Pregnancy-associated Glycoproteins Research Articles

In vitro regulation of gene expression of pregnancy-associated proteins and cytokines in bovine endometrial epithelial cells and bovine trophoblastic cells by infection with Neospora caninum

Abortion caused by the parasite Neospora caninum is an important threat to the livestock industry worldwide. Trophoblasts and caruncular cells play major roles in initiating innate immune responses and controlling parasite infection at the fetal-maternal interface. In the present study, bovine uterine epithelial cells (BUECs) and bovine trophoblastic (BT) cells treated with bovine interferon-gamma (IFN-γ), IFN-alpha (IFN-α) and IFN-tau (IFN-τ) followed by infection with N. caninum were examined by measuring the mRNA expression levels of numerous pregnancy-associated proteins and observing parasite growth to elucidate the host-parasite interaction at the uteroplacental region. N. caninum infection increased the expression of prolactin-related protein 1 (PRP1), pregnancy-associated glycoprotein 1 (PAG1), and cytokines (TNF-α, IL-8 and IL-10) in BUECs and of IL-8 in BT cells. Bovine IFN-γ inhibited IL-8 and TNF-α expression in BUECs and IL-8 in BT cells. In contrast, the expression of the interferon-stimulated gene OAS1 was significantly increased by treatment of the infected BT cells with IFN-γ. However, treatment with bovine IFNs did not inhibit N. caninum growth in either cell line. In conclusion, our results suggest that bovine IFN-γ plays a crucial role in control of pathogenesis in uterus and induction of inflammatory response in the placental region following N. caninum infection, rather than growth inhibition of the parasites.

Evaluating estrus synchronization and early pregnancy detection in Ossimi sheep: The influence of fluorogestone acetate treatment duration and dosage

Estrus synchronization is important for improving sheep reproduction. To enhance sheep reproduction efficiency, this study investigated the impact of different durations (7 vs. 14 days) and fluorogestone acetate (FGA) doses in intravaginal sponges on estrus synchronization and early pregnancy detection in Ossimi sheep. Two hundred ewes were evenly divided into two groups, each receiving a full 40 mg or a halved 20 mg FGA sponge for their respective durations. The study aimed to optimize breeding efficiency by examining the effectiveness of these treatments in synchronizing estrous cycles and by evaluating the use of serum levels of pregnancy-associated glycoprotein 1 (PAG1) and progesterone (P4) as markers for early pregnancy identification. Prostaglandin F2α and equine chorionic gonadotropin were administered to enhance the synchronization process. Results highlighted that the 7-day treatment protocol significantly improved estrus, pregnancy, and lambing rates compared to the 14-day protocol. Furthermore, pregnant ewes demonstrated elevated levels of PAG1 and P4, with PAG1 levels particularly higher in ewes with multiple pregnancies. The findings underscore that the shorter duration of FGA treatment is more effective for reproductive management in Ossimi sheep without significantly affecting PAG1 levels based on the dose or duration of FGA. PAG1 also proved to be a reliable marker for early pregnancy detection, offering a promising approach to identifying fetal numbers early in pregnancy. This research suggests optimizing FGA sponge use could be cost-efficient for improving reproductive efficiency and early pregnancy management in sheep.

Recent advances in early pregnancy loss diagnosis in dairy cows: New approaches.

Early pregnancy loss is a primary cause of low reproductive rates in dairy cows, posing severe economic losses to dairy farming. The accurate diagnosis of dairy cows with early pregnancy loss allows for oestrus synchronization, shortening day open, and increasing the overall conception rate of the herd. Several techniques are available for detecting early pregnancy loss in dairy cows, including rectal ultrasound, circulating blood progesterone, and pregnancy-associated glycoproteins (PAGs). Yet, there is a need to improve on existing techniques and develop novel strategies to identify cows with early pregnancy loss accurately. This manuscript reviews the applications of rectal ultrasound, circulating blood progesterone concentration, and PAGs in the diagnosis of pregnancy loss in dairy cows. The manuscript also discusses the recent progress of new technologies, including colour Doppler ultrasound (CDUS), interferon tau-induced genes (ISGs), and exosomal miRNA in diagnosing pregnancy loss in dairy cows. This study will provide an option for producers to re-breed cows with pregnancy loss, thereby reducing the calving interval and economic costs. Meanwhile, this manuscript might also act as a reference for exploring more economical and precise diagnostic technologies for early pregnancy loss in dairy cows.

