Pregnancy-associated Glycoprotein Concentrations Research Articles

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.

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.

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.

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.

151 Reproductive transcriptome of bovine pregnancies with high, low concentrations of pregnancy-associated glycoproteins following Artificial Insemination

151 Reproductive transcriptome of bovine pregnancies with high, low concentrations of pregnancy-associated glycoproteins following Artificial Insemination

Is pregnancy loss initiated by embryonic death or luteal regression? Profiles of pregnancy-associated glycoproteins during elevated progesterone and pregnancy loss

Because progesterone (P4) is essential for pregnancy establishment and maintenance, we investigated the effect of increased concentrations of P4 on embryonic attachment and concentrations of pregnancy-associated glycoproteins (PAG). Additionally, we investigated the relationships among luteal regression, pregnancy loss, and PAG concentrations in cows undergoing pregnancy loss by d 33 of pregnancy. Lactating dairy cows were allocated into control (n = 40) and human chorionic gonadotropin (hCG; 3,300 IU on d 7 and 13 to promote greater circulating P4; GnRH = d 0; n = 46) groups. Progesterone was measured daily from d 7 to 33, and PAG was measured daily from d 17 to 33; both hormones were also measured on d 47 and 61. An increase in PAG >10% compared with d 17 was considered a marker for pregnancy. The gold standard for pregnancy diagnosis was ultrasound evaluation of embryonic heartbeat on d 33. Statistical analyses were done with PROC MIXED from SAS Institute Inc. Concentrations of P4 were greater from d 8 onward in the hCG group. Concentrations of PAG did not differ between groups from d 17 to 33, suggesting no effect of increased P4 on hastening embryonic attachment and placental development. Nevertheless, PAG was greater in the hCG group on d 47 and 61, suggesting greater placental area or PAG secretory capacity. Pregnancy loss between d 20 and 33 occurred in 24.6% of cows. About 50% of pregnancy loss was due to luteal regression and about 50% was due to conceptus failure; that is, a decrease in PAG in the absence of luteal regression. In conclusion, increased P4 does not hasten embryonic attachment or early placental development but it leads to increased PAG in the second half of the second month of gestation. Additionally, pregnancy loss seems to be initiated by either corpus luteum regression or conceptus failure.

Use of GnRH Treatment Based on Pregnancy-Associated Glyco-Proteins (PAGs) Levels as a Strategy for the Maintenance of Pregnancy in Buffalo Cows: A Field Study.

Simple SummaryEmbryonic mortality (EM) is one of the leading causes of infertility in modern breeding. This results in a reduction in reproductive performance, significant economic losses and can affect the environmental impact. The introduction of intensive breeding systems and the selection of high-production animals have accentuated EM, also in buffalo. This study aimed to verify if GnRH treatment at 35 days after artificial insemination of subjects identified at risk of embryonic mortality on the basis of pregnancy-associated glycoprotein (PAG) levels, could reduce EM in buffalo.The aim of the study was to evaluate the effects of GnRH administrated at day 35 after artificial insemination (AI) on the reproductive performance of buffalo cows. In ten buffalo farms in the period January–February, 481 buffalo cows were subjected to estrus synchronization protocol and fixed-time artificial insemination (Ovsynch–TAI program). Radioimmunoassays (RIA) for pregnancy-associated glycoproteins (PAGs) were used to detect pregnancy at day 28 after AI (cut-off value: ≥1 ng/mL). Among pregnant subjects, those with PAG values between 1 and 2.5 ng/mL were considered at risk of embryonic mortality (EM) and were assigned into two groups: treated (T; n = 57) control (C; n = 57). Treated buffaloes received 0.01 mg of buserelin acetate intramuscularly on day 35 after AI, whereas control buffaloes received no treatment. The pregnancy diagnosis was confirmed at day 60 through PAGs level and rectal palpation. The treatment with GnRH had a significant effect (p < 0.001) in reducing EM. Between days 28 and 60 after AI, the animals that experienced EM were only 2/57 in the T group, while were 13/57 in the C group. Moreover, GnRH treatment produced a significant increase (p < 0.001) in the PAG concentration between day 28 and day 60. Administration of GnRH at day 35 after AI in animals considered at risk of low embryo survival based on PAG levels allowed a reduction in pregnancy losses and improved the pregnancy rate during low-breeding season in buffalo.

