TY - JOUR
AU - Gordon, R. K.
AB - ABSTRACT 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 (rs = 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. INTRODUCTION Exercise-induced reduction in sperm quality due to elevated core and scrotal temperatures is a concern to segments of the equine industry where stallions are being trained or competed and bred simultaneously. Hot and humid environmental temperatures pose a challenge to stallion managers, as ambient temperatures (AMBT) become extremely high (24 to 36°C) during the peak breeding and show season [National Oceanic and Atmospheric Administration (NOAA), 2010]. These temperatures, coupled with high relative humidity during exercise, may compromise the efficiency of mechanisms responsible for cooling the body. Extreme ambient temperatures affect spermatogenesis in many species (Casady et al., 1953; McNitt and First, 1970; Marai et al., 2002; Konavongkrit et al., 2005; Perez-Crespo et al., 2008; Momen et al., 2010), and exercise may affect stallion reproductive variables (Dinger et al., 1986; Lange et al., 1997; Davies Morel and Gunnarsson, 2000; Janett et al., 2006; Staempfli et al., 2006). Normal spermatogenesis requires efficient testicular thermoregulation; however, a threshold for scrotal and body temperature (measured rectally) and length of elevated temperature has not been defined above which spermatogenesis is compromised, resulting in a reduction in sperm quality. Many stallions endure rigorous training during the breeding season, which could cause a prolonged increase in body temperature resulting in thermal insult to the testes. Possible consequences might include reduced semen quality and lowered fertility. Therefore, the objective of the current study was to use subcutaneous thermal sensors to measure intramuscular neck and subcutaneous scrotal temperatures to determine the effect of exercise-induced heat stress on sperm quality in stallions. MATERIALS AND METHODS Project approval was granted by the Texas A&M University Institutional Agricultural Animal Care and Use Committee using guidelines set forth by the Federation of Animal Science Societies (1999). Stallions, Housing, and Diets Eight mature Miniature Horse stallions (4 to 16 yr, 72.1 to 110.7 kg) were used in a 12-wk study conducted from early July through early October 2010. Stallions were allowed an acclimation period of 90 d before the start of the study during which they were confined to individual stalls (1.8 m × 1.8 m) at the Texas A&M University Horse Center, allowed visual access to other horses, and provided free exercise every other day in dry-paddock runs (7.3 m × 1.8 m). In addition, all stallions were trained to mount a breeding phantom and ejaculate into an artificial vagina. Each stallion was measured weekly for BW using a digital platform scale (CAS Corp., Seoul, Republic of Korea), for rump fat thickness using an ultrasound instrument (Aloka SSD-500V, Aloka Inc., Tokyo, Japan) as previously described by Westervelt et al. (1976), and for BCS to aid in assessment of adequacy of their diet. Coastal Bermuda grass hay and a concentrate (Producers Cooperative, Bryan, TX) were fed to all stallions twice daily to maintain a BCS of 5 (Henneke et al., 1983). Stallions were allowed access to fresh water ad libitum. Thermal Sensor Implants Two subdermal thermal sensory devices (Digital Angel Corp., St. Paul, MN) were implanted into each stallion by a veterinary surgeon. Before implantation, an intramuscular tetanus toxoid (1 mL) was administered in addition to intravenous (IV) flunixin meglumine (1 mg/kg BW) to control inflammation and pain. Xylazine (1 mg/kg BW, IV) was administered as a sedative before induction of anesthesia with ketamine (2 mg/kg BW, IV), and stallions were placed in dorsal recumbency. A 10 × 10 cm2 area on the left aspect of the midneck region over the trapezius muscle was clipped, and the neck area and scrotum were aseptically prepared for surgical implantation of microchips using a Betadine and alcohol scrub. A sterile, prepackaged telemetric microchip (2-cm length, 0.25-cm diameter) was preloaded into a sterile syringe with a 12-gauge needle. The most ventral portion of the scrotum was grasped and lifted to tent the skin, and the needle was inserted into the scrotal fascia where the sensor was deposited. The skin surrounding the needle was tightly grasped as the needle was withdrawn to avoid removing the microchip with the needle. Sensor placement within the ventral scrotal fascia was verified by manual palpation. A second microchip was placed beneath the skin and subcutaneous tissue into the trapezius muscle on the left side of the neck using a similar technique. No closure of skin was performed at either site. Sensor scanners (Pocket Reader EX, Digital Angel Corp.) were used to read internal temperatures