Pregnancy-associated Glycoprotein Levels Research Articles

Pregnancy maintenance and fetal loss assessment in Holstein cows through analyzing pregnancy-associated glycoproteins in milk

Fetal loss (FL) from the 45th day of gestation until calving can impose a significant economic burden on dairy farmers, resulting in lost profits and increased production costs. Pregnancy-associated glycoprotein (PAG) is commonly used for detecting pregnancy in cows. PAG is secreted by binucleated trophoblast cells of the placenta and regulated by more than 24 genes. The purpose of this study was to determine the PAG threshold for FL and a probability of pregnancy maintenance until calving based on milk PAG levels. Our results reveal that primiparous and multiparous cows that maintained pregnancy until the 40th week exhibited higher PAG sample-negative (SN) values in their milk in the 6th week of gestation than did those that experienced FL later in gestation. Pregnant cows with higher PAG SN values in the 6th week of gestation were more likely to maintain their pregnancies. The area under the receiver operating characteristic curve for predicting the probability of pregnancy maintenance was 0.722 for our prediction model. On the other hand, a milk PAG SN value of <0.192 indicated 95 % confidence that FL would occur between the 7th and 40th weeks of gestation. Milk PAG testing is a noninvasive sampling technique that does not induce additional stress in lactating cows. The study reveals that PAG SN values increase significantly in Holstein cows during the 6th week of gestation. The predictive model developed was effective in forecasting pregnancy outcomes up to the 40th week of gestation or calving. The model's performance is moderately good for field application and could be a useful tool for dairy producers.

Serum pregnancy-associated glycoprotein profiles during early gestation in Karya and Konya Merino sheep.

Reported as being expressed by mono- and binucleate placental cells, pregnancy-associated glycoproteins (PAGs) are released into the blood circulation from the ruminant placenta. Circulating gestational PAGs levels may differ between sheep breeds. This study was aimed at the close monitoring of the serum PAGs profiles of Karya and Konya Merino sheep during early pregnancy. Fifteen Karya and 15 Konya Merino ewes were synchronized by a 12-day treatment with progesterone-impregnated intravaginal sponges. After the sponges were withdrawn, the ewes were administered 400IU of equine chorionic gonadotropin. The ewes were allowed to mate naturally, and all animals were sampled for blood as of the day of mating (day 0) at weeks 1, 2, 3, 4 and 5. Pregnancy diagnoses were made by transabdominal ultrasonography at week 5. At weeks 6, 7, 8, 9 and 11, blood samples were collected only from the pregnant ewes. The blood samples were centrifuged at 3000×g, and extracted sera were stored at -20°C until being used for laboratory analyses. Serum PAGs levels were determined with the aid of a commercial PAG-enzyme-linked immunosorbent assay test originally developed for pregnancy diagnosis in cattle. Differences in the between the PAGs levels throughout pregnancy and the group effect (Karya and Konya Merino) were determined with a two-way mixed analysis of variance. Pairwise comparisons were made using a Bonferroni adjustment. PAGs levels showed a linear increase with the advance of pregnancy in both Karya and Konya Merino sheep. No difference was detected between the breeds for serum PAGs levels. The serum PAGs levels of the pregnant and non-pregnant ewes differed as of the fourth week. The serum PAGs levels of the Karya and Konya Merino ewes were similar during the first 11 weeks of gestation, and pregnancy diagnosis could be made based on serum PAGs levels as of the 4th week in both breeds.

Plasma profile of Pregnancy Associated Glycoproteins during Postpartum period in Konya Merino ewes lambing single and twin

This study investigated the plasma profile of Pregnancy Associated Glycoproteins (PAGs) after lambing in Konya Merino ewes using specific cattle pregnancy test kit. A total of 16 Konya Merino ewes were used as a material. Four groups were set up, ewes birthing a male lamb (SM group, n=4), a female lamb (SF group, n=4), twin male lambs (TM group, n=4) or twin female lambs (TF group, n=4). Blood plasma samples were taken on days 0 (lambing day), 3, 7, 10, 14 and then weekly until day 35. All samples were analysed using a bovine pregnancy test kit to detect of PAGs level. It was determined that there was a strong negative correlation between PAGs levels in peripheral blood and days after lambing (r2=0.969; P&lt;0.01). However, no relationship was found between PAGs level and lamb gender or birth type. In conclusion, plasma PAGs level decreases rapidly in Konya Merino ewes regardless of lamb gender and birth type. Moreover, the results showed that the plasma profile of PAGs after lambing in Konya Merino ewes can be monitored with a bovine commercial ELISA–based pregnancy test kit, and the test results can be used in decisions and assessments based on the levels of PAG molecules.

