Thoroughbred stallion fertility is significantly associated with FKBP6 genotype but not with inbreeding or the contribution of a leading sire.

This is a follow-up study to validate the previously detected association of the FKBP6 gene with stallion subfertility. Using a select cohort of 150 Thoroughbred stallions with detailed breeding records, we confirm significant association (P < 0.0001) between low per-cycle pregnancy rates (≤50%) and a combined A/A-A/A genotype of SNPs chr13:11 353 372G>A and chr13:11 353 436A>C in FKBP6 exon 5. We also show that stallion subfertility and the combined genotype A/A-A/A are not associated with the level of genetic diversity based on 12 autosomal microsatellite markers, or with pedigree-based inbreeding rate, or the extent of contribution of a leading Thoroughbred sire, Northern Dancer, in a stallion's pedigree. We develop a TaqMan allelic discrimination assay for the two SNPs to facilitate accurate and high-throughput genotyping. We determine allele, genotype and combined genotype frequencies of FKBP6 exon 5 SNPs in a global cohort of 518 Thoroughbreds (76% stallions or geldings and 24% mares) and show that the frequency of the A/A-A/A genotype is 4%. Because there is no similar association between the FKBP6 exon 5 genotype and stallion subfertility in Hanoverians, we suggest that the two SNPs are not causative but rather tagging a breed-specific haplotype with genetic variants unique to Thoroughbreds. Further WGS-based research is needed to identify the molecular causes underlying the observed genotype-phenotype association in Thoroughbred stallions.

Effect of artificial vagina lubricants on stallion sperm quality.

Commercially available vaginal lubricants, typically labeled as non-spermicidal, are used to lubricate equine artificial vaginas prior to semen collection. Improper type or amount of lubricant might affect stallion sperm quality, either after short-time exposure or following cooled storage of extended semen previously exposed to lubricant. The aim of this study was to evaluate stallion sperm quality following exposure to lubricant-containing extender for 1 h (T1h) or 24 h (T24h). Three ejaculates were collected from each of four stallions using a small volume of petrolatum to lubricate artificial vaginas, and gel-free semen was diluted to 30 × 106 sperm/mL in extender containing: no lubricant (control), or 1 or 5% (v/v) HR® Lubricating Jelly (HR1 or HR5); K-Y® Jelly (KY1 or KY5); Therio-gel® (TG1 or TG5); Priority Care® Sterile Lubricating Jelly (PC1 or PC5); or Clarity® A.I. Lubricating Jelly (CL1 or CL5). Sperm were evaluated at T1h and T24h for percentages of: total and progressive sperm motility (TMOT and PMOT); curvilinear velocity (VCL; µm/s); and straightness (STR; %); viable acrosome intact sperm (VAI); sperm with abnormal DNA (COMP-αt); viable lipid peroxidation negative sperm (VLPN); and sperm with no detectable DNA oxidative injury [8OHdG(-)]. Following short-term exposure of sperm to lubricants, KY5 reduced TMOT, PMOT, VCL, VAI, VLPN, and COMP-αt in comparison with controls (i.e., P < 0.05). PC5 reduced TMOT, PMOT, VCL, VAI, and 8OHdG(-), and KY1 reduced TMOT, VAI, VLPN in comparison to controls (P < 0.05). Lubricant CL1, HR1 and HR5 yielded similar values to controls for all 8 endpoints, and CL5 yielded similar values to controls for all 8 endpoints (P > 0.05), except for VCL. Following long-term exposure, KY5 decreased TMOT, PMOT, VCL, VAI, VLPN, and COMP-αt as compared to controls (i.e., P < 0.05), PC5 decreased TMOT, VCL, VAI, and 8OHdG(-)as compared to controls in PC5, and KY1 decreased TMOT, VAI, VLPN, and COMP-αt (P < 0.05). TG5 decreased TMOT, PMOT, and VCL as compared to controls (P < 0.05). Lubricant CL5 decreased VCL (P < 0.05), and CL1, HR5, HR1, PC1, and TG1 were similar to controls for all 8 endpoints (P > 0.05). Overall, lubricant KY was the most detrimental to sperm quality, with most profound changes detected at a 5% concentration. Lubricants CL and HR were generally similar to controls and were less affected by lubricant concentration.

The effects of antibiotic type and extender storage method on sperm quality and antibacterial effectiveness in fresh and cooled-stored stallion semen.

