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

Hormone secretion by the maternal ovaries, trophoblast/placenta and fetus occurs sequentially, creating distinct steroid metabolomic 'signatures' in systemic blood of pregnant mares that vary with gestational stage. Algorithms were developed to predict the gestational day (GD) from the maternal steroid metabolome (nine steroids; pregnenolone (P5), progesterone (P4), 5α-dihydroprogesterone (DHP), 17α-hydroxyprogesterone, allopregnanolone, 20α-hydroxy-DHP, 3ß,20α-dihydroxy-DHP, DHEA and androstenedione) determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS) of eight thoroughbred mares sampled longitudinally throughout pregnancy. A physiologically based model was developed to infer rates of steroid secretion during chorionic gonadotropin secretion, the luteo-placental shift and by the equine feto-placenta unit, demonstrating more variability in P5 and DHP than P4. The average of four empirical models, using nine steroids to predict GD, was calibrated (five mares, R2 = 0.94, RMSE = 20 days) and validated (three mares, R2 = 0.84, RMSE = 32 days). Validation performance was improved using paired samples taken 14 or 30 days apart (RMSE = 29 and 19 days, respectively). A second validation used an independent dataset (single serum samples from 56 mixed breed mares, RMSE = 79 days) and an additional longitudinal subset from the same population sampled monthly throughout gestation (seven mares, RMSE = 42 days). Again, using paired samples improved model performance (RMSE = 32.5 days). Despite less predictive performance of the mixed breed than the thoroughbred datasets, these models demonstrate the feasibility and potential for using maternal steroid metabolomic algorithms to estimate the stage of gestation in pregnant mares and perhaps monitor fetal development.

Prenatal diagnosis of foetal hydrocephalus and suspected X-linked recessive inheritance of cleft lip in a Chihuahua.

A 3.5-year-old, 2.9 kg, multiparous Chihuahua presented with abdominal distension; pregnancy was diagnosed. On Day 7 before parturition, prenatal sonograms showed anechoic bilateral dilated cerebral lateral ventricles, suggesting fluid-filled regions (ventriculomegaly) in one foetus. A Caesarean section was performed and the male newborn had an abnormally enlarged dome-shaped head and a cleft lip, and died 6 days after birth. According to the family pedigree, the X-linked recessive inheritance of an orofacial cleft from the unaffected mother was suggested. This report clearly demonstrates that canine foetal ventriculomegaly (hydrocephalus) can be diagnosed in utero. For dog breeds predisposed to congenital ventriculomegaly, early detection is important for the prediction of perinatal survival and adequate supportive care can be applied at delivery.

IFPA meeting 2018 workshop report I: Reproduction and placentation among ocean-living species; placental imaging; epigenetics and extracellular vesicles in pregnancy.

Workshops are an important part of the IFPA annual meeting as they allow for discussion of specialized topics. At IFPA meeting 2018 there were nine themed workshops, four of which are summarized in this report. These workshops discussed new knowledge and technological innovations in the following areas of research: 1) viviparity in ocean-living species; 2) placental imaging; 3) epigenetics; and 4) extracellular vesicles in pregnancy.

Evaluating Information Obtained from Diagnosis of Pregnancy Status of Beef Herds.

Good reproductive efficiency is critical for economic sustainability of beef herds. Standard measurements of reproductive success are the percentage of cows exposed to bulls at the start of the breeding season that are pregnant at midgestation and the percentage of pregnant cows that give birth to a live calf. Converting fetal age data to a reproductive profile displaying pregnancy percentages by 21-day periods provides information for the diagnosis of suboptimal reproductive efficiency and guides intervention strategies. The value can be amplified by evaluating reproductive profiles by animal age or management group.