77 Assessing abundance of pregnancy markers among fertility-classified beef cows

Abstract In beef cattle, for a single pregnancy opportunity, only 60% of cows will remain pregnant. It is possible that uterine dysfunction prevents adequate embryo signaling and subsequent placentation. Our aim was to compare the abundance of molecular markers associated with pregnancy retention among embryo recipients classified for fertility. The hypothesis was that expression of interferon-stimulated genes (ISG15 and RSAD2) in lymphocytes and concentration of pregnancy associated glycoproteins (PAGs) in serum increase according to cow fertility. Cyclic, Bos Indicus-influenced, primiparous cows (n = 50) were submitted to five consecutive rounds of estrus synchronization, embryo transfer (ET), pregnancy termination and recovery. The ET was conducted 7 days after estrus (D7). Each cow received two embryos per transfer from a randomly assigned bull pair (BP) containing one embryo from each bull A, B or C (BP1 = A+B; BP2 = A+C; BP3 = B+C). Pregnancy diagnostic was conducted on D32 and D45 using B-mode ultrasonography. Blood samples were collected on D20 to quantify expression of ISG15 and RSAD2 in peripheral blood mononuclear cells and on D25, D32, and D39 to quantify serum concentration of PAGs. Based on D45 pregnancy outcome (pregnant or open), cows were classified as Fertile (Fert; n = 14), Sub-fertile (SF; n = 16) or Infertile (IF; n = 15) if they were pregnant 3, 2, or 1 out of the 5 experimental rounds, respectively. Five cows that never sustained a pregnancy until D45 were excluded. The overall prevalence of twin-pregnancies was 7.8% (n = 13/166). Expression of ISG15 was increased in Fert compared to IF cows (P = 0.01; 0.0196 ± 0.0042 vs 0.0164 ± 0.0052, respectively). There was no effect of fertility classification on the expression of RSAD2. Serum concentrations of PAGs increased over time (P < 0.0001), regardless of fertility classification (day x fertility classification; P > 0.4). There was an effect of BP on the probability of pregnancy on D32 and D45 (P < 0.05). For D32, BP2 (84%) was 3 times more likely to maintain a pregnancy than BP1 (52%), while BP3 (74%) was not significantly different. On D45, BP2 (80%) was 2.8 times more likely to remain pregnant than BP1 (50%), and BP3 (62%) was not different than either BP1 or BP2. The interaction bull pair by fertility classification on the probability of pregnancy on D45 approached significance (P = 0.1). While pregnancy to BP2 was similar across all fertility classifications (80%), pregnancy to BP1 decreased dramatically as classifications changed from Fert to IF (88% vs. 14%, respectively). In conclusion, IF cows were less able to respond to IFN-t, likely because of a reduced secretion of IFN-t from the elongating conceptus. Also, IF cows are more susceptible to the genetics of the embryo than FERT cows.

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

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

The pregnancy-associated protein glycodelin as a potential sex-specific target for resistance to immunotherapy in non-small cell lung cancer

Lung cancer has been shown to be targetable by novel immunotherapies which reactivate the immune system and enable tumor cell killing. However, treatment failure and resistance to these therapies is common. Consideration of sex as a factor influencing therapy resistance is still rare. We hypothesize that the success of the treatment is impaired by the presence of the immunosuppressive pregnancy-associated glycoprotein glycodelin that is expressed in patients with non-small-cell lung cancer (NSCLC). We demonstrate that the glycan pattern of NSCLC-derived glycodelin detected by a lectin-based enrichment assay highly resembles amniotic fluid-derived glycodelin A, which is known to have immunosuppressive properties. NSCLC-derived glycodelin interacts with immune cells in vitro and regulates the expression of genes associated with inflammatory and tumor microenvironment pathways. In tumor microarray samples of patients, high glycodelin staining in tumor areas results in an impaired overall survival of female patients. Moreover, glycodelin colocalizes to tumor infiltrating CD8+ T cells and pro-tumorigenic M2 macrophages. High serum concentrations of glycodelin prior to immunotherapy are associated with a poor progression-free survival (p < 0.001) of female patients receiving PD-(L)1 inhibitors. In summary, our findings suggest that glycodelin not only is a promising immunological biomarker for early identification of female patients that do not benefit from the costly immunotherapy, but also represents a promising immunotherapeutic target in NSCLC to improve therapeutic options in lung cancer.

