Pregnancy-associated Glycoproteins Research Articles

Size and position of the reproductive tract impacts fertility outcomes and pregnancy losses in lactating dairy cows

There are multiple factors that contribute to reduced fertility in lactating dairy cows. Recently, a reproductive tract size and position score (SPS) system was developed as a management tool to identify dairy cows with decreased fertility. The aim of this study was to evaluate the association between the SPS on fertility outcomes such as ovulation failure, pregnancy per artificial insemination (P/AI), concentration of pregnancy-associated glycoproteins (PAGs), and pregnancy loss in lactating dairy cows. Primiparous and multiparous lactating Holstein cows (n = 869) were enrolled at two locations. Location 1 (Loc. 1) in Minas Gerais, Brazil (n = 613) and location 2 (Loc. 2) in Agassiz, British Columbia, Canada (n = 256). At the time of AI (d 0), cows were classified as SPS (small [SPS1], medium [SPS2], or large [SPS3] sized reproductive tract) and ovulation failure was determined at 48 h and 7 d post-AI via ultrasonography (Loc. 2 only). Blood samples were collected on d 24 and 31 of gestation for quantification of PAGs and pregnancy diagnosis was performed via ultrasonography at d 31 and 60 post-AI (Loc. 1) and at d 31 ± 3 and 60 ± 3 post-AI (Loc. 2). Cows diagnosed pregnant at d 31 post-AI but not pregnant at d 60 were defined to have undergone late embryonic pregnancy loss. Parity was found to impact SPS (P < 0.01), as primiparous cows had a higher frequency of SPS1 and lower frequency of SPS3 when compared with multiparous cows (SPS1: 42.6 vs. 15.0%; SPS3: 7.0 vs. 22.0%, respectively). Cows classified as SPS3 had greater ovulation failure at 48 h (P = 0.04) and 7 d post-AI (P = 0.05). Cows classified as SPS1 had greater P/AI when compared to SPS2 and SPS3 (45.9 ± 3.3 vs. 37.4 ± 2.6 and 29.1 ± 3.5%, respectively; P = 0.004). There was no interaction between parity and SPS on P/AI. Pregnancy loss between 31 and 60 d post-AI was increased in cows classified as SPS3 compared to SPS2 and SPS1 (24.3 ± 0.05 vs. 11.6 ± 0.02 and 9.4 ± 0.02%, respectively; P = 0.04). Cows classified as SPS1 and SPS2 had greater concentrations of PAGs at 31 d post-AI when compared to SPS3 at both Loc.1 (P < 0.01) and Loc. 2 (P < 0.01). There was no interaction between SPS and pregnancy loss on PAGs at 24 and 31 d post- AI for either Loc. 1 (P = 0.75 and P = 0.76, respectively) or Loc. 2 (P = 0.61 and P = 0.81, respectively). In conclusion, cows that were classified as SPS3 had greater ovulation failure, reduced P/AI, similar concentrations of PAG on d 24, but decreased on d 31, and a greater incidence of pregnancy loss. Thus, size and position of the reproductive tract is associated with fertility and this scoring system could be used to make reproductive management decisions on dairy operations.

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.

Induced prostaglandin release alters steroid concentrations but not pregnancy survival in cows

Embryonic mortality (EM) is a major factor limiting reproductive efficiency in cattle, and despite negative connotations related to reproductive performance, prostaglandin F2α (PGF2α) is capable of being released by the uterus by Day 30 of gestation. Therefore, the objective was to evaluate differences in PGF2α release after an oxytocin challenge between cows with high circulating concentrations of pregnancy-associated glycoproteins (PAGs) vs low PAG because of the association of increased PAG concentrations with pregnancy success. At Day 30 of gestation, pregnant cows were divided into oxytocin treatment (OT; n = 13) and control (CON; n = 12) groups. Treatment cows were further subdivided by circulating PAG concentration (high PAG, n = 7; and low PAG, n = 6). Blood samples were collected every 30 min beginning 1 h before oxytocin administration and continuing for 4 h. Prostaglandin F2α metabolite (PGFM), progesterone, estradiol-17β (E2), and PAG concentrations were quantified. The peak concentration of PGFM occurred 2 h after oxytocin injection in treatment animals and returned to baseline levels by 4 h. No correlations were observed between PAG and PGFM, progesterone, or E2 concentrations (P > 0.05). There was no difference in initial or final PGFM concentrations between groups (P > 0.05). Progesterone and E2 concentrations decreased in cows after treatment of oxytocin (P < 0.05); however, only progesterone returned to basal concentrations by the end of the sampling period. In summary, cows with high vs low PAG concentrations at Day 30 of gestation have a similar PGFM response to oxytocin challenge.

