General and comparative aspects of endometritis in domestic species: A review.

Endometritis is a leading cause of sub- and infertility in domestic animal species. The healthy uterus is colonized by commensal bacteria, viruses and yeast/fungi that represent the nonpathogenic microbiota. A shift in the number or type of organisms accompanied by immune dysfunction, however, may trigger uterine infection and inflammation. Metritis is associated with inflammation of all uterine layers (endometrium, myometrium and perimetrium), whereas endometritis is a more superficial inflammation involving solely the endometrium. Endometritis generally occurs at two time points in domestic animal species, postpartum and postmating. Postpartum endometritis may chronically persist, either as a low-grade disease that often manifests as a vaginal discharge but not a systemic illness (in some species termed clinical endometritis) or sometimes subclinical where features are only detected by endometrial sampling. Contamination of the uterus at the time of mating occurs by direct deposition of semen (ejaculated or artificially inseminated) into the uterus. Improper drainage of the ejaculatory fluid or an inadequate immune response may result in persistent mating-induced endometritis. Both postpartum and postmating endometritis interferes with fertility by creating a suboptimal environment for embryo development and placentation, and chronic endometritis may have an impact on sperm survival and fertilization ability. In the postpartum animal, there may also be changes in milk production and maternal behaviour, which can affect offspring health and survival. Preventive strategies for endometritis largely depend on monitoring their known risk factors, which are sometimes specific with regard to the species. Effective, nonantibiotic therapy for endometritis is not available to date. Overall, extensive research has been performed in cattle and horses to unravel key aspects of endometritis, but in sows and bitches, the available literature is scant. Thus, the need and opportunity to investigate the condition vary considerably among domestic species and necessitate their comparative assessment. This article reviews general and comparative aspects of the diagnosis and classification, pathogenesis, preventive strategies and therapeutics of endometritis in domestic species with a specific focus on cows, mares, sows and bitches.

Using egg yolk in a TRIS-Equex STM paste extender for freezing of dog semen is superior to egg yolk plasma, also after addition of lecithin and catalase.

We compared the results of using egg yolk plasma (EYP) instead of egg yolk (EY) in a TRIS-based Equex STM Paste freezing extender system for dog semen [25]. We also tested whether the addition of lecithin and catalase to the EYP extenders would improve results. Fractionated semen collection was done in 17 stud dogs and the sperm rich fraction diluted with different extenders in 2 steps: (I) TRIS-fructose-citric acid extender (TRIS) containing 20% egg yolk (EY) and 3% glycerol [25], (II) TRIS containing 20% egg yolk plasma (EYP) and 3% glycerol, and (III) TRIS containing 20% EYP and 0.8% lecithin (EYP-L) and 3% glycerol. After equilibration the second dilution step was done: samples with (I) were diluted with TRIS-EY with 7% glycerol and 1% Equex STM paste [25]; samples with (II) and (III) were divided in 2 aliquots each, and one part diluted with TRIS-EYP or TRIS-EYP-L, both containing 7% glycerol and 1% Equex STM paste, and the other one part with the same extenders containing additionally 300 I.U./mL catalase. After freezing and thawing, samples were analyzed by CASA and sperm chromatin structure assay (SCSA); reactive oxygen species (ROS), degree of apoptosis and zona binding ability were determined. Semen samples with TRIS-EY with a final concentration of 5% glycerol and 0.5% Equex STM paste [25] showed best post thaw progressive motility (P), most intact cells, lowest percentage of ROS, acrosome damages, dead and apoptotic cells. Curvilinear velocity (VCL), DNA fragmentation, morphological abnormalities and zona binding ability did not differ between groups. Replacement of egg yolk by EYP increased the ROS and late apoptotic cells. Addition of lecithin and catalase to EYP containing extenders decreased motility and increased complete apoptosis. We conclude that egg yolk is superior to EYP in the here investigated extenders. The TRIS-based extender [25] with EYP could not be improved by addition of lecithin and catalase; however, in-vivo fertilization capacity of the here examined extenders remains to be investigated.

