Dexamethasone acutely regulates endocrine parameters in stallions and subsequently affects gene expression in testicular germ cells.

Testicular steroidogenesis and spermatogenesis are negatively impacted by stress-related hormones such as glucocorticoids. The effects of two injections of a therapeutic dose of dexamethasone (a synthetic glucocorticoid, 0.1mg/kg; i.v.) given 24h apart to each of three stallions were investigated and compared to three saline-injected control stallions. Dexamethasone decreased circulating concentrations of cortisol by 50% at 24h after the initial injection. Serum testosterone decreased by a maximum of 94% from 4 to 20h after the initial injection of dexamethasone. Semen parameters of the dexamethasone-treated stallions were unchanged in the subsequent two weeks. Two weeks after treatment, stallions were castrated. Functional genomic analyses of the testes revealed that, of eight gene products analyzed, dexamethasone depressed concentrations of heat shock protein DNAJC4 and sperm-specific calcium channel CATSPER1 mRNAs by more than 60%. Both genes are expressed in germ cells during spermiogenesis and have been related to male fertility in other species, including humans. This is the first report of decreased DNAJC4 and CATSPER1 mRNA concentrations in testes weeks after dexamethasone treatment. Concentrations of these mRNAs in sperm may be useful as novel markers of fertility in stallions.

The development and evaluation of a mathematical nutrition model to predict digestible energy intake of broodmares based on body condition changes.

Mathematical nutrition models have been developed for beef and dairy cattle to estimate dietary energy intake needed to change BCS. Similar technology has not been used to improve nutrition and feeding strategies for horses. An accurate equine nutrition model may enhance feeding management and reduce the costs of unnecessary overfeeding and promote an optimal level of fatness to achieve reproductive efficiency. The objectives of this study were to develop and evaluate a mathematical nutrition model capable of accurately predicting dietary energy changes to alter BW, rump fat (RF) thickness, and overall body fat (BF), which is needed to maximize profitability and productivity of mares. Model structure was similar to a previously developed model for cattle, and literature data for Quarter Horse mares were used to parameterize the horse model in predicting DE requirement associated with BCS changes. Evaluation of the horse model was performed using an independent dataset comprising 20 nonlactating Quarter Horse mares. Pretrial BCS was used to assign mares to 1 of 4 treatment groups and fed to alter BCS by 1 unit as follows: from 4 to 5 (Group 1), 5 to 4 (Group 2), 6 to 7 (Group 3), and 7 to 6 (Group 4). The BCS, RF thickness, and BW were measured for each mare before the commencement of the feeding trial and once per week thereafter for the duration of a 30-d feeding trial. Initial and target BCS, percent BF, and BW data were collected from each mare and inputted into the model. Mares were individually fed according to the DE suggestions proposed by the model to achieve the targeted BCS change within 30 d. The coefficient of determination of observed and model-predicted values (model precision) was 0.907 (P < 0.001) for BCS, 0.607 (P < 0.001) for percent BF, and 0.94 (P < 0.001) for BW. The BCS was highly correlated to percent BF (r = 0.808; P = 0.01). We concluded the reparameterized model was reliable to predict changes in BW and BCS, but more work is needed to improve the predictions of initial and final body composition.

Thermoregulation of the testicle in response to exercise and subsequent effects on semen characteristics of stallions.

Stallions (n = 8) were implanted with a thermal sensory device in the muscle of the neck and the subcutaneous tissue of the scrotum and then assigned to either a nonexercise (Non-EX; n = 4) or exercise (EX; n = 4) group. A motorized equine exerciser was used to work EX stallions 30 min/d for 4 d/wk during a 12-wk period from July through October 2010. Temperatures (subcutaneous scrotal, intramuscular neck, and rectal) were recorded at 0, 22, and 30 min after the start of exercise, as well as 60 and 120 min post-exercise. Hourly ambient temperature and relative humidity data were also obtained. Semen was collected at 0, 4, 8, and 12 wk and analyzed for volume, sperm concentration, total sperm numbers, percentages of total and progressively motile sperm, sperm morphologic characteristics, and sperm DNA quality. No effect (P > 0.05) of exercise was observed on any of the measured semen variables. Implantation of thermal sensory devices had no demonstrable acute or chronic effects on the scrotal or neck tissue, indicating that the thermal sensory devices are a safe and effective way to measure subcutaneous scrotal and neck temperatures. At 22 and 30 min of exercise, rectal and neck temperatures increased (P < 0.0001) approximately 1.9 and 2.4°C, respectively, and scrotal temperatures simultaneously increased, although not significantly (P = 0.33), approximately 0.8°C. Correlations existed between scrotal, neck, rectal, and ambient temperatures, with the correlation between scrotal and rectal temperatures being greatest (r(s) = 0.76; P < 0.0001). Although moderate exercise for a short duration in extreme heat and humidity did significantly increase core body temperatures in stallions, scrotal temperatures did not significantly increase, and sperm parameters were unaffected.

