Effect of ambient lead on progesterone and pregnancy-associated glycoprotein 1 and their relationship with abortion in Zaraibi goats: a field study.

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.

Review: Maintenance of the ruminant corpus luteum during pregnancy: interferon-tau and beyond.

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.

Comparative evaluation of two commercial pregnancy-associated glycoproteins tests for early detection of pregnancy in dairy 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.

Evaluating the use of luteal color Doppler ultrasonography and pregnancy-associated glycoproteins to diagnose pregnancy and predict pregnancy loss in Bos taurus beef replacement heifers.

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.

Using Pregnancy-Associated Glycoproteins (PAGs) to Improve Reproductive Management: From Dairy Cows to Other Dairy Livestock.

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.

Validation of pregnancy associated glycoproteins-based ELISA kits to determine early pregnancy status in lactating Nili-Ravi buffaloes.

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.

Selecting specific aptamers that bind to ovine pregnancy-associated glycoprotein 7 using real serum sample-assisted FluMag-SELEX to develop magnetic microparticle-based colorimetric aptasensor.

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.

Luteal color doppler ultrasonography and pregnancy-associated glycoproteins as early pregnancy diagnostic tools and predictors of pregnancy loss in Bos taurus postpartum beef cows.

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.

American Bison (Bison bison) reproductive endocrinology: serum Pregnancy Associated Glycoproteins (PAG), Progesterone, Estrone and Estrone-Sulfate in non pregnant animals and during gestation.

This study describes concentrations of Pregnancy Associated Glycoproteins (PAG), progesterone (P4), estrone (E1) and estrone-sulfate (E1S) in American Bison sera. In 2 ranches, mature American Bison were sampled once a year for 2 yr. Subsequent American Bison cows calving days were reported. PAG concentration was determined by Radio-Immuno Assay, whereas P4, E1 and E1S were assayed using Liquid Chromatography and Mass Spectrometry. Concentrations were compared between American Bison bulls (B, n = 7), Nonpregnant cows (NP, n = 32), first (1TP, n = 3), second (2TP, n = 26) and third (3TP, n = 15) trimester of pregnancy. Seven American Bison bulls and 92 cows were sampled, 51 calved during these 2 yr. Calving occurred mostly in spring (74.5%), but also in summer (13.7%) and fall (11.8%). PAG and P4 were higher in 2TP and 3TP than B and NP (P< 0.0001). P4 was non-basal in B and NP. E1 and E1S were correlated (P< 0.0001; r = 0.76) and increased in 2TP and 3TP when compared with B and NP (P< 0.01). Moreover, E1S was higher in 3TP than in 2TP (P< 0.0001) and correlated to pregnancy day (P< 0.0001; r = 0.60). Breeding American Bison in Belgium induces a calving seasonality loss. P4 slowly increases in 1TP and remains steady and high in 2 and 3TP. P4 non-basal and variable concentrations in B or NP disable its use as gestation marker. American Bison produce PAG in the 2 and 3TP, but Estrone-sulfate assay seems to be the best pregnancy marker during the 2 last trimesters as it could help to estimate the gestation period.

Resynchronizing the first eligible estrus in dairy cattle after a prior insemination and fertility of the prior insemination after gonadotropin-releasing hormone and progesterone treatments.

