Intrauterine infusion of killed semen adversely affects uterine blood flow and endometrial gene expression of inflammatory cytokines in mares susceptible to persistent breeding-induced endometritis.

Persistent breeding-induced endometritis (PBIE) is a leading cause of infertility in mares. The objective of the study was to assess genital perfusion and endometrial gene expression of inflammatory cytokines in mares classified as susceptible (n = 5) or resistant (n = 5) to PBIE. Ten mares were examined daily during estrus until 6 d after hCG-induced ovulation for two estrous cycles. Twenty-four hours after application of 1500 IU hCG, 4 mL of killed (by repeated freezing in liquid nitrogen and thawing at 50 °C) deep-frozen semen or sterile saline was instilled into the uterine body and examinations were carried out immediately before and 3, 6, and 12 h after intrauterine infusion. Examinations included blood sampling to determine plasma progesterone (P4) concentrations, and transrectal ultrasonography in B- and color Doppler mode to determine follicular and luteal size and blood flow, the extent of intrauterine fluid, as well as time-averaged maximum velocity (TAMV), blood flow volume (BFV), and blood flow resistance (expressed as pulsatility index, PI) of the uterine arteries. Additionally, endometrial biopsies were obtained at 24 h before, and 2 and 7 d after infusion, and mRNA expressions of IL1B, IL6, IL8, IL10, TNF, CASP3, and COX2 were determined by qRT-PCR. Statistical analyses were performed with mixed models. Intrauterine fluid retention (diameter >20 mm for at least 3 d) was found after infusion of killed semen in five susceptible mares. There was no treatment effect (semen vs saline; P > 0.05) on genital blood flow, plasma P4 concentration, and endometrial gene expression. In comparison to resistant mares, susceptible mares had an increased (P = 0.04) BFV of the uterine arteries at 24 h before intrauterine infusion of killed semen, and an increased (P = 0.03) PI at 2 d after infusion. The TAMV, plasma P4 concentrations, and follicular and luteal size and blood flow did not differ (P > 0.05) between resistant and susceptible mares. Endometrial mRNA expression of IL1B increased (P = 0.05) at 2 d after the infusion of killed semen in the susceptible mares, and the expression of IL10 increased (P = 0.003) at 7 d after the infusion within the resistant mares. Interleukin 6 mRNA was increased (P = 0.05) in susceptible compared to resistant mares at 2 d after infusion. In summary, an intrauterine infusion of killed semen increases uterine blood flow resistance and alters endometrial gene expression of inflammatory cytokines for at least 7 d but does not affect ovarian blood supply and luteal function in mares susceptible to PBIE.

Influence of long-term treatment with equine somatotropin (EquiGen) on gonadal function in stallions with poor semen quality.

The aim of the present study was to investigate the spermatogenic and Leydig cell activity in stallions with impaired semen quality after treatment with equine somatotropin. Experiments were performed using 18 adult clinically healthy stallions with poor semen quality which did not pass breeding soundness evaluation. The animals were randomly divided into a treatment (n = 9) and a control (n = 9) group. Over a period of 90 days, nine stallions received a daily intramuscular injection of 10 mg recombinant equine somatotropin (EquiGen, BresaGen Limited, Adelaide, Australia) and 9 control animals were injected with the same amount of physiological saline solution. During and until 2 months after treatment, semen characteristics and daily sperm output as well as plasma testosterone concentrations were determined monthly in all stallions. In addition, testosterone concentration measurement after stimulation with hCG was performed in all animals immediately before and at the end of the treatment period as well as 2 months later. Our results demonstrate that equine somatotropin (EquiGen) given daily in a dose of 10 mg per animal during 90 days had no significant effect neither on plasma testosterone concentrations and hCG-induced testosterone release nor on semen quality parameters in adult stallions with poor semen characteristics.

Treatment of chronic proximal suspensory desmitis in horses using focused electrohydraulic shockwave therapy.

