Pregnancy-specific Protein B Research Articles

Hormonal profiles and biomarkers leading to parturition in cattle.

This study aimed to understand the physiological mechanisms regulating parturition and to identify potential biomarkers to predict onset of birth. Additionally, we compared hormone profiles between cows with shorter and longer gestation lengths. Twenty-eight days before due date until 3d postpartum, cows (n = 18) were blood sampled daily. Circulating concentrations were measured for progesterone (P4) and estradiol (E2) by RIA, testosterone, prostaglandin F2α metabolite (PGFM), cortisol, pregnancy-specific protein B (PSPB) by ELISA and lactate concentrations by colorimetric assay. At end of gestation, P4 decreased from d-14 to d-4 (from 3.6 to 1.4ng/mL), most likely from rapid loss of placental P4 production (64% of decline in 24h). A second rapid decrease in P4 to undetectable concentrations was observed from d-2 to parturition (from 1.4 to 0.1ng/ml; most likely luteal origin) corresponding to increase in PGFM from d-2 to parturition (249.7 to 2868.4pg/mL). Estradiol and PSPB increased ~8-fold from ~13d before parturition with acute rise in E2 but not PSPB (45% vs 13% in first 24h). Testosterone decreased slightly during the same period. Cortisol and lactate increased only at calving. Comparison of cows with shorter vs longer gestation, when data were normalized to parturition day, a difference was detected in circulating E2 and PGFM patterns, but not P4 and PSPB. Thus, the first significant hormonal changes associated with parturition begin at d-14 with E2 and PSPB as two clear biomarkers of impending parturition. Cows with shorter and longer gestation had hormonal differences indicative of identifiable earlier placental maturation.

Estrogen to progesterone ratio is associated with conceptus attachment in dairy cows receiving artificial insemination after Double-Ovsynch but not estrus†.

Prediction of pregnancy survival in lactating dairy cows can be determined by the conceptus attachment timeframe via daily pregnancy-specific protein B (PSPB) monitoring. All factors contributing to reduced fertility in dairy cows receiving AI following estrus detection remain unclear. This study aimed to determine differences in time to conceptus attachment in lactating cows treated with the fertility program Double-Ovsynch compared to cows that were detected in estrus. Additionally, we investigated various pre- and post-conception factors potentially influencing fertility outcomes. We hypothesized that AI following a natural estrus detected with automated activity monitors would lead to an extended time to conceptus attachment and lower PSPB concentrations post-attachment compared to Double-Ovsynch. There were no differences in the average time to conceptus attachments between treatments. However, cows inseminated post-estrus that experienced pregnancy loss between conceptus attachment and 60-66days post-AI exhibited diminished PSPB concentrations on Days 2 and 3 following conceptus attachment. Steroid hormone interactions were assessed with radioimmunoassay to determine the ratios of estrogen to progesterone concentrations on the day of the luteinizing hormone (LH) surge. Notably, estrogen to progesterone ratio proved to predict conceptus attachment in cows subjected to Double-Ovsynch but not in those inseminated post-estrus detection surge. In conclusion, the estrogen to progesterone ratio measured around the time of the pre-ovulatory LH surge emerges as a potentially effective tool for estimating the fertility potential of lactating dairy cows undergoing timed AI, particularly in the context of the Double-Ovsynch program.

Development and evaluation of a lateral flow-based portable optical system for determination of the pregnancy status of dairy cows