Genetic parameters of pregnancy loss in dairy cows estimated from pregnancy-associated glycoproteins in milk

This study examined the feasibility of using pregnancy-associated glycoproteins (PAG) in milk within breeding for pregnancy maintenance and assessed the genetic variation in pregnancy loss traits. A total of 374,206 PAG samples from 41,889 Swedish Red (SR) and 82,187 Swedish Holstein (SH) cows were collected at monthly test-day milkings in 1,119 Swedish herds. Pregnancy status was defined based on PAG levels and confirmed by data on artificial insemination (AI), calving, and culling from d 1 postinsemination to calving. Pregnancy loss traits were defined as embryonic loss (diagnosed 28 d to 41 d after AI), fetal loss (42 d after AI until calving), and total pregnancy loss. Least squares means (± standard error, %) and genetic parameters were estimated using mixed linear models. Heritability was estimated to be 0.02, 0.02, and 0.03 for embryonic loss, fetal loss, and total pregnancy loss, respectively. Cows with pregnancy loss had lower PAG concentrations than cows which successfully maintained pregnancy and calved. PAG recording was limited to monthly test-day milking, resulting in low estimated embryonic loss (17.5 ± 0.4 and 18.7 ± 0.4 in SR and SH, respectively) and higher fetal loss (32.8 ± 0.5 and 35.1 ± 0.5 in SR and SH, respectively). Pregnancy loss might have occurred earlier but remained undetected until the next test-day milking, when it was recorded as fetal loss rather than embryonic loss. Estimated genetic correlation between embryonic and fetal pregnancy loss traits and classical fertility traits were in general high. Identification of novel genetic traits from PAG data can be highly specific, as PAG are only secreted by the placenta. Thus, PAG could be useful indicators in selection to genetically improve pregnancy maintenance and reduce reproductive losses in milk production. Further studies are needed to clarify how these results could be applied in breeding programs concurrent with selection for classical fertility traits.

Use of GnRH Treatment Based on Pregnancy-Associated Glyco-Proteins (PAGs) Levels as a Strategy for the Maintenance of Pregnancy in Buffalo Cows: A Field Study.

Simple SummaryEmbryonic mortality (EM) is one of the leading causes of infertility in modern breeding. This results in a reduction in reproductive performance, significant economic losses and can affect the environmental impact. The introduction of intensive breeding systems and the selection of high-production animals have accentuated EM, also in buffalo. This study aimed to verify if GnRH treatment at 35 days after artificial insemination of subjects identified at risk of embryonic mortality on the basis of pregnancy-associated glycoprotein (PAG) levels, could reduce EM in buffalo.The aim of the study was to evaluate the effects of GnRH administrated at day 35 after artificial insemination (AI) on the reproductive performance of buffalo cows. In ten buffalo farms in the period January–February, 481 buffalo cows were subjected to estrus synchronization protocol and fixed-time artificial insemination (Ovsynch–TAI program). Radioimmunoassays (RIA) for pregnancy-associated glycoproteins (PAGs) were used to detect pregnancy at day 28 after AI (cut-off value: ≥1 ng/mL). Among pregnant subjects, those with PAG values between 1 and 2.5 ng/mL were considered at risk of embryonic mortality (EM) and were assigned into two groups: treated (T; n = 57) control (C; n = 57). Treated buffaloes received 0.01 mg of buserelin acetate intramuscularly on day 35 after AI, whereas control buffaloes received no treatment. The pregnancy diagnosis was confirmed at day 60 through PAGs level and rectal palpation. The treatment with GnRH had a significant effect (p < 0.001) in reducing EM. Between days 28 and 60 after AI, the animals that experienced EM were only 2/57 in the T group, while were 13/57 in the C group. Moreover, GnRH treatment produced a significant increase (p < 0.001) in the PAG concentration between day 28 and day 60. Administration of GnRH at day 35 after AI in animals considered at risk of low embryo survival based on PAG levels allowed a reduction in pregnancy losses and improved the pregnancy rate during low-breeding season in buffalo.

