The effects of urine concentration, and cushion centrifugation to remove urine, on the quality of cool-stored stallion sperm.

Urine-contaminated stallion semen is a clinical problem due to a variety of causes. The effect of the level of urine contamination on the longevity of sperm quality has not been evaluated. The aim of this study was to determine the effects of urine concentration level (0%, 10%, 20%, 30%, and 40%) and cushioned centrifugation and resuspension of the sperm pellet in fresh extender, on measures of sperm quality, immediately after semen collection (T0), after 1 hour of storage at room temperature (T1), and after 24 hours of cooled storage (T24). In general, most sperm quality measures declined with increasing urine concentration starting at T0. Cushioned centrifugation (CC), but not simple dilution, generally maintained sperm quality at T24 as compared with T1. At T24, total sperm motility was higher in all urine-contaminated CC samples compared with uncentrifuged samples (P < 0.05); sperm viability was lower in CC than uncentrifuged at a urine concentration of 20%, but higher at 30% and 40% (P < 0.05); and DNA quality was decreased (higher % cells outside the main population) in all urine concentrations (P < 0.05). Immediate extension in semen extender, followed by cushioned centrifugation and resuspension of the sperm pellet in fresh extender, provided the best option for preserving sperm quality of urospermic semen.

The "dilution effect" in stallion sperm.

Dilution of semen to less than 20 × 10(6) sperm/mL has been reported to decrease sperm quality in multiple species, a phenomenon known as the semen "dilution effect." Critical evaluation of stallion semen diluted to these concentrations, however, has not been reported. This study evaluated sperm motion characteristics (percent total motility [TMOT], percent progressive motility [PMOT], curvilinear velocity [µm/s], and percent straightness) and plasma membrane integrity (percent plasma membrane intact [PMI]) in semen samples diluted to 2.5 × 10(6) sperm/mL with the addition of 0%, 7.5%, or 25% seminal plasma (groups T-2.5/0, T-2.5/7.5, and T-2.5/25, respectively), or after simple dilution to 30 × 10(6) sperm/mL (group T-30), or simple dilution to a ratio of 3:1 (extender:semen; group T-3:1SD). Evaluations were performed immediately after semen collection (T0), and after 24 and 48 hours of cooled storage (T24 and T48, respectively). The PMI and TMOT were the highest in group T-3:1SD at T0. At T24, the PMI in groups T-30, T3:1SD and T3:1/30, and T-2.5/0 were higher than that in the other groups (P < 0.05), whereas TMOT in group T-3:1SD was higher (P < 0.05) than that in all other groups except T-30. By T48, no difference was detected for PMI among groups T-3:1SD, T-30, and T-2.5/0; for TMOT among groups T-3:1SD, T-30, and T-2.5/0, and T-2.5/7.5 (P > 0.05), whereas PMOT was the highest in groups T-2.5/0 and T-2.5/7.5 (P < 0.05). These findings revealed that treatments in which semen was diluted to a concentration of 2.5 × 10(6) sperm/mL had lower initial PMI, TMOT, and PMOT, but semen quality did not decline after 24 and 48 hours of cooled storage. In this study, TMOT and PMI in dilute semen were less than those in more concentrated semen at T0. This effect, while significant, was small and less apparent after cooled storage.

Effect of non-sperm cells removal with single-layer colloidal centrifugation on myeloperoxidase concentration in post-thaw equine semen.

Myeloperoxidase (MPO) is a pro-oxidant enzyme contained in and released by neutrophils during degranulation or after lysis. Post-thaw semen contains MPO and its concentration is associated with decreased sperm motility. Recently, MPO concentration in post-thaw semen was shown to be associated with the presence of non-sperm cells (NSC). The objective of this study was to evaluate the effect of a single-layer colloidal centrifugation before cryopreservation on NSC and MPO concentrations in equine semen. The experimental design consisted of freezing semen with or without previous centrifugation through two concentrations of single-layer colloid media. Non-sperm cells and MPO concentrations were assessed in pellet and upper layer at each step of the procedure and MPO was detected in cells by immunocytochemistry. Single-layer colloid centrifugation decreased NSC and MPO concentrations in post-thaw semen. The MPO concentration was correlated with concentration of NSC in the upper layer of the supernatant. In post-thaw semen, with or without previous single-layer colloid centrifugation, MPO concentration was correlated with concentration of NSC. Overall, neutrophils were rarely observed and NSC were mainly epithelial cells or cellular debris, as demonstrated by MPO immunocytochemistry. At all steps of the semen processing and cryopreservation, MPO immunostaining was clearly identified only on NSC. In conclusion, our study shows that NSC present in fresh semen release MPO during freezing.

