Involvement of somatotrophic hormones in the postpartum regulation of ovarian activity in mares.

Mares resume ovarian activity rapidly after foaling. Besides follicle-stimulating hormone (FSH) and luteinizing hormone (LH), the pituitary synthesizes prolactin and growth hormone which stimulate insulin-like growth factor (IGF) synthesis in the liver. We tested the hypothesis that follicular growth is initiated already antepartum, mares with early and delayed ovulation differ in IGF-1 release and that there is an additional IGF-1 synthesis in the placenta. Plasma concentrations of LH, FSH, IGF-1, IGF-2, activin and prolactin. IGF-1, IGF-2, prolactin and their receptors in placental tissues were analyzed at the mRNA and protein level. Follicular growth was determined from 15 days before to 15 days after foaling in 14 pregnancies. Mares ovulating within 15 days postpartum formed group OV (n=5) and mares not ovulating within 15 days group NOV (n=9). Before foaling, follicles with a diameter >1 cm were present in all mares and their number increased over time (p<0.05). Follicle growth after foaling was more pronounced in OV mares (day p<0.001, group p<0.05, day x group p<0.05) in parallel to an increase in LH concentration (p<0.001, day x group p<0.001) while FSH increased (p<0.001) similarly in both groups. Plasma concentrations of IGF-1 and prolactin peaked one day after foaling (p<0.001). The IGF-1 mRNA abundance was higher in the allantochorion but lower in the amnion of OV versus NOV mares (group p=0.01, localization x group p<0.01). The IGF-1 receptor mRNA was most abundant in the allantochorion (p<0.001) and IGF-1 protein was expressed in placental tissue without differences between groups. In conclusion, follicular growth in mares is initiated before foaling and placental IGF-1 may enhance resumption of ovulatory cycles.

Endocrine changes induced by GnRH immunisation and subsequent early re-stimulation of testicular function with a GnRH agonist in stallions.

CONTEXT: Resumption of testicular function after gonadotrophin-releasing hormone (GnRH) immunisation varies among individual animals and some stallions regain fertility only after a prolonged time. AIMS: This study evaluated endocrine effects of GnRH immunisation and early subsequent re-stimulation with a GnRH agonist. We hypothesised that GnRH agonist treatment advances resumption of normal endocrine function in GnRH-vaccinated stallions. METHODS: Shetland stallions were assigned to an experimental and a control group (n =6 each). Experimental stallions were GnRH-immunised twice, 4weeks apart. Each experimental stallion was hemicastrated together with an age-matched control animal when testosterone concentration decreased below 0.3ng/mL. Three weeks later, daily treatment with the GnRH agonist buserelin was initiated (4µg/day for 4weeks followed by 8µg/day). The remaining testicle was removed when testosterone concentration exceeded 0.5ng/mL in vaccinated stallions. Blood was collected for LH, FSH, oestradiol and anti-müllerian hormone (AMH) analyses, and testicular and epididymal tissue were conserved for real-time qPCR and histology. KEY RESULTS: GnRH vaccination reduced blood concentrations of LH and FSH, with a structural deterioration of testicular tissue and disruption of spermatogenesis. Daily buserelin treatment for approximately 60days partially restored gonadotropin secretion and induced a recovery of the functional organisation of the testicular tissue with effective spermatogenesis. CONCLUSIONS: Endocrine testicular function can be restored in GnRH-vaccinated stallions by daily low-dose buserelin treatment. The buserelin treatment protocol may potentially be improved regarding the dose, interval and duration. IMPLICATIONS: Daily buserelin treatment can be recommended for treatment of GnRH-vaccinated stallions with prolonged inhibition of testicular function.

Low progesterone concentration in early pregnancy is detrimental to conceptus development and pregnancy outcome in horses.

High progesterone concentrations in the early luteal phase support pregnancy, whereas subphysiological progesterone concentrations delay embryonic development at least until placentation. In this study, fetal growth and development of pregnancy was investigated in pregnancies with prostaglandin F2α-induced low progesterone concentrations (PGF) in the early luteal phase and control pregnancies (CON) in the same mares (n = 12). Mares were inseminated and in PGF pregnancies received the prostaglandin F2α analogue cloprostenol (62.5 µg) on days 0-3 after ovulation to induce subphysiological progesterone concentrations; CON pregnancies remained untreated. Mares were assigned to PGF or CON treatments in alternating order and received the opposite treatment in the following year. Blood was collected and conceptus size determined repeatedly by transrectal (≤day 101) and transabdominal (>day 101) ultrasonography. After birth, foals were weighed, measured and submitted to a clinical examination. Treatment PGF resulted in fewer pregnancies than CON treatment. All foals born from CON pregnancies were healthy and mature, whereas 4/7 PGF pregnancies were either lost (one embryonic death, one abortion) or resulted in the birth of compromised foals (P = 0.018). Size of the conceptus (e.g., diameter day 49: PGF 6.6 ± 0.7, CON 7.7 ± 0.7 cm, P = 0.006) and embryo proper (e.g., crown rump length day 54; PGF 4.4 ± 0.8, CON 5.8 ± 0.6 cm, P = 0.015) differed between treatments. These size differences decreased over time and at birth PGF foals did not differ significantly from CON foals. In conclusion, reduced progesterone concentration in the early luteal phase leads to delayed conceptus growth beyond placentation and increased pregnancy loss.