Early embryo losses, progesterone and pregnancy associated glycoproteins levels during summer heat stress in dairy cows.

Objectives of this study were to characterize the effects of heat stress on pregnancy associated glycoproteins (PAG) and progesterone and its involvement in embryo survival. In trial 1, blood samples collected from days 29 to 36 post insemination were examined for the comparison of PAG concentrations between winter (n = 3721) and summer (n = 2388). In trial 2, embryo losses were assessed in winter (n = 144) and in summer (n = 133), in days 31 or 32 of pregnancy. Pregnancy diagnosis was carried out by ultrasonography on days 24 or 25, and it was repeated a week later; in the second occasion PAG concentration was also determined. In trial 3 the PAG and progesterone concentrations were assessed in days 33 to 36 in winter and summer. In trial 1 PAG levels did not differ between winter and summer, the conception rate and the proportion of uncertain pregnancies were higher in winter than summer. The likelihood of pregnancy was 10 to 15% higher in winter. In trial 2, the embryo death rate was higher in summer, but the PAG levels of cows that had embryo loss in summer were higher than those in winter. In both seasons, lower PAG levels were associated with higher risk of pregnancy loss, while embryo death was five times more likely to occur in summer than in winter and lower PAG concentrations were positively associated with higher risk of embryo loss. In trial 3, mean PAG levels were higher and of progesterone were lower during the summer than during the winter. We infer that despite the devastating effects of heat stress on cows' fertility, those early embryos that survive under continuous heat stress can form a well-functioning placenta; hence, the high embryo mortality rate observed during the summer months could be mainly attributed to luteal insufficiency.

Pregnancy-associated glycoprotein concentrations during early gestation in pregnant Awassi sheep

This study was aimed at determining the serum pregnancy-associated glycoprotein (PAG) profile of pregnant Awassi sheep during early gestation. A total of 122 Awassi ewes were used, which were in days 28-56 (28, 30, 32, 36, 42, 49, and 56) of gestation. Blood samples were taken via jugular venepuncture. Out of these animals, 104 ewes that had delivered (singleton lambs, n = 60; twin lambs, n = 44), according to lambing records, were selected for the assessment of serum PAG levels. The serum PAG level was measured using a commercial enzyme-linked immunosorbent assay (ELISA) kit (IDEXX Bovine Pregnancy Test). Whereas serum PAG levels (mean ± standard error) on days 28, 30, 32, 36 and 42 of gestation were found to be similar (0.237 ± 0.031, 0.402 ± 0.067, 0.329 ± 0.036, 0.397 ± 0.075, and 0.409 ± 0.060, respectively), the levels measured on days 49 and 56 of gestation were significantly higher (0.712 ± 0.088 and 0.798 ± 0.115, respectively) (p &lt; 0.05). The serum PAG levels of the twin-pregnant ewes on days 28, 30 and 42 of gestation were higher than those of the singlepregnant ewes (twin-pregnant ewes: 0.314, 0.648 and 0.594, respectively; single-pregnant ewes: 0.186, 0.258 and 0.308, respectively; p &lt; 0.05). With a cut-off sample minus negative (S–N) value set at &gt; 0.300, the sensitivity of serum PAG level-based pregnancy diagnosis was 51.95% between days 28 and 42 of gestation and 92.69% between days 49 and 56 of gestation. On the other hand, with a cut-off S–N value set at &gt; 0.100, the sensitivity of the PAG ELISA test was 94.81% between days 28 and 42 of gestation and 100% between days 49 and 56 of gestation. In conclusion, the serum PAG levels of the pregnant Awassi sheep remained similar between days 28 and 42 of gestation, but progressively increased thereafter. The serum PAG levels of the twin-pregnant Awassi ewes were significantly higher than those of the single-pregnant sheep on days 28, 30 and 42 of gestation, but they were not different on days 32, 36, 49 and 56 of gestation.

