Diagnosis In Cattle Research Articles

Small intestinal incarceration caused by external herniation can be diagnosed clinically in cattle, but laparotomy is required to confirm internal incarceration.

To describe the clinical, laboratory and ultrasonographic findings, treatment, and outcome of cattle with small intestinal incarceration (SII) through internal and external hernias. The medical records of 85 cattle with SII admitted between January 1, 1987, and December 31, 2019, were retrospectively reviewed. The long-term outcome was determined 2 years after discharge. 85 cattle had herniation of the small intestine through congenital or acquired openings in mesentery or omentum (internal herniation; n = 60) or the abdominal wall (external herniation; 25). The most common findings were little or no feces in the rectum (77 of 85 [90.6%]), reduced or absent intestinal motility (76 of 85 [89.4%]), and hypocalcemia (36 of 44 [81.8%]). Thirteen (15.3%) cattle died or were euthanized without surgery. Of the remaining 72 (84.7%) cattle that underwent surgery, 42 survived the procedure. Overall, 52 of 85 cattle (61.2%; 95% CI, 50% to 72%) did not survive to hospital discharge and 33 (38.8%; 95% CI, 28% to 50%) were discharged alive. Of these, 11 (33.3%; 6 with and 5 without hernias closed completely) were still productive in their respective herds 2 years later. The diagnosis of an incarcerated external hernia is usually straightforward, whereas internal SII necessitates laparotomy or postmortem examination for a definitive diagnosis. Internal herniation should be part of the differential diagnosis in cattle with signs of ileus. Immediate surgical treatment is paramount in cattle with SII.

AI-aided cardiovascular disease diagnosis in cattle from retinal images: Machine learning vs. deep learning models

Cardiovascular diseases (CVD) in animals can severely impact the heart and circulatory systems, like those in humans. Early diagnosis and treatment are crucial for improving animal welfare and lifespan. Traditional diagnostic methods face challenges such as insufficient anamnesis information, high costs of biochemical and hematological tests, and increasing data complexity. This study aims to address these issues by developing AI-based diagnostic systems for fast and accurate CVD diagnosis in cattle using retinal images. A total of 1118 retinal images from 100 cattle were collected, with 52 diagnosed with CVD and 48 as non-CVD. The dataset is publicly available on Kaggle. We evaluated three machine learning methods (Extreme Learning Machine, K-Nearest Neighbors, and Support Vector Machine) and four deep learning models (DenseNet201, ResNet101, SqueezeNet, and InceptionV3) for their diagnostic capabilities. ResNet101 emerged as the most effective model, achieving an accuracy of 96.1 ± 3.15 %, sensitivity of 97.3 ± 2.96 %, specificity of 94.9 ± 4.07 %, and an F1-score of 96.4 ± 0.03. This study demonstrates that AI-based systems, particularly deep learning models, can significantly improve the accuracy of CVD diagnosis in animals, marking a critical advancement in veterinary healthcare.

Lumpy skin disease diagnosis in cattle: A deep learning approach optimized with RMSProp and MobileNetV2.

Lumpy skin disease (LSD) is a critical problem for cattle populations, affecting both individual cows and the entire herd. Given cattle's critical role in meeting human needs, effective management of this disease is essential to prevent significant losses. The study proposes a deep learning approach using the MobileNetV2 model and the RMSprop optimizer to address this challenge. Tests on a dataset of healthy and lumpy cattle images show an impressive accuracy of 95%, outperforming existing benchmarks by 4-10%. These results underline the potential of the proposed methodology to revolutionize the diagnosis and management of skin diseases in cattle farming. Researchers and graduate students are the audience for our paper.