Effect of housing system on reproductive behaviour and on some endocrinological and seminal parameters of donkey stallions.

Reproductive management of male donkeys employed for artificial breeding has been poorly studied. The aim of this study was to evaluate the effect of housing system, with the animals grouped together in a paddock or kept in individual boxes, on sexual behaviour, cortisol and testosterone concentration and seminal characteristics of adult male donkeys. The study included four Amiata donkey jacks (stallions) from which ejaculates, saliva and blood were collected during two distinct 3 weeks periods, one in the group and one in the box housing system. Time needed for semen collection was shorter when donkeys were kept in paddocks compared to when they were kept in single boxes (14:57 ± 07:27 and 20:52 ± 09:31 min, p < .05). Native semen characteristics were not influenced by housing system, while cooled preservation in an Equitainer® showed that sperm motility parameters were significantly higher during the paddock period compared to the box period. Salivary cortisol was influenced by housing system, both before and 60 min after ejaculation, being statistically higher when donkeys were housed in paddocks. On the contrary, overall and basal testosterone concentrations were significantly higher when animals were kept in boxes. In conclusion, in the present study, good quality semen could be successfully collected from donkeys irrespective of the housing system despite some differences in hormone concentrations.

Evaluation of SmartFlare probe applicability for verification of RNAs in early equine conceptuses, equine dermal fibroblast cells and trophoblastic vesicles.

Live cell RNA imaging has become an important tool for studying RNA localisation, dynamics and regulation in cultured cells. Limited information is available using these methods in more complex biological systems, such as conceptuses at different developmental stages. So far most of the approaches rely on microinjection of synthetic constructs into oocytes during or before fertilisation. Recently, a new generation of RNA-specific probes has been developed, the so named SmartFlare probes (Merck Millipore). These consist of a central 15-nm gold particle with target-specific DNAs immobilised on its surface. Because of their central gold particle, SmartFlare probes are detectable by transmission electron microscopy. The aim of the present study was to investigate the uptake and distribution of SmartFlare probes in equine conceptuses at developmental stages suitable for embryo transfer (Days 6-10), equine trophoblast vesicles and equine dermal fibroblast cell cultures, and to determine whether differences among these cell types and structures exist. Probe uptake was followed by transmission electron microscopy and fluorescence microscopy. Although the embryonic zona pellucida did not reduce uptake of the probe, the acellular capsule fully inhibited probe internalisation. Nanogold particles were taken up by endocytosis by all cell types examined in a similar manner with regard to time and intracellular migration. They were processed in endosomal compartments and accumulated within lysosomal structures after longer incubation times. In conclusion, the SmartFlare probe is applicable in equine conceptuses, but its use is limited to the developmental stages before the formation of the embryonic capsule.

Influence of partial or total replacement of glycerol by alternative cryoprotectants in Ghent freezing extender on post-thaw sperm quality in stallions.

Although glycerol is the cryoprotectant most commonly used in stallions, it has also a considerable toxicity for equine sperm. It was the aim of this study to analyse the quality of frozen-thawed stallion semen after complete or partial replacement of glycerol in the freezing extender by alternative cryoprotectants. We hypothesized that partial or total replacement of glycerol by cryoprotectants occurring in cold-resistant frog, insect or plant species results in similar or better semen quality after freezing-thawing. As basic medium, the commercial Ghent basic extender was used and either supplemented with glucose and urea, trehalose and proline, or trehalose and betaine. Based on a series of preliminary experiments, semen was frozen in either commercial Ghent cryopreservation extender (Ghent control), Ghent glucose-urea extender or a Ghent combined extender (glucose-urea, trehalose-betaine and trehalose-proline; volume ratio of 2:1:2) in a computer-controlled rate freezer. After freezing-thawing, semen was analysed for motility, membrane integrity, phosphatidylserine translocation, mitochondrial membrane potential and chromatin condensation. No differences between Ghent control and Ghent glucose-urea extender were seen, while all endpoints except DNA integrity were negatively affected in Ghent combined extender (e.g., progressive motility: Ghent 49.2 ± 3.7, Ghent glucose-urea 46.5 ± 4.6, Ghent combined 24.4 ± 2.8%; p < .001). In conclusion, glycerol concentration in a commercial freezing extender for equine spermatozoa can be successfully reduced when urea as an additive cryoprotectant is added and the glucose concentration is elevated. However, total glycerol replacement with urea, betaine, proline and trehalose was less successful.

