Determination of Salivary Cortisol in Donkey Stallions.

Salivary cortisol provides information about free plasma cortisol concentration, and salivary sampling is a noninvasive well-tolerated procedure. The aim of this study was to validate a commercial enzyme immunoassay for the determination of salivary cortisol in donkeys. Saliva samples were collected in four donkey stallions on 13 nonconsecutive days at 8:30 AM to avoid circadian variation. Animals were already accustomed to be handled. Saliva was collected using a swab inserted at the angle of the lips, placed onto the tongue for 1 minute and returned into a polypropylene tube. Tubes were centrifuged, and at least 1 mL of saliva was aspirated from each sample and frozen at -20°C until analysis. A commercial enzyme immunoassay kit without extraction was used for determination of cortisol in saliva. Median cortisol concentrations with minimum and maximum value were calculated. Recovery of cortisol standard in donkey saliva was between 97.3% and 99.7%, and serial dilution of donkey saliva samples with assay buffer resulted in changes in optical density parallel to the standard curve. Cross-reactivity of the antiserum was 10.4% with 11-deoxycortisol, 5.2% with corticosterone, 0.4% with 11-deoxycorticosterone, 0.2% with cortisone, and <0.1% with testosterone, progesterone, and estradiol. The intra-assay coefficient of variation was 10.7%, the interassay variation was 8.0%, and the minimal detectable concentration was 0.01 ng/mL. The results of the present study demonstrate the validity of a commercial kit to determine the concentration of cortisol in donkey saliva as already reported in other species.

Anti-Müllerian hormone profiling in prepubertal horses and its relationship with gonadal function.

Anti-Müllerian hormone (AMH) has gained increasing interest as a biomarker for assessment of gonadal activity. The ability to predict the ovarian follicular reserve of prepubertal female horses (fillies) or to identify stallions with testicular pathologies already during their prepubertal life has not been analyzed so far. Both would help to select fertile horses and reduce costs associated with keeping animals. The objectives of the present study were to (1) assess AMH, LH, FSH, progesterone (females) and testosterone (males) dynamics in prepubertal horses from birth onwards and (2) determine whether AMH concentrations detected in plasma of prepubertal female and male horses are correlated with postpubertal gonadal development. Warmblood foals (n = 30, 14 females, 10 normal males and 6 males with abnormal testicular development) born between February and May of two consecutive years (n = 28 in the first year and n = 2 the next year), were included in the study. Information on gestational length, parity of the dam and placental weight was collected for all foals. Blood samples for hormone analysis were collected from birth onwards every four weeks up to the age of one year. At two years, blood samples were collected on the day when antral follicle count (AFC) and total testicular volume (TTV) were assessed. AMH was detectable in the plasma of all animals from birth onwards and its concentration was significantly higher (P < .001) in males than in females, regardless of testicular development. In males, AMH and testosterone concentration were similar for all animals during the first year of life, regardless of testicular development. At two years, AMH concentration was higher (P < .05) in males with abnormal testicular development than in those with normal testes. In females, AMH concentration at two years was correlated with AMH concentration at birth (P < .05) and with AFC (P < .001). At birth, LH concentration was lower (P < .05) in stallions with abnormal testes (0.3 ±â€¯0.2 ng/ml) than in controls (0.6 ±â€¯0.2 ng/ml). A high negative correlation between AMH concentration and gestation length was observed in males during the first eight weeks of life (P < .01, r = -0.64 to -0.71). Elevated progesterone concentrations over 1 ng/ml were observed in several females starting with 20 weeks of age. This was paralleled by an increase in AMH concentration and was preceded by FSH and LH increases. In conclusion, AMH determination can be reliably used from two years onwards to identify stallions with abnormal testicular development, but it is inconclusive before puberty. In female horses, determination of AMH concentration at a prepubertal age allows for prediction of AMH and AFC after puberty. We suggest that premature luteinisation occurs before the onset of puberty in female horses and that LH secretion in the perinatal period is involved in testicular development and descent in the horse.

Effects of sex, pregnancy and season on insulin secretion and carbohydrate metabolism in horses.