Characterization of placentome vascular perfusion in relation to pregnancy associated glycoproteins throughout gestation in pregnant beef heifers

During pregnancy, blood flow to the uterus changes to support fetal demand. Placentomes serve as vascular attachment sites on the placenta for exchange of gases, nutrients, and metabolic products. Non-invasive methods of ultrasonography and biomarkers have been described to assess placental health and fetal viability. Pregnancy associated glycoproteins (PAGs) are produced by the ruminant placenta and are detected in maternal circulation. In cattle, changes in circulating PAG concentrations are associated with embryonic and fetal outcomes. The objective of this study was to determine the association between placentome blood perfusion and circulating PAG concentrations as they relate to the health of the developing fetus. We hypothesized that placentome perfusion and PAG concentration will be positively correlated and associated with neonatal outcome. A prospective, observational study was designed using 26 pregnant, nulliparous, Angus heifers in which PAG concentration and placentome blood perfusion were assessed throughout gestation, with assessment of calving characteristics following parturition. Placentome blood perfusion was visualized at 30-day intervals via transrectal Doppler ultrasonography with power flow function. Ultrasound images were analyzed using ImageJ software to determine the percent area of perfusion and integrated pixel densities. Venous blood was collected and PAG concentrations were determined via serum PAG enzyme-linked immunoassay. Mean placentome blood perfusion increased as gestation advanced. PAG concentrations demonstrated the expected temporal trend, increasing with gestation length, and were positively linearly correlated with placentome perfusion (P < 0.0001). The relationship identified between circulating PAG concentration and placentome blood perfusion suggests the use of transrectal power flow Doppler ultrasonography as a noninvasive technique to determine placental blood flow morphometrics to assess conceptus wellbeing throughout pregnancy.

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.

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

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

Comparative Protein Profiling of Blood and Milk of Early Pregnant Buffaloes Using SDS-PAGE

Conventional methods for detecting pregnancy in buffalo are inefficient before 30-days after artificial insemination. A proteomic approach for high-resolution analysis of blood plasma and milk proteins by SDS polyacrylamide gel electrophoresis (PAGE) is of great importance. Blood and milk samples were collected on days 0 (before insemination) and 20 and 25 after insemination from retrospective pregnant (n = 6) and non-pregnant (n = 6) animals. An equal volume of samples was loaded in (12%-4%) SDS gel to resolve major plasma and milk proteins. Plasma proteins showed three distinct zones – high, medium, and low molecular weight (MW). In the high MW zone (135 - 250 kDa), ~208 kDa and ~190 kDa proteins were visible. In the moderate MW zone (60 -75 kDa), 5-6 proteins like albumin (~70 kDa), 75 kDa, and 63 kDa consisting of pregnancy-associated glycoproteins, ~73 kDa being dominant. In the low MW zone (25 – 35 kDa), a clear band of 27 kDa was visible. Proteins in milk supernatants showed four distinct zones of proteins – very high, high, medium, and low molecular weight (MW) zones. In the very high MW zone (135 - 250 kDa), ~208 kDa and ~190 kDa proteins were visible. In the high MW zone (60 -75 kDa), proteins like lactoferrin (~78.2 kDa), 73 kDa, albumen (~66 kDa), and a heavy chain of immunoglobulin (IgG) (~54 kDa) were detected. In the medium MW zone (25 – 35 kDa), clear bands of milk caseins, namely thick alpha S2 casein (29 kDa), and other caseins. In the low MW zone (11 – 20 kDa), two thick bands of 18 kDa and 12 kDa were visible. The mean gray values (sum of the gray values of all the pixels in the peak divided by the number of pixels) of the 73 kDa protein peak were analyzed in two-way ANOVA using GraphPad (ver 8.0). Repeated measures of ANOVA were selected where pregnancy status (pregnant and non-pregnant) defined column factor and time (0-, 20- and 25-day) defined row factor. The expression of 73 kDa protein in blood plasma was higher (P = 0.04) in pregnant women than in non-pregnant buffaloes at 20- and 25-days after insemination. However, in milk, there was no difference in the expression of the 73 kDa protein between pregnant and non-pregnant buffaloes across time. Variation in animals within the groups was significantly high. The differential expression of ~73 kDa protein in plasma and precise identification of the protein can be a diagnostic marker for early pregnancy detection in buffaloes.

Corrigendum to: 65 Functional ablation of pregnancy-associated glycoprotein 7 affects attachment and growth of trophectoderm cell lines.