at both sites of implantation to confirm that the microchips were present and operable before the stallions were recovered from anesthesia. The scanners were used to read internal temperatures measured by the implanted microchips throughout the study. Blocking and Treatment Groups Testicular volume (TV) was computed from ultrasonographic measurements of width, height, and length of the testes (Love et al., 1991). Ejaculates from each stallion were collected once daily for 5 d, which was the time period deemed sufficient to stabilize extragonadal sperm reserves in these stallions and thus calculate daily sperm output (DSO) for Miniature Horse stallions; this was based on modification of previous DSO determination calculations for full-sized stallions (Love et al., 1991). The mean value for sperm output during the final 2 d of semen collection was used to represent actual DSO, as the numbers of sperm no longer varied significantly (Gebauer et al., 1974; Thompson et al., 2004). No semen was collected for at least 7 d before each DSO determination. Baseline DSO variables for each stallion were determined at 0 wk. Subsequently, stallions were blocked by age, TV, total number of sperm/ejaculate, and percentage of normal sperm before being randomly assigned to either a nonexercised (Non-EX; control; n = 4) or exercised (EX; treatment; n = 4) group. Exercise Protocol Stallions in the EX group were exercised on a high-speed motorized equine exerciser (Freestyle Equine Exercisers, Summertree Co., Inc., Shawnee, OK) 4 d/wk (Monday, Tuesday, Friday, and Saturday) during a 12-wk period (between DSO determinations at 0, 4, 8, and 12 wk). Exercise protocol was divided into 3 phases: phase I (8-min walking warm-up), phase II (15-min extended trot/canter), and phase III (8-min walking cool-down; Table 1). Exercise began at approximately 1200 h. A target heart rate (HR) of 150 to 160 beats per minute (bpm) was achieved and maintained during phase II. Subcutaneous scrotal temperatures (SQST), intramuscular neck temperatures (IMNT), and rectal temperatures (RCT) were digitally recorded before exercise, immediately after phase II, immediately after phase III, and at 60 and 120 min post-exercise. Ambient temperatures were recorded hourly via iButton temperature loggers (Maxim DS1923 iButton, Maxim Integrated Products, Inc., Sunnyvale, CA) located in the shaded stall area (where pre- and post-exercise temperatures were measured) and in the sun near the motorized equine exerciser (where phase II and III temperatures were measured). Hourly humidity (HUM) percentages were obtained later from NOAA (2010). Non-exercised stallions were tied in the sun while EX stallions were being exercised in an attempt to subject both groups to consistent ambient temperatures. Table 1. Exercise protocol used for stallions (n = 4) exercised 4 d/wk during a 12-wk period Time points  Description  Duration  Intensity  Location  0  Resting      Shade  —  Phase I1  8 min  Walk  Sun  22  Phase II  15 min  Long trot/canter  Sun  30  Phase III  7 min  Walk  Sun  60 min post-exercise  Recovery  60 min    Shade  120 min post-exercise  Recovery  60 min    Shade  Time points  Description  Duration  Intensity  Location  0  Resting      Shade  —  Phase I1  8 min  Walk  Sun  22  Phase II  15 min  Long trot/canter  Sun  30  Phase III  7 min  Walk  Sun  60 min post-exercise  Recovery  60 min    Shade  120 min post-exercise  Recovery  60 min    Shade  1Phase I of exercise began at 1200 h each day View Large Table 1. Exercise protocol used for stallions (n = 4) exercised 4 d/wk during a 12-wk period Time points  Description  Duration  Intensity  Location  0  Resting      Shade  —  Phase I1  8 min  Walk  Sun  22  Phase II  15 min  Long trot/canter  Sun  30  Phase III  7 min  Walk  Sun  60 min post-exercise  Recovery  60 min    Shade  120 min post-exercise  Recovery  60 min    Shade  Time points  Description  Duration  Intensity  Location  0  Resting      Shade  —  Phase I1  8 min  Walk  Sun  22  Phase II  15 min  Long trot/canter  Sun  30  Phase III  7 min  Walk  Sun  60 min post-exercise  Recovery  60 min    Shade  120 min post-exercise  Recovery  60 min    Shade  1Phase I of exercise began at 1200 h each day View Large Semen Collection and Processing Daily sperm output was determined for each stallion at 0, 4, 8, and 12 wk, with 0 wk representing baseline values. Ejaculates were collected at the Texas A&M University Horse Center breeding facility using a breeding phantom and a 33-cm length Missouri-model artificial vagina (Nasco, Ft. Atkinson, WI), along with an intact Miniature Horse mare in behavioral estrus for sexual stimulation. Semen analysis was performed in the Theriogenology Laboratory in the College of Veterinary Medicine at Texas A&M University, located approximately 2 km (5 min) from the Texas A&M University Horse