Purification of pregnancy-associated glycoproteins from late-pregnancy Bubalus bubalis placentas and development of a radioimmunoassay for pregnancy diagnosis in water buffalo females.

BACKGROUND: Pregnancy-associated glycoproteins (PAGs) were first described as placental antigens present in the blood serum of the mother soon after implantation. Here, we describe the purification of several pregnancy-associated glycoproteins from water buffalo placenta (wbPAGs). A specific radioimmunoassay (RIA) was developed for early pregnancy diagnosis in buffalo species. RESULTS: Amino-terminal microsequencing of immunoreactive placental proteins allowed the identification of eleven wbPAGs sequences [Swiss-Prot accession numbers: P86369 to P86379]. Three polyclonal antisera (AS#858, AS#859 and AS#860) were raised in rabbits against distinct wbPAG fractions. A new RIA (RIA-860) was developed and used to distinguish between pregnant (n=33) and non-pregnant (n=26) water buffalo females. CONCLUSIONS: Our results confirmed the multiplicity of PAG expression in buffalo placenta. In addition, the RIA-860 system was shown to be sensitive, linear, reproducible, accurate and specific in measuring PAG concentrations in buffalo plasma samples from Day 37 of gestation onwards.

Comparison of commercial ELISA blood tests for early pregnancy detection in dairy cows.

The objective of the present study was to compare two commercially available blood-based pregnancy tests, namely BioPRYN, an ELISA for pregnancy-specific protein B (PSPB), and an ELISA for pregnancy-associated glycoprotein (PAG), for early pregnancy diagnosis in dairy cattle using transrectal ultrasonography as a gold standard. Transrectal ultrasonography was conducted 26-58 days after artificial insemination (AI) in 197 cattle from 19 farms. Concurrently, a blood sample was collected for determination of serum PSPB and PAG. Transrectal palpation was performed approximately 120 days after AI to verify that pregnancy was maintained. For PSPB and PAG, there were no significant differences (P>0.05) in sensitivity (98.0 and 97.8%), specificity (97.1 and 91.2%), positive predictive values (99.3 and 97.8%), negative predictive values (91.9 and 91.2%) and accuracy (97.8 and 96.4%). In conclusion, the two blood pregnancy assays were equally efficacious and were highly accurate (based on transrectal ultrasonography as the gold standard).

Immunological detection of pregnancy: Evidence for systemic immune modulation during early pregnancy in ruminants.

Mammalian pregnancy creates unique challenges for immune systems highly evolved to detect and eliminate invading pathogens. Recognition of the challenges created by gestating a semi-allogeneic fetus evolved from the discipline of transplantation biology and were informed by studies on the unique natural parabiosis that occurs when female calves are gestated with twin male fetuses. These pregnancies typically result in an intersex female termed a freemartin, which revealed insights into development of the male and female reproductive tracts. However, they also uncovered important clues on immune tolerance with wide-ranging implications to reproductive biology, transplantation biology and autoimmune disease. Many studies focused on identifying mechanisms through which the fetus evades maternal immune detection and elimination. These included studies characterizing immune interactions between the fetus and mother at the nourishing interface of the placenta and uterine endometrium. This immunological forbearance only occurs under high concentrations of circulating progesterone. Beyond the requirement for progesterone, there has been considerable progress towards understanding the effects of conceptus signals on maternal immune function. One common theme is that pregnancy induces a T helper 2 immune bias as shown in several mammalian species, including domestic ruminants. However, a growing body of evidence shows that the fetus not only evades, but also provokes immune responses locally in the uterus and in peripheral tissues. This is perhaps most dramatically illustrated by domestic ruminants where the conceptus secretes a unique interferon in the opening salvo of hormonal communication with the maternal immune system. The role of interferon tau in regulating expression of genes of the innate immune system in the uterus has been extensively studied. More recently, it was determined that these same genes are also induced in peripheral immune cells and other tissues throughout the body. In addition to interferon tau and progesterone, pregnancy associate glycoproteins and chaperonin 10 (aka Early Pregnancy Factor) are implicated in altering immune function both locally and systemically during pregnancy. While it is tempting to speculate that this activation of innate immunity is designed to counteract selective immunosuppression, knowledge of the importance of local and systemic immune activation to the success of pregnancy remains incomplete. This area remains fertile ground for developing better approaches to diagnose and treat infertility in domestic farm species and humans alike.

