Pregnancy-associated Glycoproteins Research Articles

Establishment of a Sandwich-ELISA for simultaneous quantification of bovine pregnancy-associated glycoprotein in serum and milk.

Bovine pregnancy-associated glycoproteins (boPAG) are expressed by trophoblast cells in the bovine placenta. The multigene family of boPAG belongs to the group of aspartic proteases. The accumulation and circulation in maternal blood and milk has made boPAG very useful and important for pregnancy diagnosis in cattle. The goal of the present study was to develop and validate a new Sandwich-ELISA which allows the detection of boPAG in maternal serum and whole milk. Therefore, 984 serum and 928 milk samples were collected monthly from 231 Holstein Friesian cows (Bos Taurus) from one week after insemination (p.i.) until six weeks postpartum. The ELISA is able to identify a cow as being pregnant at day 30 p.i. in serum and at day 40 p.i in milk with threshold values of 1.0 ng/ml in serum and 0.0165 ng/ml in milk. The postpartum half-life of boPAG was estimated to be 6.4 days in serum and 7.1 days in milk. The boPAG profile established during pregnancy in serum and milk showed a typical pattern. The amount of boPAG found in milk was 1.5 % of the amount of boPAG present in serum. In conclusion, a Sandwich-ELISA has been developed to quantify boPAG in serum and in whole milk simultaneously with the same test procedure. This is time saving for farmers and more efficient for laboratories.

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;< 30, 2;30–90, 3;91–178, and 4; >178 d) in the model. There was an effect of age, gestational age, and age by gestational age interaction (P< 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>0.37), but group 4 had greater PAG concentrations than all other groups (P< 0.01). Among cows, PAG concentrations decreased from group 1 to 2 (P< 0.01), and then increased throughout gestation (P< 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< 0.01; r2=0.25) and cows (gestational age 30 and greater P< 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.

Resynchronizing the first eligible estrus in dairy cattle after a prior insemination and fertility of the prior insemination after gonadotropin-releasing hormone and progesterone treatments

The hypothesis of this study tested whether application of designed treatments to synchronize estrus in nonpregnant previously inseminated lactating dairy cows increased the proportion of nonpregnant cows in estrus before early pregnancy diagnosis on Day 32 after the previous insemination (Day 0) and increase fertility of the pretreatment insemination. A progesterone insert (CIDR) and GnRH were applied to cows after insemination to resynchronize the returning estrus of cows that failed to conceive on Day 0. The combination of GnRH (Day 14) and a CIDR insert (d 17 through 24) in experiment 1 (n = 347 cows) did not increase (P = 0.13) the proportion of nonpregnant cows returning to estrus before pregnancy diagnosis, but increased (P < 0.01) the synchrony of their return by 24.4% points, and delayed (P < 0.01) that return by 2.3 ± 0.3 d compared with controls. Ovulation risk after GnRH treatment on Day 14 was only 10%. For cows that failed to return to estrus before Day 32, progesterone concentration on Days 14 and 17 were less than that in cows that returned to estrus by Day 32 and in pregnant cows. Cows that returned to estrus had larger follicles and fewer numbers of CL on Day 21 than pregnant cows and cows that failed to return to estrus, but concentrations of pregnancy-associated glycoproteins on Day 28 indicated that cows failing to return to estrus were likely pregnant but suffered embryo death. In experiment 2 (n = 881), use of GnRH alone (Day 7), a CIDR insert alone (Days 14 through 21), or in combination, failed to increase the proportion of nonpregnant cows in estrus before pregnancy diagnosis on Day 32 compared with controls. Cows receiving the CIDR insert had increased (P < 0.01) synchrony of estrus by 24–34% points compared with cows that did not receive a CIDR insert. More cows receiving GnRH had 2 or more CL on Days 14 and 21 compared with controls. Ovulation risk after GnRH on Day 7 was greater than 66%. In both experiments combined, treatments with GnRH or GnRH + CIDR insert increased (P = 0.015) pretreatment pregnancy per AI by 7.1% points, but did not affect pregnancy loss. Although administering GnRH with or without a CIDR insert synchronized returns to estrus, treatments failed to increase the proportion of nonpregnant cows reinseminated before pregnancy diagnosis, but increased pretreatment pregnancy risk.