Our objectives were to develop and evaluate an integrated system consisting of a lateral-flow immunoassay (LFIA) and an electronic portable imaging device for determination of pregnancy status of cows based on plasma concentrations of pregnancy-specific protein B (PSPB). Experiment 1 was conducted to test the performance of the LFIA for PSPB (PSPB-LFIA) whereas experiment 2 was conducted to evaluate the performance of the integrated system including both the LFIA and imaging device. The PSPB-LFIA strips were made of nitrocellulose membrane with polystreptavidin, anti-mouse antibody, Europium-anti-PSPB conjugates, and biotin-PSPB. After adding buffer and plasma in a 96-well plate, strips were dipped to initiate flow and were read in a fluorescence microscope to estimate PSPB concentrations based on the test-to-control line signal (T/C ratio). The T/C ratio of standards was linearly associated with PSPB (R2 = 0.99 in both experiments) concentrations. To test the ability to identify pregnant cows of the PSPB-LFIA only or the integrated system, plasma samples were collected and transrectal ultrasonography (TUS) was conducted 29 to 35 d post AI in lactating Holstein cows (Experiment 1: n = 83; Experiment 2: n = 205). A cow was considered pregnant (Preg) if concentrations of PSPB in plasma obtained by ELISA were ≥2 ng/mL or if an embryo was visible by TUS. In Experiment 1, the accuracy of the PSPB-LFIA compared with ELISA was 92.7% (91.2% Se; 96.1% Sp; 98.1% PPV; 83.3% NPV) and compared with TUS was 90.4% (100% Se; 78.9% Sp; 84.9% PPV; 100% NPV). The agreement between LFIA and ELISA (kappa = 0.84; 95%CI 0.71-0.96) or LFIA and TUS (kappa = 0.80; 95%CI 0.67-0.93) as methods to classify cows as Preg or Non-Preg was high. In Experiment 2, the accuracy of the PSPB-LFIA compared with ELISA was 96.1% (93.8% Se; 100% Sp; 100% PPV; 90.5% NPV) and compared with TUS was 92.2% (99.0% Se; 84.7% Sp; 87.6% PPV; 98.8% NPV). The agreement between LFIA and ELISA (kappa = 0.92; 95%CI 0.86-0.97) or LFIA and TUS (kappa = 0.84; 95%CI 0.77-0.92) as methods to classify cows as Preg or Non-Preg was high. We conclude that a system integrating a fluorescence-based LFIA and an optical reader was effective for classifying cows as pregnant or not pregnant based on estimations of plasma concentrations of PSPB. This novel system serves as a platform for further development of on-farm pregnancy testing tools based on measurement of biomarkers of pregnancy in bodily fluids of cattle.

Design of a handheld and portable fluorescence imaging system for quantitative detection of pregnancy-specific biomarkers in cattle.

Reproductive management significantly impacts dairy farm productivity, necessitating accurate timely pregnancy detection in cattle. This paper presents a novel handheld and portable fluorescence imaging system designed for quantitative assessment of pregnancy-specific biomarkers, addressing the limitations of current detection methods. The objective was to develop a cost-effective, at-farm solution for detecting pregnancy-specific protein B (PSPB) in bovine plasma samples. The system integrates an imaging module and a custom software application, enabling image capture, data processing, and PSPB concentration determination. Calibration utilizing known PSPB concentrations achieved a 0.6ng/mL limit of detection. Validation encompassed a comparison with a standard ELISA method using 100 bovine plasma samples; minimal bias and good agreement were observed within the linear range of the calibration curve for both methods. The system offers portability, user-friendliness, and potential for multiplex detection, promising real-time, at-farm reproductive management. This study demonstrates the successful development and validation of a portable fluorescence imaging system, offering an efficient and accurate approach to detecting pregnancy-specific biomarkers in cattle. Its implications extend to improving dairy farm productivity by enabling timely and reliable reproductive management practices.

VALIDATION OF A NOVEL ENZYME-LINKED IMMUNOSORBENT ASSAY FOR DETECTING PREGNANCY-SPECIFIC PROTEIN B IN MASAI GIRAFFE (GIRAFFA CAMELOPARDALIS TIPPELSKIRCHI).

The Masai giraffe (Giraffa camelopardalis tippelskirchi) is endangered in the wild, and successful reproduction in managed care is important to help maintain assurance populations of this highly charismatic subspecies. Detection of pregnancy in giraffes using hormonal monitoring requires multiple samples and cannot distinguish pregnancy from pseudopregnancy. A novel enzyme-linked immunosorbent assay that can detect pregnancy-specific protein B (PSPB) for pregnancy diagnosis with a single serum sample was developed from a reticulated giraffe (Giraffa camelopardalis reticulata) placenta. Seventy-eight serum samples were analyzed from three female Masai giraffes before and during five gestation periods that resulted in live calf births. Using an optical density cutoff of 0.2, the assay showed a sensitivity of 93% and specificity of 100% for all samples tested. At 59 d of gestation, sensitivity increased to 100%. The earliest pregnancy detection was at 40 d of gestation. This study documents the successful development of a blood-based PSPB assay for pregnancy diagnosis in Masai giraffe, which can help advance conservation efforts in this endangered species.

