Factors affecting the plasma membrane function of cooled-stored stallion spermatozoa

The spermatozoon is a highly specified cell that has the abilities of active motility and fertilization of the ovum. Damage to the sperm plasma membrane results in the irreversible loss of its functions. Because of the high content of unsaturated fatty acids in the plasma membrane, mammalian sperm are sensitive to oxidative stress. While mild peroxidation appears to promote capacitation of the sperm cell, excessive peroxidation will damage the plasma membrane and results in loss of motility and fertility. The functional integrity of the sperm plasma membrane can be determined by functional tests (determination of motility, resistance against hypoosmotic media) or different staining methods. Today, fluorescent dyes that allow the evaluation of membrane-intact cells are preferred. Computer-assisted evaluation or the use of flow cytometry has improved the precision of these methods. The use of cooled-shipped semen has become a routine method in the equine industry and semen stored at 5 degrees C for about 24 h maintains fertility close to that of fresh semen. According to the suitability of their ejaculates for cooled-storage, stallions may be classified as "good coolers" or "poor coolers". Semen processing involves a number of factors that may damage the sperm plasma membrane. This includes addition of semen extender, centrifugation, cooling and storage at a temperature of 4-6 degrees C. Extender media and their ingredients protect the sperm plasma membrane against environmental influences, but unsuitable composition of the extender can also promote membrane damage. Recent advantages in extender composition are based on the substitution of undefined factors such as milk or egg yolk by more defined and stable components.     

Effects of antioxidants on motility and membrane integrity of chilled-stored stallion semen

The use of chilled-stored stallion semen is limited by its relatively short-term fertilizing capacity. An important reason for the decrease in fertility during storage is the peroxidation of sperm membrane lipids. In this study, effects of the antioxidants ascorbic acid (0.45 and 0.9 g/L) and catalase (0.45 x 10(6) and 1.8 x 10(6) units/L) on chilled-stored stallion semen were investigated. Semen was collected by artificial vagina from 7 stallions and was diluted with skim milk extender or glycin extender. Sperm motility and membrane integrity were investigated after dilution and after 24, 48 and 72 h at 5 degrees C. Ascorbic acid significantly increased the percentage of membrane-intact spermatozoa at 24, 48 and 72 h at 5 degrees C when compared with that of the controls (P < 0.05), irrespective of the extender. Ascorbic acid decreased the percentage of progressively motile spermatozoa (P < 0.05) at a concentration of 0.9 g/L in glycin extender. Catalase decreased (P < 0.05) progressively motile spermatozoa after 24, 48 and 72 h at 5 degrees C in skim milk extender at a concentration of 1.8 x 10(6) units/L. Catalase decreased (P < 0.05) the percentage of membrane-intact spermatozoa at 24 h. Motility and membrane integrity of spermatozoa after dilution with glycin extender containing catalase did not differ from the controls. In conclusion, ascorbic acid has protective effects on sperm membrane integrity in diluted stallion semen.     

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.     

Comparison of ticarcillin and piperacillin in Kenney's semen extender

Ticarcillin and piperacillin were compared to determine their effect on sperm motility and bacterial growth of equine semen samples diluted in Kenney's glucose skim milk semen extender. Each ejaculate (n=11) was divided into three portions and glucose skim milk semen extender solution was added. The control semen extender solution contained extended semen and no antibiotic, whereas ticarcillin and piperacillin solutions contained extended semen plus 1.0mg/mL of ticarcillin or piperacillin, respectively. An aliquot was removed (1h after collection) to evaluate sperm motility and microbial concentration. All three solutions were stored at 4 degrees C and aliquots were obtained at 24 and 48 h to determine sperm motility and microbial concentration. Mean percentages of motile and progressively motile sperm did not differ significantly among control and antibiotic-containing solutions after storage. Control-extended semen samples from ejaculates of stallions (n=11) were contaminated with aerobic gram-positive and gram-negative bacteria. In solutions that contained either antibiotic, growth of these microbes was inhibited after 1, 24, and 48 h at 4 degrees C. Semen samples from stallions (n=5) were extended with Kenney's glucose skim milk extender containing no antibiotic, ticarcillin or piperacillin and then inoculated with approximately 5 x 10(2)CFU/mL Klebsiella pneumoniae or Pseudomonas aeruginosa; there was no significant difference between antibiotics in the inhibition of microbial growth. In conclusion, piperacillin was an appropriate alternative to ticarcillin in extenders for equine semen.     