Intra-uterine exposure of horses to Sarcocystis spp. antigens / Exposição intrauterina ao Sarcocystis spp. em equinos

The aim of this study was to examine the intra-uterine exposure to Sarcocystis spp. antigens, determining the number of foals with detectable concentrations of antibodies against these agents in the serum, before colostrum ingestion and collect data about exposure of horses to the parasite. Serum samples were collected from 195 thoroughbred mares and their newborns in two farms from southern Brazil. Parasite specific antibody responses to Sarcocystis antigens were detected using the indirect immunofluorescent antibody test (IFAT) and immunoblot analysis. In 84.1% (159/189) of the pregnant mares and in 7.4% (14/189) of foals we detected antibodies anti-Sarcocystis spp. by IFAT. All samples seropositive from foals were also positive in their respective mares. Serum samples of seropositive foals by IFAT, showed no reactivity on the immunoblot, having as antigens S. neurona merozoites. In conclusion, the intra-uterine exposure to Sarcocystis spp. antigens in horses was demonstrated, with occurrence not only in mares, but also in their foals, before colostrum ingestion these occurrences were reduced.

O objetivo deste estudo foi avaliar a exposição intrauterina ao Sarcocystis spp., para determinar o número de potros que possuem concentrações detectáveis de anticorpos contra esses agentes no soro, antes da ingestão do colostro, por meio da coleta de dados sobre a exposição a esses protozoários nos equinos. Amostras de soro foram coletadas de 195 éguas puro-sangue e seus respectivos potros recém-nascidos, em duas fazendas localizadas na região Sul do Brasil. Os testes utilizados na detecção de anticorpos específicos para o Sarcocystis foram a reação de imunofluorescência indireta (RIFI) e análise por meio de immunoblot. Pela RIFI, em 84,1% (159/189) das éguas e em 7,4% (14/189) dos potros foram detectados anticorpos anti-Sarcocystis spp. Todas as amostras soropositivas dos potros também foram positivas para suas respectivas mães. As amostras de soro dos potros soropositivos na RIFI, não apresentaram reatividade no immunoblot, tendo como antígenos merozoítos de S. neurona. Em conclusão, foi demonstrada a exposição intrauterina de Sarcocystis spp. em equinos, com ocorrência em éguas, porém, em seus respectivos potros, antes da ingestão de colostro a ocorrência foi reduzida.

In utero phenotyping of mouse embryonic vasculature with MRI.

The vasculature is the earliest developing organ in mammals and its proper formation is critical for embryonic survival. MRI approaches have been used previously to analyze complex three-dimensional vascular patterns and defects in fixed mouse embryos. Extending vascular imaging to an in utero setting with potential for longitudinal studies would enable dynamic analysis of the vasculature in normal and genetically engineered mouse embryos, in vivo. In this study, we employed an in utero MRI approach that corrects for motion, using a combination of interleaved gated acquisition and serial coregistration of rapidly acquired three-dimensional images. We tested the potential of this method by acquiring and analyzing images from wildtype and Gli2 mutant embryos, demonstrating a number of Gli2 phenotypes in the brain and cerebral vasculature. These results show that in utero MRI can be used for in vivo phenotype analysis of a variety of mutant mouse embryos.

Defining fetal growth restriction in mice: A standardized and clinically relevant approach.

The increasing number of mouse models of fetal growth restriction (FGR) make it crucial to standardize the way FGR is defined. By constructing growth curves in the placental-specific Igf2 knockout mouse (P0) it was demonstrated that 93% of P0 fetuses fell below the 5th centile of wild-type weights at E18.5, up from 44% at E16.5. This analysis, coupled with anthropomorphic measurements showing evidence of head sparing in P0 fetuses, allows clinical comparisons of FGR in mice through the use of clinically relevant growth curves. We suggest this as a standardized approach to defining FGR in mouse, and other animal models.

Bovine fetal DNA in the maternal circulation: Applications and implications.