Comparability of Pregnancy Specific Protein-B (PSPB) Concentration in Serum and Urine of Kedah-Kelantan (KK) Cattle

This study aimed to investigate the effect of serum and urine PSPB levels based on different biomarkers, such as serum and urine. Serum PSPB levels significantly increased from day 0 to day 164 (P<0.05). However, the PSPB urine concentration through the ELISA test showed higher concentration and reached the sensitivity of the ELISA test from Day 0 until Day 164 after AI. The results of urine biomarkers at Day 0, Day 42, and Day 164 were not significant (P>0.05). Serum PSPB production begins to be established after day 24 of parturition due to embryonic development. Furthermore, the urine PSPB concentration is considered an abundant protein circulating in the animal body and ready to be excreted in the urine. In this study, the ELISA test was unsuitable for detecting pregnancy. However, urine biomarkers can potentially become biomarkers by studying the range of concentrations of PSPB that can be considered pregnancy. In addition, the PSPB protein allows for more prolonged survival in the bovine body and decreases gradually from the 22nd to the 54th day after calving. The results demonstrate that PSPB can detect pregnancy early in pregnancy using biomarkers and evolution. Therefore, this study could be improved by examining PSPB levels of pregnant and non-pregnant cattle.

Review: Initial increase in pregnancy-specific protein B in maternal circulation after artificial insemination is a key indicator of embryonic survival in dairy cows.

Lactating dairy cows have reduced chances for pregnancy survival following 1st parturition. It appears that timing of conceptus attachment, defined as an initial daily rise in pregnancy-specific protein B (PSPB) postartificial insemination (AI), is key to the survival of the embryo. PSPB is a protein produced from binucleate cells of the trophectoderm of the conceptus. Once the conceptus has attached to the uterine epithelial cells, this large protein is detectable in maternal circulation. Daily sampling of serum allows for detection of the initial increase of PSPB. A percent increase threshold for PSPB was determined from results from each study using a conservative three-day minimum increase. Thus far, a 10 or 12.5% daily increase for three consecutive days had the greatest sensitivities and specificities for studies reported in this review. Nulliparous heifers receiving AI following estrus appear to have a reduced time to conceptus attachment compared to lactating cows that received either Ovsynch or AI following estrus. Increasing progesterone post-AI did not reduce time to conceptus attachment in lactating cows. Multiparous cows treated post-AI with human chorionic gonadotropin had a reduced percent with conceptus attachment in addition to greater time to conceptus attachment. Increased time to conceptus attachment increased the chances of multiparous cows having embryonic death before 35 d post-AI. Concentrations of PSPB are consistently reduced from conceptus attachment until d 28 post-AI in cows with pregnancy loss compared to cows that maintain pregnancy on d 34 post-AI. This novel model to determine timing of conceptus attachment provides a new perspective on percent of cows pregnant around d 20-23 post-AI and the potential for a greater understanding of subsequent pregnancy loss.

EVALUATING THE EFFICACY OF NONINVASIVE FECAL SAMPLING FOR PREGNANCY DETECTION IN ELK (CERVUS CANADENSIS).

