Pregnancy Detection In Cattle Research Articles

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

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

The potential of seed germination inhibition test for early pregnancy detection and improved reproductive efficiency of cattle in Zambia

Early pregnancy diagnosis is an important management practice for reducing calving interval, increasing cattle reproductive efficiency, and the overall herd productivity. This study was undertaken to assess the viability of seed germination inhibition technique (Punyakoti test) for early pregnancy detection in cattle under the tropical rearing conditions. Twenty-four randomly selected cows were used for the experiment. Urine samples were collected and subjected to Punyakoti test, using maize seeds, within 6 hours of collection. Descriptive statistics employing means and standard error were used to analyse data, also, inferential statistics including analysis of variance and t-tests were employed to ascertain differences between the variables under study. Urine from pregnant cows had the highest (80.03±3.99) inhibitory effect while the distilled water group had the least (7.50±3.81) mean seed germination inhibition. The means of germinated maize seeds in pregnant and non-pregnant cow urine treatment groups were significantly different. The means of germinated maize seeds in unstripped and stripped pregnant cow urine were significantly different, while the mean shoot length values for the same treatments were not significantly different. Urine from 42 days’ pregnant cows had the highest germination inhibition (80.21±3.59) while the least (25.00±4.35) was observed on day 10. The Punyakoti test reliably detected pregnancy starting from 26 days after insemination. In conclusion, this technique can be used for detecting pregnancy as early 26 days after insemination. The potential role of steroids and involvement of abscisic acid (ABA) in seed germination inhibition requires further investigation.

The Role of Transrectal Sonography in Pregnancy Diagnosis in Cattle

Objective: A reproduction management strategy is an essential component of any cattle production operation. Options include palpation per rectum, transrectal sonography, or biochemical blood tests. To determine the best method, veterinary costs must be weighed against operational costs of maintaining open cattle. Materials and Methods: An online literature search using PubMed, Medline, CINAHL, EBSCO, and Google Scholar was completed to find relevant articles regarding pregnancy detection in cattle, costs of reproductive management in cattle, and pregnancy loss in cattle. Priority was given to original research articles pertaining to palpation per rectum, transrectal ultrasound, fetal gender, and pregnancy loss. Results: Transrectal sonography can detect a pregnancy up to 15 days earlier than palpation per rectum and has sensitivities and specificities as high as 97% when performed between days 21 and 35 post artificial insemination. Conclusion: Cattle producers can be confident that transrectal sonography is a useful tool in the beef and dairy cattle industry. Its use facilitates reproductive management decisions, such as re-breeding or culling, thus reducing expenses, and increasing profitability.

Diagnostic Evaluation of Immunoassay Kits for Early Pregnancy Detection in Cattle

Diagnostic Evaluation of Immunoassay Kits for Early Pregnancy Detection in Cattle

37 Early pregnancy detection in cattle by analysis of maternal circulating microRNA expression

Abstract Pregnancy detection in cattle is performed by rectal palpation, transrectal ultrasonography or detection of pregnancy associated glycoproteins at approximately d 28 of gestation. Majority of embryonic loss in cattle occurs much earlier. MicroRNAs (miRNAs) are excellent markers for physiological states including pregnancy. The objective of this experiment was to assess expression of miRNAs in maternal serum to serve as markers for pregnancy detection at d 18 post-artificial insemination. Cows of varying age and parity (n = 80) were synchronized using a 7-d Co Synch and CIDR estrous synchronization protocol. One insemination by a single sire was performed 60 to 66 h following CIDR removal. Blood was collected via jugular venipuncture on d 0 and 18 post-insemination. Total RNA isolation was performed using PAXgene Blood RNA kit (PreAnalytiX, Hombrechtikon, Switzerland) or by miRNeasy (Qiagen, Hilden, Germany). Ultrasound was performed d 30 of gestation. Taqman microRNA assay utilizing primers for Let7a, mir-16,15b, 25, and 26 was performed on RNA from whole blood and serum in pregnant (n = 10) and open (n = 10) cows. Analysis of variance and pair-wise student’s t-test among LSMeans for relative abundance were performed with fixed effects for pregnancy, day, isolation, and their interactions. MicroRNA abundance did not differ due to pregnancy. Relative abundance for miR-16 increased in d 18 (1.86 ± 0.49) compared to d 0 samples (3.42 ± 0.48), regardless of pregnancy status or isolation method (P = 0.03). Let-7a (serum, 0.99 ± 0.98; PAXgene, 6.25 ± 0.87), miR-15b (serum, 0.59 ± 1.67; PAXgene, 12.44 ± 1.48) and miR-26 (serum, 1.44 ± 1.2; PAXgene, 7.64 ± 1.06) relative abundance was higher in serum compared to PAXgene isolated samples (P < 0.05). These results disagree with previous reports and indicate that these candidate miRNAs are not a reliable indicator for early pregnancy detection in cattle.