Technical note: A rapid enzyme-linked immunosorbent assay blood test for pregnancy in dairy and beef cattle.

The ruminant trophoblast produces pregnancy-associated glycoproteins (PAG) that can be detected in the blood of pregnant animals. The objective was to determine the accuracy of a rapid ELISA PAG-based test for the purpose of pregnancy detection in cattle. Blood was sampled from dairy cattle (539 Holstein cows, 173 Holstein heifers, 73 Guernsey cows, 22 Guernsey heifers, and 12 Jersey heifers) and crossbred beef cattle (145 cows and 46 heifers) that were >or=25 d after insemination (range = 25 to 45 d for dairy and 29 to 56 d for beef). Cattle were examined by ultrasonography for detection of pregnancy within 2 d of blood collection. Whole blood or plasma was incubated in a polystyrene tube coated with a monoclonal PAG antibody for 15 min. The tubes were then washed and subjected to sequential incubations with a biotinylated polyclonal PAG antibody (15 min, followed by wash), a horseradish peroxidase-streptavidin solution (15 min, followed by wash), and a peroxidase substrate. Tubes were visually assessed for color after 15 min (clear solution = PAG negative, not pregnant; blue solution = PAG positive, pregnant). Total assay time was approximately 90 min. The ultrasound examination was used as the standard for pregnancy diagnosis. The sensitivity (99.8 +/- 0.2%), specificity (91.7 +/- 1.4%), and negative predictive value (99.7 +/- 0.3%) for the PAG test used in dairy cattle were similar for different breeds and for cows and heifers. The positive predictive value for the test was greater in dairy heifers than in dairy cows (96.5 +/- 1.4% vs. 90.5 +/- 1.7%, respectively). In beef cattle, the sensitivity (100%), specificity (92.3 +/- 3.0%), positive predictive value (95.0 +/- 2.0%), and negative predictive value (100%) for the PAG test were similar for cows and heifers. The accuracy of the test was not different for dairy and beef cattle. In conclusion, the rapid ELISA pregnancy test based on PAG was highly sensitive and specific for pregnancy detection in dairy and beef cattle.

Measurement of EPF for detection of cow pregnancy using rosette inhibition test.

Early embryonic death of calves due to sub-fertility in cows is of great economic concern to dairy industry. Early pregnancy factor (EPF) is a secretory protein with pregnancy associated immunosuppressive properties. Rosette inhibition test (RIT) was used to detect EPF in inseminated dairy cows. Blood samples were collected at two intervals, 1-3 and 5-7 days after insemination from 23 inseminated and 18 non-inseminated control cows for RIT and pregnancy diagnosis performed between 42 and 45 days on palpation. The study indicates that RIT (P<0.05) has the potential to distinguish pregnant from non-pregnant dairy cows in the first week of pregnancy.

Field experiences with early pregnancy diagnosis by progesterone-based ELISA in sows.

In four Kenyan pig breeding units the pregnancy diagnosis of sows has been carried out in two groups: Group 1 (n = 1911): the sows were transrectaly pregnancy tested between Days 17-22 post-mating by ultrasound. Sows testing non-pregnant immediately received one dose of 400 IU pregnant mare serum gonadotropin (PMSG) (equine chorion gonadotropin, eCG) and 200 IU human chorion gonadotropin (hCG). On showing signs of oestrous, the animals were subsequently artificially inseminated (AI). Group 2 (n = 1923): sows were pregnancy tested by serum progesterone (P4)-based enzyme-linked immunosorbent assay (ELISA) on Day 17 post-breeding. P4 concentrations were categorized as positive (> 5 ng/ml) or negative (< 5 ng/ml). Sows testing nonpregnant immediately received one dose of 400 IU PMSG and 200 IU hCG by injection, and were subsequently artificially inseminated. The following parameters were evaluated: sows diagnosed non-pregnant, days from first post-weaning insemination until the sows were inseminated at their first return to oestrus; farrowing rate and total piglets born and number of live-born piglets in litters. The percentage of sows diagnosed non-pregnant in the two groups, as well as the totals of born piglets and of live-born piglets in litters did not differ significantly between the two groups. The number of days from the first post-weaning mating until the sows were artificially inseminated at their first return to oestrus and the administration of eCG and hCG was shorter (P < 0.01) and farrowing rate was higher (P< 0.01) in the ELISA-tested sows.

