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Early diagnosis of pregnancy is directly related to cost-effective livestock production. We produced a rat monoclonal antibody (mAb) against synthesized porcine early pregnancy factor (pEPF) using conventional hybridoma technology and used it as a tool for the detection of early pregnancy in Duroc sows. The rat pEPF-mAb showed reactivity to uterine tissues of pregnant sows 20 or 30 days post-mating (day 0 defined as the day of mating) and non-pregnant sows (confirmed signs of estrus) in western blotting. Immunohistochemical analysis confirmed that pEPF was located in the stromal and grand epithelial tissues of pregnant sows 20 or 30 days post-mating. In the enzyme-linked immunosorbent assay, pEPF expression in urine and blood showed similar results, with the highest expression observed in pregnant sows 20 days post-mating, whereas there was no significant difference in expression levels between non-pregnant sows and pregnant sows 30 days post-mating. The pEPF-mAb-based pregnancy diagnostic kit can be applied to pig urine samples non-invasively collected at 20 days post-mating with 70 % accuracy. Further improvements to the kit's diagnostic performance may lead to substantial benefits for the swine industry, facilitating more efficient and accurate reproductive management., Competing Interests: Declaration of competing interest No potential conflict of interest was reported by the author(s)., (Copyright © 2024 Elsevier Inc. All rights reserved.)

This study aimed to compare the accuracy of IFN-τ stimulated gene abundance (ISGs) in peripheral blood mononuclear cells (PBMCs), CL blood perfusion by Doppler ultrasound (Doppler-US), plasma concentration of P4 on Day 21 and pregnancy-associated glycoproteins (PAGs) test on Day 25 after timed-artificial insemination (TAI) for early pregnancy diagnosis in dairy cows and heifers. Holstein cows (n = 140) and heifers (n = 32) were subjected to a hormonal synchronization protocol and TAI on Day 0. On Day 21 post-TAI, blood samples were collected for PBMC isolation and plasma concentration of P4. The CL blood perfusion was evaluated by Doppler-US. Plasma samples collected on Day 25 were assayed for PAGs. The abundance of ISGs (ISG15 and RSAD2) in PBMCs was determined by RT-qPCR. Pregnancy was confirmed on Days 32 and 60 post-TAI by B-mode ultrasonography. Statistical analyses were performed by ANOVA using the MIXED procedure and GLIMMIX in SAS software. The pregnancy biomarkers were used to categorize the females as having undergone late luteolysis (LL); early embryonic mortality (EEM); late embryonic mortality (LEM); or late pregnancy loss (LPL). The abundance of ISGs, CL blood perfusion by Doppler-US, and concentrations of P4 on Day 21, and PAGs test on Day 25 were significant (P < 0.05) predictors of early pregnancy in dairy cows and heifers. Dairy cows had a greater (P = 0.01) occurrence of LL than heifers, but there was no difference (P > 0.1) for EEM, LEM, and LPL in heifers compared to cows. Cows with postpartum reproductive issues had a greater (P = 0.008) rate of LEM and a lesser (P = 0.01) rate of LPL compared to cows without reproductive issues. In summary, the CL blood perfusion by Doppler-US had the highest accuracy and the least number of false negatives, suggesting it is the best predictor of pregnancy on Day 21 post-TAI. The PAGs test was the most reliable indicator of pregnancy status on Day 25 post-TAI in dairy heifers and cows. The application of machine learning, specifically the MARS algorithm, shows promise in enhancing the accuracy of predicting early pregnancies in cows., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Transrectal ultrasonography is known as the gold standard for pregnancy detection, but requires costly equipment and technical skills; therefore, access to an inexpensive and more user-friendly method with similar accuracy could benefit cattle producers. Detection of pregnancy-associated glycoproteins can accurately determine pregnancy in ruminants; however, usually requires specialized equipment for the assay. Thus, the objectives of these studies were to 1) validate the IDEXX Alertys OnFarm Pregnancy Test (lateral flow) and compare the accuracy of all three commercial PAG assays to transrectal ultrasonography and 2) to determine the postpartum interval necessary for clearance of pregnancy-associated glycoproteins from the previous pregnancy to avoid false positives. In study 1, blood samples from previously identified pregnant Bos taurus females from six different herds (nulliparous n = 1,205 and multiparous n = 1,539; samples collected between d 27 to 285 of gestation over a three-year period) were utilized. In study 2, postpartum females (primiparous n = 48 and multiparous n = 66) from one herd were utilized: (n = 1,066; samples collected weekly for up to 12 weeks postpartum). In study 1, level of agreement between different methods of pregnancy detection was determined by Pearson's correlation and Kappa scores. In study 2, data were analyzed as a repeated measure using the MIXED procedure of SAS with main effects of parity, days postpartum (dpp), and parity by days postpartum, then data were analyzed further using the REG procedure of SAS. In study 1, transrectal ultrasonography and lateral flow were positively correlated (r = 0.77; P <0.01), with 92.4% agreement. In study 2, the abundance of absorbance of PAGs rapidly decreased from 0 to 50 days postpartum, then continued to gradually decrease (P <0.01; r = 0.90). Prior to 42 days postpartum, PAG concentrations were sufficiently elevated resulting in false positive readings in all assays. In conclusion, there is very good agreement between transrectal ultrasonography and PAG assays, but likelihood of false positive results are highif assays are performed fewer than 42 days postpartum., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: Jim Rhoades is employed by IDEXX., (Copyright: © 2024 Kline et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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 0.6 ng/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., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH, DE part of Springer Nature.)

The most reliable methods for pregnancy diagnosis in dairy herds include rectal palpation, ultrasound examination, and evaluation of plasma progesterone concentrations. However, these methods are expensive, labor-intensive, and invasive. Thus, there is a need to develop a practical, non-invasive, cost-effective method that can be implemented on the farm to detect pregnancy. This study suggests employing microwave dielectric spectroscopy (MDS, 0.5-40 GHz) as a method to evaluate reproduction events in dairy cows. The approach involves the integration of MDS data with information on milk solids to detect pregnancy and identify early embryonic loss in dairy cows. To test the ability to predict pregnancy according to these measurements, milk samples were collected from (i) pregnant and non-pregnant randomly selected cows, (ii) weekly from selected cows ( n = 12) before insemination until a positive pregnancy test, and (iii) daily from selected cows ( n = 10) prior to insemination until a positive pregnancy test. The results indicated that the dielectric strength of Δ ε and the relaxation time, τ , exhibited reduced variability in the case of a positive pregnancy diagnosis. Using principal component analysis (PCA), a clear distinction between pregnancy and nonpregnancy status was observed, with improved differentiation upon a higher sampling frequency. Additionally, a neural network machine learning technique was employed to develop a prediction algorithm with an accuracy of 73%. These findings demonstrate that MDS can be used to detect changes in milk upon pregnancy. The developed machine learning provides a broad classification that could be further enhanced with additional data.

Primiparous and multiparous dairy cattle were evaluated for pregnancy using both transrectal ultrasonography and a point-of-care pregnancy test (Alertys OnFarm Test), which measures pregnancy-associated glycoproteins through lateral diffusion, between Days 28-34 of pregnancy results were compared. A total of 637 animals were included in this study. Pregnancy was confirmed via manual palpation between Days 57-64. Data on parity, calving, and time of artificial insemination (AI) were also collected and evaluated. Overall the accuracy of the lateral diffusion test was 93.1% with 98.9% sensitivity, 88.7 % specificity, 86.8 % positive predictive value, and 99.1% negative predictive value. In heifers, the Alertys OnFarm Test had 100% sensitivity and 81.6% specificity. In contrast, the test had a sensitivity and specificity of 98.5 and 89.5%, respectively for multiparous cows. The pregnancy loss between early diagnosis and confirmation increased with parity. Heifers suffered losses as low as 2.6%, whereas animals in the third parity had significantly more losses (17.9%). Season also affected losses with spring pregnancy losses being considerably higher than autumn losses. The veterinary workload was also evaluated. Using the combined method of pregnancy testing, animals were first tested with the lateral diffusion test; then, the test-negative animals were scanned again, and the number of scanned animals was reduced. The results ranged between 3274% on farms. We concluded that veterinary workload could be reduced by using the point-of-care test. However, farm-level differences may have affected the results of the present study., Competing Interests: Declaration of competing interest There were no conflicts of interests associated with the study., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Early pregnancy detection in alpacas, whose breeding season is limited to the rainy season and has a long gestation period, is important for reproductive management. Conventional detection methods such as ultrasonography cannot be used to detect pregnancy before 30 days after mating. In this study, we examined the feasibility of using fecal steroid hormones as an early detection method in pregnant and non-pregnant alpacas. Fecal and blood samples were collected from pregnant and non-pregnant alpacas after mating. Progesterone (P4) and estradiol 17-β were extracted and quantified from blood and fecal samples. A positive correlation exists between the steroid hormones in serum and feces, indicating that serum steroid hormone concentrations can be estimated from fecal steroid hormones. Within 10 days after mating, both pregnant and non-pregnant alpacas had fecal P4 concentrations greater than 1.0 ng/mg dry matter (DM), but by 15 days after mating, fecal P4 concentrations decreased to the pre-mating concentration in non-pregnant alpacas. From 15 days after mating, non-pregnant alpacas had a low fecal P4 concentration (< 1 ng/mg DM), whereas a high fecal P4 concentration indicated the possibility of pregnancy, suggesting that this test is clinically beneficial as a supportive test for pregnancy detection., (© 2024 Japanese Society of Animal Science.)