Plasmatic Profile of Pregnancy-Associated Glycoprotein (PAG) during Gestation and Postpartum in Sarda and Lacaune Sheep Determined with Two Radioimmunoassay Systems.

Simple SummaryNowadays the need to optimize and maximize the productivity of dairy sheep leads farmers to plan lambing in specific periods in order to avoid economic loss due to nonproductive animals. The goal is to diagnose pregnancy at early gestation in order to minimize the costs of unproductive animals and to properly formulate rations for the energy requirements of gestating or lactating animals at the same time. Moreover, early pregnancy diagnosis, as well as the possibility of distinguishing between single and multiple gestations, enables farmers to plan the management of lambing well in advance thus minimizing manpower requirements. This study showed, for the first time, the plasmatic profile of PAG (pregnancy-associated glycoproteins) in Sarda and Lacaune ewes during gestation and the postpartum period using two different radioimmune assay (RIA) systems, thus enhancing our knowledge regarding PAG concentrations in domestic ruminants. Moreover, it showed that for both breeds these RIA systems are capable of distinguishing pregnant from nonpregnant ewes starting from day 18 of gestation. Furthermore, the rapid disappearance of PAG concentration following lambing did not require the use of a cut-off limit in postpartum animals as a means for detecting a new pregnancy.This study was carried out to determine ovine pregnancy-associated glycoprotein (oPAG) levels in the plasma of Sarda and Lacaune ewes throughout gestation and in the first month postpartum, using two heterologous radioimmunoassays (RIA-706 and RIA-srPool) and to study the correlations between PAG levels and fetal gender and number. On Day 18 of pregnancy, PAG concentrations were detected in 90.1% and 80.8% of Sarda pregnant ewes with RIA-706 and RIA-srPool, respectively; and in 90% and in 75% of Lacaune pregnant ewes with RIA-706 and RIA-srPool, respectively. From Day 30, PAG concentrations were detected in all pregnant ewes by using both RIA methods. In the postpartum period, the PAG concentrations in Sarda ewes decreased rapidly reaching minimal levels (<1 ng/mL) on day 28 using both RIA-706 and RIA-srPool. In Lacaune ewes, PAG-706 levels were higher than PAG-srPool from parturition until the last day of observation (Day 28 postpartum). It was also observed that mean concentrations were higher in multiple than in single pregnancies in Sarda and Lacaune ewes. Moreover, due to the rapid disappearance of PAG concentration following lambing, a cut-off limit in postpartum animals was not required as a means for detecting a new pregnancy.

Assessment of pregnancy-associated glycoprotein profile in milk for early pregnancy diagnosis in goats.

ObjectiveThis study was conducted to assess the level of pregnancy-associated glycoprotein (PAG) in whole and skim milk samples, and its suitability for early pregnancy diagnosis in goats.MethodsA two-step sandwich enzyme-linked immunosorbent assay (ELISA) system for estimation of milk PAG was developed and validated, which employed caprine-PAG specific polyclonal antisera. Whole and skim milk samples (n = 210 each) from fifteen multiparous goats were collected on alternate days from d 10 to d 30, and thereafter weekly till d 51 post-mating. PAG levels in milk samples were estimated by ELISA and the pregnancies were confirmed at d40 post-mating by transrectal ultrasonography (TRUS).ResultsThe level of PAG in whole and skim milk samples of both pregnant and non-pregnant goats remained below the threshold values until d 24 after mating. Thereafter, PAG concentration in whole and skim milk increased steadily in pregnant goats, whereas it continued below the threshold in non-pregnant does. The PAG profiles in whole and skim milk of pregnant goats were almost similar and exhibited strong positive relationship (r = 0.891; p<0.001). Day 26 post-mating was identified as the first time-point for significantly (p<0.05) higher milk PAG concentration in pregnant goats than to non-pregnant goats. When compared to TRUS examination for pregnancy diagnosis, the accuracy and specificity of PAG ELISA using whole and skim milk samples were 94.5% and 95.4%; and 95.3% and 100%, respectively. The high values of area-under-curve (0.904 [whole milk] and 0.922 [skim milk]), demonstrate outstanding discrimination ability of the milk assays. Among the sampling dates chosen, d 37 post-mating was identified as the best suitable time point for collection of milk samples to detect pregnancy in goats.ConclusionThe PAG concentration in whole and skim milk of goats collected between days 26 and 51 post-breeding can be used for the accurate prediction of pregnancy and may be useful for assisting management decisions in goat flocks.

