ng/ml are an indication of the presence of corpus luteum, while cows below 1 ng/ml will be in anoestrus.     

Plasma progesterone concentration and reproductive function relative to thyroid activity in postpartum buffaloes (Bubalus bubalis)

The lowest value for plasma protein bound iodine (PBI) (3.47 g%) in 20 freshly calved Murrah buffaloes was observed on day 1 postpartum. The values then increased consistently up to day 36 and were almost constant thereafter. The plasma progesterone concentrations were lower in animals with relatively low PBI from day 22 to 57 postpartum, except on day 36. The mean values ± SE (ng/ml) in the high and low PBI groups were 0.93±0.09 and 0.72±0.15. The correlation coefficient between PBI and progesterone was 0.80 (p<0.01). Relatively poor reproductive efficiency, in terms of a higher initiation of follicular development interval, postpartum oestrus interval, service period and number of services for conception, was observed in animals in the low PBI group.     

Milk progesterone enzyme-linked immunosorbent assay as a tool to investigate ovarian cyclicity of water buffaloes in relation to body condition score and milk production

Background

Application of assisted reproductive technologies in buffaloes is limited to some extent by farmers’ inability to detect oestrus because of its poor expression. The present study aimed at investigating reliability of a milk progesterone enzyme-linked immunosorbent assay (ELISA) to assess the ovarian cyclicity during post partum, oestrus and post-breeding periods in water buffaloes.

Methods

Progesterone concentrations were measured by an ELISA in milk of 23 postpartum buffaloes in relation to oestrus, pregnancy, body condition score (BCS) and milk production. Two milk samples were taken at 10 days intervals, every month starting from day 30 and continued to day 150 post partum. BCS and milk production were recorded during sample collection. Milk samples from bred buffaloes were collected at Day 0 (day of breeding), Days 10–12 and Days 22–24. Defatted milk was preserved at −80°C until analysis. Pregnancy was confirmed by palpation per rectum on Days 70–90.

Results

Seventeen buffaloes had 47 ovulatory cycles, one to four in each, 13 were detected in oestrus once (28 % oestrus detection rate). Progesterone concentration ≥1 ng/ml in one of the two 10-day-interval milk samples reflected ovulation and corpus luteum formation. The intervals between calving to first luteal activity and to first detected oestrus varied from 41 to 123 days (n = 17) and 83 to 135 (n = 13) days, respectively. Eight buffaloes were bred in the course of the study and seven were found pregnant. These buffaloes had a progesterone profile of low (<1 ng/ml), high (≥ 1 ng/ml) and high (≥ 1 ng/ml) on Day 0, Days 10–12 and Days 22–24, respectively. Buffaloes cycling later in the postpartum period had fewer missed oestruses (P < 0.05). Buffaloes with a superior BCS had a shorter calving to oestrus interval and produced more milk (P < 0.05).

Conclusions

Milk progesterone ELISA is a reliable tool for monitoring ovarian cyclicity and good BCS may be an indicator of resuming cyclicity in water buffalo.     

Evaluation of oestrus observation and conception rates in suckling beef cows using whole milk progesterone concentration

A 2-sample regime was used to measure whole milk progesterone concentration on the day of oestrus and insemination (Day 0) and 6 days later (Day 6) in a sample of 50 primiparous and 100 multiparous suckling beef cows. Exposure to teaser bulls and observation by cattlemen identified the occurrence of oestrus. Three sets of criteria used to define ovulatory oestrus were compared: a) milk progesterone concentration less than 6 nmol/l on Day 0; b) milk progesterone less than 6 nmol/l on Day 0 and rising to greater than 6 nmol/l on Day 6; c) milk progesterone less than 6 nmol/l on Day 0 and rising to greater than 6 nmol/l on Day 6, or cow diagnosed pregnant to 1st insemination. Using only a single milk sample on Day 0 (criterion a) would have resulted in the positive predictive value of heat detection being estimated at 98.7%. Using a pair ed measurement (criterion b) resulted in a significantly lower estimate of 84.7%. The inclusion of cows that conceived despite not showing a marked rise in milk progesterone concentration (criterion c) resulted in a more accurate estimate of 89.3 %. Use of a 2-sample regime also allowed calculation of conception rates while eliminating the effect of heat detection errors. In the cows sampled, of those in ovulatory oestrus that were inseminated, 73.1% conceived to the 1st insemination. These results demonstrate that artificial insemination within a limited breeding season can be successful if nutrition is optimal and management is intensive. The use of a 2-sample milk progesterone test may be a valuable tool in investigating heat detection and conception problems in beef herds in which artificial insemination is used.     

Relationship between rectal findings of corpus luteum and whole milk progesterone levels in postpartum dairy cows

The reliability of clinical ovarian findings was assessed as an indicator of luteal function in primiparous dairy cows. The postpartum period of 103 cows following their first parturition was studied by thrice weekly rectal palpation of ovaries and whole milk progesterone assay from 1 week after parturition to the first insemination. The relationship between milk progesterone levels and 1101 ovarian findings was compared during the follicular phases, short luteal phases and during the early, mid and late thirds of normal luteal phases. The compatibility between elevated progesterone and palpable corpus luteum was 71%, and between low progesterone and lack of corpus luteum 77%. In 10% of all rectal examinations the finding was unspecified; i.e. the clinician could not differentiate between luteal and follicular activity. During the acyclic period prior to the initiation of luteal function, the proportion of false corpus luteum findings was 11%. The corpora lutea of the short oestrous cycles were more difficult to palpate than those of normal cycles. During early dioestrus the corpus luteum was significantly more difficult to palpate than during the rest of dioestrus. The percentage of unspecified findings was highest during early dioestrus.The paper discusses the reliability of rectal examination as a method of diagnosing cyclicity and of evaluating the responsiveness of a cow to prostaglandin treatment.     