The influence of trehalose, taurine, cysteamine and hyaluronan on ram semen Microscopic and oxidative stress parameters after freeze-thawing process

There is a lack of information regarding lipid peroxidation and antioxidant capacity in cryopreserved ram semen, and cryopreservation is associated with the production of reactive oxygen species (ROS) which lead to lipid peroxidation (LPO) of sperm membranes, resulting in a loss of motility, viability and fertility of sperm. The aim of this study was to determine the influence of certain additives and their different doses on standard semen parameters, lipid peroxidation and antioxidant activities after the cryopreservation/thawing of ram semen. Ejaculates collected from four Akkaraman rams, a native breed of sheep, were evaluated and pooled at 33 degrees C. Semen samples which were diluted with a Tris-based extender containing additives including trehalose (50, 100mM), taurine (25, 50mM), cysteamine (5, 10mM), and hyaluronan (0.5, 1mg/ml), and an extender containing no additives (control) were cooled to 5 degrees C and frozen in 0.25ml French straws, being stored in liquid nitrogen. Frozen straws were thawed individually at 37 degrees C for 20s in a water bath for evaluation. The use of a Tris-based extender supplemented with 50mM trehalose, 25mM taurine, and 5 and 10mM cysteamine led to higher percentages of post-thaw motility, in comparison to the control group (P<0.01). No significant differences were observed in the percentages of acrosome and total abnormalities, and the hypoosmotic swelling test upon the supplementation of the freezing extender with antioxidants after the thawing of semen. In biochemical assays, the addition of antioxidants did not cause significant differences in levels of malondialdehyde (MDA), glutathione (GSH), and glutathione peroxidase (GSH-Px), after thawing, when compared to groups with no additives. In this study, catalase (CAT) activities were higher in the group that was applied 25mM taurine as an antioxidant, than in all of the other groups (P<0.001). Compared to the controls, antioxidant treatment with 100mM trehalose, 50mM taurine, 5mM cysteamine and 0.5mg/ml hyaluronan, significantly elevated vitamin E (vit E) levels in samples (P<0.001).     

Motility and fertility of equine spermatozoa in a milk extender after 12 or 24 hours at 20 degrees C

The effects of extender and storage at 20 degrees C on equine spermatozoa were evaluated in two experiments using embryo recovery as the end point. In both experiments, inseminations were every other day, starting on Day 2 or 3 of estrus or after a 35-mm follicle was detected, with 250 x 10(6) progressively motile cells (based on initial evaluation). In Experiment 1, semen from two stallions was used to compare the motility and fertility of spermatozoa maintained in a) heated skim milk extender at 37 degrees C with insemination in <1 h; b) E-Z Mixin extender at 37 degrees C with insemination in <1 h; and c) E-Z Mixin extender at 37 degrees C with cooling to 20 degrees C and insemination after storage for 12 h at 20 degrees C. The percentage of motile spermatozoa was 34% after 12 h compared to 55% at 0 h (P < 0.05). However, the percentage of mares from which an embryo was recovered 6.5 d after ovulation was 62, 56, and 50% for Treatments A, B, and C (P > 0.05). In Experiment 2, semen from three stallions was used to compare the motility and fertility of spermatozoa in a) E-Z Mixin extender at 37 degrees C with insemination in <1 h or b) E-Z Mixin extender at 37 degrees C with cooling to 20 degrees C and insemination after storage for 24 h at 20 degrees C. The percentage of motile spermatozoa was 17% after 24 h compared to 54% at 0 h (P < 0.05). There was no difference between treatments (P > 0.05) in the percentage of mares from which an embryo was recovered 6.0 d after ovulation (68 vs 62%) or among stallions. Thus, stallion semen extended in E-Z Mixin was held at 20 degrees C for 24 h without a marked decline in fertility.     

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.     

Comparison of three containers used for the transport of cooled stallion semen

Three containers commonly used to transport cooled equine semen (Equitainer, ExpectaFoal and a Swedish-designed semen-transport container, previously called the Salsbro Box and now called Equine Express) were compared, using four ejaculates from each of three stallions. Each ejaculate was diluted to a spermatozoal concentration of 25 x 10(6)/ml with a nonfat dry milk-glucose extender containing amikacin sulfate (1 mg/ml) and potassium penicillin G (1000 units/ml). Extended semen was divided into three 40-ml aliquots for placement in each of the three semen-transport containers. The extended semen was stored in the containers for 24 h prior to analysis. Stored semen was warmed for 15 min at 37 degrees C, then video records of sperm motility were obtained for evaluation using a Hamilton-Thorne motility analyzer equipped with a stage warmer set at 37 degrees C. The temperature of 40-ml aliquots of semen extender stored in each container was also measured for 60 h using a copper-constantan thermocouple placed in the center of the stored samples. Intervals from onset of storage until sample temperature exceeded 10 degrees C during the warming phase were 27.5, 33.5 and 53 h, for the Expecta-Foal, Equine Express and Equitainer, respectively. Semen extender stored in the Equitainer compared most favorably to ideal cooling rates and storage temperatures published previously. Following a 24-h storage period, the mean percentages of motile, progressively motile, and rapidly motile spermatozoa, as well as the mean spermatozoal curvilinear velocity were similar (P > 0.05) among the three containers.     

