Effects of cholesterol-loaded cyclodextrins on the quality of frozen-thawed equine epididymal sperm

Equine epididymal sperm are known to be severely sensitive to cryopreservation, in terms of sperm quality and pregnancy rate. The objective of this study was to examine the effects of cholesterol loaded cyclodextrins (CLCs) on the quality of stallion epididymal sperm during cryopreservation.In experiment I, sperm were treated with different concentrations of CLCs: (1) 0 mg (control), (2) 1.5 mg, (3) 3 mg, and (4) 6 mg per 120 × 10⁶ sperm. The sperm viability and amount of cholesterol were determined at 15, 30 and 45 min after CLC treatment using viability markers (Ethidium homodimer-1 and Calcein AM) and gas chromatography, respectively. In experiment II, CLC treated sperm (1.5 mg CLC per 120 × 10⁶ sperm) were fixed and stained with filipin to examine the cholesterol distribution. In experiment III, sperm were treated with CLCs at concentrations of 1.5, 3.0, 6.0 mg per 120 × 10⁶ sperm for 15 min, then equilibrated with freezing extender at 4 °C for 1 h prior to cryopreservation. Epididymal sperm without CLC loading (0 mg) were used as the control group. The sperm quality was examined at post-equilibration and 10 min, 2 h and 4 h after freezing and thawing.The cholesterol was successfully loaded into the plasma membrane of stallion epididymal sperm. The amount of cholesterol was increased in a manner of dose and time dependence, and the filipin–sterol complexes were increasingly labeled over the sperm head. CLCs at 1.5 mg/120 × 10⁶ sperm significantly improved sperm quality during sperm equilibration and cryopreservation compared to other doses of CLCs and non-CLC control. An increasing concentration and incubation time of CLCs was detrimental to sperm quality.It is concluded that cholesterol loading to the sperm plasma membrane via CLCs decreases chilling sensitivity and also improves epididymal sperm cryopreservability.     

Impact of supplementation of semen extender with antioxidants on the quality of chilled or cryopreserved Arabian stallion spermatozoa

The aim of the present study was to evaluate the effects of supplementation of semen extender with various non-enzymatic antioxidants on the quality of cooled or cryopreserved Arabian stallion spermatozoa. Semen collected from four pure Arabian stallions was centrifuged at 600g for 15 min. Spermatozoa were then diluted in INRA-82 extender supplemented with bovine serum albumin (BSA; 0, 10, 15 and 20 mg/mL) or trehalose (0, 75, 100 and 150 mM) or zinc sulphate (0, 100, 150 and 200 μM). The diluted semen was then either cooled at 5 °C or cryopreserved in 0.5–ml plastic straws. After cooling or thawing, sperm motility, viability, sperm abnormalities, viability index, and plasma membrane integrity were evaluated. The results showed that supplementation of semen extender with 150 mM trehalose or with 200 μM zinc sulphate significantly (P < 0.05) improved motility, viability, sperm membrane integrity and acrosome status in Arabian stallion spermatozoa after cooling or after freezing and thawing compared with controls (non-supplemented media) or with those supplemented with other concentrations of trehalose or zinc sulphate. Supplementation of semen extender with BSA did not improve sperm motility or cryosurvival of Arabian stallion spermatozoa after cooling or after freezing and thawing. In conclusion, supplementation of semen extender with non-enzymatic antioxidants (trehalose or zinc sulphate) improved the quality of chilled and frozen/thawed Arabian stallion spermatozoa. The most beneficial effects occur when semen diluent was supplemented with 150 mM trehalose or 200 μM zinc sulphate.     

Adding cholesterol to the stallion sperm plasma membrane improves cryosurvival

Cryopreservation induces partially irreversible damage to equine sperm membranes. Part of this damage occurs due to membrane alterations induced by the membrane changing from the fluid to the gel-state as the temperature is reduced lower than the membrane transition temperature. One way to prevent this damage is to increase the membrane fluidity at low temperatures by adding cholesterol to the membrane. Different concentrations of cholesterol-loaded-cyclodextrins (CLC) were added to stallion sperm to determine the CLC concentration that optimizes cryosurvival. Higher percentages of motile sperm were maintained after thawing when 1.5 mg CLC was added to sperm from stallions whose sperm do not survive freezing well, compared to control sperm from those same stallions (67% vs. 50%; P<0.05). Addition of CLCs increased the percentages of membrane intact sperm surviving cryopreservation compared to untreated sperm for all stallions (P<0.05). The amount of cholesterol that incorporated into the membranes of the sperm cells increased in a polynomial fashion (R2=0.9978) and incorporated into all sperm membranes. In addition, there was a significant loss of cholesterol from sperm membranes after cryopreservation; however, addition of CLCs to sperm prior to cryopreservation maintained higher cholesterol levels in the sperm after freezing and thawing than untreated sperm (P<0.05). Addition of CLCs also resulted in more sperm binding to the zona pellucida of bovine oocytes after cryopreservation than control sperm (48 vs. 15; P<0.05). In conclusion, CLCs improved the percentage of post-thaw viability in equine sperm as well as increased the number of sperm that bind to zona pellucida. Addition of CLCs to stallion sperm prior to cryopreservation is a simple procedure that increases the cryosurvival of cells.     