The aim of this study was to determine and compare the diagnostic accuracy of two pregnancy-associated glycoproteins tests (IDEXX on-farm pregnancy test [OFPT] and IDEXX rapid visual pregnancy test [RVPT]) for early pregnancy diagnosis in dairy cattle. Blood samples were collected from Holstein cows (n = 317) by coccygeal venipuncture 28-31 days after artificial insemination (AI). The OFPT and RVPT were performed on the farm within 2 h after blood collection using whole blood or blood serum. Transrectal ultrasonography (USG) was performed for pregnancy diagnosis on day 32 post-AI as a gold standard. One-hundred fourteen cows were diagnosed as pregnant and 203 were determined to be nonpregnant. Furthermore, embryonic mortality was detected in four of the nonpregnant animals, based on fragmented/dispersed embryonic membranes and the absence of heartbeat. Sixteen and 14 false positive results (13 jointly for both tests) were obtained for the RVPT and OFPT, respectively. Three false negative results were obtained for the RVPT. The sensitivity, specificity, positive predictive value, negative predictive value and accuracy for the OFPT were 100%, 93.1%, 89.1%, 100% and 95.6%, and for RVPT were 97.4%, 92.1%, 87.4%, 98.4% and 94.0%, respectively. The ability of both tests to distinguish between pregnant and open cows was very good (AUC of both tests above 0.9). The OFPT and RVPT almost perfectly agreed with each other. According to McNemar's analysis, false positive results have been caused difference between the two pregnancy tests and USG. In conclusion, both the RVPT and OFPT proved to be reliable and practical methods for pregnancy diagnosis 28-31 days after AI in dairy cows. However, the results of both pregnancy tests were affected by the occurrence of embryonic mortality around the time of their employment., Competing Interests: Declaration of competing interest None'., (Copyright © 2023 Elsevier Inc. All rights reserved.)

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., (© Wildlife Disease Association 2023.)

Hormone secretion by the maternal ovaries, trophoblast/placenta and fetus occurs sequentially, creating distinct steroid metabolomic 'signatures' in systemic blood of pregnant mares that vary with gestational stage. Algorithms were developed to predict the gestational day (GD) from the maternal steroid metabolome (nine steroids; pregnenolone (P5), progesterone (P4), 5α-dihydroprogesterone (DHP), 17α-hydroxyprogesterone, allopregnanolone, 20α-hydroxy-DHP, 3β,20α-dihydroxy-DHP, DHEA and androstenedione) determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS) of eight thoroughbred mares sampled longitudinally throughout pregnancy. A physiologically based model was developed to infer rates of steroid secretion during chorionic gonadotropin secretion, the luteo-placental shift and by the equine feto-placenta unit, demonstrating more variability in P5 and DHP than P4. The average of four empirical models, using nine steroids to predict GD, was calibrated (five mares, R2 = 0.94, RMSE = 20 days) and validated (three mares, R2 = 0.84, RMSE = 32 days). Validation performance was improved using paired samples taken 14 or 30 days apart (RMSE = 29 and 19 days, respectively). A second validation used an independent dataset (single serum samples from 56 mixed breed mares, RMSE = 79 days) and an additional longitudinal subset from the same population sampled monthly throughout gestation (seven mares, RMSE = 42 days). Again, using paired samples improved model performance (RMSE = 32.5 days). Despite less predictive performance of the mixed breed than the thoroughbred datasets, these models demonstrate the feasibility and potential for using maternal steroid metabolomic algorithms to estimate the stage of gestation in pregnant mares and perhaps monitor fetal development.

Infrared thermography is a non-invasive technique which allows to distinguish between pregnant and non-pregnant animals. Detecting accurate body surface temperatures can be challenging due to external factors altering thermograph measurements. This study aimed to determine the associations between the ambient temperature, the hair coat features and the temperatures of mares' abdomens. It compared pregnant and non-pregnant mares throughout 11 months. The research was carried out on 40 Konik Polski mares, which were divided into pregnant and non-pregnant groups. The temperature (Tmax, maximal; Taver, average; Tmin, minimal) of the mares' abdomen was evaluated in two regions of interest: the whole area of the lateral surface of the mares' abdomen (Px1) and the flank area of the lateral surface of mares' abdomen (Px2). During the increasing period, the slopes in the linear regression equation did not differ significantly for ambient (Tamb) and surface temperatures in both groups. In the decreasing period, the slopes did not differ significantly for Tamb and Tmax in the non-pregnant group. They also did not differ for Tamb and Taver in Px1 and Tamb and Tmin in Px1 in both pregnant and non-pregnant groups respectively. Other slopes varied significantly (p < .001). There was no evidence of parallel changes in hair coat features and measured temperatures. The flank area appears more suitable for thermal imaging in pregnant mares due to the seasonal fluctuations in hair coat lengths., (© 2021 The Authors. Reproduction in Domestic Animals published by Wiley-VCH GmbH.)

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.

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.

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 30 days after AI. The results of the Fassisi®BoviPreg test on serum and whole blood samples, respectively, on 30 days 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 ≥ 5 ng/mL P4 (P < 0.001). In conclusion, the results from the Fassisi®BoviPreg tests on cows on 30 and 50 days 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 50 days after AI.

We aimed to evaluate the accuracy of Interferon-tau stimulated genes (ISG) abundance in peripheral blood polymorphonuclear cells (PMNs) on D20 after fixed-time artificial insemination (FTAI; D0) as a pregnancy diagnosis method against CL evaluation by Doppler ultrasonography and progesterone (P4) concentrations on D20, as well as Pregnancy Associated Glycoproteins (PAG) concentrations on D25. Additionally, we evaluated the potential of ISG abundance in PMNs as pregnancy loss predictors. Nelore heifers (n = 103) and cows (n = 144) underwent estrous synchronization and were artificially inseminated on D0. Pregnancy was diagnosed by B-mode ultrasonography on D30 and D70, and after the final diagnosis, females were classified in four groups: Pregnant; Non-pregnant; Functional CL on D20 but non-pregnant (CL-NP) and Pregnancy loss between D30 and D70 (PL). After determining cutoff values, the Sensitivity (SE), Specificity (SP), Positive Predictive Value (PPV), Negative Predictive Value (NPV) and Accuracy (ACC) were determined for each method. All methods were classified as significant (P < 0.05) predictors of pregnancy. Both ISG expression and PAG concentrations were greater (P < 0.05) in pregnant females than in non-pregnant and CL-NP females but did not differ (P > 0.05) from the PL group. ISG15 expression was greater (P < 0.05) in heifers than in cows, but this difference was not found in OAS1 expression and PAG concentrations. All the methods evaluated were proven to be adequate predictors of pregnancy, but greater accuracies were obtained through PAG concentrations and Doppler-US, due to the decreased number of false positive and false negative results., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Objective: To assess the effect of transrectal palpation (TRP) performed with the fetal membrane slip (FMS) technique for early pregnancy diagnosis on the proportion and type of associated pregnancy losses (PLs) in dairy cattle., Animals: 580 healthy pregnant cattle., Procedures: Data for artificially inseminated females with 1 or 2 viable embryos detected by transrectal ultrasonography (TRUS) at approximately 30 days of gestation were retrospectively assessed. Cattle were assigned to 1 of 2 groups on the basis of whether they did or did not undergo TRP once between 34 and 41 days of gestation (palpation and control group, respectively). At approximately 45 and 60 days of gestation, all cattle were reevaluated by TRUS; PL was categorized as type I (FMS detectable by TRP and TRUS-confirmed evidence of embryo or fetus degeneration and a functional corpus luteum) or type II (FMS undetectable by TRP and no TRUS-confirmed evidence of an embryo or fetus or of a functional corpus luteum)., Results: Of the 580 healthy pregnant cattle, 271 underwent TRP and 309 did not. In the palpation and control groups, PL occurred in 40 (14.8%) and 47 (15.2%) cattle, respectively. Among the palpation group's PLs, 17 (43%) were type I and 23 (58%) were type II. Among the control group's PLs, 27 (57%) were type I and 20 (43%) were type II. The prevalance and type of PL did not differ between groups., Conclusions and Clinical Relevance: TRP with the FMS technique for early pregnancy diagnosis did not increase the prevalence of PL in dairy cattle or alert the proportion of type I versus type II PL.