The objective of the present clinical report was to investigate the short- and long-term outcomes of chronic proximal suspensory desmitis (PSD) treated with Extracorporeal Shockwave Treatment (ESWT). Fifty-two horses with chronic PSD in the forelimb (34 cases) or hindlimb (22 cases) were included in the study. Three horses had lesions in both hindlimbs and one in both forelimbs. The origin of the suspensory ligament was treated every three weeks for a total of three treatments using 2000 impulses applied by a focused ESWT device (Equitron) at an energy flux density of 0.15 mJ/ mm2. This treatment regime was followed by box rest and a controlled exercise program of 12 weeks duration. The horses were assessed 3, 6, 12, 24 weeks and one year after the first treatment. Of the 34 cases with forelimb PSD, 21 (61.8%) had returned to full work by six months after diagnosis and 19 cases (55.9%) were still in full work one year after ESWT. Of the 22 horses with hindlimb PSD, 9 (40.9%) had returned to full work by six months and 4 (18.2%) were still in full work one year after diagnosis. There was no association (chi-square test) between the outcome and the severity of the initial ultrasonographic and/or radiographic findings. Compared with the results of other clinical studies, these findings suggest that in horses with PSD of fore- and hindlimb, the prognosis for returning to full work six months after diagnosis can be improved when ESWT and a controlled exercise program are used. However, a high rate of recurrence occurred in the hindlimb despite treatment.

Effect of exercise and suspensory on scrotal surface temperature in the stallion.

In this study, the effect of exercise (treadmill, riding) on scrotal surface temperature (SST) in the stallion with and without suspensory was evaluated. Experiments were carried out between September and November 2004 using 12 Franches-Montagnes stallions from the National Stud in Avenches (Switzerland). Each stallion performed a standardized incremental treadmill and a ridden test with and without suspensory. The intensity of exercise was monitored by heart rate and blood lactate concentration. For SST measurements, special thermistors were developed and affixed to the most ventral part of the scrotum over each testis. SST was recorded telemetrically at 1min intervals. Our results show that type of exercise (treadmill/ridden) and suspensory (with/without) significantly influenced SST. The mean SST level was higher during treadmill (32.2+/-0.02 degrees C) than during ridden exercise (30.4+/-0.03 degrees C) and mean SST differences between stallions with and without suspensory were smaller in treadmill (0.4 degrees C) than in ridden (2 degrees C) exercise. These findings clearly demonstrate that ambient airflow, which was higher during ridden exercise, is important and effective in SST regulation. In order to prevent possible thermal damage to spermatogenic cells we recommend removing the suspensory immediately after exercise.

Influence of immunization against GnRH on reproductive cyclicity and estrous behavior in the mare.

The aim of this study was to evaluate the effect of active immunization against GnRH on ovarian activity, plasma progesterone and estradiol concentrations and on estrous behavior in adult mares. Eighteen cyclic mares were randomly divided into a treatment and control group. Nine mares were immunized twice with 2 mL (400 microg GnRH-protein conjugate) of a GnRH-vaccine (Improvac, CSL Limited, Australia) administered intramuscularly, 4 weeks apart. Control mares received the same amount of saline solution. Ovaries and uterus of all mares were examined weekly by ultrasonography from 3 weeks before to 60 weeks after first immunization. Thereafter, vaccinated mares were evaluated monthly until 100 weeks after first vaccination. In addition, mares were teased with a stallion for assessment of estrous behavior and blood was collected for progesterone, estradiol-17beta and GnRH antibody titer determination. Results demonstrate that vaccination against GnRH significantly (P<0.05) influenced all parameters, except estradiol-17beta concentration. All vaccinated mares ceased reproductive cyclicity (plasma progesterone <1 ng/mL, follicles <3 cm) within 8 weeks after the first injection and ovarian activity remained suppressed for a minimum of 23 weeks. Five mares resumed cyclicity (follicles >3 cm, progesterone >1 ng/mL) while three mares showed only follicular activity (follicles >3 cm) and one mare remained completely suppressed for the entire duration of the study. In spite of ovarian suppression, four mares expressed sporadic and one mare continuous estrous behavior. In conclusion, reproductive cyclicity in adult mares can be successfully suppressed by immunization against GnRH but the timing of resumption of cyclicity is highly variable and estrous behavior may occur in spite of ovarian suppression.

