Pregnancy Diagnosis In Cows Research Articles

The thermographic monitoring in early pregnancy detection in Holstein cows and heifers

This study aimed to conduct a comprehensive investigation to demonstrate early pregnancy detection using thermography in heifers and cows. A total of 60 heifers (n: 30) and cows (n: 30) were divided into two groups as pregnant (n: 15 heifers, n: 15 cows) and non-pregnant (n: 15 heifers, n: 15 cows) according to the day 28 of gestation. Thermographic images were taken from the vulvar and anal regions on alternate days from D0 to D20. Blood samples were collected to determine estrogen and progesterone concentrations. The mean temperature difference between the anal and vulvar regions (ΔT °C) was used in the statistical analyses. Based on the hormonal profiles, no abnormalities were observed for follicular waves or luteal profiles in heifers and cows. The ΔT °C values between heifers and cows and between days were statistically significant (P < 0.001). In thermographic analyses, the differences observed in other main effects and interactions of the group, sampling time, and pregnancy were not statistically significant (P > 0.05). In receiver operating characteristic (ROC) analyses, it was concluded that the ΔT °C value of ≤ 2.9 °C (100% Se - 61.9% Sp) was highly correlated with pregnancy diagnosis in cows on day six after artificial insemination (AI) (P < 0.001). In conclusion, it was determined that the clinical application of thermography can be used for the detection of pregnancy on day six after AI in cows. However, further studies are needed to determine heifers' thermographic characteristics and profiles.

Retrospective Study of Cattle Fetal Wastage at Hadeja Abattoir, Jigawa ‎State: Economic Implication and Seasonal Variation

Fetal wastage usually occurs due to indiscriminate slaughter of pregnant cows without proper ante mortem assessment of the pregnancy status prior to slaughter in most of the abattoir in Jigawa state. Therefore, the current retrospective study was aimed to determine the extent of cattle fetal wastage and its economic implications for a period of 6 years (2016-2021) in Hadeja abattoir Jigawa state. The total number of cows slaughtered in Hadeja abattoir and the fetal wastage were evaluated. Variation in the number of fetal wastages during dry season and rainy season were determined. Economic implications of fetal wastage were evaluated. The results revealed the number of fetuses wasted were 3,763 within a six-year period. The highest number of fetal wastages 2777 (73.8 %/) were recorded in the dry season with (P &lt; 0.05), compared to the number of fetal wastages 986 (26.2 %) recorded in the rainy season. The financial losses due to fetal wastages was estimated at ₦2,162,160,000 with an annual loss of ₦ 360,360,000. The yearly average fetal wastage across the study period was 627 (16.7 %). Large number of fetal wastages were recorded during the dry season with significant economic losses. Hence, effective ante mortem inspection and pregnancy diagnosis in cows is necessary in order to avert this problem at the abattoir. Similarly, reinforcement of legislation law against indiscriminate slaughter of pregnant cows should be enforced at the abattoir.

Production of Polyclonal Antibodies against Pregnancy Associated Glycoprotein in Cattle

Pregnancy-associated glycoproteins (PAGs) are considered to be a large gene family in the trophoblasts of ruminants. PAGs determination precisely, PAG-1 in serum has been as the solid ground for pregnancy diagnosis in cow. Unluckily the PAG-1 and the antigenically related PAGs show a long half-life in serum less than 8 days and can be assessed 80 to 100 das postpartum, thereafter giving false results in cows bred within 60 days of calving. This study involves using novel polyclonal antibodies after immunizing rabbits and purification of the produced antibodies. The cattle are pregnant when the PAG hormone level is measured in a high level in a serum sample from the animal. It could give a precise method of detecting pregnancy at the early with few false positive results.