The early pregnancy diagnosis allows optimizing production and timely management correction, with a greater reproductive output of livestock. The Idexx Rapid Visual Pregnancy Test® 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 cattle. Therefore, the aim of this study was to evaluate this rapid test and to verify the most appropriate day for the diagnosis of pregnancy in the ovine species. For this purpose, 98 serum samples from pregnant sheep and 36 from non-pregnant were used, with duplicate samples, and diagnosis was confirmed by ultrasound examination, used as the gold standard. The numbers of positive samples obtained at 26, 28, and 30 days of pregnancy were 26, 27, and 45, respectively. The Rapid Visual Pregnancy Test correctly identified 100% of the samples as positive at pregnancy of days 26, 28, and 30. The sensitivity and specificity of the test were also 100%. The Idexx Rapid Visual Pregnancy Test, initially indicated for cattle, is effective for the detection of pregnancy in the ovine species, enabling diagnosis of pregnant sheep from the 26th day of pregnancy.

Pregnancy diagnosis is an essential part of successful breeding programs on dairy farms. Milk composition alters with pregnancy, and this is well documented. Fourier-transform mid-infrared (MIR) spectroscopy is a rapid and cost-effective method for providing milk spectra that reflect the detailed composition of milk samples. Therefore, the aim of this study was to assess the ability of MIR spectroscopy to predict the pregnancy status of dairy cows. The MIR spectra and insemination records were available from 8,064 Holstein cows of 19 commercial dairy farms in Australia. Three strategies were studied to classify cows as open or pregnant using partial least squares discriminant analysis models with random cow-independent 10-fold cross-validation and external validation on a cow-independent test set. The first strategy considered 6,754 MIR spectra after insemination used as independent variables in the model. The results showed little ability to detect the pregnancy status as the area under the receiver operating characteristic curve was 0.63 and 0.65 for cross-validation and testing, respectively. The second strategy, involving 1,664 records, aimed to reduce noise in the MIR spectra used as predictors by subtracting a spectrum before insemination (i.e., open spectrum) from the spectrum after insemination. The accuracy was comparable with the first approach, showing no superiority of the method. Given the limited results for these models when using combined data from all stages after insemination, the third strategy explored separate models at 7 stages after insemination comprising 348 to 1,566 records each (i.e., progressively greater gestation) with single MIR spectra after insemination as predictors. The models developed using data recorded after 150 d of pregnancy showed promising prediction accuracy with the average value of area under the receiver operating characteristic curve of 0.78 and 0.76 obtained through cross-validation and testing, respectively. If this can be confirmed on a larger data set and extended to somewhat earlier stages after insemination, the model could be used as a complementary tool to detect fetal abortion., (The Authors. Published by FASS Inc. and Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).)

A prediction method for early pregnancy status (pregnant or non-pregnant) in cattle that can be used within 3 weeks after insemination is desired. Interferon-stimulated genes (ISGs) in peripheral blood leukocytes (PBLs) have been examined as prediction molecules for determination of pregnancy status. Relative abundances of ISG15 and MX2 gene transcripts in PBLs were suitable biomarkers for the prediction of pregnancy status when there were assessments of Holstein cattle. In the present study, it was determined whether ISG biomarkers are applicable for predicting gestation in Japanese-Black (JB) cattle and evaluation of the applicability of receiver operating characteristic (ROC) analysis procedures for this purpose. There was assessment of the reliability of using average ISG values in PBLs collected during the estrous cycle (AVE) as a cutoff compared to the Youden index cutoff values. Application of AVE to assessment of pregnancy status in JB cattle indicated there was reliable predictions for pregnancy status when using ISG15 and MX2 values on day 21 after insemination, which coincided with the time of assessment in the previous study with Holstein cattle. The area under the curve values of the ROC curves confirmed the reliability of using ISGs to predict pregnancy from days 18 to 21 after insemination. Comparing AVE with Youden index values, there was confirmation of the accuracy of AVE for predicting gestation. The average mRNA transcript abundance values of ISG15 and MX2 may serve as excellent pregnancy biomarkers for cattle within 3 weeks of insemination., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interests., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The objective of this experiment was to determine if circulating concentrations of pregnancy associated glycoproteins (PAG) on day 24 of gestation can be utilized to diagnose pregnancy and embryo viability in beef cattle. Postpartum beef cows (n = 677) and heifers (n = 127) were exposed to a 7-day CO-Synch + CIDR estrus synchronization protocol followed by fixed-time AI (FTAI) on day 0. Blood samples were collected at day 24 after TAI to assess circulating concentrations of PAG utilizing an in-house ELISA. Pregnancy diagnosis was performed 30 and 100 days after FTAI via transrectal ultrasonography. Mean circulating PAG concentration at day 24 differed (P < 0.001) between animals diagnosed pregnant and non-pregnant at day 30 (1.69 ± 0.10 ng/mL vs 0.30 ng/mL ± 0.07 ng/mL; mean ± SEM; respectively). Pregnant heifers had increased PAG concentration at day 24 compared with pregnant cows (P < 0.01; 3.29 ± 0.36 ng/mL vs 1.39 ± 0.10 ng/mL, respectively). Based on receiver operating characteristic (ROC) curve analysis, serum concentration of PAG at day 24 ≥ 0.33 ng/mL in cows and ≥0.54 ng/mL in heifers was 95% accurate at determining pregnancy status at day 30. Heifers that experienced late embryonic mortality between day 30 and 100 of gestation had decreased circulating concentrations of PAG on day 24 (2.02 ng/mL ± 0.73) compared with heifers that maintained an embryo until day 100 (3.69 ng/mL ± 0.39; P = 0.02). However, there was no difference in day 24 PAG concentration (P = 0.39) between cows that maintained or lost a pregnancy (1.31 ng/mL ± 0.25 vs 0.92 ng/ml ± 0.50). In summary, circulating PAG concentration on day 24 of gestation may be a useful marker for early pregnancy detection in beef cattle, and might be a potential marker for predicting embryonic loss., (Copyright © 2019 Elsevier Inc. All rights reserved.)

For the timed re-insemination at the minimal interbreeding interval, cows were treated with a progesterone (P 4 )-releasing intravaginal device from Days 13-15 to 21 post-insemination (Day 0 = estrus), followed by plasma P 4 assay on Day 23 and then subjected to the Experiments 1 and 2. In Experiment 1, of 18 cows, 6 cows were determined as luteolysis with low (<1 ng/ml) plasma P 4 concentrations on Day 23 and ovulated on Days 24 (3 cows), 25 (1 cow), and 26 (1 cow) except a cow affected by ovarian quiescence. In Experiment 2, all cows were treated with GnRH on Day 23. Cows with low (<1 ng/ml) plasma P 4 concentrations on Day 23 were diagnosed as non-pregnant and subjected to the re-insemination in the morning of Day 24 irrespective of estrous signs. Of 36 cows, 15 cows were diagnosed as being non-pregnant on Day 23. Fourteen cows of the non-pregnant animals were re-inseminated in the morning of Day 24 irrespective of estrous signs and the pregnancy rate of re-insemination was 36%. The conception rates of initial and re-inseminations were 50% (18/36) and 36% (5/14), respectively. The overall pregnancy rate by adding the rates of initial and re-inseminations was 64% (23/36)., (© 2020 The Authors. Animal Science Journal published by John Wiley & Sons Australia, Ltd on behalf of Japanese Society of Animal Science.)

One of the effective reproductive management programs in dairy cattle is the accurate detection of pregnancy. A total of 204 non-descriptive cows were examined for pregnancy before slaughter in Sulaimani abattoir. Examinations were done by rectal palpation and enzyme-linked immunosorbent assay (ELISA) to measure the levels of progesterone and bovine pregnancy- -associated glycoproteins (bPAGs) in their blood. Detection of a live conceptus in the uterus of slaughtered cows was used as the gold standard to determine the accuracy of the three pregnancy detection methods. The results showed that the accuracies of rectal palpation, progesterone assay, and bPAGs assay in the diagnosis of pregnancy were 87.2%, 84.8%, and 97.05%, respectively. The bPAGs assay scored the highest sensitivity (100%) for detection of pregnancy, followed by the progesterone assay (92.3%) and rectal palpation (84.6%). In addition, the specificity of the bPAGs assay was the highest (96.0%), while progesterone assay exhibited the lowest specificity (80.1%) and rectal palpation showed a specificity rate of (88.8%). In conclusion, the best method for the detection of either for early or late pregnancy in cows was the bPAGs assay, which gave the lowest number of false-positive and false-negative results., (Copyright© by the Polish Academy of Sciences.)