Elk (Cervus canadensis) were reintroduced to Tennessee, USA in the early 2000s, with limited reproductive monitoring since initial release. We assessed the efficacy of noninvasive sampling for determining pregnancy using invasive (capture) and noninvasive (fecal collection in the field) techniques at the North Cumberland Wildlife Management Area (NCWMA), Tennessee. We captured 20 female elk 2019-2020, used pregnancy-specific protein B (PSPB) in blood to determine pregnancy and compared results to fecal progesterone metabolite (FPM) concentrations using two commercially available enzyme immunoassay (EIA) kits. Based on PSPB concentrations, 8/11 and 3/4 of captured adult elk (≥2.5 yr of age) were pregnant in 2019 and 2020, respectively; no 1.5-yr-old elk were pregnant (n=5). Using the progesterone EIA kit, FPM concentrations were x̄=192.84±38.63 ng/g (95% CI, 96.48-289.20) for nonpregnant and x̄=536.17±74.98 ng/g (95% CI, 375.97-696.36) for pregnant captured females. For the progesterone metabolite kit, FPM concentrations were x̄=188.16±43.39 ng/g (95% confidence interval [CI], 76.63-299.69) for nonpregnant and x̄=693.52±126.52 ng/g (95% CI, 407.31-979.72) for pregnant captured females. From February to May 2019, we collected 357 fecal samples in 65 areas across 489.62 km2 of the NCWMA. Using extracted DNA and analysis of 15 microsatellites, we identified 62 unique individuals from 128 female fecal samples collected on the landscape. We categorized females from landscape-collected feces as nonpregnant (35.5-40.3%; Metabolite-EIA kits), undetermined (1.6-6.5%; Metabolite-EIA kits), or pregnant (62.9-53.2%; Metabolite-EIA kits) based on a 95% CI of captured female FPM concentrations, giving an overall pregnancy rate of 53.2% using the recommended EIA kit. The pregnancy rate in sexually mature females may be higher, as it was not possible to distinguish age classes of landscape-collected fecal samples; therefore, some may have been from younger age classes not expected to be pregnant. Analysis of FPM may be useful at a population level to detect pregnancy.

Late embryo mortality in Holstein cows inseminated with Holstein or Limousine frozen semen.

The objective of this study was to compare the late embryo mortality (LEM) rate (losses approximately between 32 and 53 days of gestation) and Pregnancy Specific Protein B (PSPB) and progesterone (P4) concentrations on day 32 post AI in Holstein cows bred with either Holstein or Limousine semen. A sample size of 1082 cows per group diagnosed pregnant between 28- and 35-days post breeding was calculated. The study consisted of evaluating LEM (%) in a cohort of Holstein cows bred with Holstein semen (HO × HO) or Limousine semen (HO × LM), to compare pregnancy loss from 28 to 35 days post breeding to 50-57 days post breeding. A logistic regression model to compare embryo losses was developed considering as main explanatory variable the cohort (HO × HO embryo vs. HO × LM embryo), correcting by lactation number, breeding season, days to breeding and AI technician. HO × HO embryos had greater LEM (15.16%) than HO × LM embryos (9.79%). Cows bred in summertime had higher LEM (15.23%) than cows bred in no-summertime (9.88%). There were no differences among AI technicians. Within summertime there was no difference in LEM (%) between groups within each lactation number; yet, within no-summertime, LEM (%) was higher in HO × HO than HO × LM within each lactation number. Pregnancy SPB optical densities were significantly greater in the HO × HO than in the HO × LM (p= .023) group; yet, the concentration of P4 was not different between groups (p> .05).

Time to increase in pregnancy-specific protein B following artificial insemination is a direct determinant of subsequent pregnancy loss in lactating dairy cows