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

The objective of this study was to evaluate the use of corpus luteum (CL) color Doppler (CD) ultrasonography and pregnancy-associated glycoproteins (PAG) for early pregnancy diagnosis and examine their ability to predict late embryonic/early fetal mortality (LEM) in Bos taurus beef replacement heifers. Beef heifers (n = 178) were exposed to a 7-d CO-Synch + CIDR protocol followed by fixed-time artificial insemination (day 0). On days 20 and 22, B-mode and CD ultrasonography were performed to evaluate CL morphometries and blood perfusion, respectively. Heifers were considered nonpregnant when CL area was <2 cm2 or estimated luteal blood perfusion was ≤30% of the total luteal area. Blood samples were collected on days 25 and 29 to estimate circulating concentrations of PAG. Conventional ultrasonography on days 29 and 94 was utilized to determine pregnancy status and considered the gold standard method for pregnancy diagnosis. Pregnant heifers had greater (P < 0.01) CL diameter, area, volume, and blood perfusion when compared with nonpregnant heifers on days 20 and 22. Accuracy of CD on days 20 and 22, and PAG on days 25 and 29 were 91%, 94%, 96%, and 98%, respectively. No false-negative results were observed for CD on both days 20 and 22 (negative predicted value = 100%) and false-positive results represented 8% and 6% of the diagnoses. Heifers that experienced LEM between days 29 and 94 of gestation had decreased luteal (P = 0.02) volume on day 20 and tended (P = 0.07) to have decreased concentrations of PAG on day 29 compared with heifers that maintained pregnancy. However, both CD and PAG failed to predict embryonic mortality. In conclusion, CD successfully detected most nonpregnant replacement heifers as early as day 20 of gestation, while resulting in no false negative diagnoses. Both CD and PAG failed to predict LEM in the present study.

Effect of Folic Acid Supplements on Progesterone Profile and Blood Metabolites of Heat-Stressed Holstein Cows during the Early Stage of Pregnancy.

Simple SummaryThe fertility of a dairy cow can be described as the female’s ability to conceive and maintain pregnancy when insemination is performed at the proper time relative to ovulation. In this context, poor estrous detection and embryonic or fetal losses are common causes for reduced reproductive performance in dairy cows. Furthermore, heat stress severely deteriorates conception and pregnancy rates in dairy farms. The aim of this study was to see how oral folic acid (FA) supplements affect progesterone levels, blood metabolites, endocrine patterns, and blood biochemical concentrations in heat-stressed pregnant cows. Oral FA supplementation (10 μg kg−1) in the first month of gestation improved the progesterone profile, as well as blood folates, pregnancy associated glycoprotein (PAG), growth hormone (GH), and Insulin-like growth factor-1 (IGF-1) concentrations in heat-stressed Holstein cows.The aim was to elucidate the impact of oral folic acid (FA) supplements on progesterone profile, blood metabolites and biochemical indices of heat-stressed Holstein cows during the early stage of pregnancy. The study lasted from the day of artificial insemination through the end of the fourth week of pregnancy. The first group (CON, n = 17) received 0 μg of FA/kg BW as a control. The second and third groups received oral FA doses of 5 (FA5, n = 19) and 10 (FA10, n = 20) μg kg−1 BW, respectively. At the 2nd and 3rd weeks of pregnancy, the FA10 group had greater progesterone levels than the CON group (p < 0.05). The FA10 group had a greater progesterone level than the FA5 and CON groups at the fourth week of pregnancy (p < 0.01). The FA10 group had higher folate levels than CON group during the first three weeks of pregnancy (p < 0.01). Both FA-supplemented groups had significantly greater serum folates than the CON group by the end of the fourth week of pregnancy (p < 0.01). At the 2nd and 4th weeks of pregnancy, the FA10 group had greater levels of serum glucose and globulin than the CON group (p = 0.028 and 0.049, respectively). Both FA-supplemented groups had greater serum growth hormone (GH) levels at the 4th week of pregnancy (p = 0.020). Additionally, the FA10 group showed significantly higher levels of IGF-1 at the 2nd and 4th week of gestation (p = 0.040 and 0.001, respectively). FA supplementation decreased the levels of non-esterified fatty acid (NEFA) at the 2nd and 4th week of gestation (p = 0.020 and 0.035, respectively). Additionally, the FA10 group showed significantly higher pregnancy-associated glycoprotein (PAG) levels at the 2nd and 4th week of gestation (p = 0.005 and 0.001, respectively). In conclusion, oral FA supplementation (10 mcg kg−1) in the first month of gestation improved the progesterone profile, as well as blood folates, PAG, GH, and IGF-1 concentrations in heat-stressed Holstein cows. These findings could be useful in developing practical strategies to keep dairy cows’ regular reproductive patterns under heat stress conditions.