Slaughter of pregnant animals is a common abattoir practice. This study was designed to compare the diagnostic capacity of pregnancy specific protein-B (PSPB) with post slaughter inspection (PSI) procedures in detecting cows pregnant at slaughter. Blood was collected from cows presenteded for slaughter at an abattoir. The uteri were examined post slaughter for the presence or absence of a foetus. Recovered foetuses were aged using crown-rump length to estimate the stage of pregnancy. Of the 361 cows examined, 72 (19.9 %) were diagnosed pregnant using the PSPB procedure, while 32 (8.9 %) were diagnosed pregnant using PSI diagnosis. Furthermore, with PSI there was a lack of pregnancy diagnosis in 42 (11.6 %) cows detected pregnant using PSPB procedure, and two (0.6 %) cows detected pregnant using PSI were not detected to be pregnant using PSPB diagnosis. Validity of the diagnostic procedures indicated that sensitivity and specificity of the PSPB was 93.8 % and 87.2 %, respectively, while with the PSI diagnosis there was a sensitivity and specificity of 41.7 % and 99.3 %, respectively. The PSPB diagnosis, had an excellent predictive value (AUC - 0.92; p < 0.001; 95 % CI - 0.856 to 0.981). Most of the pregnancy wastage (n = 22; 68.8 %) diagnosed using the PSI method were in the second trimester. The results of this study indicate that PSPB is reliable and a more sensitive diagnostic method than PSI . It is therefore recommended that the PSPB test be incorporated in routine screening for pregnancy status of cows before slaughter., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Uterine examinations provide an inexpensive and reliable postmortem alternative to monitor pregnancy rates in free-ranging elk ( Cervus canadensis ). However, this technique may be insensitive during early pregnancies (i.e., <20 d postconception), relies on proper collection of tissues, and may not be comparable to antemortem approaches used throughout the rest of the year. To circumvent some of these issues, the sensitivity and specificity of a commercially available serum pregnancy-specific protein B (PSPB) enzyme-linked immunosorbent assay (ELISA) was determined relative to uterine examination. From 2013 to 2017, paired serum samples and uteri were collected from 245 harvested free-ranging cow elk in Pennsylvania, US in November. Uteri were examined to determine whether the cow was pregnant, and, if so, gestation age was estimated based on embryo crown-rump (CR) length. The serum PSPB ELISA testing was then performed. Since harvested elk could not be retested, samples with optical densities close to the threshold for pregnancy determination (i.e., high-recheck samples) were considered as both not pregnant and pregnant, and analyses were performed separately under each scenario. Overall, the PSPB ELISA had a sensitivity of 95% (high-recheck considered pregnant) and 93% (high-recheck considered not pregnant), and a specificity of 91% (high-recheck considered pregnant) and 93% (high-recheck considered not pregnant) relative to uterine examinations. Based on CR length, gestation age was <14 to 55 d. Our results indicated the PSPB ELISA was an accurate serum-based pregnancy test for elk.

Transrectal palpation of the reproductive tract is the most common method for pregnancy determination in cattle and is considered a veterinary skill that new veterinary medicine (DVM) graduates should perform proficiently. However, using privately owned cattle to train students can be difficult because producers may believe that transrectal palpation by inexperienced students increases the risk of pregnancy wastage compared with examination by an experienced clinician. We used a randomized field trial of 1,216 healthy Holstein and Jersey cattle in 2 commercial dairy herds to estimate the effect of veterinary student transrectal palpation on early pregnancy loss. All cattle were determined to be pregnant using transrectal ultrasonography at approximately 37 d after artificial insemination. Cattle were then allocated into 2 groups based upon their ear tag number (study group = 598; control group = 618). Cattle in the study group were immediately palpated after ultrasonography by a fourth-year veterinary student, whereas control cattle were not subject to any additional pregnancy assessment. For analysis, the student palpators were divided into 2 groups: students who had previously had formal palpation training via an elective bovine palpation class (n = 30) and students who had not had palpation training (n = 134). All cattle were reevaluated using transrectal ultrasonography approximately 70 d after artificial insemination. A total of 53 (4.36%) animals lost their pregnancy between the first and second pregnancy assessments. Of the animals that lost their pregnancy, 26 (4.35%) were study group cows and 27 (4.37%) were control cows. Of the 26 cows documented to have had pregnancy loss within the study group, 20 out of 378 (5.3%) had been palpated by students who had not taken the palpation elective and 6 out of 220 (2.7%) had been palpated by students who had completed the elective. We found no difference in pregnancy loss between student-palpated and clinician-ultrasounded cattle, supporting the safety of using privately owned animals for student bovine palpation and pregnancy diagnosis training without affecting early pregnancy loss., (Copyright © 2019 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

The objective was to compare the use of corpus luteum (CL) vascular perfusion to CL diameter and/or echogenicity to diagnose pregnancy at 21 d after timed-AI. Ovaries of Nelore heifers were assessed using ultrasonography in B-mode and color Doppler simultaneously 21 d after timed-AI (n = 113). Objective evaluations were performed using an image processing software to extract the number of colored pixels (ColorPix), diameter (mm) and echogenicity/mm² (EchoPix) of the CL. Subjective evaluations of the CL were performed by five evaluators using scores of estimated vascular perfusion area of color Doppler scan videos and estimated CL size and qualitative echogenicity of B-mode scan videos. The reference pregnancy diagnosis was performed 33 d after timed-AI using an ultrasonic device. Corpus luteum ColorPix, diameter and EchoPix were highly correlated (P < 0.001) with pregnancy. Pregnancy diagnosis accuracy, sensitivity, and negative predictive value were not different for CL ColorPix and diameter and was less with use of EchoPix compared to the other parameters. Size and vascular perfusion scores were correlated to the greatest extent (0.88-0.94) with the respective objective values within evaluator. The results from the ROC curve analysis indicated a smaller area under the curve for qualitative echogenicity compared to CL size and vascular perfusion. Corpus luteum vascular perfusion was the only subjective evaluation that when assessed there were no false negative pregnancy diagnoses. In conclusion, the use of the objective CL diameter resulted in the same efficacy as CL vascular perfusion evaluations for early pregnancy diagnosis in Nelore heifers., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Besides chemical testing, thermographic imaging is a possible method to determine pregnancy in some animals. Hilsberg (2001) defined the unilateral warming at the end of a pregnancy, caused by the metabolizing fetus and the uterus near the outer skin, as the pregnancy field. Its appearance was observed in various species at the end of the gestation period. No studies were yet carried out to investigate the changing of thermal patterns during pregnancy. This study monitored the thermographic changes of one pregnant babirusa (Babyrousa babyrussa) sow and one babirusa male by taking thermal images once a week from the estimated 3rd pregnancy week until parturition in the 23rd pregnancy week. Images were searched manually for heat areas correlated to pregnancy as well as analysed automatically for median and maximum temperatures of the animal's side. Additionally, the locations of all pixels, which were not more than 2 K below the maximum temperature, were evaluated. In the female, there was a positive correlation between maximum temperatures of left and right side to pregnancy weeks (R = 0.61, R = 0.66, respectively; p < 0.05). The female's median temperatures and all temperatures of the male were not correlated to pregnancy weeks. Differences between median and maximum temperatures were significantly different during the 2nd, and 3rd trimester in the female, but not in the male. Temperature differences between 1st and 2nd trimester were not significant in both animals. The location of the warmest areas in the female shifted from thorax and flank to be mainly localised on the teats. In the male, the warmest areas remain localised on thorax and flank. No distinct pregnancy field was observed, but the warming of the teats could be seen in the images and the evaluated data. The teats are less insulated in most animals. Contraindications for thermal imaging like insulation could therefore become less important, allowing thermal pregnancy diagnosis for a larger range of animals than before. Based on these findings we suggest that the warming of the teats could be used for thermal pregnancy diagnosis when a pregnancy field is not observable., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The in-line milk analysis system (IMAS) is an automated biosensor technology that samples and quantifies milk progesterone concentrations (P4c) at frequent intervals starting early postpartum until pregnancy. The objective was to validate the use of pregnancy notifications (PregN) generated by an IMAS based on P4c profiles after artificial insemination (AI) to determine pregnancy and nonpregnancy status in dairy cows. Records of 1,821 AI events from 715 Holstein cows that had milk P4c (ng/mL) measured every 2.2 ± 1.9 d (mean ± standard deviation) between 24.5 ± 8.2 and 173.4 ± 49.3 d in milk through a real-time IMAS (Herd Navigator, DeLaval International, Tumba, Sweden) were evaluated. Based on variations in adjusted milk P4c (< vs. ≥ the 5.0 ng/mL threshold), the system determined the sampling frequency, onset and cessation of luteal phases, and pregnancy. If a luteal phase initiated (P4c increased to ≥5.0 ng/mL) after AI and remained uninterrupted, a PregN was generated starting at (mean ± standard deviation) 31.0 ± 4.3 d until 53.4 ± 7.9 d after AI, when sampling stopped, unless a decline in P4c (to <5.0 ng/mL) occurred indicating nonpregnancy and imminent estrus. The assessment of IMAS PregN at 4 weekly intervals was tested, and a confirmed calving occurrence between 262 and 296 d after AI, with no other subsequent AI recorded, was the gold standard for pregnancy. In total, 14.1 (256/1,821), 41.0 (746/1,821), and 50.7% (924/1,821) of AI events were followed by a decline in P4c before 19, 23, and 30 d after AI, respectively. Frequency of the last 3 sampling events preceding P4c decline was greater if P4c decline occurred between 18 and 25 d after AI (1.4 ± 0.5 samples per day) compared with before 17 or beyond 26 d after AI (1.0 ± 0.5 samples per day). At 30 ± 3 (27 to 33) d after AI, PregN occurred in 46.8% (853/1,821) of AI events, of which 15.2% (130/853) had a decline in P4c between 30 and 55 d after AI and 17.1% (146/853) was later confirmed nonpregnant based on the gold standard. A total of 40.7% (742/1,821) of AI events was confirmed pregnant by the gold standard, which was no different than the proportion of PregN at 51 ± 3 (48 to 54) d (40.9%; 744/1,821). At any time point between 27 and 54 d after AI, sensitivity and negative predictive values for PregN were greater than 95.0 and 96.0%, respectively, whereas specificity values were less than 90.0% for PregN before 40 d but greater than 94.0% for PregN beyond 41 d after AI. In conclusion, IMAS is able to diagnose pregnancy based on P4c profiles with high precision and determine early nonpregnancy based on the spontaneous cessation of the luteal phase. However, for accuracy greater than 95.0%, pregnancy declaration based on IMAS notifications alone should occur no earlier than 41 d after AI., (Copyright © 2019 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