Increasing progesterone (P4) during early conceptus development may be crucial for establishment of pregnancy in dairy cattle. The objective of this study was to determine if human chorionic gonadotropin (hCG) at various times after ovulation will increase serum P4 during elongation and increase the chances for, and reduce variability to, initial increase in pregnancy-specific protein B (PSPB) following artificial insemination (AI). Time to PSPB increase was defined as the first day of increase in concentrations of PSPB between d 18 and 28 after ovulation in cows with ≥12.5% increases for 3 consecutive days compared with baseline. Lactating cows (n = 368) synchronized to Double-Ovsynch (first service) or Ovsynch (second or greater service) received one of 4 treatments: no hCG (control), or 3,000 IU of hCG on d 2 (D2), 2 and 5 (D2+5), or 5 (D5) after ovulation. All cows were examined via ultrasound on d 5 and 10 postovulation to determine percentage of cows with hCG-induced accessory CL (aCL) and to quantify and measure all luteal structures. Samples for serum P4 were collected on d 0, 5, 19, and 20 postovulation. The P4 was increased in D2, D2+5, and D5 groups compared with control. The D2+5 and D5 treatments increased aCL and P4 compared with D2 and control. The D2 treatment increased P4 on d 5 after ovulation compared with control. Serum PSPB samples were collected daily from all cows on d 18 through 28 after ovulation for determination of d of PSPB increase. Pregnancy diagnoses were performed via ultrasound examination on d 35, 63, and 100 after ovulation and AI. The D5 treatment reduced percentage of cows with, and increased the time to, PSPB increase. Primiparous cows with ipsilateral aCL had reduced pregnancy loss before d 100 postovulation compared with cows with contralateral aCL. Cows that had PSPB increase >21 d postovulation had 4× greater chances of pregnancy loss compared with cows that had PSPB increase on d 20 or 21. The highest quartile of P4 on d 5, but not on d 19 and 20, was associated with reduced time to PSPB increase. Time to PSPB increase appears to be an important measurement to understand reasons for pregnancy loss in lactating dairy cows. Increasing P4 utilizing hCG after ovulation did not enhance early pregnancy or reduce pregnancy losses in lactating dairy cows.

Dairy heifers have an earlier increase in serum pregnancy-specific protein B compared with lactating dairy cows. Is this an indicator of earlier conceptus attachment?

This study was designed to use within-individual daily increases in circulating pregnancy-specific protein B (PSPB) to determine time to increase in serum PSPB in nulliparous heifers and multiparous dairy cows following artificial insemination (AI). Weekly cohorts of lactating Holstein cows (n = 56) averaging 122 ± 7 d in milk at AI and nulliparous Holstein heifers (n = 23) averaging 16 ± 0.24 mo old at AI were used in this study. Lactating cows received AI to either Ovsynch (n = 38) or at ~12 h following observed standing estrus (n = 18). All heifers were inseminated ~12 h after observed standing estrus. Blood samples for measurement of PSPB were collected daily from d 15 through d 35 postovulation. Nulliparous heifers had increases in serum PSPB earlier compared with both primiparous and multiparous cows. Day of increase in serum PSPB was defined as the day serum PSPB optical density levels initially increased ≥10% from baseline and continued to increase from baseline of ≥10% the following 2 d. Average PSPB were greater in pregnant heifers compared pregnant cows from d 23 through 29. Early lactation nonpregnant cows maintained greater average optical density of serum PSPB from 15 to 35 d postovulation compared with nonpregnant heifers (n = 38). In summary, fertility differences in heifers versus lactating cows may be due to the differences in timing of increases in serum PSPB. This appears to be one of the first publications that used daily PSPB sampling to investigate possible differences in fertility in heifers versus lactating cows.

Influence of parity and live weight on the concentration of Pregnancy-Specific Protein B (PSPB) in Kedah Kelantan (KK) cattle

The pregnancy-specific protein B (PSPB) is the protein of binucleated cells in the ruminant’s placenta. It contains several antigenic variants, which contributed to the ability to detect pregnancy from day 20 of pregnancy until parturition. In this research, the main objective is to determine the concentration of PSPB in serum and urine and their relation to Kedah-Kelantan (KK) cattle’s parity and live weight. Ten cattle (n=10) were selected for this study. The blood serum (2 ml) and urine samples were collected at days 0, 24, 42, and 164 post artificial insemination (AI). All the samples were centrifuged at 3000 rpm for 15 minutes at 4°C. The serum and urine were analyzed with competitive ELISA test kit and read at 450 nm wavelength speed. The standard OD value provided by the kit created the equation to calculate the concentration of PSPB in the serum and blood. Then, the correlation and multiple linear regression of parity and live weight were analyzed with a significant level of P < 0.01. Through the finding, the live weight and parity were positive correlate with PSPB in serum. The relationship between live weight and PSPB concentration in urine showed a negative correlation but there no correlation between parity and PSPB concentration in urine. The regression analysis result was 35% and 36% for PSPB concentration in serum and urine, respectively. This study suggests that cattle parity and live weight did not significantly affect the PSPB concentration.