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 48kDa 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 = 11.7~40.2nM) were identified. The selected aptamers were successfully used in enzyme-linked aptamer assay (ELAA) to detect bPAG4 at a detection limit of 0.09ng/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.

Production of Polyclonal Antibodies against Pregnancy Associated Glycoprotein in Cattle

Pregnancy-associated glycoproteins (PAGs) are considered to be a large gene family in the trophoblasts of ruminants. PAGs determination precisely, PAG-1 in serum has been as the solid ground for pregnancy diagnosis in cow. Unluckily the PAG-1 and the antigenically related PAGs show a long half-life in serum less than 8 days and can be assessed 80 to 100 das postpartum, thereafter giving false results in cows bred within 60 days of calving. This study involves using novel polyclonal antibodies after immunizing rabbits and purification of the produced antibodies. The cattle are pregnant when the PAG hormone level is measured in a high level in a serum sample from the animal. It could give a precise method of detecting pregnancy at the early with few false positive results.

Sire contribution to pregnancy loss in different periods of embryonic and fetal development of beef cows

The objective was to evaluate the effect of service sire on pregnancy results during different periods of embryonic and fetal development. Angus cows (n = 658) were timed artificially inseminated (TAI) on day 0 after synchronization of estrus using one of eight Angus sires. Estrus expression prior to TAI was evaluated using estrus breeding indicators. Blood samples were collected on days 0, 24 and 31 for quantification of circulating pregnancy-associated glycoprotein (PAG). Pregnancy was diagnosed at day 24 based on blood-based test, and on days 31 and 60 by transrectal ultrasonography to determine early embryonic mortality (EEM; between days 24 and 31) and late embryonic/early fetal mortality (LEM; between days 31 and 60). Sires were retrospectively classified according to the amount of pregnancy loss in each evaluated interval. Overall EEM was 5.54%, with values ranging from 1.8 to 11.7% among sires, whereas LEM was 6.7% with values ranging from 2.3 to 12.6% among sires. Individual sires had different phenotypes in regard to pregnancy loss during different developmental periods, indicating the importance of evaluating multiple milestones of embryonic development when classifying sire fertility. No difference (P ≥ 0.05) was observed in serum concentrations of PAG in cows bred with sires of different phenotypes. Pregnancy rate by sire was also influenced by estrus expression. Sires with a greater number of cows pregnant without estrus expression, had similar incidence of early (P = 0.71) but greater incidence of late embryonic mortality (P = 0.05). These results are significant to characterize sire contribution to pregnancy maintenance and establishment in beef cows and to serve as basis of studies to identify markers to improve sire fertility evaluation.

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 2.5nM). 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 134ng/mL with a detection limit of 0.037ng/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.

Efficiency of Two Different Synchronization Protocols in Conception in Simmental Heifers

This study aimed to reveal the effects of timed artificial insemination applied after the synchronization protocol known as day5 (D5) and the prostaglandin F2α (PGF2α) protocol applied in 11-day intervals (D11) on pregnancy rates in Simmental heifers. A total of 88 heifers were divided into two treatment groups. In the D5 group (n: 56), after gonadotrophin releasing hormone (GnRH) application, PGF2α injection was made on the 5th day and the second PGF2α injection was given one day later. The second GnRH was applied at the time of TAI and at the 44th hour of the second PGF2α application. In the D11 group (n: 32), double-dose PGF2α was given in 11-day intervals. GnRH was applied at the time of TAI and at the 81st hour following the second PGF2α injection. Pregnancy-associated glycoprotein visual test was carried out for determining to pregnancy on the 28th day. Additionally, for the purpose of determining embryonic deaths, the pregnancies were checked again by ultrasonography on the 63rd day following TAI. The 28th-day pregnancy rates in the synchronized heifers were 51.8% (29/56) in the D5 group and 43.8% (14/32) in the D11 group P>0.05. The 63rd-day pregnancy rates were 48.21% (27/56) in the D5 group and 40.62% (13/32) in the D11 group P>0.05. Consequently, although there was no statistically significant difference in terms of pregnancy rates between the D5 and D11 protocols, there was a numerical increase in the D5 protocol. Considering its short application time, D5 may be used for TAI in Simmental heifers.

