Pregnancy Rates of Holstein Friesian Cows with Cavitary or Compact Corpus Luteum.

Cavitary corpora lutea are commonly observed during the estrous cycle in bovines. Since the quality of the corpus luteum (CL) is extremely important during embryo transfer when embryos are implanted into the recipient, the ultrasonographic examination of the CL is becoming more and more important in terms of the outcome of the procedure. In the present study, a total of 2477 ultrasonographic transrectal diagnoses were performed, and data were collected between the years of 2018 and 2020 in a large-scale Holstein Friesian dairy farm in Hungary. In 91.1% (n = 2257) and in 8.9% (n = 220) of the cases, compact CLs and cavitary CLs, respectively, were diagnosed at pregnancy diagnosis. The presence of a cavitary CL on the ovary at pregnancy diagnosis increased the odds of remaining open after pregnancy by 21 times compared to the presence of a compact CL (OR = 21.0, p < 0.001) in the cows. The presence of cavitary CL was not influenced either by month or season. Ovarian cysts were detected in 196 cases (8.0%) in the examined animals. The presence of a cavitary CL decreased by 9 times when an ovarian cyst was also diagnosed (OR = 9.0, 1.6% vs. 9.5%, p < 0.001). The presence of an ovarian cyst decreased the odds of established pregnancy by 81 times (OR = 81.1, p < 0.001). Based on our results, the presence of a cavitary CL between days 31 and 42 after artificial insemination is associated with a smaller chance of conception in Holstein Friesian cows. The presence of an ovarian cyst decreases the occurrence of cavitary CL and the chance of conception.

Microbial Profiling of Amniotic Fluid, Umbilical Blood and Placenta of the Foaling Mare.

The presence of a microbiome/microbiota in the placenta is hotly debated. In previous studies, the presence of bacteria in equine amniotic fluid and umbilical blood was independent of foal health. The objective of the present study was to determine if the same bacteria are present in the equine placenta as in amniotic fluid and umbilical blood. Samples were obtained from 24 parturient mares and foals. Placental bacterial DNA was extracted, and the microbiome was identified using 16S rRNA sequencing. All amniotic fluid samples contained some polymorphonucleocytes; bacteria were isolated from four samples. Aerobic or anaerobic growth was found in 18 and 3 umbilical blood samples, respectively. Serum amyloid A was <5 mg/L in all 24 samples, total WBC varied between 2900 and 10,700/µL, and fibrinogen varied between 0 and 5.16 g/L. In jugular blood, serum amyloid A was <5 mg/L in all 24 foals, total white blood count was 3200 to 8100/µL, and fibrinogen was 0.44 to 4.42 g/L. The diversity of bacterial microbiota was similar in all placental regions at the phylum level but differed at the genus level; the most abundant phyla were Proteobacteria (42-46.26%) and Actinobacteria (26.91-29.96%). In conclusion, bacteria were found in the fetal compartments and placenta of healthy equine pregnancies; however, we can neither prove nor disprove the hypothesis that the placenta has its own microbiome.

Post-thaw viability of mouse preantral follicles after cryopreservation with cryotube freezing and OPS vitrification procedures.

In the field of reproductive science, there is an increased interest in the application of ovarian preantral follicles. Since the ovary contains a great amount of preantral follicles (PAF), the cryopreservation and in vitro culture of such follicles support the fertility preservation of domestic animals with high genetic value, endangered or zoo animals, and women before anticancer therapy. To date, no standard freezing or vitrification protocol is available in human or animals. The aim of the present study was to examine the viability of preantral follicles cryopreserved using freezing or vitrification protocols: cryotube freezing or OPS vitrification.

The role of selenium and vitamin E in a Transylvanian enzootic equine recurrent rhabdomyolysis syndrome.