Copper and zinc balance in exercising horses fed 2 forms of mineral supplements.

Studies comparing the absorption and retention of various forms of trace minerals in horses have yielded mixed results. The objective of this study was to compare Cu and Zn absorption and retention in exercising horses where the mineral was supplemented in the sulfate or organic chelate form. Nine mature horses were used in a modified switchback design experiment consisting of seven 28-d periods. Horses were fed a diet consisting of 50% concentrate and 50% hay that was balanced to meet the energy, protein, Ca, and P requirements for horses performing moderate-intensity exercise. Horses were subjected to a controlled mineral repletion-depletion diet sequence before feeding the experimental diet to standardize mineral status across horses. The experimental diet was designed to provide 90% of the 1989 NRC for Cu and Zn, with supplemental mineral provided in the inorganic sulfate form (CuSO(4) and ZnSO(4)) or the organic chelate form (Cu-Lys and Zn-Met). Feed, fecal, urine, and water samples collected during a total collection during the last 4 d of the experimental diet periods were analyzed to determine apparent absorption and retention of Cu and Zn from the 2 mineral forms. A formulation error caused horses receiving the organic chelate diet to consume about 3 times the amount of Cu and Zn compared with those fed the sulfate-supplemented diet. Copper and Zn intake and fecal excretion were greater (P < 0.05) for horses consuming the organic chelate-supplemented diet. Apparent absorption values for all horses were negative. Apparent Cu absorption and retention as a percentage of intake were greater for horses fed the organic chelate diet (P < 0.05). It is unknown why excretion of Cu and Zn by the horses during the total collection exceeded the mineral intake. Although Cu-Lys seemed to be better absorbed than CuSO(4) and absorption of Zn-Met and ZnSO(4) were not different, these results are tempered by the observation of abnormally high fecal and urinary excretion values for Cu and Zn in the present study.

Heat shock protein 70 gene expression in equine blastocysts after exposure of oocytes to high temperatures in vitro or in vivo after exercise of donor mares.

Heat above homeothermy can be detrimental to embryonic development, and cells may produce heat shock proteins to try to mitigate these effects. The authors examined the developmental competence of equine oocytes after a single heat exposure (42 degrees C, 2 or 4 h) during early or late stages of in vitro maturation. Rates of nuclear maturation, cleavage after intracytoplasmic sperm injection, and advanced embryonic development (morula or blastocyst) were compared to those for unexposed controls. Concentrations of heat shock protein 70 (HSPA1A) mRNA were determined by real-time RT-PCR in resulting blastocysts, and were compared to those for embryos derived in vivo from control or exercised mares. Exposure of oocytes to heat at the onset of in vitro maturation did not affect any measured end point. However, exposure to 42 degrees C late in maturation culture reduced rates of oocyte nuclear maturation for both the 2 h (43/105 (43%) compared to control 70/103 (68%); P < 0.01), and 4 h (47/106 (44%) compared to control 60/103 (59%); P < 0.05) groups. Additionally, late heat exposure reduced development to morulae and blastocyst stages after intracytoplasmic sperm injection (ICSI; 18/89 (20%) compared to control 43/128 (34%); P < 0.05). Seven days after oocyte heat exposure, resultant blastocysts had a higher abundance of HSPA1A gene transcripts, relative to those for 18S rRNA. In vitro-produced embryos and lower-quality in vivo-produced embryos had significantly higher relative HSPA1A mRNA (lower 18S rRNA) concentrations than did higher-quality in vivo-produced embryos. The authors concluded that equine oocytes were sensitive to heat during late in vitro maturation, and responded to thermal shock with an increased ratio of HSPA1A:18S gene expression that was measurable in the resulting blastocyst. Embryos produced in vitro (including controls) had increased levels of HSPA1A mRNA relative to 18S rRNA compared to in vivo-produced embryos, suggesting a response to environmental insult.

Embryo recovery from exercised mares.