Center. Therefore, semen was initially processed at the Horse Center and transported within 30 min of collection to the Theriogenology Laboratory for analysis. To standardize ambient temperature for semen transport, a cooler (Koolatron PC3, Koolatron, Brantford, Ontario, Canada) was set at 16°C. This resulted in an approximate cooler temperature, after frequent opening and closing of the cooler, of 22°C (room temperature), thereby limiting exposure of sperm to the harmful effects of ultraviolet light and high environmental temperatures (Davies Morel, 2008). Immediately after ejaculation, gel-free volume (VOL) was measured in the Horse Center Laboratory using a 10-mL graduated cylinder. Two 0.5-mL aliquots of raw semen were prepared; 1 aliquot was placed in a 16°C cooler until analysis for concentration, and the second aliquot was frozen in liquid nitrogen and maintained at −80°C for later assessment of DNA quality using the sperm chromatin structure assay (SCSA). Raw semen was also preserved in buffered formol saline for pending morphological analysis. Three additional 1.5-mL aliquots of raw semen were diluted 1:4 (semen:extender) with a commercial semen extender (INRA-96, IMV, Maple Grove, MN). One aliquot was placed in the cooler set at 16°C for later analysis of sperm motion characteristics, and the other 2 aliquots were stored in separate cooling devices (Equitainer-11, Hamilton-Thorne Biosciences, Beverly, MA), with 1 sample analyzed after 24 h (T24) and the other after 48 h (T48) of storage. End points measured for T24 and T48 samples were total motile (TMOT) and progressively motile (PMOT) sperm and DNA quality. All materials used in processing ejaculates were maintained at 37°C in an incubator until time of use. Sperm Analysis Upon arrival at the Theriogenology Laboratory, fresh (T0) samples were removed from the cooler. Raw semen was analyzed for sperm concentration using a fluorescence-based commercial instrument (NucleoCounter SP-100, ChemoMetec, Allerød, Denmark). All extended semen samples were analyzed for percentages of TMOT and PMOT sperm using a computer-assisted sperm motion analysis instrument (CASMA; IVOS version 12.2 L, Hamilton-Thorne Biosciences). Before analysis of sperm motion characteristics, extended semen samples were further diluted with INRA-96 extender to achieve a concentration of approximately 30 × 106 sperm/mL. Samples were then placed in a 37°C incubator for 15 min before sperm motility analysis. Warmed (37°C) analysis chambers (fixed height of 20 μm) affixed to microscope slides (Leja Standard Count 2 Chamber slides, Leja Products, B.V., Nieuw-Vennep, the Netherlands) were loaded with 6 μL of extended semen and inserted into the CASMA instrument for evaluation. A total of 10 microscopic fields and a minimum of 500 sperm were examined per sample. Fresh semen samples preserved in buffered formol saline were evaluated for morphological characteristics using a wet-mount preparation and differential interference contrast microscopy (Olympus BX60, Olympus America, Inc., Melville, NY; 1,250× magnification). Specifically, a 1.5-μL drop was placed on a microscope slide, and a cover slip was applied before analysis. A total of 100 cells was counted per sample. The percentage of morphologically normal sperm was identified as well as specific morphologic abnormalities, including abnormal heads, abnormal acrosomes, abnormal midpieces, detached heads, bent tails, coiled tails, and premature germ cells (Kenney et al., 1983). Percentages of each abnormality were determined by counting all abnormalities present on a sperm. The DNA quality was assessed using the SCSA as previously described (Love and Kenney, 1998). Briefly, semen samples were thawed in a 37°C water bath, and aliquots (approximately 5 μL) of thawed semen were mixed with 195 μL of a TNE buffer solution (500-mL mixture of 0.79 g Trizma HCl, 4.38 g NaCl, and 0.186 g EDTA), which was then combined with 400 μL of a low-pH (∼1.2) Triton X-100 detergent solution (500-mL mixture of 4.38 g NaCl, 20 mL 2N HCl, and 0.5 mL Triton-X detergent) for 30 s. Acridine orange was added (1.2 mL at 4.0 μg/mL) to the sample, which was processed after 30 s on a flow cytometer (FACScan, Becton Dickinson, Mountain View, CA). Quantification of DNA denaturation in each cell was determined by the term alpha-t (αt), defined as the ratio of red:(red + green fluorescence). The αt describes the relationship between the amounts of green (native, double-stranded DNA) and red (single-stranded DNA) fluorescence. The end point evaluated was the percentage of cells with abnormal DNA or the cells outside the main population. This was determined by selecting those sperm cells to the right of the main population and represents the number of sperm cells outside the main population as a percentage of the total number of sperm