Pregnancy-associated glycoprotein profile in milk and its relationship with the circulating level during early pregnancy in goats

The level of pregnancy-associated glycoprotein (PAG) in milk and its relationship with the concentration in peripheral circulation during early pregnancy remain uncertain in goats. Therefore, the objectives of present study were to investigate temporal profile of PAG in milk (whole and skim milk) and blood, and to determine the relationship between milk and circulating PAG level during early pregnancy in goats. Milk and plasma samples were collected from multiparous goats (n = 5) on alternate day from d 26 to 30, and thereafter weekly till d 51 of gestation to estimate PAG level using commercial ELISA tests. Pregnancies were confirmed at d 37 of gestation by transrectal ultrasonography (USG). The eff ;ects of days of pregnancy and animal status (pregnant vs. non-pregnant) on PAG levels in milk and plasma were analyzed using linear mixed model. Compared to USG, assay accuracy were 94.4%, 88.9% and 93.3% for whole milk, skim milk and plasma ELISA tests, respectively. Maternal PAG level in milk (both whole and skim milk) and circulation progressively increased with the days of pregnancy during early gestation (P < 0.001). On the contrary, the PAG level of corresponding milk and plasma samples of non-pregnant goats remain unchanged (P > 0.05) and below to cut-off values throughout the experiment. PAG levels in whole and skim milk samples of pregnant goats were similar (P > 0.05) at all-time points with approximately 2-folds lower PAG compared to the plasma samples. A strong positive correlation and high agreement of plasma PAG with PAG levels in whole and skim milk were detected. By using milk, the ELISA showed sensitivity of 100% after d 28 of gestation. In conclusion, the PAG level in milk and peripheral circulation is affected by the day of gestation during early pregnancy and the optimal time to carry out first pregnancy diagnosis using milk or plasma based ELISA is on and after d 28 of gestation. The strong relationship and high agreement of milk PAG with its level in peripheral circulation supports that milk (both whole and skim milk) is a good alternative to plasma for early pregnancy diagnosis from d 28 to d 51 and can effectively be used as a marker of placental functions and fetal health in goats.

Genetic and nongenetic profiling of milk pregnancy-associated glycoproteins in Holstein cattle

Pregnancy-associated glycoproteins (PAG) are secreted by the trophoblast and are detectable in maternal circulation around the time of attachment of the fetal placenta, as well as in blood and milk throughout gestation. The understanding of the genetic mechanisms controlling PAG levels can confer advantages for livestock breeding programs given the precocity and the ease of obtaining this phenotype from routine pregnancy diagnosis. The aim of this study was to characterize PAG levels by estimating genetic parameters and correlations with other dairy traits, and to identify genomic regions and candidate genes associated with PAG levels in milk. The PAG data consisted of pregnancy diagnoses using commercial assays from 2012 to 2017, and genotype data consisted of 54,123 SNP markers for 2,352 individuals (embryos and dams). The model included contemporary group (herd, year, and season) and embryo age as fixed effects, and random embryonic (direct) and maternal (indirect) genetic effects. Using genomic data, the estimated heritability for direct and maternal genetic effects (± standard deviations) were 0.23 ± 0.05 and 0.11 ± 0.05, respectively. The genetic correlation between these effects was almost zero (0.001 ± 0.06). A preliminary analysis revealed low correlations between milk PAG levels and other dairy traits. The genome-wide association analysis was performed using 2 approaches: single-marker and single-step using all markers. Four genomic regions with direct genetic effects were detected on Bos taurus autosome (BTA) 6, BTA7, BTA19, and BTA29 of the embryonic genome. The BTA29 locus was within the bovine PAG gene cluster, suggesting a cis-regulatory quantitative trait locus on the PAG expression. However, other associations, without an obvious link to PAG expression, could be related to the transportation of PAG and their concentration in milk. Only 1 region from the maternal genome, on BTA4, had a significant indirect effect, where WNT2 is a candidate gene related to placenta vascularization and gestation health. Collectively, our results suggest a moderate genetic control of milk PAG levels from both maternal and fetal genomes, but larger studies are needed to fully evaluate the usefulness of milk PAG in the genetic evaluation of fetal growth and cow fertility.

