Pregnancy-specific Protein B Concentrations Research Articles

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.

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.

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.

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.

A negative energy balance during the peri-implantational period reduces dam IGF-1 but does not alter progesterone or pregnancy-specific protein B (PSPB) or fertility in suckled cows

The aim of this study was to evaluate the effect of a negative energy balance during the first third of gestation on metabolic, endocrine, and pregnancy recognition parameters in 2 beef cattle breeds adapted to semiextensive conditions. Seventy-five lactating Parda de Montaña and 40 Pirenaica multiparous cows rearing calves were synchronized and timed artificial inseminated (TAI) on day 76 postpartum. Cows were assigned to one of 2 diets (CONTROL or SUBNUT; 100% or 65% of their requirements supplied) until day 82 of gestation. Pregnancy was diagnosed 37 d post-TAI using ultrasound. Blood samples were obtained to determine metabolic (glucose, NEFA, β-hydroxybutyrate, cholesterol, and urea) and endocrine (IGF-1) status throughout the first third of gestation and to determine the concentrations of progesterone and pregnancy-specific protein B (PSPB) in the peri-implantational period. Undernutrition affected both cow and calf performance. The CONTROL cows maintained BCS and BW, whereas SUBNUT cows had negative daily gains. The CONTROL lactating calves had higher BW gains than SUBNUT. These negative effects were more evident in the Pirenaica breed, which was more sensitive to undernutrition. The negative energy balance was reflected in the cows’ metabolic profiles, with higher NEFA values and lower IGF-1 concentrations in SUBNUT cows. However, undernutrition did not affect dam pregnancy/TAI or pregnancy recognition and maintenance, confirming that during periods of undernourishment pregnant dams prioritize the allocation of dietary energy toward reproductive functions. Progesterone concentration on day 21 post-TAI (with a 4.8 ng/mL cut-off value) and PSPB on day 26 post-TAI (with a 0.57 ng/mL cut-off value) were determined as the earliest indicators to accurately establish dam pregnancy status, regardless of breed or nutrition treatment. In summary, early undernutrition affected cow performance and metabolic profiles and impaired lactating calf growth, but did not affect progesterone or PSPB concentrations or the pregnancy/TAI rate in suckled cows.

Effect of manipulating progesterone before timed artificial insemination on reproductive and endocrine outcomes in high-producing multiparous Holstein cows

Our objective was to evaluate the effect of manipulating progesterone (P4) concentrations before timed artificial insemination (TAI) on reproductive and endocrine outcomes in high-producing Holstein cows. Multiparous lactating Holstein cows (n = 80) were synchronized for first TAI using a Double-Ovsynch protocol and were randomly assigned to receive 25 mg of PGF2α 1 d after the first GnRH treatment of the Breeding-Ovsynch protocol that included a once-used P4 insert (low-P4 group) or to receive 2 new P4 inserts during the Breeding-Ovsynch protocol (high-P4 group). Blood samples were collected thrice weekly from -10 to 32 d relative to TAI for all cows and from 32 to 67 d after TAI for pregnant cows and were analyzed for P4 and pregnancy-specific protein B (PSPB) concentrations. Expression of IFNτ-stimulated gene 15 (ISG15) was assessed in blood leukocytes 18 and 20 d after TAI. As expected, P4 concentrations were greater for high-P4 cows than for low-P4 cows from 3 to 8 d before TAI. Incidence of double ovulation was 3-fold greater for low-P4 cows than for high-P4 cows (33 vs. 10%), which resulted in more twin pregnancies 32 d after TAI for low-P4 cows than for high-P4 cows (29 vs. 0%). Low-P4 cows had larger preovulatory follicles at the last GnRH treatment of the Double-Ovsynch protocol and greater P4 concentrations than high-P4 cows after TAI. Relative expression of ISG15 mRNA 18 and 20 d after TAI was greater for low-P4 cows than for high-P4 cows and for pregnant cows than for nonpregnant cows. Overall, PSPB concentrations tended to be greater for low-P4 cows than for high-P4 cows, and pregnant cows had greater P4 concentrations than nonpregnant cows. In summary, cows with low P4 before TAI had increased preovulatory follicle diameter, PSPB concentrations, relative expression of ISG15 mRNA 18 and 20 d after TAI, double ovulations, and twinning compared with cows with high P4 before TAI. Increasing P4 before TAI may effectively decrease double ovulation and twinning in high-producing multiparous Holstein cows.