Re-stimulation of testicular function in GnRH-vaccinated stallions by daily GnRH agonist treatment.

In stallions temporarily not intended for breeding, reversible suppression of testicular function by vaccination against GnRH can be of interest. In the present study, effects of GnRH agonist treatment on the resumption of testicular function after GnRH vaccination were investigated. Testis size, testosterone release, semen characteristics and behavior were evaluated. We hypothesized that GnRH agonist treatment would restore testicular function. Shetland stallions were assigned to an experimental and a control group (n = 6 each). Experimental stallions were GnRH-immunized twice, four weeks apart. Ejaculates for semen analysis and blood for analysis of testosterone concentration and GnRH antibody titers were collected. Each experimental stallion was hemicastrated together with an age-matched control animal when testosterone concentration decreased below 0.3 ng/mL. Three weeks thereafter, daily treatment with the GnRH agonist buserelin was initiated (4 µg/day for 4 weeks followed by 8 µg/day). The remaining testicle was removed when testosterone concentration exceeded 0.5 ng/mL in vaccinated stallions. Time from exposure to a mare until mounting increased in GnRH-vaccinated stallions and decreased with buserelin treatment. Total sperm count decreased after vaccination but increased only slightly in response to buserelin. Sperm motility and percentage of membrane-intact spermatozoa decreased after vaccination and returned to pre-vaccination values with buserelin treatment. Testosterone concentration and testis volume decreased after GnRH vaccination and started to increase with buserelin treatment. In conclusion, the downregulation of testicular function by GnRH vaccination can be counteracted with buserelin. This approach may be useful in GnRH-vaccinated stallions with prolonged suppression of testicular function.

Epigenetic Changes in Equine Embryos after Short-Term Storage at Different Temperatures.

In embryos subjected to assisted reproductive techniques, epigenetic modifications may occur that can influence embryonic development and the establishment of pregnancy. In horses, the storage temperature during transport of fresh embryos before transfer is a major concern. The aim of this study was, therefore, to determine the effects of two storage temperatures (5 °C and 20 °C) on equine embryos, collected at day seven after ovulation and stored for 24 h, on: (i) morphological development; (ii) expression of candidate genes associated with embryo growth and development, maternal recognition of pregnancy, methylation and apoptosis, and (iii) gene-specific and global DNA methylation. Embryos (n = 80) were collected on day seven or day eight after ovulation and assigned to four groups: day seven control (E7F, fresh); day seven, stored for 24 h at 5 °C (E5C); day seven, stored for 24 h at 20 °C (E20C) and day eight control (E8F, fresh 24h time control). The embryos and the storage medium (EquiHold, holding medium, Minitube, Tiefenbach, Germany) from all treatment groups were analyzed for (i) medium temperature, pH, and lipid peroxidation (malondialdehyde; MDA) and (ii) embryo morphology, mRNA expression and DNA methylation (immunohistochemistry and gene-specific DNA methylation). The size of embryos stored at 5 °C was larger (p < 0.01), whereas embryos stored at 20 °C were smaller (p < 0.05) after 24 h. There were no changes in pH and MDA accumulation irrespective of the group. The mRNA expression of specific genes related to growth and development (POU5F1, SOX2, NANOG), maternal recognition of pregnancy (CYP19A1, PTGES2), DNA methylation (DNMT1, DNMT3A, DNMT3B) and apoptosis (BAX) in the E5C and E20C were either up or downregulated (p < 0.05) when compared to controls (E7F and E8F). The immune expression of 5mC and 5hmC was similar among treatment groups. Percentage of methylation in the CpG islands was lower in the specific genes ESR1, NANOG and DNMT1 (p < 0.001) in E20C embryos when compared to E8F (advanced embryo stage). Therefore, our study demonstrates for the first time the gene-specific and global DNA methylation status of fresh equine embryos collected on days seven and eight after ovulation. Although our results suggest some beneficial effects of storage at 20 °C in comparison to 5 °C, the short-term storage, regardless of temperature, modified gene expression and methylation of genes involved in embryo development and may compromise embryo viability and development after transfer.