Efficiency of short-term storage of equine semen in a simple-design cooling system

Five experiments tested the efficiency of a simple, low-cost system (CP) for cooling and storing equine semen at 2.0 degrees C for 24 h and 48 h. Pantaneiro stallions of known fertility were used. Semen quality was evaluated for progressive motility (PM), plasma membrane integrity (PMI), and pregnancy rate. Experiment 1 showed that PM and PMI were similar between CP and the control (Equitainer) in cooled semen. In Experiment 2, the influence was evaluated of combinations (four treatments) of two volumes (50/100 ml) and two sperm concentrations (500/750x10(6)) on sperm quality of semen cooled and preserved by CP (cooling system replaced at 24 h). While PM decreased gradually from before cooling to 24 h and 48 h, PMI decreased only at the least and greatest sperm volume and concentrations. Storage time did not affect PMI. Results from Experiment 3 showed that CP maintained semen PM>or=30% in all samples 24 h after cooling and decreased to about 70% 42 h after cooling. Results from Experiments 4 and 5 confirmed semen quality after cooling and storage (24 h and 48 h, respectively), achieving a 69% pregnancy rate in the first estrous cycle when insemination occurred. Thus, the CP system is satisfactory for cooling and preserving equine semen for up to 48 h.     

Osmotic tolerance limits and membrane permeability characteristics of stallion spermatozoa treated with cholesterol

Stallion spermatozoa exhibit osmotic damage during the cryopreservation process. Recent studies have shown that the addition of cholesterol to spermatozoal membranes increases the cryosurvival of bull, ram and stallion spermatozoa, but the exact mechanism by which added cholesterol improves cryosurvival is not understood. The objectives of this study were to determine if adding cholesterol to stallion sperm membranes alters the osmotic tolerance limits and membrane permeability characteristics of the spermatozoa. In experiment one, stallion spermatozoa were treated with cholesterol-loaded cyclodextrin (CLC), subjected to anisotonic solutions and spermatozoal motility analyzed. The spermatozoa were then returned to isotonic conditions and the percentages of motile spermatozoa again determined. CLC treatment increased the osmotic tolerance limit of stallion spermatozoa in anisotonic solutions and when returned to isotonic conditions. The second and third experiments utilized an electronic particle counter to determine the plasma membrane characteristics of stallion spermatozoa. In experiment two, stallion spermatozoa were determined to behave as linear osmometers. In experiment three, spermatozoa were treated with CLC, incubated with different cryoprotectants (glycerol, ethylene glycol or dimethyl formamide) and their volume excursions measured during cryoprotectant removal at 5° and 22 °C. Stallion spermatozoa were less permeable to the cryoprotectants at 5 °C than 22 °C. Glycerol was the least permeable cryoprotectant in control cells. The addition of CLC’s to spermatozoa increased the permeability of stallion spermatozoa to the cryoprotectants. Therefore, adding cholesterol to spermatozoal membranes reduces the amount of osmotic stress endured by stallion spermatozoa during cryopreservation.     

Quality of chilled and cold-stored (5 °C) canine spermatozoa submitted to different rapid cooling rates

The aim of this study was to evaluate the sperm quality in chilled canine semen using di?erent cooling rates from room temperature (23 °C) to 5 °C and subsequently cold-stored at 5 °C for up to 96 hours. In experiment 1, semen samples from five dogs were pooled, diluted in Tris-fructose-citrate extender with 20% egg yolk and split into four aliquots that were chilled to 5 °C using di?erent cooling rates of 2.25, 0.9, 0.45, and 0.2 (control) °C/min. In experiment 2, semen from five dogs was processed individually as described above and split into two aliquots that were chilled to 5 °C using rates of either 2.25 °C/min or 0.2 °C/min. In both experiments, the sperm quality (i.e., sperm motility and viability) was evaluated before cooling and after 0, 24, 48, 72, and 96 hours of storage at 5 °C. The total motility, progressive motility, and quality of movement parameters were assessed using computer-assisted analysis system, and the percentage of viable spermatozoa was determined using ?ow cytometry (H-42/PI//FITC-PNA). The cooling rate did not in?uence the sperm quality parameters at any of the evaluation times. All evaluated males showed the same response to chilling semen at a rapid cooling rate. Storage time negatively in?uenced (P < 0.05) sperm motility, regardless of the cooling rate used. In conclusion, canine sperm could be chilled and stored for 96 hours at 5 °C in a Tris-fructose extender with 20% egg yolk using rapid cooling rates, with values for sperm quality similar to those from a conventional protocol.     