136 Quantitative and qualitative analysis of interferon stimulated genesMX2, OAS1, and ISG15 for detection of pregnancy in cattle

Abstract Pregnancy in cattle can be determined using transrectal ultrasonography or by detection of pregnancy associated glycoproteins present in maternal circulation begging at approximately d 28 of gestation; however, the majority of pregnancy loss occurs prior to this date. Interferon stimulated genes (ISG) have been observed to be upregulated in pregnant cattle and measurable at d 18 of gestation. The objective of this study was to assess ISG expression as an indicator of pregnancy at d 18 post-insemination. A 7-d Co Synch and CIDR estrous synchronization protocol was utilized on cows of varying age and parity (n = 80) and insemination to a single sire performed 60 to 66 h following removal of the CIDR. On d 0 and d 18 post-insemination, blood was collected via jugular venipuncture for isolation of total RNA by PAXgene Blood RNA kit (PreAnalytiX, Hombrechtikon, Switzerland). Pregnancy detection was performed on d 30 of gestation by transrectal ultrasonography. Differences in ISG expression between d 0 and d 18 samples in both pregnant (n = 10) and open (n = 10) cows, according to d 30 ultrasound, were assessed. Primers for ISGs (MX2, OAS1, and ISG15) and housekeeping, cyclophilin were generated for use in end-point PCR and qRT-PCR. Data for qRT-PCR were normalized to cyclophilin expression. Analysis of variance and pair-wise student’s t-test among LSMeans were performed with fixed effects for pregnancy status, day, and their interactions. End-point products in all ISGs, regardless of day or pregnancy status, were observed. Expression of MX2 and ISG15 were upregulated in open cows and in d 0 samples (P < 0.05). Expression of OAS1 was increased in open cows (P = 0.001). These data disagree with previous findings in which ISGs were upregulated in pregnant animals, and indicate expression of these ISGs may not be good indicators for pregnancy.

Differential neutrophil gene expression in blood and milk during pre-implantation pregnancy in Karan Fries cattle

ABSTRACT The blood leucocytes, neutrophils being the first line of defense, detect the implanting embryo, which is semi-allogenic, and show changes in their gene expression. It is possible to diagnose cattle as non-pregnant around 21–24 days post insemination by progesterone (P4) assay. Concentration of progesterone level in the inseminated animals may not be an accurate method for early pregnancy diagnosis. Identification of suitable methods that allow pregnancy diagnosis around or before day 20 after artificial insemination (AI) will be of considerable value. The present study has been carried out to understand the differential gene expression of IFI16 (interferon-inducible protein 16), MX1 (Myxovirus resistance 1), OAS1 (2'-5'-Oligoadenylate Synthetase 1) and TNFα (tumor necrosis factor alpha) genes on 0, 4, 8, 12, 15, 18 and 21 days post AI, respectively, from Karan Fries cattle as a tool for early pregnancy diagnosis in neutrophil cells isolated from blood and milk. Statistical analysis between pregnant and non-pregnant animals in both blood and milk samples revealed that IFI16 gene was upregulated in the milk samples of pregnant animals on 8th day post-AI and found to be significantly different (P < 0.05) between pregnant and non-pregnant cattle and may be used as a biomarker for early pregnancy detection in cattle.