The hypothesis of this study tested whether application of designed treatments to synchronize estrus in nonpregnant previously inseminated lactating dairy cows increased the proportion of nonpregnant cows in estrus before early pregnancy diagnosis on Day 32 after the previous insemination (Day 0) and increase fertility of the pretreatment insemination. A progesterone insert (CIDR) and GnRH were applied to cows after insemination to resynchronize the returning estrus of cows that failed to conceive on Day 0. The combination of GnRH (Day 14) and a CIDR insert (d 17 through 24) in experiment 1 (n = 347 cows) did not increase (P = 0.13) the proportion of nonpregnant cows returning to estrus before pregnancy diagnosis, but increased (P < 0.01) the synchrony of their return by 24.4% points, and delayed (P < 0.01) that return by 2.3 ± 0.3 d compared with controls. Ovulation risk after GnRH treatment on Day 14 was only 10%. For cows that failed to return to estrus before Day 32, progesterone concentration on Days 14 and 17 were less than that in cows that returned to estrus by Day 32 and in pregnant cows. Cows that returned to estrus had larger follicles and fewer numbers of CL on Day 21 than pregnant cows and cows that failed to return to estrus, but concentrations of pregnancy-associated glycoproteins on Day 28 indicated that cows failing to return to estrus were likely pregnant but suffered embryo death. In experiment 2 (n = 881), use of GnRH alone (Day 7), a CIDR insert alone (Days 14 through 21), or in combination, failed to increase the proportion of nonpregnant cows in estrus before pregnancy diagnosis on Day 32 compared with controls. Cows receiving the CIDR insert had increased (P < 0.01) synchrony of estrus by 24-34% points compared with cows that did not receive a CIDR insert. More cows receiving GnRH had 2 or more CL on Days 14 and 21 compared with controls. Ovulation risk after GnRH on Day 7 was greater than 66%. In both experiments combined, treatments with GnRH or GnRH + CIDR insert increased (P = 0.015) pretreatment pregnancy per AI by 7.1% points, but did not affect pregnancy loss. Although administering GnRH with or without a CIDR insert synchronized returns to estrus, treatments failed to increase the proportion of nonpregnant cows reinseminated before pregnancy diagnosis, but increased pretreatment pregnancy risk.

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.

Approaches to Identify Pregnancy Failure in Buffalo Cows.

The aim of this work was to find the best strategy to diagnose pregnancy failures in buffalo. A total of 109 animals belonging to a buffalo herd subjected to a synchronization and artificial insemination (AI) program were enrolled in this study. Blood samples were collected at days 0, 14, 25, 28 and 40 after AI for the determination of progesterone (P4) and pregnancy-associated glycoproteins (PAGs) by the radioimmunoassay (RIA) method. Transrectal ultrasonography was performed on day 25, 28 and 40 after AI to monitor pregnancy. The animals included in the data analysis were assigned ex post in pregnant (n = 50) and mortality (n = 12) groups. By ultrasonography, the predictive sign of mortality was the heartbeat. At day 25, the PAGs concentration was significant in predicting embryonic mortality with respect to ultrasonography and P4, at the cut-off of 1.1 ng/mL. At day 28, either PAGs, at a cut-off of 2.2 ng/mL, or ultrasonography, with no detection of heartbeat, were highly predictive of embryonic mortality. PAGs were the best marker (p < 0.05) for predicting embryonic mortality between 25 and 40 days of gestation in buffalo. Its utilization as a diagnostic tool can influence management decisions in order to improve farm reproductive management.

Preconceptional diet manipulation and fetus number can influence placenta endocrine function in sheep.

Changes in maternal nutrition during pregnancy can result in profound effects on placental function and fetal development. Although the preconceptional period holds the potential to reprogram embryonic and placental development, little is known regarding the effects of premating nutritional manipulation on placental function and fetal and postnatal offspring growth. To test this, Polypay-Dorset sheep (n = 99) were assigned to 1 of 3 nutritional treatments (n = 33/treatment) receiving 50% (UN: undernutrition), 100% (C: control), or 200% (ON: overnutrition) of maintenance energy requirements for 21 d before mating during April-May (increasing photoperiod). Thereafter, diets were the same across groups. We evaluated maternal reproductive variables and maternal and offspring weight and body mass index through weaning. Maternal plasma was collected through pregnancy until postnatal day 1 to assay pregnancy-associated glycoproteins (PAGs) and progesterone. Fertility rate was similar among treatments, but ON females had a higher reproductive rate (UN: 82%; C: 100%, ON: 145%). When correcting by total birth weight, twin pregnancies had lower PAGs and progesterone versus singleton pregnancies (P < 0.001). At birth, UN lambs were heavier than C lambs regardless of birth type (P < 0.01). Growth velocity, daily gain, and weaning weight were similar, but UN and ON females grew faster and were heavier at weaning versus C females. We demonstrated that a 3-wk preconceptional maternal undernutrition or overnutrition, when correcting by total birth weight, results in lower endocrine capacity in twin pregnancies. Preconceptional maternal undernutrition and overnutrition increased postnatal female lamb growth, suggestive of reprogramming of pathways regulating growth before conception. This highlights how preconceptional nutrition can result in marked sex-specific differences.

Selection, identification, and application of DNA aptamers against bovine pregnancy-associated glycoproteins 4.