In pregnant mares, peripheral insulin antagonism channels glucose preferentially to the foetus. On the other hand, horses reduce their metabolic activity in winter. Taking these aspects of equine pregnancy and metabolism together, we hypothesized that glucose clearance from blood and the insulin response to glucose do not only change throughout gestation but also with season. To test this hypothesis, the glucose and insulin response to an oral glucose test and relative insulin release were analysed in pregnant mares (n=12) and in geldings (n=10) as controls. Animals were tested in June, September, December, and in March (geldings) and on day 320 of gestation (mares). Furthermore, the 6 mares foaling early and 6 foaling later in the year were compared. In mares and geldings, plasma glucose concentration increased after glucose feeding (p<0.001). The increase was more pronounced in mares (p<0.05) and increased from June to December in mares (p<0.001) but not geldings (month x group p<0.05). This indicates constant glucose clearance in geldings but reduced clearance in pregnant mares. A partial insulin resistance is thus induced by pregnancy independent from season. Insulin release increased after glucose feeding (p<0.001) similarly in geldings and mares. The insulin response to glucose and relative insuslin release increased from June to December (p<0.001) indicating seasonal changes in ß-cell sensitivity. Glucose and insulin concentration did not differ between early and late foaling mares. In conclusion, in horses, ß-cell sensitivity to glucose is affected by season while insulin sensitivity during pregnancy decreases independent from season.

Effects of environmental temperature and season on hair coat characteristics, physiologic and reproductive parameters in Shetland pony stallions.

We hypothesized that housing of stallions in a thermoneutral temperature zone during autumn and winter does not only influence metabolism and hair shedding but also improves the characteristics of raw and processed semen. Fertile Shetland pony stallions were followed from October to June. This time coincided with the seasons autumn, winter and spring. Ponies were kept in outside paddocks (group CON, n = 8) or in indoor stables (group ST, n = 8) from October to March when ST stallions returned to outdoor paddocks, but ponies remained in the same groups. The rectal temperature was measured once weekly. Heart rate, heart rate variability, testosterone and cortisol concentration in blood as well as quality and length of the coat were determined. Semen was collected once weekly and raw semen characteristics were analyzed. The characteristics of cooled-stored and cryopreserved semen were determined once monthly. During the stabling period, environmental temperature for group ST averaged 13.6 ± 2.3 and for group CON 5.6 ± 4.2 °C. The mean rectal temperature was higher (p < 0.05) in ST than in CON stallions. All hair coat parameters underwent seasonal changes (p < 0.001) and differed between groups (p < 0.05) with shorter guard hair, slower hair regrowth and earlier hair change in ST stallions. Season influenced heart rate which was highest in autumn, lowest in winter and intermediate in spring but did not differ between groups. Testosterone and cortisol concentrations in blood as well as sexual behavior underwent seasonal changes but did not differ between CON and ST stallions. Gel-free semen volume and total sperm count were influenced by season (p < 0.01) and showed a more pronounced increase from winter to spring in CON than in ST stallions (p < 0.05) while no differences with regard to sperm concentration in raw semen were detected. Progressive motility of spermatozoa in raw semen was highest in spring (p < 0.05) but not affected by group. In cooled-stored and cryopreserved semen, neither season nor group affected total motility, progressive motility or membrane integrity. In conclusion, environmental temperature during autumn and winter had clear results on body temperature as well as hair coat characteristics in Shetland stallions. Simultaneously determined effects on semen characteristics were minimal indicating that reproductive function in the horse is more dependent on day length i.e. the geophysical year than on other environmental factors.

Oxytocin treatment does not change cardiovascular parameters, hematology and plasma electrolytes in parturient horse mares.