Influence of repeated treadmill exercise on quality and freezability of stallion semen.

The objective of this study was to investigate changes of quality and freezability of stallion semen in response to repeated acute treadmill exercise. Ejaculates from 11 stallions were collected, evaluated and frozen weekly during four periods of 4 weeks each defined as before (period 1), during (period 2) and after (periods 3 and 4) intense exercise. In fresh semen the gel-free volume, sperm concentration, motility, normal sperm and sperm with major defects (acrosome defects, nuclear vacuoles, abnormal heads, midpiece defects and proximal droplets) were evaluated. In frozen-thawed semen, motility as well as viability (SYBR-14/PI) were examined. In period 2, all stallions were exercised on an indoor high speed treadmill twice a week (total of eight sessions) using an incremental workload test. Heart rate was monitored telemetrically during exercise and blood samples were taken for determination of cortisol, testosterone and lactate. Results of our investigation demonstrate that heart rate and the plasma concentrations of cortisol, testosterone and lactate significantly (P < 0.05) increased during each exercise session. Furthermore, significantly more major sperm defects were present in periods 3 (69.5+/-2.1%) and 4 (66.8+/-2.1%) than in periods 1 (62.2+/-2.4%) and 2 (62.5+/-2.2%). Acrosome defects increased towards the end of exercise but improved 3 weeks later to values observed before exercise. In frozen-thawed semen, motility was significantly lower in period 2 (45.4+/-2.3%) compared to period 4 (51.6+/-1.7%) and viability was significantly lower in period 2 (49.2+/-2.0%) than in periods 1 (53.8+/-2.1%) and 4 (53.7+/-1.6%). Our results clearly demonstrate that in the stallion repeated strenuous treadmill exercise can negatively influence semen quality and freezability.

Influence of vedaprofen (Quadrisol) on quality and freezability of stallion semen.

The objective of this study was to evaluate the effect of the non-steroidal anti-inflammatory drug (NSAID) vedaprofen (Quadrisol) on quality and freezability of stallion semen. Experiments were performed using 22 Franches Montagnes stallions from the National Stud in Avenches (Switzerland) randomly divided into a control and test group. Vedaprofen was given orally to all stallions of the test group at the recommended therapeutic dose (initial dose of 2mg/kg followed by 1mg/kg body weight every 12h) for 14 days. Control animals received the same amount of carrier substance. During treatment, blood samples of five stallions in both test and control group were collected for PGF(2 alpha)-metabolite (PG-metabolite) determination. Ejaculates from all stallions were collected and cryopreserved weekly for 14 weeks from September to December. Concentrations of PG-metabolite, PGF and PGE were measured in the seminal plasma of ejaculates collected 2 weeks before, during and 2 weeks after treatment. In fresh semen the volume, concentration, motility and number of normal sperm and sperm with major defects (acrosome defects, abnormal heads, nuclear vacuoles, proximal droplets, midpiece defects) were evaluated. In frozen-thawed semen samples motility as well as viability (SYBR-14/PI) were tested and the hypoosmotic swelling test (HOS) was performed. Results demonstrate that vedaprofen had no effect on blood plasma concentration of PG-metabolite but significantly inhibited both, PGF and PGE concentrations in seminal plasma. Furthermore, all quality parameters in fresh and frozen-thawed semen were not affected by vedaprofen treatment but the time of semen collection had a significant (P<0.05) effect on motility, normal sperm and sperm with nuclear vacuoles in fresh semen.