Interleukin-6 supplementation improves post-transfer embryonic and fetal development of in vitro-produced bovine embryos

The use of in vitro produced embryos in dairy and beef cattle has increased in recent years, but compromised post-transfer pregnancy success prevents producers from capturing all the benefits this technology can provide. This study explored whether supplementing interleukin-6 (IL6) during in vitro embryo development influences post-transfer development of the embryo-proper, fetus and placenta during early gestation in cattle. Slaughterhouse-derived cumulus oocyte complexes underwent IVM (day −1) and IVF (day 0). On day 5 post-fertilization, embryos were treated with either 0 (CONT) or 100 ng/mL recombinant bovine IL6. No difference in blastocyst formation was detected on day 7.5 post-fertilization, but an increase (P < 0.05) in inner cell mass cell numbers and tendency for increased (P = 0.08) trophectoderm cell numbers were detected in IL6-treated blastocysts. A subset of the blastocysts was loaded individually into transfer straws, and embryo transfer (ET) was completed using estrous cycle stage-matched, nonlactating commercial beef and dairy cows. A subset of cows from each group underwent timed artificial insemination (TAI). Pregnancy rates were similar among all three treatment groups at day 28 and 70. No differences in crown-rump length (CRL), crown nose length (CNL), abdominal diameter (AD), or placental fluid volume (PFV) were detected between TAI and ET-IL6 groups. Reductions (P < 0.05) in CRL and AD were detected at day 56 and a tendency for a reduction (P = 0.08) in PFV was detected on day 35 when comparing the ET-CONT group with the TAI group. Reductions (P < 0.05) in CRL and PFV on day 28 and CNL and AD on day 56 as well as a tendency for a reduction (P = 0.08) in PFV on day 35 were detected when contrasting ET-CONT with ET-IL6. Circulating plasma pregnancy-associated glycoprotein concentrations were similar among all treatment groups. In summary, IL6 treatment to IVP embryos before ET produced pregnancies that more closely resembled TAI-generated pregnancies than pregnancies generated using conventionally cultured embryos. These findings failed to find any adverse effects of IL6 supplementation on early development of the embryo-proper and fetus or on placental activity. Rather, these observations suggest that IL6 treatment may normalize the developmental trajectory of the embryo-proper and fetus for in vitro produced embryos.

Early embryo losses, progesterone and pregnancy associated glycoproteins levels during summer heat stress in dairy cows.

Objectives of this study were to characterize the effects of heat stress on pregnancy associated glycoproteins (PAG) and progesterone and its involvement in embryo survival. In trial 1, blood samples collected from days 29 to 36 post insemination were examined for the comparison of PAG concentrations between winter (n = 3721) and summer (n = 2388). In trial 2, embryo losses were assessed in winter (n = 144) and in summer (n = 133), in days 31 or 32 of pregnancy. Pregnancy diagnosis was carried out by ultrasonography on days 24 or 25, and it was repeated a week later; in the second occasion PAG concentration was also determined. In trial 3 the PAG and progesterone concentrations were assessed in days 33 to 36 in winter and summer. In trial 1 PAG levels did not differ between winter and summer, the conception rate and the proportion of uncertain pregnancies were higher in winter than summer. The likelihood of pregnancy was 10 to 15% higher in winter. In trial 2, the embryo death rate was higher in summer, but the PAG levels of cows that had embryo loss in summer were higher than those in winter. In both seasons, lower PAG levels were associated with higher risk of pregnancy loss, while embryo death was five times more likely to occur in summer than in winter and lower PAG concentrations were positively associated with higher risk of embryo loss. In trial 3, mean PAG levels were higher and of progesterone were lower during the summer than during the winter. We infer that despite the devastating effects of heat stress on cows' fertility, those early embryos that survive under continuous heat stress can form a well-functioning placenta; hence, the high embryo mortality rate observed during the summer months could be mainly attributed to luteal insufficiency.