Protein Synthesis by Day 16 Bovine Conceptuses during the Time of Maternal Recognition of Pregnancy.

Interferon Tau (IFNT), the conceptus-derived pregnancy recognition signal in cattle, significantly modifies the transcriptome of the endometrium. However, the endometrium also responds to IFNT-independent conceptus-derived products. The aim of this study was to determine what proteins are produced by the bovine conceptus that may facilitate the pregnancy recognition process in cattle. We analysed by mass spectrometry the proteins present in conceptus-conditioned media (CCM) after 6 h culture of Day 16 bovine conceptuses (n = 8) in SILAC media (arginine- and lysine-depleted media supplemented with heavy isotopes) and the protein content of extracellular vesicles (EVs) isolated from uterine luminal fluid (ULF) of Day 16 pregnant (n = 7) and cyclic (n = 6) cross-bred heifers on day 16. In total, 11,122 proteins were identified in the CCM. Of these, 5.95% (662) had peptides with heavy labelled amino acids, i.e., de novo synthesised by the conceptuses. None of these proteins were detected in the EVs isolated from ULF. Pregnancy-associated glycoprotein 11, Trophoblast Kunitz domain protein 1 and DExD-Box Helicase 39A were de novo produced and present in the CCM from all conceptuses and in previously published CCM data following 6 and 24 h. A total of 463 proteins were present in the CCM from all the conceptuses in the present study, and after 6 and 24 h culture in a previous study, while expression of their transcripts was not detected in endometrium indicating that they are likely conceptus-derived. Of the proteins present in the EVs, 67 were uniquely identified in ULF from pregnant heifers; 35 of these had been previously reported in CCM from Day 16 conceptuses. This study has narrowed a set of conceptus-derived proteins that may be involved in EV-mediated IFNT-independent embryo–maternal communication during pregnancy recognition in cattle.

Applied use of interferon-tau stimulated genes expression in polymorphonuclear cells to detect pregnancy compared to other early predictors in beef cattle

We aimed to evaluate the accuracy of Interferon-tau stimulated genes (ISG) abundance in peripheral blood polymorphonuclear cells (PMNs) on D20 after fixed-time artificial insemination (FTAI; D0) as a pregnancy diagnosis method against CL evaluation by Doppler ultrasonography and progesterone (P4) concentrations on D20, as well as Pregnancy Associated Glycoproteins (PAG) concentrations on D25. Additionally, we evaluated the potential of ISG abundance in PMNs as pregnancy loss predictors. Nelore heifers (n = 103) and cows (n = 144) underwent estrous synchronization and were artificially inseminated on D0. Pregnancy was diagnosed by B-mode ultrasonography on D30 and D70, and after the final diagnosis, females were classified in four groups: Pregnant; Non-pregnant; Functional CL on D20 but non-pregnant (CL-NP) and Pregnancy loss between D30 and D70 (PL). After determining cutoff values, the Sensitivity (SE), Specificity (SP), Positive Predictive Value (PPV), Negative Predictive Value (NPV) and Accuracy (ACC) were determined for each method. All methods were classified as significant (P < 0.05) predictors of pregnancy. Both ISG expression and PAG concentrations were greater (P < 0.05) in pregnant females than in non-pregnant and CL-NP females but did not differ (P > 0.05) from the PL group. ISG15 expression was greater (P < 0.05) in heifers than in cows, but this difference was not found in OAS1 expression and PAG concentrations. All the methods evaluated were proven to be adequate predictors of pregnancy, but greater accuracies were obtained through PAG concentrations and Doppler-US, due to the decreased number of false positive and false negative results.

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.

