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.

Intrafollicular oocyte transfer in the horse: effect of autologous vs. allogeneic transfer and time of administration of ovulatory stimulus before transfer.

PURPOSE: To assess meiotic and developmental competence after transfer of immature cumulus-oocyte complexes (COCs) to the preovulatory follicles of mares (intrafollicular oocyte transfer (IFOT)). METHODS: In Experiment 1, mares received an ovulatory stimulus at IFOT. Thirty hours later, COCs were recovered from the follicle, and mature oocytes underwent ICSI and embryo culture. In Experiments 2 and 3, autologous vs. allogeneic COCs were used. The mares were inseminated and embryos were recovered. In Experiment 3, the ovulatory stimulus was administered 9 h (autologous) and 15 h (allogeneic) before IFOT. In Experiment 4, only allogeneic COCs were used; the ovulatory stimulus was administered 9 or 15 h before IFOT. Excess embryos (autologous) and parentage-verified embryos (allogeneic) were considered IFOT-derived. RESULTS: In Experiment 1, 36/54 IFOT oocytes (67%) were recovered, of which 56% were mature, vs. 49% of in vitro matured oocytes (P > 0.1). After ICSI, blastocyst rates were 25% and 18%, respectively (P > 0.1). In Experiment 2, 0/6 autologous and 2/6 allogeneic IFOT yielded IFOT-derived embryos. In Experiment 3, 0/7 autologous and 2/5 allogeneic IFOT yielded IFOT-derived embryos. The proportion of mares yielding IFOT-derived embryos was lower after autologous vs. allogeneic IFOT (0/13 vs. 4/11; P < 0.05). In Experiment 4, 1/8 9-h and 1/7 15-h IFOT yielded IFOT-derived embryos. CONCLUSIONS: Transferred oocytes mature within the follicle and can maintain developmental competence. Allogeneic IFOT was more efficient than was autologous IFOT. The time of ovulatory stimulation did not affect embryo yield. The IFOT procedure is still not repeatable enough to be recommended for clinical use.

Epigallocatechin-3-gallate (EGCG) and green tea polyphenols do not improve stallion semen parameters during cooling at 4°C.

Stallion semen storage for artificial insemination is mainly based on liquid cooled storage. In many stallions this technique maintains sperm quality for an extended period of time (24-72 hr) at 7°C. While this technique is commonly used in the horse industry, there can be a decline in fertility in some stallions, due to an inability of their sperm to tolerate the cool storage process. The aim of the present work was to evaluate the effect of two natural antioxidants (epigallocatechin-3-gallate (EGCG) at 20, 60 and 120 µm and green tea polyphenols, and p at .001, .01 and .1 mg/ml) on some sperm parameters (sperm motility, viability/acrosome integrity and DNA quality) in extended semen immediately after its collection (T0) and after 2, 6, 24 and 48 hr of cool storage. Two ejaculates from three trotter stallions were analysed after 48 hr of storage at 4°C. No beneficial effect on the analysed parameters was observed: the two antioxidants were not able to improve sperm quality after 48 hr of storage. These results are in agreement with previous findings on the effect of different antioxidants reported by other researches, who have demonstrated that stallion semen keeps good antioxidant capacity after dilution for 24 hr. In conclusion, the positive effect exerted by antioxidant molecules in other species is not confirmed in the equine one.

Characteristics of high-quality Asian elephant (Elephas maximus) ejaculates and in vitro sperm quality after prolonged chilled storage and directional freezing.

The in vitro quality of spermatozoa from one elephant (Elephas maximus) was examined after chilled storage and directional freezing (DF). High-quality, non-contaminated ejaculates (77.6±6.0% progressive motility, 3.9±1.5 µg creatinine mL(-1) raw semen, 2.7±0.6% detached heads) were cryopreserved after 0 (0hStor), 12 (12hStor) and 24 h (24hStor) of chilled storage. At 0 h and 6h post-thawing, total motility, plasma membrane integrity, acrosome integrity, mitochondrial activity and normal morphology were similar (P>0.05) across treatments. In contrast, progressive motility, rapid velocity and several kinematic parameters were lower (P<0.05) for 24Stor compared with 0hStor at 0 h post-thaw. By 6 h post-thaw, amplitude of lateral head displacement and velocity parameters (average pathway, straight-line and curvilinear velocity) were lower (P<0.05) for 24hStor compared with 0hStor and 12hStor. DNA integrity was high and remained unchanged (P>0.05) across all groups and processing stages (1.6±0.6% of cells contained fragmented DNA). Results indicate that DF after up to 12 h of chilled storage results in a post-thaw sperm population of acceptable quality for artificial insemination. These findings have implications for the cryopreservation of sex-sorted spermatozoa, which typically undergo more than 12 h of chilled storage prior to sorting and preservation.