Effects of linear cooling rate on motion characteristics of stallion spermatozoa

Three experiments were designed to analyze the effects of cooling rate on survival of stallion spermatozoa in a milk-based extender, at 0 to 96 hours after reaching the desired temperature. The samples were warmed to 37 degrees C and were evaluated by computer-assisted analysis of sperm motility. In Experiment 1, rate of cooling between 37 and 20 degrees C was evaluated. Sperm motion was not affected by cooling at plunge, -0.42 or -0.28 degrees C/minute. However, storage of spermatozoa at 5 degrees C after slow cooling below 20 degrees C was superior to storage at 20 degrees C. In Experiment 2, 3 cooling rates from 37 degrees to 5 degrees C were evaluated. Cooling at either -0.05 or -0.7 degrees C/minute was superior (P<0.05) to plunging spermatozoa to 5 degrees C. Cooling at -0.05 degrees C/minute rather than -0.7 degrees C/minute maximized the percentage of motile spermatozoa and their curvilinear velocity. In Experiment 3, cooling rates from 20 to 5 degrees C were evaluated, with all samples cooled at -0.7 degrees C/minute from 37 to 20 degrees C. Sperm motion was similar (P>0.05) after cooling below 20 degrees C at -0.012, -0.05 or -0.10 degrees C/minute, and the 2 slower rates were superior (P<0.05) to cooling at -0.3 degrees C/minute. It was concluded that stallion spermatozoa can be cooled rapidly from 37 to 20 degrees C, but should be cooled at 相似文献   

Lipase activity in stallion seminal plasma and the effect of lipase on stallion spermatozoa during storage at 5 degrees C

Previous studies have demonstrated a detrimental effect of seminal plasma on the maintenance of motility of cooled equine spermatozoa; however, the mechanism for the adverse effect of seminal plasma during cooled storage remains undetermined. In goats, a glycoprotein component of bulbourethral gland secretion contains lipase activity that is detrimental to sperm motility when stored in skim milk-based extenders. The objective of the current study was to determine the amount of lipase activity in stallion seminal plasma and to determine the effect of added lipase on spermatozoal motility during cooled semen storage. In the first experiment, seminal plasma (1.0 ml) was assayed for lipase activity based upon hydrolysis of triglycerides (olive oil substrate) into free fatty acids and subsequent titration of pH change (SigmaDiagnostic Lipase Kit). Lipase activity in stallion seminal plasma was 0.36 +/- 0.02 Sigma units/ml, (mean + S.E.M.; n = 16 ejaculates from six stallions). In the second experiment, equine semen (three ejaculates from each of four stallions) was divided into five treatment aliquots. In Treatment 1, semen was extended 1:3 with nonfat dried skim milk extender (NFDSM). In treatment groups 2 through 5, spermatozoa were washed by centrifugation (300 x g for 15 min) and resuspended in NFDSM to a final concentration of 25 x 10(6) spermatozoa/ml. Porcine pancreatic lipase (pPL) was added to Treatment 3 (10 pPL units/ml), Treatment 4 (100 pPL units/ml) and Treatment 5 (100 pPL units/ml, heat inactivated at 100 degrees C for 5 min) while Treatment 2 had no pancreatic lipase added and served as the control. Samples were cooled slowly to 5 degrees C, and stored at 5 degrees C until evaluation. Sperm motility was evaluated at time 0, 24, 48 and 72 h by computerized semen analysis, and data were analyzed via repeated measures ANOVA. The addition of 100 units/ml but not 10 units/ml of pPL decreased (P < 0.01) total and progressive motility of stored sperm. Heat-inactivated pPL (Treatment 5) did not significantly decrease motility of spermatozoa during storage. Because the lipase activity assayed (Sigma units) and the lipase activity added to cooled semen (pPL units) were not equivalent, pPL was assayed in the Sigma Diagnostic Lipase assay. The relationship between Sigma Units (Y) and pPL units (X) appeared to be a log-linear relationship with log(Y) = -0.912 + 0.007X; R2 = 0.90. Mean lipase activity assayed in stallion seminal plasma was equivalent to approximately 64 pPL units/ml. These data suggest that endogenous lipase activity in stallion seminal plasma may be a factor in the adverse effects of seminal plasma on cooled spermatozoa in some stallions.     