Validation of Buffalo PAG Antisera for Early Pregnancy Diagnosis in Bovine

This study was designed ability using antibody against buffalo PAG to detect concentration of pregnancy-associated glycoprotein (PAG) in bovine plasma samples. Successful purification of buffalo PAG molecules with high enrichment degree by using Vicia villosa aûnity chromatography was recently reported. In this research, the RIA system using novel anti buffalo-PAG was used for bovine pregnancy diagnosis. In a total 437 blood sampling of dairy cow in Belgium were used. Three PAG-RIA systems were undertaken to measure PAG concentration in bovine routine blood samples: AS#497 (or RIA-1) using rabbit antisera against purified bovine PAG67kDa, AS#706 (or RIA-2) using rabbit antisera against caprine PAG55+62kDa and new AS#859 (or RIA-3) using rabbit antisera against buffalo PAG59.5-75.8kDa). The measured PAG concentrations were similar for the systems As#497 and As#859; although the dilution was shown parallel, the measured PAG concentrations were significantly different between systems As#706 and As#859. On the other hand, the coefficients of correlations between concentrations obtained by the use of As#859 and As#497 were highly comparable to those between As#859 and As#706 or As#497 and As#706. In conclusion, the present study clearly shows that antisera against buffalo PAG can be used to develop new PAG-RIA system for bovine pregnancy diagnosis with high comparable results.

Pregnancy-associated glycoproteins as a potential marker for diagnosis of early pregnancy in goats: A scoping reviewing

Early diagnosis of pregnancy plays an important role to minimize reproductive losses in farm animals. There are several methods for pregnancy diagnosis like profiling of reproductive hormones (such as progesterone and estrone sulfate), but sometimes they provide false-positive results. Embryo specific pregnancy markers, which delineate the presence and viability of the embryo, are considered as perfect for pregnancy determination. Pregnancy-associated glycoproteins are distinguished as the best indicator for the determination of early pregnancy, fetal number, and birth weight of kids. Pregnancy-associated glycoproteins are structurally correlated to aspartic proteinase and are communicated in the external epithelial cell layer of the placenta. They have been found to share about half amino acid sequence identity with pepsinogen, pepsin, cathepsin D and E. Dislike different individuals from aspartic proteinase family, numerous pregnancy-associated glycoproteins appear to be latent compound as a result of amino acid substitutions in and around the catalytic site. This review is to discuss the scope and prospects of pregnancy-associated glycoproteins as a pregnancy marker in farm animals, more specifically in goats.

Shearing during late pregnancy increases size at birth but does not alter placental endocrine responses in sheep

Shearing during the latter half of pregnancy is a common practice to improve flock health and productivity. Previous studies have demonstrated that shearing pregnant ewes at mid or late pregnancy is associated with an increase in lamb birth weight. In the present study, we used singleton Polypay × Dorset pregnant sheep, to investigate the potential roles of placental function and changes in maternal metabolism in underlying this increased birth weight response. Two groups were randomly established and blocked at enrollment by animal BW, body condition score and subcutaneous adipose tissue depth. The groups were shorn (SH; n = 18) or not (C; n = 20) at gestational day (GD) 107 ± 1 (mean ± SEM). Weekly maternal plasma samples were collected between shearing and birth, but only six samples were assayed for progesterone, pregnancy-associated glycoproteins (PAG1), glucose and non-esterified fatty acids (NEFAs). At birth, sex, birth weight, and newborn body mass index (BMI) were recorded. Maternal BW during mid- to late-pregnancy was similar between groups. Shearing resulted in increased lamb birth weight and BMI (P < 0.05) regardless of fetal sex but did not affect the maternal concentration of PAG1 or progesterone from GDs 100 to 142. After shearing (GD100) and up to lambing, shorn females had higher circulating glucose concentrations (P < 0.05), but not NEFA, compared to the control group. Maternal circulating PAG1, progesterone, glucose or NEFA concentration across pregnancy did not differ according to lamb sex. Across pregnancy, birth weight was positively associated with PAG1 (P < 0.001), but not with progesterone concentrations. In conclusion, weight and BMI at birth were higher in both sexes upon shearing in singleton pregnancies. Despite PAG1 being associated with birth weight, late-pregnancy shearing did not alter the placental endocrine response. Whether other placental factors are altered upon shearing and may influence the increase in birth weight and BMI remain to be investigated.