Proteomics and transcriptomics study reveals the utility of ISGs as novel molecules for early pregnancy diagnosis in dairy cows

An early and precise diagnosis of pregnancy in cows is critical to short the calving interval and to improve their reproductive efficiency. Neutrophils are the first blood cells to sensitize the embryo in the uterus and participate in maternal recognition of pregnancy after getting induced by interferon tau (IFNτ). To study the protein abundance ratio, blood samples were collected on 0th, 10th, 18th and 36th day post-artificial insemination (AI) from crossbred Karan Fries cows. Neutrophils were isolated through density gradient centrifugation and studied for protein abundance by high-performance liquid chromatography coupled with mass spectrometry (LC–MS). Protein abundance ratios for Myxovirus resistance (MX1 and MX2) were found to be higher (P < 0.05) on day 10 and day 18 post-AI, whereas Oligoadenylate synthetase-1 (OAS1) and Interferon stimulated gene-15 ubiquitin-like modifier (ISG15) proteins were more abundant on day 18 post-AI. The relative mRNA expressions of these molecules were also studied by qPCR. The gene expression of ISG15, MX1, MX2 and OAS1 was found to be higher (P < 0.05) on day 10th, 18th and 36th post-AI compared to day 0. The study indicates that ISGs on blood neutrophils are essential for the establishment of pregnancy and may be targeted as potential biomarkers for pregnancy diagnosis in cows.

Duplex Quantitative Polymerase Chain Reaction of ISG15 and RSAD2 Increases Accuracy of Early Pregnancy Diagnosis in Dairy Cows

Abstract Early diagnosis of pregnancy is important in livestock production, but there is no reliable technology used for pregnancy diagnosis within the first three weeks after insemination. During early pregnancy, the expression of interferon-stimulating genes (ISGs) in peripheral blood leukocytes (PBL) is significantly increased. However, due to different strains, detection sample types, detection methods, threshold value, etc. the specific effectiveness of early pregnancy diagnosis using ISGs is worth further study. The purpose of this study was to test interferon-stimulated protein 15 (ISG15), 2'–5'-oligoadenylate synthetase 1 (OAS1) and radical S-adenosyl methionine domain containing 2 (RSAD2) for early pregnancy diagnosis in dairy cows. The expression of ISG15, OAS1, and RSAD2 in PBL of pregnant and non-pregnant heifers on days 0, 14, 18, 21 and 28 after artificial insemination (AI) was detected by fluorescence quantitative polymerase chain reaction (PCR). The sensitivity and specificity of the pregnancy diagnosis was analyzed using expression of these three genes separately or in combination by receiver operating characteristic curve. The combination with the highest accuracy used probe primers and duplex fluorescence quantitative PCR. The single quantitative PCR results showed that expression of ISG15, OAS1 and RSAD2 on day 18 after AI was significantly higher in pregnant than in non-pregnant cows. When these three genes were used separately, or in combination, for early pregnancy diagnosis, the sensitivity for the RSAD2 gene was 100%, and the combination of ISG15 with RSAD2 was 94.7%. The duplex quantitative PCR showed that, although the sensitivity of ISG15 alone was 100%, its specificity was only 88.2% (cut-off value 1.402). The sensitivity of RSAD2 alone was 89.5%, and the specificity was 88.2%; however, when the two genes were used in combination, the sensitivity, specificity and diagnostic cut-off value were consistent with the results of single quantitative PCR. These results indicated that a duplex quantitative PCR assay system for early pregnancy diagnosis in cows using ISG15 and RSAD2 was established. Simultaneous detection of expression of ISG15 and RSAD2 by duplex quantitative PCR can effectively improve the diagnostic accuracy for dairy cows.

Subsidiary methods of pregnancy diagnosis in diary cattle – alternative for the future

Early pregnancy diagnosis in cows is one of the key elements of effective reproduction management of this species. Still imperfect effectiveness of procedures performed motivates veterinarians for further exploration of new techniques for the diagnosis of pregnancy and their new application. The following article aims to give existing laboratory methods and possibilities for their use in the diagnosis of pregnancy before the 3rd day of insemination. In particular, substances that can be identified as soon as possible after insemination are presented: early pregnancy factor (EPF), progesterone (P4), estrogen sulfate and pregnancy-associated glycoproteins (PAG). A detailed description of these substances and their role in the formation and maintenance of celibacy in cows are given. The possibility of using laboratory methods in modern milk reproduction protocols is shown. From the perspective of modern literature sources, the most effective laboratory methods for diagnosing pregnancy in dairy farming today are the definition of an early pregnancy factor (EPF) and pregnancy-associated glycoproteins (PAG). Description of the methods available on the market and a new compilation (protocols) above techniques may be of interest for veterinarian involved in cattle reproduction. Despite the rapid development and the presence of more and more effective ultrasound devices, a practical veterinarian should not forget about the existence of indirect methods of diagnosis of pregnancy, which in large herds can significantly improve the economic conditions for the management of reproduction of cows in the future.