Effect of treatment with human chorionic gonadotropin 7 days after artificial insemination or at the time of embryo transfer on reproductive outcomes in nulliparous Holstein heifers

Our objective was to assess the effect of treatment with human chorionic gonadotropin (hCG) 7 d after artificial insemination (AI) or at the time of in vitro-fertilized (IVF) embryo transfer on reproductive outcomes, including progesterone (P4), interferon-tau stimulated gene 15 (ISG15), pregnancy-specific protein B (PSPB), and pregnancies per AI (P/AI) or pregnancies per embryo transfer (P/ET), in nulliparous Holstein heifers. Heifers in experiment 1 were randomly assigned to receive no treatment (control; n = 129) or 2,000 IU of hCG 7 d after AI to a detected estrus (estrus = experimental d 0; hCG; n = 132). Heifers in experiment 2 were randomly assigned to receive no treatment (control; n = 143) or 2,000 IU of hCG (hCG; n = 148) at transfer of an IVF embryo 7 d after the last GnRH treatment of a 5-d controlled internal drug release-synch protocol (last GnRH = experimental d 0). Blood samples were collected from a subgroup of heifers (experiment 1, n = 82; experiment 2, n = 104) at d 7, 11, 18, 20, 25, 28, and 32, and blood samples from heifers diagnosed pregnant were collected on d 35, 39, 46, 53, 60, and 67. Blood samples were assayed for P4 by RIA and for PSPB by ELISA, and expression of ISG15 was assessed in mRNA isolated from blood leukocytes on d 18 and 20. Data were analyzed by ANOVA and logistic regression using the MIXED and GLIMMIX procedures. In both experiments, treatment with hCG increased P4 concentrations from d 11 to 32; however, treatment did not affect P/AI or P/ET at d 32 or 67, PSPB concentrations from d 11 to 67 of pregnancy, or relative ISG15 mRNA concentrations on d 18 or 20. Heifers diagnosed not pregnant at d 32 in experiment 2 with an extended luteal phase (>20 d) and treated with hCG had greater relative ISG15 mRNA concentrations on d 20 than control heifers. Treatment with hCG did not affect pregnancy loss in experiment 1, whereas heifers treated with hCG at the time of IVF embryo transfer had fewer pregnancy losses from d 32 to 67 than control heifers. We concluded that treatment with 2,000 IU of hCG 7 d after AI or at the time of embryo transfer increased P4 concentrations without affecting P/AI or P/ET in nulliparous Holstein heifers.

Effects of polyunsaturated fatty acids supplementation on reproductive parameters associated with the performance of suckled beef cows

