Evaluation of pregnancy associated glycoproteins assays for on farm determination of pregnancy status in beef cattle.

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.

Accuracy of early pregnancy diagnosis and determining pregnancy loss using different biomarkers and machine learning applications in dairy cattle.

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.

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 0.6 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.

Microwave Dielectric Response of Bovine Milk as Pregnancy Detection Tool in Dairy Cows.

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 Δε 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.

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.

Comparative evaluation of two commercial pregnancy-associated glycoproteins tests for early detection of pregnancy in dairy cattle.

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.

Algorithms predicting gestational stage from the maternal steroid metabolome of mares.

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, 3ß,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.

Thermal features, ambient temperature and hair coat lengths: Limitations of infrared imaging in pregnant primitive breed mares within a year.

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.

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

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

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

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

Development of the Timed Re-Insemination (TRI-synch) program re-inseminating 24 days after the initial service in dairy cows.

For the timed re-insemination at the minimal interbreeding interval, cows were treated with a progesterone (P4 )-releasing intravaginal device from Days 13-15 to 21 post-insemination (Day 0 = estrus), followed by plasma P4 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 P4 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 P4 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).

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

The objective of this study was to determine the efficacy of the Fassisi®BoviPreg visual test kit (Fassisi®BoviPreg) in determining early pregnancies in cows by measuring the pregnancy-associated glycoprotein (PAG) in whole blood and blood serum. The study was conducted on 50 cows, the artificial insemination (AI) dates of which were designated as day 0. Pregnancy diagnosis was performed with transrectal ultrasonography (USG), and serum samples were simultaneously collected and used with Fassisi®BoviPreg to determine pregnancies on 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.

Applied use of interferon-tau stimulated genes expression in polymorphonuclear cells to detect pregnancy compared to other early predictors in beef cattle.

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.

Effects of transrectal palpation with the fetal membrane slip technique for early pregnancy diagnosis on the proportion and type of associated pregnancy loss in dairy cattle.

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.

Use of a prediction method for early pregnancy status utilizing receiver operating characteristic curve analysis of peripheral blood leukocyte interferon-stimulated genes in Japanese-Black cattle.

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.

Diagnosing the pregnancy status of dairy cows: How useful is milk mid-infrared spectroscopy?

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.

Evaluation of a cattle rapid test for early pregnancy diagnosis in sheep.

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.

Using pregnancy associated glycoproteins (PAG) for pregnancy detection at day 24 of gestation in beef cattle.

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 ±â€¯0.10 ng/mL vs 0.30 ng/mL ±â€¯0.07 ng/mL; mean ±â€¯SEM; respectively). Pregnant heifers had increased PAG concentration at day 24 compared with pregnant cows (P < 0.01; 3.29 ±â€¯0.36 ng/mL vs 1.39 ±â€¯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 ±â€¯0.73) compared with heifers that maintained an embryo until day 100 (3.69 ng/mL ±â€¯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 ±â€¯0.25 vs 0.92 ng/ml ±â€¯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.

Accuracy of bovine pregnancy-associated glycoproteins (bPAGs) in the diagnosis of pregnancy: A comparative study of three pregnancy diagnostic methods.

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.

Comparative diagnosis of pregnancy wastage in cows at slaughter using pregnancy specific protein-B and post slaughter inspection diagnostic procedures.

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.