The aim of this study was to investigate the interferon tau (IFNt) concentration in the peripheral maternal blood during the early phase of pregnancy in buffalo cows and improve the knowledge on the physiological importance of circulating IFNt, evaluating the possible interaction with pregnancy-associated glycoproteins (PAGs) and progesterone (P4). Blood samples were taken from buffalo cows on day 0 (day of AI), 7, 14, 18, 28, and 40 post insemination for the IFNt, PAG, and P4 analysis and to determine the IFNt mRNA expression. The animals were categorized ex post into Pregnant, Non-pregnant and Embryo mortality groups. The interferon value was influenced by group (p = 0.003), being always higher in pregnant buffalo cows than in non-pregnant ones, while the embryo mortality group showed intermediate values between those for pregnant and non-pregnant animals. The mRNA expression of IFNt was not influenced by groups or any time points. The regression analysis that included IFNt as the independent variable showed that PAGs, from day 18 (p < 0.01), and P4, from day 28 (p < 0.05), were positively associated with IFNt values. The close associations among IFNt, PAGs and P4 demonstrate that all three molecules work together for fetal-placental well-being and pregnancy support. Unfortunately, the great individual variability in circulating IFNt makes this analysis unsuitable for early pregnancy diagnosis.

Preventing dystocia can stabilise beef cattle management. This study aimed to investigate the relationship between serum pregnancy-associated glycoproteins (PAGs) S-N values and estrone sulphate (E1S) concentrations during pregnancy and the calf birth weight in beef cattle and to evaluate their usefulness as new predictive parameters for dystocia due to foetal overgrowth. Thirty-eight pregnant Japanese Black cattle were used. Blood samples were collected at 40, 70, 100, 150, 200, 250, 280, and 285 days after artificial insemination (AI), and birth weight of the offspring was measured. Serum PAGs S-N values and E1S concentrations were measured, and the area under the curve (AUC) and the ratio of change based on 70 days after AI were calculated, followed by calculation of the correlation coefficient with the birth weight of the offspring and comparison between the eutocia (n = 32) and dystocia (n = 6) groups. The birth weight of the offspring was moderately positively correlated with the AUC of serum PAGs S-N values and E1S concentrations in the second (r = 0.425, P < 0.01) and third (r = 0.595, P < 0.01) trimesters, respectively. The ratio of change in serum E1S concentrations between 70 and 280 days after AI was greater (P < 0.05) in the dystocia group (1276.6 ± 229.1 %) than in the eutocia group (852.6 ± 69.6 %). These results suggest that blood PAGs S-N values at mid-pregnancy (100-199 days after AI) and the ratio of changes in blood E1S concentrations between 70 and 280 days after AI may be new parameters for predicting dystocia.

This study aimed to investigate the impact of ambient lead (Pb) exposure on progesterone (P4) and pregnancy-associated glycoprotein 1 (PAG1) and their relationship with abortion in Egyptian Zaraibi goats (C. hircus). To achieve this, 40 female goats (does) were mated with highly fertile male goats, resulting in a total of 28 pregnant goats. Eight of them aborted, and each of the 12 pregnant goats gave birth to one kid, whereas the remaining eight gave birth to twins. The levels of PAG1, P4, and Pb in serum were estimated by enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and inductively coupled plasma mass spectrometry (ICP-MS) respectively. Statistically, the repeated measure two-way ANOVA, regression analysis, correlation coefficient, and receiver operating characteristic (ROC) curves were applied. The current data demonstrated that the levels of blood Pb in aborted goats were significantly higher than those in non-aborted goats at the early, mid, and late gestations, and this was followed by significant decreases in serum PAG1 and P4. Furthermore, there were substantial inverse associations between blood Pb concentration and levels of PAG1 and P4, with markedly negative correlation coefficients of - 0.88 and - 0.77, respectively, in aborted goats. The threshold level of Pb required to cause abortion was ≥ 32.08 μg/dl, but for PAG1 and P4 were respectively ≤ 0.95 ng/ml and ≤ 0.48 ng/ml. Additionally, threshold levels of ≥ 12.34 ng/ml and ≥ 31.52 ng/ml for P4 and PAG1, respectively, were needed to deliver twins. In conclusion, pollution-induced increases in Pb bioavailability resulted in dramatic decreases in P4 and PAG1 levels, leading to abortions. PAG1 and P4 levels are also key factors in determining whether Zaraibi goats will give birth to twins.