Corrigendum to: 65 Functional ablation of pregnancy-associated glycoprotein 7 affects attachment and growth of trophectoderm cell lines.

Immunohistochemical examination of the utero-placental interface of cows on days 21, 31, 40, and 67 of gestation.

What we understand about early stages of placentation in cattle is based on an elegant series of electron microscopic images that provide exquisite detail, but limited appreciation for the microanatomy across the utero-placental interface. In order to achieve a global perspective on the histology of bovine placentation during critical early stages of gestation, i.e., days 21, 31, 40, and 67, we performed immunohistochemistry to detect cell-specific expression of pregnancy-associated glycoprotein (PAG), cytokeratin, epithelial (E)-cadherin, and serine hydroxymethyltransferase 2 (SHMT2) at the intact utero-placental interface. Key findings from the immunohistochemical analyses are that there are: (1) PAG-positive cells with a single nucleus within the uterine luminal epithelial (LE) cells; (2) PAG-positive cells with two nuclei in the LE; (3) PAG-positive syncytial cells with more than three nuclei in the LE; (4) LE cells that are dissociated from one another and dissociated from the basement membrane in regions of syncytialization within the LE layer; (5) replacement of the mononuclear LE with a multi-layer thick population of PAG-positive cells invading into the uterine stroma of caruncles, but not into the stroma of intercaruncular endometrium; and (6) PAG-, E-cadherin- and SHMT2-positive mononuclear cells at the leading edge of developing cotyledonary villi that eventually represent the majority of the epithelial surface separating caruncular stroma from cotyledonary stroma. Finally, the utero-placental interface of ruminants is not always uniform across a single cross-section of a site of placentation which allows different conclusions to be made depending on the part of the utero-placental interface being examined.

Profiles of interferon-stimulated genes in multiple tissues and circulating pregnancy-associated glycoproteins and their association with pregnancy loss in dairy cows†.

Pregnancy loss (PL) in lactating dairy cows disrupts reproductive and productive efficiency. We evaluated the expression of interferon-stimulated genes (ISG) in blood leukocytes, vaginal and cervical epithelial cells, luteolysis-related genes, progesterone, and pregnancy-associated glycoprotein (PAG) profiles in lactating dairy cows (n = 86) to gain insight about PL. Expression of ISG on d17, d19, and d21 was greater in cows that maintained the pregnancy (P33) compared to nonpregnant with no PL (NP). Greater ISG differences between groups were observed in the cervix (96.7-fold) than vagina (31.0-fold), and least in blood leukocytes (5.6-fold). Based on individual profiles of ISG and PAG, PL was determined to occur either before (~13%) or after (~25%) d22. For cows with PL before d22, ISG expression was similar on d17 but by d21 was lower and OXTR was greater than P33 cows and similar to NP; timing of luteolysis was similar compared to NP cows suggesting embryonic failure to promote luteal maintenance and to attach to the endometrium (no increase in PAG). For cows with PL after d22, ISG expression was similar to P33 cows on d17, d19, and d21 and luteolysis, when it occurred, was later than NP cows; delayed increase in PAG suggested later or inadequate embryonic attachment. In conclusion, PL before d22 occurred due to embryonic demise/failure to signal for luteal maintenance, as reflected in reduced ISG expression by d21. Alternatively, embryos with PL between d22 and 33 adequately signaled for luteal maintenance (ISG) but had delayed/inadequate embryonic attachment and/or inappropriate luteolysis causing PL.

65 Functional ablation of pregnancy-associated glycoprotein 7 affects attachment and growth of trophectoderm cell lines

65 Functional ablation of pregnancy-associated glycoprotein 7 affects attachment and growth of trophectoderm cell lines

2 Profiles of pregnancy-associated glycoproteins for Holstein embryo recipients with pregnancy losses in different periods during the first two months of gestation

2 Profiles of pregnancy-associated glycoproteins for Holstein embryo recipients with pregnancy losses in different periods during the first two months of gestation

Characterizing pregnancy losses in Bos indicus beef females receiving a fixed-timed artificial insemination protocol