First Case of Autochthonous Equine Theileriosis in Austria.

A 23-year-old pregnant warmblood mare from Güssing, Eastern Austria, presented with apathy, anemia, fever, tachycardia and tachypnoea, and a severely elevated serum amyloid A concentration. The horse had a poor body condition and showed thoracic and pericardial effusions, and later dependent edema and icteric mucous membranes. Blood smear and molecular analyses revealed an infection with Theileria equi. Upon treatment with imidocarb diproprionate, the mare improved clinically, parasites were undetectable in blood smears, and 19 days after hospitalization the horse was discharged from hospital. However, 89 days after first hospitalization, the mare again presented to the hospital with an abortion, and the spleen of the aborted fetus was also PCR-positive for T. equi. On the pasture, where the horse had grazed, different developmental stages of Dermacentor reticulatus ticks were collected and subjected to PCR, and one engorged specimen was positive for T. equi. All three amplicon sequences were identical (T. equi genotype E). It is suspected that T. equi may repeatedly be transmitted in the area where the infected mare had grazed, and it could be shown that transmission to the fetus had occurred. Due to the chronic nature of equine theileriosis and the possible health implications of infection, it is advised to include this disease in the panel of differential diagnoses in horses with relevant clinical signs, including horses without travel disease, and to be aware of iatrogenic transmission from inapparent carrier animals.

Stress Response of Beagle Dogs to Repeated Short-Distance Road Transport.

This study aimed to characterize the response of transport-naïve dogs to one and two-hour road transports based on cortisol in saliva and blood plasma, heart rate, heart rate variability (HRV), neutrophil to lymphocyte (N/L) ratio and behavior. Two persons familiar to the dogs were present during transports and control experiments. We hypothesized that transport elicits a stress response, which decreases with repeated transports. Beagle dogs were allocated to three groups (n = 6 each). Group 1 served as control in the stable in week 1 and was transported for one hour in weeks 2, 3 and 4. Groups 2 and 3 served as controls in a non-moving vehicle and in the stable, respectively, in week 2. All three groups were transported for two hours in week 6. Cortisol concentration increased during transports (p < 0.001), and this increase remained constant with repeated transports. Cortisol release during two-hour transports was not affected by transport experience. Cortisol concentration increased twofold in plasma and eightfold in saliva, indicating an increase in free cortisol. The N/L ratio increased during transport (p < 0.05). Heart rate increased at the beginning of transport while HRV decreased (p < 0.001). Heart rate and HRV neither differed among weeks nor between animals with different transport experience. During transports, but also in the stationary vehicle, dogs were mostly sitting, and time spent standing decreased during experiments (p < 0.001). Licking the mouth was the most frequent behavior during transports but not in the stationary vehicle (p < 0.01). In conclusion, a transport-induced stress response was evident in dogs. There was no habituation with repeated transports, and transported dogs may suffer from motion sickness.

Differences in Endocrine and Cardiac Changes in Mares and Her Fetus before, during, and after Parturition in Horses of Different Size.

Equine fetomaternal monitoring is based on endocrine and cardiac parameters which may differ among small, medium-size, and full-size horses. Therefore, Shetland (n = 6), Haflinger (n = 8), and Warmblood pregnancies (n = 9) were studied during late gestation and at foaling. Weight of mares, foals and placenta, plasma progestin and cortisol concentration, heart rate and heart rate variability (HRV) were determined. Foal weight always approximated 10% of mare weight but relative placenta weight was highest in full-size mares (p < 0.05). Progestin (p < 0.001) and cortisol (p < 0.05) concentration was highest in full-size mares. Progestin concentration decreased towards parturition (p < 0.001) while cortisol concentration increased (p < 0.01). Maternal heart rate increased before foaling with the most pronounced increase in small mares (p < 0.001). The HRV increased during foaling and decreased when delivery was completed (p < 0.001). Changes were most pronounced in full-size mares (p < 0.001). Atrio-ventricular blocks regularly occurred in parturient full-size mares but only occasionally in medium-size and small mares (time p < 0.05, time × group p < 0.05). This may reflect breed differences in cardiovascular efficiency. Fetal heart rate decreased towards birth (p < 0.001) with the most pronounced decrease in full-size horses (p < 0.01). Fetal HRV showed no consistent changes before birth but increased when the foal was born (p < 0.001), this increase being most pronounced in full-size foals (p < 0.05). In conclusion, this study demonstrates both similarities and differences in peripartum endocrine and cardiac changes in horses of different size.

Road Transport of Late-Pregnant Mares Advances the Onset of Foaling.