The effect of exercise on mare reproductive efficiency was evaluated by comparing rates of embryo recovery from mares assigned to either an exercise regimen or a non-exercise (control) regimen. Exercised mares were worked daily for 30 min under average ambient conditions of >30 degrees C and >50% humidity. Mares were inseminated during estrus and subjected to uterine flush for embryo recovery on d 7 after ovulation for two consecutive cycles. After this, mares were allocated to the opposite group and allowed an estrous cycle without reproductive manipulation; then insemination and uterine flushing were conducted on two more consecutive cycles. Prostaglandin F(2alpha) was administered on the day of uterine flush. Mare rectal temperature increased during exercise from a mean of 38 degrees C to a mean of 39.9 degrees C. Mares had ovulations from smaller follicles when exercised than they did under control conditions (39.8+/-0.5 compared with 41.5+/-0.5mm diameter; P<0.05), and had an increased time from PGF(2alpha) administration to subsequent ovulation (8.47+/-0.337 compared with 9.27+/-0.294 d; P<0.05). Embryo recovery from control mares was 22 of 35 (63%). Fewer embryos were recovered from exercised mares (11 of 32, 34%; P<0.05). The proportion of embryos classified as Grade 1 tended to be less in exercised than in non-exercised mares (4 of 11, 36% compared with 16 of 22, 73%; P=0.051). These data indicate that exercising mares in a hot and humid environment are associated with changes in ovarian follicle development and ovulation, and a reduction in embryo recovery.

Reproductive performance in mares subjected to examination by diagnostic ultrasound.

Mares were subjected to frequent examination by diagnostic ultrasound and data were compiled with respect to reproductive efficiency. The data were collected over a 3-yr period on 1032 light horse mares. The cummulative pregnancy rate at 35 d post-ovulation was 96.8% and the pregnancy rate per cycle was 76.0% as determined by ultrasound examination. The average number of cycles per conception was 1.43, with an average of 2.29 inseminations per cycle. The incidence of early embryonic death was 7.8%. Mares were subjected to an average of 5.04 scans during the follicular phase of the cycle. The average number of ultrasound examinations per mare (including pregnancy examinations) was 9.99. Although these data were obtained from an experiment that did not use both control and treated mares, there was no indication that preovulatory oocytes or embryos were damaged by routine ultrasound examination. Comparisons with existing data from commercial facilities are difficult to make concerning any improvement in reproductive efficiency resulting from the routine use of ultrasonography, but these data do suggest relative safety in equine reproductive management when ultrasound examinations are conscientiously used.

Methods for collecting follicular oocytes from mares.

A series of experiments was conducted to develop a procedure for consistent, repeatable collection of oocytes from the preovulatory follicle of the mare. In one experiment, in situ follicular aspiration with a needle and syringe was performed on 19 mares. From 37 aspirations, four oocytes were recovered (10% recovery rate). In a second experiment, ovaries were visualized via standing flank laparotomy during which two different aspiration techniques were used. Use of a needle and syringe as in the first experiment resulted in successful oocyte recovery in one of seven (14%) attempts. Aspiration via a continuous irrigation vacuum system (CIV), developed for use during laparotomy, resulted in collection of oocytes from six of 10 (60%) attempts. In the third experiment, oocytes were recovered from seven of 18 (38%) attempts at in situ follicular aspiration using a double-lumen needle attached to the CIV. In each experiment, some mares were subjected to stimulation of follicular maturation by exogenous hormones. Oocyte recovery was significantly increased in treated mares as compared with nontreated mares. Results indicate that collection of equine follicular oocytes by in situ aspiration is possible with moderate success. Oocytes apparently are not physically damaged by the procedure, as most retained either the corona radiata or the entire cumulus cell mass.

Fine structure of the follicular oocyte of the horse.

Oocytes recovered by follicular aspiration were evaluated by light and transmission electron microscopy. Of the 22 oocytes, 4 exhibited characteristics of degeneration, and the remaining 18 were in various stages of meiotic development. Of the non-degenerate oocytes, 14 were in the germinal vesicle stage, 2 had undergone nuclear membrane disintegration, 1 displayed chromosomes in late metaphase I-early anaphase I, and 1 oocyte was in the process of extrusion of the first polar body. Although some oocytes retained complete cumulus cell investments, oocytes were predominantly enclosed only by the corona radiata. Ultrastructural evaluation revealed that follicular oocytes of the horse are similar to those of other mammals. An abundance of vesicular agranular endoplasmic reticulum in horse oocytes was the most obvious difference. Variation in intracellular organization seemed to be primarily dependent upon stage of meiotic development.