Using pregnancy-associated glycoproteins to provide early pregnancy diagnosis in Nelore cows

Pregnancy-associated glycoproteins (PAG) are expressed in the placenta of ungulate mammals. Ruminants secrete PAGs continuously from embryonic implantation until delivery. In this study, we evaluated the feasibility of using ELISA to detect PAG levels as a method for early pregnancy diagnosis in Zebu (Nelore) beef cows. We compared the efficacy of this method with transrectal ultrasonography (US) in detecting pregnancy on the 25th and 28th days after timed artificial insemination (TAI) of 130 crossbred Zebu cows. Blood samples were taken during these two time points for PAG determination; ultrasound examinations were also performed on both days. Rectal palpation was used on the 80th day to definitively verify pregnancy. PAG ELISA accuracy was equal across both time points, with 100% sensitivity, 92.86% specificity, 96.70% positive predictive value, and 100% negative predictive value. In contrast, US had 62.50% and 93.18% sensitivity, 95.24% specificity, 96.49% and 97.63% positive predictive value, coupled with 54.79% and 86.96% negative predictive value on the 25th and 28th days, respectively. Thus, the two diagnosis methods differed significantly in negative predictive value and sensitivity, but not positive predictive value or specificity. In conclusion, PAG ELISA was just as specific as US, but more sensitive. Its safety (compared with US and rectal palpation) and high accuracy in identifying non-pregnant cows make PAG ELISA a good option for early pregnancy diagnosis (beginning 25 days post-TAI) in Zebu females.

Storage strategies and processing of bovine milk samples for pregnancy-associated glycoproteins detection test

ABSTRACTThree experiments were performed to test if the time and storage conditions of milk samples, the preheating of samples in a water bath, as well as the carryover effect in laboratory analysis equipment could affect the pregnancy-associated glycoproteins (PAG) levels, and consequently the results of a pregnancy test. The pregnancy test used in both experiments uses the enzyme-linked immunosorbent assay method to measure the concentrations of PAG and classify the samples as pregnant, nonpregnant or suspicious. As a result, PAG levels showed no variation when the samples were analyzed up to 9 days after collection, whether stored in ambient temperature or refrigerated. The preheating of the samples in a water bath and prior to the analysis of SCC and composition also did not affect the levels of PAG, allowing the same sample used in the quality analysis to be used for the pregnancy test.

Short communication: Bovine milk sampling efficiency for pregnancy-associated glycoproteins (PAG) detection test

Two experiments were conducted to verify whether the time of day at which a milk sample is collected and the possible carryover in the milking system may affect pregnancy-associated glycoproteins (PAG) levels and, consequently, the pregnancy test results in dairy cows. In experiment one, we evaluated the effect of time of day at which the milk sample is collected from 51 cows. In experiment two, which evaluated the possible occurrence of carryover in the milk meter milking system, milk samples from 94 cows belonging to two different farms were used. The samples were subjected to pregnancy test using ELISA methodology to measure PAG concentrations and to classify the samples as positive (pregnant), negative (nonpregnant), or suspicious (recheck). We found that the time of milking did not affect the PAG levels. As to the occurrence of carryover in the milk meter, the PAG levels of the samples collected from Farm-2 were heavily influenced by a carryover effect compared with the samples from Farm-1. Thus, milk samples submitted to a pregnancy test can be collected during the morning or the evening milking. When the sample is collected from the milk meters, periodic equipment maintenance should be noted, including whether the milk meter is totally drained between different animals’ milking and equipment cleaning between milking is performed correctly to minimize the occurrence of carryover, thereby avoiding the effect on PAG levels and, consequently, the pregnancy test results. Therefore, a single milk sample can be used for both milk quality tests and pregnancy test.