The use of earwax to determine livestock exposure to teratogenic lupine

Lupines (Lupinus spp.) are a common plant species on western U.S. rangelands with several lupine species containing alkaloids that can be toxic and/or teratogenic to livestock. In North America, more than 150 lupine species are recognized with some ranches or grazing allotments containing multiple species. One or more of these lupine species may contain alkaloids that are teratogenic to cattle. Previous work has shown that lupine alkaloids can be detected in earwax of cattle grazing lupine infested rangelands. Our hypothesis is that earwax can be used to determine if cattle have been exposed to teratogenic alkaloids from multiple lupine species. Two lupine species, L. sericeus and L. polyphyllus, were present on a rangeland in east-central Idaho. The teratogen, anagyrine, was detected in L. sericeus and the teratogen, ammodendrine, was detected in L. polyphyllus plants collected on this rangeland. In this study, earwax was collected from 69 pregnant cows that had previously grazed a rangeland containing two different lupine species containing alkaloids that cause crooked calf syndrome (CCS). Anagyrine was detected in the earwax of all 69 cows sampled. Ammodendrine, was detected in the earwax of 28 of the 69 cows sampled. Earwax is a good non-invasive sample to aid in the diagnosis of cattle that have consumed lupine and does appear, in this case, to be a good diagnostic tool to differentiate between more than one lupine species that may be the cause of CCS. Concentrations of anagyrine or ammodendrine did not correlate with the incidence of CCS.

Diagnóstico clínico e anatomopatológico em bovinos acometidos por úlceras do abomaso perfuradas e com comorbidades

This study describes the findings of the clinical and anatomopathological diagnosis in cattle affected by perforated abomasal ulcers and with comorbidities. Twenty-five cattle were evaluated, young (n=8, aged less than two years) and adults (n=17, aged greater than two and a half years), diagnosed with perforated abomasal ulcers. Epidemiological information was retrieved from the medical records. Blood, ruminal fluid, and peritoneal fluid (PI) samples were collected for laboratory examination. Ultrasound examination, exploratory laparotomy, and post mortem examinations were performed for diagnostic purposes. The main clinical findings of perforated ulcers in the present study were: apathy, dehydration, intestinal hypomotility, diarrheal stools, hypomotile or atonic rumen, tachycardia, tachypnea, abdominal distention with bilateral bulging, and increased abdominal tension. The most frequent abnormal hematological findings were hypoproteinemia and neutrophilic leukocytosis with a left shift. The peritoneal fluid (PL) showed an inflammatory reaction in the cytology, characteristic of an exudate. A confirmatory diagnosis of peritonitis by ultrasonography was possible in five cases, and in two cases at exploratory laparotomy. In the post mortem examination, the ulcers were characterized as type 3, 4, or 5, with comorbidities present in 68% of the cases. It is necessary to combine ultrasonography with abdominal centesis and exploratory laparotomy procedures to suggest perforated ulcers. However, post mortem examination better characterizes the type of perforated ulcer present, particularly type 5.

First Report of Bovine Coenurosis in North Macedonia

Abstract Coenurosis is a cystic parasitic disease affecting ruminants and other mammals worldwide. The disease is caused by Coenurus cerebralis, the larval stage of the tapeworm Taenia multiceps. Sheep and goats are frequently infected, while cases in cattle are rare. Here we describe the first recorded case of bovine coenurosis in North Macedonia. The diagnosis was based on the neurological clinical signs and the postmortem findings of a 1-year-old bull with symptoms typical for coenurosis, i.e. ataxia, circling movements, mild depression and impaired vision. Postmortem, a cyst was found in the left cerebral hemisphere and was confirmed as fertile Coenurus cerebralis by parasitological analysis. These findings suggest that coenurosis should be considered part of the differential diagnosis in cattle with neurological symptoms. This highlights the need for routine surveillance of this disease in livestock and proactive tracking of the parasite in the final hosts to enhance disease management.

