Milk fat progesterone concentrations in goats and early pregnancy diagnosis

Blood and milk samples from foremilk during afternoon milking, were simultaneously collected from 285 dairy goats. In experiment 1, fiva cyclic goats were sampled daily for 21 days. In experiment 2, 280 females from 9 flocks were submitted to sampling 21 days after insemination. In addition, some milk samples were divided in two parts, after which one was frozen and the other kept at +4 degrees C until assay. Progesterone concentrations were measured in blood, whole milk and milk fat by radioimmunoassay. No difference in whole milk or fat progesterone levels was found between frozen and refrigerated milk samples. Milk butterfat progesterone concentrations paralleled those in plasma or whole milk throughout the estrous cycle and ranged from about 20 ng/ml at estrus to about 400 ng/ml in mid-luteal phase. The ratio of mid-luteal phase progesterone levels to those seen in the estrous period was over 20 in fat and in blood. This ratio was very much lower in whole milk. Consequently the determination of pregnant and non-pregnant goats from the samples collected 21 days after service was very much easier and accuracy was better when the progesterone content was assayed from milk fat than from whole milk. It was concluded that early pregnancy diagnosis in goats can be done routinely by determination of progesterone levels in milk fat.     

The accuracy of enzyme-linked immunosorbent assay and latex agglutination progesterone test for the validation of estrus and early pregnancy diagnosis in dairy cattle

The accuracy of the enzyme-linked immunosorbent assay (ELISA) and the latex agglutination (LA) on-farm progesterone kit for detecting estrus and diagnosing early pregnancy was investigated in this study. Italian Friesian dairy cows (n=82) from 6 dairy herds were used for the collection of foremilk samples at the time of breeding and at 19, 21, and 23 days post insemination. Pregnancy status was ascertained by uterine palpation per rectum 40 to 60 days post insemination. Progesterone levels were affected by herd, percentage of milk fat, and the day of testing x diagnosis interaction. Validation of estrus by qualitative on-farm tests was 74.6% (LA) and 100.0% (ELISA) accurate using 0.5 ng/ml of progesterone as the RIA estimate for estrus. The accuracy rate for early pregnancy diagnosis by RIA was 68.4 to 83.8% for day 19 and day 21, respectively, while the detection rate for nonpregnancy was 84.6 to 100% on day 19 and day 21, respectively, as compared with uterine palpation per rectum. The average accuracy rate for early pregnancy diagnosis ranged from 84.7 to 92.3% for the LA and ELISA tests, respectively; the nonpregnancy rate was correctly predicted 93.9% to 68.2% for the LA and ELISA tests, respectively.     

Comparison of radioimmunoassay and gas-liquid chromatography analyses of progesterone concentrations in cow's milk.

Progesterone concentrations in milk were not significantly different when quantitated by different methods (RIA vs. GLC). There was a significant day effect (P less than 0.05) on milk progesterone level due apparently to gradually decreasing values as pregnancy advanced over days 30, 120 and 210. The means for the percent fat content were different (P less than 0.05) for all comparisons among four times of collection (immediately premilking, milking pool, immediately postmilking, and 3 hr postmilking). For progesterone concentration, the main effect of time and the three-way interaction (time times antiserum times purification method) were significant (P less than 0.005); all other main effects and interactions were not significant. Within each of 4 assay groups (2 antisera times 2 purifications), the concentration of progesterone was greater (P less than 0.05) for the samples which were collected immediately postmilking than for any of the other times of collection. The three-way interaction seemed due primarily to difference in progesterone determinations among the four assay groups in the samples which were taken immediately postmilking. Over all milk samples within each assay group, the percent fat content and the concentration of progesterone were positively correlated (r = 0.71, P less than 0.01).     

Milk progesterone levels to monitor reproductive status of Murrah buffalo

Concentration of progesterone in whole milk was used to diagnose pregnancy in 44 lactating buffaloes. Milk samples were taken from days 19 to 27 post breeding and analysed for progesterone by radio-immunoassay. A level exceeding 10 ng/ml of milk was taken as an indication of pregnancy. Using this criterion, the accuracy of the pregnancy test from a single milk sample on 19, 21, 23, 25 and 27 days after insemination ranged from 71.42 to 86.95% and 86 to 87.50% for pregnant and non-pregnant animals, respectively. Milk progesterone concentrations were significantly higher (P / 0.001) in pregnant compared with those in non-pregnant buffaloes on day 19, 21, 23, 25 and 27 post-insemination.     