To evaluate the effects of a polyunsaturated fatty acids (PUFA) supplement on reproductive parameters of suckled beef cows, two experiments were conducted. In Experiment (Exp.) 1, 60 primiparous cows were randomly assigned to one of two treatments: CTRL – 1.36 kg/day of corn gluten feed (CGF) and MEGR – 1.36 kg/day of CGF and 0.23 kg/day of calcium salts of soybean oil. Supplementation occurred from 30 days before fixed-time artificial insemination (TAI) until 7 days post-TAI. The expression of interferon-stimulated genes (ISG) was measured on days 18 and 21. Pregnancy rates were diagnosed on days 30 and 100. Treatment altered plasma fatty acid profile (P<0.05), however, did not change cow BW (P=0.52) or body condition score (BCS) (P=0.52). Treatment did not alter (P=0.12) pregnancy rates to TAI or final pregnancy rates (P=0.56). Treatments did not impact messenger RNA (mRNA) expression of the ISG OAS1 or MX2 on days 18 (P=0.67; P=0.96, respectively) or 21 (P=0.72; P=0.17, respectively). Length of gestation was greater (P=0.02) for MEGR, however, treatments did not alter calf birth weight (P=0.20). In Exp. two, 66 multiparous cows were assigned to one of two treatments: MEG – 0.65 kg/day of CGF+0.23 kg/day of calcium salts of palm oil and MEGR – 0.65 kg/day of CGF+0.23 kg/day of Ca salts of soybean oil. Cows were supplemented from 30 days prepartum to 30 days postpartum. On day 35 after TAI, pregnancy status, embryo crown-to-rump length (CRL), and plasma concentrations of pregnancy-specific protein-B (PSPB) were evaluated. Treatment altered plasma fatty acid profile (P<0.05). In addition, cows from the MEG treatment had greater BW (P<0.01) and BCS (P<0.01) than those in the MEGR treatment, as well as heavier calves at weaning (P=0.03). Treatment did not affect resumption of estrous cycle (P=0.29). There were no differences in pregnancy rates to TAI (P=0.87) or final pregnancy rates (P=0.29). No differences between treatments were detected on CRL (P=0.24) and plasma concentrations of PSPB (P=0.46). Birth weight (P=0.12) and calving distribution (P=0.52) were not altered. We concluded that PUFA supplementation altered plasma fatty acid profile, however, did not impact the remaining reproductive parameters evaluated.

Assessment of ovulation synchronization protocols in goats and use of pregnancy specific protein B (PSPB) enzyme linked immunsorbent assay (ELISA) to determine kid number at birth

The efficacy of several protocols for ovulation synchronization and timed artificial insemination (TAI) in goats was examined. In addition, the relationship between levels of pregnancy specific protein B (PSPB) during gestation assessed with a commercially available ELISA and the number of offspring at birth was determined. In Experiment 1, 70 does were randomized into four treatments: (1) breed by estrus [BBE], (2) 6-d treatment with a new [C6N], (3) once-used [C61], or (4) twice-used Controled Internal Drug Release (CIDR) device [C62)]. BBE does received two 15 mg doses of prostaglandin-F2α (PGF) at a 10-d interval and were bred 12 h after estrus onset. CIDR groups received a CIDR for 6 d with 15 mg PGF given at CIDR removal. TAI was performed 48 h after CIDR removal and does were given 50 μg GnRH. All does were inseminated with a single dose of frozen semen using a non-surgical, transcervical technique. Pregnancy rates for the BBE, C6N, C61 and C62 treatment groups were 39% ± 12%, 64% ± 12%, 77% ± 12% and 57% ± 12%, respectively, and did not differ. Reuse of CIDRs, even with reuse extending for a total of 21 d, was as effective as new CIDRs for synchronization of ovulation. In Experiment 2, 68 does were randomized into four treatments: (1) BBE, (2) C6N, (3) NC.Synch [NCS], (4) modified NCS [NCSM]. The BBE and C6N groups were as described for Experiment 1. The NCS and NCSM groups received 15 mg PGF on Day 1, 50 μg GnRH on Day 8 and 15 mg PGF on Day 15 (NCS) or Day 15.5 (NCSM). Does were bred by TAI at 72 h (NCS) or 60 h (NCSM) after the second PGF injection. All does in the NCS and NCSM groups received 50 μg GnRH at TAI. Pregnancy rates were 53% ± 12%, 30% ± 11%, 50% ± 11% and 41% ± 12% for does in the BBE, C6N, NCS and NCSM group, respectively, and did not differ. In Experiment 3, 62 does pregnant to TAI were bled at Days 48 and 85 post-insemination for PSPB. Data on kid numbers and birth weights were subsequently recorded. At Day 48 of gestation, PSPB levels for does birthing singletons were lower than for does birthing twins or triplets (25.0 ± 0.1a, 28.8 ± 0.1b and 30.7 ± 0b ng/mL, respectively, abP<0.05). At Day 85 of gestation, PSPB levels were progressively greater for does birthing singletons versus twins versus triplets (27.0 ± 0.1a, 28.5 ± 0.1b and 31.6 ± 0c ng/mL, abcP<0.05). In conclusion, PSPB concentrations detected using a commercially available ELISA at Day 48 or 85 of gestation could distinguish does carrying single versus multiple fetuses.