Development of enzyme-linked immunosorbent assay for early pregnancy diagnosis in cattle

The prerequisite for enhancing reproductive efficacy and successful calving is an early and accurate diagnosis of pregnancy. A highly sensitive enzyme-linked immunosorbent assay (ELISA) utilizing the second antibody coating technique and the horseradish peroxidase conjugate as a label for the determination of Interferon-stimulated protein, 15 kDa (ISG15) in dairy cows was developed. To validate a neutrophil lysate based ELISA for early pregnancy diagnosis, blood samples from healthy multiparous Karan Fries (KF) cows were collected on day 0 (day of AI), 10, 14, 16 and 21 post artificial insemination. The pregnancy and non-pregnancy in cows were confirmed by plasma progesterone assay, ultrasonography and per rectal palpation. The detection range of the assay was 0.312 to 25 ng/ml with a sensitivity of 0.13 ng/ml. The inter-assay and intra-assay coefficient of variation was 12.1% and 10.1%, respectively. The parallelism for measured and expected concentrations had an r2 value of 0.90 and 0.93, respectively. It was observed that ISG15 concentration was greater (P < 0.01) in pregnant as compared to non-pregnant cows, the greatest being 9.36 ± 0.50 ng/ml on day 16 in pregnant cows. With the use of immunocytochemistry, there was a positive staining for ISG15 with greater staining in the neutrophils of day 16 pregnant cows. The study reveals that ISG15 protein in a neutrophil lysate can be utilized as a biomarker for early pregnancy diagnosis in cows.

Ultrasonic Assessment of Corpora Lutea and Plasma Progesterone Levels in Early Pregnant and Non Pregnant Cows

Corpus luteum cross sectional (by ultrasonography) and plasma progesterone (by DELFIA) were estimated in early pregnant and non pregnant cows on days 14th and 20th to 23rd post insemination. On day 14th, corpus luteum sectional area was 348.43 mm2 in pregnant and 387.84mm2 in non pregnant cows. Within days 20th to 23rd, corpus luteum sectional area ranged between 342.06 and 367.90 mm2 in pregnant and between 193.85 and 270.69 mm2 in non pregnant cows. Plasma progesterone level was 2.43 ng/ml in pregnant and 2.46 ng/ml in non pregnant cows on day 14th , while during days 20th to 23rd the level ranged between 2.47 and 2,84 ng/ml in pregnant and between 0.53 and 1.17 ng/ml in non pregnant cows. Results of both luteal tissue areas as well as plasma progesterone levels were highly significantly deferent (P<0.01) between pregnant and non pregnant cows during days 20th to 23rd, but there were no significant differences on day 14th. The correlation between CL cross sectional area and plasma progesterone level was 0.4 in pregnant cows and 0.99 in non pregnant cow. It is clear, from this study, that ultrasonic assessment of corpora lutea is a viable alternative to determine plasma progesterone levels for early pregnancy diagnosis in cows.

A new dried milk sampling technique and its application for progesterone detection in cows

A new method for milk sample collection and storage, based on a dried milk sampling technique, is proposed. The method includes application of a whole milk sample to a porous membrane followed by drying. One hundred whole milk samples (dried and liquid) taken on day 21 post insemination were analysed for progesterone by ELISA and results for both dried and liquid samples were well correlated (r=0.911). Milk progesterone ELISA accuracy for pregnancy diagnosis in cows was 87%.