Season does not influence embryo recovery rate and conceptus size until day 14 after ovulation in the horse.

Although the horse is a seasonal breeding species, a considerable number of mares continue to cycle throughout autumn and winter. Slower equine embryo growth during the non-breeding season has been hypothesized, and because smaller embryo size is beneficial for cryopreservation, embryo collection outside the breeding season could be an interesting approach for the production of frozen horse embryos. In the present retrospective study, we have therefore analysed embryo recovery rates and conceptus size in mares (n = 30) throughout the year. Conceptus diameter was either size determined after collection with a microscopic scale (day 7-10 after ovulation) or determined by transrectal ultrasound immediately before collection (day 11-14 after ovulation). In 19 of the 30 mares (63%), ovulatory cycles were detected throughout the year. A total of 352 embryo collections with a mean recovery rate of 64.2% were performed and not affected by season. The size was analysed in a total of 165 conceptuses. Conceptus diameter significantly increased (p < 0.001) with day of pregnancy (e.g. day 7: 0.3 ± 0.04, day 10: 4.1 ± 0.2, day 12: 10.1 ± 0.5, day 14: 17.4 ± 0.9 mm), but was not influenced by season. In conclusion, successful embryo collection is possible throughout the year in spontaneously cyclic mares. Under these conditions, neither collection rates nor embryo growth appeared to be affected by season.

Stress Response of Veterinary Students to Gynaecological Examination of Horse Mares - Effects of Simulator-Based and Animal-Based Training.

Invasive procedures in animals are challenging for veterinary students who may perceive a gynaecological examination of mares as stressful. Simulator-based training may reduce stress. In this study, students received equine gynaecology training 4 times either on horses (group H; n = 14) or a teaching simulator (group SIM; n = 13). One day and 14 days thereafter, their diagnostic skills were tested on horses (skills tests 1 and 2). During the skills tests, the students' stress response was analysed by heart rate, heart rate variability (HRV) parameters SDRR (standard deviation of beat-to-beat [RR] interval) and RMSSD (root-mean-square of successive RR differences), and salivary cortisol. In addition, students answered a questionnaire on their perceived stress. Sympathetic activation with increased heart rate (p < 0.001) occurred in both skills tests. In test 1, this increase was more pronounced in SIM than in H students (time × group p < 0.01). HRV decreased in students of both groups (p < 0.001). In skills test 1, this decrease was more pronounced for SIM than for H students (between groups and time × group p < 0.01 for SDRR and p < 0.05 for RMSSD). High cortisol concentrations before the skills tests may indicate an anticipatory stress response. Subjective stress perception of students was higher in skills test 1 vs 2 (p < 0.01). In skills test 2, H students felt more stressed than SIM students (p < 0.01). Self-assessment thus differed from physiological stress parameters. In conclusion, gynaecological examination of mares evoked a moderate stress response in veterinary students, which was more evident after simulator-based than animal-based training.

Immunolocalization of the cholesterol transporters ABCA1 and ABCG1 in canine reproductive tract tissues and spermatozoa.

