Short-term equine interaction for reducing test anxiety and facilitating coping skill development in college students during examination periods: A preliminary study.

Mental health concerns are common among college students and more prevalent during examination periods when stress and anxiety are heightened. While the benefits of equine interaction have been reported, the impact of short-term equine interaction on coping skills valuable for managing test anxiety have yet to be studied. Therefore, the purpose of this preliminary study was to evaluate the impact of short-term equine interaction during the college examination period on development of coping skills and anxiety levels associated with testing, both self-reported and those reflected in physiological measures. Full-time college students engaged in a one-hour equine interaction session held on campus at the onset of final exam week. Participants completed a survey targeting perception of anxiety levels and coping skills. Heart rate and salivary cortisol measurements were collected pre- and post-session. Paired t-tests were performed between pre- and post- session measures and correlations between survey answers and physiological measures determined. Significant reductions were found in all areas of anxiety (Overall Academics P = 0.0003, Final Exam Preparation P = 0.0003, Taking Final Exam P = 0.0003). The reduction in exam-related anxiety was positively correlated to cortisol concentrations (r = 0.69, P = 0.03) with lower concentrations (P = 0.05) and increased heart rate (P = 0.003) post-session. Regarding coping skills, improvements were observed post-session in assertiveness (P = 0.008). Skill development coupled with reduced anxiety supports the potential benefits short-term equine interaction can offer students during the high stress periods, but these findings should be confirmed with more in-depth study.

Effect of cryopreservation techniques on proliferation and apoptosis of cultured equine ovarian tissue.

Preservation of cellular integrity and its mechanisms after ovarian tissue cryopreservation (OTC) and in vitro culture (IVC) procedures are crucial aspects for the success of preservation and recovery of female fertility. This study aimed to evaluate the effects of two cryopreservation methods (slow-freezing, SF, and vitrification, VIT) on the equine ovarian tissue after 1, 3, and 7 days of IVC by assessing: (i) preantral follicle morphology and distribution of follicle classes; (ii) protein expression of markers of cell proliferation for EGFR and Ki-67; (iii) markers of apoptosis for Bax and Bcl-2; and (iv) DNA fragmentation. Percentages of normal primordial follicles were similar (P > 0.05) among SF-control, VIT-control, and fresh control groups. After 7 days of culture, VIT-IVC7 had a greater (P < 0.05) total percentage of normal preantral follicles when compared with SF-IVC7, but both had a lower (P < 0.05) percentage than fresh IVC7 group. Prior to and after 7 days of culture, expression of EGFR and Ki-67 were similar (P > 0.05) among fresh, SF, and VIT groups. After 7 days of culture, VIT had higher (P < 0.05) Bax expression than the fresh and SF tissues, but Bcl-2 was similar (P > 0.05) among groups. Prior to IVC, TUNEL signals were similar (P > 0.05) among groups; however, VIT-IVC7 had greater (P < 0.05) TUNEL signals when compared with the fresh IVC7 group. In conclusion, findings demonstrated: (i) similar efficiency between SF and VIT compared with fresh control to preserve morphologically normal follicles; and (ii) similar tissue functionality and cell proliferation capability after equine OTC by either SF and VIT methods following IVC for 7 days. The results herein presented shed light on equine fertility preservation programs using OTC techniques.

In vivo antral follicle wall biopsy: a new research technique to study ovarian function at the cellular and molecular levels.

BACKGROUND: In vivo studies involving molecular markers of the follicle wall associated with follicular fluid (FF) milieu are crucial for a better understanding of follicle dynamics. The inability to obtain in vivo samples of antral follicle wall (granulosa and theca cells) without jeopardizing ovarian function has restricted advancement in knowledge of folliculogenesis in several species. The purpose of this study in mares was to develop and validate a novel, minimally invasive in vivo technique for simultaneous collection of follicle wall biopsy (FWB) and FF samples, and repeated collection from the same individual, during different stages of antral follicle development. We hypothesized that the in vivo FWB technique provides samples that maintain the normal histological tissue structure of the follicle wall layers, offers sufficient material for various cellular and molecular techniques, and allows simultaneous retrieval of FF. METHODS: In Experiment 1 (ex vivo), each follicle was sampled using two techniques: biopsy forceps and scalpel blade (control). In Experiment 2 (in vivo), FWB and FF samples from 10-, 20-, and 30-mm follicles were repeatedly and simultaneously obtained through transvaginal ultrasound-guided technique. RESULTS: In Experiment 1, the thickness of granulosa, theca interna, and theca externa layers was not influenced (P > 0.05) by the harvesting techniques. In Experiment 2, the overall recovery rates of FWB and FF samples were 97 and 100%, respectively. However, the success rate of obtaining samples with all layers of the follicle wall and clear FF varied according to follicle size. The expression of luteinizing hormone receptor (LHR) was mostly confined in the theca interna layer, with the estradiol-related receptor alpha (ERRα) in the granulosa and theca interna layers. The 30-mm follicle group had greater (P < 0.05) LHR expression in the theca interna and ERRα in the granulosa layer compared to the other groups. The overall expression of LHR and ERRα, and the intrafollicular estradiol were higher (P < 0.05 - P < 0.0001) in the 30-mm follicle group. CONCLUSION: The in vivo technique developed in this study can be repeatedly and simultaneously used to provide sufficient FWB and FF samples for various cellular and molecular studies without jeopardizing the ovarian function, and has the potential to be translated to other species, including humans.