Two experiments were conducted to evaluate the effects of antibiotic-containing extender of on sperm quality and control of bacterial growth. In Experiment 1, ejaculates were diluted in extender containing no antibiotics, potassium penicillin G-amikacin disulfate (PEN-AMIK), ticarcillin disodium-potassium clavulanate (TICAR-CLAV), piperacillin sodium/tazobactam sodium (PIP-TAZ), or meropenem (MERO). In freshly extended semen, only slight differences were detected among some antibiotic treatments for total sperm motility, curvilinear velocity, and viable acrosome-intact sperm (P < 0.05). In cool-stored semen, slight differences were also detected among certain antibiotic treatments for curvilinear velocity and chromatin integrity (P < 0.05). In Experiment 2, ejaculates were diluted in extender and subjected to no bacterial spiking, or inoculated with lower or higher doses of K. pneumoniae or P. aeruginosa. Following cooled storage of semen, colony forming units/ml (CFU/mL) were less in PEN-AMIK (706 ±â€¯244) and MERO (1576 ±â€¯1076) treatment groups than in TICAR-CLAV (4678 ±â€¯1388) or PIP-TAZ (8108 ±â€¯3198) treatment groups (P < 0.05). The CFU/mL were lower in all antibiotic-containing treatment groups than the control group (18478 ±â€¯4374; P < 0.05). The percentage of culture plates containing no bacterial growth in unspiked semen was greater in PEN-AMIK (75%) than PIP-TAZ (15%) or TICAR-CLAV (20%; P < 0.05). The percentages of culture plates containing no bacterial growth in semen spiked with a lower doses of K. pneumoniae or P. aeruginosa were higher in PEN-AMIK (70% and 50%, respectively) then in all other treatment groups (0-40% and 0-15% for K. pneumonia and P. aeruginosa, respectively; P < 0.05); however, complete control of bacterial load was only modest even with PEN-AMIK. In both experiments, freezing and thawing extender prior to use did not have any appreciable detrimental effect on sperm quality or antibiotic efficacy. In summary, all antibiotics tested had minimal effects on measures of sperm quality in fresh or cool-stored semen extenders; however, PEN-AMIK, followed by MERO, yielded the best results in terms of antimicrobial efficacy. None of the antibiotic types controlled bacterial growth, in comparison with the antibiotic-free control group, when extended semen was spiked with a high concentration of Pseudomonas aeruginosa. Cooled storage of extended semen reduced bacterial growth in comparison with freshly extended semen.

Effects of seminal plasma and flash-freezing on DNA structure of stallion epididymal sperm exposed to different potentiators of DNA damage.

The tolerance of sperm DNA structure to seminal plasma and freezing conditions has both clinical and basic biologic relevance. In this study, fresh (FS) or flash-frozen (FZ) stallion epididymal sperm were exposed (SP+) or unexposed (SP-) to seminal plasma. Sperm were then evaluated to monitor the degree of change in DNA structure following challenge with chemical (dithiothreitol-DTT), oxidative (iron sulfate; FeSO4) or enzymatic (DNase I) potentiators of DNA damage. For sperm not treated with potentiators (controls), there was no effect of SP treatment (SP- vs. SP+) or freezing treatment (FS vs. FZ; non-significant) on measures of any DNA assays (i.e., 8-hydroxy, 2'deoxyguanosine [8OHdG], TUNEL, or sperm chromatin structure [SCSA] assays). Group FZ was more susceptible than Group FS to potentiators of DNA damage. Percent 8OHdG-positive sperm was higher in Group FZ/SP- treated with FeSO4 than all other groups (P < 0.05). Percent TUNEL-positive sperm was similar among FZ/SP- groups treated with DTT, FeSO4, or DNase (non-significant) and was higher in these groups than all other treatments (P < 0.05). Percent COMP-αt was higher following treatment with DNase or DTT, as compared to their respective controls, regardless of prior exposure to SP (P < 0.05). Overall, sperm DNA structure was unaffected by seminal plasma or freezing treatment when samples were not exposed to potentiators of sperm DNA damage; however, marked differences were identified in DNA structure when sperm were challenged with chemical, oxidative or enzymatic treatments. These results highlight the importance of challenging DNA structure prior to analysis. The use of potentiators of DNA damage provided a model to evaluate sperm DNA structure following exposure of sperm to various experimental treatments.

The effect of flash-freezing temperature on stallion sperm DNA structure.