Accuracy of a pregnancy-associated glycoprotein ELISA to determine pregnancy status of lactating dairy cows twenty-seven days after timed artificial insemination.

To determine the accuracy of a pregnancy-associated glycoprotein (PAG) ELISA in identifying pregnancy status 27 d after timed artificial insemination (TAI), blood samples were collected from lactating Holstein cows (n = 1,079) 27 d after their first, second, and third postpartum TAI services. Pregnancy diagnosis by transrectal ultrasonography (TU) was performed immediately after blood sample collection, and pregnancy outcomes by TU served as a standard to test the accuracy of the PAG ELISA. Pregnancy outcomes based on the PAG ELISA and TU that agreed were considered correct, whereas the pregnancy status of cows in which pregnancy outcomes between PAG and TU disagreed were reassessed by TU 5 d later. The accuracy of pregnancy diagnosis was less than expected when using TU 27 d after TAI (93.7 to 97.8%), especially when pregnancy outcomes were based on visualization of chorioallantoic fluid and a corpus luteum but when an embryo was not visualized. The accuracy of PAG ELISA outcomes 27 d after TAI was 93.7, 95.4, and 96.2% for first, second, and third postpartum TAI services, respectively. Statistical agreement (kappa) between TU and the PAG ELISA 27 d after TAI was 0.87 to 0.90. Pregnancy outcomes based on the PAG ELISA had a high negative predictive value, indicating that the probability of incorrectly administering PGF(2alpha) to pregnant cows would be low if this test were implemented on a commercial dairy.

Evaluation of early conception factor lateral flow test to determine nonpregnancy in dairy cattle.

The early conception factor (ECF) lateral flow test was evaluated for its ability to accurately determine nonpregnant status in dairy cattle. Results of 2 field trials involving 191 cows and 832 tests indicated the probability that a cow can be correctly diagnosed as nonpregnant by using the ECF test is only about 50%. Agreement of test results between milk and serum obtained from the same cow was 57.5%. The ECF test was not consistent in identifying nonpregnancy when the same cows were tested repeatedly over a period of 4 weeks. We conclude that the ECF lateral flow test does not accurately identify nonpregnancy in dairy cattle.

Evaluation of a rosette inhibition test for pregnancy diagnosis in pigs.

A rosette inhibition test was developed using pig lymphocytes and sheep red blood cells. Antilymphocyte serum (ALS) in the presence of complement inhibited rosette formation by greater than 95% at 1/250 declining to no inhibition at 1/8000. Sera obtained from a total of 14 pregnant sows before and 1, 2, 3 and 4 wk after mating were tested for their ability to augment the rosette depression caused by ALS. In one experiment in which the responses of 4 pregnant sows were compared to 4 non-pregnant sows by discriminant analysis, sera were classified correctly in 83% of the samples taken from either pregnant or non-pregnant sows. When the more usual method of calculating the rosette inhibition titre was used, the responses of sera from pregnant pigs were classified with 31% accuracy and those from non-pregnant pigs with 80% accuracy. In a second experiment, sera from 10 pregnant sows were classified with 25% accuracy using the rosette inhibition titre. Thus 4 of these pigs were classified as non-pregnant by this method. Data from the second experiment were not suitable for discriminant analysis. It was concluded that there was some factor present in the sera of pregnant pigs, particularly by 3 or 4 wk post-mating, which could be detected by the rosette inhibition test. However, the test is not sensitive enough to allow specific diagnosis of early pregnancy in pigs.

Milk progesterone radioimmunoassay using radioiodinated tracers: a rapid and reliable assay system.

Both the validity and practicability of a direct progesterone radioimmunoassay based on radioiodinated progesterone tracers were studied. The results obtained show the reliability of the assay; when compared with assays based on 3H-progesterone tracers there are fewer steps for assay execution, saving time and reducing the number of reagents used. Various commercially available 125I-progesterone tracers were assayed, and only those with an 11 alpha-hemisuccinate bridge were suitably bound by antisera raised against progesterone-bovine serum albumin conjugates having identical bridge structure. The bridge effect caused no observable alteration in validity parameters. Finally, our results support the utility of this assay as a practical method of early diagnosis of pregnancy and as a reliable experimental technique to monitor cow ovarian function.