Evaluation of synchronization protocols and methods of early pregnancy diagnosis in dairy cattle

The studies aimed to evaluate the pregnancy rate (PR) for timed artificial insemination (TAI) after G7G-Ovsynch, modified G7G-Ovsynch (MG7G-Ovsynch) and Ovsynch protocols and to assess the accuracy of using pregnancy-associated glycoproteins (PAGs) and plasma progesterone (P4) in pregnancy diagnosis compared with ultrasonography (US). In study 1, Holstein cows (n = 37) were bred by TAI following the G7G-Ovsynch protocol (n = 19) or MG7G-Ovsynch (n = 18). Pregnancy was evaluated by US at days 31, 59, and 87 after breeding. The PR was not different for the G7G-Ovsynch and MG7G-Ovsynch. Blood and milk samples were collected on day 3 after insemination and then weekly through day 59 post TAI in cows diagnosed as not pregnant on day 31 and through day 87 in pregnant cows. PAGs were measured using ELISA and P4 by radioimmunoassay (RIA). In the second study, Holstein cows (n = 212) were bred by TAI following G7G-Ovsynch protocol (n = 110) or standard Ovsynch (n = 102). Cows were subjected to pregnancy diagnosis on days 30, 60, and 90. A subset (n = 15 in each group) was subjected to blood and milk samples on days 30, 45, 60, 75, and 90 to measure PAGs and P4. In study 2, PR was not significantly different between synchronization protocols on days 30, 60, and 90. Pregnancy loss averaged 15% between day 30 and day 90. The use of PAGs and P4 proved equally effective in diagnosis of pregnancy. Thus, G7G-Ovsynch was deemed the protocol of choice in postpartum cows, and PAGs assayed in milk or plasma could be used to diagnose pregnancy.

Prevalence, risk factors, and effects on fertility of cytological endometritis at the time of insemination in Norwegian Red cows

The present study aimed to assess the occurrence of cytological endometritis (CYTO), a nonsymptomatic inflammation of the endometrium, at first artificial insemination (AI) postpartum in Norwegian Red cows. Further, risk factors for CYTO manifestation and its effect on reproductive success and late embryo loss were evaluated. In total 1,648 cows located in 116 herds were included in the study. On mainly spontaneous estrus, endometrial cytology samples were collected using a cytotape technique, and a total of 300 representative epithelial cells and polymorphonuclear neutrophils (PMN) were counted at 400× magnification. Vaginal mucus obtained by Metricheck (Simcro) and body condition score were recorded. Milk samples for progesterone analysis were collected at AI and 21 d later. Pregnancy was diagnosed by rectal palpation or analysis of pregnancy-associated glycoproteins. Based on the constructions of a receiver operator characteristics curve, the cut-off level for PMN defined as CYTO was set to 3.0%, representing the level at which the PMN occurrence affected pregnancy outcome, with the highest summation of sensitivity (32.4%) and specificity (74.9%). Three logistic models with herd included as random factor were constructed. The outcome for the first model was the likelihood for CYTO based on the endometrial samples, in the second model pregnancy to first AI, and in the third model embryo loss. The proportion of CYTO was 28.0% (461/1,648). The average interval in days to first AI was 71.7 d (standard error ± 0.7) and the overall pregnancy incidence to first AI was 59.8% (866/1,449). The likelihood for CYTO at first AI was associated with AI personnel, calving to first AI interval, vaginal mucus characteristics, amount of red blood cells in sample, season, and barn type. Pregnancy to first AI was lower in CYTO-positive cows (odds ratio = 1.51, confidence interval = 1.17-1.94). Other factors affecting pregnancy to first AI were AI personnel, test day milk yield, barn type, and obstetrical conditions or fertility treatments before first AI. The proportion of late embryo loss and abortion was 8.6% (82/948) and 2.8% (24/866), respectively. Late embryo loss was associated with treatment against fertility disorders before first AI, but not associated with CYTO. Overall, our results suggest that even if Norwegian Red cows show a fairly high prevalence of CYTO in the endometrium at first AI, it does not seem to have a major effect on the reproductive performance. The Norwegian Red breeding program has emphasized fertility and health for decades, and a genetically advantageous uterine immunology might be one of the preserved mechanisms.