27 Transcriptome Comparison of Parthenogenetic vs. Biparental Embryos in Beef Cattle

Abstract This experiment aimed to determine differences in circulating pregnancy-associated glycoprotein (PAG) concentrations, differentially expressed genes (DEGs), and comparative gene ontology profiles between parthenogenetic embryos (PA; absence of paternal genetics) and biparental control embryos (CON). PA embryos were produced in vitro using a validated chemical activation method, and CON embryos were produced in vitro using industry standard techniques. A blood sample was collected on day 31, and PAG concentrations were determined by an in-house ELISA. Transrectal ultrasonography was performed daily to monitor conceptus development. Cows (n = 30) were synchronized, underwent embryo transfer [ET; 7 days after onset of estrus (day 0)] and were euthanized at approximately day 31 of gestation. Tissues from endometrium (EM), trophectoderm (TE), and corpus luteum (CL) were collected and sequenced from both groups (3 PA; 3 CON). Average PAG concentrations between PA and CON on day 31 were 0.81±0.44 ng/mL and 5.23±0.44 ng/mL (P = 0.01), respectively. When comparing PA to CON, there were 969 DEG in the EM (599 genes upregulated; 269 genes downregulated; 101 nonannotated genes) and 250 DEG in the TE (163 genes upregulated; 47 downregulated; 40 nonannotated genes). Comparative gene ontology profiles between DEGs showed an upregulation in immune system-associated activities and extracellular matrix interactions and a downregulation in redox reactions and proteolytic activities involving PAGs for PA as compared to CON. Similarly, the TE comparisons showed an upregulation in prolactin (PRL) inhibiting pathways and a downregulation in structural molecule activity and signaling for PA as compared to CON. The DEGs observed in PAs show significant decreases in conceptus-derived products and gene ontology profiles associated with pregnancy. Overall, these findings demonstrate that uniparental embryos can be a useful model to better understand pregnancy development and loss in cattle.

Pregnancy maintenance following sequential induced prostaglandin pulses in beef cows

The relationship between the maternal endocrine environment and late embryonic mortality (> 28 d of gestation) in cattle is poorly defined. A definitive rise and alterations in secretion patterns of prostaglandin F2α (PGF2α) concentration without luteal regression is a trademark of this period. The objective was to evaluate whether consecutively induced PGF2α pulses would alter steroid hormone production and luteal blood perfusion potentially influencing pregnancy success. Pregnant beef cows (n = 12) were selected to receive either an oxytocin injection (OT, n = 8) or saline injection (CON, n = 4) on d 30 and 31 of gestation to stimulate sequential prostaglandin releases 24 h apart. Blood samples were collected every 30 min for 1 h before and continuing for 4 h post oxytocin administration. Luteal blood perfusion was measured via Doppler ultrasound at the beginning and end of the OT challenge. Concentrations of prostaglandin F2α metabolite (PGFM) were quantified to show effectiveness of the treatment while concentrations of progesterone, estradiol and pregnancy-associated glycoproteins (PAG) were measured to examine the effect of PGF2α release. Control animals exhibited no changes in any quantified hormone and an expected numerical increase in circulating PAG concentrations. Peak concentrations of PGFM in OT cows were observed 2 h post OT administration and concentrations returned to basal levels by the end of the sampling period. Peak concentrations of PGFM were decreased on d 31 compared to d 30. Following OT administration, progesterone and estradiol concentrations did not change in response to PGF2α release but were decreased on d 31 compared to d 30. There were no changes in luteal blood perfusion in response to PGF release on d 30 or d 31. Repeated PGF2α release may alter steroid hormone production; however, it does not negatively affect pregnancy status during the transition between early and late embryonic development.