This experiment evaluated pregnancy losses from day 30 of gestation to calving in Bos indicus females, and evaluated if serum haptoglobin concentrations during early gestation impacts subsequent pregnancy losses. A total of 4926 Nelore females were used, being 1802 nulliparous heifers inseminated as yearlings (precocious heifers), 1356 nulliparous heifers inseminated at 24 months of age (conventional heifers), 887 suckling primiparous cows, and 881 suckling multiparous cows. Cows were assigned to an ovulation synchronization + fixed-time artificial insemination (FTAI) protocol from day −11 to 0 of the experiment. Pregnancy status was verified using transrectal ultrasonography on days 30 and 60 after FTAI, via transrectal palpation on day 150 after FTAI, and according to calf birth. Blood samples were collected from all animals diagnosed pregnant on day 30 after FTAI, and analyzed for serum concentrations of haptoglobin and pregnancy associated glycoproteins (PAG). Pregnancy loss was greater (P < 0.01) from day 60–150 of gestation (10.2 %) compared with day 30–60 of gestation (6.0 %) and with day 150 of gestation to calving (7.4 %), and differed (P = 0.04) between these latter two periods. Pregnancy loss from day 30–60 of gestation did not differ (P = 0.26) among parities, whereas total pregnancy losses (day 30 to calving) were greater (P < 0.01) in precocious (28.4 %) and conventional (27.1 %) heifers compared with primiparous (16.4 %) and multiparous (13.0 %) cows. Serum PAG concentrations on day 30 after FTAI were less (P ≤ 0.03) in cows that lost the pregnancy (5.63 ng/mL) from day 30–60 of gestation, as well as those that lost the pregnancy from day 30 to calving (8.59 ng/mL) compared with cohorts that maintained the pregnancy (9.39 and 9.32 ng/mL, respectively). No differences in serum PAG concentrations on day 30 after FTAI were noted (P ≥ 0.23) according to pregnancy losses from day 60 to calving. Serum haptoglobin concentration on day 30 after FTAI also did not differ (P ≥ 0.48) between cows that maintained or lost the pregnancy. This experiment provides novel information about pregnancy losses after day 30 of gestation in B. indicus cattle, with most losses occurring as fetal mortality and not affected by systemic inflammation during early gestation. Pregnancy losses were nearly doubled in precocious and conventional heifers compared with parous cows, demonstrating the relevance of this reproductive failure to B. indicus replacement heifers.

Associations of postpartum health with progesterone after insemination and endocrine signaling during early pregnancy in dairy cows

Our objective was to investigate associations of postpartum health with serum progesterone (P4) concentrations after first artificial insemination (AI), expression of interferon-stimulated genes (ISG) in blood leukocytes, and serum pregnancy-associated glycoprotein (PAG) during early pregnancy in dairy cows. In this observational study, Holstein cows (n = 475) from 2 commercial herds were enrolled 3 wk before expected parturition. Body condition score (BCS) and lameness were assessed at enrollment, and total Ca, haptoglobin, and nonesterified fatty acids (NEFA) were measured in serum at 2 and 6 ± 2 d in milk (DIM). Blood β-hydroxybutyrate and metritis were evaluated at 4, 8, 11, and 15 ± 2 DIM, and purulent vaginal discharge (PVD) and endometritis based on endometrial cytology were diagnosed at 35 DIM. Onset of cyclicity was assessed by biweekly P4 measurements. The BCS was assessed at enrollment and at 63 DIM, and lameness at enrollment and at 21 and 49 DIM. First AI was based on estrus detection until ∼75 DIM, or synchronization for timed AI thereafter. Serum P4 was measured at d 8 and 12 after first AI; expression of ISG (ISG15 and RTP4) in blood leukocytes was assessed at d 19; and PAG in serum of pregnant cows was measured at d 29, 33, and 40. Multivariable linear regression models were built including health variables (with metabolites categorized via receiver operating characteristic curve analysis) and covariates (season, milk yield, AI method, DIM), accounting for pregnancy status (for ISG and PAG models), repeated measures (for P4 and PAG models), and herd as random. Results are reported comparing cows affected versus unaffected by each predictor. Serum P4 concentrations were lesser at d 8 after AI in cows that had total Ca ≤2.09 mM (3.6 vs. 4.0 ± 0.2 ng/mL) at 2 DIM or a clinical disease (3.7 vs. 4.0 ± 0.2 mg/mL), and P4 tended to be lesser at d 8 in cows that had PVD (3.6 vs. 4.0 ± 0.2 ng/mL) or any uterine disease (3.7 vs. 4.1 ± 0.2 ng/mL). Among cows pregnant at d 29, relative ISG15 expression (fold change) at d 19 was greater in cows that had lameness (5.10 [4.18-6.18] vs. 3.14 [3.10-3.19]) but tended to be lesser in cows that had endometritis (3.20 [2.89-3.54] vs. 4.29 [4.20-4.35]), and relative expression of RTP4 tended to be lesser in cows that had displaced abomasum (1.02 [0.62-1.67] vs. 1.75 [1.73-1.78]). Serum PAG was lesser at d 29 (3.8 vs. 4.2 ± 0.2 sample-to-positive ratio [S:P]) in cows that had NEFA ≥0.73 mM at 6 DIM, and lesser at d 33 (3.9 vs. 4.5 ± 0.2 S:P) and 40 (3.1 vs. 3.7 ± 0.2 S:P) in cows that had prepartum BCS ≥3.75. These findings suggest long-term effects of metabolic or reproductive tract disorders on luteal function after first AI, pregnancy recognition signaling, and placental function during early pregnancy. These associations may partially explain some of the pathways linking postpartum health and fertility in dairy cows.