Early pregnancy diagnosis in water buffaloes through detection of Pregnancy-associated glycoprotein (PAG) in milk using Enzyme-link immunosorbent assay

Early pregnancy diagnosis in water buffaloes through detection of Pregnancy-associated glycoprotein (PAG) in milk using Enzyme-link immunosorbent assay

Characterization of pregnancy-associated glycoproteins and progesterone as a predictor of twins and conceptus loss in high-risk-pregnancy Holstein cows

The objective of this study was to characterize plasma concentrations of pregnancy-associated glycoprotein (PAG) and progesterone (P4) as predictors of twins and pregnancy loss in Holstein cows with high-risk pregnancies. High-risk pregnancy was characterized using transrectal ultrasonography 37 d after artificial insemination (AI) based on the following criteria: small embryo size <15 mm, n = 10), slow heartbeat (<60 beats per minute, n = 11), and extra-amniotic membrane (additional amniotic membrane, n = 3). A cohort of twins (n = 41) diagnosed at d 37 post-AI was also enrolled. Each high-risk and twin pregnancy cow was paired with a cow of the same parity carrying a normal singleton at d 37 post-AI (control, n = 65). Blood samples were collected to measure PAG and P4 at d 37, 44, and 51 post-AI. Statistical analysis was performed using ANOVA, logistic regression, and receiver operator characteristic (ROC) curve with JMP software (SAS Institute Inc., Cary, NC). Pregnancy loss at d 51 post-AI was greater in high-risk pregnancies than in controls and twins (control = 1.5%; high-risk = 87.5%; twins = 12.2%). Concentration of PAG at d 37 post-AI did not differ among groups (control = 5.3 ± 0.7; high-risk = 4.8 ± 1.2; twins = 4.0 ± 0.9 ng/mL). Concentration of P4 at d 37 post-AI was greater in twins than in high-risk pregnancies and control, and lesser in high-risk pregnancies than in controls (control = 7.0 ± 0.3; high-risk = 5.9 ± 0.4; twins = 8.4 ± 0.3 ng/mL). Regression and ROC analysis for PAG at d 37 post-AI did not find a threshold to predict pregnancy loss or twins. Regression and ROC analysis for P4 at d 37 post-AI found that a threshold of 6.5 ng/mL predicted pregnancy loss with an area under the curve (AUC) of 0.64, and a threshold of 7.2 ng/mL predicted twins with AUC of 0.71. In summary, pregnancy loss and twins were predicted with only moderate accuracy by P4 concentration at d 37 post-AI, and the variability in PAG concentrations at d 37 post-AI was not sufficient to generate a threshold to predict pregnancy loss and twins in Holstein lactating cows.

Approaches to Identify Pregnancy Failure in Buffalo Cows.

Simple SummaryEmbryonic mortality and pregnancy failures still represent a major issue in domestic livestock production, particularly in dairy cattle. Despite the presence of extensive work in this research area, there is still no effective, accurate and practical method able to determine timing and viability of embryo specifically during early gestation. Indeed, technologies and techniques for predicting pregnancy success must continue to be developed. The aim of this work was to find the best strategy to diagnose pregnancy failures in buffalo cows in order to improve farm reproductive management. Among the methods compared in this study (ultrasonography, progesterone, PAGs), pregnancy-associated glycoproteins (PAGs) seem to be the best marker for predicting embryonic mortality between 25 and 40 days of gestation to be utilized as a diagnostic tool to improve reproductive management in buffalo farms.The aim of this work was to find the best strategy to diagnose pregnancy failures in buffalo. A total of 109 animals belonging to a buffalo herd subjected to a synchronization and artificial insemination (AI) program were enrolled in this study. Blood samples were collected at days 0, 14, 25, 28 and 40 after AI for the determination of progesterone (P4) and pregnancy-associated glycoproteins (PAGs) by the radioimmunoassay (RIA) method. Transrectal ultrasonography was performed on day 25, 28 and 40 after AI to monitor pregnancy. The animals included in the data analysis were assigned ex post in pregnant (n = 50) and mortality (n = 12) groups. By ultrasonography, the predictive sign of mortality was the heartbeat. At day 25, the PAGs concentration was significant in predicting embryonic mortality with respect to ultrasonography and P4, at the cut-off of 1.1 ng/mL. At day 28, either PAGs, at a cut-off of 2.2 ng/mL, or ultrasonography, with no detection of heartbeat, were highly predictive of embryonic mortality. PAGs were the best marker (p < 0.05) for predicting embryonic mortality between 25 and 40 days of gestation in buffalo. Its utilization as a diagnostic tool can influence management decisions in order to improve farm reproductive management.

EVALUATION OF A VISUAL ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA) FOR PREGNANCY DETECTION IN FOUR UNGULATE SPECIES.