MicroRNA of whole milk samples are not suitable for pregnancy detection in cattle

Common pregnancy detection methods in dairy cattle have limits in sensitivity and timing. Thus, we aimed at finding new early pregnancy biomarkers in milk. A previous study in milk cells and skim milk suggested the possible use of several miRNAs for this purpose. In order to make the sampling procedure more convenient for daily use and to include the milk fat into analysis, here, a similar study was repeated using whole fresh milk of days 4, 12, 18 and 21 of pregnancy and non-pregnant day-matched controls, respectively. The microRNAs (miRNAs) were extracted from milk samples and sequenced on a high throughput sequencer (RNAseq). Promising miRNAs were validated via RT-qPCR. Several miRNAs were significantly regulated and six miRNAs (bta-miR-221, bta-miR-223, bta-miR-93, bta-miR-200c, bta-miR-125b and the closely related bta-miR-15b) showed a continuous trend, similar as in the milk cells of the previous study. However, pregnant and cyclic animals could not be separated completely and RT-qPCR validation of the RNAseq data was unsuccessful. Thus, miRNA of bovine whole fresh milk samples failed to show suitability for early pregnancy diagnosis.

DISEMINASI TEKNOLOGI DETEKSI KEBUNTINGAN DINI “DEEA GESTDECT” TERHADAP SAPI POTONG DI KINALI KABUPATEN PASAMAN BARAT

Daerah Pasaman Barat merupakan daerah yang memiliki potensi lahan perkebunan maupun pertanian yang cukup luas. Kinali merupakan salah satu nagari di Pasaman Barat dengan luas wilayah 489.64 km₂ merupakan salah satu nagari di Kabupaten ini yang memiliki daya dukung lahan yang luas dan berpotensi dalam pengembangan usaha peternakan sapi. Kegiatan pengabdian kepada masyarakat ini dilakukan dengan tujuan untuk memberikan informasi pada peternak dan masyarakat tentang optimalisasi penggunaan “DEEA GestDect” sebagai deteksi kebuntingan dini pada sapi rakyat dan meningkatkan pengetahuan serta keterampilan peternak dalam penangan reproduksi/ kebuntingan ternak di daerah Kinali Pasaman Barat. Metoda yang digunakan dalam kegiatan ini adalah (1) Penyuluhan (ceramah), dan (2) Demontrasi pelaksanaan Inseminasi Buatan (IB) dan desiminasi teknologi penggunaan “DEEA GestDect” sebagai alat deteksi kebuntingan dini pada sapi rakyat. Dari hasil kegiatan pengabdian yang telah dilakukan maka dapat diambil beberapa kesimpulan sebagai berikut: Pengetahuan dan keterampilan peternak dan masyarakat di daerah Kinali Pasaman Barat sebelum dilaksanakan kegiatan pengabdian ini belum mengenal deteksi kebuntingan dini pada sapi. Namun demikian motivasi atau keinginan peternak dan masyarakat untuk melakukan usaha budidaya ternak sapi sangat besar. Hal ini dapat dilihat dari keinginan peternak dan masyarakat untuk menguasai teknologi teknologi deteksi kebuntingan dini “DEEA GestDect” terhadap sapi rakyat sangat tinggi. Lebih jauh dari kegiatan pengabdian pada masyarakat ini dapat menambah pengetahuan dan keterampilan masyarakat dalam penangan reproduksi/ kebuntingan dini ternak di daerah Kinali Pasaman Barat.

Molecular Cloning and Characterization of Pregnancy Specific Protein Uteroglobin can Lead to Pregnancy Detection in Cattle- A Review

Molecular Cloning and Characterization of Pregnancy Specific Protein Uteroglobin can Lead to Pregnancy Detection in Cattle- A Review

Can milk cell or skim milk miRNAs be used as biomarkers for early pregnancy detection in cattle?