PREGNANCY DIAGNOSIS IN OKAPI (OKAPIA JOHNSTONI) USING BIOPRYN ENZYME-LINKED IMMUNOSORBENT ASSAY FOR DETECTION OF PREGNANCY-SPECIFIC PROTEIN-B (PSPB)

Banked serum samples from seven okapi (Okapia johnstoni) with known pregnancy status were evaluated using the BioPRYN wild enzyme-linked immunosorbent assay to detect pregnancy-specific protein B (PSPB). Thirty-six serum samples, 18 from known pregnant and 18 from nonpregnant okapi, were analyzed. Using optical density cutoffs, the BioPRYN wild assay demonstrated a sensitivity of 88% (95% confidence interval, 65%-98%) and a specificity of 100% (95% confidence interval, 81%-100%). In one sample, this test confirmed pregnancy as early as 21 days of gestation; however, two pregnant okapi were reported to be not pregnant at 23 and 38 days of gestation, suggesting sensitivity may be lower in early gestation. Sensitivity improved to 100% when samples were evaluated in okapi at 116 days or greater of gestation. Analysis of PSPB can be used to augment pregnancy diagnosis in okapi, a species that is of high conservation value and has documented pregnancy-associated morbidity.

110 Pregnancy rate and embryo viability in response to chorionic gonadotrophins given for oestrus induction and gonadotrophin-releasing hormone 5 days after timed laparoscope-aided insemination of lactating and non-lactating goats

Gonadotrophins are included in oestrous/ovulation synchronization (E/OS) regimens when breeding early in the transitional phase to promote follicular development. However, the time of use has been a matter of debate. Hence, the objective of this study was to determine whether equine and human chorionic gonadotrophins (eCG and hCG, respectively) given 24h before or 24h after concurrent removal of intravaginal progestagen (P4) and prostaglandin (PGF2α) delivery influences pregnancy rate (PR) and/or embryo viability (EV). Relatedly, the influence of gonadotrophin-releasing hormone (GnRH) 5 days post-breeding (dpb) on PR and/or EV was also considered. A prospective randomised control trial was conducted using lactating and non-lactating Alpine breed goats of different parity, average±s.e. age, and bodyweight (3.7±2.6), and (52.5±7), respectively. Goats were time-bred at 48h after P4 removal by laparoscopy (LAI; n=75) or by natural service (NS; n=29) during the early transitional breeding phase in mid-September through mid-December (corresponding to 12:12h daylight/darkness). E/OS used an intravaginal device containing 300mg of P4 for 12±1d. At P4 removal, 2mL of PGF2α was administered IM. Twenty-four hours before (E) or 24h after (L) P4 removal, an IM dose of 1.5mL containing 120IU of eCG and 60IU of hCG was given. A third control (C) group (n=38) was not exposed to gonadotrophins. GnRH analogue was given (n=51) 5 days after insemination, and the remaining goats (n=53) received an IM dose of 2mL of saline solution. PR was evaluated at 18 to 24 (dpb) by non-return to oestrus (NRE), at 30 dpb by pregnancy-specific protein B (PSPB), and at 40 dpb by ultrasound imaging (UI). EV was determined from the difference between pregnancy outcomes for NRE and PSPB, and PSPB and UI. Statistical analysis (JMP/SAS vs.11; SAS Institute Inc.) were obtained using logistic regression. All concomitant variables (i.e. parity, bodyweight, days of P4 exposure, and lactation were not influenced by PR or EV; P&amp;gt;0.07). The overall PR was 71% for LAI and 69% for NS and, as shown in Table 1, was similar (P&amp;gt;0.49) at all times (at 18 to 24, 30, and 40 dpb). Likewise, the breeding procedure did not influence the levels of EV at 30 or 40 dpb. E/OS protocol or the use of GnRH 5 dpb did not affect PR or EV at any of the time points evaluated. Table 1. Main effect mean comparisons and logistic model probabilities for pregnancy rate (PR) and embryo viability (EV) by main treatment effect Response variable (days post-breeding) Breeding procedure E/OS Protocol Gonadotrophin LAI NS P&amp;gt; Control Early Late P&amp;gt; GnRH Placebo P&amp;gt; PR at 18–24 77 72 0.49 76 73 78 0.83 78 74 0.49 PR at 30 68 66 0.65 66 65 70 0.82 71 64 0.43 PR at 40 69 69 0.78 66 69 73 0.77 73 66 0.43 EV at 30 89 90 0.94 86 94 90 0.65 92 87 0.46 EV at 40 88 95 0.42 86 89 94 0.73 92 87 0.43