The mammalian sperm membrane undergoes cholesterol efflux during maturation and fertilization. Although ATP-binding cassette (ABC) transporters are known to transport cholesterol through cell membranes in other organs, their presence in canine testis, epididymis and sperm has not been proven to date. Hence, the aim of the present study was to localize the ABC transporters ABCA1 and ABCG1 in canine testicular and epididymidal tissue as well as in spermatozoa membranes. To this end, semen samples from 12 dogs as well as testicles and epididymides of four young and healthy dogs were prepared for immunohistochemistry, respectively. Capacitation and acrosome reaction (AR) were induced in aliquots of the semen samples before immunostaining to assess changes in the expression of ABCA1 and ABCG1. Evaluation by confocal microscopy revealed the presence of both ABCA1 and ABCG1 in canine testicles and of ABCA1 in the epididymides. In spermatozoa, only ABCA1 immunoreactivity was detected, mainly in the region of the acrosome and midpiece. After induction of capacitation, ABCA1 signal persisted in the acrosome but disappeared after AR, indicating a loss of ABCA1 with the loss of the acrosome. We conclude that ABCA1 and ABCG1 are expressed in canine testis, whereas only ABCA1 is expressed in epididymis and spermatozoa membrane, both transporters probably contributing to the regulation of membrane cholesterol content.

Physiological and behavioral stress parameters in calves in response to partial scrotal resection, orchidectomy, and Burdizzo castration.

Establishing artificial cryptorchids by partial scrotal resection without removing the testicles is a technique for castration of bull calves that recently has gained new interest. In contrast to orchidectomy and Burdizzo castration, the stress response of calves to shortening of the scrotum is unknown. In this study, partial scrotal resection in bull calves was compared with orchidectomy, Burdizzo castration, and controls without intervention (n=10 per group, ages 56 ± 3 d). Procedures were performed under xylazine sedation and local anesthesia. We hypothesized that partial scrotal resection is least stressful. Salivary cortisol, heart rate, heart rate variability, behavior, and locomotion were analyzed. Cortisol concentration peaked 60 min after start of the procedures. Cortisol release was at least in part xylazine induced and none of the experimental procedures released additional cortisol. Heart rate increased in calves of all groups with initial handling, but immediately after xylazine sedation decreased to 30% below initial values and was not modified by surgical procedures. The heart rate variability variables standard deviation of beat-to-beat interval and root mean square of successive beat-to-beat differences increased when calves were placed on the surgery table but effects were similar in calves submitted to surgeries and control calves. Locomotion increased, whereas lying time decreased in response to all surgeries. Locomotion increase was most pronounced after orchidectomy. Plasma fibrinogen concentrations increased after orchidectomy only. With adequate pain medication, orchidectomy, Burdizzo castration, and partial scrotal resection do not differ with regard to acute stress and, by inference, pain. Partial scrotal resection when carried out under xylazine sedation and local anesthesia thus is an acceptable castration technique in bull calves.

Applicability of a new cell culture device for cooled-storage of stallion semen.

A new device for storage and shipping of cell cultures--the Petaka G3 cell management device--was tested for its applicability for cooled-storage of equine semen. Semen from three stallions was processed with EquiPro extender either without antibiotics (three ejaculates per stallion) or with gentamicin (250 mg/l; three ejaculates per stallion). Semen was either stored at five (anaerobic conditions) or 15 °C (aerobic conditions) in syringes or cell culture devices. Total and progressive motility, as well as membrane integrity of spermatozoa, were evaluated from days 1 to 7 after collection with computer-assisted semen analysis. In experiment 1 (extender without antibiotics), total motility, progressive motility and viability of spermatozoa significantly decreased over time (p < 0.05). The decrease was significantly faster at 15 °C than at 5 °C (p < 0.05). In the presence of gentamicin (experiment 2), this difference was no longer present. It can be concluded that cooled-storage of equine semen in sophisticated devices for cell culture is not advantageous to syringes for successful maintenance of semen longevity.

Expression of 11ß-hydroxysteroid dehydrogenase type 1 and glucocorticoid receptors in reproductive tissue of male horses at different stages of sexual maturity.