Effects of body condition score (BCS) on steroid- and eicosanoid-metabolizing enzyme activity in various mare tissues during winter anoestrus.

The objective of this study was to determine the activity of steroid- and eicosanoid-metabolizing enzymes in horses with varying BCSs. The BCSs of twenty non-pregnant, anoestrous mares were determined prior to euthanasia, and tissue samples were collected from the liver, kidney, adrenal gland, ovary and endometrium. Cytochrome P450 1A (CYP1A), 2C (CYP2C), 3A (CYP3A) and uridine 5'-diphospho-glucuronosyltransferase (UGT) activities were determined using luminogenic substrates. The MIXED procedure of SAS was used to test the effect of BCS on enzyme activity and differences between tissues. Activity of CYP1A in adrenals was increased (p ≤ .05) in BCS 5 versus BCSs 4 and 6. Activity of CYP1A in the liver was increased (p = .05) in BCS 4 versus BCSs 5 and 6. Activity of CYP1A was 100-fold greater (p < .0001) in the liver than in the adrenal, ovary and kidney. Activity of CYP2C was 100-fold greater (p < .0001) in the liver than in the adrenal, ovary and endometrium. Activity of CYP3A was only detectable in the liver. Activity of UGT in the kidney was decreased (p = .02) in BCS 4 versus BCSs 5 and 6. Activity of UGT was threefold greater (p < .0001) in the liver than in the kidney, whereas activity of UGT was ninefold greater (p < .0001) in the kidney than in the ovary and endometrium. In general, BCS did not alter the activity of steroid- and eicosanoid-metabolizing enzymes in horses. However, tissue differences in these enzymes indicated abundant hepatic metabolism in horses, which is similar to other livestock species.

Body fat of stock-type horses predicted by rump fat thickness and deuterium oxide dilution and validated by near-infrared spectroscopy of dissected tissues.

Body condition score and percent body fat (BF; %) of horses are positively correlated with reproductive efficiency and are indicative of metabolic issues. However, BF in horses may be poorly predicted because current procedures are either subjective or dependent on one anatomical location. Therefore, the objectives of the current study were to compare 2 methods of predicting BF using rump fat thickness (RFT) and deuterium oxide (DO) dilution with actual tissue fat analysis by near-infrared spectroscopy (NIRS) in stock-type horses and to identify the relationship between BF and BCS. Twenty-four stock-type horses were selected to be humanely euthanized based on 3 primary criteria: geriatric, crippled, and/or unsafe. Approximately 20 h before slaughter, horses were weighed and BCS assessed to be 1 ( = 1; 433 kg), 2 ( = 1; 415 kg), 3 ( = 1; 376 kg), 4 ( = 7; 468 ± 13 kg), 5 ( = 10; 455 ± 11 kg), and 6 ( = 4; 493 ± 12 kg) and RFT was measured using ultrasonography. Blood samples were collected immediately before and 4 h after DO infusion (0.12 g/kg BW). Deuterium oxide concentration of plasma was determined by gas isotope ratio mass spectrometry. Horses were housed in a dry lot overnight before being individually sedated (1.1 mg xylazine/kg BW) and anesthetized using a jugular venipuncture (2.2 mg ketamine/kg BW), and potassium chloride (KCl) solution was administered to cease cardiac function before exsanguination. After euthanasia, horse carcasses were processed and dissected and tissues were collected for NIRS analysis. Body fat predicted by DO dilution was correlated with BF measured by NIRS analysis on various weight bases ( = 0.76 to 0.81, < 0.001), whereas no correlation was observed for BF predicted by RFT. A paired -test indicated that both DO dilution and RFT overestimated BF by 2.48 to 3.26% ( < 0.001) and 5.81 to 6.59% ( < 0.001), respectively, compared with NIRS analysis. Body condition scores affected BF measured by NIRS analysis ( ≤ 0.038) and predicted by DO dilution ( < 0.001) and RFT ( = 0.042). The current study provided evidence that DO dilution was a better prediction method for BF than RFT and that BCS may serve as a BF predictor in stock-type horses.