In mares, foaling is associated with changes in hematology, plasma electrolytes, blood pressure and heart rate and it has been hypothesized that these are induced by oxytocin. To test this hypothesis, mares (n = 8-14/group) were treated with oxytocin (OT; 20 I.U.) or saline (CON) at 1 h (test A) and 12 h after foaling (test B) and during first postpartum diestrus (test C). Heart rate, heart rate variability (HRV), atrioventricular blocks, salivary cortisol concentration, blood pressure, plasma electrolytes and blood count were determined. Heart rate decreased from test A to C (P < 0.001) but at no time differed between groups. The HRV, blood pressure and occurrence of atrioventricular blocks did not change in response to oxytocin. Cortisol concentration decreased from test A to C (P < 0.001). Oxytocin induced a cortisol release in test B (time x treatment P < 0.001, time x test P < 0.001). Plasma sodium and chloride concentrations decreased from test A to C (P < 0.001) but did not differ between groups. In test A, potassium concentration increased in CON but not OT mares (time P < 0.01, time x test P < 0.01, time x treatment P < 0.05). Polymorphnuclear cell (PMN) numbers in blood decreased from test A to C (P < 0.001) while lymphocytes increased (P < 0.05). At no time PMN and lymphocytes differed between groups. Oxytocin treatment had no effect on skin temperature. In conclusion, except for a limited effect on cortisol release, oxytocin was without effect and the hypothesis of oxytocin-induced alterations in cardiac parameters, plasma electrolytes and hematology of foaling mares was not verified.

Cooled-storage of equine semen does not induce major changes in sperm DNA methylation.

A decrease in fertility of equine semen during cooled-storage so far has mainly been attributed to changes in sperm membrane function. In the present study we hypothesized that cooled-storage also changes the sperm DNA methylation level. For this purpose, semen was collected from 10 fertile stallions and processed for cooled-storage at 5 °C. Two final concentrations, 50 × 106 and 100 × 106 cells/mL, were used. Semen was analyzed for total motility, progressive motility, membrane integrity, phosphatidylserine translocation (PST), mitochondrial membrane potential and chromatin condensation, immediately after processing and at 24 h-intervals until 72 h of storage. DNA cytosine methylation was assessed by ELISA after DNA extraction and denaturation. DNA methylation did neither change over time nor was affected by semen concentration. Total motility, progressive motility, membrane integrity, PST, mitochondrial membrane potential and chromatin condensation changed over storage time, but no differences between semen concentrations could be demonstrated. The results demonstrate that cooled-storage of equine semen does not induce changes in sperm DNA cytosine methylation. In cooled-semen of good quality, a concentration of 100 × 106 sperm/mL does not affect semen longevity. It can be concluded that a better fertility of cooled-stored than cryopreserved stallion semen is at least in part a result of only minor influences of cooled-storage on DNA integrity and methylation.

Anti-Muellerian hormone, inhibin A, gonadotropins, and gonadotropin receptors in bull calves after partial scrotal resection, orchidectomy, and Burdizzo castration.

Eight-week-old calves were either castrated by partial scrotal resection (SR) without removing the testes (n = 10), Burdizzo (BZ) clamp (n = 10), orchidectomy (OR; n = 10), or were left gonad intact as controls (CO; n = 10). Concentrations of anti-Muellerian hormone (AMH), inhibin A, luteinizing hormone (LH), and follicle-stimulating hormone (FSH) in plasma were determined from 16 to 48 weeks of age. At 18 months, testes of SR, BZ, and CO bulls were obtained and the immunolocalization of LH and FSH receptors and AMH analyzed. Concentration of AMH in plasma of CO and SR bulls decreased with increasing age (P < 0.001). A similar AMH profile in CO and SR indicates that SR did not induce a true cryptorchid state. In groups OR and BZ, AMH was undetectable. Plasma inhibin concentration was higher in groups CO and SR than BZ and OR (P < 0.001). Plasma LH and FSH concentrations decreased over time (P < 0.001) and were higher in groups BZ and OR than SR and CO (P < 0.001). In the testes, immunolabeling for AMH existed in Sertoli cells of CO and SR but not BZ bulls. FSH receptors were localized in Sertoli cells, Leydig cells, spermatocytes, and the epididymis of CO and SR animals, whereas LH receptors were restricted to Leydig cells. In BZ animals, FSH and LH receptors and AMH were absent, indicating complete testicular degeneration. In conclusion, AMH is a more reliable marker for the presence of testicular tissue in bulls than inhibin. Scrotal resection did not induce a true inguinal cryptorchid state but affected testicular responsiveness to gonadotropic stimulation.