Early pregnancy diagnosis using a commercial ELISA test based on pregnancy-associated glycoproteins in Holstein-Friesian heifers and lactating cows

In this study, we aimed to investigate the efficacy of a commercial ELISA test kit for detecting pregnancy-associated glycoproteins (PAGs) in peripheral blood for early pregnancy diagnosis and compare plasma PAG levels during early pregnancy in both Holstein-Friesian heifers and lactating cows. A total of 231 plasma samples were collected from heifers and lactating cows on days 25, 28, and 32 after insemination. Pregnancies were confirmed 30 days after the collection of plasma samples. Plasma PAG levels were measured using a commercial ELISA test kit for diagnosing bovine pregnancy. The effects of examination date and animal status (heifers vs. lactating cows) on PAG levels in heifers and lactating cows were analyzed using two-way ANOVA. Plasma PAG levels were significantly higher in heifers than in lactating cows; levels also increased significantly with the date of examination, i.e. days 25, 28, and 32 for heifers (P > 0.001), but not for lactating cows (P > 0.05). Although the sensitivity, specificity, and accuracy of pregnancy diagnosis using the ELISA test were acceptable in both groups, the performance of the test was superior in pregnant heifers compared to lactating cows.

Evaluation of a bovine pregnancy-associated glycoprotein enzyme-linked immunosorbent assay kit for serological diagnosis of pregnancy in sheep

ABSTRACT: Pregnancy diagnosis is an important tool for farm management. Ultrasonography is the main technique used for pregnancy diagnosis in ewes. As an alternative, radioimmunoassay (RIA) allows accurate and early detection of pregnancy-associated glycoproteins (PAGs) in sheep blood. However, radioactive-based techniques, as RIA, have been increasingly inadvisable due to environmental risk. Homology between ovine and bovine PAGs is high, and ELISA kits used for PAGs detection in cattle are safer than RIA. Thus, this study aimed to evaluate the feasibility of PAGs detection for pregnancy diagnosis in sheep serum samples using an ELISA kit produced for cattle. The sensitivity and specificity of the ELISA kit were 93.5% and 98.9%, respectively, whereas positive and negative predictive values were 99.0% and 93.1%, respectively, in comparison to ultrasonography diagnostic (control). PAGs reached consistently detectable concentrations in ovine serum around 33 days after mating. Accuracy of the ELISA test was 96.1% from 33 days of pregnancy until lambing. After parturition, PAGs were still detectable seven days post-lambing. However, from 21 days post-parturition, PAGs from the previous pregnancy were no longer detected in serum samples. In conclusion, the bovine ELISA kit can accurately detect pregnancy in sheep 33 days following mating, while PAGs levels from the previous gestation are no longer detected from 21 days post-partum. The evaluated ELISA test is a reliable tool for pregnancy diagnosis in sheep at random stages and as a complementary exam at early gestation.

Pregnancy outcomes based on milk pregnancy-associated glycoprotein levels

Timely diagnosis of pregnancy and pregnancy losses is economically important. A commercially available pregnancy-associated glycoprotein (PAG) milk assay (IDEXX Laboratories, Inc.) is offered through routine Dairy Herd Improvement (DHI) testing for diagnosis of pregnancy. The objective of this observational study was to describe the relationship between PAG at various stages of gestation and the likelihood of successful calving. The hypothesis was that higher PAG levels would be associated with successful calving.

Evaluation of norgestomet Crestar® on oestrus synchronization and reproductive performance of dairy cows in Algeria

Abstract Objective To evaluate the effectiveness of norgestomet Crestar® for synchronization of oestrus and its effect on pregnancy rate in cows. Methods after a waiting period (∼90 days postpartum), Holstein-Frisian females kept in different dairy farms were inseminated for the detection of estrous behavour ( n =133). Females were allocated randomly to treated ( n =69) or control ( n =64) groups. The treated animals received the Crestar® treatment during 9–10 days. After implant removal, the treated animals were artificially inseminated (AI) for the detection of standing estrous behaviour. Blood samples were collected on Days 0, 21 and 30 after service. Plasma progesterone (P4) and pregnancy-associated glycoprotein (PAG) levels were determinate by radioimmunoassay. Results the study showed a higher rate of heat synchronization in the treated group (94%) compared to the control group (86%). Out of 133 cows inseminated, thirteen (4 treated and 9 control) had a plasma progesterone concentrations higher than 1.0 ng/mL at service. The plasma PAG levels at Day 30 after AI were significantly lower in the treated group (7.13 ± 2.29) compared to the control group (8.37 ± 2.30). Pregnancy rate was 28% (16/57) and 42% (23/54) for treated and untreated group, respectively. There were no significant differences in the pregnancy rates of different parity in both groups. Conclusion The norgestomet Crestar® treatment carried out during 9–10 days did not improve reproductive performance in dairy cattle. However, these data demonstrate that this hormonal protocol can be used to obtain high oestrus cow rates to initiate correct timing of insemination.