The bovine pregnancy-associated glycoproteins (bPAGs) have been widely used as robust markers for early diagnosis of pregnancy in the cattle. The current immune recognition methods for detecting bPAGs are limited and, to a certain extent, are associated with high costs and poor stability of the antibody. Aptamers that are more stable and easily synthesized than antibodies might serve as suitable candidates for the development of rapid detection methods. This paper describes selection and characterization of bPAG4 aptamers and theirs applicability to detect bPAG4 in the serum. In this work, the recombinant bovine pregnancy-associated glycoproteins 4 (bPAG4) with a relative molecular mass of about 48 kDa was successfully expressed in human embryonic kidney 293 (HEK 293) cells. Subsequently, the ssDNA aptamers were selected by systematic evolution of ligands by exponential enrichment (SELEX) using magnetic beads (MB) coated with bPAG4 as target. After 9 rounds of selection, three aptamers with high affinity to bPAG4 (Kd = 11.7~40.2 nM) were identified. The selected aptamers were successfully used in enzyme-linked aptamer assay (ELAA) to detect bPAG4 at a detection limit of 0.09 ng/mL. Meanwhile, it has been successfully applied for the detection of bPAG4 in serum samples. This work demonstrated that the selected aptamers could be used as promising affinity probes in the development of inexpensive, simple, and sensitive analysis methods for detecting bPAGs. Graphical abstract.

Colorimetric enzyme-linked aptamer assay utilizing hybridization chain reaction for determination of bovine pregnancy-associated glycoproteins.

DNA aptamers that bind to bovine pregnancy-associated glycoproteins (bPAGs) were selected by the systematic evolution of ligands by exponential enrichment (SELEX) procedure coupled to surface plasmon resonance (SPR) and high-throughput sequencing (HTS) technology. After seven rounds of selection using carboxylated magnetic beads (MB) coated with bovine pregnancy-associated glycoproteins 9 (bPAG9) and bovine serum albumin (BSA) as target and counter targets, respectively, two aptamers designated as A1 and A24 showed high affinities to bPAG9 (Kd = 1.04 and 2.5 nM). Moreover, the specificity was determined by testing the non-targets bPAG4, bPAG6, bPAG16, BSA, and ovalbumin (OVA). Results showed that two aptamers demonstrated broad group specificity to bPAG family. Subsequently, a colorimetric sandwich enzyme-linked aptamer assay was developed for ultrasensitive detection of bPAG9 based on hybridization chain reaction (HCR) amplification strategy. The method exhibited a broad determination from 0.134 to 134 ng/mL with a detection limit of 0.037 ng/mL. The method has been successfully applied to determine bPAGs in real samples. The results demonstrate that the developed aptamers could be used as promising molecular probes for the development of pregnancy diagnostic tools. Graphical abstract In this study, we first selected aptamers against bovine pregnancy-associated glycoproteins (bPAGs) as molecular recognition elements and then developed a colorimetric enzyme-linked aptamer assay utilizing hybridization chain reaction to detect bPAGs in the serum.The GA can't be deleted, the modified GA can not upload. So themodified GA and figures will be send to CorrAdmin3@spi-global.com.

Using pregnancy associated glycoproteins (PAG) for pregnancy detection at day 24 of gestation in beef cattle.

The objective of this experiment was to determine if circulating concentrations of pregnancy associated glycoproteins (PAG) on day 24 of gestation can be utilized to diagnose pregnancy and embryo viability in beef cattle. Postpartum beef cows (n = 677) and heifers (n = 127) were exposed to a 7-day CO-Synch + CIDR estrus synchronization protocol followed by fixed-time AI (FTAI) on day 0. Blood samples were collected at day 24 after TAI to assess circulating concentrations of PAG utilizing an in-house ELISA. Pregnancy diagnosis was performed 30 and 100 days after FTAI via transrectal ultrasonography. Mean circulating PAG concentration at day 24 differed (P < 0.001) between animals diagnosed pregnant and non-pregnant at day 30 (1.69 ±â€¯0.10 ng/mL vs 0.30 ng/mL ±â€¯0.07 ng/mL; mean ±â€¯SEM; respectively). Pregnant heifers had increased PAG concentration at day 24 compared with pregnant cows (P < 0.01; 3.29 ±â€¯0.36 ng/mL vs 1.39 ±â€¯0.10 ng/mL, respectively). Based on receiver operating characteristic (ROC) curve analysis, serum concentration of PAG at day 24 ≥ 0.33 ng/mL in cows and ≥0.54 ng/mL in heifers was 95% accurate at determining pregnancy status at day 30. Heifers that experienced late embryonic mortality between day 30 and 100 of gestation had decreased circulating concentrations of PAG on day 24 (2.02 ng/mL ±â€¯0.73) compared with heifers that maintained an embryo until day 100 (3.69 ng/mL ±â€¯0.39; P = 0.02). However, there was no difference in day 24 PAG concentration (P = 0.39) between cows that maintained or lost a pregnancy (1.31 ng/mL ±â€¯0.25 vs 0.92 ng/ml ±â€¯0.50). In summary, circulating PAG concentration on day 24 of gestation may be a useful marker for early pregnancy detection in beef cattle, and might be a potential marker for predicting embryonic loss.