Comparison of a solid-phase,no-extraction radioimmunoassay for progesterone with an extraction assay for monitoring luteal function in the mare,bitch, and cow

Equine, canine, and bovine plasma samples were assayed for progesterone (P₄) using extracted plasma in a liquid-phase assay and unextracted plasma in a solid-phase ¹²⁵I direct assay developed for human serum. The direct assay was also used to monitor P₄ levels in defatted milk. Results indicated that the direct-assay method was as reliable as the extraction assay for monitoring changes in plasma P₄ during the estrous cycle and early pregnancy. The regression equation specifying the relationship for the two methods was exponential. In this study, correlation coefficients from data subsets ranged from 0.93 to 0.99. The direct assay for P₄ did result in higher values than the extraction assay in plasma from diestrous animals. The use of diluted standards with stripped plasma from the species being assayed gave values that correspond more closely to the extraction assay. The comparisons in this study indicate that the direct radioimmunoassay (RIA) method offered a convenient, reliable method for monitoring luteal function in the species that were evaluated.     

Luteal function and conception in lactating cows and some factors influencing luteal function after first insemination

The objective of this study was to investigate the types and incidence of luteal sub-function in lactating cows after artificial insemination (AI) and their relationship with pregnancy, and to clarify the relationship between luteal function and parity, body condition score (BCS), milk yield, and dietary intake. In 19 cows, milk samples were collected daily from AI to confirmation of pregnancy. Milk progesterone concentrations were determined by EIA. Based on peak progesterone concentration and the day of onset of luteal phase, 15 of 30 progesterone profiles (50%) were normal, with progesterone concentration reaching 1.0 ng/ml within 5 days after insemination and > or =2.0 ng/ml thereafter. In addition, 6 (20%) were insufficient, (progesterone concentration remained < 2.0 ng/ml), 5 (17%) were delayed (progesterone reached 1.0 ng/ml after 5 days), 2 (7%) were both delayed and insufficient, one (3%) was short (progesterone >1.0 ng/ml for only 7 days), and one (3%) remained basal. Cows with a normal profile had a higher (P < 0.05) pregnancy rate than those with an abnormal profile (87% versus 33%, respectively). The amount of progesterone secreted in milk after first AI, as indicated by progesterone area under curve (AUC), was negatively correlated with milk yield (r = -0.83, P < 0.01), dry matter intake (r = -0.81, P < 0.05), total digestible nutrients (r = -0.82, P< 0.05), and digestible crude protein (r = -0.79, P <0.05). Cows that produced more milk and consumed more dry matter had less progesterone during the luteal phase. In conclusion, abnormal luteal function was associated with reduced pregnancy rates and high milk production and increased dietary intake during breeding were associated with reduced progesterone concentrations.     

Use of milk progesterone enzyme immunoassay for early pregnancy diagnosis in cows

An enzyme immunoassay using beta-galactosidase as the tracer was applied to determine milk progesterone level in cows. The novel method was reliable and practicable and required only a spectrophotometer and a centrifuge as major equipment. The milk progesterone enzyme immunoassay successfully diagnosed early pregnancy in cows. Milk samples were collected from 268 Holstein-Friesian cows in commercial dairy herds on 20, 21 or 22 days(usually 21 days) after insemination. Progesterone level in skim milk higher than 1.0 ng/ml indi-cated pregnancy. Pregnancy was confirmed by rectal palpation on 60 to 120 days after insemination. The accuracy of the milk progesterone test was 60.0 % (132 220 ) for a positive diagnosis and 100 % (48 48 ) for a negative result. A high incidence of embryonic death, 27.9 % (51 183 ), may have reduced accuracy for a positive test result. The enzyme immunoassay may be an alternative to radioimmunoassay in milk progesterone analysis for pregnancy diagnosis.     