Glucocorticoids (GCs) as mediators of the stress response may affect Leydig cell function by inhibiting either luteinizing hormone receptor expression or testosterone biosynthesis. The isozymes 11ß-hydroxysteroid dehydrogenase (11ßHSD) 1 and 11ßHSD2 control the intracellular cortisol levels. Little is known about the effects of stress on fertility in the equine. The objective of the present study was to determine the presence and cellular localization of glucocorticoid receptors (GCR) and glucocorticoid-metabolizing enzymes (11ßHSD1 and 11ßHSD2) in equine epididymal and testicular tissue with special regard to sexual maturation. Testicular and epididymal tissue was collected from 21 healthy stallions, and four age groups were designed: pre-pubertal, young, mature and older horses. Immunohistochemistry (IHC) analysis and quantitative real-time PCR (qRT-PCR) were used. Pre-pubertal horses showed higher testicular gene expression of 11ßHSD1, 11ßHSD2 and GCR than horses of all other groups (p < 0.05). A positive intranuclear immunoreaction for GCR was seen in epithelial cells of caput, corpus and cauda epididymidis and in Leydig cells. Significant differences (p < 0.05) between age groups occurred. The number of Leydig cells staining positive for GCR was highest in immature stallions (p < 0.05). The enzyme 11ßHSD1 was localized in epithelial cells of the caput and corpus epididymidis and in Leydig cells. As determined by enzyme assay, nicotinamide adenine dinucleotide (NAD)-dependant dehydrogenase (oxidation) activity was not detected in testicular tissue from immature stallions but in all other age groups (n = 3 per group). Results of this study suggest a contribution of GCs to maturation of male reproductive tissue in horses. In mature stallions, expression of 11ßHSD enzymes and the oxidative 11ßHSD activity in Leydig cells and epididymal basal and principal cells suggest a protective role on these tissues contributing to physiological intracellular glucocorticoid concentrations.

Investigations on the endometrial response to intrauterine administration of N-acetylcysteine in oestrous mares.

In mares, mating-induced persistent endometritis contributes to low fertility. The condition is in part related to delayed clearance of mucus accumulated within the uterine lumen. The objective of this study was to investigate the endometrial response of healthy mares to intrauterine (i.u.) treatment with N-acetylcysteine (NAC). Oestrous mares (n = 12) were randomly assigned to a treatment (TM) or control (C) group and received an i.u. infusion of 5% NAC and saline (total volume 140 ml), respectively. Endometrial biopsies were collected in five of the mares 24 h after treatment, in the remaining seven mares 72 h after treatment. Endometrial biopsies were evaluated for integrity of the luminal epithelium, number of polymorphonuclear neutrophils (PMN), staining for cyclooxygenase 2 (COX2), staining with Kiel 67 antigen (Ki-67), lectins and periodic acid-Schiff (PAS). The integrity of endometrial epithelial cells was not affected by treatment (no statistical differences between groups or times). At 24 h after treatment, the mean number of PMN in endometrial biopsies from NAC- and C-mares did not differ, but at 72 h after treatment, number of PMN was significantly higher (p < 0.05) in C (3.9 ± 0.6 PMN/field) compared with NAC-treated mares (2.3 ± 0.2 PMN/field). At 72 h after treatment, the intensity of staining for COX2 was significantly higher after saline than after NAC treatment (p < 0.05). In the epithelium, no differences in staining for the proliferation marker Ki-67 were seen with respect to time and treatment. Score for the lectin wheat germ agglutinin (WGA) was slightly higher in NAC-treated mares than in C-mares 72 h after treatment (p < 0.05). Score for PAS staining of mucus in deep uterine glands differed significantly between groups at 24 h after treatment (p < 0.05). The present study demonstrates that NAC does not adversely affect the endometrial function. Moreover, an anti-inflammatory effect on the equine endometrium was observed.

Cooled storage of canine semen: in vitro effects of different concentrations of an antibiotic combination on growth of mollicutes.

