Pregnancy-associated Glycoproteins Research Articles

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.

Development and Validation of a Solid Phase Radioimmunoassay System for the Determination of Pregnancy-Associated Glycoproteins in Bovine Serum

Development and Validation of a Solid Phase Radioimmunoassay System for the Determination of Pregnancy-Associated Glycoproteins in Bovine Serum

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.

Plasma profile of Pregnancy Associated Glycoproteins during Postpartum period in Konya Merino ewes lambing single and twin

This study investigated the plasma profile of Pregnancy Associated Glycoproteins (PAGs) after lambing in Konya Merino ewes using specific cattle pregnancy test kit. A total of 16 Konya Merino ewes were used as a material. Four groups were set up, ewes birthing a male lamb (SM group, n=4), a female lamb (SF group, n=4), twin male lambs (TM group, n=4) or twin female lambs (TF group, n=4). Blood plasma samples were taken on days 0 (lambing day), 3, 7, 10, 14 and then weekly until day 35. All samples were analysed using a bovine pregnancy test kit to detect of PAGs level. It was determined that there was a strong negative correlation between PAGs levels in peripheral blood and days after lambing (r2=0.969; P&lt;0.01). However, no relationship was found between PAGs level and lamb gender or birth type. In conclusion, plasma PAGs level decreases rapidly in Konya Merino ewes regardless of lamb gender and birth type. Moreover, the results showed that the plasma profile of PAGs after lambing in Konya Merino ewes can be monitored with a bovine commercial ELISA–based pregnancy test kit, and the test results can be used in decisions and assessments based on the levels of PAG molecules.

Single-cell insights into development of the bovine placenta†.

A central determinant of pregnancy success is proper development of the conceptus (embryo/fetus and associated extraembryonic membranes including the placenta). Although the gross morphology and histology of the bovine placenta have been well studied, the cellular and molecular mechanisms regulating placenta development and trophoblast differentiation and function remain essentially undefined. Here, single-cell transcriptome (scRNA-seq) analysis was performed on the day 17 bovine conceptus and chorion of day 24, 30, and 50 conceptuses (n = 3-4 samples per day) using the 10X Genomics platform. Bioinformatic analyses identified cell types and their ontogeny including trophoblast, mesenchyme, and immune cells. Loss of interferon tau-expressing trophoblast uninucleate cells occurred between days 17 and 30, whereas binucleate cells, identified based on expression of placental lactogen (CSH2) and specific pregnancy-associated glycoprotein genes (PAGs), first appeared on day 24. Several different types of uninucleate cells were present in day 24, 30, and 50 samples, but only one (day 24) or two types of binucleate cells (days 30 and 50). Cell trajectory analyses provided a conceptual framework for uninucleate cell development and binucleate cell differentiation, and bioinformatic analyses identified candidate transcription factors governing differentiation and function of the trophoblasts. The digital atlas of cell types in the developing bovine conceptus reported here serves as a resource to discover key genes and biological pathways regulating its development during the critical periods of implantation and placentation.

Identification of genomic regions associated with reproductive longevity in the Rubia Gallega beef cattle breed using a censored threshold model

In beef cattle, the economic viability of farms is heavily influenced by the cow's ability to survive subsequent pregnancies. To understand the genetic basis of reproductive longevity in Rubia Gallega beef cattle breed, a ssGWAS was performed by back-solving the output of ssGBLUP under a censored threshold model. It considers the number of parities each cow reaches during its productive life as a phenotypic trait. The results of the study showed that the main source of variation of reproductive longevity is the herd. However, the posterior mean of the heritability of reproductive longevity was 0.173, indicating the potential for an appropriate genetic response to selection. Furthermore, it is shown that four genomic regions in chromosomes 2, 11 and 29 explain a large proportion of the additive genetic variance. The most important signal was detected on chromosome 2 in the vicinity of the MSTN (myostatin) gene that is associated with double muscling, and that it is segregating in the Rubia Gallega population. Some other interesting genes located within these regions encoded for several PAGs (Pregnancy-associated glycoprotein), LHCGR (luteinizing hormone/choriogonadotropin receptor), FSHR (follicle stimulating hormone receptor), PROKR1 (prokineticin receptor 1) and EHD3 (Eps15 homology domain-containing protein 3). This confirms the relevance of the reproductive performance in the reproductive longevity of cows. These findings provide valuable insights for the Rubia Gallega breeding program, as they can be used to define future selection strategies to improve reproductive longevity of the breed.

Genetic parameters of pregnancy loss in dairy cows estimated from pregnancy-associated glycoproteins in milk

This study examined the feasibility of using pregnancy-associated glycoproteins (PAG) in milk within breeding for pregnancy maintenance and assessed the genetic variation in pregnancy loss traits. A total of 374,206 PAG samples from 41,889 Swedish Red (SR) and 82,187 Swedish Holstein (SH) cows were collected at monthly test-day milkings in 1,119 Swedish herds. Pregnancy status was defined based on PAG levels and confirmed by data on artificial insemination (AI), calving, and culling from d 1 postinsemination to calving. Pregnancy loss traits were defined as embryonic loss (diagnosed 28 d to 41 d after AI), fetal loss (42 d after AI until calving), and total pregnancy loss. Least squares means (± standard error, %) and genetic parameters were estimated using mixed linear models. Heritability was estimated to be 0.02, 0.02, and 0.03 for embryonic loss, fetal loss, and total pregnancy loss, respectively. Cows with pregnancy loss had lower PAG concentrations than cows which successfully maintained pregnancy and calved. PAG recording was limited to monthly test-day milking, resulting in low estimated embryonic loss (17.5 ± 0.4 and 18.7 ± 0.4 in SR and SH, respectively) and higher fetal loss (32.8 ± 0.5 and 35.1 ± 0.5 in SR and SH, respectively). Pregnancy loss might have occurred earlier but remained undetected until the next test-day milking, when it was recorded as fetal loss rather than embryonic loss. Estimated genetic correlation between embryonic and fetal pregnancy loss traits and classical fertility traits were in general high. Identification of novel genetic traits from PAG data can be highly specific, as PAG are only secreted by the placenta. Thus, PAG could be useful indicators in selection to genetically improve pregnancy maintenance and reduce reproductive losses in milk production. Further studies are needed to clarify how these results could be applied in breeding programs concurrent with selection for classical fertility traits.