In vivo fertilizing ability of stallion spermatozoa processed by single layer centrifugation with Androcoll-E™

A colloid with a species specific silane-coated, silica-based formulation, optimized for stallion (Androcoll-E™), enables a better sub-population of spermatozoa to be selected from stallion ejaculates. However, such a practice has not been critically evaluated in stallions with fertility problems. In this study we evaluate whether single-layer centrifugation (SLC) through Androcoll-E™ could be used to enhance fertility rates in a subfertile stallion. Ejaculates were obtained from two different stallions, one Lusitano (fertile) and one Sorraia (subfertile), with distinct sperm characteristics and fertility. Motility, morphology, plasma membrane structural (eosin-nigrosin) and functional integrity (HOS test), mitochondrial functionality (Δψm; JC-1) and longevity (motility after 72 h cooling) after centrifugation in Androcoll-E™, as well as pregnancy rates obtained after artificial insemination (AI), with and without (control group) SLC-treated sperm were assessed. The effect of SLC on sperm characteristics, and fertility results were evaluated by ANOVA and Fisher procedures, respectively. Our results showed that SLC-selected sperm did not differ from the raw semen in terms of viability, morphology, response to hypo-osmotic conditions (HOS test) and mitochondrial membrane potential (↑ΔΨmit; JC-1). Sperm motility in cooled samples was not improved by SLC treatment. Our data show that SLC through Androcoll-E™ has no effect on pregnancy rates in the stallions used in this trial.     

Advances in cooled semen technology.

In the horse industry, milk or milk-based extenders are used routinely for dilution and storage of semen cooled to 4-8 degrees C. Although artificial insemination (AI) with chilled and transported semen has been in use for several years, pregnancy rates are still low and variable related to variable semen quality of stallions. Over the years, a variety of extenders have been proposed for cooling, storage and transport of stallion semen. Fractionation of milk by microfiltration, ultrafiltration, diafiltration and freeze-drying techniques has allowed preparation of purified milk fractions in order to test them on stallion sperm survival. Finally, a high protective fraction, native phosphocaseinate (NPPC), was identified. A new extender, INRA96, based on modified Hanks' salts, supplemented with NPPC was then developed for use with cooled/stored semen.Four experiments were conducted to compare INRA96 and milk-based extenders under various conditions of storage. The diluted semen was maintained under aerobic conditions when stored at 15 degrees C, and anaerobic conditions when stored at 4 degrees C. In experiment 1, split ejaculates from 13 stallions were diluted either in INRA96 extender then stored at 15 degrees C or diluted in Kenney or INRA82 extenders and then stored at 4 degrees C for 24h, until insemination. In experiment 2, semen from two stallions was extended in INRA96 then inseminated immediately or stored at 15 degrees C for 3 days until insemination. In experiment 3, semen from three stallions was diluted in INRA96 then stored at 15 or 4 degrees C for 24h until insemination, finally, in experiment 4, split ejaculates from four stallions were diluted in INRA96 or E-Z Mixin extenders then stored at 4 degrees C for 24h until insemination. Experiment 1 demonstrated that at 15 degrees C, INRA96 extender significantly improved pregnancy rate per cycle compared to Kenney or INRA82 extenders at 4 degrees C after 24h of storage (57%, n=178 versus 40%, n=171, respectively; P<0.01). Experiment 2 showed that semen stored at 15 degrees C for 3 days can achieve pregnancy at a fertility rate per cycle of 48% (n=52) compared to 68% (n=50, immediate insemination, P=0.06). Experiment 3 demonstrated that INRA96 extender can be as efficient at 15 degrees C (54%, n=37) as at 4 degrees C (54%, n=35) after 24h of storage. Finally, experiment 4 showed that INRA96 extender used at 4 degrees C (59%, n=39) seems to improve fertility per cycle compared to E-Z Mixin at 4 degrees C (49%, n=39, P=0.25), but this result has to be confirmed.These results demonstrate that semen diluted in INRA96 extender and stored at 15 degrees C can be an alternative to semen diluted in milk-based extenders and stored at 4 degrees C for "poor cooler" stallions. Furthermore, INRA96 extender can be as efficient at 15 degrees C as at 4 degrees C, for preserving sperm motility and fertility.     

Effect of cholesterol-loaded cyclodextrins on bull and goat sperm processed with fast or slow cryopreservation protocols

Cholesterol-loaded cyclodextrins (CLC) added to the sperm before cryopreservation enhance sperm quality after freeze-thawing in several cold shock-sensitive species, including cattle and goats. However, all studies conducted to date have used conventional protocols, in which sperm are cooled slowly to 5°C before freezing. As cholesterol plays a significant role in sperm cold shock resistance, it is possible that CLC-treated sperm can withstand cooling damage when the sperm are not cooled slowly to 5°C before freezing. In this study, we determined whether CLC-treated goat (1 mg CLC/120×10⁶ sperm) and bull (2 mg CLC/120×10⁶ sperm) sperm quality, after thawing, was different for sperm frozen using conventional protocols (including a slow cooling phase to 5ºC) and protocols in which the sperm were frozen from room temperature, without cooling the sperm slowly to 5°C before freezing. CLC-treated sperm exhibited higher percentages of plasma membrane-intact sperm than control sperm when cryopreserved using conventional protocols. In addition, CLC treatment enhanced both sperm motility and plasma membrane integrity when sperm were frozen directly from room temperature. However, this treatment did not fully prevent the damage of the sperm after cooling rapidly and subsequent freezing, as the sperm quality was lower than that presented by the samples frozen using the conventional protocol. The results are promising, but studies to optimize the protocols for freezing sperm directly from room temperature need to be conducted, as well as studies to determine how cryopreserving sperm in this manner affects other sperm functions.     