Influence of bacteria and gentamicin on cooled-stored stallion spermatozoa

This study investigated effects of bacteria from the genital tract of horses and the effect of gentamicin in semen extender on spermatozoal function in cooled-stored stallion semen. Semen was collected from healthy stallions and processed with a milk-based extender with or without gentamicin (1g/l). Pseudomonas (Ps.) aeruginosa, Staphylococcus (St.) aureus, Streptococcus (Sc.) equi subsp. equi (Sc. equi), Sc. equi subsp. zooepidemicus (Sc. zooepidemicus), Sc. dysgalactiae subsp. equisimilis (Sc. equisimilis) or culture medium alone (control) were added. Immediately after addition of bacteria and after storage at 5 degrees C for 24, 48 and 72h, motility, velocity and membrane integrity of diluted semen were determined with a CASA system. After 24h, semen with Ps. aeruginosa and Sc. equisimilis showed significantly lower motility and velocity compared to all other groups; after 72h these differences still existed for Ps. aeruginosa (p<0.05). The percentage of membrane-intact spermatozoa was significantly lower after 24h of storage in spermatozoa incubated with Sc. equisimilis and after 72h with Sc. equisimilis and Ps. aeruginosa. Addition of gentamicin to extender resulted in decreased motility and velocity in semen without addition of bacteria and did not improve motility parameters in semen with bacteria added. In conclusion, certain bacteria may have detrimental effects on semen quality during cooled-storage. These effects are not reduced by addition of gentamicin. Gentamicin can negatively affect spermatozoal function in extended semen during cooled-storage and therefore, optimal concentrations have to be tested for the respective extender medium.     

Fertility of stallion semen processed, frozen and thawed by a new procedure

The fertility of frozen-thawed and fresh semen from three stallions was compared in a trial using a randomized block design and 90 mares for 108 cycles. Semen was collected every third day, diluted to 50 x 10(6) sperm/ml with a citrate-based centrifugation medium, and centrifuged. The cells were resuspended at 700 x 10(6) progressively motile sperm/1.0 ml of added lactose-EDTA-egg yolk extender containing 4% glycerol, packaged by placing 0.55 ml into polypropylene straws, and frozen. Semen was thawed by immersion in 75 degrees C water for 10 sec. All of the 43 ejaculates collected were frozen, but 21 were discarded because progressive sperm motility was <35% immediately after thawing or <40% after 30 min of incubation at 37 degrees C. semen from the same stallions was collected daily for inseminations with fresh semen. Semen containing 200 x 10(6) progressively motile sperm was added to 10 ml of heated skimmilk extender. Mares were inseminated daily starting on the third day of estrus or when a >/=4-cm follicle was detected, whichever came later, and continuing through the end of estrus or for nine days. Based on palpation per rectum on day 50 postovulation, the pregnancy rates from inseminations during one estrus were 50, 56 and 61% with frozen semen and 67, 67 and 61% with fresh semen (P>0.05) from the three stallions, respectively. Thus, mean pregnancy rate with frozen semen was 86% of the rate attained with fresh semen.     

Effect of different extenders and storage temperatures on sperm viability of liquid ram semen

Semen was collected with an artificial vagina from four adult rams. The ejaculates were pooled and diluted, using a split-sample technique, in four different extenders: one for milk (Mi), one for sodium citrate (Na), and two for Tris-based extenders (T1 and T2) including egg yolk. Thereafter, the diluted semen was stored at 5 and 20 degrees C, respectively. We evaluated sperm viability after 0, 6, 12, 24 and 30 h of storage. We assessed sperm motility subjectively, and we determined sperm membrane integrity using both the hypo-osmotic resistance test (ORT) and a fluorophore staining (SYBR-14 and propidium iodide) technique. We evaluated acrosomal status with Spermac and capacitation status with Chlortetracycline (CTC assay). All sperm viability parameters were influenced by storage time and extender, while sperm motility was the only evaluated parameter that was influenced by the interaction between extender and temperature. Semen that was diluted and stored in the commercially available Tris-based extender (T2) maintained sperm motility for a longer period of time, and acrosome and membrane integrity was higher during storage for up to 30 h as compared to the other extenders independent of storage temperature. In general, however, storage of ram semen at 5 degrees C seemed to influence sperm viability parameters less than storage at 20 degrees C. In conclusion, the results of the present study indicate that Tris-based extenders, especially T2, preserved sperm viability better than both the sodium citrate- and the milk-based extender did when liquid ram semen was stored up to 30 h at 5 and 20 degrees C. Whether the differences found between the extenders will be reflected in the fertility results after AI is yet unknown and needs to be further studied.     