Azoospermia in stallions: determining the cause.

Determining the cause of failure to ejaculate sperm can be a diagnostic dilemma. The first diagnostic step is to ascertain whether the stallion is ejaculating. If the stallion appears to ejaculate, but there is azoospermia (absence of sperm in the seminal fluid), testing alkaline phosphatase (ALP) activity in seminal plasma can determine whether testicular and epididymal fluids are present. If ALP activity is low, the possibility of either blockage to sperm outflow in the excurrent duct system or retrograde ejaculation should be pursued diagnostically. If ALP activity is high, the possibility of a testicular defect should be pursued diagnostically. In some cases (notably plugged ampullae or transient, thermally induced testicular degeneration), treatment or the passage of time may restore a stallion's fertility.

Effect of feeding a DHA-enriched nutriceutical on the quality of fresh, cooled and frozen stallion semen.

Eight stallions were used in 2 x 2 crossover study to determine if feeding a nutriceutical rich in docosahexaenoic acid (DHA) would improve semen quality. Stallions were randomly assigned to one of two treatment groups (n = 4 per group). Stallions were fed their normal diet (control) or their normal diet top-dressed with 250 g of a DHA-enriched nutriceutical. Feeding trials lasted for 14 week, after which a 14-week washout period was allowed and the treatment groups were reversed for another 14 week feeding trial. Feeding the nutriceutical resulted in a three-fold increase in semen DHA levels and 50% increase in the ratio of DHA to DPA in semen. Sperm motion characteristics in fresh semen were unaffected by treatment. After 24 h of cooled semen storage in an Equitainer, total and progressive motility did not differ between treatment groups, but sperm from stallions fed the nutriceutical exhibited higher velocity and straighter projectory (P < 0.05). After 48 h of cooled storage, increases in the percentages of sperm exhibiting total motility (P = 0.07), progressive motility (P = 0.06) and rapid motility (P = 0.04), were observed when stallions were being fed the nutriceutical. For a subset of four stallions, whose progressive sperm motility was <40% after 24 h of cooled storage when fed the control diet, feeding the nutriceutical resulted in improvements in mean progressive motility of sperm after 24 h (P = 0.10) and 48 h (P = 0.03) of storage. Feeding the nutriceutical resulted in similar improvements in motion characteristics being observed in frozen-thawed semen. While it appears that feeding the nutriceutical may improve the motion characteristics of cool-stored stallion semen, it may be most beneficial for stallions of marginal fertility whose sperm do not tolerate the rigors of cooling and storage. The nutriceutical also appeared to improve the freezability of semen. More dramatic improvements in semen quality may be observed if modifications in the main fat content of the diet are incorporated with the DHA supplement.

A Bayesian approach to prediction of stallion daily sperm output.

In equine breeding, the number of spermatozoa ejaculated is considered an important factor in fertility. Methods for predicting the number of spermatozoa have been derived from semen collection procedures. A once-daily collection period for 10 days is a standard recommendation to predict long-term daily sperm output (DSO). The first objective of this study was to determine the precision or repeatability of these DSO predictions. Semen was collected and evaluated daily during four periods for 10 days, for 15 different stallions. The analytical methods utilized hierarchal Bayesian modeling as implemented by Gibbs Sampling. The overall population model showed an initial decline in total sperm number of 1.54 billion spermatozoa per day until the observed mean change point of 4.71 days, at which time mean DSO was estimated at 5.28 billion spermatozoa per day. The hierarchal model showed standard deviations in DSO within-stallion of 0.67 billion spermatozoa per day and among-stallion of 1.86 billion spermatozoa per day. The study's second objective was to determine how testicular size affected DSO models. When the model was extended to include testicular size, the optimal prediction of DSO was that DSO = 0.79 + 0.018 x testicular size (in milliliters). Testicular size explained 36.5% of the among-stallion standard deviation in DSO, but was not significantly related to the mean number of collection-days required to reach DSO.

Gene expression in the spermatogenically inactive "dark" and maturing "light" testicular tissues of the prepubertal colt.