Effects of periovulatory gonadotrophin treatment on luteal function and endometrial expression of selected genes in cyclic pony mares.

Progestin concentration in plasma during the early luteal phase is crucial for endometrial function and conceptus development. We hypothesized that periovulatory gonadotrophin treatment via support of luteal function affects endometrial gene expression in horses. Effect of age was analyzed as well. Shetland mares (n = 8, age 4-25 years) were assigned to the following treatments during five consecutive cycles in alternating order following a cross-over design: treatment hCG/-: preovulatory injection of hCG, but no gonadotrophin injection at detection of ovulation, treatment -/hCG: no preovulatory gonadodrophin injection, but injection of hCG at detection of ovulation, treatment eCG/-: preovulatory injection of eCG, but no gonadotrophin injection at detection of ovulation, treatment -/eCG: no preovulatory gonadotrophin injection, but injection of eCG at detection of ovulation, treatment control: no treatment. Concentration of progestin was analyzed by ELISA from the day of ovulation until Day 10. On Day 10, endometrial cells were collected transvaginally by cytobrush technique. Expression of mRNA of cyclooxygenase-2 (COX-2), prostaglandin F2α-synthase, prostaglandin E-synthase, progesterone receptor (PR), estradiol receptor (E2R), acyl-CoA-dehydrogenase (ACAD), uteroglobin (UGB), uteroferrin, and uterocalin was analyzed by RT qPCR. Immunohistological staining of endometrial tissue, obtained via biopsy, was performed for COX-2, PR and UGB. The P4 concentration was influenced by day of cycle (P < 0.01), but not by treatment. No effects of age on gene expression were determined. Neither of the periovulatory gonadotrophin treatments nor age influenced mRNA expression of the genes of interest. Treatment did also not affect immunohistological staining of the endometrium. In contrast, age affected the percentage of PR positive stromal cells (e.g. mare 1 (4 years): 65.5 ± 2.6, mare 2 (24 years): 82.7 ± 2.2%, P < 0.05) and COX-2 positive stained ciliated cells (e.g. mare 1: 15.8 ± 2.9, mare 2: 33.4 ± 6.0%, P < 0.05). In conclusion, no effects of periovulatory gonadotrophin treatment and age on endometrial gene expression in luteal phase pony mares were reported. A lack of treatment effects on luteal function and expression of PRs in the endometrium can at least in part be explained by differences in the reproductive physiology between horses and ponies.

Stress response and cardiac activity of term and preterm calves in the perinatal period.

This study tested the hypothesis of gestational age affecting fetal cardiac activity and the stress response at birth. Heart rate (HR), heart rate variability variables, SD of the beat-to-beat interval and root mean square of successive beat-to-beat differences, and postnatal salivary cortisol concentration were studied in calves born at term (Term, n = 7, gestation length 286.3 ± 2.1 days) or after induction of parturition (Preterm, n = 7, gestation length 279.6 ± 0.2 days). Observation periods covered the last month of gestation (phase A), the last hours before birth including the first stage of labor (phase B), and the neonatal period (phase C). Fetal HR decreased in phase A (P < 0.001) and did not differ between groups. During phase B, HR increased (P < 0.05) and was higher in Preterm than in Term calves in phases B (P < 0.05) and C (P < 0.01). In Term calves, heart rate variability increased from Day 6 until birth (P < 0.05). At birth, SD of the beat-to-beat interval was higher in Term than in Preterm calves (P < 0.01). On Day 1 after birth (phase C), HR accelerations were more frequent in Term than Preterm calves (P < 0.01), whereas decelerations were more frequent in Preterm calves (P < 0.05). Cortisol concentration increased postnatally (P < 0.001) and was correlated with gestation length (r ≥ 0.68, P < 0.01). Because of a certain degree of immaturity, the ability to cope with the stress of birth may be impaired in calves born 1 week before term.

Cytosine methylation of sperm DNA in horse semen after cryopreservation.