The most critical phase of pregnancy is the first three weeks following insemination. During this period about 50% of high yielding lactating cows suffer embryonic loss prior to implantation, which poses a high economic burden on dairy farmers. Early diagnosis of pregnancy in cattle is therefore essential for monitoring breeding outcome and efficient production intervals. Regulated microRNAs (miRNAs) that reach easily accessible body fluids via a ‘liquid biopsy’ could be a new class of pregnancy predicting biomarkers. As milk is obtained regularly twice daily and non-invasively from the animal, it represents an ideal sample material. Our aim was to establish a pregnancy test system based on the discovery of small RNA biomarkers derived from the bovine milk cellular fraction and skim milk of cows. Milk samples were taken on days 4, 12 and 18 of cyclic cows and after artificial insemination, respectively, of the same animals (n = 6). miRNAs were analysed using small RNA sequencing (small RNA Seq). The miRNA profiles of milk cells and skim milk displayed similar profiles despite the presence of immune cell related miRNAs in milk cells. Trends in regulation of miRNAs between the oestrous cycle and pregnancy were found in miR-cluster 25~106b and its paralog cluster 17~92, miR-125 family, miR-200 family, miR-29 family, miR-15a, miR-21, miR-26b, miR-100, miR-140, 193a-5p, miR-221, miR-223, miR-320a, miR-652, miR-2898 and let-7i. A separation of cyclic and pregnant animals was achieved in a principal component analysis. Bta-miRs-29b, -221, -125b and -200b were successfully technically validated using quantitative real-time PCR, however biological validation failed. Therefore we cannot recommend the diagnostic use of these miRNAs in milk as biomarkers for detection of bovine pregnancy for now.

Technical note: A rapid enzyme-linked immunosorbent assay blood test for pregnancy in dairy and beef cattle

The ruminant trophoblast produces pregnancy-associated glycoproteins (PAG) that can be detected in the blood of pregnant animals. The objective was to determine the accuracy of a rapid ELISA PAG-based test for the purpose of pregnancy detection in cattle. Blood was sampled from dairy cattle (539 Holstein cows, 173 Holstein heifers, 73 Guernsey cows, 22 Guernsey heifers, and 12 Jersey heifers) and crossbred beef cattle (145 cows and 46 heifers) that were ≥25 d after insemination (range = 25 to 45 d for dairy and 29 to 56 d for beef). Cattle were examined by ultrasonography for detection of pregnancy within 2 d of blood collection. Whole blood or plasma was incubated in a polystyrene tube coated with a monoclonal PAG antibody for 15min. The tubes were then washed and subjected to sequential incubations with a biotinylated polyclonal PAG antibody (15min, followed by wash), a horseradish peroxidase-streptavidin solution (15min, followed by wash), and a peroxidase substrate. Tubes were visually assessed for color after 15min (clear solution = PAG negative, not pregnant; blue solution = PAG positive, pregnant). Total assay time was approximately 90min. The ultrasound examination was used as the standard for pregnancy diagnosis. The sensitivity (99.8±0.2%), specificity (91.7±1.4%), and negative predictive value (99.7±0.3%) for the PAG test used in dairy cattle were similar for different breeds and for cows and heifers. The positive predictive value for the test was greater in dairy heifers than in dairy cows (96.5±1.4% vs. 90.5±1.7%, respectively). In beef cattle, the sensitivity (100%), specificity (92.3±3.0%), positive predictive value (95.0±2.0%), and negative predictive value (100%) for the PAG test were similar for cows and heifers. The accuracy of the test was not different for dairy and beef cattle. In conclusion, the rapid ELISA pregnancy test based on PAG was highly sensitive and specific for pregnancy detection in dairy and beef cattle.

The establishment of an ELISA for the detection of pregnancy-associated glycoproteins (PAGs) in the serum of pregnant cows and heifers

The pregnancy-associated glycoproteins (PAGs) are a large gene family expressed in trophoblast cells of ruminant ungulates. The detection of PAGs (more specifically, PAG-1) in maternal serum has served as the basis for pregnancy detection in cattle. Unfortunately, PAG-1 and/or antigenically-related PAGs exhibit a long half-life in maternal serum (>8 d) and can be detected 80–100 d post-partum, thereby producing false positives in animals bred within 60-d of calving. The goal of the present studies was to develop a monoclonal-based assay that targeted early-pregnancy PAGs whose persistence in maternal serum post-partum might be relatively short-lived. Three anti-PAG monoclonal antibodies that recognized distinct subsets of PAGs were selected and used as trapping reagents in a ‘sandwich’ type of enzyme-linked immunosorbant assay (ELISA). A polyclonal antiserum with broad specificity was used for detecting bound PAGs. A total of 42 cows and heifers were bled daily on day 15, days 22 to 28, and then weekly throughout pregnancy and for 10 weeks (∼70 d) into the post-partum period. The ELISA was able to detect PAG in maternal serum of all animals unambiguously by day 28 post-insemination (PAG concentration: 8.75 ± 3.04 ng/mL). In maternal serum, PAG concentrations peaked during the week of parturition at 588.9 ± 249.9 ng/mL, and after calving, PAG was completely cleared (half-life: 4.3 d) by eight-week post-partum in 38 of 40 of the animals tested and was at very low concentrations in the remaining two (1.4 and 4.9 ng/mL, respectively). In summary, a monoclonal-based assay has been established that is sensitive enough to detect PAG in maternal serum by the forth week of pregnancy, but does not suffer from carry-over of antigen from a previous pregnancy.