In this issue – August 2009

Post‐weaning mortality of Merino sheep ▪ Transcutaneous ultrasound over the right flank for pregnancy diagnosis in cows ▪ Right flank transcutaneous vs transrectal ultrasound for pregnancy diagnosis in cows ▪ Ultrasound of soft tissue tumours in dogs ▪ Idiopathic hypereosinophilic syndrome in a Rottweiler ▪ Toxicity from accidental oral dosing of a topical endectocide ▪ Scrub‐itch mite in an endangered wallaby ▪ Cerebellar cortical degeneration in a koala

314 EARLY PREGNANCY DIAGNOSIS USING A NOVEL TRANSRECTAL ULTRASONOGRAPHY PROTOCOL IN JAPANESE BLACK COWS

The present study aims to establish a novel practical protocol for early pregnancy diagnosis in cows by using transrectal ultrasonography. The protocol is based on measurements of corpus luteum (CL) cross-sectional area (CL c-s area) change performed at 2 separate days before the coming estrus after AI. Fourteen cows were inseminated artificially, and transrectal ultrasonographical observation of the ovaries and blood collection for measurement of peripheral plasma progesterone (P4) concentration were carried out daily from Days 12 to 23 (Day 0 = the day of onset of estrus). Thereafter, cows were routinely diagnosed for pregnancy at Day 30 by transrectal ultrasonography. The largest CL c-s area was obtained at Day 14 in both pregnant and non-pregnant cows. Seven out of 8 non-pregnant cows showed significant CL c-s area regression between Days 14 and 20 (422 ± 112 v. 249 ± 63 mm2), whereas no regression was observed between Days 14 and 20 in pregnant cows (416 ± 65 v. 402 ± 78 mm2). The regression in the CL c-s area between pregnant and non-pregnant cows was significantly different during Day 18 (424 ± 65 v. 288 ± 88 mm2) to Day 23 (402 ± 71 v. 139 ± 64 mm2). P4 concentration was significantly low (less than 1 ng mL–1) at Day 20 in 3 out of 8 non-pregnant cows, whereas the pregnant cows showed significant increase of P4 between Days 14 and 20 (2.6 ± 0.2 v. 3.4 ± 0.5 ng mL–1). The pregnant cows showed significantly higher P4 concentration starting from Day 18 than non-pregnant cows. However, in non-pregnant cows, 4 cows returned to estrus on Day 20 or after, 3 cows showed no signs of estrus, and 1 cow came in estrus as early as Day 18 after AI. In conclusion, the results of the present study suggest that measuring the change in the CL c-s area at Days 14 and 20 makes it possible to detect the non-pregnant cows at Day 20 after AI. However, it was also indicated that measuring the change of P4 concentrations on the same days did not always successfully detect non-pregnant cows. The new protocol based on CL c-s area regression rate can detect almost certainly non-pregnant cows at Day 20 after AI. It is suggested that this method is advantageous in research and industrial breeding.

Pregnancy associated glycoproteins (PAG) in postpartum cows, ewes, goats and their offspring

Determination of plasma concentrations of pregnancy associated glycoproteins (PAG) has been used for early pregnancy diagnosis in cows. However, this is complicated by the presence of PAG in plasma for an extended period postpartum. The main objective of the present study was to investigate the postpartum elimination rates of pregnancy associated glycoproteins (PAG) in sheep, goats and cows in order to gain background information applicable to the use of PAG for pregnancy diagnosis in domestic ruminants. A second objective was to investigate whether PAG are transferred to the foetus and newborn, by measuring plasma PAG concentrations in calves, lambs and goat kids before and after colostrum feeding. PAG in the blood at parturition were eliminated by a first order process in the cows and ewes, while a two-step log-linear decline occurred in the goats. Estimated postpartum half-life of plasma PAG in the cows and ewes was 9 and 4.5 days, respectively. In the goats, half-lives were 3.6 and 7.5 days in the initial fast and terminal slow phase. Basal levels were reached 80–90 days postpartum in cows. Plasma PAG concentration can be used for pregnancy diagnosis from day 28 after AI, provided that the time interval from calving to AI is >60 days. Using a heterologous antibody RIA, we found 4 ng/mL to be the appropriate cut-off. Due to the presence of PAG residues from the previous gestation, the interval from AI to pregnancy diagnosis should increase by approximately 0.5 days beyond 28 days for each day of AI closer to calving than 60. Measurements in newborn ruminants suggested that PAG enter the foetal blood in utero and that colostral PAG are transferred to the newborn. Following the peak plasma concentration observed 1 day after birth in most of the animals, PAG were rapidly eliminated in a log-linear fashion.