A severe form of recurrent exertional rhabdomyolysis occurs enzootically in a well-defined region of Transylvania, Harghita county. At the highest lying two settlements (more than 800 m above sea level), the prevalence of equine rhabdomyolysis is between 17 and 23%, while in the neighbouring villages in the valley it is less than 2%. The objective of our study was to clarify the role of selenium and vitamin E in the high prevalence of rhabdomyolysis in that region. Soil and hay samples were collected from each area to evaluate mineral content. Ten horses from the non-affected and 20 horses from the affected area were tested for serum selenium, vitamin E, glutathione peroxidase (GSH-Px), muscle enzymes, lactate and electrolytes. Hay samples collected from the affected area had lower selenium content. Horses in the affected regions had significantly lower serum selenium (P = 0.006) and GSH-Px levels than animals living in the non-affected regions. A good correlation between erythrocyte GSH-Px and serum selenium concentration could be demonstrated (r = 0.777, P < 0.001). Serum vitamin E levels were low independently of the origin of the horse. Based on our results, selenium deficiency possibly has a role in the Transylvanian enzootic equine recurrent rhabdomyolysis syndrome.

Family aggregation analysis shows a possible heritable background of equine grass sickness (dysautonomia) in a Hungarian stud population.

Equine grass sickness (also known as dysautonomia) is a life-threatening polyneuropathic disease affecting horses with approx. 80% mortality. Since its first description over a century ago, several factors, such as the phenotype, intestinal microbiome, environment, management and climate, have been supposed to be associated with the increased risk of dysautonomia. In this retrospective study, we examined the possible involvement of genetic factors. Medical and pedigree datasets regarding 1,233 horses with 49 affected animals born during a 23-year period were used in the analysis. Among the descendants of some stallions, the proportion of animals diagnosed with dysautonomia was unexpectedly high. Among males, the odds of dysautonomia were found to be higher, albeit not significantly, than among females. Significant familial clustering (genealogical index of familiality, P = 0.001) was observed among the affected animals. Further subgroups were identified with significant (P < 0.001) aggregation among close relatives using kinship-based methods. Our analysis, along with the slightly higher disease frequency in males, suggests that dysautonomia may have a genetic causal factor with an X-linked recessive inheritance pattern. This is the first study providing ancestry data and suggesting a heritable component in the likely multifactorial aetiology of the disease.

Sex determination using circulating cell-free fetal DNA in small volume of maternal plasma in elephants.

The genetic sexing of animals having long gestation periods offers significant benefits in regard to breeding management among their populations living in captivity. In our study, a new increased-sensitivity PCR method for fetal sexing was developed and tested successfully on elephants, from only a small volume of maternal plasma. Suitable sensitivity was obtained by using short, reduced amplicon lengths with fluorescent labelling for capillary electrophoresis detection. The fundamental principle for this technique was based on the detection of two Y-specific markers (AmelY and SRY), the presence of which indicates the mother is carrying a male fetus and the absence of these markers designates a female fetus. As a reaction control, the X-chromosomal marker (PlpX) was used. To the best of our knowledge, this is the first report on this topic, confirming the presence of fetal cell-free DNA from the plasma of a pregnant captive elephant, and demonstrating a new opportunity for non-invasive assessment in fetal sex determination.

Introduction of a new fetal examination protocol for on-field and clinical equine practice.

Prepartum fetal surveillance techniques have been widely used in both human and veterinary obstetrics, although these tests differ in their sensitivity for the assessment of fetal well-being. In equine reproduction, the 'gold standard' examination technique is the equine biophysical profile (EBP) described by Virginia Reef in 1995. Although this fetal assessment has many benefits, its accuracy, sensitivity and specificity have not been evaluated so far. In this study, 129 late-term pregnant mares were scanned twice in their last month of gestation using two different protocols (the EBP and a new, rapid examination protocol, REP) in order to determine the accuracy of these tests. The REP included the determination of fetal heart rate, fetal aortic diameter and the combined thickness of the uteroplacental unit. Twenty-seven fetuses proved to be compromised and 102 mares had healthy asymptomatic foals. Statistical analyses showed slight differences in test sensitivity (81.48% and 85.19%), specificity (83.3% and 87.25%) and accuracy (82.95% and 86.82%) for EBP and REP, respectively. Furthermore, a statistically significant interchangeability could be demonstrated between the two methods. It is concluded that, depending on the prevalence of the disease (here: compromised state), both methods are useful for the diagnosis of fetal abnormalities and make it possible to provide satisfactory care for broodmares and their fetuses.