In the testis of the 1.5-year-old horse, spermatogenesis initiates locally in grossly light, central areas that contrast with grossly dark, peripheral areas that are as yet inactive in spermatogenesis. Gene expression was compared between "light" and "dark" tissues of 1.5-year-old horse testes to identify mechanisms important to the initiation of spermatogenesis. Microarrays containing human cDNAs were used to assess expression levels of 9132 genes simultaneously in matched pairs of dark and light testis tissues from 3 prepubertal colts. In all 3 analyses, dysferlin (DYS), down-regulated in ovarian cancer 1 (DOC1), and Golgi apparatus protein 1 (GLG1) genes were preferentially expressed in dark tissues, while outer dense fiber of sperm tails (ODF2) and phosphodiesterase 3B (PDE3B) genes were more highly expressed in light testis tissue (>1.7 balanced difference value, Incyte GEM tools software). Expression levels of 88 additional genes appeared to be different between dark and light tissues in 2 of the 3 microarray analyses. The preferential expression of DYS, DOC1, ODF2, and PDE3B genes in dark or light testis tissues was confirmed on Northern blots and localized to cell types by in situ hybridization. Future studies to determine the role of genes regulated during the initiation of spermatogenesis may aid in elucidating molecular mechanisms during this critical time as well as in identifying new therapies for enhancing male fertility.

Germ cell apoptosis in the testes of normal stallions.

Apoptosis in testicular germ cells has been demonstrated in many mammalian species. However, little is known about the stallion (Equus caballus) and rates of apoptosis during spermatogenesis. Morphological and biochemical features of apoptosis reported in other species were used to confirm that the TdT-mediated dUTP Nick end labeling (TUNEL) assay is an acceptable method for identification and quantification of apoptotic germ cells in histological tissue sections from stallion testis. Seminiferous tubules from eight stallions with normal testis size and semen quality were evaluated according to stage of seminiferous epithelium to determine the germ cell types and stages where apoptosis most commonly occurs. Spermatogonia and spermatocytes were the most common germ cell types labeled by the TUNEL assay. A low rate of round and elongated spermatids were labeled by the TUNEL assay. Mean numbers of TUNEL-positive germ cells per 100 Sertoli cell nuclei were highest in stages IV (15.5 +/- 1.0) and V (13.5 +/- 1.1) of the seminiferous epithelial cycle (P < 0.001). An intermediate level of apoptosis was detected in stage VI (P < 0.02). These stages (IV-VI) correspond to meiotic divisions of primary spermatocytes and mitotic proliferation of B1 and B2 spermatogonia. Establishing basal levels of germ cell apoptosis is a critical step towards understanding fertility and the role of apoptosis in regulating germ cell numbers during spermatogenesis.

Evaluation of administration of West Nile virus vaccine to pregnant broodmares.

OBJECTIVE: To determine whether administration of killed West Nile virus vaccine was associated with pregnancy loss among broodmares. DESIGN: Retrospective cohort study. ANIMALS: 595 mares. PROCEDURE: Records of pregnant mares with known vaccination history from 4 farms were reviewed. Information obtained from 595 mares included mare's identification; farm; age; breed; reproductive status; last breeding date; date last known pregnant; vaccination date; age of conceptus at vaccination; vaccination during the early embryonic, early fetal, and late fetal periods; and whether an early embryonic death (EED), early fetal loss (EFL), or late fetal loss (LFL) occurred. The relationships between the dichotomous outcomes of loss (eg, EED, EFL, LFL) and independent categoric variables (eg, vaccination during the early embryonic, early fetal, or late fetal periods) were examined. RESULTS: Vaccination of pregnant mares during any period of gestation was not associated with increased incidence of pregnancy loss. CONCLUSIONS AND CLINICAL RELEVANCE: Many mares are already pregnant at the onset of mosquito season, when mares are more likely to be vaccinated than at other times. Our findings provide evidence that vaccine administration will not compromise pregnancy in horses.

Pregnancy rates in mares following hysteroscopic or transrectally-guided insemination with low sperm numbers at the utero-tubal papilla.