Development of a direct enzyme immunoassay of milk progesterone and its application to pregnancy diagnosis in cows

The purposes of this study were to develop an enzyme immunoassay (EIA) for determination of progesterone in unextracted whole milk and to apply it to pregnancy diagnosis. Paper fibers covalently coupled to antibody specifically and competitively bound ³H-progesterone and 11α-hydroxyprogesterone hemisucccinate-horseradish peroxidase and were stable for 9 mo at ?20°C. The sensitivity, recovery, precision, and cross reactivity of the EIA and a radioimmunoassay (RIA) of milk progesterone were evaluated, and showed that this EIA was comparable to the RIA. Milk samples were collected once daily for one estrous cycle from ten lactating Holstein cows and the progesterone levels were determined by RIA. Milk progesterone in 70 samples measured by EIA were highly correlated (r = 0.90) with the values of RIA for the same samples. Milk samples for pregnancy diagnosis by EIA of milk progesterone were obtained daily from days 20 to 24 after 115 artificial inseminations of 85 lactating Holstein cows. Pregnancy diagnosis by EIA was confirmed by rectal palpation at 30 to 60 days after insemination or return to estrus. The accuracy based on single, two, three, four, and five consecutive samples was from 67.2 to 80.0%, 77.3 to 84.0%, 79.2 to 87.5%, 82.0 to 85.4%, and 85.4%, respectively, for pregnancy diagnosis; and from 95.0 to 98.3% for nonpregnancy.     

Use of progesterone 11-glucuronide-alkaline phosphatase conjugate in a sensitive microtitre-plate enzymeimmunoassay of progesterone in milk and its application to pregnancy testing in dairy cattle

A simple direct-addition microtitre plate enzymeimmunoassay (EIA) for progesterone in whole milk is described. The assay used antiserum raised against 11 alpha-hydroxyprogesterone 11-hemisuccinate (progesterone 11-hemisuccinate) and a heterologous label prepared by conjugation of 11 alpha-hydroxyprogesterone 11-glucuronide (progesterone 11-glucuronide) with alkaline phosphatase using an active ester procedure. The sensitivity, analytical recovery, linearity of response and precision of the assay compared favourably with radioimmunoassay (RIA). Results from EIA of milk samples were compared with determinations made after isolation of progesterone by HPLC (r = 0.910). Milk samples (200) were assayed by RIA at both the Milk Marketing Board and the Cattle Breeding Centre and the results were correlated with EIA performed at the Cattle Breeding Centre (r = 0.890 and r = 0.833 respectively). Calving data were obtained from a further 110 cows for which the milk progesterone EIA had provided a pregnancy test 24 days after AI; 46 cows were correctly identified as non-pregnant and 58 as pregnant and there were 4 false positive and 2 inconclusive results.     

Radioimmunoassay of ovine and bovine serum progesterone without extraction and chromatography

A procedure for the radioimmunoassay of ovine and bovine serum progesterone is described which does not require extraction and chromatography. Serum samples are assayed directly, and a highly specific antiserum that was prepared in rabbits against 11 alpha-hydroxyprogesterone conjugated to bovine serum albumin is used. Interference from serum binding proteins is alleviated by use of a phosphate buffer containing 5% BSA and separation of bound and free tritiated progesterone by a double antibody procedure. Serum samples are assayed in a mini-vial, the bound fraction (double antibody precipitate) is mixed with scintillation solution and the radioactivity is counted in the same vial. The assay procedure is sensitive (10 pg, 100 pg/ml) and has acceptable accuracy and precision. Because there is no extraction or chromatography, serum progesterone is not lost. Most important, the procedure is specific for progesterone and measures serum progesterone concentrations in the ewe and cow which are comparable with those obtained with conventional assay techniques. The progesterone assay described herein provides a rapid, economical procedure that can facilitate the study of ovarian cyclicity and aid in the early diagnosis of pregnancy.     

Development and validation of an enzyme immunoassay for progesterone in bovine milk or blood plasma