Evaluation of motility, membrane status and DNA integrity of frozen-thawed bottlenose dolphin (Tursiops truncatus) spermatozoa after sex-sorting and recryopreservation.

Artificial insemination (AI) with sex-sorted frozen-thawed spermatozoa has led to enhanced management of ex situ bottlenose dolphin populations. Extended distance of animals from the sorting facility can be overcome by the use of frozen-thawed, sorted and recryopreserved spermatozoa. Although one bottlenose dolphin calf had been born using sexed frozen-thawed spermatozoa derived from frozen semen, a critical evaluation of in vitro sperm quality is needed to justify the routine use of such samples in AI programs. Sperm motility parameters and plasma membrane integrity were influenced by stage of the sex-sorting process, sperm type (non-sorted and sorted) and freezing method (straw and directional) (P<0.05). After recryopreservation, sorted spermatozoa frozen with the directional freezing method maintained higher (P<0.05) motility parameters over a 24-h incubation period compared to spermatozoa frozen using straws. Quality of sperm DNA of non-sorted spermatozoa, as assessed by the sperm chromatin structure assay (SCSA), was high and remained unchanged throughout freeze-thawing and incubation processes. Though a possible interaction between Hoechst 33342 and the SCSA-derived acridine orange was observed in stained and sorted samples, the proportion of sex-sorted, recryopreserved spermatozoa exhibiting denatured DNA was low (6.6±4.1%) at 6 h after the second thawing step and remained unchanged (P>0.05) at 24 h. The viability of sorted spermatozoa was higher (P<0.05) than that of non-sorted spermatozoa across all time points after recryopreservation. Collective results indicate that bottlenose dolphin spermatozoa undergoing cryopreservation, sorting and recryopreservation are of adequate quality for use in AI.

Production of live foals via intracytoplasmic injection of lyophilized sperm and sperm extract in the horse.

Work with lyophilized sperm helps delineate the factors required for successful fertilization. We investigated the use of lyophilized sperm in equine embryo production. In Experiment 1, sperm DNA fragmentation index was not affected by three freeze/thaw or lyophilization cycles. In Experiment 2, oocytes injected with lyophilized sperm or with sperm from a treatment in which lyophilized sperm were suspended in sperm cytoplasmic extract (SE) yielded blastocyst development rates of 0 and 28% respectively (P < 0.05). In Experiment 3, blastocyst development rate was 6-11% after injection of sperm lyophilized from fresh or frozen-thawed semen, suspended in SE. In Experiment 4, sperm lyophilized 3.5 months or 1 week previously, suspended in SE, yielded similar blastocyst rates (6 and 3% respectively). Rates of normal pregnancy after transfer were 7/10 and 5/7 for embryos from control and lyophilized sperm treatments respectively. Three pregnancies from the lyophilized sperm treatments were not terminated, resulting in two healthy foals. Parentage testing determined that one foal originated from the lyophilized sperm; the other was the offspring of the stallion providing the sperm extract. Further testing indicated that two of five additional embryos in the lyophilized sperm treatment originated from the stallion providing the sperm extract. We conclude that both lyophilized stallion sperm and stallion sperm processed by multiple unprotected freeze-thaw cycles (as for sperm extract) can support production of viable foals. To the best of our knowledge, this is the first report on production of live offspring by fertilization with lyophilized sperm in a non-laboratory animal species.

Equine fetal genotyping via aspiration of yolk-sac fluid at 22-28 days of gestation.

Fetal genotyping has important applications in the horse, but currently necessitates embryo recovery and biopsy. We investigated whether fetal genotyping could be performed on yolk-sac fluid recovered from pregnant mares via transvaginal aspiration. Fluid was collected before Day 30 to provide results before establishment of the endometrial cups (Day 37). Genotyping and assessment of maternal DNA contamination was performed by analyzing histograms of PCR results for 19 loci. In Exp. 1, mares underwent yolk-sac aspiration on Days 22-28 of gestation. Fluid (0.56-1.02 mL) was recovered from five of seven mares. Four of the five mares maintained pregnancy. One pregnancy was electively terminated at Day 75; the other three mares delivered healthy foals. Extraction of DNA from the fluid sample followed by direct PCR allowed the highest rate of determination of fetal alleles. Fetal genotype was correctly determined in three samples, and for 14/19 alleles in one sample. In Exp. 2, we evaluated whether recovery of more fluid (up to 1.6 mL), and fractionation of the sample, would minimize maternal DNA contamination. One of four mares maintained pregnancy. Evaluation at informative loci showed no difference in maternal contamination among fractions. We determined that mares can maintain pregnancy after aspiration of yolk-sac fluid, and that fetal genotype can be accurately determined from the sample obtained. Further work is needed on factors affecting maintenance of pregnancy after the procedure. The ability to access the yolk sac in early pregnancy opens the door to novel potential clinical and research applications.