Assessment of equine alpha-fetoprotein levels in mares and newborn foals in the periparturient period.

Alpha-fetoprotein (AFP) is best known in human obstetrics for its association with fetal anomalies recognized in the 1970s. Although this fetal protein had been shown to be present in the sera of many mammalian species, its possible diagnostic role in the detection of abnormalities was evaluated only later, when a research laboratory published variable levels of AFP in different groups of mares with pregnancy problems (twins, conception failure, placentitis, embryonic loss), and subsequently differences were demonstrated in its serum levels between aborted and healthy mares. In this study, peri- and intrapartal AFP levels were measured in maternal serum, amniotic fluid, neonatal blood, and umbilical blood samples. The mean levels of AFP were lower in umbilical blood and amniotic fluid samples than in foal and maternal blood. Older mares had lower AFP levels correlated with their age in years. The time remaining until foaling had a significant, non-linear effect on AFP levels: an elevation could be detected in the last two weeks of pregnancy, followed by a decline after foaling. Also, AFP levels were found to be elevated in the hot summer months. There was a significant individual variation in AFP levels in the population studied.

Applicability of fetal thoracic aortic diameter measurement in the prediction of birth weight in Holstein-Friesian cows - Short communication.

Transabdominal ultrasonography has been shown to be a useful and reliable method for assessing fetal well-being in horses and cattle. To test the applicability of fetal aortic diameter measurement in cattle, 44 late-term pregnant cows and heifers were examined 21 to 0 days prior to calving. Mean fetal aortic diameter was 2.07 ± 0.14 cm and mean fetal heart rate (FHR) was 109 ± 17 bpm. Three dead calves were dissected and their aortic diameter was measured in a water bath. The mean birth weight (n = 44) was 39.9 ± 5.8 kg. There was a significant negative correlation between FHR and fetal aortic diameter. However, although some studies have shown that fetal aortic diameter strongly correlates with birth weight in near-term horses and cattle, in this study there was no correlation between fetal aortic diameter and birth weight in Holstein-Friesian cows and heifers irrespective of whether the fetus was born alive or dead.

Pregnancy-associated changes of serum biochemical values in Lipizzaner broodmares.

The aims of this study were to detect physiological changes in blood biochemical parameters throughout gestation, to compare the findings in nonpregnant and pregnant Lipizzaner mares in early-mid and late pregnancy, and to provide reference values for clinical chemistry parameters in this horse breed. A total of 136 venous blood samples were collected from 20 pregnant and 10 nonpregnant (control) asymptomatic Lipizzaner broodmares for biochemical analyses. Twelve parameters (albumin, total protein, urea, triglycerides, glucose, creatinine, alkaline phosphatase, aspartate transaminase, glutamate dehydrogenase, gammaglutamyltransferase, creatine kinase and lactate dehydrogenase) were measured. For the statistical analyses, correlation, analysis of variance and Kruskal-Wallis H-test were used to evaluate the possible associations between parameters. Serum triglyceride levels proved to be significantly different in pregnant mares compared to the control group. Total protein and urea levels significantly decreased, while glucose, triglyceride and glutamate dehydrogenase values increased from approx. the fifth month of gestation until parturition. Four biochemical parameters (albumin, aspartate transaminase, total protein and urea) were lower and three other variables (glucose, alkaline phosphatase and creatinine) were significantly higher in late-term pregnant mares than in mares in early or mid-gestation. It is concluded that reference values not only reflect the species, breed and sex but also the reproductive status of animals.

Equine alpha-fetoprotein levels in Lipizzaner mares with normal pregnancies and with pregnancy loss.