Factors associated with pregnancy-associated glycoprotein (PAG) levels in plasma and milk of Holstein cows during early pregnancy and their effect on the accuracy of pregnancy diagnosis

Lactating Holstein cows (n=141) were synchronized to receive their first timed artificial insemination (TAI). Blood and milk samples were collected 25 and 32d after TAI, and pregnancy status was determined 32d after TAI using transrectal ultrasonography. Cows diagnosed pregnant with singletons (n=48) continued the experiment in which blood and milk samples were collected and pregnancy status was assessed weekly using transrectal ultrasonography from 39 to 102d after TAI. Plasma and milk samples were assayed for pregnancy-associated glycoprotein (PAG) levels using commercial ELISA kits. Compared to ultrasonography, accuracy was 92% for the plasma PAG ELISA test and 89% for the milk PAG ELISA test 32d after TAI. Plasma and milk PAG levels for pregnant cows increased from 25d to an early peak 32d after TAI. Plasma and milk PAG levels then decreased from 32d after TAI to a nadir from 53 to 60d after TAI for the plasma PAG assay and from 46 to 67d after TAI for the milk PAG assay followed by an increase from 74 to 102d after TAI. Overall, plasma PAG levels were approximately 2-fold greater compared with milk PAG levels, and primiparous cows had greater PAG levels in plasma and milk compared with multiparous cows. The incidence of pregnancy loss from 32 to 102d after TAI based on ultrasonography was 13% for cows diagnosed with singleton pregnancies, and plasma and milk PAG levels decreased to nonpregnant levels within 7 to 14d after pregnancy loss. Both plasma and milk PAG levels were negatively correlated with milk production for both primiparous and multiparous cows. We conclude that stage of gestation, parity, pregnancy loss, and milk production were associated with plasma and milk PAG levels after TAI similarly. Based on plasma and milk PAG profiles, the optimal time to conduct a first pregnancy diagnosis is around 32d after AI, coinciding with an early peak in PAG levels. Because of the occurrence of pregnancy loss, all pregnant cows should be retested 74d after AI or later when plasma and milk PAG levels in pregnant cows have rebounded from their nadir.

A pregnancy detection assay using milk samples: Evaluation and considerations

Two experiments were conducted to evaluate a pregnancy-detection assay based on the measurement of pregnancy-associated glycoproteins (PAG) in milk samples. In experiment 1, milk samples were collected on the day of first pregnancy check (33–52 d postinsemination; n=119) or second check (60–74 d postinsemination; n=60). The accuracy in identification of pregnant and nonpregnant cows was 99% at first check. Only 6% of samples were found to be within an intermediate range of PAG concentrations and classified as requiring recheck by the assay. At second check, the accuracy of the assay was 98%. Fifteen percent of these samples were classified as requiring recheck. In experiments 2a (n=17 cows) and 2b (n=16 cows), milk and plasma samples were collected from cows at weekly intervals beginning 2 (experiment 2a) or 4 d (experiment 2b) after insemination. The earliest time point at which pregnant cows were accurately classified as pregnant by the assay was on d 30 postinsemination. A transient decline in PAG levels into the intermediate range was observed on d 46 to 72 postinsemination. This coincides with the time of recheck in experiment 1. Results obtained with the plasma samples were essentially the same. The accuracy of pregnancy identification based on milk samples from nonpregnant and pregnant cows was 99%. Levels of PAG in milk were useful in identifying 6 incidences of embryonic mortality. No consistent relationship was noted between the timing of the decline in PAG levels and the timing of luteal regression in this small number of cows.