Semen processing may contribute to epigenetic changes in spermatozoa. We have therefore addressed changes in sperm DNA cytosine methylation induced by cryopreservation of stallion semen. The relative amount of 5-methylcytosine relative to the genomic cytosine content of sperm DNA was analyzed by ELISA. In experiment 1, raw semen (n = 6 stallions, one ejaculate each) was shock-frozen. Postthaw semen motility and membrane integrity were completely absent, whereas DNA methylation was similar in raw (0.4 ± 0.2%) and shock-frozen (0.3 ± 0.1%) semen (not significant). In experiment 2, three ejaculates per stallion (n = 6) were included. Semen quality and DNA methylation was assessed before addition of the freezing extender and after freezing-thawing with either Ghent (G) or BotuCrio (BC) extender. Semen motility, morphology, and membrane integrity were significantly reduced by cryopreservation but not influenced by the extender (e.g., total motility: G 69.5 ± 2.0, BC 68.4 ± 2.2%; P < 0.001 vs. centrifugation). Cryopreservation significantly (P < 0.01) increased the level of DNA methylation (before freezing 0.6 ± 0.1%, postthaw G 6.4 ± 3.7, BC 4.4 ± 1.5%; P < 0.01), but no differences between the freezing extenders were seen. The level of DNA methylation was not correlated to semen motility, morphology, or membrane integrity. The results demonstrate that semen processing for cryopreservation increases the DNA methylation level in stallion semen. We conclude that assessment of sperm DNA methylation allows for evaluation of an additional parameter characterizing semen quality. The lower fertility rates of mares after insemination with frozen-thawed semen may at least in part be explained by cytosine methylation of sperm-DNA induced by the cryopreservation procedure.

The PGF2α agonists luprostiol and d-cloprostenol reliably induce luteolysis in luteal phase mares without evoking clinical side effects or a stress response.

In the present study we have evaluated a possible stress reaction in response to two different PGF2α analogs-luprostiol and D-cloprostenol--and their effects on estrous cycle characteristics. In a cross-over-design eight mares received in alternating order either luprostiol (Treatment LUP; 3.75 mg im), D-cloprostenol (Treatment CLO; 22.5µg im) or saline (Treatment CON; NaCl 0.9% 0.5ml im) on day 8 after ovulation. Injection of either LUP or CLO, but not of CON resulted in a significant decline of progesterone concentration in plasma to baseline concentrations within two days (time: p<0.001, treatment: p<0.01, time × treatment: p<0.05). The treatment to ovulation interval was significantly shorter in LUP and CLO than in CON cycles (LUP: 9.4 ± 0.4 d; CLO: 9.4 ± 1.3 d; CON: 16.1 ± 0.8 d; p<0.001). Injection of either LUP or CLO, but not of CON significantly increased salivary cortisol concentration (immediately before injection: CON 1.3 ± 0.2, LUP 1.4 ± 0.3, CLO 1.4 ± 0.3 ng/ml; 60 min after injection: CON 1.0 ± 0.3, LUP 8.0 ± 1.4, CLO 4.2 ± 0.7 ng/ml; time: p<0.01, treatment: p<0.001, time × treatment: p<0.001). Heart rate decreased over time (p<0.05) independent of treatment and no changes in heart rate variability were detected. Injection of the PGF2α analogs CLO and LUP reliably induced luteolysis and apart from a transient increase in salivary cortisol concentration no signs of a physiological stress response or apparent side effects occurred.

Sympathoadrenal balance and physiological stress response in cattle at spontaneous and PGF2α-induced calving.

Increased cortisol release in parturient cows may either represent a stress response or is part of the endocrine changes that initiate calving. Acute stress elicits an increase in heart rate and decrease in heart rate variability (HRV). Therefore, we analyzed cortisol concentration, heart rate and HRV variables standard deviation of beat-to-beat interval (SDRR) and root mean square of successive beat-to-beat intervals (RMSSD) in dairy cows allowed to calve spontaneously (SPON, n = 6) or with PGF2α-induced preterm parturition (PG, n = 6). We hypothesized that calving is a stressor, but induced parturition is less stressful than term calving. Saliva collection for cortisol analysis and electrocardiogram recordings for heart rate and HRV analysis were performed from 32 hours before to 18.3 ± 0.7 hours after delivery. Cortisol concentration increased in SPON and PG cows, peaked 15 minutes after delivery (P < 0.001) but was higher in SPON versus PG cows (P < 0.001) during and within 2 hours after calving. Heart rate peaked during the expulsive phase of labor and was higher in SPON than in PG cows (time × group P < 0.01). The standard deviation of beat-to-beat interval and RMSSD peaked at the end of the expulsive phase of labor (P < 0.001), indicating high vagal activity. Standard deviation of beat-to-beat interval (P < 0.01) and RMSSD (P < 0.05) were higher in SPON versus PG cows. Based on physiological stress parameters, calving is perceived as stressful but expulsion of the calf is associated with a transiently increased vagal tone which may enhance uterine contractility.