Equine ovarian tissue viability after cryopreservation and in vitro culture.

Ovarian tissue cryopreservation allows the preservation of the female fertility potential for an undetermined period. The objectives of this study were to compare the efficiency of cryoprotective agents (CPAs; dimethyl sulfoxide, DMSO; ethylene glycol, EG; and propylene glycol, PROH) using slow-freezing and vitrification methods, and evaluate the viability of cryopreserved equine ovarian tissue after 7 days of culture. Fresh and cryopreserved ovarian fragments were evaluated for preantral follicle morphology, stromal cell density, EGFR, Ki-67, Bax, and Bcl-2 protein expression, and DNA fragmentation. Vitrification with EG had the highest rate of morphologically normal preantral follicles, while DMSO had the lowest (76.1 ± 6.1% and 40.9 ± 14.8%, respectively; P < 0.05). In slow-freezing, despite that DMSO had the highest percentage of morphologically normal follicles (77.7 ± 5.8%), no difference among the CPAs was observed. Fluorescence intensity of EGFR and Ki-67 was greater when vitrification with EG was used. Regardless of the cryopreservation treatment, DMSO had the highest (P < 0.05) Bax/Bcl-2 ratio; however, DNA fragmentation was similar (P > 0.05) among treatments after thawing. After in vitro culture, the percentage of normal follicles was similar (P > 0.05) between slow-freezing and vitrification methods; however, vitrification had greater (P < 0.05) stromal cell density than slow-freezing. In summary, equine ovarian tissue was successfully cryopreserved, increasing the viability of the cells in the ovarian tissue after thawing when using DMSO and EG for slow-freezing and vitrification methods, respectively. Therefore, these results are relevant for fertility preservation programs.

The relationship between sperm quality in cool-shipped semen and embryo recovery rate in horses.

The relationship between the quality of cool-shipped stallion semen and fertility has not been adequately described. This study evaluated sperm quality of cool-shipped semen from 459 ejaculates (N = 130 stallions) that were used for insemination of 196 embryo donor mares (n = 496 estrous cycles). Embryo recovery rate (ERR; %) increased, as all sperm measures (e.g., motility, viability, DNA quality, morphology, concentration, and total number) increased. Threshold values are reported for each sperm quality measure (e.g., total sperm motility ≥ 65%) that separate two ERR groups (e.g., average: ∼50% ERR; high: ∼65% ERR).

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.

Effect of surgical technique for unilateral orchiectomy on subsequent testicular function in Miniature Horse stallions.

REASONS FOR PERFORMING STUDY: Management decisions on unilateral orchiectomy are often influenced by the potential for post operative return to successful breeding. The effects of 2 surgical methods (first intention [FI] vs. second intention [SI] incision healing) for unilateral orchiectomy on resulting semen quality and scrotal temperature were evaluated. OBJECTIVE: To evaluate the effects of 2 surgical unilateral orchiectomy techniques on scrotal healing, size of the remaining testis and post operative sperm quality. MATERIALS AND METHODS: Unilateral orchiectomy was performed on mature Miniature Horse stallions. Semen was collected prior to and up to 60 days after, unilateral orchiectomy. Semen parameters, scrotal and body temperatures, testis volume and days to incision healing were evaluated. RESULTS: There was no effect of treatment or time on percent total sperm motility. Total sperm numbers were higher (P < 0.05) 60 days after unilateral orchiectomy compared with 14 and 30 days. Percent viable sperm were higher (P < 0.05) 30 and 60 days compared with pre- and 14 day post unilateral orchiectomy. Scrotal temperatures were lower after unilateral orchiectomy compared with preoperative values ( < or = 0.003). Higher scrotal temperatures were recorded in Group IF, as compared with Group IS, during recoveryfrom anaesthesia and at 1 and 2 h after surgery (P = 0.02). Mean time to incision healing was less in Group II (10.0 days) than in Group II (21.5 days; P = 0.05). CONCLUSION: In this study, total sperm motility was maintained and size of the remaining testis, total sperm numbers and percent viable sperm increased after unilateral orchiectomy. Incision healing time was shorter in Group II; however, surgical technique did not have an effect on semen quality at 30 and 60 days post unilateral orchiectomy. POTENTIAL RELEVANCE: These data suggest that surgical technique for unilateral orchiectomy may not dramatically influence function of the remaining testis.