The aim of this study was to examine effects of an antibiotic combination at different concentrations on growth of mycoplasma and ureaplasma during cooled storage of canine semen (n = 20). Semen aliquots were diluted with Tris-citric acid-fructose-egg yolk extender containing either 1.0 g/l streptomycin and 0.6 g/l benzylpenicillin (control) or a combination of gentamycin, tylosin, lincomycin and spectinomycin (GTLS-1: 0.25, 0.05, 0.15 and 0.3; GTLS-2: 0.5, 0.1, 0.3 and 0.6; GTLS-3: 1.0, 0.2, 0.6 and 1.2 g/l). Samples were assessed for motility and membrane integrity by computer-assisted sperm analysis immediately after dilution and at 24, 48 and 72 h of cooled storage. Morphologically, normal spermatozoa were determined, and bacterial culture was performed at 24 and 72 h. Mycoplasma spp. were detected in 14 of 20 ejaculates (70%) with severe growth in 12 samples. A reduction but not total elimination of mycoplasma growth occurred in all GTLS extenders with the most pronounced reduction in group GTLS-3 (control vs GTLS-1 and GTLS-2 p < 0.05, control vs GTLS-3 p < 0.001). Ureaplasmas were detected in four ejaculates, and growth was reduced to the same extent in GTLS and control extender. Progressive motility in all groups, total motility in groups GTLS 1-3 and percentage of membrane-intact spermatozoa in groups GTLS 2 and 3 decreased slightly (p < 0.05) over time. In conclusion, dilution of canine semen with GTLS extender has no major detrimental effects on spermatozoa during cooled storage. It reduced the growth but did not totally eliminate mycoplasmas and ureaplasmas from cooled-stored dog semen.

The levels of zearalenone and its metabolites in plasma, urine and faeces of horses fed with naturally, Fusarium toxin-contaminated oats.

Concentration profile of zearalenone (ZON) and its metabolites in plasma, urine and faeces samples of horses fed with Fusarium toxin-contaminated oats is described. In plasma, ß-zearalenol (ß-ZOL) was detected at high levels on day 10 of the study (3.21-6.24 µg/l). ß-Zearalenol and α-zearalenol were the major metabolites in urine. Zearalenone, α-ZOL and ß-ZOL were predominantly found in faeces. Zearalanone could also be detected in urine (1.34-5.79 µg/l) and faeces (1 µg/kg). The degree of glucuronidation was established in all sample types, approximately 100% in urine and plasma. Low per cent of glucuronidation (4-15%) was found in faeces samples. The results indicate the main conversion of ZON into ß-ZOL in horse. This finding could explain why horse is not susceptible to ZON in comparison with swine which produce α-ZOL as a predominant metabolite.

Cortisol release, heart rate and heart rate variability, and superficial body temperature, in horses lunged either with hyperflexion of the neck or with an extended head and neck position.

Bringing the head and neck of ridden horses into a position of hyperflexion is widely used in equestrian sports. In our study, the hypothesis was tested that hyperflexion is an acute stressor for horses. Salivary cortisol concentrations, heart rate, heart rate variability (HRV) and superficial body temperature were determined in horses (n = 16) lunged on two subsequent days. The head and neck of the horse was fixed with side reins in a position allowing forward extension on day A and fixed in hyperflexion on day B. The order of treatments alternated between horses. In response to lunging, cortisol concentration increased (day A from 0.73 ± 0.06 to 1.41 ± 0.13 ng/ml, p < 0.001; day B from 0.68 ± 0.07 to 1.38 ± 0.13 ng/ml, p < 0.001) but did not differ between days A and B. Beat-to-beat (RR) interval decreased in response to lunging on both days. HRV variables standard deviation of RR interval (SDRR) and RMSSD (root mean square of successive RR differences) decreased (p < 0.001) but did not differ between days. In the cranial region of the neck, the difference between maximum and minimum temperature was increased in hyperflexion (p < 0.01). In conclusion, physiological parameters do not indicate an acute stress response to hyperflexion of the head alone in horses lunged at moderate speed and not touched with the whip. However, if hyperflexion is combined with active intervention of a rider, a stressful experience for the horse cannot be excluded.

In vivo dynamics of pro-inflammatory factors, mucins, and polymorph nuclear neutrophils in the bovine oviduct during the follicular and luteal phase.