Influences of a diet supplemented with linseed oil and antioxidants on quality of equine semen after cooling and cryopreservation during winter

Seasonal changes in the reproductive physiology of stallions contribute to a decrease in the quality of frozen-thawed semen during late winter. Changes in the lipid composition of the sperm plasma membrane may contribute to this phenomenon. In the present study, we have, therefore, investigated the effects of adding linseed oil (LO) in combination with antioxidants to the diet of breeding stallions on the motility and membrane integrity of cooled–stored and cryopreserved semen. Starting in November, the diet of LO stallions (n = 6) but not control (C) stallions (n = 5) was supplemented with LO (100 mL once daily) plus an antioxidant (Myostem Protect; Audevard, Clichy, France) for a total of 84 days. Before (November) and at the end of this period (February), ejaculates were processed for cryopreservation (n = 3 ejaculates per stallion) and cooled shipping at 5 °C. Frozen-thawed and cooled–shipped semen was sent to the laboratory for computer-assisted semen analysis of total motility, progressive motility, and velocity parameters (average path velocity [VAP], curved line velocity [VCL], and straight-line velocity [VSL]) and evaluation of membrane integrity. The quality of frozen-thawed semen decreased (P < 0.05) from November (e.g., total motility LO 69 ± 3% and C 67 ± 3%) to February (total motility: LO 55 ± 4% and C 59 ± 3%) independent of treatment (P > 0.05). A decrease in the velocity parameters VAP, VCL, and VSL was more pronounced in LO stallions than in C stallions (e.g., VSL: November LO 67 ± 1 μm/s, C 64 ± 2 μm/s; February LO 59 ± 2 μm/s, C 63 ± 2 μm/s; interaction month by treatment, P < 0.05). In cooled–stored semen, total motility, progressive motility, and membrane integrity were lower in February than in November (P < 0.001 for all parameters). Supplementation of the diet with LO and antioxidants attenuated this decrease (e.g., Day 1 of cooled storage = 24 hours after semen collection: total motility in November LO 88 ± 1% and C 87 ± 3%; in February LO 83 ± 2% and C 73 ± 11%; interaction month by treatment: P < 0.05). Velocity parameters VAP, VCL, and VSL were significantly lower in February than in November (P < 0.001), but this decrease was not affected by treatment. In summary, dietary supplementation of stallions with LO plus antioxidants attenuated a decline in motility and membrane integrity of cooled–stored stallion semen during winter. This may improve the fertility of cooled–shipped semen. In contrast, the treatment did not counteract the decrease in quality of frozen-thawed semen that occurs in late winter.     

Effect of sod (superoxide dismutase) protein supplementation in semen extenders on motility, viability, acrosome status and ERK (extracellular signal-regulated kinase) protein phosphorylation of chilled stallion spermatozoa

New studies are underway to find new methods for supporting longer storage of cooled stallion semen. It is known that high concentrations of reactive oxygen species (ROS) cause sperm pathology. The metalloprotein superoxide dismutase (SOD) is responsible for H₂O₂ and O₂ production, by dismutation of superoxide radicals. The aim of this study is to assess the quality of chilled stallion semen processed with extenders containing SOD at different concentrations as antioxidant additives. A total of 80 ejaculates collected from 5 standardbred stallions was divided into 5 aliquots treated as: native semen (control 1); native semen diluted 1:3 with Kenney semen extender (control 2); spermatozoa diluted after centrifugation in extender without (control 3) or with SOD at 25 IU/ml (experimental 1) or 50IU/ml (experimental 2). Each sample was analyzed for motility, viability and acrosome status, immediately after semen preparation and again after storage at 5 °C for 24h, 48h and 72h.Acrosome integrity was evaluated by Chlortetracycline (CTC) and Fluorescent-labeled peanut lectin agglutinin (PNA-FITC conjugated staining). A proteomic approach of quantifying extracellular signal regulated kinase (ERK) was also evaluated as an indirect indicator of oxidative stress. In all samples sperm progressive motility and sperm acrosomal integrity showed a significant reduction between fresh and cooled spermatozoa at 24h, 48h and 72h. Quality parameters of sperm were significantly higher (Progressive Motility P < 0.01; Viability P < 0.001) in aliquots supplemented with SOD. ERK phosphorylation was statistically higher (P < 0.01) in aliquots without SOD. The Authors concluded that addition of SOD to semen extenders improves the quality of chilled equine semen and reduces ERK activation.     