As sheep produce similar pregnancy-associated glycoproteins (PAGs) to cattle, a commercially available bovine visual pregnancy test based on the detection of PAGs (visual-PAG-test) was evaluated in sheep. The test was performed with whole blood (WhB), plasma (P) and serum (S) of 163 pregnant and 153 non-pregnant ewes. Additionally, 11 pregnant ewes were tested weekly from day 14 to 49 of gestation and monthly from day 60 to day 149. Ten ewes were sampled weekly from the date of lambing until day 63 post-partum (p.p.). The sensitivity in mid-pregnancy (n = 163) was 98.16% (WhB), 99.39% (P) and 99.39% (S) compared to transabdominal ultrasonography as the gold standard while the specificity (n = 153) was 94.12% (WhB), 76.47% (P) and 93.46% (S), respectively. During early pregnancy, all 11 ewes were correctly identified as pregnant on day 42 (100%); however, the test sensitivity decreased to 54.6% (WhB) and 63.6% (S and P, respectively) at day 49. The ewes were again consistently identified as pregnant on day 63 (P) or on day 119 (S, WhB). The test was consistently negative from day 42 p.p. onwards in eight out of ten ewes. Two ewes remained consistently positive until the last sample on day 63 p.p. In conclusion, the test could be used to accurately select pregnant ewes at day 42 with a drop in sensitivity at day 49. The sensitivity of the visual-PAG-test was good in mid to late pregnancy, and early detection of pregnancy was possible in individual animals. In some ewes, the PAGs were however detectable for more than 63 days p.p.-the previous breeding history should therefore always be taken into account for correct interpretation of the test results., (© 2018 Blackwell Verlag GmbH.)

Confirmation of the pregnancy establishment at the very earliest day post-insemination increases the reproduction efficiency of high yielding dairy cows and farm profitability by allowing rebreeding of the non-pregnant cows. Inaccuracies in the currently available pregnancy detection tools to detect pregnancy establishment within the first 3 weeks post insemination extends the inter-calving interval and have contributed to the decline in profitability. Thus, development of non-invasive early pregnancy detection biomarkers could be proposed as alternative tools. MicroRNAs (miRNAs), a subclass of small non-coding RNAs are abundantly expressed in virtually all bio fluids circulation and have been associated with various pregnancy-related pathophysiological conditions. The study aimed to determine the expression of circulatory miRNAs in serum samples of pregnant and non-pregnant cows at day 19 and 24 post-insemination. Lactating Holstein-Friesian cows were estrous synchronized and inseminated with frozen semen. Blood samples were taken 19 and 24 days post-insemination. Serum samples were retrospectively categorized according to the pregnancy status of cows diagnosed 35 later using ultrasonography. Total RNA enriched with miRNAs was isolated from pooled (4 animals/pool) serum samples of pregnant and non-pregnant cows and subjected to cDNA synthesis. The expression of circulatory miRNAs was performed using PCR array containing primers 748 mature miRNAs. Results showed that a total of 302 and 316 miRNAs were detected in day 19 pregnant and non-pregnant cows, respectively. Similarly, 356 and 325 miRNAs were detected in day 24 pregnant and non-pregnant cows, respectively. Principal component analysis showed clear separation between pregnant and non-pregnant cows both at 19 and 24 days. We identified 8 and 23 differentially expressed miRNAs in the serum of pregnant cows of day 19 and 24, respectively. Interestingly, miR-433 and 4 other miRNAs (miR-487b, miR-495-3p, miR-376b-3p, and miR-323a-3p), which are homologous to the human pregnancy-associated C14MC miRNAs were among the differentially expressed miRNAs in day 19 and 24 pregnant cows, respectively. The adherens junction and ECM-interaction are among the pathways significantly enriched by predicted target genes of differentially expressed miRNAs. In conclusion, the expression of circulatory miRNAs in maternal blood serum of pregnant and non-pregnant cows showed distinct expression pattern and could suggest their potential involvement in early pregnancy establishment., (Copyright © 2018. Published by Elsevier Inc.)

The usefulness of a radiotelemetric pedometer for estrus detection in standing (ST) heat, or in silent heat without ST events, but in which ovulation is observed, in Japanese Black cattle was investigated. The duration of an increase in steps in ST heat was 11.8 ± 1.3 hr, and it was similar to that of ST events (duration: 10.1 ± 0.8 hr). Even in silent heat, the change pattern and the duration (11.6 ± 0.2 hr) of the period with an increase in steps during estrus were not different compared with ST heat. When artificial insemination (AI) was performed at 15.5 ± 0.6 hr from the onset of estrus detected by the pedometer in ST heat cases, the conception rate was 57.1% (8/14). Furthermore, fertility in cattle that underwent silent heat was evaluated. When AI was performed at 14.4 ± 2.0 hr from the onset of estrus detected by the pedometer, the conception rate was 60% (3/5) in silent heat cases. The overall results suggest that the radiotelemetric pedometer is a valid device for detecting estrus and it can even detect silent heat in Japanese Black cattle. Moreover, even silent heat cattle are fertile when AI is performed at the appropriate time., (© 2018 Japanese Society of Animal Science.)

The early detection of pregnancy and the determination of fetal numbers have economic benefits in sheep production because of the seasonal breeding patterns where missing a breeding opportunity means the loss of one productive year. The purpose of this study was to evaluate the efficacy of the B6-HRP ELISA for ovine pregnancy-specific protein B (oPSPB) measurement in the detection of pregnancy and estimation of fetal numbers in different sheep breeds. BioPRYN® ELISA assay kit was used for the detection of pregnancy in the experimental animals. Ninety-three ewes of three breeds (British Milksheep - BM, Lacaune - L and Transylvanian Racka - TR), each from three farms in Hungary, were included in the study. BM and L ewes were artificially inseminated (AI). Thirty-five days after AI, all ewes were examined by transabdominal ultrasound. The TR flock was mated naturally over a six-week period. At the end of the mating period, the ewes were similarly examined by ultrasound. Blood samples were taken from all pregnant ewes twice (35 and 65 days after AI), and serum samples were assayed by the BioPRYN test. It can be concluded that the detection of serum PSPB by ELISA is a much easier, safer, less expensive and highly accurate method for the detection of ovine pregnancy. Although some breed-related differences were detectable at 35 and 65 days post breeding, no differences in oPSPB levels were found in pregnant ewes carrying different numbers of fetuses.