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

The objective of this study was to evaluate the use of luteal color doppler (CD) ultrasonography and plasma concentrations of pregnancy-associated glycoproteins (PAG) for early pregnancy diagnosis in Bos taurus beef cows. Additionally, CD and PAG were evaluated as potential predictors of late embryonic/early fetal mortality (LEM). Postpartum beef cows (n = 212) were exposed to estrus synchronization followed by fixed-time artificial insemination (day 0). On days 20 and 22, B-mode and CD ultrasonography were performed to evaluate corpus luteum (CL) morphometries and blood perfusion. Moreover, blood samples were collected on days 25 and 29 to quantify circulating concentrations of PAG. Conventional ultrasonography on days 29 and 100 was utilized as the gold-standard method for pregnancy diagnosis. Cows that experienced pregnancy loss between days 29 and 100 were classified as LEM. Pregnant cows had larger and more vascularized CL compared with nonpregnant cows on days 20 and 22 (P < 0.001 for all response variables). Accuracy for CD on days 20 and 22 were 87% and 92%, respectively. Accuracy for PAG on days 25 and 29 were 84% and 99%, respectively. No false negative (FN) results were observed for CD on both days 20 and 22; however, there were 7.1% FN results for PAG on day 25. Cows that experienced LEM had decreased (P = 0.04) circulating PAG on day 29 of gestation compared with cows that maintained pregnancy; however, there were no differences in luteal blood perfusion on days 20 and 22 (P ≥ 0.53) or circulating PAG on day 25 (P = 0.46) between LEM cows and cows that maintained pregnancy. Sensitivity and specificity of PAG on day 29 as predictors of LEM were 83% and 77%, respectively. In conclusion, CD resulted in accurate pregnancy diagnosis in B. taurus beef cows on both days 20 and 22 of gestation, while having no FN results. Circulating concentrations of PAG were decreased in cows that experienced LEM; however, further research is required to utilize PAG as a predictor of LEM commercially.

Using commercial enzyme immunoassay for measuring pregnancy-associated glycoproteins to diagnose pregnancy in dairy cows under field conditions in Algeria

The purpose of the present work was to study effectiveness for early pregnancy diagnosis in cattle of the new enzyme immunoassay (EIA) sandwich kit commercially available based on the measurement of pregnancy-associated glycoproteins (PAGs). 120 Holstein-Friesian cattle of mixed age and parity were comprised from different dairy herds. The pregnant females (n = 68) were diagnosed by ultrasonography at day 35-40 after artificial insemination and confirmed by transrectal exploration at 2-3 months after AI. The non-pregnant females (n = 52) were housed in the absence of males during the experimental period. Blood samples were collected from coccygeal vessels of females into EDTA tubes. The serum was obtained by centrifugation and the serum was stored at - 20 C until assay. The PAG concentrations in pregnant and non-pregnant females were determined in serum by EIA kit. The reproducibility inter- and intra-assay of the PAG-EIA is satisfactory (2.78 and 13.19 %, respectively). The accuracy ( 94.8 %) and the test of parallelism were largely acceptable. No cross-reaction was observed with the different hormones tested at different dilutions. PAG-EIA system gave 100 % sensitivity and negative predictive values. Whereas, specificity and positive predictive value were 91.93 and 71.15 %, respectively. The accuracy of pregnancy diagnosis by PAG-EIA was 87.5 %. In conclusion, the present study shows clearly that the EIA kit can be used to measure PAG in serum cows for the detection of gestation in Algeria. Therefore, this alternative technique could be recommended to replace the radioactive methods in immunoassays to improve the reproductive performances and an efficient tool for reproductive management of dairy cattle.