Replacing egg yolk with soybean lecithin in the cryopreservation of stallion semen

The objective of this study was to determine whether replacing the egg yolk with soybean lecithin in the Botu-Crio? cryodiluent would maintain the fertility of cryopreserved stallion sperm. Two experiments were performed to evaluate cell freezability. In experiment 1, sperm from 15 stallions were frozen in Botu-Crio? (BC) or Botu-Crio? which contained 45g/L soybean lecithin (BCLS45) in place of the egg yolk. In experiment 2, we compared different concentrations of soybean lecithin: 0, 10.0, 12.5, 15.0, 17.5 and 20.0g/L (BC, BCLS10, BCLS12.5, BCLS17.5 and BCLS20, respectively). In experiment 1, sperm frozen in BC and BCLS45 exhibited similar (P>0.05) percentages of total motile sperm (61% and 61%, respectively); progressively motile sperm (27% and 27%, respectively) and sperm with intact plasma membranes (IMP; 53% and 57%, respectively). Similarly, sperm frozen in BC or BC containing any concentration of soybean lecithin maintained similar (P>0.05) percentages of total motile sperm (61-68%) and progressively motile sperm (27-31%). In the first fertility trial, we used cryopreserved semen from a single stallion was inseminated into mares. The semen from the sperm that were frozen in BC diluent resulted in a higher fertility rate (66%, 16/24) compared to the sperm that were frozen in BCLS45 diluent (17%, 5/29; P<0.01). Similarly, in a second fertility trial, the mares that were inseminated with the sperm that were frozen in BC diluent exhibited a higher fertility rate (66%, 16/24) compared to the mares that were inseminated with the sperm that were frozen in BCLS20 (40%, 10/25; P<0.05). Finally, in a third trial, the sperm that were frozen in BC resulted in a higher fertility rate in mares (75%, 18/24) compared to the sperm that were frozen in BCLS10 (41%, 10/24; P<0.05). Although replacing the egg yolk in the BC cryodiluent with soybean lecithin provided similar laboratory results for stallion sperm, after cryopreservation, the sperm that was frozen with soybean lecithin in the diluent correlated with lower fertility rates. Based on these results, we concluded that the use of BCLS can be used as an alternative diluent for cryopreserving stallion sperm. However, the resulting reduced fertility rate is a matter of concern. Further studies are necessary to clarify the reasons for this decrease in fertility and to determine the optimal lecithin concentration for diluents to freeze stallion sperm.     

Effect of semen collection practices on sperm characteristics before and after storage and on fertility of stallions

This study analyzed effects of different methods and intervals of semen collection on the quantity and quality of fresh, cool-stored, and frozen-thawed sperm and fertility of AI stallions. In Experiment 1, ejaculates were obtained from six stallions (72 ejaculates per stallion) using fractionated versus non-fractionated semen collection techniques. Initial sperm quality of the first three jets of the ejaculate was not different from that of total ejaculates. Centrifugation of sperm-rich fractions before freezing improved post-thaw motility and sperm membrane integrity when compared to non-centrifuged sperm-rich fractions or non-fractionated centrifuged ejaculates (P<0.05). In Experiment 2, semen from four stallions (60-70 ejaculates per stallion) was collected either once daily or two times 1h apart every 48 h. The first ejaculates of double collections had significantly higher sperm concentrations, percentages of progressively motile sperm (PMS) after storage for 24h at 5 degrees C and lower percentages of midpiece alterations than single daily ejaculates. Semen collected once daily showed significantly lower values of live sperm after freezing and thawing than the first ejaculate of two ejaculates collected 1h apart every 48 h. In Experiment 3, semen was collected from 36 stallions (> or =12 ejaculates per stallion) during the non-breeding season and the time to ejaculation and the number of mounts was recorded. When time to ejaculation and the number of mounts increased, volume and total sperm count (TSC) also increased (P<0.05), whereas a decrease was observed in sperm concentration, percentage of PMS after storage for 24 h at 5 degrees C, percentage of membrane-intact sperm in fresh semen (P<0.05) as well as motility and percentage of membrane-intact sperm of frozen-thawed sperm (P<0.05). In Experiment 4, AI data of 71 stallions were retrospectively analyzed for the effect of number of mounts per ejaculation and frequency, time interval of semen collections on pregnancy, and foaling rates (FRs) of mares. Semen volume increased, but sperm concentration and percentage of PMS after 24-h cool-storage decreased with increasing number of mounts on the phantom (P<0.05). A statistically significant inter-relationship was demonstrated between frequency and interval of semen collection and FR. Mares inseminated with stallions from which semen was collected frequently (> or =1 on an average per day) showed significantly higher FRs than mares inseminated with semen from stallions with a daily collection frequency of 0.5-1 or <0.5. FR of mares inseminated with stallions having 0.5-1 days between semen collections was significantly better than FR of mares that were inseminated with stallions having semen collection intervals of 1-1.5 days or >2.5 days.     