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.

Factors impacting equine sperm recovery rate and quality following cushioned centrifugation.

Two experiments were conducted to investigate modifications in cushioned centrifugation of stallion semen. Specifically, the effects of tube type, centrifugation medium, cushion type, and centrifugation force on post-centrifugation sperm recovery rate and quality were evaluated. In Experiment 1, sperm recovery rate was higher (P<0.05) in conventional plastic conical-bottom tubes (103%) than in newly developed glass nipple-bottom tubes (96%) following cushioned centrifugation; however, several measures of semen quality (i.e., % total motility [MOT], % progressive motility [PMOT], curvilinear velocity, and average-path velocity) yielded higher values following centrifugation in nipple-bottom tubes (P<0.05). Sperm recovery rate following cushioned centrifugation was similar between semen previously diluted in optically clear centrifugation extender (100%) and semen diluted in opaque centrifugation extender (100%); however, MOT and PMOT were higher in semen subjected to cushioned centrifugation in opaque extender (P<0.05). An extender by tube-type interaction was not detected for recovery rate or post-centrifugation semen quality. In Experiment 2, sperm recovery rate following cushioned centrifugation in nipple-bottom tubes was similar when forces of 400xg or 600xg were applied (90 and 90%, respectively; P>0.05), and no resulting differences in semen quality were detected between these treatment groups (P>0.05). The type of iodixanol cushion medium used (i.e., OptiPrep, Eqcellsire Component B, or Cushion Fluid did not impact post-centrifugation semen quality, based on the laboratory values measured (P>0.05). In conclusion, cushioned centrifugation of stallion semen in either conical-bottom or nipple-bottom tubes yielded a high sperm harvest, while maintaining sperm function. An optically opaque extender, commonly used in the equine breeding industry, can be used to achieve this goal.

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 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.

Effect of urine contamination on stallion semen freezing ability.

Urospermia is a common ejaculatory dysfunction of stallions. Current practice suggests that urine contaminated semen should not be used for cryopreservation. The aim of this study was to determine effects of urine contamination on semen freezing. Sixty-five ejaculates from eight stallions were divided into no urine (CONT), low (20% urine, LOW), and high (50% urine, HIGH) samples. Semen was extended with a commercial cooling extender, cushion-centrifuged, resuspended to 200 million/mL in a commercial egg-yolk based extender, and cryopreserved in liquid nitrogen. A subset of ejaculates (n = 20) were split in two after cushion-centrifugation, and one half of the ejaculate was submitted to a single-layer gradient centrifugation before cryopreservation. Sperm motility parameters were assessed pre- and post-freezing with an automated sperm analyzer. Semen pH, creatinine, and urea concentrations were assessed in raw samples, after urine contamination and after centrifugation and extension. Statistical analyses were performed with ANOVA and Tukey's posthoc. There were significant reductions in total and progressive sperm motilities (i.e., %TM and %PM, respectively) with increasing urine contamination pre-freezing (%TM 67 ±â€¯1.7, %PM 50 ±â€¯2.2, CONT), (%TM 60.3 ±â€¯1.7, % PM 42.5 ±â€¯2.1, LOW), and (%TM 41.3 ±â€¯2, %PM 21.3 ±â€¯1.5, HIGH). Post-thaw motilities for CONT (%TM 54 ±â€¯2.3, %PM 40.8 ±â€¯3.3) and LOW (%TM 51.7 ±â€¯1.8, %PM 36.2 ±â€¯2.1) were not different, but were higher than the HIGH (%TM 31.5 ±â€¯1.2, %PM 17.1 ±â€¯1.0) (p < 0.05). Post-thaw sperm viability was significantly lower in the HIGH (54.7 ±â€¯2.4) than in the CONT (63.8 ±â€¯2.3) or LOW (64.6 ±â€¯3.4) groups. Semen creatinine and urine levels were significantly higher with increasing urine contamination and were significantly decreased after centrifugation and resuspension in freezing extender. Pre-treatment semen pH was significantly lower than semen contaminated with low or high amounts of urine, and pH decreased significantly after centrifugation and resuspension. Gradient centrifugation did not improve %TM in the control group, but it did improve pre-freeze %TM and %PM in the low and high groups and improved significantly post freezing %TM and %PM in the high urine contaminated group. Semen contaminated with a small amount of urine may be suitable for freezing, whereas highly contaminated semen might not be usable. Although urine was mostly removed in this fashion, the initial exposure to high quantities was sufficient to decrease sperm motility pre- and post-freezing, whereas low urine contamination was not as detrimental.