PSXII-21 Pregnancy associated glycoproteins for pregnancy diagnosis on day 25 of gestation

Abstract The objective of this study was to evaluate the use of plasma concentrations of pregnancy associated glycoproteins (PAG) for pregnancy diagnosis in beef cows on day 25 of gestation. Additionally, differences in circulating concentrations of PAG among cows that experienced late embryonic/early fetal mortality (LEM) were evaluated. Beef cows (n = 214) were exposed to the 7-d CO-Synch + CIDR protocol followed by fixed-time artificial insemination (day 0). Blood samples were collected on days 25 and 29 to determine circulating concentrations of PAG and ultrasonography on day 29 was used as the gold-standard method for pregnancy diagnosis. On days 25 and 29, PAG concentrations were greater (P &amp;lt; 0.001) in pregnant compared with non-pregnant cows. Sensitivity, specificity, positive predicted value (PPV), negative predicted value (NPV), and accuracy on day 25 were 87, 80, 83, 84, and 83%, respectively. Sensitivity, specificity, PPV, NPV, and accuracy for PAG on day 29 were 99, 94, 96, 97, and 99%, respectively. Pairwise comparison of receiver operating characteristics curve analysis indicated greater area under the curve for PAG on day 29 compared with day 25 (P &amp;lt; 0.001). There were no differences in plasma concentrations of PAG on day 25 among cows that experienced LEM compared to cows that maintained pregnancy (P = 0.46). Concentrations of PAG on day 29 were reduced (P = 0.03) in cows that experienced LEM compared with cows that maintained pregnancy, and were not different between LEM and non-pregnant cows (P = 0.12). In summary, cows that experienced LEM had decreased plasma concentrations of PAG compared with cows that maintained pregnancy. The PAG assay utilized in this study yielded accurate pregnancy diagnosis results on day 29 of gestation: however, the presence of false negative results (NPV = 84%) limits its use on day 25 of gestation.

236 Factors Influencing Clearance of Pregnancy-associated Glycoproteins in Postpartum Beef Cattle

Abstract Blood pregnancy tests can determine pregnancy status in cattle; however, pregnancy-associated glycoproteins (PAGs) have a long half-life (80 to 100 d). If tested too soon after calving, false positive readings can occur due to elevated concentrations from the previous pregnancy. This study’s objective was to determine factors that impact clearance of PAGs in postpartum beef females. Blood samples were collected from postpartum Angus and Angus-cross females (primiparous n = 46 and multiparous n = 58); once a week for up to 14 weeks after calving (range of first to last sample 1–7 to 98–105 days postpartum; dpp). Serum was tested in duplicate using the IDEXX Alertys Pregnancy Test. Data were analyzed as a repeated measure using the MIXED procedure of SAS (9.4) with parity, calf sex (sex), days postpartum, and all interactions in the model. Data were analyzed using the REG procedure in SAS. There was a significant effect of dpp (P &amp;lt; 0.01) on PAG concentrations; however, PAG concentrations were not influenced by parity (P = 0.24), sex (P = 0.49), parity by dpp (P = 0.12), parity by sex (P = 0.65), sex by dpp (P = 0.37), or parity by sex by dpp (P = 0.99). Concentrations of PAGs rapidly decreased from d 0 to 50 postpartum and then continued to gradually decrease to d 100. Days postpartum accounted for 67.53% of the variation in PAG concentrations. Prior to 50 dpp, PAG concentrations were sufficiently elevated which resulted in false positive readings (S – N ≥ 0.3). In summary, concentrations of PAGs remained elevated after calving, but the clearance of PAGs were not impacted by parity or sex of the calf. Clearance of PAGs were significantly influenced by days postpartum. Additionally, using a blood pregnancy test prior to d 50 postpartum would increase the likelihood of false positive readings due to residual PAG concentrations from the previous pregnancy.

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

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

Changes in milk and plasma progesterone and pregnancy-associated glycoprotein and their relationships with the foetal number during early pregnancy in Jakhrana goats