Is twin pregnancy, calving and pregnancy loss predictable by serum pregnancy-specific protein b (pspb) concentration 28-35 days after ai in dairy cows?

Double ovulation occurs more frequently in multiparous cows with high milk production than in primiparous cows and the rate of twin pregnancy/calving is increasing worldwide. Diagnosis of twin pregnancy is possible by ultrasound at the time of early pregnancy examination [28-34 days after artificial insemination (AI)]. Pregnancy proteins are also well-known indicators of gestation. The risk of pregnancy loss during the first trimester of gestation for cows carrying twins is three to nine times higher than for cows carrying singletons. Pregnancy-specific protein B (PSPB) is a good indicator not only of pregnancy but also of pregnancy loss. The aims of this study were (a) to collect calving data in some Hungarian Holstein-Friesian herds (n = 7,300) to compare PSPB serum concentrations (measured 29-35 days post insemination) in twin- and singleton-calving cows (Trial 1), and (b) to check the predictive value of PSPB serum concentration for twin pregnancy and pregnancy loss in high-producing Spanish Holstein-Friesian cows (n = 98; Trial 2). Our results showed almost 7% twin calving rate. Although hormonal treatments are commonly believed to be major causes of twin pregnancies, our data do not support this hypothesis. The only exception is the single PGF injection, which significantly increased twin calving. No effect of milk production on the risk of twin pregnancy was found, and twin pregnancy increased with parity. The AI bull, the bull's sire, the bull's grandfather and the cow's father also affected twin calving (P ≤ 0.02). We found much higher frequency of twin calving in cows diagnosed pregnant with higher than 3 ng/ml serum PSPB concentrations at 29-35 days after insemination. In Trial 2, non-significant but well-marked differences were found in PSPB serum concentration between singleton- and twin-pregnant cow samples (2.1 and 2.9 ng/ml) at different bleeding times. Probably the small size of the study population and the effects of milk production on PSPB values may explain this lack of significance.

Embryo Mortality Around the Period of Maintenance of the Corpus Luteum Causes Alterations to the Ovarian Function of Lactating Dairy Cows.

Objectives were to identify cows with embryo mortality (EM) around the period of corpus luteum maintenance by interferon tau (IFNT) and to characterize ovarian function in cows that underwent EM. Lactating Holstein cows received artificial insemination (AI) (Day = 0) with semen or extender only. From Day 14 to 42 transrectal ultrasonography was performed daily to monitor ovarian dynamics and uterine contents whereas blood was collected every 48 h to determine ISG15 and MX2 mRNA abundance in blood mononuclear cells (Day 14 to 22 only) and determination of hormone concentrations. Cows were classified in the following reproductive status groups: cyclic (inseminated with extender; n = 15), pregnant (embryo present on Day 42; n = 23), no embryo (n = 23), and EM (n = 14). EM was defined as the presence of an embryo based on interferon-stimulated genes (ISG) mRNA abundance and concentrations of pregnancy-specific protein B (PSPB) above specific cutoff points but no embryo visualized by ultrasonography. Within the EM group, early EM (up to Day 22) was when ISG fold changes were above specific cutoff points from Day 18 to 22 and PSPB below 0.7 ng/ml on and after Day 24, whereas late EM (after Day 22) was when PSPB was above 0.7 ng/ml on or after Day 24 regardless of ISG expression. This experiment provided evidence that the combination of ISG expression patterns and PSPB concentrations is a reasonable method to determine EM around the period of corpus luteum maintenance by IFNT because cows with evidence of EM had patterns of ISG expression more similar to pregnant than cyclic cows or cows with no embryo. Within the EM group, only cows with late EM had delayed luteal regression and longer interovulatory intervals. No major alterations in follicular function were observed after the onset of luteolysis. Our results suggest that embryo development needs to continue beyond 22 days after AI to effectively prevent luteolysis and extend the luteal phase.