Identification of 1093-bp intron A, SNPS and indels discovered in the porcine pregnancy-associated glycoprotein 2-like (pPAG2-L) gene subfamily in pigs

The objective of this study was to identify the intron A sequence (between exons 1 and 2) of pPAG2-L, novel single nucleotide polymorphisms (SNPs) and mutations (InDels) within intron A in the crossbreed (Landrace x Large White), Hirshmann hybrid and Duroc pigs. Genomic templates were isolated from leukocytes, amplified, and the gel-out were purified and then sequenced. Positive amplification control included CH242-60C13 BAC clone (Duroc) containing pPAG1-L and pPAG2-L. This is the first report that describes the 1093-bp intron A sequence from the pPAG2-L (Acc. No. KF471015; GenBank), which increased general knowledge of the porcine genome. Novel SNPs/InDels were identified within the intron A of the pPAG2-L in the crossbreeds (72), Duroc (45) and Hirshmann hybrids (17). This is a pioneer study describing identification of the intron A and SNPs/InDels in crossbreeds that provides a novel major pattern that represents a large portion of the genetic variation within the porcine genome. This information should be valuable when genotyping (coding and noncoding regions) multiparous sows from many breeds, in which reproductive phenotypes are known.

PSII-33 Differences in prostaglandin release in cows with high vs low circulating concentrations of pregnancy associated glycoproteins

Abstract Embryonic mortality (EM) is a major factor limiting pregnancy rates in cattle and occurs early (&amp;lt; day 28) or late (≥ day 28) during gestation. However, causes and mechanisms associated with late EM remain unknown. Despite negative connotations related to pregnancy, prostaglandin F2α (PGF) is capable of being released by the bovine uterus between days 28 and 32 of gestation. Therefore, the objective was to evaluate differences in PGF release between cows with high circulating concentrations of pregnancy associated glycoproteins (PAG) vs low PAG following an oxytocin (OT) challenge. Increased concentrations of PAG are associated with pregnancy success. At day 29 of gestation, pregnant cows were divided into high (n = 10) and low (n = 10) PAG groups and received either 100 I. U. of oxytocin intramuscularly (OT) or saline (control). Blood samples were collected every 30 minutes beginning 1 hour before and continuing for 4 hours after treatment. Prostaglandin F2α metabolite (PGFM), progesterone (P4), estradiol (E2) and PAG concentrations were determined. Peak concentration of PGFM occurred 2 hours after OT injection (average high PAG group peak: 345.6 ± 73.6 pg/mL; low PAG group peak: 326.4 ± 61.4 pg/mL, P &amp;gt; 0.05) and returned to baseline levels by 4 hours. No correlations were observed between PAG and PGFM concentrations (P &amp;gt; 0.05). There was no difference in initial or final PGFM concentrations between high or low PAG groups and control animals (P &amp;gt; 0.05). Furthermore, there was no difference in P4 or E2 concentration between treatment and control animals (P &amp;gt; 0.05) despite a luteolytic release of PGF2α. In summary, cows with high vs low PAG concentrations at day 30 of gestation have a similar PGFM response to OT challenge. This project supported by Agriculture and Food Research Initiative Competitive Grant no. 2017-67015-26457 from USDA-NIFA.

383 Effect of trace mineral source on heifer reproductive performance

Abstract Trace minerals are known to play important roles in early embryo development. The study objective was to determine effects of trace mineral source on heifer reproductive performance. Over two years, beef heifers (n = 130) were randomly assigned to two pens and two treatments, within each pen, based on breed, age, and BW. From weaning through the breeding season, all heifers were individually fed a basal diet of grass hay, corn silage, and a supplement pellet (soybean hulls, dried distiller’s grains plus solubles, minerals, vitamins and monensin). Cobalt, Cu, Mn, and Zn were supplemented with either complexed sources (Availa-4) or inorganic sources (Cu, Mn, and Zn hydroxychlorides; Intellibond C, M, and Z) and Co as CoSO4. Blood samples were collected bi-weekly, and a reproductive tract score (RTS) was collected 30 d prior to breeding to determine pubertal status. All animals were synchronized and artificially inseminated (AI). Pregnancy status was determined by lymphocyte gene expression on d 17, 19, and 21; by circulating concentrations of pregnancy associated glycoproteins (PAGs) on d 22, 23, 24, 25, 26, 27, and 28; and by transrectal ultrasonography on d 30 and 60 after AI. Embryonic loss was defined as when a previously pregnant animal was subsequently diagnosed not pregnant. Data were analyzed using the MIXED procedure in SAS. Puberty (P = 0.44), pelvic area (P = 0.74), RTS (P = 0.49), and estrus expression (P = 0.82) were not influenced by treatment. There was no effect of treatment (P = 0.37) or treatment by time (P = 0.19) on pregnancy, but there was a weak tendency (P = 0.13) for decreased embryonic loss among heifers supplemented with complexed trace minerals (27±6%) compared to inorganic minerals (38±6%). In summary, source of trace mineral did not affect puberty, RTS, pelvic area, or overall pregnancy success, but feeding complexed trace minerals tended to increase embryo survival from d 17 to 60.