This manuscript reviews the mechanisms that maintain the corpus luteum (CL) of pregnancy in ruminants. In mammals, ovulation and luteinization of the remaining cells in the CL are due to a surge in Luteinizing Hormone (LH). In cattle, continued secretion of pulses of LH is essential for full development and function of the CL during the estrous cycle (LH pulses), however, the few studies on the CL after d20 of pregnancy do not indicate that LH is essential for maintaining the CL of pregnancy. The first essential step in maintaining the CL of pregnancy in ruminants is overcoming the mechanisms that cause regression of the CL in non-pregnant ruminants (d18-25 in cattle; d13-21 in sheep). These mechanisms have a uterine component involving oxytocin-induced prostaglandin F2α (PGF2A) pulses and a luteal component involving decreased progesterone production and luteal cell death. There is a critical role for embryonic interferon-tau (IFNT) in suppressing the uterine secretion of PGF2A during early pregnancy (d13-21 in sheep; d16-25 in cattle) and preventing luteolysis. There are also effects of IFNT on the expression of interferon-stimulated genes in other tissues including the CL but the physiologic role of these interferon-stimulated genes is not yet clear. After the IFNT period, there is another mechanism that maintains the CL of pregnancy in ruminants since embryonic IFNT is inhibited as attachment occurs and trophoblastic binucleate/giant cells begin secretion of pregnancy-associated glycoproteins. The second mechanism for luteal maintenance has not yet been defined but acts in a local manner (ipsilateral to pregnancy), and remains functional from d25 until just before parturition. The most likely mechanisms mediating later maintenance of the CL of pregnancy are increased uterine blood flow or decreased prostaglandin transporter expression in the utero-ovarian vasculature, preventing PGF2A reaching the CL. Finally, implications of these ideas on pregnancy loss in cattle are explored, highlighting the importance of inappropriate regression of the CL of pregnancy as a mechanism for pregnancy loss in cattle.

The aim of this study was to determine and compare the diagnostic accuracy of two pregnancy-associated glycoproteins tests (IDEXX on-farm pregnancy test [OFPT] and IDEXX rapid visual pregnancy test [RVPT]) for early pregnancy diagnosis in dairy cattle. Blood samples were collected from Holstein cows (n = 317) by coccygeal venipuncture 28-31 days after artificial insemination (AI). The OFPT and RVPT were performed on the farm within 2 h after blood collection using whole blood or blood serum. Transrectal ultrasonography (USG) was performed for pregnancy diagnosis on day 32 post-AI as a gold standard. One-hundred fourteen cows were diagnosed as pregnant and 203 were determined to be nonpregnant. Furthermore, embryonic mortality was detected in four of the nonpregnant animals, based on fragmented/dispersed embryonic membranes and the absence of heartbeat. Sixteen and 14 false positive results (13 jointly for both tests) were obtained for the RVPT and OFPT, respectively. Three false negative results were obtained for the RVPT. The sensitivity, specificity, positive predictive value, negative predictive value and accuracy for the OFPT were 100%, 93.1%, 89.1%, 100% and 95.6%, and for RVPT were 97.4%, 92.1%, 87.4%, 98.4% and 94.0%, respectively. The ability of both tests to distinguish between pregnant and open cows was very good (AUC of both tests above 0.9). The OFPT and RVPT almost perfectly agreed with each other. According to McNemar\'s analysis, false positive results have been caused difference between the two pregnancy tests and USG. In conclusion, both the RVPT and OFPT proved to be reliable and practical methods for pregnancy diagnosis 28-31 days after AI in dairy cows. However, the results of both pregnancy tests were affected by the occurrence of embryonic mortality around the time of their employment.