First Report of Bovine Coenurosis in North Macedonia

Abstract Coenurosis is a cystic parasitic disease affecting ruminants and other mammals worldwide. The disease is caused by Coenurus cerebralis, the larval stage of the tapeworm Taenia multiceps. Sheep and goats are frequently infected, while cases in cattle are rare. Here we describe the first recorded case of bovine coenurosis in North Macedonia. The diagnosis was based on the neurological clinical signs and the postmortem findings of a 1-year-old bull with symptoms typical for coenurosis, i.e. ataxia, circling movements, mild depression and impaired vision. Postmortem, a cyst was found in the left cerebral hemisphere and was confirmed as fertile Coenurus cerebralis by parasitological analysis. These findings suggest that coenurosis should be considered part of the differential diagnosis in cattle with neurological symptoms. This highlights the need for routine surveillance of this disease in livestock and proactive tracking of the parasite in the final hosts to enhance disease management.

Diagnostic significance of plasma gastrin concentration for the diagnosis of type 1 abomasal ulcer in water buffalo: A preliminary case control study

The type 1 abomasal ulcer (AU1) does not have specific clinical signs, so there is a need to identify some early biochemical markers for its diagnosis in cattle and buffaloes. The plasma gastrin is reported to reflect the gastric mucosa damage but its utility for the diagnosis of AU1 in buffaloes has not been evaluated. The objective of this study was to investigate the test performance of plasma gastrin to distinguish between healthy buffaloes and the buffaloes with AU1. Twenty-three buffaloes with AU1 and six buffaloes without any abomasal ulcer were used. The blood samples were collected from buffaloes, slaughtered in a buffalo specific slaughterhouse for estimation of the plasma gastrin. After slaughter the abomasa were examined for the presence of AU1 and the blood samples were collected for estimation of plasma gastrin. The mean plasma gastrin concentration of the ulcer-positive buffaloes was significantly (P<0.05) higher than the ulcer-negative buffaloes. The receiver operating characteristic curve analysis suggested that the optimal value of plasma gastrin for the diagnosis of AU1 was 106.2 pg/mL. This preliminary work suggests that plasma gastrin could be a valid diagnostic test for the detection of AU1 in the buffaloes. The sensitivity, specificity, positive predictive value and negative predictive value of the plasma gastrin to diagnose AU1 in the buffaloes were 78.3, 100, 100 and 69.9 pg/mL, respectively.

Serum pregnancy-associated glycoprotein profiles during early gestation in Karya and Konya Merino sheep.

Reported as being expressed by mono- and binucleate placental cells, pregnancy-associated glycoproteins (PAGs) are released into the blood circulation from the ruminant placenta. Circulating gestational PAGs levels may differ between sheep breeds. This study was aimed at the close monitoring of the serum PAGs profiles of Karya and Konya Merino sheep during early pregnancy. Fifteen Karya and 15 Konya Merino ewes were synchronized by a 12-day treatment with progesterone-impregnated intravaginal sponges. After the sponges were withdrawn, the ewes were administered 400IU of equine chorionic gonadotropin. The ewes were allowed to mate naturally, and all animals were sampled for blood as of the day of mating (day 0) at weeks 1, 2, 3, 4 and 5. Pregnancy diagnoses were made by transabdominal ultrasonography at week 5. At weeks 6, 7, 8, 9 and 11, blood samples were collected only from the pregnant ewes. The blood samples were centrifuged at 3000×g, and extracted sera were stored at -20°C until being used for laboratory analyses. Serum PAGs levels were determined with the aid of a commercial PAG-enzyme-linked immunosorbent assay test originally developed for pregnancy diagnosis in cattle. Differences in the between the PAGs levels throughout pregnancy and the group effect (Karya and Konya Merino) were determined with a two-way mixed analysis of variance. Pairwise comparisons were made using a Bonferroni adjustment. PAGs levels showed a linear increase with the advance of pregnancy in both Karya and Konya Merino sheep. No difference was detected between the breeds for serum PAGs levels. The serum PAGs levels of the pregnant and non-pregnant ewes differed as of the fourth week. The serum PAGs levels of the Karya and Konya Merino ewes were similar during the first 11 weeks of gestation, and pregnancy diagnosis could be made based on serum PAGs levels as of the 4th week in both breeds.