This study was designed to investigate milk and circulating profiles of progesterone (P4) and pregnancy-associated glycoprotein (PAG) during early pregnancy and to determine their associations with the foetal numbers in Jakhrana goats. For this, milk (whole and defatted milk) and blood samplings from10 pregnant and 3 non-pregnant Jakhrana goats were continued from the day 7 until day 51 post-mating. The PAG profile in both milk and plasma increased gradually from day 26 to 51 of gestation. Whereas, circulating P4 remains unchanged during early pregnancy in pregnant goats. The P4 and PAG concentrations in blood plasma, whole milk and defatted milk were 1.30, 1.24 and 2.04, 1.98, 1.99 and 1.88 folds higher in twin foetus than the single foetus bearing does, respectively. The P4 and PAG in plasma and milk samples were positively correlated with the foetal number. However, the results of stepwisemultivariate linear regression analyses indicated milk and circulating P4 as better predictors of foetal numbers than plasma PAG. The defatting of milk samples resulted in about 2-folds decline in the P4 concentration, however, the PAG level remains unaffected. In conclusion, milk and plasma profiles of P4 and PAG were affected by the foetal number and P4 is a reliable predictor of foetal number during early pregnancy in Jakhrana goats.

Impact of trace mineral source on beef replacement heifer growth, reproductive development, and biomarkers of maternal recognition of pregnancy and embryo survival.

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. Beef heifers (n = 129) were randomly assigned to one of two treatments. From weaning through breeding, all heifers were individually fed a basal diet supplemented with cobalt (Co), copper (Cu), manganese (Mn), and zinc (Zn) either from organic sources (COMP; Cu, Mn, and Zn amino acid complexes and Co glucoheptonate; Availa-4, Zinpro Corporation, Eden Prairie, MN) or inorganic sources (INORG; Cu, Mn, and Zn hydroxychlorides; Intellibond C, M, and Z, Micronutrients, Indianapolis, IN) and Co as CoSO4. Blood samples and a reproductive tract score (RTS) were collected to determine pubertal status. All animals were synchronized and artificially inseminated. Pregnancy status was determined by lymphocyte gene expression, circulating concentrations of pregnancy-associated glycoproteins (PAGs), and by transrectal ultrasonography after artificial insemination. 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 tendency (P = 0.13) for decreased embryonic loss among COMP heifers (27 ± 6%) compared to INORG heifers (38 ± 6%). There was a treatment by pregnancy status by time interaction (P < 0.01) on circulating PAG concentrations with PAG concentrations tending (P = 0.08) to be greater on day 25 among heifers in the COMP treatment compared to heifers in the INORG group. 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 circulating PAG concentrations and embryo survival.

PSV-2 Use of Pregnancy Associated Glycoproteins to Determine Fetal Age Throughout Gestation

Abstract Blood pregnancy tests have gained popularity as there is no need for a costly ultrasound machine or special training; however, blood pregnancy tests only provide an answer of pregnant or open. Conversely, palpation and transrectal ultrasonography can determine gestational age. The objective of this study was to determine if a commercially available blood pregnancy test could detect differences in pregnancy-associated glycoprotein (PAG) concentrations indicative of gestational age. Previously identified pregnant females were grouped by age (heifers n=173, cows n=512); blood samples were collected between d 27 and 190 of gestation. Serum was tested in duplicate using a commercially available blood pregnancy test, IDEXX Alertys Ruminant Pregnancy Test. Procedures were adapted to allow concentrations to be within detectible range of the assay. Data was analyzed using MIXED procedure of SAS with age and gestational age (animals grouped into four gestational groups 1;&amp;lt; 30, 2;30–90, 3;91–178, and 4; &amp;gt;178 d) in the model. There was an effect of age, gestational age, and age by gestational age interaction (P&amp;lt; 0.01). Heifers had greater PAG concentrations compared to cows. Among heifers, PAG concentrations did not differ between gestational groups 1, 2, and 3 (P&amp;gt;0.37), but group 4 had greater PAG concentrations than all other groups (P&amp;lt; 0.01). Among cows, PAG concentrations decreased from group 1 to 2 (P&amp;lt; 0.01), and then increased throughout gestation (P&amp;lt; 0.01). Within age, group data were analyzed using REG procedure of SAS. There was a positive correlation between gestational age and PAG concentrations among both heifers (P&amp;lt; 0.01; r2=0.25) and cows (gestational age 30 and greater P&amp;lt; 0.01; r2=0.64). In summary, among heifers circulating PAG concentrations increased with gestational age, but gestational age only accounted for 25% of the variation. Among cows, gestational age (d 30–190) accounted for 64% of variation in PAG concentrations, thus a modified blood pregnancy test may allow for determining gestational age.