This study was conducted to evaluate two methods for insemination of a low number of sperm in the tip of the uterine horn, and to determine whether prebreeding intrauterine treatment with prostaglandin E(2) would improve pregnancy rates. Estrus was synchronized in 36 fertile Quarter Horse and Thoroughbred broodmares. When a dominant follicle >or=33 mm diameter was present, mares were treated with 2500 units hCG intravenously and were assigned to one of four treatment groups for insemination with five million total sperm in 200 microl extender the next day as follows: (1) Group PGE-HYS (n=9): 0.25mg PGE(2) in 1 ml 0.9% NaCl solution infused into the tip of the uterine horn ipsilateral to the dominant follicle 2h prior to hysteroscopic-guided inseminate placement onto the oviductal papilla; (2) Group SAL-HYS (n=9): 1 ml 0.9% NaCl solution infused into the tip of the uterine horn ipsilateral to the dominant follicle 2h prior to hysteroscopic-guided inseminate placement onto the oviductal papilla; (3) Group PGE-PIP (n=9): 0.25mg PGE(2) in 1 ml 0.9% NaCl solution infused into the tip of the uterine horn ipsilateral to the dominant follicle 2h prior to transrectally-guided pipette placement of the inseminate into the tip of the uterine horn; and (4) Group SAL-PIP (n=9): 1 ml 0.9% NaCl solution infused into the tip of the uterine horn ipsilateral to the dominant follicle 2h prior to transrectally-guided pipette placement of inseminate into the tip of the uterine horn. Mares in estrus were evaluated daily by transrectal ultrasonography to monitor follicular status and confirm ovulation. If mares had not ovulated within 2 days of insemination, the assigned treatment was repeated. Pregnancy status was evaluated by transrectal ultrasonography 12-14 days postovulation, and pregnancy rates were compared. No interaction between prebreeding treatment (SAL:PGE) and insemination protocol (HYS:PIP) on pregnancy rates occurred (P>0.10). Pregnancy rates did not differ between mares inseminated by HYS (12/18; 67%) or PIP (10/18; 56%) (P>0.10). Pregnancy rates did not differ between mares treated prior to breeding with PGE (11/18; 61%) or SAL (11/18; 61%) (P=1.00). In summary, satisfactory pregnancy rates were obtained when a low number of sperm were either placed directly onto the oviductal papilla using hysteroscopy or placed in the tip of the uterine horn using a transrectally-guided uterine pipette. Infusion of 0.25mg PGE(2) in the tip of the uterine horn 2h prior to insemination did not improve pregnancy rates.

Evaluation of early fetal losses on four equine farms in central Kentucky: 73 cases (2001).

OBJECTIVE: To determine features of an early fetal loss (EFL) syndrome and evaluate potential risk factors for EFL in Thoroughbred broodmares on 4 farms in central Kentucky. DESIGN: Retrospective study. ANIMALS: 288 pregnant broodmares. PROCEDURE: Year-2001 breeding records for 288 Thoroughbred broodmares were examined. Early fetal loss was defined as loss of a fetus that was viable at > or = 40 days of gestation but was subsequently lost by 5 months of gestation. RESULTS: Overall 2001 EFL rate was 25% (73/288), median gestational age at time of fetal loss was 77 days, and median date of loss was May 7. Mares on 1 farm had significantly fewer fetal losses (5%) than mares on the other 3 farms (26 to 35%). Fetal losses were higher for maiden (42%) and barren (42%) mares than for foaling mares (18%). Fetal losses were greater in young than in older mares. Effects of broodmare farm, mare age, and reproductive status were all significant. Fetal losses were not associated with sire used for mating or stud farm. CONCLUSIONS AND CLINICAL RELEVANCE: Greatest risk for EFL occurred during the period from late April to May (ie, in mares bred during February through March). Higher incidence of EFL in maiden and barren mares and lower incidence of EFL on 1 farm suggest management or environmental influences may have affected outcome. Risk factors that should be investigated include environmental differences among farms and differences in management procedures used for lactating versus nonlactating mares.

Uterine involution, day and variance of first postpartum ovulation in mares treated with progesterone and estradiol-17beta for 1 or 2 days postpartum.

The effects of a single or double regimen of exogenous progesterone and estradiol-17beta (P/E, total dose 300 mg P/20 mg E) were investigated in 50 postparturient Quarter Horse mares. In Trial 1, at 1 and 24 h after foaling, mares were injected with progesterone (150 mg) and estradiol-17beta (10 mg) (n = 7) or 0.9% NaCl (control, n = 13). In Trial 2, within 12 h after foaling, mares were injected with progesterone (300 mg) and estradiol-17beta (20 mg) (n = 13) or 0.9% NaCl (control, n = 17). Mares were examined daily by palpation per rectum and transrectal ultrasonography to determine the day of ovulation. The largest cross sectional diameters of each uterine horn and uterine body were measured ultrasonographically on Day 15 postpartum. Mean uterine diameters did not differ between treatment groups (P > 0.05) in Trial 1, Trial 2 or for combined data for both Trials 1 and 2. For mares bred on the first postpartum estrus pregnancy rates did not differ (P > 0.05) between treatment groups (16/18, 89%) and controls (22/30, 81%) nor was there a difference in mean day to first postpartum ovulation (P > 0.05) between treated and control groups in Trial 1, Trial 2 or Trials 1 and 2 combined. However, fewer (P < 0.05) total P/E treated mares (0/20) ovulated prior to Day 10 postpartum than did control mares (6/30). Variance in days to ovulation was lower (P < 0.05) for P/E treated mares (var = 3.73 days) than for control mares (var = 7.64 days) for data combined from Trials 1 and 2.