We have developed and validated a sensitive, simple and direct (i.e. without extraction) enzyme immunoassay (EIA) system for the measurement of progesterone in bovine milk and blood plasma. Progesterone (P) has been analysed by a microtiterplate EIA, employing polyclonal antibodies against P-7α-carboxyethylthioether-BSA as the antigen. The enzyme used as a label is horseradish peroxidase (HRP) and the chromogen is tetramethylbenzidine (TMB). Sensitivity of the EIA has been greatly improved by introduction of a heterologous tracer, in which progesterone is coupled to HRP at the 6β position. Compared to the radioimmunoassay (RIA) in which the same antiserum has been used, the sensitivity is 20 times greater. The detection limit of the assay is 0.4 pg per well. The working range of the standard curve is 0–20 pg per well (i.e. 0–40 ng per ml), and 50% reduction of the initial binding is obtained with 2.5-5.0 pg. Results can be obtained either by spectrophotometric measurement at 450 nm, or by naked eye. Total time needed for the assay of 40 replicate samples is approximately 3 h. Comparison of the EIA system with a previously validated RIA system gave a regression line EIA = 0.85 RIA + 2.11 (r = 0.93, n = 400 milk samples). Application of the milk-progesterone EIA to pregnancy testing (n = 66) gave an accuracy of 79.6% for positive diagnoses and 100% for negative diagnoses.     

Early pregnancy diagnosis in the ewe, based on milk progesterone levels.

Plasma and milk progesterone concentrations in pregnant sheep (18--22 days after mating) were similar, about 3.7 ng/ml whereas values in non-pregnant sheep were less than 1 ng/ml. Lambing results indicated identical accuracy for both methods (82 and 84% in 2 flocks). The accuracy was 92--100% for ewes diagnosed non-pregnant in the breeding season, but for ewes tested in the non-breeding season the diagnosis of non-pregnancy according to milk progesterone levels was only 50% accurate.     

Concentrations of progesterone in milk as a monitor of early pregnancy diagnosis in dairy cows

The objectives of the experiment were to evaluate the efficacy of using progesterone concentrations in milk and palpation per rectum on days 21 or 22 postbreeding to estimate pregnancy and evaluate management practices; and to investigate physiological occurrences leading to incorrect diagnosis of pregnancy when serial samples of milk were collected. Of particular interest were indications of early embyronic death and insemination of cows not in estrus. Milk samples were collected at the afternoon milking of days 0 or 1 (day 0 = day of estrus), 9 or 10, 21 or 22 and 27 or 28 following breeding in 200 lactating dairy cows. Tentative diagnosis of pregnancy was made based on concentrations of progesterone in milk on days 21 and 22 alone and on days 21 or 22 and 27 or 28. In addition all cows were palpated per rectum on days 21 or 22 postbreeding and a tentative pregnancy diagnosis was made. Pregnancy was confirmed by examination of the genital tract per rectum between 35 and 50 days after breeding. Values of 4 ng/ml or greater and/or the presence of a mature corpus luteum were considered positive signs of pregnancy. Progesterone in milk ranged from 0.1 to 18 ng/ml. On days 0 or 1, 9 or 10, 21 or 22 and 27 or 28 concentrations of progesterone in milk averaged 1.5 +/- 0.3, 11.1 +/- 0.5, 12.0 +/- 0.4 12.5 +/- 0.5 ng/ml for pregnant cows. Corresponding samples from nonpregnant cows averaged 1.2 +/- 0.2, 10.3 +/- 0.4, 3.0 +/- 0.4, 6.8 +/- 0.6 ng/ml, respectively. Ninety-six and 104 cows were classified as pregnant and nonpregnant on days 21 or 22 as compared to 78 and 118 cows diagnosed as pregnant and nonpregnant on days 21 or 22 and 27 or 28 combined. Pregnancy detection by progesterone in milk on days 21 or 22 with pregnancy determined via rectal palpation 35 to 50 days postbreeding was 77 and 100% accurate for positive and negative diagnosis, respectively. The percent agreement using progesterone in milk on days 21 or 22 and 27 or 28 combined was 95 and 100%, respectively, for positive and negative diagnosis. Diagnosis based on rectal palpation 21 or 22 days postbreeding was 63 92 (69%) and 76 88 (87%) for pregnant and nonpregnant cows, respectively. Ten of the 200 cows had progesterone concentratins in milk of > 4 ng/ml at the time of breeding. Six of these cows were pregnant from a previous insemination. The other four cows were nonpregnant and were inseminated during the luteal phase of the cycle. In conclusion, measurement of progesterone in milk is a useful tool in early detection of pregnant and nonpregnant cows and may be useful in detecting reproductive problems in a dairy herd. It will probably be most useful when used in combination with later pregnancy diagnosis per rectum .     