Teaching of diagnostic skills in equine gynecology: simulator-based training versus schooling on live horses.

Transrectal palpation and ultrasonography of the genital tract in mares are first-day skills for equine veterinarians. In this study, the learning outcome in equine gynecology after four times training on horses (group H4, n = 8), training on horses once (group H1, n = 9), and four times simulator-based training (group Sim, n = 8) was assessed in third-year veterinary students with two tests in live mares 14 days apart. The students of group H4 always scored better for transrectal palpation than students of group Sim and H1 (P < 0.05). Overall, the students reached better results for palpating the left versus the right ovary (P < 0.001), but group H1 students were least successful in obtaining correct ovarian findings (P < 0.05 vs. both other groups). Students' self-assessment reflected test results with palpation of the right ovary experienced as most difficult for group H1 students (P < 0.01 vs. both other groups). Groups did not score differentially for ultrasound examinations. Sim students were nearly as successful in transrectal palpation of the genital tract in mares as H4 students, and for most parameters assessed, they performed better than H1 students. After training four times on horses, students scored best but nevertheless the overall effect of intensive training was limited. Repeated simulator-based training is a useful tool to prepare veterinary students for transrectal palpation of the genital tract in mares and is more effective than one training session on horses.

Increased cortisol release and transport stress do not influence semen quality and testosterone release in pony stallions.

The use of breeding stallions for equestrian competitions requires that fertility is not negatively affected by competition or transport to the competition site. In this study, effects of cortisol release induced by road transport (600 km), adrenocorticotropic hormone (ACTH) administration (3 × 0.5 mg synthetic ACTH) and placebo treatment on semen quality and testosterone release were investigated in Shetland stallions (N = 13) using a crossover design. Saliva for cortisol and blood for testosterone analysis were collected for 10 weeks after treatments. Semen was collected daily for 5 days directly after treatments and twice weekly for another 9 weeks. Total sperm count, sperm morphology, motility, and membrane integrity were analyzed. We hypothesized that elevated cortisol decreases testosterone concentration and semen quality. Cortisol concentrations increased in response to transport and ACTH (P < 0.001) but not control treatments (peak concentration, transport: 7.6 ± 2.4, ACTH: 13.7 ± 1.5, control: 3.8 ± 0.9 ng/mL). No treatment effects on testosterone existed. Total sperm count decreased with daily semen collections in week 1 (P < 0.01) but did not differ between the treatments. The percentage of motile, progressively motile, membrane-intact, and morphologically defective spermatozoa did not change over time from Days 2 to 6, and there existed no differences between the treatments. In conclusion, road transport evoked a stress response which was mimicked by ACTH treatment. Both treatments had no effect on testosterone release and semen quality. Testicular function in stallions is apparently well protected against transiently elevated cortisol concentrations, and stallions can be transported over longer distances without negatively affecting their fertility.

Treatment with human chorionic gonadotrophin before ovulation increases progestin concentration in early equine pregnancies.