Dynamic functional changes in the oviductal microenvironment are the prerequisite for the establishment of pregnancy. The objective of this study was to gain the first insights into oestrous cycle-dependent dynamics of polymorph nuclear neutrophils (PMN) and the mRNA abundance of selected genes and their correlations in the oviduct of living cows. Mini-cytobrush samples were taken from the oviducts of healthy heifers (n = 6) and cows (n = 7) during the follicular (FOL) and luteal phase (LUT) by transvaginal endoscopy. Total RNA was isolated from the samples and subjected to reverse transcription-quantitative PCR for selected pro-inflammatory factors, glycoproteins, and a metabolic marker. The percentage of PMN was determined by cytological examination. The mean PMN percentage was 2.8-fold greater during LUT than FOL. During LUT, significantly greater mRNA abundance of the pro-inflammatory factors IL1B, CXCL1, CXCL3, and CXCL8 was observed. The OVGP1 mRNA abundance was twice as high during FOL than in LUT. Pearson correlation, principal component analysis and heatmap analyses indicated characteristic functional patterns with strong correlations among investigated factors. Using this novel approach, we illustrate complex physiological dynamics and interactions of the mRNA expression of pro-inflammatory factors, mucins, OVGP1, and PMN in the oviduct during the oestrous cycle.

Gene expression of ACTH, glucocorticoid receptors, 11ßHSD enzymes, LH-, FSH-, GH receptors and aromatase in equine epididymal and testicular tissue.

Glucocorticoids (GCs) are important mediators of the stress response and have been implicated in the function and regulation of testicular functions in different species. In many tissues, intracellular glucocorticoid activity is controlled by either or both of the two known isoforms of 11ß-hydroxysteroid dehydrogenase (11ßHSD) type 1 and 2, which interconvert active and inactive GCs. Little is known about the effects of stress on fertility in the equine species. The main objective of the present study was to investigate the expression of receptors for GCs and adrenocorticotropic hormone [ACTH, melanocortin 2 receptor (MC2R)] as well 11ßHSD1 and 11ßHSD2 in male equine epididymal and testicular tissue. In addition, expression of aromatase P-450 and receptors for luteinizing hormone (LHR), follicle stimulating hormone (FSHR) and growth hormone (GHR) was studied. Reverse transcriptase PCR and quantitative real-time PCR were performed in tissue from the epididymis (caput and cauda) and testes collected from nine healthy mature stallions (age 4-10 years). mRNA for ACTH and GC receptors as well as 11ßHSD1 and -2 were found in epididymal and testicular tissue. Expression of the genes studied was always positive in testicular tissue, while it was inconsistent in epididymal tissue. Quantitative gene expression in relation to ß-actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was significantly correlated (R = 0.403, p < 0.001). Quantitative PCR in relation to ß-actin revealed significant differences in the gene expression of 11ßHSD1, 11ßHSD2, LHR, FSHR, MC2R and aromatase between tissue collected from caput epididymidis, cauda epididymidis and testicular parenchyma (p < 0.05). With GAPDH, differences between tissues were significant for 11ßHSD1, 11ßHSD2 and MC2R (p < 0.05) In addition, high concentrations of mRNA of aromatase and receptors of LH and FSH were found in testicular tissue, while a pronounced expression of GH receptor was present in epididymal tissue. The results support the hypothesis of an interaction between the pituitary-adrenal axis and testicular function in the stallion.

Effects of oral supplementation with ß-carotene on concentrations of ß-carotene, vitamin A and α-tocopherol in plasma, colostrum and milk of mares and plasma of their foals and on fertility in mares.