cells evaluated. Statistical Analysis Mean values were determined using PROC MEANS (SAS Inst. Inc., Cary, NC) on all temperature measurements (RCT, IMNT, SQST, AMBT, and HUM) at each time point (0, 22, 30, 60, and 120 min) during exercise. Temperature data were then analyzed using a nonparametric method that made no assumptions based on distribution of the data, as the data were not normally distributed. A Spearman's rank correlation test was performed to determine relationships among SQST, IMNT, RCT, AMBT, and HUM during exercise. Measures of sperm quality were normally distributed; therefore, these data were subjected to parametric analysis by repeated measures using PROC MIXED of SAS. The model included group (Non-EX or EX), week (0, 4, 8, or 12), semen collection sample (T0, T24, and T48), and all interactions, with stallion as the subject of the repeated measures. Level of significance was set at P ≤ 0.05. RESULTS No fever, local edema, or other deleterious effects were noted after the subdermal implantation of thermal sensory devices into the neck or scrotum of the stallions in this study. Ambient Temperature and Humidity No effect of week on hourly AMBT measurements from iButton temperature loggers fixed in the stall area (shade) or exercise area (sun) was observed; therefore, the mean AMBT at each time point during exercise was reported. Ambient temperatures to which the stallions were exposed differed (P < 0.0001) during the exercise protocol, as stallions were moved from the shaded stall area at 0 min to the heat of the sun at 22 and 30 min and back to the shaded stall area at 60 and 120 min postexercise. Mean AMBT increased 9.8°C (P < 0.0001) from 34.6°C at 0 min of exercise and 60 and 120 min post-exercise to a peak of 44.4°C at 22 and 30 min of phases I to III. A slight, but insignificant, decrease (P = 0.43) in mean HUM was observed between exercise time points. Stallion Temperatures, Sperm Analysis, and Correlations The mean and peak HR of EX stallions were 149 and 164 bpm, respectively. Rectal and IMNT of EX stallions increased (P < 0.0001) by approximately 1.9°C and 2.4°C, respectively, after 22 min of exercise (Figure 1). Although not significant, EX stallions experienced a mean increase (P = 0.33) in SQST of 0.8°C from 0 to 22 min of exercise (Figure 1). Analysis of T0, T24, and T48 semen samples showed no effect (P > 0.05) of treatment or treatment by week interaction for any of the sperm quality and quantity end points measured (Table 2). Table 2. Mean (± SD) semen characteristics for all semen samples of nonexercised (Non-EX; n = 4) and exercised (EX; n = 4) stallions1 Semen end points2  WEEK 0  WEEK 4  WEEK 8  WEEK 12  Non-EX  EX  Non-EX  EX  Non-EX  EX  Non-EX  EX  VOL      T0  9.1 ± 4.1  9.1 ± 4.3  12.0 ± 7.7  7.7 ± 4.8  11.8 ± 5.2  11.9 ± 2.6  7.9 ± 5.5  10.1 ± 8.2  CONC      T0  140 ± 36  193.5 ± 39.0  172.1 ± 29.6  203 ± 51  100.5 ± 24.1  169.8 ± 35.1  102.6 ± 61.0  218.1 ± 127.7  TOTCELL      T0  1.2 ± 0.3  1.6 ± 1.1  1.6 ± 1.2  1.5 ± 0.8  1.1 ± 0.6  1.9 ± 0.9  0.7 ± 0.4  1.6 ± 0.6  NORMCELL      T0  61 ± 18  72.5 ± 5.8  58.8 ± 16.0  74.5 ± 3.7  51.8 ± 7.3  73.3 ± 6.1  57.8 ± 4.6  71.3 ± 6.5  TMOT      T0  72 ± 7  75.8 ± 6.1  71.5 ± 5.8  77.5 ± 4.7  63.3 ± 5.9  70.1 ± 3.8  58.3 ± 15.4  76.0 ± 6.8      T24  68.6± 12.6  73.4 ± 9.9  67.4 ± 12.7  74.9 ± 9.9  55.5 ± 8.2  65.9 ± 4.6  49.6 ± 12.7  64.1 ± 9.2      T48  49.9 ± 13.4  56.3 ± 11.0  44.1 ± 20.7  56.6 ± 15.0  35.3 ± 8.1  47.6 ± 14.7  40.1 ± 14.3  56.3 ± 13.9  PMOT      T0  48 ± 11  52.9 ± 8.8  46.1 ± 3.6  53.5 ± 8.7  36.8 ± 7.2  46.3 ± 9.1  35.6 ± 10.9  55.1 ± 8.5      T24  37.8 ± 14.2  50.3 ± 11.8  39.4 ± 15.3  50.4 ± 13.4  26.6 ± 8.4  42.9 ± 6.4  23.1 ± 10.7  34.6 ± 13.6      T48  24.4 ± 14.7  31.5 ± 10.7  23.5 ± 15.3  34.4 ± 15.4  11.6 ± 6.1  27.5 ± 12.7  17.5 ± 7.1  32.6 ± 13.0  COMP-αt      T0  12.7 ± 8.3  10.8 ± 6.7  13.3 ± 6.3  8.3 ± 0.8  12.8 ± 3.3  11.2 ± 6.4  12.1 ± 4.5  8.8 ± 2.9      T24  20.9 ± 11.2  16.0 ± 5.1  20.4 ± 8.3  13.9 ± 3.7  21.9 ± 3.7  16.9 ± 5.2  19.6 ± 3.9  15.1 ± 4.0      T48  22.7 ± 13.5  18.0 ± 3.2  22.9 ± 7.0  13.8 ± 1.4  25.2 ± 4.4  18.0 ± 4.4  22.3 ± 3.4  16.9 ± 4.3  Semen end points2  WEEK 0  WEEK 4  WEEK 8  WEEK 12  Non-EX  EX  Non-EX  EX  Non-EX  EX  Non-EX  EX  VOL      T0  9.1 ± 4.1  9.1 ± 4.3  12.0 ± 7.7  7.7 ± 4.8  11.8 ± 5.2  11.9 ± 2.6  7.9 ± 5.5  10.1 ± 8.2  CONC      T0  140 ± 36  193.5 ± 39.0  172.1 ± 29.6  203 ± 51  100.5 ± 24.1  169.8 ± 35.1  102.6 ± 61.0  218.1 ± 127.7  TOTCELL      T0  1.2 ± 0.3  1.6 ± 1.1  1.6 ± 1.2  1.5 ± 0.8  1.1 ± 0.6  1.9 ± 0.9  0.7 ± 0.4  1.6 ± 0.6  NORMCELL      T0  61 ± 18  72.5 ± 5.8  58.8 ± 16.0  74.5 ± 3.7  51.8 ± 7.3  73.3 ± 6.1  57.8 ± 4.6  71.3 ± 6.5  TMOT      T0  72 ± 7  75.8 ± 6.1  71.5 ± 5.8  77.5 ± 4.7  63.3 ± 5.9  70.1 ± 3.8  58.3 ± 15.4  76.0 ± 6.8      T24  68.6± 12.6  73.4 ± 9.9  67.4 ± 12.7  74.9 ± 9.9  55.5 ± 8.2  65.9 ± 4.6  49.6 ± 12.7  64.1 ± 9.2      T48  49.9 ± 13.4  56.3 ± 11.0  44.1 ± 20.7  56.6 ± 15.0  35.3 ± 8.1  47.6 ± 14.7  40.1 ± 14.3  56.3 ± 13.9  PMOT      T0  48 ± 11  52.9 ± 8.8  46.1 ± 3.6  53.5 ± 8.7  36.8 ± 7.2  46.3 ± 9.1  35.6 ± 10.9  55.1 ± 8.5      T24  37.8 ± 14.2  50.3 ± 11.8  39.4 ± 15.3  50.4 ± 13.4  26.6 ± 8.4  42.9 ± 6.4  23.1 ± 10.7  34.6 ± 13.6      T48  24.4 ± 14.7  31.5 ± 10.7  23.5 ± 15.3  34.4 ± 15.4  11.6 ± 6.1  27.5 ± 12.7  17.5 ± 7.1  32.6 ± 13.0  COMP-αt      T0  12.7 ± 8.3  10.8 ± 6.7  13.3 ± 6.3  8.3 ± 0.8  12.8 ± 3.3  11.2 ± 6.4  12.1 ± 4.5  8.8 ± 2.9      T24  20.9 ± 11.2  16.0 ± 5.1  20.4 ± 8.3  13.9 ± 3.7  21.9 ± 3.7  16.9 ± 5.2  19.6 ± 3.9  15.1 ± 4.0      T48  22.7 ± 13.5  18.0 ± 3.2  22.9 ± 7.0  13.8 ± 1.4  25.2 ± 4.4  18.0 ± 4.4  22.3 ± 3.4  16.9 ± 4.3  1No differences (P > 0.05) were observed in any of the end points measured. 2VOL = volume, mL; CONC = sperm concentration, million/mL; TOTCELL = total sperm/ejaculate, billions; NORMCELL = morphologically normal sperm, %; TMOT = total sperm motility, %; PMOT = progressively motile sperm, %; COMP-αt = sperm with abnormal DNA, %. T0 = fresh; T24 = 24 h cooled; T48 = 48 h cooled. View Large Table 2. Mean (± SD) semen characteristics for all semen samples of nonexercised (Non-EX; n = 4) and exercised (EX; n = 4) stallions1 Semen end points2  WEEK 0  WEEK 4  WEEK 8  WEEK 12  Non-EX  EX  Non-EX  EX  Non-EX  EX  Non-EX  EX  VOL      T0  9.1 ± 4.1  9.1 ± 4.3  12.0 ± 7.7  7.7 ± 4.8  11.8 ± 5.2  11.9 ± 2.6  7.9 ± 5.5  10.1 ± 8.2  CONC      T0  140 ± 36  193.5 ± 39.0  172.1 ± 29.6  203 ± 51  100.5 ± 24.1  169.8 ± 35.1  102.6 ± 61.0  218.1 ± 127.7  TOTCELL      T0  1.2 ± 0.3  1.6 ± 1.1  1.6 ± 1.2  1.5 ± 0.8  1.1 ± 0.6  1.9 ± 0.9  0.7 ± 0.4  1.6 ± 0.6  NORMCELL      T0  61 ± 18  72.5 ± 5.8  58.8 ± 16.0  74.5 ± 3.7  51.8 ± 7.3  73.3 ± 6.1  57.8 ± 4.6  71.3 ± 6.5  TMOT      T0  72 ± 7  75.8 ± 6.1  71.5 ± 5.8  77.5 ± 4.7  63.3 ± 5.9  70.1 ± 3.8  58.3 ± 15.4  76.0 ± 6.8      T24  68.6± 12.6  73.4 ± 9.9  67.4 ± 12.7  74.9 ± 9.9  55.5 ± 8.2  65.9 ± 4.6  49.6 ± 12.7  64.1 ± 9.2      T48  49.9 ± 13.4  56.3 ± 11.0  44.1 ± 20.7  56.6 ± 15.0  35.3 ± 8.1  47.6 ± 14.7  40.1 ± 14.3  56.3 ± 13.9  PMOT      T0  48 ± 11  52.9 ± 8.8  46.1 ± 3.6  53.5 ± 8.7  36.8 ± 7.2  46.3 ± 9.1  35.6 ± 10.9  55.1 ± 8.5      T24  37.8 ± 14.2  50.3 ± 11.8  39.4 ± 15.3  50.4 ± 13.4  26.6 ± 8.4  42.9 ± 6.4  23.1 ± 10.7  34.6 ± 13.6      T48  24.4 ± 14.7  31.5 ± 10.7  23.5 ± 15.3  34.4 ± 15.4  11.6 ± 6.1  27.5 ± 12.7  17.5 ± 7.1  32.6 ± 13.0  COMP-αt      T0  12.7 ± 8.3  10.8 ± 6.7  13.3 ± 6.3  8.3 ± 0.8  12.8 ± 3.3  11.2 ± 6.4  12.1 ± 4.5  8.8 ± 2.9      T24  20.9 ± 11.2  16.0 ± 5.1  20.4 ± 8.3  13.9 ± 3.7  21.9 ± 3.7  16.9 ± 5.2  19.6 ± 3.9  15.1 ± 4.0      T48  22.7 ± 13.5  18.0 ± 3.2  22.9 ± 7.0  13.8 ± 1.4  25.2 ± 4.4  18.0 ± 4.4  22.3 ± 3.4  16.9 ± 4.3  Semen end points2  WEEK 0  WEEK 4  WEEK 8  WEEK 12  Non-EX  EX  Non-EX  EX  Non-EX  EX  Non-EX  EX  VOL      T0  9.1 ± 4.1  9.1 ± 4.3  12.0 ± 7.7  7.7 ± 4.8  11.8 ± 5.2  11.9 ± 2.6  7.9 ± 5.5  10.1 ± 8.2  CONC      T0  140 ± 36  193.5 ± 39.0  172.1 ± 29.6  203 ± 51  100.5 ± 24.1  169.8 ± 35.1  102.6 ± 61.0  218.1 ± 127.7  TOTCELL      T0  1.2 ± 0.3  1.6 ± 1.1  1.6 ± 1.2  1.5 ± 0.8  1.1 ± 0.6  1.9 ± 0.9  0.7 ± 0.4  1.6 ± 0.6  NORMCELL      T0  61 ± 18  72.5 ± 5.8  58.8 ± 16.0  74.5 ± 3.7  51.8 ± 7.3  73.3 ± 6.1  57.8 ± 4.6  71.3 ± 6.5  TMOT      T0  72 ± 7  75.8 ± 6.1  71.5 ± 5.8  77.5 ± 4.7  63.3 ± 5.9  70.1 ± 3.8  58.3 ± 15.4  76.0 ± 6.8      T24  68.6± 12.6  73.4 ± 9.9  67.4 ± 12.7  74.9 ± 9.9  55.5 ± 8.2  65.9 ± 4.6  49.6 ± 12.7  64.1 ± 9.2      T48  49.9 ± 13.4  56.3 ± 11.0  44.1 ± 20.7  56.6 ± 15.0  35.3 ± 8.1  47.6 ± 14.7  40.1 ± 14.3  56.3 ± 13.9  PMOT      T0  48 ± 11  52.9 ± 8.8  46.1 ± 3.6  53.5 ± 8.7  36.8 ± 7.2  46.3 ± 9.1  35.6 ± 10.9  55.1 ± 8.5      T24  37.8 ± 14.2  50.3 ± 11.8  39.4 ± 15.3  50.4 ± 13.4  26.6 ± 8.4  42.9 ± 6.4  23.1 ± 10.7  34.6 ± 13.6      T48  24.4 ± 14.7  31.5 ± 10.7  23.5 ± 15.3  34.4 ± 15.4  11.6 ± 6.1  27.5 ± 12.7  17.5 ± 7.1  32.6 ± 13.0  COMP-αt      T0  12.7 ± 8.3  10.8 ± 6.7  13.3 ± 6.3  8.3 ± 0.8  12.8 ± 3.3  11.2 ± 6.4  12.1 ± 4.5  8.8 ± 2.9      T24  20.9 ± 11.2  16.0 ± 5.1  20.4 ± 8.3  13.9 ± 3.7  21.9 ± 3.7  16.9 ± 5.2  19.6 ± 3.9  15.1 ± 4.0      T48  22.7 ± 13.5  18.0 ± 3.2  22.9 ± 7.0  13.8 ± 1.4  25.2 ± 4.4  18.0 ± 4.4  22.3 ± 3.4  16.9 ± 4.3  1No differences (P > 0.05) were observed in any of the end points measured. 