Buffalo early pregnancy biomarker coding sequence cloning and partial length expression in E. coli after codon optimization.

Pregnancy-associated glycoproteins (PAGs) secreted from conceptus specific trophoblast cells are widely accepted biomarkers of ruminants. Limited information of PAGs in buffalo warrants further investigation for the development of sensitive homologous early pregnancy-specific diagnostic immunoassay. This experiment was thus designed to identify and clone the predominantly expressed early placentome-specific buffalo PAG (buPAG) isoform; to express this PAG isoform and verify its antigenicity by developing antisera and testing immuno-reactivity with recombinant proteins. Results indicated PAG 7 (buPAG 7) was the predominant isoform in buffalo early pregnant placentome. Attempt to express the full native glycosylated protein in the pcDNA3.3 vector and FreeStyle HEK 293F host was not successful. However, a partial 124 amino acid sequence selected from the non-glycosylated region of buPAG 7 could be expressed in E. coli BL21 (DE3) cells after codon optimization however; the yield was low. Antigenicity of the expressed protein was confirmed by successful immuno-reaction in rabbits indicating possibilities of using 124 aa partial PAG 7 protein as a putative antigen for monoclonal antibody production and development sensitive homologous immunoassay. In conclusion, our results confirmed the findings that buPAG 7 as the predominant early pregnancy-specific transcript. A selected partial 124 amino acid sequences of it could even be expressed in a heterologous host (E. coli). Based on our data presented here, we anticipate that the expressed recombinant protein can be useful as an antigen suitable for the development of PAG specific immunoassays in buffalo.

Pregnancy associated glycoproteins (PAGs) and pregnancy loss in high vs sub fertility heifers.

Reproductive inefficiency and infertility are major financial burdens to domestic livestock. Variables associated with these reproductive losses during early gestation include contributions from the oocyte, uterus, sperm, embryo and placenta. Bovine pregnancy associated glycoproteins (PAG) are produced by the binucleate cells of the ruminant placenta and can be used to diagnose pregnancy. Increased circulating concentrations of PAG early in gestation have been correlated with pregnancy success and decreased concentrations are predictive of impending embryonic mortality in both beef and dairy cattle. The objectives of the current study were to determine whether: 1) heifer fertility status is associated with circulating concentrations of PAG and pregnancy loss; and 2) PAG concentrations within the same animal are repeatable across multiple pregnancies. We hypothesized maternal PAG concentrations would be increased in high fertility compared to subfertile heifers but not repeatable across subsequent pregnancies in the same heifer. Serial embryo transfer (ET; n = 4 rounds) was used to classify predominately Angus heifers (n = 92) as highly fertile (HF = 30; 100% pregnancy success) or subfertile (SF = 62; average = 33%; range = 25-75% pregnancy success) based on day 28 ultrasound diagnosis. Blood samples were collected at both day 28 and 44 for quantification of circulating PAG concentrations by an in house PAG ELISA with antibodies raised against early secreted PAGs. Pregnancy was terminated at day 44 of gestation and heifers were allowed 30 days recovery before synchronization for the next ET. Only heifers that were diagnosed pregnant by ultrasound were used in this study (HF: n = 30, SF: n = 62). Serum concentrations of PAGs were not different between HF (5.90 ±â€¯0.27 ng/mL) and SF (5.56 ±â€¯0.31 ng/mL; P = 0.16) heifers at day 28 of gestation nor was there a difference at day 44 of gestation (P = 0.32). Subfertile heifers had increased pregnancy loss between days 28 and 44 of gestation. Based on odds ratio analysis, SF heifers had a 2.41 times chance to undergo pregnancy loss between day 28-44 compared to HF heifers (P < 0.05). There was no correlation (P > 0.05) in maternal circulating concentrations of PAG between pregnancies on day 28 or 44 of gestation in samples obtained from HF heifers. In summary, circulating concentrations of PAG are not different between HF and SF heifers; however, HF classified heifers have decreased pregnancy loss between days 28 and 44 of gestation.