MX2 gene mRNA expression as potential biomarker for early pregnancy diagnosis in cattle

Early pregnancy diagnosis is vital for economic sustainability of dairy farms and maintaining the reproductive efficiency of the herd. There are many techniques including progesterone assay, pregnancy specific proteins and interferon stimulated genes have been explored for early pregnancy diagnosis but, they are associated with varying level of efficacy. In the present experiment, interferon stimulated gene (Myxovirus resistance gene 2/MX2) expression pattern was used as a potential biomarker for early pregnancy in cattle. The association of MX2 gene expression in relation to progesterone assay was studied to explore its potential use as biomarker of early pregnancy. The plasma progesterone concentration in conceived animals on day 7 (2.26±0.19 ng/ml), 17 (5.42±0.35 ng/ml) and 21(6.38±0.39 ng/ml) was recorded to be significantly higher as compared to respective values in non-conceived animals, i.e. 1.55±0.09 ng/ml, 4.14±0.14 ng/ml and 0.81±0.06 ng/ml. The sudden decrement in plasma progesterone concentration after day 17th discriminates conceived and non-conceived animals. MX2 expression levels were observed to spike in blood due to release of interferon tau (τ) after implantation of embryo. The relative mRNA expression of MX2 gene showed a 9.5 to 28.64-fold higher expression on 17 days post insemination in pregnant animals as compared to non-pregnant animals. Thus, MX2 gene can be used as a reliable biomarker for the early detection of pregnancy.

Inquérito epidemiológico e estudo de casos da brucelose canina por Brucella abortus

The objective of this research was to identify brucellosis by Brucella abortus and the possible risk factors associated with the transmission of the infection in dogs in the urban area of the municipality of Viçosa-AL, as well as to alert health authorities about the risk of this potential zoonosis, create a booklet to alert the population about the risks of the disease and the possibility of creating a test protocol in clinical care at the Veterinary Hospital of the Federal University of Alagoas. A study was carried out in the city of Viçosa-AL to investigate the presence of B. abortus in 30 adult male dogs. The animals that showed symptoms suggestive of reproductive disease were referred for evaluation of the hematological pattern and clinical and surgical treatment. Parallel to the blood collection, a questionnaire was applied to the owners of these dogs. Buffered acid antigen (AAT) techniques were used as a screening test and 2-Mercaptoethanol (2-ME) as a confirmatory test for AAT positives with symptoms of brucellosis. The study aimed to analyze the number of positive dogs, the clinical and anatomopathological changes and the risk factors for the occurrence of brucellosis in dogs in the municipality of Viçosa-AL. As dogs are a means of transmission for humans, health authorities must pay special attention to this disease, including the control of dogs in the Program for Control and Eradication of Brucellosis of the Ministry of Agriculture. In addition to intensifying control of vaccination and diagnosis in cattle.

Are rapid tests and confirmatory western blot used for cattle and small ruminants TSEs reliable tools for the diagnosis of Chronic Wasting Disease in Europe?

The first case of CWD in Europe was detected in a Norwegian reindeer in 2016, followed later by two CWD cases in Norwegian moose. To prevent the potential spread of CWD to the EU, the European Commission (Regulation EU 2017_1972) implemented a CWD surveillance programme in cervids in the six countries having reindeer and or moose (Estonia, Finland, Latvia, Lithuania, Poland, and Sweden). Each country had to test a minimum of 3000 cervids for CWD using diagnostic rapid tests approved by the EC Regulation. Experimental transmission studies in rodents have demonstrated that the CWD strains found in Norwegian reindeer are different from those found in moose and that these European strains are all different from the North American ones. Data on the performances of authorised rapid tests are limited for CWD (from North America) and are currently minimal for CWD from Europe, due to the paucity of positive material. The aim of this study was to evaluate the diagnostic performances of three of the so-called "rapid" tests, commercially available and approved for TSE diagnosis in cattle and small ruminants, to detect the CWD strains circulating in Europe. The performances of these three tests were also compared to two different confirmatory western blot methods. Using parallel testing on the same panel of available samples, we evaluated here the analytical sensitivity of these methods for TSE diagnosis of CWD in Norwegian cervids tissues. Our results show that all the methods applied were able to detect the CWD positive samples even if differences in analytical sensitivity were clearly observed. Although this study could not assess the test accuracy, due to the small number of samples available, it is conceivable that the rapid and confirmatory diagnostic systems applied for CWD surveillance in Northern Europe are reliable tools.