2VOL = volume, mL; CONC = sperm concentration, million/mL; TOTCELL = total sperm/ejaculate, billions; NORMCELL = morphologically normal sperm, %; TMOT = total sperm motility, %; PMOT = progressively motile sperm, %; COMP-αt = sperm with abnormal DNA, %. T0 = fresh; T24 = 24 h cooled; T48 = 48 h cooled. View Large Figure 1. View largeDownload slide Mean (± SD) rectal, neck, and scrotal temperatures recorded for non-exercised (Non-EX) and exercised (EX) stallions at each time point (0, 22, and 30 min) during exercise protocol and post-exercise recovery (60† and 120† min) over a 12-wk period (n = 4/group). Neck temperatures of EX stallions (solid circles) and rectal temperatures of EX stallions (open triangles) increased (P < 0.0001) after 22 and 30 min of exercise. Figure 1. View largeDownload slide Mean (± SD) rectal, neck, and scrotal temperatures recorded for non-exercised (Non-EX) and exercised (EX) stallions at each time point (0, 22, and 30 min) during exercise protocol and post-exercise recovery (60† and 120† min) over a 12-wk period (n = 4/group). Neck temperatures of EX stallions (solid circles) and rectal temperatures of EX stallions (open triangles) increased (P < 0.0001) after 22 and 30 min of exercise. Significant, positive correlations existed between SQST, IMNT, RCT, and AMBT; humidity was not highly correlated with any of the measured temperatures in this study (Table 3). Table 3. Spearman's rank correlations (rs) between stallion temperatures and other end points measured during a 12-wk exercise period1 End point  SQST  IMNT  RCT  IMNT  0.476*      RCT  0.761*  0.453*    AMBT  0.625*  0.513*  0.558*  HUM  −0.074*  −0.263*  −0.025  End point  SQST  IMNT  RCT  IMNT  0.476*      RCT  0.761*  0.453*    AMBT  0.625*  0.513*  0.558*  HUM  −0.074*  −0.263*  −0.025  1End points measured: SQST = subcutaneous scrotal temperature; IMNT = intramuscular neck temperature; RCT = rectal temperature; AMBT = ambient temperature; HUM = relative humidity. *P < 0.0001. View Large Table 3. Spearman's rank correlations (rs) between stallion temperatures and other end points measured during a 12-wk exercise period1 End point  SQST  IMNT  RCT  IMNT  0.476*      RCT  0.761*  0.453*    AMBT  0.625*  0.513*  0.558*  HUM  −0.074*  −0.263*  −0.025  End point  SQST  IMNT  RCT  IMNT  0.476*      RCT  0.761*  0.453*    AMBT  0.625*  0.513*  0.558*  HUM  −0.074*  −0.263*  −0.025  1End points measured: SQST = subcutaneous scrotal temperature; IMNT = intramuscular neck temperature; RCT = rectal temperature; AMBT = ambient temperature; HUM = relative humidity. *P < 0.0001. View Large DISCUSSION Many stallions are subjected to moderate or intense exercise regimens to maintain a high level of fitness to be competitive in their respective disciplines. Stallions exercised in the current study achieved a mean peak RCT of approximately 39.2°C, and other research has shown that excessive exercise can elicit even higher core body temperatures approaching 41.1°C (Webb et al., 1987; Scott, 1992; Kohn et al., 1999). However, it is unknown whether these temperature increases may be a factor contributing to decreased sperm quality or reduced fertility in stallions. The current study subjected EX stallions to a moderate exercise regimen for a relatively short period of time when compared with a study by Janett et al. (2006), in which stallions were exercised until exhaustion (when stallions could no longer maintain treadmill speed) on a high-speed equine treadmill. Janett et al. (2006) reported an increase in acrosome defects and nuclear vacuoles during the exercise period as well as an increase in the percentage of major sperm defects immediately after the exercise period. Paradoxically, the motility of fresh semen was least 4 to 8 wk after the end of the exercise treatment, whereas postthaw sperm motility and viability were greatest during that time period (Janett et al., 2006). Stallion temperatures were not monitored during exercise; therefore, the cause of the decline in sperm parameters was unclear and could have been attributed to either increased body temperatures or stress induced by exercising the stallions until exhaustion. Therefore, the current study sought to record both rectal and subcutaneous scrotal temperatures resulting from a moderate exercise regimen and to determine the subsequent effects on sperm parameters. Most studies have used scrotal skin temperature (SST) as the end point to quantify the degree of thermal insult to the testes (McNitt and First, 1970; Ross and Entwistle, 1979; Freidman et al., 1991; Vogler et al., 1993; Barth and Bowman, 1994; Love and Kenney, 1999; Blanchard et al., 2000). However, Kastelic et al. (1995) used intratesticular thermistors to measure the temperature within the testicular parenchyma and revealed a low correlation between SST and intratesticular temperature (ITT). Radiative and evaporative cooling mechanisms as well as the location of the testes acutely affect SST, whereas thermoregulation of deeper testicular tissue is regulated primarily by metabolic factors and circulatory anatomy, which results in a more uniform temperature throughout the testis (Kastelic et al., 1997). Because SQST displayed a small to moderate correlation with