The effect of flash-freezing storage temperature on stallion sperm DNA has not been evaluated. Commonly, sperm are flash-frozen at various temperatures to preserve sperm DNA prior to analysis. It is unclear whether the temperature at which sperm are frozen and stored may affect the results of DNA assays. In this study, the neutral comet assay was used to evaluate the effect of flash-freezing storage temperature (freezer [-60 °C], dry ice [-78.5 °C], liquid nitrogen [-196 °C]) compared to fresh sperm DNA structure. In addition, intra- and inter-assay and intra- and inter-stallion variabilities were determined. All comet tail measures were higher following any flash-freezing method, as compared to fresh sperm DNA (P < 0.05), with no difference among flash-frozen treatments (P > 0.05). For most comet variables, intra- and inter-assay variabilities were <10%. Intra- and inter-stallion variabilities revealed that comet head length (HL) and width (CW) were less variable as compared to comet tail values, i.e., % comet tail DNA (T-DNA), tail length (TL), tail moment (OTM), and tail migration (TM). Certain comet tail values in fresh (% T-DNA, and OTM) and flash-frozen sperm (OTM, % T-DNA, TL, and TM) were correlated to the Sperm Chromatin Structure Assay (SCSA) variable, COMP-αt. The comet tail measures were negatively correlated to % morphologically normal sperm (P < 0.05) and positively correlated to % abnormal heads and premature germ cells (P < 0.05). Variables COMP-αt and % total sperm motility were not correlated to any morphologic sperm feature in this group of stallions (P > 0.05). While significant differences in the structure of the sperm DNA were identified in the flash-frozen as compared to the fresh sperm DNA with the neutral comet assay, it cannot be assumed that these changes are fertility limiting.

The effect of dual-hemisphere breeding on stallion fertility.

Breeding records were analyzed from 24 Thoroughbred stallions that were subjected to dual-hemisphere breeding (DH), including novice (first-year; NOV; n = 11) and experienced (EXP; n = 13) stallions. Fertility variables included seasonal pregnancy rate, pregnancy rate per cycle, and first-cycle pregnancy rate. In addition, values for book size, total number of covers, distribution of mare type (maiden, foaling, and barren) within a stallion's book, cycles per mare, and mare age were examined. Some data were also categorized by mare type (maiden-M, foaling-F, and barren-B). Five separate analyses of the data were performed. For Analyses 1-3, the effects of hemisphere (northern hemisphere [NH] vs. southern hemisphere [SH]) and breeding order (refers to the first [O1] or second [O2] season within the first year of dual-hemisphere breeding) were examined for all stallions (combined group [CG]), NOV stallions only, and EXP stallions only, respectively. Fertility values were generally higher in the SH than the NH (P < 0.05), whereas book size, total number of covers, and cycles per mare were higher in the NH than the SH (P < 0.05). Book size and total covers were negatively correlated to first cycle pregnancy rate (r = -0.57, r = -0.71, respectively; P < 0.05) for NOV stallions. Pregnancy rate per cycle was also negatively correlated with total covers (r = -0.58; P < 0.05) for NOV stallions. Similar trends were noted for Groups CG and EXP, but the relationship was not as marked as for NOV stallions. The fertility of O1 was generally similar to O2 (P > 0.05). For Analysis 4, fertility of DH breeding seasons was compared to single hemisphere (SIN) breeding seasons within the same 16 stallions and was found to be similar between the two groups (P > 0.05). For Analysis 5, the effect of the number of consecutive DH breeding seasons on fertility was examined and was found to remain unchanged (P > 0.05). In summary, no adverse effects of DH breeding on fertility were detected. Fertility was higher when stallions were bred in the SH, as compared to the NH. Potential reasons for higher fertility achieved in the SH were smaller book sizes and better mare reproductive quality.

The effect of two levels of hemospermia on stallion fertility.

Hemospermia can occur consistently or intermittently in stallion ejaculates and may cause a reduction in the fertility of the affected ejaculate. It is unknown what amount of blood in an ejaculate leads to subfertility. This study investigated the effect of higher and lower levels of hemospermia (50% and 5%, respectively) on fertility using 24 reproductively normal mares inseminated over three consecutive estrous cycles with fresh extended semen. Mares inseminated with a 5% blood-contaminated ejaculate became pregnant at the same rate (75% per cycle; 18 of 24) as the mares inseminated with blood-free (control) semen (75% per cycle; 18 of 24). The ejaculates containing 50% blood were sterile (0% per cycle, 0 of 24). We concluded that it is the amount of blood, not the mere presence of blood, in an ejaculate that impacts fertility.

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).