Factors influencing the accuracy of early pregnancy detection in cattle by real-time ultrasound scanning of the uterus

Transrectal real-time ultrasound scanning (5MHz linear array probe) examinations of the uterus were conducted to determine the accuracy of detecting pregnancy and nonpregnancy in 200 Holstein cows and heifers at 16 to 31 d following artificial insemination under field conditions. Two operators separately conducted the examinations. Overall diagnostic accuracy was 62.5%, with a sensitivity of 55.6%, specificity of 70.2%, and a positive predictive value of 67.8%. Diagnostic accuracy and sensitivity were lower (P<0.05) for examinations performed early (16 to 22 d) than those performed at a later stage (23 to 31 d) after insemination. Diagnostic accuracy and sensitivity of the ultrasound examinations between Days 16 and 22 were 50.0 and 25.0%, respectively, and 70.2 and 68.8%, respectively, between Days 23 and 31. Between operators, no difference was noted in diagnostic accuracy, sensitivity, specificity, or predictive value. However, the sensitivity of one operator decreased slightly when his results were evaluated over three time periods. Results for the heifers examined were not different from those for the cows (P>0.05) Determination of pregnancy and nonpregnancy, using ultrasound scanning examinations between Days 16 and 31 days postinsemination proved to be significantly less accurate than using palpation per rectum after Day 35 postinsemination. Palpation proved to be 99% accurate in diagnosing pregnancy when compared with breeding and calving records.

4895804 Bovine antigen glycoprotein, related antibody, and use in detection of pregnancy in cattle: Eileen F Bostwick, Alan Hunter assigned to University of Minnesota

4895804 Bovine antigen glycoprotein, related antibody, and use in detection of pregnancy in cattle: Eileen F Bostwick, Alan Hunter assigned to University of Minnesota

4895804 Bovine antigen glycoprotein, related antibody, and use in detection of pregnancy in cattle: Eileen F Bostwick, Alan Hunter assigned to University of Minnesota

4895804 Bovine antigen glycoprotein, related antibody, and use in detection of pregnancy in cattle: Eileen F Bostwick, Alan Hunter assigned to University of Minnesota

Lactate dehydrogenase isoenzyme patterns as a method of pregnancy detection in cattle

The objective of this study was to evaluate serum lactate dehydrogenase isoenzyme patterns in open and pregnant Holstein and Hereford cows as a method of detecting pregnancy. Serum samples were collected from 26 Holstein and 13 Hereford cows and lactate dehydrogenase isoenzyme patterns were examined by electrophoresis and quantitated by scanning densitometry. Lactate dehydrogenase isoenzyme 4 and LDH 5 were found in higher concentration (P ≤ .05) in pregnant versus open Holstein cows. Twelve of 14 and 10 of 12 Holstein cows were correctly diagnosed as being pregnant and open, respectively by examining differences in serum concentrations of isoenzymes LDH 4 and LDH 5. Pregnancy was detected as early as 36 days of gestation. No differences were found in serum LDH isoenzyme patterns between pregnant and open Hereford cows. However, differences (P ≤ .05) in concentration of all five LDH isoenzymes were observed between Holstein and Hereford cows, which may lend insight into the metabolic significance of the elevation of LDH 4 and LDH 5 in pregnancy. The results suggest that elevation of serum LDH 4 and LDH 5 may be used as a means of pregnancy detection in the Holstein but not the Hereford cow.