The aim of the present study was to compare a Bovine Pregnancy Rapid Test (Ubio quickVET; BPRT) with a commercial ELISA-PAG test (Bovine Pregnancy Test DG29®) for early pregnancy diagnosis based on the presence of the PAG (pregnancy-associated glycoprotein) in dairy cattle between 30 and 40 days after artificial insemination (AI). Blood samples were collected from 212 cows between 30 and 40 days after artificial insemination (AI) to quantify the concentrations of PAG in each sample. Transrectal ultrasonography (TRUS) diagnosis of pregnancy was conducted at day 45 ± 3 after AI as the reference standard for the two PAG tests. The results indicated the sensitivity (Se) of the BPRT and DG29 for diagnosing pregnant cattle were 89.4% and 100%, respectively while the specificity (Sp) of the two tests for diagnosis of non-pregnant animals was 89.8% and 81.3%, respectively. Based on these results, the BPRT has a slightly lesser sensitivity and greater specificity than the DG29 test. Moreover, the accuracy of both tests was 94% and 90% respectively for DG29 and BPRT. This implies that the BPRT test could be considered an accurate test when compared to PAG-ELISA test and TRUS at days 30-40 after AI. The BPRT test, therefore, can be used as an alternative to the PAG-ELISA test with some constraints that need to be considered with its use., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Interferon tau plays an important role in establishing bovine pregnancy. Interferon-stimulated genes (ISGs) have been examined to identify a suitable indicator for the diagnosis of early gestation in cows. Although ISGs can be specifically detected in peripheral white blood cells during early gestation, its reliability remains to be validated. In the current study, a predictive threshold level of ISGs to determine pregnancy in cows during Days 20-22 of gestation was verified by analyzing the expression of ISGs in granulocytes and peripheral blood leucocytes (a total of 57 cows were used, 28 of which were pregnant and 29 were non-pregnant). Four genes, interferon-stimulated gene 15 ubiquitin-like modifier (ISG15), MX dynamin like GTPase (MX) 1, MX2, and 2'-5'-oligoadenylate synthetase 1 (OAS1), were analyzed via quantitative RT-PCR and a receiver operating characteristic (ROC) curve was produced to visualize diagnostic accuracy measures. The expression values of the four ISGs during the estrous cycle (100 collection points from 65 cattle) were used to determine a pregnancy prediction cutoff value. Pregnancy status was determined using these cutoff values and then confirmed by ultrasonography. ROC analysis was then applied to confirm the accuracy of the pregnancy statuses (positive and negative) statistically. The statistical evaluation of the diagnostic accuracy measurements suggested that the average values of ISG15 and MX2 in granulocytes were reliable indicators of pregnancy within the three weeks after insemination with 80% accuracy. Average ISG15 and MX2 levels during the estrous cycle were more reliable biomarkers for the prediction of gestation. They predicted negative and positive pregnancies efficiently within three weeks after artificial insemination., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The present study was conducted to determine the difference in plasma prostaglandin F 2 α metabolite concentrations following oxytocin (OT) challenge between pregnant and non-pregnant cows. Experiment 1: cows were subjected to the OT challenge test on days 12, 14 or 16 (day of estrus = day 0) with or without prior insemination and plasma 13,14-dihydro-15-keto prostaglandin F 2 α (PGFM) concentrations were measured from -30 to 180 min after OT injection. On day 16, the increment of plasma PGFM concentrations in response to OT injection was significantly smaller in pregnant than that in cyclic cows. On days 12 and 14, there was little OT-induced PGFM secretion and no difference in PGFM increase between the pregnant and cyclic cows. Experiment 2: cows were inseminated on day 0 and subjected to the OT challenge test on day 16. Cows were classified into non-pregnant/early embryonic death (NP/EED), late embryonic death (LED) and pregnant (PREG) groups. The increment of PGFM concentrations in response to OT injection was less in both PREG and LED groups than that in the NP/EED group. In conclusion, plasma PGFM secretion induced by OT is suggested as the base of pregnancy diagnosis prior to returning estrus in cows., (© 2017 Japanese Society of Animal Science.)

Various methods are being employed to detect early pregnancy in domestic animals. This study aimed to predict early pregnancy in buffaloes via measuring the corpus luteum (CL) diameter, the luteal blood flow (LBF) area, the uterine blood flow (UBF) vascularization area, and progesterones in saliva and serum for non-pregnant (NPBs, N = 12) and pregnant (PBs, N = 12) buffaloes. The results revealed that the CL diameter and the luteal color blood flow blue and red (P = 0.0001) areas of the pregnant animals kept increasing from day 1 to day 35 of the gestation period, but it decreased in NPBs on day 21 after reaching a peak from ovulation to day 18. Interestingly, the UBF of the pregnant buffaloes (PBs) kept increasing (P = 0.0001) from ovulation to day 42. The difference of the CL diameter (P = 0.03) and the LBF color blue vascularization area (P = 0.002) between PBs and NPBs became clear from day 14 after ovulation, though the difference of UBF between PBs and NPBs became markedly obvious from day 7 after breeding. Both saliva (P = 0.001) and serum (P = 0.0001) progesterones of PBs continued increasing (P = 0.0001) from day 14 to day 35, but those of NPBs started decreasing (P = 0.0001) from day 14 and reached low values on day 21. Therefore, measuring saliva progesterone in addition to the high LBF (day 14) and UBF (day 7) of the pregnant buffaloes using a Doppler ultrasound could be applicable as noninvasive methods to detect early pregnancy and to improve reproductive management of buffaloes.

Aims: To determine the sensitivity (Se) and specificity (Sp) of pregnancy diagnosis using transrectal ultrasonography and an ELISA for pregnancy-associated glycoprotein (PAG) in milk, in lactating dairy cows in seasonally calving herds approximately 85-100 days after the start of the herd's breeding period., Methods: Paired results were used from pregnancy diagnosis using transrectal ultrasonography and ELISA for PAG in milk carried out approximately 85 and 100 days after the start of the breeding period, respectively, from 879 cows from four herds in Victoria, Australia. A Bayesian latent class model was used to estimate the proportion of cows pregnant, the Se and Sp of each test, and covariances between test results in pregnant and non-pregnant cows. Prior probability estimates were defined using beta distributions for the expected proportion of cows pregnant, Se and Sp for each test, and covariances between tests. Markov Chain Monte Carlo iterations identified posterior distributions for each of the unknown variables. Posterior distributions for each parameter were described using medians and 95% probability (i.e. credible) intervals (PrI). The posterior median estimates for Se and Sp for each test were used to estimate positive predictive and negative predictive values across a range of pregnancy proportions., Results: The estimate for proportion pregnant was 0.524 (95% PrI = 0.485-0.562). For pregnancy diagnosis using transrectal ultrasonography, Se and Sp were 0.939 (95% PrI = 0.890-0.974) and 0.943 (95% PrI = 0.885-0.984), respectively; for ELISA, Se and Sp were 0.963 (95% PrI = 0.919-0.990) and 0.870 (95% PrI = 0.806-0.931), respectively. The estimated covariance between test results was 0.033 (95% PrI = 0.008-0.046) and 0.035 (95% PrI = 0.018-0.078) for pregnant and non-pregnant cows, respectively. Pregnancy diagnosis results using transrectal ultrasonography had a higher positive predictive value but lower negative predictive value than results from the ELISA across the range of pregnancy proportions assessed., Conclusions and Clinical Relevance: Pregnancy diagnosis using transrectal ultrasonography and ELISA for PAG in milk had similar Se but differed in predictive values. Pregnancy diagnosis in seasonally calving herds around 85-100 days after the start of the breeding period using the ELISA is expected to result in a higher negative predictive value but lower positive predictive value than pregnancy diagnosis using transrectal ultrasonography. Thus, with the ELISA, a higher proportion of the cows with negative results will be non-pregnant, relative to results from transrectal ultrasonography, but a lower proportion of cows with positive results will be pregnant.

A controlled field trial was conducted to assess the potential influence of practitioner inexperience during early pregnancy diagnosis with ultrasound (PD-US) on the risk of pregnancy loss. A veterinarian with more than 10 years' experience in PD-US (Vet-A) and a veterinarian with fewer than 12 months' experience at the start of the study (Vet-B) visited the same dairy farm once a week for 33 and 26 weeks, respectively. The two veterinarians did not interact with each other at any time during the study, nor did they know that their data would later be used in this study. Using the same farm scanner, they performed PD-US at 28-34 day after breeding, together diagnosing 915 pregnancies. All cows were re-checked at 49-56 day after artificial insemination, and cows no longer pregnant were recorded as having suffered pregnancy loss. Although Vet-A and Vet-B diagnosed a similar proportion of pregnancies (58.44 ± 16% vs 56.96 ± 18%, p > .05), the rate of pregnancy loss was significantly higher among cows diagnosed by Vet-B (10.41 ± 11.2% vs 4.87 ± 9.0, p = .029). In addition, among cows diagnosed by Vet-B, the rate of pregnancy loss was significantly higher among cows diagnosed, while he had fewer than 12 months' PD-US experience (11.17 ± 12.14%) than among cows that he diagnosed later (7.14 ± 11.01%, p = .038); in fact, this latter loss rate was comparable to that among cows diagnosed by Vet-A during the same period (3.51 ± 9.83%, p = .620). These results suggest that inexperience with PD-US during the late embryonic period can increase risk of early pregnancy loss, supporting the need for proper training., (© 2017 Blackwell Verlag GmbH.)

The objective of this study was to evaluate the effect of storage temperature and time from sample collection to analysis on test classification of a commercially available ELISA for diagnosis of pregnancy using the measurement of pregnancy-associated glycoproteins (PAG) in milk samples from dairy cows. Few studies have evaluated the effects of sample handling on milk PAG results. Using a repeated-measures study design, we evaluated sample storage at 5 temperatures: 37°C, 22°C, 4°C, -20°C, or -80°C. Sample aliquots from 45 cows (20 with a pregnant test result, 10 open, and 15 recheck) were stored for 4, 7, 14, 28, 60, 90, or 365 d. The measured PAG level was influenced by storage duration and condition. Samples stored for 365 d had a slightly increased PAG level, whereas samples stored for all other durations showed a slight decline in PAG level compared with the initial result. The reason for an increase in PAG level following long-term storage is not known. This will not affect dairy producers using the test but may be important in samples stored for research applications. The changes in PAG level were small and within the expected variation for this test. Fewer than 6% of samples changed in classification and, as expected, they were samples near the test interpretation cut-points., (Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Early detection of gestation is important in the bovine industry. New methods have been developed to detect gene expression in leucocytes induced by interferon-tau (IFNT) as gestation biomarkers. However, it is debatable which blood cell is suitable for detecting gene expression. This study was aimed at confirming whether granulocytes respond to IFNT specifically. Granulocytes and mononuclear cells (MNCs) from cows, and several types of bovine cultured cells, were treated with recombinant (r) IFNT and gene expression was analysed by quantitative real-time reverse transcriptase (RT)-PCR and microarray analysis. Expression levels of IFN receptors (R1 and R2) were approximately 30- to 900-fold higher in granulocytes than in other cultured cells, and 1.5- to 2.5-fold higher in MNCs than in granulocytes. Microarray analysis following a 2h recombinant IFNT (rIFNT) treatment revealed expression changes for 900 genes in granulocytes. Genes with expression changes included known IFN-stimulated genes (ISGs; ISG15, OAS1, MX1, and MX2). Eighteen genes were selected following granulocyte microarray analysis and their expression changes were confirmed in early gestation, which revealed that nine genes had significantly higher expression levels in pregnant than in non-pregnant animals. In conclusion, granulocytes specifically responded to rIFNT treatment and the resulting gene expression changes correlated with those in vivo. Microarray analysis indicated that various genes showed expression changes in rIFNT-treated granulocytes, which may result in the identification of alternate candidate genes for the early detection of gestation. These results strongly indicate that gene expression in granulocytes is a suitable tool to determine pregnancy status., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