Sperm DNA quality evaluated by comet assay and sperm chromatin structure assay in stallions after unilateral orchiectomy.

Unilateral orchiectomy (UO) may interfere with thermoregulation of the remaining testis caused by inflammation surrounding the incision site, thus altering normal spermatogenesis and consequently sperm quality. Two measures of sperm DNA quality (neutral comet assay and the sperm chromatin structure assay [SCSA]) were compared before UO (0 days) and at 14, 30, and 60 days after UO to determine whether sperm DNA changed after a mild testis stress (i.e., UO). The percent DNA in the comet tail was higher at 14 and 60 days compared to 0 days (P < 0.05) after UO. All other comet tail measures (i.e., length, moment, migration) were higher at all time periods after UO compared to 0 days (P < 0.05). Two SCSA measures (mean-αt, mode-αt) increased at 14 days after UO (P < 0.05), whereas two measures (SD-αt and COMP-αt) did not change. This study identified a decrease in sperm DNA quality using both the neutral comet assay and the SCSA, which was not identified using traditional measures of sperm quality.

Effect of calcium, bicarbonate, and albumin on capacitation-related events in equine sperm.

Repeatable methods for IVF have not been established in the horse, reflecting the failure of standard capacitating media to induce changes required for fertilization capacity in equine sperm. One important step in capacitation is membrane cholesterol efflux, which in other species is triggered by cholesterol oxidation and is typically enhanced using albumin as a sterol acceptor. We incubated equine sperm in the presence of calcium, BSA, and bicarbonate, alone or in combination. Bicarbonate induced an increase in reactive oxygen species (ROS) that was abolished by the addition of calcium or BSA. Bicarbonate induced protein tyrosine phosphorylation (PY), even in the presence of calcium or BSA. Incubation at high pH enhanced PY but did not increase ROS production. Notably, no combination of these factors was associated with significant cholesterol efflux, as assessed by fluorescent quantitative cholesterol assay and confirmed by filipin staining. By contrast, sperm treated with methyl-ß-cyclodextrin showed a significant reduction in cholesterol levels, but no significant increase in PY or ROS. Presence of BSA increased sperm binding to bovine zonae pellucidae in all three stallions. These results show that presence of serum albumin is not associated with a reduction in membrane cholesterol levels in equine sperm, highlighting the failure of equine sperm to exhibit core capacitation-related changes in a standard capacitating medium. These data indicate an atypical relationship among cholesterol efflux, ROS production, and PY in equine sperm. Our findings may help to elucidate factors affecting failure of equine IVF under standard conditions.

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.

Stallion fertility: a focus on the spermatozoon.

Stallion fertility is a vast subject, with a wide array of permutations that can impact reproductive performance in either positive or negative ways. This review is intended to address a mere segment of the male fertility issue, but the very essence of the male contribution to fertilisation, that of the spermatozoon. Spermatozoal ultrastructure and form-to-function are detailed and spermatozoal metabolism is discussed, with specific reference to distinctive characteristics of stallion spermatozoa. Lastly, methods for assessment of spermatozoal function are considered, with emphasis on spermatozoal motility, the acrosome reaction and spermatozoon-oocyte interactions. Closing comments address the need for development and standardisation of molecular-based assays for use with spermatozoa of stallions whose subfertility cannot be explained with conventional tests.

Dense spermatozoa in stallion ejaculates contain lower concentrations of mRNAs encoding the sperm specific calcium channel 1, ornithine decarboxylase antizyme 3, aromatase, and estrogen receptor alpha than less dense spermatozoa.

Stallions are unique among livestock in that, like men, they commonly receive medical treatment for subfertility. In both species, about 15% of individuals have normal semen parameters but are subfertile, indicating a need for novel analyses of spermatozoa function. One procedure for improving fertilizing capability of stallions and men is isolation of dense spermatozoa from an ejaculate for use in artificial insemination. In the current study, dense and less dense spermatozoa were purified by density gradient centrifugation from individual ejaculates from seven reproductively normal adult stallions. The RNA isolated from the spermatozoa seemed to be naturally fragmented to an average length of 250 bases, consistent with reports of spermatozoa RNA from other species. The DNAse treatment of RNA prepared from spermatozoa removed any genomic DNA contamination, as assessed by PCR with intron spanning primers for the protamine 1 (PRM1) gene. Concentrations of seven mRNAs in spermatozoa, correlated with the fertility of men and bulls, were quantified by reverse transcription polymerase chain reaction in dense and less dense spermatozoa. Concentrations of four mRNAs were two- to four-fold lower in dense spermatozoa compared with less dense spermatozoa: Encoding the spermatozoa-specific calcium channel (P < 0.03), ornithine decarboxylase antizyme 3 (P < 0.02), aromatase (P < 0.02), and estrogen receptor alpha (P < 0.08). In contrast, concentrations of three other mRNAs, encoding PRM1 and heat shock proteins HSPA8 and DNAJC4, were not different (P > 0.1). These results identify new differences in mRNA concentrations in populations of spermatozoa with dissimilar densities.