378 Awardee Talk - Impact of fetal versus maternal contributions of Bos indicus and Bos taurus genetics on embryonic and fetal development

Abstract 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 in order to generate 55 pregnancies (n = 55). Recipient cows were randomly assigned to 1) a diet that met daily energy maintenance requirements (MAINT), or 2) 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 28d. Recipients remained on the dietary scheme until d91 of gestation, and were then comingled and fed a common diet that met their requirements. Measurements included pregnancy establishment at d28 of gestation, pregnancy-associated glycoproteins (PAG; using 2 commercial [A1 and A2] and 1 in-house assay), and fetal size (CRL). Recipients in the RESTR diet had lower BW and BCS (diet×day; P &amp;lt; 0.01) than MAINT recipients. Energy-restricted AN recipients experienced greater (recipient breed×diet, P &amp;lt; 0.01) pregnancy failure by d28 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). Brangus embryos resulted in greater plasma concentrations of PAG in both A1 (embryo breed×day, P &amp;lt; 0.01) and A2 (embryo breed P &amp;lt; 0.01). Alternatively, recipients that received AN embryos had greater plasma concentrations of PAG for the in-house assay (embryo breed×day; P &amp;lt; 0.01). In addition, fetuses from AN recipients had greater CRL on d91 (breed×day, P &amp;lt; 0.01). In summary, Bos taurus cows experienced greater pregnancy failure when nutrient restricted. Furthermore, fetal size and profile of PAG production during early gestation differed between Bos indicus-influenced and Bos taurus cattle.

64 Prediction of pregnancy and early embryo loss through OAS-1 expression, concentrations of pregnancy-associated glycoproteins, and Doppler ultrasonography in beef cattle

We aimed to compare three methods to detect pregnancy based on interferon-tau stimulated gene expression in peripheral blood polymorphonuclear cells and Doppler ultrasonography (Doppler-US) 20 days after timed AI and the concentrations of pregnancy associated glycoproteins (PAGs) 25 days after timed AI. Our second objective was to compare interferon-tau stimulated genes and PAGs as early embryo loss (EEL) predictors. Nelore cows (n=144) and heifers (n=103) were submitted to timed AI (Day (D) 0). On D20, polymorphonuclear cells were isolated from blood samples by Ficoll gradient (GE Healthcare), and the RNA was extracted. Expression of the interferon-tau stimulated gene (OAS-1) was quantified by qPCR and normalized to reference genes (GAPDH and ACTB). On D25, blood was collected from the jugular vein and PAG concentrations were measured by an in-house enzyme-linked immunosorbent assay. Pregnancy diagnoses were performed on D20 by luteolysis detection with Doppler-US and on D30 by detection of an embryo by heartbeat. Animals were classified as pregnant (P; fetus on D30), non-P (NP; no active corpus luteum on D20), and EEL (active corpus luteum on D20 but NP on D30). Expression of OAS-1 and PAG concentrations were analysed by analysis of variance using PROC MIXED of SAS considering the effects of group, category, and their interaction. Receiver operating characteristic curves were created, and the area under the curve (AUC), accuracy, specificity, and sensitivity were calculated for pregnancy predictions on D20 (OAS-1 and Doppler-US) and D25 (PAGs) compared with the standard diagnosis method on D30. Expression of OAS-1 and PAG concentrations were greater (P&amp;lt;0.01) in the P (2.4±0.2 and 4.8±0.2ngmL−1, respectively) compared with the NP (0.50±0.1 and 1.2±0.2ngmL−1, respectively) and EEL (0.8±0.2 and 1.0±0.2ngmL−1, respectively) groups. Receiver operating characteristic analysis indicated that OAS-1, Doppler, and PAGs were significant (P&amp;lt;0.01) predictors of pregnancy in heifers (AUC=0.86, 0.92, and 0.94, respectively) and cows (AUC=0.82, 0.94, and 0.95, respectively). The PAGs and Doppler-US presented higher accuracy on diagnosing pregnancy (92 and 89% for heifers, 94 and 93% for cows, respectively) than OAS-1 (81% for heifers and 75% for cows). Doppler-US was 100% sensitive to detect nonpregnant females, which avoided false negative results, whereas PAG concentrations presented a higher specificity (86% for heifers and cows) when compared with Doppler-US and OAS-1 (76 and 65% for heifers, 85 and 75% for cows, respectively). On D25, PAGs were 90% accurate to detect heifers and cows that would experience EEL, whereas on D20, OAS-1 was 50% accurate. In conclusion, Doppler-US can be used as early as D20 with high accuracy and sensitivity. Likely, the use of PAG concentrations on D25 is highly accurate and is more effective at detecting EEL. Pregnancy diagnosis through OAS-1 expression is not as accurate as the other methods and could not efficiently predict EEL. This research was supported by FAPESP (2015/106069; 2017/134729).