Evaluation of A Rapid Visual Pregnancy (Bio- RPD) test for detection of Pregnancy Specific Protein B (PSPB) in Cow Serum

The early pregnancy diagnosis allows optimizing production and timely management correction, with a greater reproductive output of livestock. The BioPRYN Rapid Visual Pregnancy Test® from Bio tracking USA consists of an ELISA for visual reading which does not require the use of readers in the laboratory, with satisfactory pregnancy diagnoses at 28 days of pregnancy in heifers and 30 days in cows. Therefore, the aim of this study is to determine the accuracy of this rapid test (Bio-RPD) based on visual observation of color change and Optical Density (OD) using BioPRYN Flex ELISA. For this purpose, 92 serum samples from cows were tested. The numbers of positive pregnant samples obtained with Biorapid (Bio-RPD) were 63 (68.5 %) while with BioPRYN Flex ELISA was 66 (71.7%). The numbers of negative not pregnant samples were 25 (27.2%) with Bio-RPD while they were 26 (28.2%0) with BioPRYN ELISA. Three samples were recorded as recheck with Bio-RPD and they were pregnant with BioPRYN Flex. Only one sample was identified as pregnant by Bio-RPD (1.1%) while it is not pregnant by BioPRYN Flex ELISA (false positive). Test results were showing that accuracy of the BioPRYN Visual Pregnancy test kit with sensitivity of 99% compared to BioPRYN ELISA Pregnancy test kit. In conclusion, we recommend that the Bio-RPD visual test could be used as a rapid accurate tool for pregnancy diagnosis on farms which has no laboratory ELISA equipment or trans rectal ultrasonography.

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.

Assessment of estrous synchronization protocols and pregnancy specific protein B concentration for the prediction of kidding rate in Lezhi black goats

• The onset of estrus is earlier in CIDR + cPGF 2α + eCG. • GnRH treated at CIDR removal showed similar reproductive performance as CIDR + eCG. • PSPB detected with ELISA can identify does carrying four fetuses. The objectives of this study were to assess the efficacy of estrous manipulation protocols and pregnancy specific protein B (PSPB) levels to determine kid number at birth in Lezhi black goats. Eighty-eight does were randomly allocated to three treatments. The PPe group (n = 29) was treated with Controlled Internal Drug Release devices (CIDR) for 7 days and 300 IU eCG and 0.2 mg PGF 2α at the time of CIDR removal. The PPG group (n = 28) was treated with a CIDR for 7 days and 0.2 mg PGF 2α at the time of CIDR removal and 15 μg GnRH at the onset of estrus. The GPG group (n = 31) was treated with 15 μg GnRH (day 0) and 0.2 mg PGF 2α on day 7, and a second GnRH treatment at the onset of estrus. Does at estrus were hand-mated using proven bucks. Estrous response ranged from 80.6%–89.7 %. The onset of estrus for the PPe, PPG and GPG groups was 38.4 ± 1.9, 45.6 ± 1.9 and 53.8 ± 1.4 h (P < 0.05), respectively. Mean kidding rates (205.9 %–227.8 %) and kidding percentages (112.9 %–146.4 %) were not different in the three treatments. At day 85 of gestation, does carrying quadruplets had a greater (P < 0.01) plasma PSPB level than does that delivered one to three kids. In conclusion, PPG may be a useful estrous synchronization protocol in goats. PSPB concentration at day 85 of gestation could be a potential diagnostic tool for predicting quadruplets.