ITT, SQST may be more representative than SST for predicting ITT, although caution must be exercised when making such inferences (Kastelic et al., 1995). Subcutaneous scrotal temperatures in the current study were significantly correlated with RCT and AMBT. Perhaps further studies will allow prediction of SQST from RCT. In the current study, no acute or chronic complications (swelling, edema, or increase in rectal temperature) were associated with implantation of the thermal sensory devices in either the neck or scrotum. In addition, this device was functional over the 12-wk trial period and has remained operable for 1 yr after implantation. Ambient temperature and HUM to which stallions were subjected during the exercise protocol of the current study were extremely high. Intensely exercised stallions may experience difficulty dissipating body heat in such climates, resulting in inefficient thermoregulation of the body and, concurrently, the scrotum. However, stallions used in the current study did not appear to have difficulty dissipating heat generated from the short-term exercise protocol to which they were subjected. Increases in RCT of stallions in the present study were not as large as increases in core temperatures previously documented in exercised horses (Webb et al., 1987; Scott, 1992; Kohn et al., 1999). Quarter horses conditioned aerobically and trained for cutting work in 45-min bouts experienced a mean RCT of approximately 38.9°C during exercise, with a greater mean temperature of 40.5°C recorded at 30-min recovery (Webb et al., 1987). Daily aerobic conditioning by galloping around an oval track daily for approximately 50 d before a final anaerobic exercise test consisting of four 600-m sprints resulted in maximal core body temperatures averaging 41°C at 15-min recovery (Scott, 1992). Thoroughbred horses also were subjected to 46-min standard exercise tests in varying AMBT and HUM on a high-speed treadmill, with RCT reaching 41.5°C in hot and humid conditions (Kohn et al., 1999). The core temperatures achieved during the exercise protocols employed in those projects approached 42°C, which is considered the critical body temperature in equines that may elicit voluntary exhaustion (Lindinger, 1999). Although mean peak RCT of 39.2°C and IMNT of 39.9°C were achieved in EX stallions, these increases were not as great as those noted in previous studies. Therefore, the exercise protocol used in the current study cannot be considered as an intense regimen capable of producing maximal core body temperatures but may represent equine industry-applicable standard exercise periods that may be used to athletically train young horses. Also, the current study did not determine the time required for RCT, IMNT, or SQST temperatures of EX stallions to return to baseline after exercise because the earliest recovery temperatures were recorded at 60 min post-exercise when temperatures had already returned to resting values. By recording stallion temperatures every 10 min for a period of 60 min post-exercise, a more defined recovery period may have been apparent, which would have helped to more precisely determine the length of time stallion temperatures remained elevated. Although the increase in SQST was not significant in either group of stallions, both groups of stallions had apparently increased RCT, IMNT, and SQST at 22 and 30 min of exercise protocol, even the group of Non-Ex stallions. Those stallions were not physically exerted but were exposed to the heat of the sun while the EX stallions were exercised, after which all stallions were returned to their shaded stalls for the 60 and 120 min postexercise measurements. This finding was unexpected but indicates that exposure to direct sun alone during the summer months elevates core and scrotal temperatures. Previous scrotal insulation studies have described the relationship between increased SST and deterioration in semen quality. The scrotal temperature increase in the insulation studies ranged from 1.0 to 3.5°C, and the period of insulation ranged from 3 to 48 h (McNitt and First, 1970; Ross and Entwistle, 1979; Freidman et al., 1991; Vogler et al., 1993; Barth and Bowman, 1994; Love and Kenney, 1999; Blanchard et al., 2000). In the present study, a mean increase in SQST of 0.8°C was achieved in EX stallions after 22 min of exercise. No adverse effects on semen parameters were observed, which is similar to results from studies in men that reported no changes in semen quality due to SQST increases of 0.8 to 1.0°C (Wang et al., 1997). However, rams subjected to a 1.4 to 2.2°C increase in SQST 16 h/d for 21 d experienced a significant decrease in concentration of sperm, percentage of live and motile sperm, and fertility (Mieusset et al., 1992). Clearly, prolonged increases in scrotal temperature due to insulation