Comparisons of eight commercial on-farm milk progesterone tests

To evaluate eight commercial on-farm milk progesterone kits, milk samples (50 ml each of foremilk and postmilk strippings) were collected during the estrous cycle from 10 cycling Holstein cows for 24 consecutive days. Relative concentrations of progesterone were classified as low or high by comparison with standard progesterone samples supplied with each kit. The concentration of progesterone in each milk sample was determined by radioimmunoassay (RIA). Accuracy of classification into low or high levels by commercial tests was determined by the percentage of similarity with RIA values using discriminant analysis. Accuracy of the eight tests ranged from 89.0 to 98.9% for low progesterone, 74.8 to 85.6% for high progesterone, and 80.3 to 87.3% for all samples (n = 238). The percentage of fat in milk or an interaction of the percentage of milkfat by day of estrous cycle influenced commercial test results for all tests except Accufirm and Calfcheck. Progesterone levels, estimated by the test-kits, were low from 1.5 +/- 0.5 to 2.8 +/- 0.9 days before estrus (X +/- SEM) and until 4.0 +/- 0.6 to 5.9 +/- 1.3 days after estrus. These data support the principle that a single low progesterone sample cannot be used to determine proper timing of insemination. All eight commercial kits can be used to determine accurately the relative concentrations of progesterone in milk samples.     

Pregnancy management in the bitch

Pregnancy management to optimize maternal and neonatal health begins with breeding management and the selection of normal, healthy brood stock in ideal body condition. After breeding, a commercial diet appropriate for reproduction and lactation should be fed. Typically these contain 29–32% protein of animal source, at least 18% fat, 20–30% carbohydrate, and essential vitamins, minerals and fatty acids. Pregnancy is confirmed approximately 25 d after breeding. A “maternity ward” and whelping box should be provided. Steady increases in caloric intake and body weight are expected as pregnancy progresses. Weight loss should not occur. Throughout pregnancy, changes in the bitch’s attitude, activity, appetite, body weight, and physical findings should be monitored by the owner. If appetite and body weight do not continue to increase, or if any signs of illness develop, maternal health should be assessed with a complete physical examination and a CBC, biochemical profile, and free-catch urinalysis. Fetal health should be assessed with ultrasonography. Maternal or fetal abnormalities will put the pregnancy at risk. Impending parturition and the progress of labor and delivery can be monitored by assessing rectal temperature, serum concentrations of progesterone, and/or uterine and fetal monitors. This article reviews the physiology of canine pregnancy and parturition, and typical schemes used to manage normal canine pregnancy to optimize maternal and puppy health.     

Level and distribution of progesterone in bovine milk in relation to storage in the mammary gland.

The study investigated the effect of the place of storage of milk in the mammary gland on progesterone concentrations in whole milk, skim milk and milk fat. Skim milk, milk fat and whole milk progesterone concentrations were lower (P < 0.05) in milk fractions obtained from the cisternal part of the mammary gland compared to those in the milk fractions from the alveoli. Mean milk fat concentrations did not mirror the changes in the mean skim milk, milk fat and whole milk progesterone concentrations. After administration of oxytocin, milk fat concentrations rose significantly (P < 0.01). At the same time, skim milk and milk fat progesterone concentrations remained unchanged (P > 0.05), compared to those in the milk fractions of alveolar origin, obtained before oxytocin administration. Skim milk and whole milk progesterone concentrations were higher (P < 0.01) in composite milk and in milk samples collected 1 h after milking, compared to concentrations in the milk samples collected before morning milking and at 3, 5, 7 and 9 h after milking. The results suggest that defatted milk, milk fat and whole milk progesterone concentrations were affected by the place of storage of the milk in the mammary gland, and that this effect is independent of milk fat content. Time of milk sampling, not the milk fat concentration, in relation to time of milking, was a critical factor in determining skim milk, milk fat and whole milk progesterone. The study also revealed that the concentrations of the other milk components, somatic cell count, lactose and protein were affected by the place of storage of milk in the mammary gland.     