In this study, effects of oral ß-carotene supplementation to mares (ß-carotene group: 1000 mg/day, n = 15; control group: n = 15) from 2 weeks before foaling until 6 weeks thereafter on concentrations of ß-carotene, vitamin A and α-tocopherol in plasma, colostrum and milk and plasma of their foals were determined. In addition, effects on fertility were studied. Beta-carotene concentrations increased in plasma and colostrum of ß-carotene-supplemented mares compared to control mares (p < 0.05). In mares of both groups, ß-carotene concentrations were higher in colostrum than in milk (p < 0.05). In foals, ß-carotene concentrations increased with colostrum uptake and were higher in foals born to supplemented mares (p < 0.05; control group: 0.0003 ± 0.0002 µg/ml on day 0, 0.008 ± 0.0023 µg/ml on day 1; ß-carotene group: 0.0005 ± 0.0003 µg/ml on day 0, 0.048 ± 0.018 µg/ml on day 1). Concentrations of vitamin A and α-tocopherol were higher in colostrum than in milk (p < 0.05) but did not differ between groups. Concentration of α-tocopherol in plasma of mares decreased over time and in foals, increased markedly within 4 days after birth. All but one mare (control group) showed oestrus within 2 weeks post-partum. Occurrence of oestrus did not differ between groups. More mares of the control group (7/7 vs. 5/12 in the ß-carotene group) became pregnant after being bred in first post-partum oestrus (p < 0.05). In conclusion, ß-carotene supplementation to mares increased ß-carotene concentrations in plasma, colostrum and milk of mares and plasma of their foals but had no positive effects on fertility.

Induction of parturition in horses - from physiological pathways to clinical applications.

Based on the marked variability in physiological equine gestation length, induction of foaling in mares often results in the birth of dysmature foals. Precise prediction of preparedness of the mare for foaling is thus essential. Treatment with glucocorticoids mimics the fetal signal that initiates birth. Repeated daily dexamethasone treatment in late gestation results in birth of mature foals but the time from initiation of treatment to foaling is highly variable and complications such as dystocia have been reported. Contrary to most expectations, treatment of prepartum mares with progestogens does not delay but advances the onset of foaling. Prostaglandin F2α (PGF2α) and its analogues are effective to induce foaling but even in mares ready for parturition, foal health remains to some extent unpredictable. This may be caused by a relatively long interval between PGF2α treatment and birth, exposing the fetus for several hours to uterine contractions. Oxytocin reliably induces foaling towards the end of pregnancy, but when given at high doses is effective also in the pre-viable period of gestation, resulting in birth of premature foals. Recent research has focused on reducing the amount of oxytocin with the aim to induce foaling only in mares prepared for foaling. Mares selected on clinical criteria receive 1 dose of 2.5 to 3.5 IU of oxytocin. Mares not responding to oxytocin are judged not yet ready for foaling and treatment is repeated the earliest after 24 h. This protocol at present is the most reliable and safest way to induce parturition in mares.

Altrenogest treatment reduces the stress response of three-year-old warmblood mares during their initial equestrian training.

Horse mares are frequently treated with the progestin altrenogest with the aim to suppress estrous behavior and its negative impact on equestrian performance. Progestogens, however, also have sedative effects in males, and females of different species. The aim of our study was therefore to investigate altrenogest-induced changes in the stress response of female horses during initial equestrian training. Three-yr-old Warmblood mares were randomly assigned to treatment with altrenogest (ALT; 0.044 mg/kg once daily; n = 6) or sunflower oil (CON; n = 5) for 12 wk during training. At predefined steps of the training program (free movement, lunging without and with side reins, lunging with saddle, mounting of a rider, free riding, riding by an unfamiliar rider) salivary cortisol concentration, and heart rate were determined from 60 min before to 120 min after training. The same procedures were performed during repeated gynecologic examinations and 2 novel object tests. Bodyweight and body condition scores (BCS) were assessed at 4-wk intervals. During all training units, salivary cortisol concentration and heart rate increased (P < 0.001), but the increase was smaller in group ALT mares (time x treatment P < 0.001). Gynecologic examinations and novel object tests induced a much smaller increase in cortisol and heart rate (P < 0.001) than equestrian training with no difference between groups ALT and CON. Initially, bodyweight, and BCS decreased during training. The subsequent increase was larger in group ALT vs CON (time x treatment P < 0.05). In conclusion, altrenogest reduced the stress response of 3-yr-old mares to equestrian training. The use of altrenogest during equestrian competitions should therefore be reconsidered.