Rectal palpation

Rectal palpation is considered by most to be a safe, accurate method for pregnancy diagnosis in cows from about day 30 to 35 of pregnancy until term. Diagnosis is possible a few days earlier in heifers. A diagnosis of nonpregnant should never be made until the uterus has been retracted and carefully palpated along its entire length.&#x0D; Differential diagnoses that should be considered for pregnancy in the cow include pyometra, fetal mummification, fetal death and/or maceration, segmental aplasia with accumulation of fluid, adhesions of the reproductive tract to other abdominal organs, uterine lymphoma, abscesses of the reproductive tract, and ovarian tumors. Mistakes involving these conditions should not be made if the examiner relies on the positive signs of pregnancy mentioned above.

Bovine salivary progesterone: Application to the assessment of ovarian function and early pregnancy diagnosis

A simple, sensitive and reproducible direct RIA method for the determination of low quantities of progesterone in bovine saliva was developed and applied for the assessment of the ovarian function and early pregnancy diagnosis in cows and heifers. The sensitivity of the method was 3.3±0.9 pg/tube, intra- and interassay CVs for low and high pools of saliva were 4.7 and 3.8% and 14.6 and 10.7%, respectively, and the mean recoveries when 200 μl of stripped saliva were supplemented with 20, 50, 100, 300 pg standard progesterone were 93.1, 94.0, 101.8 and 100.9%. The ovarian function was assessed in matched blood and saliva samples from pregnant and early postpartum cows. It was found that salivary levels measured by this method reflected the changes in plasma progesterone concentrations. The saliva/plasma progesterone ratio × 100 during the follicular phase was 15% while during the luteal phase it was 12%. The salivary progesterone content was used to diagnose pregnancy in cows 21 days after service. The accuracy of the method was 100% for the negative and 73.7% for the positive cases.

Volatile fatty acids in urine and vaginal secretions of cows during reproductive cycle

Levels of volatile fatty acids (VFA) in vaginal secretions (VS) of cows were nearly independent of concentration changes of acids in the gastrointestinal tract, while levels in urine showed marked dependence. During three-week cycles, the VFA concentrations in VS increased shortly before heat and decreased to one third on days 19-22. Some cows showed an immediate increase after heat while in others the increase was slow and gradual. Moderately increased levels at midcycle were probably related to interovulatory follicle growth. Content of VFA in VS of pregnant cows was significantly above the level of the postestrus drop. Some pregnant cows in the first two thirds of gravidity had substantially higher levels of VFA than the other cows. Changes in VFA levels have a potential value for estrus detection and pregnancy diagnosis in cows. The gas chromatographic method used is too slow and tedious for practical application, but a reliable semiquantitative test for rapid proof of VFA or acetic acid in VS would be a very useful device for field use. It would have a substantial impact on the economy of cattle production by minimizing the loss of time due to delayed breeding.

Pregnancy diagnosis in cows with linear-array real-time ultrasound scanning: a preliminary note.

Pregnancy diagnoses were performed under farm conditions in 201 cows (Hungarian Red Pied and crossbreds with Holstein) between 21 and 70 days after last insemination date, using a linear-array real-time ultrasound scanner with a 3.0 MHz rectal transducer. Identification of (a) sharply demarcated black area(s) and/or an embryo within the uterus were used as criterions for pregnancy. Pregnancy was confirmed by rectal examination at 6 and/or 8 weeks after A.I. From the total number of correct positive diagnoses (n = 129), incorrect positive diagnoses (n = 3, made on days 36, 40 and 44 after A I), correct negative diagnoses (n = 62) and incorrect negative diagnoses (n = 7, made on days 25, 28, 28, 29, 30, 31, and 33 after A I), a sensitivity of 94.8%, a specificity of 95,3%, a positive predictive value of 97.7% and a negative predictive value of 89.8% were calculated. From these preliminary results it was concluded that real-time ultrasound scanning is a useful and reliable technique for early pregnancy diagnosis in cows. It is suggested that a combination of milk/plasma progesterone estimation between days 21 and 24 and real-time ultrasound scanning between days 25 and 45 would allow the confirmation and quantification of (late) embryonic mortality in the cow.