Early pregnancy diagnosis in swine by direct radioimmunoassay for progesterone in blood spotted on filter paper.

A direct radioimmunoassay method for the measurement of progesterone in blood dried on filter paper has been developed for the early pregnancy diagnosis in sows, as well as for monitoring progesterone levels during the oestrous cycle. Pregnancy diagnosis was performed with 95 sows on Days 17-22 after artificial insemination (AI). The cut-off value for pregnancy diagnosis of 7.5 ng/ml was calculated (mean+/-2S.D.) from the progesterone concentrations measured on the same days from non-inseminated animals. There were 85 cases considered pregnant on the basis of progesterone concentration, leaving 10 animals non-pregnant. The accuracy for the positive cases was 98.8%. Two of the 10 sows considered as negative subsequently farrowed, giving an accuracy of 80%. The overall accuracy of the method was 96.8%. The blood-spot assay may be a useful tool for early pregnancy diagnosis in swine, with respect to sampling, simplicity, speed and accuracy.

Pregnancy-associated glycoproteins (PAG) detection in milk samples for pregnancy diagnosis in dairy goats.

In ruminants, glycoproteins synthesized in the outer layers of the trophoblast enter the maternal bloodstream. The assay of their concentration in plasma or in serum can be used for pregnancy diagnosis and to indicate feto-placental health status. In this study, concentrations of the pregnancy-associated glycoprotein (PAG) were investigated in the milk of dairy goats. The mean concentrations of PAG in milk were significantly higher in pregnant than in non-pregnant goats throughout the sampling period. Positive milk PAG levels were found in all pregnant animals from Day 32 after breeding, but remained at negative values in nonpregnant ones. This study reports for the first time a pregnancy diagnosis based on milk concentrations of PAG, that could be of great use on the farm.

Evaluation of the early conception factor (ECF) test for the detection of nonpregnancy in dairy cattle.

The ability to detect conception and/or conception failure in cattle would be beneficial to producers in formulating reproductive management plans. A new diagnostic test, the early conception factor (ECF) test, has been developed forthis application yetthe accuracy of this test has not been adequately determined. The objectives of this study were to evaluate the effectiveness of the ECF test for detecting the nonpregnant cow, and to compare the reliability of serum versus milk ECF tests relative to actual pregnancy rates. In Trial 1, Holstein heifers were synchronized, the animals were bred (timed-AI), and serum ECF tests were performed 72 h later. Heifers exhibiting a negative ECF test after AI were re-synchronized, bred again, and re-tested for ECF for up to three services. Relative to actual pregnancy rates, a negative ECF test was correct (i.e., true negative) 38.5% of the time over the three services. In Trial II, Holstein heifers were bred (AI) after observed estrus and serum ECF tests conducted between Days 1 and 3 and Days 7 and 9 after AI. In this trial, only 44.4% and 55.6% of the confirmed nonpregnant heifers were identified correctly by serum ECF analysis at Days 1 to 3 and Days 7 to 9 post-AI respectively. In Trial III, 40 lactating cows were synchronized, the animals were bred (AI), and serum and milk ECF tests were performed on Days 3, 9, 15, 21 and 30 after AI. Pregnancy diagnosis (ultrasound on Day 30 and palpation on Day 51) confirmed that 50% of the cows were pregnant to AI, while serum and milk ECF analysis indicated a 100% and 37.5% predicted pregnancy rate, respectively, at 30 d post-AI. Moreover, results of the serum and milk ECF tests disagreed with one another 36.9% of the time overall, while agreement between ECF and actual pregnancy rates were 50.6% and 45.6% for milk and serum respectively. Additionally in Trial III, a negative ECF result only identified 5% and 28.8% of nonpregnant cows overall for serum and milk tests respectively (i.e., true negatives), with a high incidence of false positive ECF results noted (47.5% and 31.3% for serum and milk, respectively). Collectively, these data indicate that the current ECF test cannot accurately identify the nonpregnant cow with the precision needed by the dairy producer.