OBJECTIVE: The main aim of the present study was to detect bovine fetal DNA in the maternal circulation, a relatively unexplored subject in the literature. STUDY DESIGN: DNA was extracted from blood of 84 primipara cows (Bos indicus) at different gestational ages (30-270 days) and from 100 adult animals (50 males and 50 non-pregnant cows). The samples were analyzed using PCR with primers for TSPY gene. RESULTS: Molecular results matched the fetal phenotypic gender in all 47 male and 37 female fetuses, including early pregnancy, and in control animals. CONCLUSIONS: These results evidence a bovine transplacental fetal DNA passage.

Fetal sex determination of macaque monkeys by a nested PCR using maternal plasma.

Non-invasive fetal sex determination is required for biomedical studies, in which some sexual difference would be expected in fetal events, in order to make a choice of male or female fetus. To detect male fetal DNA of the sex-determining region Y gene (SRY) in maternal macaque plasma, nested real-time PCR using the SYBR Green system was developed. In all cases of pregnant macaques with male fetuses, a nested PCR product of SRY was amplified from the mother's plasma, while no amplicon was detected in any case of pregnancy with a female fetus. Interestingly, fetal SRY DNA appeared to be cleared rapidly from the maternal blood after parturition. The current method is sensitive and can be performed with a regular PCR machine.

The freemartin syndrome: an update.

The freemartin condition represents the most frequent form of intersexuality found in cattle, and occasionally other species. This review considers the current state of knowledge of freemartin biology, incidence, experimental models, diagnosis, uses for freemartins in cattle herds, occurrence in non-bovine species, effects on the male, and highlights potential new research areas. Freemartins arise when vascular connections form between the placentae of developing heterosexual twin foeti, XX/XY chimerism develops, and ultimately there is masculinisation of the female tubular reproductive tract to varying degrees. With twinning rates in Holstein cows increasing, there will be greater economic importance to establish early diagnosis of the freemartin and the detection of the less common single born freemartin. New diagnostic methods based on the detection of Y-chromosome DNA segments by polymerase chain reaction (PCR) show improved assay sensitivity and efficiency over karyotyping and clinical examination. The implications for the chimeric male animal born co-twin to the freemartin are contentious as to whether fertility is affected; if germ cell chimerism does indeed occur; and, if there are any real effects on the sex ratio of offspring produced. In beef cattle, the freemartin carcass has similar characteristics to normal herdmates. Hormonal treatment of freemartins for use as oestrous detectors has been used to obtain salvage value. The biology of freemartin sheep has recently been studied in detail, and the condition may be increasing in prevalence with the introduction of high fecundity genes into flocks. Potential new research areas are discussed, such as detection of foetal DNA in maternal circulation for prenatal diagnosis and investigation of the anti-tumour properties of Mullerian inhibiting substance (MIS). The freemartin syndrome will always be a limiting factor in cattle and to a lesser extent in sheep production systems that have the goal to produce multiple reproductively normal female offspring from a single dam without using sex predetermination.

Percutaneous collection of fetal fluids for detection of bovine viral diarrhea virus infection in cattle.

OBJECTIVE: To develop a method for percutaneous collection of fetal fluid from cattle in the late stages of gestation and determine whether bovine viral diarrhea virus (BVDV) can be isolated from such fluids. DESIGN: Case series. ANIMALS: 169 pregnant beef cattle. PROCEDURE: Animals were restrained in a squeeze chute, and hair was clipped from a region of the right flank. Pregnancy was confirmed, and fetal fluids were identified by means of abdominal ultrasonography. Fetal fluid was collected with a spinal needle. Virus isolation was performed on fetal fluids, WBC lysates from 160 live calves, and tissues from 12 calves that died or were aborted. Blood samples collected from adult cattle were assayed with an immunoperoxidase monolayer assay. RESULTS: Fourteen animals aborted or delivered premature calves within 3 weeks after fetal fluid collection; however, it could not be determined whether this was a complication of the procedure or attributable to other factors. Results of BVDV isolation from fetal fluid samples were negative for 168 animals. However, a noncytopathic BVDV was isolated from fetal fluid obtained from a 2-year-old heifer; results of the immunoperoxidase assay of serum from this heifer were also positive, and a noncytopathic BVDV was isolated from tissue specimens from a stillborn calf produced by this heifer. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that fetal fluids can be collected percutaneously from cattle in the late stages of gestation and that virus isolation performed on fetal fluids can be used to identify fetuses infected with BVDV in utero. However, safety of the procedure could not be evaluated.