adversely affect reproductive parameters, as heat dissipation is compromised. Stallions in the current study did experience elevated RCT and IMNT during exercise, but the increases only lasted a maximum of 30 min/d for 4 d/wk, and SQST never increased significantly. The implemented exercise protocol more appropriately mirrors current industry standard daily exercise regimens for performance horses, and only intermittent increases in temperature due to exercise were noted. Scrotal temperatures were maintained even when the EX stallions experienced a marked increase in rectal temperature. Alterations in SST are quickly transferred to superficial testicular veins, which are regulated by the arteriovenous countercurrent heat exchange mechanism at the pampiniform plexus (Waites and Moule, 1961). Although arteriovenous blood temperatures were not measured in the current study, the authors can surmise that the efficient thermoregulatory action of the pampiniform plexus combined with sweat being generated by the scrotum to dissipate heat was remarkable. Additionally, the testes were likely dropped due to tunica dartos and cremaster muscle relaxation during recovery from exercise, which would allow for optimal cooling (Staempfli et al., 2006). However, testicular position was not recorded during the current study. Research has demonstrated that sperm quality parameters and sperm production per unit of testis of Miniature Horse stallions are similar to other breeds of horses (Paccamonti et al., 1999). The authors of the current study acknowledge that because Miniature Horse stallions were used, effects of breed differences as well as body and testicular size may exist. However, it can be assumed that the thermoregulatory mechanisms involving scrotal surface area and testicular volume would mimic those of full-sized horses, relative to their body size. Therefore, results of the current study suggest that daily short-term exercise in hot and humid climates has little effect on RCT and SQST and no adverse effects on sperm quality in horses with normally functioning thermoregulatory mechanisms. Furthermore, results of the current study are consistent with reports by Dinger et al. (1986) that evidenced no change in semen parameters of stallions subjected to minimal or short-term daily exercise. Another report claimed that stallions trained for intermediate and advanced levels of dressage or show jumping for 1 to 2 h/d had significantly greater motility of spermatozoa than stallions used for breeding alone (Lange et al., 1997). However, these stallions were not blocked into groups according to age or reproductive parameters, and no specific exercise protocol was defined. Further contradictory literature reported a decrease in the percentage of normal cells and motility of fresh and frozen-thawed semen during and up to 1 mo after intense exercise until exhaustion for 2 d/wk for 4 wk (Janett et al., 2006). The exercise regimen used by Janett et al. (2006) was more strenuous than that used in the current study; however, neither rectal nor scrotal temperatures were measured. Therefore, it cannot be determined whether the detriment to spermatozoal parameters was due to increased temperatures, stress levels, or other existing factors. Future research on this topic might include a longer exercise protocol. In the current study, the stallion scrotum appeared to efficiently dissipate heat during and after exercise; however, a prolonged or more intense exercise protocol would likely result in sustained elevated body temperatures, which might have a more profound effect on reproductive variables. Staempfli et al. (2006) recorded peak SST values when horses were at rest after exercise; the faster the horse moved, the more efficient the scrotal cooling. Stallions performing in events such as roping and cutting are subjected to intense but interrupted exercise regimens, stopping between performances and sometimes being restrained in direct sunlight. These horses likely experience elevated temperatures during exercise, followed by a period of standing at rest, followed again with exercise. During rest periods, the scrotum may not receive optimal air flow; therefore, efficient scrotal cooling could be hindered. In summary, the current study is the first to determine the effects of a moderate exercise regimen on RCT, IMNT, and SQST and sperm quality of Miniature Horse stallions. 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TI - Thermoregulation of the testicle in response to exercise and subsequent effects on semen characteristics of stallions
JF - Journal of Animal Science
DO - 10.2527/jas.2011-4543
DA - 2012-08-01
UR - https://www.deepdyve.com/lp/oxford-university-press/thermoregulation-of-the-testicle-in-response-to-exercise-and-p9EkMlqtIB
SP - 2532
EP - 2539
VL - 90
IS - 8
DP - DeepDyve
ER -