Comparison of Pregnancy Diagnosis by Milk Progesterone on Day 21 and Day 24 Postbreeding: Field Study in Dairy Cattle

Milk samples collected at 21 and 24 d after breeding were used to diagnose cows as pregnant, questionable, or not pregnant with high, intermediate, or low progesterone in the milk. Total agreement of pregnancy diagnosis in cows by milk progesterone at 21 d postbreeding and diagnosis by return to estrus or palpation of reproductive organs was 85.8% (77.4% on pregnant progesterone diagnosis and 97.2% on not pregnant progesterone diagnosis). Total agreement of diagnosis by progesterone at 24 d postbreeding and diagnosis by estrus or palpation was 88.4% (83.5% on pregnant progesterone diagnosis and 95.3% on not pregnant progesterone diagnosis). Although total accuracy of cows diagnosed both pregnant and not pregnant by milk progesterone on d 24 postbreeding was greater than diagnosis on d 21 postbreeding when cows in estrus by d 24 were deleted from the data, other comparisons for effect of day on accuracy of diagnosis were not different. Total accuracy of diagnosis on d 24 was not increased by using milk progesterone on both d 21 and 24 Samples collecteds on the day of breeding indicated that 18 of 190 (1.9%) had intermediate or high progesterone. Fourteen (1.5) additional cows had progesterone above levels expected for a cow in estrus.

Pregnancy diagnosis in cows and heifers by determination of oestradiol-17α in faeces

The concentration of unconjugated oestradiol-17α was measured in faeces of 21 non-pregnant animals and of 39 cows and heifers between the 10th and 25th week of gestation. After the 14th week of gestation all pregnant animals secreted significantly more oestradiol-17α in the faeces than non-pregnant animals. This is a possible method therefore for the confirmation of pregnancy not only in cows but also in heifers.

Development of a direct enzyme immunoassay of milk progesterone and its application to pregnancy diagnosis in cows

The purposes of this study were to develop an enzyme immunoassay (EIA) for determination of progesterone in unextracted whole milk and to apply it to pregnancy diagnosis. Paper fibers covalently coupled to antibody specifically and competitively bound 3H-progesterone and 11α-hydroxyprogesterone hemisucccinate-horseradish peroxidase and were stable for 9 mo at −20°C. The sensitivity, recovery, precision, and cross reactivity of the EIA and a radioimmunoassay (RIA) of milk progesterone were evaluated, and showed that this EIA was comparable to the RIA. Milk samples were collected once daily for one estrous cycle from ten lactating Holstein cows and the progesterone levels were determined by RIA. Milk progesterone in 70 samples measured by EIA were highly correlated (r = 0.90) with the values of RIA for the same samples. Milk samples for pregnancy diagnosis by EIA of milk progesterone were obtained daily from days 20 to 24 after 115 artificial inseminations of 85 lactating Holstein cows. Pregnancy diagnosis by EIA was confirmed by rectal palpation at 30 to 60 days after insemination or return to estrus. The accuracy based on single, two, three, four, and five consecutive samples was from 67.2 to 80.0%, 77.3 to 84.0%, 79.2 to 87.5%, 82.0 to 85.4%, and 85.4%, respectively, for pregnancy diagnosis; and from 95.0 to 98.3% for nonpregnancy.

Use of milk progesterone enzyme immunoassay for early pregnancy diagnosis in cows

An enzyme immunoassay using β-galactosidase as the tracer was applied to determine milk progesterone level in cows. The novel method was reliable and practicable and required only a spectrophotometer and a centrifuge as major equipment. The milk progesterone enzyme immunoassay successfully diagnosed early pregnancy in cows. Milk samples were collected from 268 Holstein-Friesian cows in commercial dairy herds on 20, 21 or 22 days(usually 21 days) after insemination. Progesterone level in skim milk higher than 1.0 ng/ml indi-cated pregnancy. Pregnancy was confirmed by rectal palpation on 60 to 120 days after insemination. The accuracy of the milk progesterone test was 60.0 % ( 132 220 ) for a positive diagnosis and 100 % ( 48 48 ) for a negative result. A high incidence of embryonic death, 27.9 % ( 51 183 ), may have reduced accuracy for a positive test result. The enzyme immunoassay may be an alternative to radioimmunoassay in milk progesterone analysis for pregnancy diagnosis.