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

During pregnancy, blood flow to the uterus and placenta changes to support fetal demand. The placentomes serve as vascular attachment sites for exchange of gases, nutrients and fetal metabolic products. In human and veterinary medicine, 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 detected in maternal circulation. Current literature suggests that changes in circulating PAG con­centrations may be a useful biomarker for assessing placental health. The objective of this study is to determine the associa­tion between placentome blood perfusion and circulating PAG concentrations as they relate to the health of the developing fetal calf. The hypothesis states that placentome perfusion and PAG concentration will be positively correlated and associated with neonatal outcome.

Pregnancy diagnosis in small ruminants

Pregnancy diagnosis in small ruminant species is crucial for the management of sheep and goats, particularly in commer­cial settings. There are many options for pregnancy diagnosis in these species, including ultrasonography and blood-based assays to detect pregnancy-associated glycoproteins. The meth­od used often depends on the availability of veterinary services to perform pregnancy diagnosis and the management goals of the herd or flock. Regardless of the method chosen, it should be economical for the producer, easily performed in field condi­tions, and highly accurate. Additional information on gestation­al age and fetal number allows for improved nutritional man­agement of the herd or flock to ensure adequate nutrition in the periparturient period to prevent metabolic disease such as pregnancy toxemia. Providing these diagnostic services can be an important source of revenue for the veterinarian while pro­viding invaluable information to aid small ruminant producers in improving profitability in their herds and flocks.

Evaluate the effectiveness of Pregnancy-Associated Glycoprotein and Progesterone in predicting the gestational status in Goats

The objective of this study was to evaluate the PAG and P4 profiles in pregnant and non-pregnant does using Enzyme Immunoassay (EIA) and enzyme-linked immunosorbent assay (ELISA), respectively. Additionally, the study aimed to compare the sensitivity (Se), specificity (Sp), and accuracy (Acc) of pregnancy diagnosis using PAG and P4 detection in serum samples. Twenty does were synchronized using P4 sponge+eCG for 12 days, followed by breeding after estrus. Blood samples were collected at different experimental periods (22, 30, 40, and 60 days post-mating) and analyzed using EIA and ELISA. The lambing and Ultrasonographic examination on day 60 were used as a golden standard for pregnancy evaluation. The results demonstrated that both PAG and P4 mean concentrations significantly increased in pregnant does compared to non-pregnant does in all experimental periods. When comparing the efficiency of each method for pregnancy prediction, the PAG assay method showed a sensitivity (Se) similar to P4 (85%) at day 22 post-mating, but reached 100% at day 30 for PAG and day 40 for P4. Additionally, the accuracy (Acc) of the PAG analysis method was higher than P4 at day 22 (80% vs. 75%) and day 30 (95% vs. 85%), and reached 100% on day 40. In contrast, the Acc of the P4 assay for pregnancy diagnosis was 95% at days 40 and 60. The specificity (Sp) was lower in both methods, but the diagnosis using the PAG assay was better than P4 on all days, reaching its optimal value on day 40, while reach to 83% for P4 assay method on day 40 and 60 PM. The present study concluded that both PAG and P4 analyses were specific and reliable methods for pregnancy determination in does starting from day 22 onward, but the PAG assay was more accurate than the P4 assay.

A Million-Cow Genome-Wide Association Study of Three Fertility Traits in U.S. Holstein Cows.

A genome-wide association study (GWAS) of the daughter pregnancy rate (DPR), cow conception rate (CCR), and heifer conception rate (HCR) using 1,001,374-1,194,736 first-lactation Holstein cows and 75,140-75,295 SNPs identified 7567, 3798, and 726 additive effects, as well as 22, 27, and 25 dominance effects for DPR, CCR, and HCR, respectively, with log10(1/p) > 8. Most of these effects were new effects, and some new effects were in or near genes known to affect reproduction including GNRHR, SHBG, and ESR1, and a gene cluster of pregnancy-associated glycoproteins. The confirmed effects included those in or near the SLC4A4-GC-NPFFR2 and AFF1 regions of Chr06 and the KALRN region of Chr01. Eleven SNPs in the CEBPG-PEPD-CHST8 region of Chr18, the AFF1-KLHL8 region of Chr06, and the CCDC14-KALRN region of Chr01 with sharply negative allelic effects and dominance values for the recessive homozygous genotypes were recommended for heifer culling. Two SNPs in and near the AGMO region of Chr04 that were sharply negative for HCR and age at first calving, but slightly positive for the yield traits could also be considered for heifer culling. The results from this study provided new evidence and understanding about the genetic variants and genome regions affecting the three fertility traits in U.S. Holstein cows.