Review on Lumpy Skin Disease Virus Characteristics and its Diagnosis in Cattle

Lumpy skin disease is characterized by its signs including eruptive, infectious and occasionally causing death to affected animals. It is caused by the family Poxviridea and genus Capripox with a strain of Neethling virus, a double stranded DNA virus. LSD clinical signs are mainly observed on skin as nodules (0.5 to 5 cm in diameter) throughout the skin surface or subcutaneous tissue involving superficial lymph nodes. Diagnosis of CaPV is based upon clinical signs with laboratory confirmation by virus isolation, polymerase chain reaction (PCR) and electron microscopy. Field diagnosis of LSD is often based on characteristic clinical signs of the disease. However, mild and subclinical forms require rapid and reliable laboratory testing to confirm diagnosis. Isolation of virus can be made from collected biopsy or at post-mortem from skin nodules, lung lesions or lymph nodes within the first week of the occurrence of clinical signs, before the development of neutralizing antibodies. There are different methods to detect LSD virus antibody from blood samples. These are serum neutralization test (SNT), indirect fluorescent antibody test (IFAT) and enzyme-linked immune-sorbent assay. LSD as a member of CaPVs, it has a single serotype, do not cause persistent infection, have a limited host range and vaccines are available that may provide long term immunity. These attributes increase the prospect of successfully implementing regional control programs, leading to the elimination of the virus and conceivably global eradication.

Viperin (RSAD2) gene expression in peripheral blood mononuclear cells of pregnant crossbred beef cows is altered by Bos indicus genetics

The expression of interferon (IFN) stimulated genes (ISGs) in lymphocytes has been used for pregnancy diagnosis in cattle. However, among-cow variability has yielded sub-optimal predictive accuracy. We hypothesized that the expression of ISGs (ISG15, OAS1, RSAD2, CLEC3B, and AKR1B1) in early pregnancy varies according to the proportion of Bos indicus (B. indicus) genetics on females. Multiparous cows were classified in three genetic groups, High Angus (HA; n = 45 [0–33% Brahman influence]), Angus-Brahman (AB; n = 30 [34–67%]), and High Brahman (HB; n = 19 [68–100%]) and submitted to a Select-Synch + CIDR protocol. Cows that displayed estrus (n = 94) were artificially inseminated (Day0; D0). On D19, blood samples were collected to obtain peripheral blood mononuclear cells (PBMC) and measure progesterone (P4) concentrations. On D30, pregnancy diagnosis was performed. The expression of RSAD2 in PBMC of pregnant cows was positively related to the proportion of B. indicus genetics of the groups, but not the expression of ISG15 and OAS1. In pregnant cows, the proportion of B. indicus genetics was negatively associated to circulating levels of P4 concentrations. The P4 concentrations were related positively with RSAD2 expression. ROC curve results determined that for cattle with B. indicus genetics lower than 67%, the CLEC3B and AKR1B1 combination was the most accurate option to predict the outcome of pregnancy. In cows with more than 68% of B. indicus genetics, RSAD2 provided the best accuracy. In conclusion, there is a relationship between the proportion of B. indicus genetics and the ISGs gene expression in PBMC during pregnancy.