Cryopreservation of donkey sperm using non-permeable cryoprotectants.

The aim of this study was to evaluate the effect of different concentrations of sucrose combined with bovine serum albumin (BSA), as non-permeable cryoprotectants, on donkey sperm parameters after cryopreservation, in comparison to a control extender containing glycerol. Semen from five Andalusian donkeys (n = 12) were centrifuged and resuspended with a commercial extender for equine sperm (Gent A, Minitube) adding 1% BSA and different concentrations (M, mol/l) of water-diluted sucrose: 0.05, 0.1, 0.25, 0.35 and 0.45. Thereafter, semen (n = 24) were diluted in the same base extender containing 0.25 M sucrose (S25) or glycerol (GLY, Gent B). Sperm were slowly cooled, filled in 0.5 ml straws and frozen in nitrogen vapours. Post-thaw samples were assessed for sperm motility, plasma membrane and DNA integrity and results were compared by ANOVA. In Experiment 1, sperm motility was significantly higher (P < 0.001) for S25 than the remaining treatments, and no differences were found for plasma membrane or DNA integrity. In Experiment 2, no differences were found between S25 or GLY for sperm motility and DNA integrity but plasma membrane integrity was significantly higher (P < 0.05) for S25. In conclusion, the extender with sucrose 0.25 M combined with BSA can be considered as an alternative to conventional extenders with glycerol for donkey sperm cryopreservation.

Cholesterol-to-phospholipid ratio in whole sperm and seminal plasma from fertile stallions and stallions with unexplained subfertility.

Semen samples were collected from six fertile stallions and seven stallions with unexplained infertility. Percentages of motile sperm (77.5 +/- 11.3 versus 67.5 +/- 12.2, P = 0.2), and progressively motile sperm (70.8 +/- 13.6 versus 60.7 +/- 14.0, P = 0.2) were similar between fertile and subfertile stallions, respectively. Morphologic characteristics in ejaculates of control and affected stallions (% normal: 60.2 +/- 18.2 versus 52.9 +/- 11.3, P = 0.4; % abnormal heads 7.3 +/- 4.8 versus 12.1 +/- 5.0, P = 0.11; and % abnormal acrosomes 1.6 +/- 2.1 versus 3.0 +/- 3.4, P = 0.4) did not differ. After incubation with the calcium ionophore A23187, acrosome reaction rate of sperm from fertile stallions was 96 +/- 2.8% whereas only 2.9 +/- 2.5% of sperm from stallions with unexplained subfertility had acrosome reacted (P < 0.001). Molar amounts of cholesterol and phospholipid in whole sperm and seminal plasma did not differ (P > 0.1) between fertile and subfertile stallions. However, the molar ratio of cholesterol-to-phospholipid was 2.5 times greater in the seminal plasma (P = 0.09) and 1.9 times greater (P = 0.009) in whole sperm of subfertile stallions compared to fertile stallions.

Ejaculate and type of freezing extender affect rates of fertilization of horse oocytes in vitro.

In vitro fertilization (IVF) was performed on in vitro-matured equine oocytes in three experiments. Frozen-thawed sperm were prepared using swim-up separation and heparin treatment. In Experiment 1, fertilization was achieved with sperm from only one frozen ejaculate of four obtained from the same stallion. Within this ejaculate, fertilization rates were higher with fresh media, as compared to media held for 6-8 days before use (39.6% versus 7.3%, respectively; P<0.001). The type of bovine serum albumin used affected fertilization rates (4% versus 39.6%; P<0.001). To determine if IVF rates were influenced by factors associated with the freezing process (Experiment 2), a single ejaculate from a second stallion was frozen using eight variations in timing of steps in the freezing protocol. There were no differences among treatments in fertilization rates (range, 0-3%). In Experiment 3, fertilization rates of semen frozen in an extender containing 21.5% egg yolk were lower than fertilization rates of semen from the same ejaculate but frozen with a 3% egg-yolk extender (0% versus 15%, respectively; P<0.01). We inferred that rates of equine IVF with frozen-thawed sperm were influenced by ejaculate, the composition and age of the media used, and freezing extender. To our knowledge, this is the first report of ejaculate or extender differences affecting in vitro fertilization in this species. These factors may help to explain the great variability in fertilization rates reported with equine IVF, both among and within laboratories.