Tolerance of brown bear spermatozoa to conditions of pre-freezing cooling rate and equilibration time

Specific protocols for the cryopreservation of endangered Cantabrian brown bear spermatozoa are critical to create a genetic resource bank. The aim of this study was to assess the effect of cooling rates and equilibration time before freezing on post-thawed brown bear spermatozoa quality. Electroejaculates from 11 mature bears were extended to 100 × 10⁶ spermatozoa/mL in a TES–Tris–Fructose–based extender, cryopreserved following performance of the respective cooling/equilibration protocol each sample was assigned to, and stored at −196 °C for further assessment. Before freezing, after thawing, and after 1 hour's incubation post-thawing at 37 °C (thermal stress test), the quality of the samples was assessed for motility by computer-assisted semen analysis, and for viability (SYBR-14/propidium iodide), acrosomal status (peanut agglutinin–fluorescein isothiocyanate /propidium iodide), and sperm chromatin stability (SCSA) by flow cytometry. In experiment 1, three cooling rates (0.25 °C/min, 1 °C/min, and 4 °C/min) to 5 °C were assessed. After thawing, total motility (%TM) was higher and percentage of damaged acrosomes (%dACR) was lower (P < 0.05) for 0.25 °C/min than for 4 °C/min. The thermal stress test data indicated equally poor quality (P < 0.05) for the 4 °C/min cooled samples in viability (%VIAB), %dACR, %TM, and progressive motility (%PM). In experiment 2, the effect of a pre-freezing equilibration period at 5 °C for 1 hour (cooling at 0.25 °C/min) was evaluated. Samples kept at 5 °C for 1 hour showed higher (P < 0.05) values than the nonequilibrated ones for both thawing (%dACR) and thermal stress test (%VIAB, %TM, and %PM). In experiment 3, samples stored without cooling and equilibration (direct freezing) were compared with the samples cooled at 0.25 °C/min and equilibrated for 1 hour (control freezing). Using thermal stress test, we observed that direct freezing causes damage in viability, acrosomal status, and motility of spermatozoa compared with the control group (P < 0.05). In conclusion, our results suggest that slow cooling rates to 5 °C and at least 1 hour equilibration time are necessary for the effective cryopreservation of brown bear sperm.     

Effects of reduced glutathione and catalase on the kinematics and membrane functionality of sperm during liquid storage of ram semen

The objective of this study was to evaluate the effects of reduced glutathione (GSH) and catalase (CAT) supplementation on the kinematics and membrane functionality of sperm during the liquid storage of ram semen, cooled at 5 °C, for up to 24 h. Semen samples from four rams were pooled, diluted with Tris-egg yolk extender without antioxidants (control) or supplemented with either CAT (100, 200, and 400 U/mL) or GSH (100, 200, and 400 mM) at a final concentration of 50 × 10⁶ sperm/mL. Sperm kinematics, which was analyzed by computer-assisted sperm analysis (CASA), and membrane functionality, which was analyzed using the hypo-osmotic swelling test (HOST), were determined after the addition of the semen samples at different processing times (fresh/diluted, 1.5, 6, 12, and 24 h, at 5 °C). No significant differences were recorded in the kinematics or membrane functionality between treatments at different times. The supplementation of diluents with 100 and 200 U/mL of CAT prevented the harmful effects of cooling on total sperm motility. No significant differences were observed in progressive sperm motility throughout processing, regardless of the treatment and time of evaluation. Supplementation with 400 mM GSH resulted in an earlier reduction (P < 0.05) of total sperm motility, a decrease in rapid sperm rate and a reduction in curvilinear velocity during incubation, at 5 °C. The cooling induced a reduction (P < 0.05) in the percentage of sperm with a functional plasma membrane (HOST), especially after 1.5 h of incubation. Based on the results of the present study, the addition of CAT (100 and 200 U/mL) reduced the deleterious effects of cooling on total motility in ram sperm maintained at 5 °C for 24 h, although it did not affect the functionality of the sperm membranes. However, the addition of 400 mM GSH caused negative effects on the velocity parameters of the sperm.     

Effects of cooling rate and storage temperature on equine spermatozoal motility parameters