Pregnancy Loss in Dairy Cattle: Relationship of Ultrasound, Blood Pregnancy-Specific Protein B, Progesterone and Production Variables.

Objectives were to determine associations between percentage pregnancy loss (PPL) in dairy cattle and: (i) pregnancy diagnosis by ultrasonography; (ii) pregnancy diagnosis by serum pregnancy-specific protein B (PSPB) concentrations, with or without serum progesterone concentrations; and (iii) production and environmental factors. This study included 149822 pregnancy diagnoses conducted over 13years in Holstein-Friesian cows in Hungarian dairy herds. The following were determined: PPL in cows diagnosed pregnant by transrectal ultrasonography 29-42days after artificial insemination (AI; n=11457); PPL in cows diagnosed pregnant by serum PSPB 29-35days after AI (n=138365); and PPL and its association with serum progesterone concentrations, PSPB and production/environmental variables. The definition of PPL was percentage of cows initially diagnosed pregnant based on ultrasonography or PSPB, but not pregnant when examined by transrectal palpation 60 -70days after AI. The PPL was lower (p<0.001) in cows following ultrasonographic vs PSPB diagnosis of pregnancy at 29-35days (8.1 vs 19.3%, respectively), but was higher in cows following ultrasonographic pregnancy diagnosis on 29-35 vs 36-42days (8.1 vs 7.1%, respectively, P<0.05). Furthermore, 72.9% of pregnancies with ultrasound-detected morphological abnormalities resulted in pregnancy loss. As a subset of PSPB data, a fully quantitative PSPB assay was used for 20430 samples; PPL in cows with a high PSPB concentration (>1.1ng/ml) was lowest (15.0%), whereas cows with low concentrations of both PSPB and progesterone (0.6-1.1 and <2ng/ml, respectively) had the highest PPL (76.3%; p<0.0001). Furthermore, PPL was higher in cows with advanced parity and with high milk production, when ambient temperatures were high, although body condition score (BCS) had no effect on PPL. Finally, there were no significant associations between serum PSPB and environmental temperatures or number of post-partum uterine treatments.

Effects of recombinant bovine somatotropin administration at breeding on cow, conceptus, and subsequent offspring performance of beef cattle.