For prevention of early conceptus loss in the horse, treatment with progestins has become common practice. In cattle, treatment with human chorionic gonadotrophin (hCG) during the early postovulatory phase stimulates endogenous progesterone synthesis, which is an important factor for maintenance of early pregnancy via stimulation of endometrial function and conceptus development. In the present study we have therefore investigated the influence of treatment with hCG either for induction of ovulation or during the early luteal phase on plasma progestin concentrations, size of the corpus luteum and size of the conceptus in early pregnant mares. We hypothesized that hCG treatment stimulates progestin secretion and conceptus development. In Experiment 1, induction of ovulation with hCG (1500 IU i.v.; n=14) significantly increased progestin concentration between days 5 and 15 after ovulation compared to untreated controls (n=28; p<0.05; e.g. day 5 hCG i.v.: 17.2 ± 1.9, control: 13.9 ± 0.8 ng/ml). A significant interaction (p<0.05) of hCG treatment with size of the conceptus between days 30 and 40 of pregnancy was detected. In Experiment 2, treatment of mares with hCG (5000 IU) on day 5 after ovulation (n=12) did neither affect progestin secretion (e.g. day 8 hCG: 15.4 ± 1.6, control: 17.6 ± 1.2 ng/ml) nor luteal tissue area (e.g. day 8 hCG: 9.0 ± 0.7, control: 7.6 ± 1.4 cm(2)) compared to untreated mares (n=9). In conclusion, treatment of mares with hCG for induction of ovulation within 48 h before ovulation but not on day 5 of the luteal phase stimulates progestin secretion and may enhance conceptus development via stimulation of endometrial function during early pregnancy.

Parturition in horses is dominated by parasympathetic activity of the autonomous nervous system.

External and internal stressors prolong parturition in different species. At parturition, sympathoadrenal activation should be avoided because an increased sympathetic tone may cause uterine atonia via ß2-receptors. We hypothesized that at physiological parturition, horses are under parasympathetic dominance, and stress-response mechanisms are not activated during delivery of the foal. To evaluate stress responses, heart rate, heart rate variability, catecholamines, and cortisol were analyzed in mares (n = 17) throughout foaling. Heart rate decreased from 2 hours before (51 ± 1 beats/minute) to 2 hours after delivery (41 ± 2 beats/minute; P < 0.05). Heart rate variability variables, standard deviation of the beat-to-beat interval, and root mean square of successive beat-to-beat differences, changed over time (P < 0.05) with the highest values within 15 minutes after delivery. The number of mares with atrioventricular blocks and the number of atrioventricular blocks per mare increased over time (P < 0.01) and were significantly elevated from 15 minutes before to 45 minutes after birth of the foal. Salivary cortisol concentrations increased to a maximum at 30 minutes after delivery (25.0 ± 3.4 ng/mL; P < 0.01). Plasma epinephrine and norepinephrine concentrations showed significant fluctuations from rupture of the allantochorion to expulsion of the fetal membranes (P < 0.01) but were not markedly elevated at any time. In conclusion, mares give birth under high parasympathetic tone. Cortisol release during and after foaling is most likely part of the endocrine pathways regulating parturition and not a labor-associated stress response.

Cortisol release, heart rate and heart rate variability in the horse and its rider: different responses to training and performance.

Although some information exists on the stress response of horses in equestrian sports, the horse-rider team is much less well understood. In this study, salivary cortisol concentrations, heart rate (HR) and heart rate variability (HRV), SDRR (standard deviation of beat-to-beat interval) and RMSSD (root mean square of successive beat-to-beat intervals) were analysed in horses and their riders (n=6 each) at a public performance and an identical rehearsal that was not open to the public. Cortisol concentrations increased in both horses and riders (P<0.001) but did not differ between performance and rehearsal. HR in horses and riders increased during the rehearsal and the public performance (P<0.001) but the increase in HR was more pronounced (P<0.01) in riders than in their horses during the public performance (from 91 ± 10 to 150 ± 15 beats/min) compared to the rehearsal (from 94 ± 10 to 118 ± 12 beats/min). The SDRR decreased significantly during the equestrian tasks in riders (P<0.001), but not in their horses. The RMSSD decreased in horses and riders (P<0.001) during rehearsal and performance, indicating a decrease in parasympathetic tone. The decrease in RMSSD in the riders was more pronounced (P<0.05) during the performance (from 32.6 ± 6.6 to 3.8 ± 0.3 ms) than during the rehearsal (from 27.5 ± 4.2 to 6.6 ± 0.6 ms). The study has shown that the presence of spectators caused more pronounced changes in cardiac activity in the riders than it did in their horses.