This study evaluated the use of a commercially available, visual enzyme-linked immunosorbent assay (ELISA) for diagnosis of pregnancy in okapi (Okapia johnstoni), gerenuk (Litocranius walleri), eastern giant eland (Tragelaphus derbianus spp. gigas), and dama gazelle (Nanger dama). This assay has been validated for use in domestic cattle, sheep, goats, and water buffalo. Unlike other blood-based pregnancy associated glycoprotein (PAG) detection methods, this assay does not require sophisticated laboratory equipment for detection or interpretation and can therefore be utilized in many settings. Banked serum samples from gerenuk (n = 11), giant eland (n = 4), dama gazelle (n = 33) and okapi (n = 3) were tested, and a pregnant and nonpregnant sample from each individual were included. The ELISA showed 100% sensitivity and specificity in gerenuk and giant eland samples, and 0% sensitivity in dama gazelle and okapi samples. Using this assay, pregnancy was detected by 7-8 wk gestation in gerenuk and 6 wk in giant eland. These results are consistent with previous studies that were able to accurately detect pregnancy in other members of the family Bovidae, but it is possible that PAGs present in okapi and dama gazelle are structurally dissimilar relative to the intended test target, and are therefore unrecognizable using this test. The faint positivity in the dama gazelle assays may be due to cross-reactivity with other proteins in the sample, or due to inconsistent binding with the dama gazelle PAG. This ELISA appears to be an accurate, rapid, and inexpensive method of point-of-care pregnancy diagnosis in gerenuk and giant eland, but not okapi and dama gazelle. Additional studies should be pursued to further characterize the limits of pregnancy detection using this assay in gerenuk and giant eland, and to investigate the validity of this test in other nondomestic ruminant species.

Alterations in pregnancy-associated glycoprotein concentrations of pregnant sheep experimentally infected with bovine viral diarrhea virus.

To compare pregnancy-associated glycoprotein 1 (PAG1) concentrations in maternal (jugular vein) and fetal (uterine vein) circulations and amniotic fluid samples between pregnant ewes that were and were not experimentally infected with bovine viral diarrhea virus (BVDV). 11 healthy pregnant yearling ewes. Before study initiation, all ewes were naïve to BVDV and confirmed pregnant by transabdominal ultrasonography at approximately 60 days of gestation. At 65 days of gestation, ewes were intranasally inoculated with a noncytopathic BVDV type 1b strain (concentration, 107 TCID50/mL; 2 mL/nostril; n = 6) or an equal volume of BVDV-free viral culture medium (control; 5). A blood sample was collected for measurement of PAG1 concentration before inoculation. At 80 days of gestation, each ewe was anesthetized and underwent an ovariohysterectomy. While sheep were anesthetized, blood samples from the jugular and uterine veins and an amniotic fluid sample were collected for measurement of PAG1 concentration. Fetal tissues underwent real-time PCR analysis for BVDV RNA, and placental specimens underwent histologic evaluation and immunohistochemical staining for BVDV antigen. At 80 days of gestation, BVDV RNA in fetal tissues and mild placentitis were detected in 5 of 6 BVDV-inoculated ewes. Mean PAG1 concentrations in the maternal and fetal circulations of BVDV-inoculated ewes were significantly less than those in control ewes. Mean amniotic fluid PAG1 concentration did not differ significantly between the 2 groups. Concentration of PAG1 in the maternal circulation may be a useful biomarker for determining placental health in sheep after viral infection of the reproductive tract.

Determination of pregnancy protein levels to distinguish between singleton and twin pregnancies in Awassi sheep

Determining the fetal number to avoid pregnant sheep management, feeding, and delivery issues is of vital importance. This study aimed to determine the levels of pregnancy-associated glycoprotein (PAG) and pregnancy-specific protein B (PSPB), which are pregnancy proteins, to accurately predict singleton and twin pregnancies in Awassi sheep. A total of 40 Awassi sheep were used for the study. According to the number of offspring, pregnant ewes were separated into two groups. The study's first group (Group 1) included singleton pregnant ewe (n=20), while the second group (Group 2) included twin pregnant ewe (n=20). Blood samples were collected from the ewes of both study groups at 30th, 45th, 60th, 75th and 90th day of gestation, which were used for PAG and PSBP evaluation along with ultrasonographic examination. Independent samples t-test and repeated measurements ANOVA were used to analyze the data. The correlations between the measures were calculated using Pearson correlation coefficients. Accordingly, a statistically significant difference was observed between single and twin pregnant sheep for all PAG and PSBP measurements at days 30-45-60-75 and 90 (p&lt;0.05). In singleton and twin pregnant ewes, there was a statistically significant difference in PAG and PSBP measurements (p&lt;0.05). In singleton pregnant ewes, a significant positive correlation was found between PAG30 and PSPB30 values (p&lt;0.05), while a significant negative correlation was found between paired measures of PAG45-PSPB90, PAG60-PSPB90, and PAG75-PSPB90 (p&lt;0.05). In conclusion, significant differences in pregnancy protein levels were found in singleton and twin pregnant ewes. It was deduced that knowledge of this difference might give sheep breeders an idea about management factors.