The effects of administration of recombinant bovine ST (bST) on plasma hormone concentrations of cows, conceptus development, and postnatal calf performance were examined. Lactating beef cows ( = 190) were exposed to a fixed-time AI (TAI) protocol from d -10 to 0 (TAI on d 0). Cows were blocked by breed and stratified by days postpartum and then randomly assigned to receive, subcutaneously 1) 2 injections of saline (1 mL of 0.9% saline), 1 on d 0 at TAI and a second injection on d 14 (CTRL; = 53); 2) an injection of 325 mg of bST on d 0 and a saline injection on d 14 (bST0; = 48); 3) a saline injection on d 0 and an injection of 325 mg of bST on d 14 (bST14; = 49); or 4) 2 injections of 325 mg of bST, 1 on d 0 and a second injection on d 14 (bST0+14; = 40). Pregnancy status, crown-to-rump length (CRL) on Day 35, and crown-to-nose length (CNL) on Day 65 were determined via transrectal ultrasonography. Blood samples were collected on d 0, 7, 14, 21, 35, and 65, relative to TAI, to determine plasma concentrations of progesterone (P4), IGF-1, and pregnancy-specific protein B (PSPB) and also on d 18 and 21 for isolation of peripheral blood leukocytes for RNA extraction and measurement of interferon-stimulated genes transcript abundance. Individual calf BW was determined at birth and every 30 d until weaning. A subset of 24 calves was randomly selected for liver biopsies at birth to determine mRNA expression of target genes. Administration of bST to cows increased ( < 0.0001) concentrations of plasma IGF-1 for 14 d after injection compared with CTRL but did not affect fetal CRL and CNL ( = 0.23). Cows receiving bST only on d 0 had a greater ( = 0.05) transcript abundance in myxovirus resistance 2 on d 21 compared with 2bST cows (2.0- and 0.8-fold for bST0 and 2bST, respectively), whereas cows receiving bST14 and CTRL were intermediate (1.2- and 0.9-fold, respectively). Calf BW did not differ ( ≥ 0.100) among treatments on d 0, 30, 60, 90, 120, and 150 relative to birth. Injection of bST only on d 0 tended ( = 0.062) to increase calf liver mRNA expression of at birth compared with the calves born to cows in other treatments. Therefore, during a TAI protocol, the administration of 1 or 2 injections of 325 mg of bST to lactating beef cows enhanced their plasma concentrations of IGF-1 but failed to improve fetal size and plasma concentrations of maternal PSPB and P4 and had no effect on postnatal calf growth performance.

Secondary Corpora Lutea Induced by hCG Treatment Enhanced Demi‐Embryo Survival in Lactating High‐Yielding Dairy Cows

Using a novel in vivo model considering a low developmental competence embryo (demi-embryo) and a subnormal fertility recipient (lactating high-yielding dairy cow), this experiment evaluated the effect of human chorionic gonadotrophin (hCG) treatment at embryo transfer (ET) on embryonic size at implantation, embryonic survival and recipient plasma progesterone (P4 ) and bovine pregnancy-specific protein B (PSPB) concentrations until day 63 of pregnancy. Embryos were bisected and each pair of demi-embryos was bilaterally transferred to recipients (n=61) on day 7 of the oestrous cycle. At ET recipients were randomly assigned to treatment with 1500IU hCG or to untreated controls. Higher (p<0.01) pregnancy rates on days 25, 42 and 63, and embryo survival rate on day 63 were observed in hCG-treated cows with secondary CL than in hCG-treated cows without secondary CL and in untreated cows. Pregnancy rates and embryo survival rate were similar in hCG-treated cows without secondary CL and untreated cows. Embryonic size on day 42 was not affected by treatment with hCG, presence of secondary CL and type of pregnancy (single vs twin). Presence of secondary CL increased (p<0.05) plasma P4 concentrations of pregnant cows on days 14, 19 and 25 but not thereafter and of non-pregnant cows on days 14-21. Treatment with hCG and presence of secondary CL had no effect on plasma PSPB concentrations, which were higher (p<0.05) in twin than in single pregnancies. In conclusion, secondary CL induced by hCG treatment at ET significantly increased plasma P4 concentrations, the survival rate of demi-embryos and the pregnancy rate of high-yielding lactating dairy cows. Embryos were rescued beyond maternal recognition of pregnancy, but later embryonic survival, growth until implantation and placental PSPB secretion until day 63 of pregnancy were not affected by treatment or presence of secondary CL.

Environmental regulation of pregnancy-specific protein B concentrations during late pregnancy in dairy cattle1