Two experiments were conducted to examine the effects of cooling rate and storage temperature on motility parameters of stallion spermatozoa. In Experiment 1, specific cooling rates to be used in Experiment 2 were established. In Experiment 2, three ejaculates from each of two stallions were diluted to 25 x 10(6) sperm/ml with 37 degrees C nonfat dry skim milk-glucose-penicillin-streptomycin seminal extender, then assigned to one of five treatments: 1) storage at 37 degrees C, 2) storage at 25 degrees C, 3) slow cooling rate to and storage at 4 degrees C, 4) moderate cooling rate to and storage at 4 degrees C, and 5) fast cooling rate to and storage at 4 degrees C. Total spermatozoal motility (TSM), progressive spermatozoal motility (PSM), and spermatozoal velocity (SV) were estimated at 6, 12, 24, 48, 72, 96 and 120 h postejaculation. The longevity of spermatozoal motility was greatly reduced when spermatozoa were stored at 37 degrees C as compared to lower spermatozoal storage temperatures. At 6 h postejaculation, TSM values (mean % +/- SEM) of semen stored at 37 degrees C, slowly cooled to and stored at 25 degrees C or slowly cooled to and stored at 4 degrees C were 5.4 +/- 1.1, 79.8 +/- 1.6, and 82.1 +/- 1.6, respectively. Mean TSM for semen that was cooled to 4 degrees C at a slow rate was greater (P<0.05) than mean TSM of semen cooled to 4 degrees C at a moderate rate for four of seven time periods (6, 24, 72 and 120 h), and it was greater (P<0.05) than mean TSM of semen cooled to 4 degrees C at a fast rate for five of seven time periods (6, 12, 24, 72 and 120 h). Mean TSM of semen cooled to 4 degrees C at a slow rate was greater (P<0.05) than mean TSM of semen cooled to 25 degrees C for five of seven time periods (24 to 120 h). A similar pattern was found for PSM. Mean SV of semen cooled to 4 degrees C at a slow rate was greater (P<0.05) than mean SV of semen cooled to 25 degrees C for all time periods. A slow cooling rate (initial cooling rate of -0.3 degrees /min) and a storage temperature of 4 degrees C appear to optimize liquid preservation of equine spermatozoal motility in vitro.     

Preservation of ejaculated and epididymal stallion spermatozoa by cooling and freezing

The suitability of ejaculated and epididymal stallion spermatozoa for cooled storage (5 degrees C) and cryopreservation was examined in 5 ejaculates from each of 6 stallions and in spermatozoa recovered from the cauda epididymidis after castration of these stallions. The percentage of progressively motile spermatozoa, examined by subjective estimation (cooled samples) or by computerized analysis (frozen-thawed samples), was used as parameter. In ejaculated semen samples containing 5 and 25% seminal plasma in a skim milk glucose extender, the lower amount of seminal plasma supported spermatozoal motility significantly better throughout storage at 5 degrees C. Addition of 5 or 25% seminal plasma to perfused epididymal spermatozoa (0% seminal plasma) resulted in a significant stimulation of spermatozoal motility by 25% seminal plasma at 0 h (P<0.05) and to a lesser extent at 24 and 48 h. Post-thaw motility of ejaculated as well as epididymal spermatozoa was not influenced by slow cooling to 15 degrees or 5 degrees C with or without glycerol prior to rapid freezing in liquid nitrogen vapor. During cooled storage, seminal plasma had a stimulatory effect on epididymal spermatozoa and depressed motility in ejaculated spermatozoa. Results on cryopreservation indicate that freezability of equine spermatozoa is already determined when spermatozoa leave the tail of the epididymis.     

Dynamics of sperm DNA fragmentation in domestic animals II. The stallion

The mixed success of equine artificial insemination programs using chilled and frozen-thawed semen is most likely associated with the variable response of the sperm cell to the preservation process and the fact that stallions are not selected on the basis of reproductive performance. We propose that the traditional indicators of sperm viability do not fully account for male factor infertility in the stallion and that knowledge of sperm DNA damage in the original semen sample and during semen processing may provide a more informed explanation of an individual stallion's reproductive potential. This study reports on the validation of a sperm DNA fragmentation test based on the sperm chromatin dispersion test (SCD) for stallion spermatozoa and on its application to semen that was chilled (4 degrees C; n=10) or frozen-thawed (n=13). Semen samples were collected by artificial vagina and the proportion of sperm with fragmented DNA determined. Seminal plasma was then removed by centrifugation and the sperm pellet re-suspended in commercial extenders prior to being chilled or cryopreserved using standard industry protocols. Chilled semen was cooled slowly to 4 degrees C and stored for 1h before commencing the analysis; cryopreserved semen was thawed and immediately analyzed. Following chilling or cryopreservation, the semen samples were incubated at 37 degrees C and analyzed for SCD after 0, 4, 6, 24 and 48 h storage. The results of this investigation revealed that there was no significant difference in the sperm DNA fragmentation index (sDFI) of sperm evaluated initially after collection compared to those tested immediately after chilling or cryopreservation. However, within 1h of incubation at 37 degrees C, both chilled and frozen-thawed spermatozoa showed a significant increase in the proportion of sDFI; after 6h the sDFI had increased to over 50% and by 48 h, almost 100% of the sperm showed DNA damage. While the sDFI of individual stallions at equivalent times of incubation was variable, an analysis of the rate of change of sDFI revealed no difference between stallions or the way in which the semen was preserved. In terms of sperm DNA fragmentation dynamics, the highest intensity of sperm DNA damage occurred in the first 6h of incubation. We suggest that the SCD test can be used as a routine assessment tool for the development and refinement of preservation protocols designed to reduce stallion sperm DNA damage.