PSIV-14 Effects of Maternal Nutrient Restriction During Late Gestation on Expelled Cotyledonary Mrna Expression in Primiparous Beef Females

Abstract To determine the effects of late gestational nutrient restriction on expelled cotyledonary mRNA expression, fall-calving Hereford × Simmental-Angus heifers [single-sired; BW: 451 ± 28 (SD) kg; BCS: 5.4 ± 0.7] bred to a single sire were allocated by fetal sex and expected calving date to receive either 100% (control; CON; n = 12) or 70% (nutrient restricted; NR; n = 11) of NASEM metabolizable energy and metabolizable protein requirements for maintenance, pregnancy, and growth. Beginning on d 160 of gestation, heifers were individually fed chopped sorghum sudan hay (1.74 Mcal ME/kg, 6.69% CP, 72.0% NDF; DM basis) and based on individual intakes, supplemented to meet targeted nutritional planes. Calving was observed for all females and placentas were collected at natural expulsion. Immediately after collection (average: 4.5 h post-calving; range: 2.3 to 12.1 h post-calving), 2 representative cotyledons on the ipsilateral side were cleaned with phosphate buffered saline, and cotyledonary tissue was flash frozen. Total RNA was extracted, and RT-qPCR was performed for mRNA expression of glucose transporters (SLC2A1, SLC2A3, SLC2A4), a fructose transporter (SLC2A5), amino acid transporters (SLC1A1, SLC3A2, SLC7A1, SLC7A5, SLC7A8, SLC38A2), angiogenic factors/receptors (VEGFA, NOS3, FLT1, KDR, GUCY1B3), and pregnancy-associated glycoprotein 2 (PAG2). Relative mRNA expression was analyzed with nutritional plane, treatment initiation date, time to cotyledon sampling, and calf sex (when P &amp;lt; 0.25) as fixed effects. We have previously reported that total uterine blood flow during late gestation was not affected (P ≥ 0.20) by nutritional plane or the nutritional plane × day of gestation interaction. Post-calving, NR dams were 64 kg BW less (P &amp;lt; 0.001) and 2.0 BCS less (P &amp;lt; 0.001), but calf birth weight was not affected (P = 0.72) by nutritional plane. Contralateral placental dry weight tended to be less (P = 0.07) in NR dams, but total placental weight was not affected (P = 0.86) by late gestational nutritional plane. Cotyledonary relative mRNA expression of SLC2A3 and SLC38A2 was greater (P ≤ 0.05) and relative expression of SLC2A1, SLC2A4, and NOS3 tended to be greater (P ≤ 0.07) in NR dams compared with CON. Late gestational nutritional plane did not affect (P ≥ 0.13) cotyledonary relative mRNA expression of FLT1, GUCY1B3, KDR, PAG2, SLC1A1, SLC2A5, SLC3A2, SLC7A1, SLC7A5, SLC7A8, and VEGFA. In summary, first parity beef females who experienced late gestational nutrient restriction maintained uterine blood flow and placental mass and had 4 nutrient transporters and 1 angiogenic factor upregulated in cotyledons, all which likely contributed to their conservation of fetal growth.