Environmental factors, such as photoperiod and heat stress, can be manipulated during the dry period to influence health, productivity, and reproductive performance of dairy cows in their subsequent lactation. The impacts of photoperiod and heat stress on subsequent lactation are related to alterations in prolactin (PRL) signaling and may affect the expression of pregnancy-specific protein B (PSPB). Additionally, exposure of cows to heat stress during the dry period decreases gestation length; however, the mechanism involved in this process is unknown. The objective of these experiments was to evaluate the influence of environmental factors (i.e., heat stress and photoperiod) during late gestation (i.e., dry period) on PSPB concentrations in plasma of dairy cows. In Exp. 1, cows were dried off in the summer months approximately 46 d before expected calving and assigned randomly to heat stress (HT; n=30) or cooling (CL; n=30) treatment. Cooling cows were housed with sprinklers, fans, and shade, whereas HT cows were provided only shade. In Exp. 2, cows were dried off at approximately 60 d before expected calving in summer/fall months and randomly assigned to 3 treatments: long day photoperiod (LDPP: 16L:8D; n=15), short day photoperiod (SDPP: 8L:16D; n=14) and SDPP+PRL implant (12 mg/d of PRL at 28 d or 16 mg/d of PRL at 39 d; n=11). In both experiments, plasma samples were collected at dry off and at -32, -18, -7, -3 and 0 d relative to calving. In Exp. 1, greater concentrations of PSPB were detected in plasma of CL versus HT cows (388.3±24.7 vs. 287.4±23.8 ng/mL; P<0.01). Concentrations of PSPB did not differ between -46 to -18 d before calving (66.0 ng/mL). However, PSPB concentrations were greater (P<0.01) for CL cows at d -7 (534.7>357.2 ng/mL), -3 (807.2>572.2 ng/mL) and 0 (800.8>563.5 ng/mL) relative to calving. Additionally, HT cows in Exp. 1 had increased PRL plasma concentrations compared with CL cows (21.01±1.6 vs. 13.78±1.6 ng/mL). In Exp. 2, no differences were detected in plasma concentrations of PSPB (ng/mL) among LDPP, SDPP, or SDPP+PRL groups on d -60 (41.5), -32 (51.7), -18 (58.5), -7 (532.9), -3 (838.2), and 0 (729.4) relative to parturition. Photoperiodic PRL concentrations were 10.81, 7.84, and 4.22 ng/mL for LDPP, SDPP+ PRL, and SDPP, respectively. Results indicate that HT alters PSPB concentrations in late pregnancy, suggesting that placental activity is altered in cows exposed to excessive elevated temperatures around the time of calving. However, the mechanism involved likely is not associated with changes in PRL secretion.

Changes in serum pregnancy-associated glycoprotein, pregnancy-specific protein B, and progesterone concentrations before and after induction of pregnancy loss in lactating dairy cows

Lactating crossbred dairy cows were synchronized to receive a timed artificial insemination (TAI), and blood samples were collected from all cows from TAI until pregnancy diagnosis 39 d after TAI (period 1), and from pregnant cows from onset of treatment until the end of the experiment (period 2). Cows diagnosed pregnant 39 d after TAI were randomly assigned to 1 of 3 treatments to receive (1) an i.m. injection of saline (CON, n=10); (2) an i.m. injection of PGF2α (PGF, n=10); or (3) an intrauterine infusion of 120mL of hypertonic saline (INF, n=9). During period 1, serum pregnancy-associated glycoprotein (PAG) concentrations began to increase in pregnant cows by 25 d after TAI and differed from those in nonpregnant cows by 27 d after TAI, whereas serum pregnancy-specific protein B (PSPB) concentrations in pregnant cows differed from those in nonpregnant cows by 22 d after TAI. During period 2, time from treatment to cessation of the embryonic heartbeat was greater for PGF than for INF cows (36.0±5.7 vs. 0.2±0.1h, respectively), and time from treatment to conceptus disappearance was greater for INF than for PGF cows (7.1±3.3 vs. 1.9±0.3 d, respectively). Overall, progesterone concentration was greater for CON and INF than for PGF cows (8.7±2.8, 8.2±3.1, and 1.0±2.3ng/mL, respectively) due to luteal regression for PGF cows and corpus luteum maintenance for CON and INF cows. Serum PAG and PSPB concentrations differed among CON cows and PGF and INF cows beginning 1 and 2.5 d after treatment for PAG and PSPB, respectively. By 9.5 d after treatment, PAG and PSPB concentrations were similar to those of nonpregnant cows. We conclude that although timing of conceptus expulsion occurred 5.2 d later for INF than for PGF cows, serum PAG and PSPB concentrations decreased at a similar rate from the onset of treatment for both models of pregnancy loss evaluated.