Impact of fetal vs. maternal contributions of Bos indicus and Bos taurus genetics on embryonic and fetal development1.

To evaluate how the inclusion of Bos indicus genotype influences early fetal development in cattle, a reciprocal embryo transfer approach was used in a completely randomized design with a 2 × 2 × 2 factorial arrangement of treatments to generate 55 pregnancies over 2 consecutive years (n = 55). Recipient cows were randomly assigned to (i) a diet that met daily energy maintenance requirements (MAINT) or (ii) a diet that restricted intake to 70% of the energy maintenance requirements (RESTR). Angus (AN) and Brangus (BN) embryo donors were superovulated and artificially inseminated with female sexed-sorted semen from the same breed. Embryos were then randomly transferred to either AN or BN recipients fed their respective diets for 28 d. Recipients remained on the dietary scheme until day 91 of gestation and were then comingled and fed a common diet that met their energy requirements until calving. Measurements included pregnancy establishment at day 28 of gestation, interferon-stimulated genes (ISG) expression in peripheral blood leukocytes, pregnancy-associated glycoproteins (PAG; using two commercial [A1 and A2] and one in-house assay), and fetal crown-to-rump length (CRL). Recipients in the RESTR diet had lower BWs and BCS (diet × day; P < 0.01) than MAINT recipients. Energy-restricted AN recipients experienced greater (recipient breed × diet, P < 0.01) pregnancy failure by day 28 than the other recipient breed × diet combinations. Restricted recipients that received AN embryos experienced greater pregnancy failure than RESTR recipients receiving BN embryos (embryo breed × diet; P = 0.03). No relevant differences were observed in ISG expression (P > 0.10). Recipients that received BN embryos had greater plasma concentrations of PAG in both A1 (embryo breed × day, P < 0.01) and A2 (embryo breed; P < 0.01). Alternatively, recipients that received AN embryos had greater plasma concentrations of PAG for the in-house assay (embryo breed × day; P < 0.01). In addition, fetuses from AN recipients had greater CRL on day 91 (breed × day, P < 0.01). In summary, Bos taurus cows experienced greater pregnancy failure when nutrient restricted. Furthermore, fetal size and the profile of PAG production during early gestation differed between B. indicus-influenced and B. taurus cattle.

Evaluation of a bovine visual pregnancy test for the detection of pregnancy-associated glycoproteins in sheep.

As sheep produce similar pregnancy-associated glycoproteins (PAGs) to cattle, a commercially available bovine visual pregnancy test based on the detection of PAGs (visual-PAG-test) was evaluated in sheep. The test was performed with whole blood (WhB), plasma (P) and serum (S) of 163 pregnant and 153 non-pregnant ewes. Additionally, 11 pregnant ewes were tested weekly from day 14 to 49 of gestation and monthly from day 60 to day 149. Ten ewes were sampled weekly from the date of lambing until day 63 post-partum (p.p.). The sensitivity in mid-pregnancy (n = 163) was 98.16% (WhB), 99.39% (P) and 99.39% (S) compared to transabdominal ultrasonography as the gold standard while the specificity (n = 153) was 94.12% (WhB), 76.47% (P) and 93.46% (S), respectively. During early pregnancy, all 11 ewes were correctly identified as pregnant on day 42 (100%); however, the test sensitivity decreased to 54.6% (WhB) and 63.6% (S and P, respectively) at day 49. The ewes were again consistently identified as pregnant on day 63 (P) or on day 119 (S, WhB). The test was consistently negative from day 42 p.p. onwards in eight out of ten ewes. Two ewes remained consistently positive until the last sample on day 63 p.p. In conclusion, the test could be used to accurately select pregnant ewes at day 42 with a drop in sensitivity at day 49. The sensitivity of the visual-PAG-test was good in mid to late pregnancy, and early detection of pregnancy was possible in individual animals. In some ewes, the PAGs were however detectable for more than 63 days p.p.-the previous breeding history should therefore always be taken into account for correct interpretation of the test results.