English

This study aimed to describe the clinical course of SA-MCF and the duration of viremia in surviving cattle and to perform the phylogenetic analysis of the tegument protein gene of OvHV-2 in cattle in the Central Balkan. A farm housing two heifers and 7 sheep with a confirmed SA-MCF case were selected for the investigation. For the estimation of the length of viremia and the virus shedding, the animals were sampled repeatedly, weekly for two months. For the phylogenetic analysis, a retrospective study was performed on 21 samples from cattle, and 7 samples from sheep, from the Central Balkan. In the blood samples of the survived heifer, the OvHV-2 genome was detected until week 7, in corneal swabs, the OvHV-2 genome was detected until week 6, and in nasal swabs until week two of the study. A retrospective study revealed that out of 21 tested cattle, OvHV–2 was detected in 15 (71.4%), and out of 7 tested sheep, three (42.9%) were positive. The sequenced samples show the highest percentage of similarity with the strains from Brasil KJ658293.1 (100%) and Germany HM216475.1 (100%).. Since there is a variety of different clinical signs similar to other notifiable diseases such as BVD, IBR, and FMD, there is a clear benefit in including SA-MCF in the differential diagnosis in cattle. As mixed farming in the Central Balkan is practiced, implementing SA-MCF monitoring in passive surveillance would allow a better understanding of the disease, ascertaining its prevalence and could provide new information regarding SA-MCF epidemiology

Cardiac Biomarkers and Biochemical Changes in Cattle with Traumatic Pericarditis

Traumatic pericarditis (TP) emerges as a result of foreign bodies pricking the heart, and these animals cannot be treated. Traumatic pericarditis is one of the most important cardiac diseases that may result in a decrease in milk yield, animal death, and fetal deaths in affected pregnant animals and lead to economic losses. Cardiac markers, which have been used in human medicine for years and have recently started to be used in veterinary medicine, can detect mild myocardial damage that may occur in the early stages of heart-related diseases. In this study it was aimed to determine the cardiac damage in cattle with TP by cardiac biomarkers. In the study, cTn-I, ANP, NT-proBNP, homocysteine, CRP, CK-MB, LDH, AST, and ALT levels were determined to be statistically higher in cattle with TP than in healthy animals (P < 0.01). NT-proBNP increased approximately 11 times, ANP about 10 times, cTn-I and Homocysteine increased 3 times, and CRP and CK-MB increased 2 times in cattle with TP. In conclusion, some cardiac markers were examined in the early detection of cardiac damage and diagnosis of cattle with TP, and it was concluded that the use of these markers would be beneficial. Moreover, it was concluded that ANP, Nt-proBNP, and homocysteine, which were the first data on cattle with TP, could be used in the detection of the severity of cardiac damage.

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.

Using commercial enzyme immunoassay for measuring pregnancy-associated glycoproteins to diagnose pregnancy in dairy cows under field conditions in Algeria

The purpose of the present work was to study effectiveness for early pregnancy diagnosis in cattle of the new enzyme immunoassay (EIA) sandwich kit commercially available based on the measurement of pregnancy-associated glycoproteins (PAGs). 120 Holstein-Friesian cattle of mixed age and parity were comprised from different dairy herds. The pregnant females (n = 68) were diagnosed by ultrasonography at day 35-40 after artificial insemination and confirmed by transrectal exploration at 2-3 months after AI. The non-pregnant females (n = 52) were housed in the absence of males during the experimental period. Blood samples were collected from coccygeal vessels of females into EDTA tubes. The serum was obtained by centrifugation and the serum was stored at - 20 C until assay. The PAG concentrations in pregnant and non-pregnant females were determined in serum by EIA kit. The reproducibility inter- and intra-assay of the PAG-EIA is satisfactory (2.78 and 13.19 %, respectively). The accuracy ( 94.8 %) and the test of parallelism were largely acceptable. No cross-reaction was observed with the different hormones tested at different dilutions. PAG-EIA system gave 100 % sensitivity and negative predictive values. Whereas, specificity and positive predictive value were 91.93 and 71.15 %, respectively. The accuracy of pregnancy diagnosis by PAG-EIA was 87.5 %. In conclusion, the present study shows clearly that the EIA kit can be used to measure PAG in serum cows for the detection of gestation in Algeria. Therefore, this alternative technique could be recommended to replace the radioactive methods in immunoassays to improve the reproductive performances and an efficient tool for reproductive management of dairy cattle.