The objective of this study was to evaluate the use of corpus luteum (CL) color Doppler (CD) ultrasonography and pregnancy-associated glycoproteins (PAG) for early pregnancy diagnosis and examine their ability to predict late embryonic/early fetal mortality (LEM) in Bos taurus beef replacement heifers. Beef heifers (n = 178) were exposed to a 7-d CO-Synch + CIDR protocol followed by fixed-time artificial insemination (day 0). On days 20 and 22, B-mode and CD ultrasonography were performed to evaluate CL morphometries and blood perfusion, respectively. Heifers were considered nonpregnant when CL area was <2 cm2 or estimated luteal blood perfusion was ≤30% of the total luteal area. Blood samples were collected on days 25 and 29 to estimate circulating concentrations of PAG. Conventional ultrasonography on days 29 and 94 was utilized to determine pregnancy status and considered the gold standard method for pregnancy diagnosis. Pregnant heifers had greater (P < 0.01) CL diameter, area, volume, and blood perfusion when compared with nonpregnant heifers on days 20 and 22. Accuracy of CD on days 20 and 22, and PAG on days 25 and 29 were 91%, 94%, 96%, and 98%, respectively. No false-negative results were observed for CD on both days 20 and 22 (negative predicted value = 100%) and false-positive results represented 8% and 6% of the diagnoses. Heifers that experienced LEM between days 29 and 94 of gestation had decreased luteal (P = 0.02) volume on day 20 and tended (P = 0.07) to have decreased concentrations of PAG on day 29 compared with heifers that maintained pregnancy. However, both CD and PAG failed to predict embryonic mortality. In conclusion, CD successfully detected most nonpregnant replacement heifers as early as day 20 of gestation, while resulting in no false negative diagnoses. Both CD and PAG failed to predict LEM in the present study.
Identifying nonpregnant females early after breeding allows cattle producers to rapidly rebreed females that failed to conceive after their first artificial insemination and consequently increases reproductive efficiency. Additionally, embryonic and fetal mortality during early gestation are the main drivers of pregnancy failure and represents a significant economic burden to both the beef and dairy industries. This study evaluated the use of color Doppler (CD) ultrasonography of specific ovarian structures and blood concentrations of pregnancy-associated glycoproteins (PAG) as potential methods to diagnose pregnancy earlier than industry-standard techniques. Moreover, the present study investigated the use of CD and blood PAG as predictor of pregnancy loss. Data generated here indicates that CD and blood PAG can accurately detect pregnancy as early as 20 and 25 d after insemination, respectively. In the present study, heifers that experienced pregnancy loss between days 29 and 94 of gestation tended to have decreased plasma concentrations of PAG during early pregnancy. Heifers experiencing pregnancy loss between days 29 and 94 of gestation were also more likely to have a luteal cavity on day 20 of gestation and had decreased luteal volume on the same day. However, results reported herein indicate that both CD and blood concentrations of PAG failed to predict pregnancy loss in replacement heifers.

Pregnancy success represents a major issue for the economic income of cattle breeders. Early detection of pregnant and non-pregnant animals, as well as the prediction of early pregnancy failure, can influence farm management decisions. Several diagnostic tools for pregnancy are currently available. Among these, pregnancy-associated glycoproteins (PAGs) have been shown to be useful for identifying the presence of vital embryos and for pregnancy follow-up monitoring. This review presents an overview of the PAGs\' functions, their pregnancy trends, and their use as a tool to improve reproductive management in bovine and other dairy livestock, such as small ruminants and buffalos.

The objective of the current study was to evaluate pregnancy-associated glycoproteins (PAGs) based enzyme-linked immunosorbent assay (ELISA) kits utilizing whole blood, serum, or milk samples for diagnosis of early pregnancy status in lactating Nili-Ravi buffaloes. Dairy buffaloes (n = 174) of mixed parity, 4-6 years of age, having mean (± standard deviation) days in milk 165 ± 87, and body condition score of 3.26 ± 0.34 were randomly enrolled in this study. Buffaloes were exposed to penile deviated bulls with 12 h interval for estrus detection during peak breeding season and eventually bred naturally at their respective standing estrus (day 0). Blood and milk samples were collected at days 24, 28, and 35 post-breeding to run a rapid visual pregnancy test® (RVPT) as a buffalo-side test or ELISA-based assay in the laboratory to detect early pregnancy status. Transrectal B-mode ultrasonography was performed to diagnose pregnancy at day 35 post-breeding and used as a gold standard to validate results of RVPT or ELISA-based tests. The RVPT is a visual readout test for pregnancy detection and had sensitivity (77.9 vs. 89.7 vs. 93.3%), specificity (77.9 vs. 89.7 vs. 93.3%), and accuracy (84.5 vs. 90.1 vs. 94.2%) at days 24, 28, and 35 post-breeding, respectively. The PAGs were assayed using ELISA kits in serum and had sensitivity (77.9 vs. 89.7 vs. 93.3%), specificity (84.2 vs. 87.7 vs. 93.9%), and accuracy (82.1 vs. 88.4 vs. 93.7%) at days 24, 28, and 35 post-breeding, respectively. Similarly, PAGs were also analysed using ELISA kits in milk samples and had sensitivity (77.6 vs 89.5 vs 95.0%), specificity (89.1 vs 91.9, vs 93.9%), and accuracy (85.1 vs 91.1 vs 94.3%) at days 24, 28, and 35 post-breeding, respectively. Overall, the Kappa values in this study exceeded 0.85 at day 35 post-breeding using RVPT or ELISA-based test kits in serum or milk samples, indicating a high level of agreement between PAGs detection method and gold standard for pregnancy diagnosis. The pregnancy outcomes based on ELISA-based PAGs detection at day ≥28 post-breeding had a high negative predictive value, indicating that the probability of incorrectly administering prostaglandins to pregnant buffaloes would be low if these tests were implemented on a commercial dairy herd. Taken together, it is concluded that PAGs-based determination of pregnancy using RVPT or ELISA in blood, serum, or milk samples can be used effectively for pregnancy diagnosis at ≥28 days post-breeding with more than 90% accuracy in Nili-Ravi buffaloes.