Pharmacokinetics and distribution of sulphadimidine in plasma,milk and uterine fluid of female buffaloes

The pharmacokinetics of sulphadimidine after a single dose (200 mg/kg i.v.) was studied in five healthy lactating buffaloes. The study revealed that the drug attained its peak concentration of 314.0±13.0, 242.4±3.0 and 100.2±2.5 g/ml at 15 min, 30 min and 12 h in plasma, milk and uterine fluid, respectively. The pharmacokinetic parameters calculated by a 2-compartment open model gave values for t₁, t₁ and vd_area of 2.10±0.36 h, 12.36±0.57 h and 1.23±0.07 L/Kg, respectively. A high vd_area as well as a value of 0.74±0.08 for K₁₂:K₂₁- (tissue Plasma) indicates better penetration of the drug into the different body fluids and tissues, which is further supported by a high concentration obtained in milk and uterine fluid. The therapeutic concentration (50 g/ml) was maintained for around 24 h in plasma and milk and 12 h in uterine fluid. The results suggest that, apart from its use in systemic infections, the drug can be effectively used by the i.v. route in uterine and mammary gland infections. The dosages for maintaining concentration of 50 g/ml, 100 g/ml and 150 g/ml at convenient dosage intervals of 12 and 24 h were also determined.     

Development of a diagnostic gene expression assay for tuberculosis and its use under field conditions in African buffaloes (Syncerus caffer)

The development of diagnostic tests for tuberculosis (TB) in exotic species is constrained by host biology and the limited availability of suitable assay reagents. As such, we evaluated a gene expression assay (GEA) which is easily modified for novel species and allows for initial sample processing under field conditions. African buffaloes (Syncerus caffer) were categorized using the single comparative intradermal tuberculin test, and blood from test-positive and test-negative animals was incubated for 20h in "Nil" tubes (containing saline) and "TB Antigen" tubes (containing Mycobacterium tuberculosis complex (MTC)-specific antigens) of a commercial human TB test, the QuantiFERON(?)-TB Gold (In-Tube) (QFT) assay. Blood samples were then stabilized in RNAlater(?) and transported to the laboratory for RNA extraction. A Custom TaqMan GEA was used to calculate the relative abundance of interferon-gamma (IFN-γ) mRNA in the TB Antigen tube compared to that in the Nil tube as a marker of immune activation in response to MTC antigen recognition. The GEA results from the two buffalo groups were compared and a cutoff value of 2.85 was calculated to differentiate between animals from these groups with a sensitivity of 80% (95% C.I.: 56-94%) and a specificity of 95% (95% C.I.: 75-100%). Further optimization of this assay could provide a highly useful tool for the diagnosis of MTC infection in exotic species.     

Evidence for different types of seasonal anoestrus in the dairy goat as revealed by progesterone determination in milk fat

Progesterone profiles were measured in milk fat of 12 goats (Deutsche Bunte Edelziege) from kidding until a new pregnancy. The goats were kept under natural light conditions without bucks. Eight of the does started to cycle from August until October without a progesterone rise in the preceding period. Three does exhibited one single luteal phase in March and resumed cycling in July and August. The occurrence of a normal corpus luteum was typically preceded by a transient progesterone “spike” in 10 out of the 12 goats. One goat additionally showed several “spikes” in June and July; normal luteal function, however, started in September. With one exception oestrus symptoms were never observed without a preceding progesterone rise.     

检测奶牛乳汁孕酮的免疫生物传感器优化

为了完善已建立的孕酮免疫生物传感器的检测性能,本试验应用碳化二亚胺法将11α-羟基孕酮半琥珀酸和OVA制备成孕酮完全抗原,其浓度达0.6 mg/m L。在此基础上建立了以乳汁为基质的孕酮生物传感器的标准曲线(y=-4.9461x+8.7982,R2=0.993 2),进一步确立了检测乳汁孕酮的线性范围为0.31 ng/m L~50ng/m L,检测限为0.31 ng/m L,灵敏度为0.15 ng/m L,而且孕酮与皮质酮、皮质醇、雄烯二酮的免疫交叉反应较低,表明特异性较好。批内、批间变异系数分别为7.7%和7.5%,回收率为95.8%~115.7%,生物传感器稳定性在4℃保存4 d。该优化的奶牛乳汁孕酮免疫生物传感器的性能参数符合规定的标准,为检测妊娠、乏情和繁殖障碍疾病的奶牛乳汁孕酮含量提供了工具。     

Submucosal vestibulo-vaginal administration of cloprostenol (Oestrophan-Spofa) in cows in relation to estrus activity, conception, and the level of progesterone in milk

Trials were performed to examine the effectiveness of 250 micrograms (1 ml) of cloprostenol (Oestrophan Spofa) implantation under the mucous membrane of the vaginal vestibule of 128 cows with a clinically pronounced corpus luteum on ovaries. Within 72 hours from administration, oestrus was observed in 112 animals (85.5%). Out of the 97 cows inseminated, 63 cows (64.94%) got in calf. The effectiveness of the luteolytic action was examined on the basis of progesterone check in milk in 56 treated cows. The submucous implantation of cloprostenol rapidly degraded the function of the corpus luteum since from the original level of 14.78 ng/ml progesterone decreased to 0.87 ng/ml within 72 hours. However, luteolysis did not affect all the corpora lutea. Hence the submucous administration of cloprostenol was found to be effective, and at the same time, highly economical, owing to a substantial reduction in the costs of reproduction control as well as the costs of production.