Analysis of circulating microRNA during early gestation in Japanese black cattle

Circulating microRNAs (miRNAs) have been used as biomarkers for various diseases and physiological conditions in humans and mice; studies in domestic animals, particularly cattle, are limited. The importance of early pregnancy diagnosis (especially within the 21-d cow estrous cycle) in the livestock industry is extremely high. This study compared the circulating miRNAs in bred non-pregnant and pregnant Japanese Black cows, explored miRNAs as biomarkers for early pregnancy diagnosis, and established a measurement system that included selecting an appropriate reference miRNA and determining the effect of hemolysis on miRNA quantification in plasma. miRNA was extracted from the plasma of Japanese Black cows on day 21 after artificial insemination and subjected to a customized bovine oligonucleotide microarray for expression analysis. Differentially expressed miRNAs and reference miRNA candidates were selected and validated using reverse transcription-quantitative PCR (RT-qPCR). An appropriate endogenous reference miRNA for normalization was selected using NormFinder software. To evaluate the effect of hemolysis on miRNA quantification, hemolyzed samples were prepared using plasma from four cows in the estrous cycle and subjected to RT-qPCR. A total of 124 miRNAs were detected in bovine plasma by microarray analysis in bred non-pregnant and pregnant cows. The levels of five circulating miRNAs were significantly higher in pregnant cows than in bred non-pregnant cows, and 24 miRNAs were detected only in the pregnant group. NormFinder analysis and RT-qPCR validation showed that miR-2455 was an appropriate reference miRNA in the plasma of bred non-pregnant and pregnant Japanese Black cows, and miR-19b, miR-25, miR-29a, and miR-148a were significantly higher in the pregnant group. These four circulating miRNAs did not change during the estrous cycle and were less affected by hemolysis. In the current study, we found four miRNAs, miR-19b, miR-25, miR-29a, and miR-148a, which were present at high levels in the plasma of pregnant Japanese Black cows. Since these miRNAs are less affected by hemolysis, they may potentially be used as biomarkers for early pregnancy diagnosis in cattle.

Establishment of a Sandwich-ELISA for simultaneous quantification of bovine pregnancy-associated glycoprotein in serum and milk.

Bovine pregnancy-associated glycoproteins (boPAG) are expressed by trophoblast cells in the bovine placenta. The multigene family of boPAG belongs to the group of aspartic proteases. The accumulation and circulation in maternal blood and milk has made boPAG very useful and important for pregnancy diagnosis in cattle. The goal of the present study was to develop and validate a new Sandwich-ELISA which allows the detection of boPAG in maternal serum and whole milk. Therefore, 984 serum and 928 milk samples were collected monthly from 231 Holstein Friesian cows (Bos Taurus) from one week after insemination (p.i.) until six weeks postpartum. The ELISA is able to identify a cow as being pregnant at day 30 p.i. in serum and at day 40 p.i in milk with threshold values of 1.0 ng/ml in serum and 0.0165 ng/ml in milk. The postpartum half-life of boPAG was estimated to be 6.4 days in serum and 7.1 days in milk. The boPAG profile established during pregnancy in serum and milk showed a typical pattern. The amount of boPAG found in milk was 1.5 % of the amount of boPAG present in serum. In conclusion, a Sandwich-ELISA has been developed to quantify boPAG in serum and in whole milk simultaneously with the same test procedure. This is time saving for farmers and more efficient for laboratories.