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.

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.

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.

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.

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.

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.

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.

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.

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.

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 ± 0.27 ng/mL) and SF (5.56 ± 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.