PSI-30 Evaluation of urine estrone sulfate (P-Test) in beef cattle as an alternative pregnancy detection method

Abstract Pregnancy detection in cattle can be expensive and labor intensive to producers. A simple, cost-efficient method of determining early pregnancy, chute side, is lacking within the industry. This study tested various proven pregnancy detection methods against an alternative method. Pregnancy was assessed by ultrasonography, blood serum pregnancy-specific protein B (PSPB), and urine estrone sulfate (“P-Test”). A fixed timed AI (FTAI) protocol was conducted on 14 predominantly British cattle housed at Tarleton State University in Stephenville, TX. Cows were synchronized using the co-synch plus 7 day CIDR protocol, with PGF injection of d 7. Heifers were synchronized similarly with the exception of GnRH injection on d 1. Estrotech patch scores (1 – 4, 1 showing little signs of estrus to 4 presenting signs of estrus) were utilized to indicate estrus activity prior to FTAI (21.4% vs. 78.6% females scored 1 vs. 4, respectively). FTAI was performed on protocol d 10 along with a 2mL injection of GnRH. Serum for PSPB analysis was collected at d 30 and 60 post insemination, while urine was sampled on d 60. Overall 53.8% (ultrasonography) and 71.4% (PSPB) of cattle were determined to be pregnant at d 30 post FTAI. Chi squared analysis proved that PSPB and ultrasound were similar at detecting pregnancy (P = 0.04). At d 60 “P-Test” color varied indicating differing levels of estrone sulfate. A t-test revealed increased PSPB from d 30–60 post FTAI (P &amp;lt; 0.0001); however, “P-Test” could not predict similar results (P = 0.655). Data suggest the “P-Test” is not a consistent method to diagnose early pregnancy. Future studies may be warranted to investigate a more desirable time point for utilizing the chute side “P-Test” as a reliable method to determine pregnancy.

Pregnancy specific protein B concentration for the prediction of lambing rate in sheep

Abstract An accurate diagnosis of pregnancy and prediction of lambing rates is a critical tool for improving efficiency in sheep flocks. The ability to manage ewe nutrition based on number of fetuses could improve animal welfare and productivity. Pregnancy Specific Protein B (PSPB) assays are commercially available and have proven a valuable tool for pregnancy diagnosis but less work has been done on accuracy of predicting fetal counts. The objective of this study was to determine if PSPB could be used to accurately predict lambing rates in sheep. Samples were collected from 54 Dorset, Katahdin, or crossbred ewes at 28 days post ram removal which was approximately 46 days post breeding. Samples were processed using the quantitative BioPRYN® assay. Ewes carrying twins had greater (P

Pregnancy per artificial insemination during summer in lactating dairy cows after treatment with aspirin

Embryonic loss in cattle may be related to a hormonal imbalance resulting in alterations in timing of prostaglandin F2α (PGF2α) secretion around the time of maternal recognition of pregnancy. The objective of this study was to examine effects of aspirin (a PGF2α inhibitor) on pregnancy per AI (P/AI), and progesterone (P4), and pregnancy specific protein B (PSPB) concentrations in lactating dairy cows inseminated more than once after parturition. Fourteen days after second or subsequent AI (Day 0 = Day of AI), 556 cows were assigned randomly to aspirin (187.2 g total; n = 277) or control (n = 279) groups. Aspirin was administered orally on Day 14 and 15, and control cows were subjected to sham bolus administration. On Day 25, blood samples were collected from a subset of cows (n = 194) to quantify P4 and PSPB, whereas pregnancy was determined in all cows at 35–42 days post-AI. Maximum daily ambient temperature ranged from 38-41 °C during the experiment. Mean parity, days in milk, and times bred before treatment (TBRD) did not differ between groups. There were no differences in P/AI between treatments (aspirin 21.6 % compared with control 27.5 %). Neither treatment, parity, TBRD, or any two-way interactions with treatment affected concentrations of P4. Moreover, there were no effects (P > 0.50) of treatment, or treatment by TBRD interaction on serum PSPB concentrations. A tendency (P = 0.07) occurred for multiparous cows to have greater serum PSPB concentrations compared with primiparous cows. Mean serum PSPB concentrations tended (P = 0.07) to be greater for second or third TBRD compared to fourth and greater TBRD. These results provide evidence that aspirin administered during periods of heat stress after the second and subsequent AI post-partum during the summer months does not improve P/AI or alter P4 and PSPB in lactating dairy cows.