The objective of this study was to evaluate the use of luteal color doppler (CD) ultrasonography and plasma concentrations of pregnancy-associated glycoproteins (PAG) for early pregnancy diagnosis in Bos taurus beef cows. Additionally, CD and PAG were evaluated as potential predictors of late embryonic/early fetal mortality (LEM). Postpartum beef cows (n = 212) were exposed to estrus synchronization followed by fixed-time artificial insemination (day 0). On days 20 and 22, B-mode and CD ultrasonography were performed to evaluate corpus luteum (CL) morphometries and blood perfusion. Moreover, blood samples were collected on days 25 and 29 to quantify circulating concentrations of PAG. Conventional ultrasonography on days 29 and 100 was utilized as the gold-standard method for pregnancy diagnosis. Cows that experienced pregnancy loss between days 29 and 100 were classified as LEM. Pregnant cows had larger and more vascularized CL compared with nonpregnant cows on days 20 and 22 (P < 0.001 for all response variables). Accuracy for CD on days 20 and 22 were 87% and 92%, respectively. Accuracy for PAG on days 25 and 29 were 84% and 99%, respectively. No false negative (FN) results were observed for CD on both days 20 and 22; however, there were 7.1% FN results for PAG on day 25. Cows that experienced LEM had decreased (P = 0.04) circulating PAG on day 29 of gestation compared with cows that maintained pregnancy; however, there were no differences in luteal blood perfusion on days 20 and 22 (P ≥ 0.53) or circulating PAG on day 25 (P = 0.46) between LEM cows and cows that maintained pregnancy. Sensitivity and specificity of PAG on day 29 as predictors of LEM were 83% and 77%, respectively. In conclusion, CD resulted in accurate pregnancy diagnosis in B. taurus beef cows on both days 20 and 22 of gestation, while having no FN results. Circulating concentrations of PAG were decreased in cows that experienced LEM; however, further research is required to utilize PAG as a predictor of LEM commercially.
Early pregnancy detection allows cattle producers to increase reproductive efficiency. Pregnancy failures associated with embryonic mortality represents an economic burden to the beef production industry. The present study evaluated the use of color doppler (CD) ultrasonography of specific ovarian structures and pregnancy-associated glycoproteins (PAG) in plasma as potential alternatives to diagnose pregnancy earlier than industry-standard methods in beef cows with Bos taurus genetics. Additionally, the present study evaluated the use of these methods to predict embryonic mortality during early gestation. The results of the present study indicate that: 1) CD can accurately recognize most nonpregnant cows with B. taurus genetics as early as 20 days after breeding. 2) Plasma concentrations of PAG resulted in suboptimal accuracy on day 25 of gestation. 3) Cows that undergo pregnancy loss between 29 and 100 d after breeding have decreased concentrations of PAG in their circulation on day 29, even though they were identify as pregnant with ultrasonography on the same day. 4) Both blood concentrations of PAG and luteal blood perfusion estimated by CD failed to accurately predict pregnancy loss.

The pregnancy-associated glycoproteins (PAGs) have been widely used as biomarkers for the early diagnosis of pregnancy in cattle and sheep. This study aimed to obtain the single-stranded DNA aptamers that specifically bound to ovine pregnancy-associated glycoprotein 7 (ovPAG7) with high affinity (Kd = 9.8-32.4 nM) using real serum sample-assisted FluMag-systematic evolution of ligands by exponential enrichment (SELEX). Subsequently, the selected aptamers were applied to fabricate an ultrasensitive colorimetric aptasensor for ovPAG7 detection based on functionalized magnetic microparticles and hybridization chain reaction. Under the optimized conditions, the colorimetric aptasensor exhibited a broad linear range (0.2-500 ng mL-1), low detection limit (0.081 ng mL-1), good recovery rate (94.5-109.1%), and high repeatability (relative standard deviation of 4.02-8.16%) in ovPAG7-spiked serum. Furthermore, this aptasensor was applied to measure the ovPAG7 in serum samples of ewes for pregnancy diagnosis. Blood samples were collected from Chinese Merino ewes on days 22, 28 after artificial insemination (AI) for ovPAG7 detection, respectively. Transrectal ultrasonography diagnosis of pregnancy 45 days after AI was the reference (gold) standard for all PAG tests. Diagnostic sensitivity, specificity, and accuracy of the proposed aptasensor were 95.8, 87.5, and 92.5% at day 22 and 95.8, 90.6, and 93.7% at day 28, respectively. The degree of agreement (Kappa) between developed aptasensor and ultrasonography diagnosis 22 and 28 day after AI were higher than 0.8. These results illustrated that the aptasensor was proved to be a sensitive, reliable and cost-effective way of measuring PAG and might be a useful means of pregnancy detection in ewes.