This study assessed the accuracy of the commercial BioPRYN ® ELISA for the detection of pregnancy-specific protein-B (PSPB) using a single blood sample to determine pregnancy status in American bison (Bison bison). A total of 49 bison cows were used in the study, and sampled at two time-points during the gestation period, fall and spring, correlating with early- to mid-term gestation (average 62.9 days post-mating) and mid- to late-term gestation (average 229.2 days post-mating), respectively. Sensitivity of the test during early- to mid-term gestation sampling period (fall) was 87.1%, while specificity was 100%; sensitivity of the test during late-term gestation sampling period (spring) was 96.3%, while specificity remained at 100%. In total, the test showed a total sensitivity of 91.4%, specificity of 100% and total accuracy of 93.8%, similar to domestic cattle. Use of the single-sample BioPRYN ® ELISA in American Bison for pregnancy diagnosis is economical and practical, minimizing animal handling time, frequency and subsequent stress while providing accurate results for pregnancy diagnosis at 62 days post-mating. This method should be considered over more traditional pregnancy diagnosis methods for use in managed bison herds., (© 2017 Blackwell Verlag GmbH.)

Improper or delayed pregnancy diagnosis has significant impact over animal production, particularly in buffaloes which inherently suffer from several reproductive inefficiencies. Thus the present study has undertaken to identify serum protein markers pertaining to early pregnancy diagnosis in buffaloes. Serum samples were collected from 10 pregnant Murrah Buffalo heifers at weekly intervals from days 0-35 post-artificial insemination and from 12 inseminated non-pregnant cyclic buffalo heifers on days 0, 7, 14 and 21. Two-dimensional gel electrophoresis and densitometric analysis revealed the presence of five protein spots showing average density fold change of ≥4 during early pregnancy. Mass spectrometry analysis identified these up-regulated proteins as anti-testosterone antibody light chain, apolipoprotein A-II precursor, serum amyloid A, cytokeratin type II, component IV isoform 1, which are have established roles in embryogenesis, but over-expression of the fifth identified protein immunoglobulin lambda light chain in pregnancy has been elucidated as a novel finding in the current study. Further, with bioinformatics analysis, potential antigenic B-cell epitopes were predicted for all these five proteins. An antibody cocktail-based approach involving antibodies against all these five up-regulated entire proteins or their epitopes could be developed for early detection of pregnancy in buffaloes. © 2016 Japanese Society of Animal Science., (© 2016 Japanese Society of Animal Science.)

Using a milk sample for pregnancy diagnosis in dairy cattle is extremely convenient due to the low technical inputs required for collection of biological materials. Determining accuracy of a novel pregnancy diagnostic test that relies on a milk sample is, however, difficult since no gold standard test is available for comparison. The objective of the current study was to estimate diagnostic accuracy of the milk PAG-based ELISA and of transrectal ultrasonographic (TUS) exam for determining pregnancy status of individual dairy cows using a methodology suited for test validation in the absence of gold standard. Secondary objectives were to evaluate whether test accuracy varies with cow's characteristics and to identify the optimal ELISA optical density threshold for PAG test interpretation. Cows (n=519) from 18 commercial dairies tested with both TUS and PAG between 28 and 45days following breeding were included in the study. Other covariates (number of days since breeding, parity, and daily milk production) hypothesized to affect TUS or PAG test accuracy were measured. A Bayesian hierarchical latent class model (LCM) methodology assuming conditional independence between tests was used to obtain estimates of tests' sensitivities (Se) and specificities (Sp), to evaluate impact of covariates on these, and to compute misclassification costs across a range of ELISA thresholds. Very little disagreement was observed between tests with only 23 cows yielding discordant results. Using the LCM model with non-informative priors for tests accuracy parameters, median (95% credibility intervals [CI]) TUS Se and Sp estimates of 0.96 (0.91, 1.00) and 0.99 (0.97, 1.0) were obtained. For the PAG test, median (95% CI) Se of 0.99 (0.98, 1.00) and Sp of 0.95 (0.89, 1.0) were observed. The impact of adjusting for conditional dependence between tests was negligible. Test accuracy of the PAG test varied slightly by parity number. When assuming false negative to false positive costs ratio≥3:1, the optimal ELISA optical density threshold allowing minimization of misclassification costs was 0.25. In conclusion, both TUS and PAG showed excellent accuracy for pregnancy diagnosis in dairy cows. When using the PAG test, a threshold of 0.25 could be used for test interpretation., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Poor reproductive performance remains a major issue in the dairy industry, with low conception rates having a significant impact on milk production through extended calving intervals. A major limiting factor is the lack of reliable methods for early pregnancy diagnosis. Identification of animals within a herd that fail to conceive within 3 weeks after insemination would allow early re-insemination and shorten calving intervals. In a previous study, we found an increase in plasma miR-26a levels in Day 16-pregnant relative to non-pregnant heifers, however changes in miRNA levels that early during pregnancy were very small which likely prevented the identification of robust biomarkers. In this study, we extended our analyses to a wider interval during pregnancy (Days 8 to 60, n = 11 heifers) with the rationale that this may facilitate the identification of additional early pregnancy miRNA biomarkers. Using small RNA sequencing we identified a total of 77 miRNAs that were differentially expressed on Day 60 relative to Day 0 of pregnancy. We selected 14 miRNAs for validation by RT-qPCR and confirmed significant differences in the expression of let-7f, let-7c, miR-30c, miR-101, miR-26a, miR-205 and miR-143 between Days 0 and 60. RT-qPCR profiling throughout Days 0, 8, 16 and 60 of pregnancy showed a distinct increase in circulating levels of miR-26a (3.1-fold, P = 0.046) as early as Day 8 of pregnancy. In summary, in contrast to earlier stages of pregnancy (≤ Day 24), marked differences in the levels of multiple miRNAs can be detected in circulation by Day 60 in cattle. Retrospective analyses showed miR-26a levels to be increased in circulation as early as Day 8, sooner than previously reported in any species, suggesting a biological role for this miRNA in the very early events of pregnancy.

The objectives of the present investigation were to evaluate the pregnancy diagnosis by detection of either the allantochorion membrane (FMS) or amniotic sac (ASP) by per rectum palpation (PRP) during late embryonic or early fetal period on pregnancy loss (PRL) at reexamination, calving rates, and abnormalities in newborn calves. A controlled randomized blind design with 800 lactating dairy pregnant cows diagnosed by transrectal ultrasonography (TRUS) between Days 35 and 57 of gestation from one dairy farm were included. The cows were randomly divided according to detection of allantochorion membrane (FMS group; n = 264), detection of amniotic sac (ASP group; n = 266), and TRUS (control [CON] group; n = 270). TRUS was considered as the criterion standard method of comparison. The entire PRP was performed by one experienced veterinarian. Then, all the cows were reexamined only by TRUS between 2 and 4 weeks later by two independent veterinarians to assess PRL. The calving rate one (number of cows calved divided by the number of cows initially pregnant) and calving rate two (number of cows calved divided by the number of cows pregnant at reexamination) for each group was calculated. All abortions and stillborns were necropsied, and calves alive were followed for 5 days. The overall initial PRL (between initial pregnant cows and reexamination) for FMS, ASP, and CON groups was 7.4% (19/258), 8.8% (23/262), and 9.2% (24/260), respectively (P = 0.75). The overall late PRL (between reexamination and calving) for FMS, ASP, and CON groups was 4.2% (9/213), 5.7% (12/209), and 4.2% (9/216), respectively (P = 0.71). The calving rate one for FMS, ASP, and TRUS groups was 79.1% (204/258), 75.2% (197/262), and 79.6% (207/260), respectively (P = 0.63). The calving rate two for the same groups was 85.4% (204/239), 82.4% (197/239), and 87.7% (207/236), respectively (P = 0.27). The number of fetuses aborted late, premature, and mature dead from FMS, ASP, and CON groups was 6, 4, and 5, respectively (P = 0.85), and no abnormalities at necropsy were detected. One stillborn male calf with atresia coli after 281 days of gestation from a cow examined by ASP at Day 51 was diagnosed. It was concluded that the use of either FMS or ASP for pregnancy diagnosis during late embryonic or early fetal period did not increase the PRL, affect calving rates, or produce calves with congenital abnormalities., (Copyright © 2016 Elsevier Inc. All rights reserved.)