Cortisol is involved in the initiation of parturition and we hypothesized that increased maternal cortisol release advances the onset of foaling. Transport is a stressor for horses and induces an increase in cortisol release. To determine stress effects on the time of foaling, late-pregnant mares were transported by road for 3 hours (n = 12) or remained in their stable as controls (n = 4). Starting on day 325 of gestation, saliva and blood samples were taken for cortisol and progestin analysis, respectively. Fetomaternal electrocardiograms were recorded repeatedly. Mares were checked for impending parturition and changes in precolostrum pH. When pH decreased to 6.5, mares were either transported or left untreated. After birth, saliva was collected repeatedly from mares and their foals and heart rate (HR) was recorded. Foals were checked for maturity and health. Gestation length was 337 ± 2 days in stressed and 336 ± 2 days in control mares. Cortisol concentration increased from 3.3 ± 0.9 to 8.4 ± 0.8 ng/mL in transported mares (P < .001) and remained constant in controls. Maternal HR and heart rate variability (HRV) did not differ between groups and neither fetal HR nor HRV changed in response to transport. In transported mares, time from precolostrum decrease to parturition was shorter (40 ± 10 hours) than the respective time in controls (134 ± 49 hours, P < .01). Neither duration of foaling nor times to first standing and suckling of foals or the postnatum increase in HR and decrease in HRV differed between groups. In conclusion, transport-induced maternal cortisol release may have advanced the onset of foaling.

Controlled delay of the expulsive phase of foaling affects sympathoadrenal activity and acid base balance of foals in the immediate postnatal phase.

Stress at foaling has been demonstrated to delay birth. In this study, we followed the hypothesis that even a short delay of foaling increases catecholamine and cortisol release in foals, induces acidosis and impairs neonatal adaptation. Foaling was prolonged for 5 min by transferring mares to an unfamiliar environment at rupture of the allantochorion (group delay, n = 6) while control mares (n = 5) were left undisturbed. In their foals, times from birth to first standing and first suckling, heart rate, heart rate variability (HRV) and salivary cortisol concentration were analysed. Blood for analysis of epinephrine, norepinephrine, hematology and blood gases was collected directly and 30 min after birth. Statistical comparisons were made by repeated measures ANOVA. Times to first standing and suckling did not differ between groups. Fetal heart rate remained unchanged during birth and increased within 15 min postnatum (p < 0.001) while HRV decreased during the first hour of life in foals of both groups (p < 0.05). Immediately after birth, actual base excess was lower in foals with delayed birth than in control foals (p < 0.05). Epinephrine concentration immediately after birth was higher in group delay foals and increased from 0 to 30 min after birth in control foals (time p < 0.001, time x group p = 0.001). Cortisol concentration peaked at 1 h after birth in both groups (p < 0.001). Leukocyte and PMN count decreased from 0 to 30 min after birth (p < 0.001). In conclusion, a 5-min delay at foaling affected epinephrine release and acid base balance, but was without further effect on neonatal adaptation.

External stress increases sympathoadrenal activity and prolongs the expulsive phase of foaling in pony mares.

Mares usually give birth when they perceive their environment as safe and therefore disturbance at foaling may inhibit labor. In this study, foaling mares were transferred to an unfamiliar environment at rupture of the allantochorion (stress, n = 6) or were left undisturbed (control, n = 5). The progress of foaling, heart rate, heart rate variability (HRV) and plasma catecholamine, oxytocin and cortisol concentration were determined. In stressed mares, time from rupture of the allantochorion to appearance of the fetal feet (5.3 ±â€¯1.1 vs. 1.6 ±â€¯0.4 min) and total length of fetal expulsion were longer than in controls (both p < 0.05). Heart rate decreased during the expulsive phase of foaling in control mares (p < 0.01) but increased transiently in stressed mares. Heart rate calculated as percentage of the baseline was higher in stressed than in control mares (p = 0.05). HRV variables SDRR (standard deviation of the beat-to-beat interval) and RMSSD (root mean square of successive beat-to-beat differences) increased during foal expulsion (SDRR p < 0.01 and RMSSD p < 0.05). The increase in HRV was delayed in stressed compared to control mares (SDRR and RMSSD time x group p < 0.05). Plasma epinephrine and norepinephrine concentrations calculated as area under the curve for the expulsive phase of foaling were higher in stressed than control mares (p < 0.05). Concentrations of oxytocin and cortisol were elevated during foal expulsion (both p < 0.001) but not different between groups. In conclusion, disturbance of mares during expulsion of the foal prolonged foaling. This effect is most likely mediated via increased sympathetic activity and not inhibition of oxytocin release.