Pregnancy-associated glycoprotein concentrations during early gestation in pregnant Awassi sheep

This study was aimed at determining the serum pregnancy-associated glycoprotein (PAG) profile of pregnant Awassi sheep during early gestation. A total of 122 Awassi ewes were used, which were in days 28-56 (28, 30, 32, 36, 42, 49, and 56) of gestation. Blood samples were taken via jugular venepuncture. Out of these animals, 104 ewes that had delivered (singleton lambs, n = 60; twin lambs, n = 44), according to lambing records, were selected for the assessment of serum PAG levels. The serum PAG level was measured using a commercial enzyme-linked immunosorbent assay (ELISA) kit (IDEXX Bovine Pregnancy Test). Whereas serum PAG levels (mean ± standard error) on days 28, 30, 32, 36 and 42 of gestation were found to be similar (0.237 ± 0.031, 0.402 ± 0.067, 0.329 ± 0.036, 0.397 ± 0.075, and 0.409 ± 0.060, respectively), the levels measured on days 49 and 56 of gestation were significantly higher (0.712 ± 0.088 and 0.798 ± 0.115, respectively) (p &lt; 0.05). The serum PAG levels of the twin-pregnant ewes on days 28, 30 and 42 of gestation were higher than those of the singlepregnant ewes (twin-pregnant ewes: 0.314, 0.648 and 0.594, respectively; single-pregnant ewes: 0.186, 0.258 and 0.308, respectively; p &lt; 0.05). With a cut-off sample minus negative (S–N) value set at &gt; 0.300, the sensitivity of serum PAG level-based pregnancy diagnosis was 51.95% between days 28 and 42 of gestation and 92.69% between days 49 and 56 of gestation. On the other hand, with a cut-off S–N value set at &gt; 0.100, the sensitivity of the PAG ELISA test was 94.81% between days 28 and 42 of gestation and 100% between days 49 and 56 of gestation. In conclusion, the serum PAG levels of the pregnant Awassi sheep remained similar between days 28 and 42 of gestation, but progressively increased thereafter. The serum PAG levels of the twin-pregnant Awassi ewes were significantly higher than those of the single-pregnant sheep on days 28, 30 and 42 of gestation, but they were not different on days 32, 36, 49 and 56 of gestation.

PSIV-25 Effect of Various Handling Methods of Plasma and Serum Samples on Pregnancy Associated Glycoprotein Concentrations

Abstract Pregnancy associated glycoproteins (PAG) can be used as a biomarker for early pregnancy diagnosis, so accurate and consistent PAG detection is critical. The objective of this study was to determine if plasma and serum PAG concentrations were altered when centrifugation occurred at different times post-collection, when subjected to repeated freezing and thawing, and when monoclonal antibodies were kept in frequently or infrequently opened containers. Plasma (n = 4) and serum (n = 4) samples were collected from two open cows and two pregnant cows 28 days after artificial insemination. Pregnancy status was determined via transrectal ultrasonography. Plasma and serum samples were evenly separated and either centrifuged on the day of collection, or placed at 4°C and centrifuged the next day. An in-house PAG ELISA was performed on all samples before freezing (NOTHAW), after being frozen for one week (INTACT), after one freeze/thaw cycle (THAW1), two freeze/thaw cycles (THAW2), and three freeze/thaw cycles (THAW3). Data were analyzed using one-way ANOVA (GLM procedure, SAS 9.4). All samples from open cows were below the baseline of the assay. For pregnant cows, plasma samples had greater PAG concentrations than serum samples (11.84 vs 3.30 ± 0.66 ng/mL, respectively, P &amp;lt; 0.05). No differences were observed for day of centrifugation in both plasma and serum samples (P = 0.50 and P = 0.60, respectively) and in handling of monoclonal antibodies (P = 0.90). Freezing and thawing did not impact PAG concentrations in plasma samples (P = 0.19), but did alter serum concentrations (P = 0.01). Specifically, THAW1 (1.98 ng/mL) and THAW2 (1.42 ng/mL) serum PAG concentrations were lower compared to NOTHAW, THAW3, and INTACT samples (4.66, 4.85, and 3.57 ng/mL, respectively). Based on these data, plasma yields more consistent results than serum, even after several freeze-thaw cycles, and handling of monoclonal antibodies or time of centrifugation has no significant effect on measured PAG.