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.

Equine blastocyst development after intracytoplasmic injection of sperm subjected to two freeze-thaw cycles.

This study was conducted to evaluate the effects of thawing, division into aliquots and refreezing on fertilizing capacity (ability to support embryo development after intracytoplasmic sperm injection; ICSI) of frozen stallion semen. Frozen semen from a fertile stallion was thawed, diluted 1:100 with freezing extender, and refrozen (2F treatment). Control semen was frozen only once. In vitro matured equine oocytes were injected with: (1) motile control spermatozoa; (2) motile 2F spermatozoa; (3) non-motile 2F spermatozoa; or (4) non-motile 2F spermatozoa, followed by injection of sperm extract. Blastocyst development after ICSI was equivalent between control spermatozoa and motile 2F spermatozoa (27 and 23%, respectively). Blastocyst development after injection of non-motile 2F spermatozoa (13%) tended (P=0.07) to be lower than that for control spermatozoa. Injection of sperm extract into oocytes that received non-motile 2F spermatozoa resulted in a significant decrease in blastocyst development (to 2%) compared with injection of non-motile 2F spermatozoa alone. Spermatozoa from a subfertile stallion was similarly processed and used for ICSI; blastocyst development for both motile control (once frozen) spermatozoa and motile 2F spermatozoa was 9%. In conclusion, frozen stallion semen may be thawed, diluted, and refrozen without effect on the ability of motile spermatozoa to initiate embryo development after ICSI. Non-motile spermatozoa from reprocessed semen may also achieve embryo development after ICSI. To our knowledge, this is the first report evaluating the ability of refrozen spermatozoa to produce embryos by ICSI in any species.

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.

Relationship of seminal plasma level and extender type to sperm motility and DNA integrity.

The relationship between seminal plasma level (0, 10, or 20%) and extender type [Kenney type (EZ-Mixin-CST) or Kenney-modified Tyrodes-KMT] to the susceptibility of sperm DNA to denaturation and sperm motility measures were investigated in cooled (5 degrees C) stallion sperm. Three ejaculates from each of three fertile stallions were collected in an artificial vagina and processed as follows: diluted one part uncentrifuged semen with four parts of extender to a final concentration of 20% seminal plasma in either CST or KMT (20% CST; 20% KMT); diluted to a final concentration of 25 million sperm/mL in either CST or KMT (10% CST; 10% KMT); centrifuged to remove virtually all seminal plasma and resuspended in either CST or KMT (0% CST-Cent; 0% KMT-Cent); centrifuged semen to remove virtually all seminal plasma and resuspended with previously filtered seminal plasma from the same stallion in either CST or KMT to a final concentration of 20% seminal plasma (20% CST-Cent; 20% KMT-Cent). Sperm motion characteristics were determined by CASA and DNA integrity (%COMP, percent of cells outside the main population) evaluated by the Sperm Chromatin Structure Assay prior to cooling, and after 24 and 48 h cooled-storage at 5 degrees C. After 48 h of storage at 5 degrees C, extenders with 0% seminal plasma (0% CST-Cent, 0% KMT-Cent) maintained highest quality DNA (P < 0.05), but 0% KMT-Cent maintained higher velocity measures (P < 0.05) than 0% CST-Cent. Total sperm motility was highest (P < 0.05) in 0% CST-Cent, 0% KMT-Cent, 10% CST, 20% CST-Cent, and 20% CST compared to the other treatment groups. Progressive sperm motility was highest (P < 0.05) after 48 h of storage in the treatment with 10% seminal plasma in Kenney extender (10% CST), despite a reduction in DNA integrity. Regardless of extender type, addition of 20% seminal plasma following centrifugation resulted in almost a two-fold increase in %COMP(alpha t), even though one of the treatments (20% CST-Cent) maintained total and progressive motility similar to treatments with no seminal plasma, suggesting that sperm motility and DNA integrity may respond independently to environmental conditions. Overall, better quality sperm features (motility and DNA) were maintained in sperm from which seminal plasma was removed followed by resuspension in either Kenney extender or modified Kenney Tyrodes-type extender.