9 Pregnancy loss in Holstein lactating recipient cows diagnosed pregnant by pregnancy-associated glycoprotein test in blood

The objective of this study was to evaluate the pregnancy loss (PL) between Day 30 (P30) and Day 80 (P80) of pregnancy in lactating Holstein recipients that received an invivo- (flush) or invitro-produced (IVF) embryo. The recipient cows were located at Maddox Dairy in Riverdale, CA, USA, a Holstein herd that milks 3500 cows with a 305-day mature-equivalent milk production of 12 800 kg. First-lactation cows were enrolled in a Presynch-Ovsynch oestrus-synchronization program and scheduled to be artificially bred for the first time at 80 days after calving or to receive an embryo 7 or 8 days after the expected heat (recipients). The data from 590 pregnancies (1045 embryo transfers) from embryos transferred between January 2018 and March 2019 was analysed. Only grade 1 embryos (from morula to hatched blastocyst stage) produced invivo or invitro from Holstein donor heifers, lactating or dry cows, were transferred fresh (invivo or invitro) or frozen-thawed (invivo), and pregnancy rates are shown in Table 1. The ruminant trophoblast produces pregnancy-associated glycoproteins (PAG) that can be detected by enzyme-linked immunoassay (ELISA) in the blood of pregnant cattle as early as 28 days after insemination. Various dairy herds in the USA have been using this test to supplement or replace the use of transrectal ultrasonography for early pregnancy diagnosis. Blood was sampled on P30 after expected heat day (23 days after embryo transfer) from the recipient cows and sent to IDEXX for the PAG Bovine Pregnancy Test, which was reconfirmed on P80 of pregnancy by transrectal ultrasonography. Pregnancy loss was considered to have occurred when a cow was pregnant on P30 but not pregnant on P80. The variable PL was analysed by binary logistic regression in the MINITAB program, and the model included effects of donor status (heifer vs. milk vs. dry) and embryo type. The total PL was 12.2% (72/590) and the details are shown in Table 1. No effect of donor status (P=0.80) was detected. However, there was effect of embryo type (P=0.004). The IVF embryos had a PL of 18.0% compared with 9.5% for the invivo-produced embryos. Further research should be performed to study heifer embryos PL, because currently more embryos are being produced from very young donor cows and sires due to intensive use of genomic testing. In conclusion, there is a higher PL in lactating dairy recipients receiving IVF fresh embryos compared with fresh or frozen invivo-produced embryos. Table 1.Embryo transfer (ET) pregnancy rates (PR, P30) in first-lactation Holstein cows (top) and pregnancy loss (PL) from Day 30 (P30) to 80 (P80) in first-lactation recipient Holstein cows (bottom) Item Heifer donor Lactating donor Dry donor Total ET P30 PR% ET P30 PR% ET P30 PR% ET P30 PR% Invivo - fresh 6 3 50.0 43 25 58.1 329 206 62.6 378 234 61.9 Invivo - frozen 75 38 50.7 221 129 58.4 296 167 56.4 IVF - fresh 123 61 49.6 91 42 46.2 157 86 54.8 371 189 50.9 Total 129 64 49.6 209 105 50.2 707 421 59.5 1045 590 56.5 P30 P80 PL% P30 P80 PL% P30 P80 PL% P30 P80 PL% Invivo - fresh 3 3 0.0 25 24 4.0 206 186 9.7 234 213 9.0 Invivo - frozen 38 35 7.9 129 115 10.9 167 150 10.2 IVF - fresh 61 48 21.3 42 35 16.7 86 72 16.3 189 155 18.0 Total 64 51 20.3 105 94 10.5 421 373 11.4 590 518 12.2