Efficacy of a commercial test kit to determine early pregnancy in cows using whole blood and blood serum.

The objective of this study was to determine the efficacy of the Fassisi®BoviPreg visual test kit (Fassisi®BoviPreg) in determining early pregnancies in cows by measuring the pregnancy-associated glycoprotein (PAG) in whole blood and blood serum. The study was conducted on 50 cows, the artificial insemination (AI) dates of which were designated as day 0. Pregnancy diagnosis was performed with transrectal ultrasonography (USG), and serum samples were simultaneously collected and used with Fassisi®BoviPreg to determine pregnancies on 30days after AI. The results of the Fassisi®BoviPreg test on serum and whole blood samples, respectively, on 30days after AI were as follows: sensitivity, 61.54% and 50.0%; specificity, 79.17% and 75%; accuracy, 70.0% and 62.0%; positive predictive values, 76.2% and 68.4%; negative predictive values, 65.5% and 58.1%; false-positive diagnoses, 23.8% and 31.6%; and false-negative diagnoses, 34.5% and 41.9%. On day 50 after AI, sensitivities were 63.64% and 50.0%, specificities were 100.0% and 100.0%, and accuracies were 75.0% and 65.62% in serum and whole blood, respectively. Higher pregnancy rates were obtained using Fassisi®BoviPreg in cows with ≥ 5ng/mL P4 (P < 0.001). In conclusion, the results from the Fassisi®BoviPreg tests on cows on 30 and 50days after AI showed that the use of blood serum is more accurate and suitable than that of whole blood. The results also showed a higher confidence level in specificity 50days after AI.

The Effectiveness of Pharmacological Synchronization of the Estrous Cycle in Hinds (Cervus elaphus L.): A Pilot Field Trial.

Simple SummaryReproductive biotechnics of Cevidae species may increase valuable traits for the agricultural sector, and improve genetic diversity in relatively small farmed populations. The first step of biotechnics in terms of suitability for embryo transfer or insemination is preparation of females for proper phase of the estrous cycle. Based on available synchronization protocols applicated in red deer and domestic ruminant females we estimated the protocol for hinds in view of future routine use. Progestin analogues in the form of an insert (CIDR) or a sponge (Chronogest) and injection of equine chorionic gonadotropin (eCG; Folligon) were used on the appropriate day for inducing the estrus. The effect of synchronization was checked by measurement of progesterone (P4) and 17-beta estradiol (E2) concentration by RIA, pregnancy-associated glycoproteins (PAG)s concentration by EIA and insemination effectiveness—pregnancy rate. The better method of the estrous cycle synchronization found down to be progestin sponge and eCG based protocol in hinds considering hormone responsiveness and pregnancy rate.The aim was to estimate the effective pharmacological method of the estrous cycle synchronization by checking the effects of synchronization by measurement of progesterone (P4) and 17-beta estradiol (E2) concentration by RIA and artificial insemination. The experiment was performed at the red deer farm in Rudzie (North-East Poland; 3 year’s old). The herd (N = 14) was kept away from bulls and was divided in two groups of seven animals. In the Group I, CIDR insert (0.3 g of P4) was applicated intravaginally for 12 days; a second insert replaced the first one for the next 12 days, and next 200 IU of equine chorionic gonadotropin (eCG) was injected intramuscularly (Folligon). Estrus was expected 48 h after eCG injection. In the Group II, Chronogest sponge (20 mg of flugestone acetate) was applicated intravaginally and after 7 days replaced with second chronogest sponge for 7 days. After removing the sponge, on the same day eCG was injected and estrus was expected after 48 h. Artificial insemination was provided with frozen-thawed semen twice: 12 and 24 h after expected estrus. The peripheral blood from the jugular vein was collected each time when the inserts or sponge were applicated and 40 days after insemination. The concentration of P4 and E2 in plasma was measured by RIA. The effectiveness of insemination was monitored by pregnancy-associated glycoproteins determination and observed by the number of calves born. Two pregnancies were confirmed in Group I and five in Group II based on PAG concentration. One newborn was observed in Group I and five in Group II. Both methods of synchronization are effective in hinds based on the received profile of steroids. Although the sponge shape in case of chronogest is better comparing with CIDR, which was not completely deposited in the vagina of hind, potentially leads to bacteria inflammation, and it disturbs the rightful endocrine regulation. Moreover, pregnancy rate and hormone responsiveness were better in Group II.