Pregnancy evaluation with a point-of-care pregnancy test in dairy cattle

Primiparous and multiparous dairy cattle were evaluated for pregnancy using both transrectal ultrasonography and a point-of-care pregnancy test (Alertys OnFarm Test), which measures pregnancy-associated glycoproteins through lateral diffusion, between Days 28–34 of pregnancy results were compared. A total of 637 animals were included in this study. Pregnancy was confirmed via manual palpation between Days 57–64. Data on parity, calving, and time of artificial insemination (AI) were also collected and evaluated. Overall the accuracy of the lateral diffusion test was 93.1% with 98.9% sensitivity, 88.7 % specificity, 86.8 % positive predictive value, and 99.1% negative predictive value. In heifers, the Alertys OnFarm Test had 100% sensitivity and 81.6% specificity. In contrast, the test had a sensitivity and specificity of 98.5 and 89.5%, respectively for multiparous cows. The pregnancy loss between early diagnosis and confirmation increased with parity. Heifers suffered losses as low as 2.6%, whereas animals in the third parity had significantly more losses (17.9%). Season also affected losses with spring pregnancy losses being considerably higher than autumn losses.The veterinary workload was also evaluated. Using the combined method of pregnancy testing, animals were first tested with the lateral diffusion test; then, the test-negative animals were scanned again, and the number of scanned animals was reduced. The results ranged between 3274% on farms. We concluded that veterinary workload could be reduced by using the point-of-care test. However, farm-level differences may have affected the results of the present study.

Associations of inflammatory and reproductive tract disorders postpartum with pregnancy and early pregnancy loss in dairy cows

Our objective was to describe associations of postpartum health with pregnancy and pregnancy loss (P-LOSS) from d 19 to 40 after first postpartum artificial insemination (AI) in lactating Holstein cows. In 2 commercial dairy herds in Ontario, Canada, 468 Holstein cows were enrolled 21 ± 3 d before expected parturition when body condition score (BCS) and lameness were assessed. Serum total Ca, haptoglobin (Hp), and nonesterified fatty acids (NEFA) were measured at 2 and 6 ± 2 d in milk (DIM). Blood β-hydroxybutyrate (BHB) measurement and metritis detection were done at 4, 8, 11, and 15 ± 2 DIM. Cows were examined for endometritis (ENDO; ≥11.5% polymorphonuclear cells in endometrial cytology) and purulent vaginal discharge (PVD) at 35 ± 3 DIM. Lameness was assessed again at 21 and 49 ± 3 DIM and BCS at 63 ± 3 DIM. First postpartum AI occurred primarily (86%) based on detection of estrus by activity monitors, on average (± standard deviation) at 65 ± 9 DIM, and the remaining cows received timed AI at 86 ± 18 DIM. Serum progesterone (P4) was measured on d 8 and 12 after AI, and pregnancy at first AI (P/AI) was estimated by the expression of ISG15 in peripheral blood leukocytes at d 19 after AI and by pregnancy-associated glycoprotein in serum at d 29, 33, and 40 after AI. Each metabolite (Ca, Hp, NEFA, and BHB) was categorized above or below a cut-point identified with receiver operating characteristic curve analysis associated with P/AI confirmed by ultrasound at d 33 from a larger data set. Data were analyzed using multivariable mixed logistic regression models, accounting for parity, health variables, covariates (season at calving and at AI, milk yield at first Dairy Herd Improvement Association test [categorized into terciles], AI method, and DIM), and herd. The proportions of cows classified pregnant at d 19, 29, 33, and 40 after AI were 64%, 54%, 50%, and 45%, respectively. At d 19 after AI, P/AI was less likely in cows diagnosed with ENDO (52% vs. 69%) or PVD (54% vs. 67%). At d 29, P/AI was less likely in cows with Hp ≥1.54 g/L at 2 DIM (38% vs. 55%) or PVD (35% vs. 56%). Both metritis and ENDO were associated with decreased P/AI at d 40 after AI. Cows diagnosed with metritis had greater risk of P-LOSS from d 19 to 29 (43% vs. 22%) or from d 33 to 40 (37% vs. 7%) than cows without metritis. From d 29 to 33, the risk of P-LOSS was greater in cows with NEFA ≥0.73 mM at 2 DIM (13% vs. 5%) or BCS ≤2.75 at 63 DIM (14% vs. 5%). The concentration of P4 on d 8 after AI was positively associated with P/AI at d 29, 33, and 40, and negatively associated with P-LOSS from d 19 to 29. Postpartum health disorders, particularly reproductive tract disease, can have detrimental effects on early pregnancy establishment and on pregnancy maintenance from d 19 to 40 after AI.