Pregnancy-specific protein B (PSPB), progesterone and some biochemical attributes concentrations in the fetal fluids and serum and its relationship with fetal and placental characteristics of Iraqi riverine buffalo (Bubalus bubalis)

This study was carried out to demonstrate the pregnancy-specific protein B (PSPB), progesterone and some biochemical parameters concentrations in amniotic fluid, allantoic fluid and fetal serum collected from slaughtered Iraqi riverine pregnant buffaloes at three different months of gestation (6th, 7th and 8th). Ten out of 22 adult buffaloes of 4.6±0.97 years old were used in this study. The buffaloes were mated naturally by monitoring the estrus cycles via appearance of vaginal fluids and mounting by bulls. Pregnancy was checked for these buffaloes by non-returning to estrus for three estrus cycles and assured by rectal palpation on day 61 post-mating (PM). Buffaloes were slaughtered at three different periods of gestation (three at 6th month, four at 7th month and three at 8th month of gestation) to verify the progesterone and PSPB as well as some blood attributes levels (glucose, cholesterol, total protein, albumin, globulins and albumin: globulins ratio) in amniotic fluid (AF), allantoic fluid (LF) and fetal serum (FS). Progesterone was higher (P<0.01) in LF at the 8th month of gestation and lower in FS during the 7th and 8th months of pregnancy. PSPB concentrations were greater in FS (6th and 8th months in particular) than in both AF and LF. The overall mean of cholesterol concentration was higher in FS (P<0.05) followed by AF and LF that had the lowest concentration. The FS exhibited higher total protein during the three gestation periods. Most of fetal and placental measurements increased as the pregnancy advanced. In conclusion, these results described, for the first time, the PSPB and progesterone concentrations and blood characteristics in fetal fluids and serum in water riverine buffaloes during different stages of pregnancy. Progesterone concentrations were greater in allantoic fluid than in other fluids. In contrast, PSPB and other blood attributes were higher in fetal serum than other fluids of Iraqi riverine buffaloes. These findings reflect the changes in hormones, proteins and other metabolites during different gestation periods.

Accuracy of pregnancy specific protein-B test for early pregnancy diagnosis in dairy cattle

The objective was to evaluate the accuracy of an ELISA for pregnancy specific protein B (PSP-B) for early pregnancy diagnosis in dairy cattle. Blood from lactating (>100 d postpartum) dairy cows (n = 738), was collected on Days 28, 30, and 35 (Day 0 = estrus), analyzed with an ELISA for PSP-B, and the cows designated as pregnant, probable, unlikely, or non-pregnant. Immediately after blood collection, transrectal ultrasonography (TRUS) was done for pregnancy diagnosis, and the results used as a criterion standard test for comparison with PSP-B. At Day 28, 46.3% were diagnosed by TRUS as pregnant. The PSP-B sensitivity was 93.9% on Day 28 and similar on Days 30 and 35. The PSP-B specificity, positive predictive value, negative predictive value, and accuracy were all >94% on Day 28 and similar on Days 30 and 35. However, the accuracy was significantly less compared to TRUS (P < 0.01). The percentage of all samples from cows that were probably pregnant or unlikely to be pregnant was 5.6%. At Days 28, 30, and 35, percentages of uncertain samples were 8.5, 4.8, and 3.3%, respectively (P < 0.01), and Kappa values were 0.92, 0.92, and 0.95. False negative and false positive results were attributed to low concentrations of PSP-B in pregnant animals and to persistence of pregnant concentrations of PSP-B in females with pregnancy loss, respectively. In conclusion, PSP-B ELISA was a sensitive, specific, and accurate test for pregnancy diagnosis (relative to TRUS) at Days 28, 30, and 35 after breeding.