Calcium-calmodulin and pH regulate protein tyrosine phosphorylation in stallion sperm.

The mechanisms leading to capacitation in stallion sperm are poorly understood. The objective of our study was to define factors associated with regulation of protein tyrosine phosphorylation in stallion sperm. Stallion sperm were incubated for 4 h in modified Whitten's media with or without bicarbonate, calcium, or BSA. When sperm were incubated in air at 30×106/ml at initial pH 7.25, protein tyrosine phosphorylation was detected only in medium containing 25 mM bicarbonate alone; calcium and BSA inhibited phosphorylation. Surprisingly, this inhibition did not occur when sperm were incubated at 10×106/ml. The final pH values after incubation at 30×106 and 10×106 sperm/ml were 7.43 ± 0.04 and 7.83 ± 0.07 (mean ± s.e.m.) respectively. Sperm were then incubated at initial pH values of 7.25, 7.90, or 8.50 in either air or 5% CO2. Protein tyrosine phosphorylation increased with increasing final medium pH, regardless of the addition of bicarbonate or BSA. An increase in environmental pH was observed when raw semen was instilled into the uteri of estrous mares and retrieved after 30 min (from 7.47 ± 0.10 to 7.85 ± 0.08), demonstrating a potential physiological role for pH regulation of capacitation. Sperm incubated in the presence of the calmodulin (CaM) inhibitor W-7 exhibited a dose-dependent increase in protein tyrosine phosphorylation, suggesting that the inhibitory effect of calcium was CaM mediated. These results show for the first time a major regulatory role of external pH, calcium, and CaM in stallion sperm protein tyrosine phosphorylation.

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.

Pregnancy rates in mares inseminated with 0.5 or 1 million sperm using hysteroscopic or transrectally guided deep-horn insemination techniques.

Placement of sperm deep in the equine uterine horn allows fewer sperm to be inseminated while maintaining acceptable fertility, and has been promoted for use in circumstances when fertility would be expected to be low if standard insemination were used (e.g., semen from a subfertile stallion, or frozen-thawed semen). Two main techniques, transrectally guided (TRG) and hysteroscopic (HYS) insemination, have been developed for this purpose; however, there is some controversy regarding their comparative efficacy. This study was conducted to compare pregnancy rates when mares were inseminated by TRG or HYS, using sperm numbers approaching and under the minimum threshold, resulting in reduced fertility. When 1 × 10(6) sperm were inseminated, pregnancy rates were not different (P > 0.10) between techniques HYS (10/13, 77%) and TRG (11/15, 73%). Similarly, when 0.5 × 10(6) sperm were inseminated, pregnancy rates were not different (P > 0.10) between techniques HYS (3/15, 20%) and TRG (4/13, 31%). Combined pregnancy rates for the two treatments were 13/28 (46%) for HYS and 15/28 (54%) for TRG (P > 0.10). Pregnancy rates using a subthreshold number of sperm were not significantly affected by a deep-horn insemination technique.

Effect of daily semen centrifugation and resuspension on the longevity of equine sperm quality following cooled storage.

An experiment was conducted to determine whether cooled semen quality could be maintained for a longer interval by conducting daily centrifugation of extended semen, with resuspension of the sperm pellet in fresh extender. Semen treatments included SP10NC and SP50NC which contained 10 and 50% seminal plasma, respectively, were not centrifuged (NC), and were stored at 4 to 7 °C for 96 h. Treatments SP10C and SP50C contained 10 and 50% seminal plasma, respectively, but were centrifuged (C) after 24, 48, and 72 h of cooled storage, with daily resuspension in fresh extender containing 10% seminal plasma. Percent total sperm motility (TMOT) and progressively motile (PMOT) was reduced (P < 0.05) in the SP50NC treatment after 24, 48, 72, and 96 h of storage, and TMOT did not differ (P > 0.05) in the SP10C, SP50C, SP10NC groups after the same storage periods. The % COMP-(αt) did not differ (P > 0.05) among treatments at any time period. Percent membrane intact sperm (SMI) was reduced in SP50NC, as compared to SP10C at 48, 72, and 96 h (P < 0.05). Daily centrifugation and resuspension of sperm exposed to 50% seminal plasma for the first 24 h (SP50C) yielded similar TMOT, PMOT, VCL, SMI, % COMP-(αt) (P > 0.05) to Groups SP10NC and SP10C after 96 h of storage. Daily centrifugation and resuspension of cool-stored equine semen in fresh extender may be a method to increase sperm longevity.