Preconceptional diet manipulation and fetus number can influence placenta endocrine function in sheep

Changes in maternal nutrition during pregnancy can result in profound effects on placental function and fetal development. Although the preconceptional period holds the potential to reprogram embryonic and placental development, little is known regarding the effects of premating nutritional manipulation on placental function and fetal and postnatal offspring growth. To test this, Polypay-Dorset sheep (n = 99) were assigned to 1 of 3 nutritional treatments (n = 33/treatment) receiving 50% (UN: undernutrition), 100% (C: control), or 200% (ON: overnutrition) of maintenance energy requirements for 21 d before mating during April–May (increasing photoperiod). Thereafter, diets were the same across groups. We evaluated maternal reproductive variables and maternal and offspring weight and body mass index through weaning. Maternal plasma was collected through pregnancy until postnatal day 1 to assay pregnancy-associated glycoproteins (PAGs) and progesterone. Fertility rate was similar among treatments, but ON females had a higher reproductive rate (UN: 82%; C: 100%, ON: 145%). When correcting by total birth weight, twin pregnancies had lower PAGs and progesterone versus singleton pregnancies (P < 0.001). At birth, UN lambs were heavier than C lambs regardless of birth type (P < 0.01). Growth velocity, daily gain, and weaning weight were similar, but UN and ON females grew faster and were heavier at weaning versus C females. We demonstrated that a 3-wk preconceptional maternal undernutrition or overnutrition, when correcting by total birth weight, results in lower endocrine capacity in twin pregnancies. Preconceptional maternal undernutrition and overnutrition increased postnatal female lamb growth, suggestive of reprogramming of pathways regulating growth before conception. This highlights how preconceptional nutrition can result in marked sex-specific differences.

Determination of fetal viability

Assessment of the feto-placental unit is needed during maternal states of health and disease. This presentation will discuss practical approaches to assess reproductive efficiency and placental health. Biomarkers including pregnancy-associated glycoproteins, and the use of ultrasonography will be described. Current literature review will be presented describing the utilization of different markers to assess placental health during times of disease or determine fetal outcome following conception. The practitioner will be exposed to cutting-edge research in the area of placental health, yet walk away with a practical understanding where these progressive techniques can be used in practice.

Temporal changes in circulating progesterone and pregnancy-associated glycoprotein concentrations in Jakhrana goats with failed pregnancy

This study aimed to evaluate the changes in circulating Progesterone (P4) and Pregnancy-Associated Glycoprotein (PAG) during pregnancy interrupted by abortion and to identify the better predictor of abortion risk in Jakhrana goats. Pluriparous goats (18) were involved in the study. Out of 15 pregnant goats, 3 goats were aborted during the third or fourth months of pregnancy. In the normal pregnancy, mean P4 concentration (13.96±0.27 ng/mL) remained unchanged throughout gestation whereas, the PAG level increased during early pregnancy and reached to the highest level (S-N=2.14±0.40) on day 51 of gestation. In non-pregnant goats, the P4 (0.3±0.03 ng/mL) and PAG (0.06±0.03) were significantly lower compared to the goats with maintained or failed pregnancy. In goats with the failed pregnancy, the PAG level started to decline about 12 days before the drop in P4 concentration (day 33.0±2.1 vs 21.3±3.5, before the occurrence of abortion). The mean PAG level in goats with failed pregnancy (0.33±0.03) was 4.8-folds lower compared to the animals with maintained pregnancy (1.58±0.08). In conclusion, the P4 and PAG concentrations in maternal circulation are changed by the pregnancy status and abortion. Plasma PAG is a more reliable predictor for the high risk of pregnancy failure than the P4 concentration in goats.