Alpha-fetoprotein has proved to be a good indicator of fetal well-being in human medicine for decades. Although this molecule is present in most of the mammalian species including horses, reference values in healthy and high-risk pregnant mares have not yet been published. The aim of the present study was to determine whether equine alpha-fetoprotein (eqAFP) is a good indicator of complicated pregnancies in Lipizzaner mares. A total of 111 serum samples from 30 mares have been analyzed for eqAFP levels throughout gestation (Days 60-325). After the pregnancy was confirmed, 23 mares had normal pregnancies with viable foals, six had late embryonic loss, and one of the mares aborted in the ninth gestational month. Equine alpha-fetoprotein concentrations significantly differed in the normal group (72.93 ± 49.25 pg/mL; mean ± standard deviation) and in the complicated pregnancy loss group (152 ± 36.48 pg/mL; mean ± standard deviation). The mares' age, gestational age, and the conception rate significantly affected the alpha-fetoprotein concentrations in the normal group. Furthermore, notable individual differences occurred in eqAFP concentrations between mares. Equine alpha-fetoprotein seems to be an important indicator of fetal well-being in horses, but there are still some unanswered questions (levels in foals of different age, ponies, and draft horses) regarding this serum protein. Large-scale studies are needed to assess the specificity, sensitivity, and reliability of this test as a possible future diagnostic tool for fetal well-being in horses.

Fetal heart rate and fetal heart rate variability in Lipizzaner broodmares.

Monitoring fetal heart rate (FHR) and fetal heart rate variability (FHRV) helps to understand and evaluate normal and pathological conditions in the foal. The aim of this study was to establish normal heart rate reference values for the ongoing equine pregnancy and to perform a heart rate variability (HRV) time-domain analysis in Lipizzaner mares. Seventeen middle- and late-term (days 121-333) pregnant Lipizzaner mares were examined using fetomaternal electrocardiography (ECG). The mean FHR (P = 0.004) and the standard deviation of FHR (P = 0.012) significantly decreased during the pregnancy. FHR ± SD values decreased from 115 ± 35 to 79 ± 9 bpm between months 5 and 11. Our data showed that HRV in the foal decreased as the pregnancy progressed, which is in contrast with the findings of earlier equine studies. The standard deviation of normal-normal intervals (SDNN) was higher (70 ± 25 to 166 ± 108 msec) than described previously. The root mean square of successive differences (RMSSD) decreased from 105 ± 69 to 77 ± 37 msec between the 5th and 11th month of gestation. Using telemetric ECG equipment, we could detect equine fetal heartbeat on day 121 for the first time. In addition, the large differences observed in the HR values of four mare-fetus pairs in four consecutive months support the assumption that there might be 'high-HR' and 'low-HR' fetuses in horses. It can be concluded that the analysis of FHR and FHRV is a promising tool for the assessment of fetal well-being but the applicability of these parameters in the clinical setting and in studs requires further investigation.

Transabdominal ultrasonographic evaluation of fetal well-being in the late-term mare and cow.

In the equine practice, attempts have been made to examine the fetus in the second and third trimester of pregnancy but all of the available methods have limitations. Until now, transabdominal ultrasonography has been regarded as the most informative examination. This method allows us to measure fetal heart rate, fetal activity as well as the quality and quantity of the fetal fluids. A modified biophysical profile for horses was used by several researchers in the USA from the 1990s as a gold standard. However, it is not sensitive enough and, in the authors' experience, professionals can face difficulties during its application (e.g. for measuring aortic diameter and fetal breathing movements). In cows, this method was first used for this purpose by a Canadian research group in 2007. They reported that transabdominal ultrasound was promising but showed low sensitivity in this species. The present studies show that birth weight cannot be predicted from fetal aortic diameter measurement in cows as suggested by other researchers. Transabdominal ultrasound needs special equipment (2-3.5 MHz convex transducer) and basic ultrasonographic knowledge; however, we suggest that in most cases it can be performed with the dam placed in a stock and without shaving the examination area. The method provides useful information within 30-40 minutes, enabling the examiner to determine whether or not the fetus is alive and to recognise placentitis or twins. This technique also allows measuring the combined thickness of the uteroplacental unit, and the authors' ongoing study showed higher normal values in Lipizzaner mares compared to values in other breeds. In conclusion, with the help of advanced techniques, simple and low-cost methods should be developed for the evaluation of the pregnant dam and its fetus to assess fetal viability in the veterinary practice.