Effects of sample handling temperatures on bovine skim milk progesterone concentrations

The effects of incubation of whole milk at various temperatures and times on the concentration of progesterone in the skim milk fraction was determined. For the study, milk samples were collected from 10 pregnant Holstein cows. The milk from each cow was transferred to culture tubes to provide 32 replicates of 3 ml volume. To begin the incubation study, all samples were placed in a 37 degrees C water bath for 4 h. The end of this incubation was designated as time 0 and a sample from each cow was centrifuged to harvest skim milk. At time 0, samples from each cow were divided among incubation temperatures of 0 degrees, 4 degrees, 20 degrees and 37 degrees C. Samples were removed from each incubation group at 30, 60, 90 and 120 min. After 120 min, all remaining samples were returned to the 37 degrees C incubation and skim milk was collected at 30, 60 and 90 min. Progesterone was measured in skim milk by radioimmunoassay. The mean +/- SE concentration of progesterone in skim milk at time 0 was 10.9 +/- 1.1 nmol/L. The mean concentration of progesterone in skim milk was higher (P < 0.05) in all samples incubated at 0 degrees and 4 degrees C, with incremental increases ranging from 34% to 67% above time 0. Progesterone in skim milk returned to time 0 concentrations in milk samples transferred from 0 degrees or 4 degrees C to 37 degrees C. There was no change in skim milk progesterone in whole milk samples incubated at 20 degrees or 37 degrees C. From this study, it can be concluded that the concentration of progesterone in skim milk is temperature dependent. Inconsistency in handling whole milk samples can have a profound effect in the concentration of progesterone on skim milk. The temperature-dependent effect was reversible and may be related to solubility of progesterone in milk fat.     

Flow-injection determination of streptomycin residues in milk using the luminol-periodate-Mn2+ chemiluminescence system.

A novel flow-injection method with chemiluminescence detection was developed for the determination of streptomycin residues in milk, based on the enhancement by streptomycin of the luminol-potassium periodate-Mn(2+) chemiluminescence system. The assay allowed analysis of streptomycin residues in whole milk samples ( approximately 3.5% fat) after sample work-up procedures. The limit of detection was 5.16 x 10(-9) mol/L for milk, and was far below the regulations of maximum residue limits (MRLs). The relative standard deviation (RSD) for 8.66 x 10(-8) mol/L streptomycin was 1.94% (n = 15) in milk. The results obtained for the assay of streptomycin residues in milk was comparable with those obtained by the official methods (ELISA) and demonstrated good accuracy and precision. The possible mechanism is briefly discussed.     

Milk progesterone measurement in dairy cows: Correlation with estrus and pregnancy determination

Progesterone levels in fore milk, determined by a highly specific radioimmunoassay, were compared for the assessment of estrus by a veteran herdsman and an experienced inseminator, in cows presented for insemination. In addition, an examination was made of the relative accuracy of using milk progesterone levels for the determination of pregnancy at 24, 40 and 44 days after insemination, as compared with rectal palpation at 45–50 days post-breeding.Fat-free fore milk progesterone levels were similar to jugular plasma levels at 24 days post-insemination and reached roughly 60% of the level of unextracted fore milk at this time. Accuracy of estrus diagnosis by herdsman, inseminator and milk progesterone level was 84%, 93% and 96%, respectively. For pregnancy diagnosis, milk progesterone determination in 85 cows showed 78% accuracy in predicting pregnancy and 100% accuracy in predicting non-pregnancy. At 40 days post-insemination false positives dropped to 10% and at 44 days only 7% of the cows were incorrectly diagnosed as pregnant. The false positives in this study were largely due to embryonic mortality as reflected by abnormal intervals of return to estrus. Two milk progesterone determinations, at 24 and either 40 or 44 days post-insemination ensure maximum reliability for early pregnancy diagnosis.     

EXPECTATIONS AND STABILITY OF PREFERENCE CHOICE

When identical milk samples are presented, only 30% of participants respond with a “no preference” rating. Stability of the “no preference” rating was studied under a variety of conditions, having consumer panelists rate both identical and different pairs of milk samples with varying fat content. The proportion of participants choosing the “no preference” option, when the samples in the pair were identical, was largely consistent despite manipulation of pretest conditions and changes in test questions and answer formats. However, when the milk preference test was preceded by a same/different test, and those responding “same” were assumed to have no preference, the percent of “no preference” was two to three times larger for identical test samples (60–69%). Thus by branching the question, the “false preference” choice for identical milks was lowered. Among those responding “different” to identical milks, the false‐alarm rate increased to 91%, suggesting that perception of (spurious or momentary) differences is driving at least part of the preference choice.