Influence of day of postpartum breeding on pregnancy rate, pregnancy loss rate, and foaling rate in Thoroughbred mares.

Records (years 2005-2007) were analyzed from a Thoroughbred stud farm in central Kentucky. Data from all breeding cycles of foaling mares were tabulated (3184 cycles of 2003 foaling mares bred between 7 and 163 days postpartum). A multiple logistic regression model employing Bayesian statistics was used to adjust for factors that significantly affected outcome; odds ratios (ORs) for pregnancy rate, pregnancy loss rate, and foaling rate were determined to examine the influence of day of postpartum breeding on these parameters. Mares bred before Day 22 (Day 0 = day of foaling) postpartum had a decreased OR for becoming pregnant (P < 0.05); the median OR for becoming pregnant (1.00) was not reached until Day 46 postpartum. Mares bred before Day 13 postpartum had an increased OR for pregnancy loss (P < 0.05). The median OR for pregnancy loss did not decline below 1.00 until Day 78 postpartum. Mares bred before Day 20 postpartum had a decreased OR for producing a foal (P < 0.05). The median OR for producing a foal (1.00) was not reached until Day 75 postpartum. We concluded that fertility (in terms of a higher OR for becoming pregnant and a lower OR for pregnancy loss, resulting in a higher OR for producing a foal) continued to improve in Thoroughbred mares for approximately 2.5 mo postpartum. These findings are of importance to management strategies directed at early postpartum breeding, and explain some of the reported drift in subsequent foaling dates of Thoroughbred mares, despite management practices that employ early postpartum breeding.

The addition of ticarcillin-clavulanic acid to INRA 96 extender for stallion semen cooling.

REASONS FOR PERFORMING STUDY: A commonly used commercial extender (i.e. INRA 96) contains antimicrobials that may have limited effectiveness. Therefore, addition of ticarcillin-clavulanic acid to this extender is a widespread procedure in the equine breeding industry in the United States. However, such practice has not been critically evaluated. OBJECTIVES: To evaluate the addition of ticarcillin-clavulanic acid to INRA 96 and different extender and antimicrobial storage conditions on sperm function and antimicrobial effectiveness. METHODS: Gel-free semen (42 ejaculates from 14 mature Quarter Horse stallions) was extended with INRA 96 and stored for 24 h in an Equitainer II. The effects of added ticarcillin-clavulanic acid and different extender storage procedures on sperm motion characteristics (by computer-assisted analysis), sperm membrane integrity (by fluorescence-based measurement) and suppression of bacterial growth (by aerobic and anaerobic culture methods) were evaluated using analysis-of-variance and Chi-square statistical methods. The P value for significance was set at < 0.05. RESULTS: Freezing and thawing of modified or unmodified extender prior to use for stallion semen resulted in reduced sperm quality post cooling for 24 h, as evidenced by a significant reduction in sperm motility (i.e. total and progressive) and sperm membrane integrity. Addition of ticarcillin-clavulanic acid to extender resulted in higher sperm velocity when the reconstituted antimicrobial was subjected to cooled storage, as compared with frozen storage, prior to use. Only 28 of 42 ejaculates (67%) yielded presence of bacteria in neat semen but addition of ticarcillin-clavulanic acid to INRA 96 was not different than INRA 96 alone for inhibiting growth of bacteria (98 vs. 94%, respectively). CONCLUSIONS: Addition of ticarcillin-clavulanic acid (1 mg/ml) to INRA 96 did not adversely affect sperm quality in extended semen after cooled storage. Extender freezing and thawing prior to use had detrimental effects on sperm quality. POTENTIAL RELEVANCE: These data suggest that INRA 96 should not be frozen and thawed prior to use. Addition of ticarcillin-clavulanic acid to INRA 96 did not impair sperm quality. All extender treatments effectively controlled the bacterial growth compared with neat semen.

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.