Sheep breeding program for the year-round production of timed pregnancies for experimental foetal surgery.

Foetal lambs of known gestation were supplied to research programs throughout the entire year by controlling the oestrous cycle of the breeding ewes. In about 55 ewes, an average of 70-3 pregnancies of known duration produced an average of 118 lambs for each of the 3 years (168% of lambs to pregnancies). The accuracy of the ram-marking information, plasma radioimmunoassay (18 days after mating) and ultrasonic flow detection (Doppler; 60 days after mating) as indicators of pregnancy is reported for the last 3 years of this program.

Diagnosis of pregnancy in marmosets (Callithrix jacchus).

3 techniques for studying reproductive function in the marmoset are described. Abdominal palpation was found to be the most successful method for diagnosis of pregnancy. Vaginal cytology and urinary immunoassays met with limited success and were of supportive value only.

Evaluation of the sub-human primate tube test for pregnancy in primates.

A haemagglutination inhibition test, developed specifically for primates, diagnoses early pregnancy in the chimpanzee, gorilla, orang-utan and baboon. The test was sensitive and reacted positively when the concentration of gonadotrophin in urine was equivalent to 0.03 i.u. human chorionic gonadotrophin per ml. This degree of sensitivity and the certitude that it cross-reacts with primate luteinizing hormone probably accounts for most of the false positive results.

Assessment of pregnancy in sheep by analysis of plasma progesterone using an amplified enzyme immunoassay technique.

An amplified enzyme immunoassay kit for progesterone analysis was used to diagnose pregnancy in a flock of 130 mule ewes. An accuracy of 100 per cent was obtained after the analysis of progesterone in plasma samples taken 15 to 16 days after mating. In mule ewes a plasma progesterone level greater than 5.9 nmol/litre was indicative of pregnancy. In the validation of the technique, duplicate ewe plasma samples and progesterone standards were compared with a radio immunoassay technique; the regression coefficient between the two techniques was r = 0.82.

The immunological approach to pregnancy diagnosis: a review.

The developing embryo/fetus bears antigens which are foreign to the mother and it could be expected that immune rejection of the conceptus would occur. One of the reasons why the fetus is not rejected is because a depression of the maternal immune response takes place during pregnancy. Serum from pregnant animals of several species has been shown to contain a factor, early pregnancy factor (EPF), which is immunosuppressive. EPF has been detected as early as six hours after mating and its detection could aid diagnosis of early pregnancy in all species.

Laboratory methods of equine pregnancy diagnosis.

Rectal examination is a reliable method of diagnosing pregnancy in the mare. Also, test kits are available for the simple quick detection of pregnant mare serum gonadotrophin. Nevertheless there is a considerable demand by practitioners for an independent laboratory service in equine pregnancy diagnosis, particularly during the gestational phase when placental gonadotrophin is concentrated in the blood. An initial attempt to provide a service by means of the agar gel diffusion test was disappointing and alternatives were sought. The primary requirements for an ideal alternative technique were defined as: accuracy, sensitivity, applicability to the optimum request period, elimination of subjective interpretation and a minimal inconclusive rate. Additional considerations included cost, practicality and test duration. The various tests available are summarised and their published accuracies are discussed. It was decided to reverse the current trend from biological to immunological techniques and to modify the test by which Cole and Hart announced the discovery of PMSG in 1930. The utero-ovarian response in the immature female mouse was quantified simply to produce a numerical result. The reason for this is explained, the method is described and its accuracy is evaluated. The primary requirements, defined above, were achieved.

[Reliability of pregnancy diagnosis in sows using direct radioimmunoassay of estrone-sulfate]. / Spolehlivost diagnostiky gravidity u prasnic prímou radioimunoanalýzou estron-sulfátu.

The pregnancy diagnosis in sows using direct radioimmunoassay of estrone-sulphate in the blood serum without sample extraction is described. It was found that for the pregnancy diagnosis the period between days 22 and 30 of pregnancy can be used since in this period the estrone-sulphate concentrations in all pregnant sows markedly exceeded those of 64 non-pregnant animals. The estrone-sulphate estimation cannot be used for the pregnancy diagnosis before day 22 and between day 30 and 40 because the estrone-sulphate concentrations in most samples collected in these periods were lower than 4 nmol.l-1.