Clinical signs and treatment of chronic uterine torsion in two mares.

Two mares were evaluated because of chronic uterine torsions of 2 and 4 weeks' duration; both were pyrectic, anemic, tachycardic, and anorectic, had signs of depression, and had an abnormal uterus and contents on transrectal examination. Both mares underwent cesarean section for lysis of adhesions from the uterus to the peritoneum, correction of the torsion, and ovariohysterectomy Both mares recovered with only minor complications and returned to be used as riding horses. Chronic uterine torsion should be considered in mares in late stages of gestation that have vague clinical signs and transrectal palpation findings that are unlike those described in typical cases of uterine torsion. Prognosis for life can be good after treatment by ventral midline celiotomy, cesarean section, correction of the torsion, and ovariohysterectomy.

Prenatal infection with Setaria marshalli (Boulenger 1921) in cattle.

A full year's epidemiological study was conducted in Japan on prenatal infection with Setaria marshalli in cattle. A total of 65 bovine fetuses of abattoir origin, both male and female of 2-9 months of age, were examined postmortem. Results showed that S. marshalli adult worms were detected in the peritoneal cavity of six bovine fetuses of 7-9 months of age during the months of October-December, but not in fetuses of any age during the months of January-September. Infection was not detected in any fetuses under the age of 7 months at any given month of the year. This is the first observation to demonstrate prenatal S. marshalli infection in fetuses. Since S. marshalli has not been detected in cattle older than 2 years, it is speculated that prenatal infection is the common type, while postnatal infection is rather uncommon. Considering fetal age and the seasonal factor, a fetus of age 4-5 months would most frequently develop prenatal infection in the June-August period, when mosquitoes are active as vectors. It is concluded, therefore, that S. marshalli prenatal infection develops during the middle stage of fetal life in summer.

Prenatal diagnosis of bovine citrullinaemia.

Holstein/Friesian embryos, progeny of citrullinaemia heterozygotes, were transferred to recipient cows. The citrullinaemia genotype of the fetus/calf was established, at birth, by estimation of citrulline concentration in plasma or by analyses of argininosuccinate synthetase exon 5 sequences in DNA from blood leucocytes. Between 115 and 125 days of gestation amniotic fluid was collected transabdominally from nine recipient cows. The mean citrulline concentration in amniotic fluid from fetuses unaffected with citrullinaemia was 28 microM, SD 9 and range 15 to 38 microM. Citrulline concentration was 107 and 130 microM in amniotic fluid from two fetuses homozygous for the citrullinaemia mutation. Only the normal bovine citrullinaemia sequence was detected in cells cultured from amniotic fluid taken from two homozygous normal and two heterozygous fetuses. Both normal and mutant sequences were found in cells cultured from amniotic fluid collected from three of five heterozygous fetuses. Only the mutant sequence was found in cells cultured from the two citrullinaemia affected fetuses. MHC class II DRB3 haplotyping revealed that in four instances cultured cells were from the surrogate mother, not the fetus, these included samples from two heterozygotes in which only the normal sequence was detected in amniotic fluid.

Prediction of calving date in beef cows by real-time ultrasonic scanning.

Three hundred pregnant beef cows between 35 and 125 days of gestation were scanned ultrasonically and their calving dates were predicted from measurements of fetal parts. The mean difference between the actual and predicted calving dates was 0.9 day with a standard deviation of 9.0 days. The accuracy and precision of the prediction of calving date were sufficient to be of benefit in the management of cows in late pregnancy and at calving.