Effect of selenium supplementation and plane of nutrition on mares and their foals: selenium concentrations and glutathione peroxidase.

To investigate the maternal plane of nutrition and role of Se yeast on muscle Se concentration, plasma glutathione peroxidase (Gsh-Px) activity, and colostrum Se concentration in mares and their foals, 28 Quarter Horse mares (465 to 612 kg of BW, and 6 to 19 yr of age) were used in a study with a randomized complete block design. Mares were blocked by expected foaling date and randomly assigned to dietary treatments within blocks. Dietary treatments were arranged as a 2 x 2 factorial with 2 planes of nutrition, pasture or pasture + grain mix (fed at 0.75% of BW on an as-fed basis) and 2 concentrations of Se yeast supplementation (0 or 0.3 mg/kg of DMI), resulting in 4 treatments: pasture, pasture + grain mix, pasture + grain mix + Se, or pasture + Se. Mares fed diets of pasture and pasture + Se received approximately 100% of the calculated NRC (2007) DE requirements, whereas mares fed diets of pasture + grain mix and pasture + grain mix + Se received 120%. Selenium supplementation began 110 d before the estimated foaling date and treatments were terminated at parturition. Blood and muscle (biopsy) samples were collected on d 0 and then every 14 or 28 d, respectively, thereafter until parturition. Additionally, BW, BCS, and rump fat (RF) were recorded every 14 d. At parturition, colostrum, foal plasma, and foal muscle samples were collected and sampling continued every 14 d for plasma and every 28 d for muscle until d 56. Mare BW, BCS, and RF were affected by plane of nutrition (P

Effect of selenium supplementation and plane of nutrition on mares and their foals: foaling data.

To investigate the maternal plane of nutrition and role of Se yeast on foaling variables and passive transfer of IgG, 28 Quarter Horse mares were used in a study with a randomized complete block design. Mares were blocked by expected foaling date and assigned randomly within block to dietary treatments. Dietary treatments were arranged as a 2 x 2 factorial with 2 planes of nutrition, pasture or pasture + grain mix (fed at 0.75% of BW on an as-fed basis) and 2 concentrations of Se yeast (0 or 0.3 mg/kg of DMI). This resulted in 4 treatments: pasture (PA), pasture + Se (PS), pasture + grain mix (PG), and pasture + grain mix + Se (PGS). Assuming DMI at 2% of BW, the mares fed PA and PS received approximately 100% of the calculated NRC (2007) DE requirements, whereas PG and PGS received 120%. Selenium supplementation began 110 d before the estimated foaling date, and all dietary treatments were terminated at parturition. At parturition, foaling variables were recorded. Additionally, placental weight was recorded and 2 samples from each placenta were collected for analysis of DNA, RNA, and protein. Colostrum was obtained for fat, protein, milk urea N, somatic cell count, and IgG analyses. Foal blood samples were collected at 0, 6, 12, 18, and 24 h after parturition for IgG analysis. There was no effect (P >or= 0.21) of Se or plane of nutrition on foaling variables; however, foal BW as a percentage of mare BW tended (P = 0.10) to be reduced in foals from mares on grain mix (PG and PGS; 7.6%) compared with mares not fed grain mix (PA and PS; 8.0%). There was also no effect (P >or= 0.20) of Se or plane of nutrition on placental cell number (mg of DNA/g), potential cellular activity (RNA:DNA), expulsion time, or weight. However, mares fed supplemental Se (PS and PGS) had decreased (P = 0.02) placental cell size (24.1 mg of protein/mg of DNA) compared with mares not fed supplemental Se (PA and PG; 32.5 mg of protein/mg of DNA). There was also no effect (P >or= 0.18) of Se or plane of nutrition on colostral fat, protein, milk urea N, or somatic cell count. However, mares fed grain mix (PG and PGS) had less (P = 0.03) colostral IgG (76.5 g/L) compared with mares not fed grain mix (PA and PS; 126.6 g/L). Foals from mares fed grain (PG and PGS) tended (P = 0.06) to have less overall serum IgG (13.6 g/L) compared with foals from mares not fed grain (PA and PS; 15.3 g/L). These data indicate that the maternal diet during the last one-third of gestation affects placental efficiency and colostral IgG.