This study investigates for the first time mRNA pregnancy-associated glycoprotein 2 (PAG-2) expression in blood cells during early pregnancy in water buffalo. The PAGs constitute a large family of glycoproteins expressed in the outer epithelial layer of the placenta in eutherian species. All PAGs are not concomitantly expressed throughout pregnancy; some of them are expressed in the earlier phases, whereas others appear later and are expressed over a shorter period. Twenty-one lactating buffaloes were analyzed-17 females were synchronized with PRID and artificially inseminated (AI), whereas four females were synchronized but not inseminated (control group). Blood was collected at Days 0, 18, 28, 40, and 75 from AI (AI = Day 0). Expression of PAG-2 mRNA in blood samples was measured with real-time polymerase chain reaction. Pregnancy diagnosis was performed on Day 28 (D28) and Day 40 (D40) after AI by ultrasonography (US) and by PAG-1 RIA method. The females diagnosed pregnant at D28 and confirmed pregnant at D40 were defined as D28(+)D40(+) group; the females diagnosed pregnant at D28 but not confirmed pregnant at D40 were defined as D28(+)D40(-) group; and the females that were diagnosed as nonpregnant on either days were defined as D28(-)D40(-) group. PAG-2 mRNA at Day 0 was not observed in any groups. The D28(+)D40(+) group showed the highest expression, starting on Day 18 and increasing progressively up to Day 75. PAG-2 mRNA was also expressed on Day 18 in both D28(+)D40(-) and D28(-)D40(-) groups, but their levels were lower than those of D28(+)D40(+) group and almost constant over time. PAG-2 mRNA was never detected in the control group. The significant difference in the expression of PAG-2 mRNA between the D28(+)D40(+) group and the D28(-)D40(-) group, starting from Day 18, suggests that these animals might have conceived, but have experienced early embryonic loss; therefore, the PAG-2 mRNA was still present in blood circulation although at lower levels, as found in the D28(+)D40(-) group. In conclusion, this study shows that PAG-2 mRNA can be detected in peripheral maternal blood cells earlier than circulating PAG-1 molecules and could be useful for studies on early pregnancy and embryonic mortality., (Copyright © 2016 Elsevier Inc. All rights reserved.)

We investigated the diagnostic reliability of pregnancy detection using changes in interferon stimulated gene (ISG) messenger RNA (mRNA) levels in circulating immune cells in ewes. Two different groups of ewes (an experimental group, experiment 1 and a farm group, experiment 2) were oestrus-synchronized and blood sampled on day 14 (D0=day of insemination in control animals, experiment 1) and day 15 (experiment 2). Real-time PCR were performed to evaluate the abundance of different ISG mRNAs. In the experimental group, peripheral blood mononuclear cells of 29 ewes born and bred in experimental facilities were isolated using a Percoll gradient method. Gene expression for Chemokine (C-X-C motif) ligand 10 (CXCL10), Myxovirus (influenza virus) resistance 1 (MX1) and Signal transducer and activator of transcription 1 (STAT1) mRNA were, respectively, 8.3-fold, 6.1-fold and 2.7-fold higher (P0.10) in CXCL10, STAT1, MX1, Myxovirus (influenza virus) resistance 2 (MX2) and ISG15 ubiquitin-like modifier (ISG15) mRNA expression were found between pregnant and non-pregnant ewes. The ROC curves and the hierarchical classification generated from the real-time PCR data failed to discriminate between pregnant and non-pregnant animals. In this group of animals, our results show a strong variability in ISG expression patterns: 17% of animals identified as non-pregnant by the five tests were in fact pregnant, only 52% of pregnant animals had at least two positive results (two genes above threshold), whereas up to five positive results (five genes above threshold) were needed to avoid misclassification. In conclusion, this study illustrates the high variability in ISG expression levels in immune circulating cells during early pregnancy and, therefore, highlights the limits of using ISG expression levels in blood samples, collected on PAXgene® tubes on farms, for early pregnancy detection in sheep.

This study aimed to assess the suitability of luteal blood flow analyses measured by color Doppler ultrasonography (CDUS), to predict pregnancy at pre- and post-embryo transfer (ET) in dairy cows, and to compare with the established criterion like luteal size and plasma progesterone (P4) concentrations. Lactating Holstein cows (n = 65) with spontaneous (n = 34) or synchronized estrus (n = 31) were examined. Cows with a CL greater than or equal to 20 mm in diameter (n = 58) received embryo transfer on Day 7 (Day 0 = estrus). Brightness mode images were captured for calculation of the CL area, luteal cavity area, and dominant follicle area on Days 3, 5, 7, and 14. Color Doppler ultrasonography examinations were conducted to determine the blood flow area (BFA) within the CL at the maximum diameter and the time-averaged maximum velocity (TAMV) of the base of the spiral artery on the same days. Plasma P4 concentrations were determined from blood samples collected at each ultrasound examination. Pregnancy was diagnosed by an ultrasound on Day 30. There was no significant difference in the proportion of cows received embryo (91.2% vs. 87.1%, P = 0.70) and pregnancy rate (58.1% vs. 59.3%, P = 1.00) between the spontaneous estrus and synchronized groups. The BFA values of the pregnant group (n = 34) were approximately 1.42 and 1.54 times higher than those of the nonpregnant group (n = 24) on Days 7 (0.54 ± 0.04 cm(2) vs. 0.38 ± 0.02 cm(2); P < 0.01) and 14 (0.80 ± 0.23 cm(2) vs. 0.52 ± 0.22 cm(2); P < 0.01), respectively. The TAMV of the pregnant group was approximately 1.45 times higher than that of the nonpregnant group on Day 14 (57.8 ± 3.5 cm/s vs. 40.0 ± 3.3 cm/s; P < 0.01). However, no differences were found in the CL area, CL tissue area, dominant follicle area, and plasma P4 concentrations among these groups. In addition, the best logistic regression model to predict pregnancy included scores for BFA on Day 7, BFA and TAMV on Day 14. Setting the cutoff value of BFA at 0.43 cm(2) yielded the highest sensitivity (79.4%) and specificity (75.0%) on Day 7, indicating the effectiveness of using BFA data for predicting pregnancy on Day 7. Furthermore, setting the cutoff value at one obtained from a sample with BFA 0.63 cm(2) and TAMV 50.60 cm/s yielded the highest sensitivity (85.3%) and specificity (91.7%) on Day 14. In conclusion, the evaluations of BFA on Day 7, and paired BFA and TAMV on Day 14 represent reliable predictors of pregnancy in the cow., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Chemical pregnancy testing is an alternative to traditional methods of pregnancy diagnosis (either manual palpation or ultrasound) in postpartum dairy cows and heifers. The objective was to validate a chemical pregnancy test that confers the advantages of using whole blood, rapid incubation times, and visual readout. Blood and milk samples were collected from Holstein dairy cows [n=320; 162±62 (mean ± SD) d in milk] on a confinement farm in northeast Missouri at 28 d after artificial insemination (AI). The samples were assayed for pregnancy-associated glycoproteins (PAG) by using a rapid visual test as well as traditional plasma- and milk-based tests. Transrectal ultrasonography diagnosis for pregnancy at 35 to 38 d after AI was the reference (gold) standard for all PAG tests. One hundred fifty-nine cows were diagnosed as pregnant by the reference standard (pregnancies per AI=49.7%). The tests were ELISA and either optical density (OD; measured with a microtiter plate reader; plasma, milk, and rapid visual tests) or visual readout (rapid visual test) were used to diagnose pregnancy. When OD was used, the percentage of pregnant cows classified correctly (sensitivity) for the plasma, milk, and rapid visual tests were 97±1, 96±2, and 95±1% (±SE), respectively. The sensitivity of the rapid visual test when assessed visually was 98±1%. The specificity (proportion of nonpregnant cows classified correctly) for the plasma, milk, and rapid visual was 94±2%, 94±2%, and 93±2% when an OD was used. When read visually, the specificity of the rapid visual test was lesser (85±3%) because some cows with faint visual signals yielded false positive diagnosis. The overall accuracy (proportion of pregnant and nonpregnant cows diagnosed correctly) was similar for all tests (plasma, milk, rapid visual OD, and rapid visual; 96±1, 95±1, 94±1, and 92±2%, respectively). In a second experiment, lactating Holstein cows (n=291) from 4 commercial confinement dairy farms in western Kentucky were tested 25 to 95 d after AI using the rapid visual test. The OD of the rapid visual test followed the known profile for PAG in circulation during the first trimester of pregnancy. The conclusion is that the rapid visual test has equal sensitivity and accuracy as existing PAG tests. A slightly lower specificity was found when the rapid visual test was read visually., (Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)