Comparison of an extender containing defined milk protein fractions with a skim milk-based extender for storage of equine semen at 5 degrees C

A problem of semen extenders based on milk or egg yolk is the fact that these biological products consist of a variety of substances. Extenders containing only components with clearly protective effects on spermatozoa would thus be an advantage. In this study, we have compared the effects of an extender containing defined caseinates and whey proteins only (EquiPro, defined milk protein extender) with skim milk extender on equine spermatozoa during cooled storage. The defined milk protein extender was used with and without the antioxidant N-acetyl cysteine (NAC). In a second experiment, semen was diluted with PBS or defined milk protein extender and was either stored directly or 90% of seminal plasma was removed by centrifugation and replaced by defined milk protein extender before storage. In both experiments, eight stallions were available for semen collections. Motility, velocity and membrane integrity of spermatozoa were determined by CASA immediately after semen processing and after 24, 48 and 72 h of storage at 5 degrees C. Total motility after 24 h of storage was lowest in semen diluted with PBS (p<0.05 versus all extenders). At 48 and 72 h, motility of spermatozoa in defined milk protein extender was significantly (p<0.05) higher than in PBS or skim milk extender. Velocity of spermatozoa after storage was highest in defined milk protein extender. Membrane integrity after storage was significantly (p<0.05) lower in semen diluted with PBS than in semen diluted with both extenders. Addition of NAC was without effect on the examined parameters. Centrifugation further increased the percentage of motile and membrane-intact spermatozoa in the defined milk protein extender (p<0.05). Velocity of spermatozoa in this extender was not negatively affected by centrifugation.     

Effect of centrifugation and partial removal of seminal plasma on equine spermatozoal motility after cooling and storage

The objective of this study was to determine if centrifugation and partial removal of seminal plasma would improve spermatozoal motility in semen from stallions whose whole ejaculates have poor tolerance to cooling and storage. Stallions were divided into two groups (n = 5/group) based on the ability of their extended semen to maintain spermatozoal motility after cooling and storage. Group 1 stallions ("good coolers") produced semen in which progressive spermatozoal motility after 24 h of cooling and storage was reduced by < or = 30% of progressive motility prior to storage. Group 2 stallions ("poor coolers") produced semen in which progressive spermatozoal motility after 24 h of cooling and storage was reduced by > or = 40% of progressive motility prior to storage. The sperm-rich portion of each ejaculate was divided into 4 aliquots. Two aliquots underwent standard processing for cooled transported semen and were examined after 24 and 48 h of cooling and storage in an Equitainer. The remaining two aliquots were diluted 1:1 with semen extender, then centrifuged at 400 x g for 12 min at room temperature. After centrifugation, approximately 90% of the seminal plasma was removed, and the sperm pellet was resuspended in extender to a final concentration of 25 to 50 x 10(6) sperm/mL. These aliquots were then packaged as for the non-centrifuged aliquots and examined after 24 and 48 h of storage. The spermatozoal motion characteristics in fresh semen and after 24 and 48 h of cooling and storage was determined via computer-assisted semen analysis. Centrifugation and partial removal of seminal plasma increased the percentage of progressively motile spermatozoa and limited the reduction in progressive spermatozoal motility of "poor cooling" stallions after 48 h of cooling and storage. Results of this study indicate that centrifugation and partial removal of seminal plasma is beneficial for stallions whose ejaculates have poor tolerance to cooling and storage with routine semen dilution and packaging techniques, especially if the semen is stored for > 24 h.     

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.     

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.     

Effect of a dietary antioxidant supplementation on semen quality in pony stallions

Lipid peroxidation contributes to the damage of the sperm plasma membrane. In different species, dietary supplementation with antioxidants has been shown to improve semen quality. Therefore, we tested effects of dietary supplementation with antioxidants and l-carnitin on semen quality in Shetland pony stallions (n=6). Semen was collected twice a week over a time period of 16 weeks. From weeks 5 to 12, a special diet for stallions containing a variety of antioxidants (STALLION, Pavo Pferdenahrung GmbH, Goch, Germany; tocopherol 300 mg/day; ascorbic acid 300 mg/day; l-carnitin 4000 mg/day; folic acid 12 mg/day) was added to the basal diet (hay, mineral supplements, water). Ejaculates were evaluated for total sperm count, semen motility (percentage of totally and progressively motile spermatozoa, longevity for 24 h at 5 degrees C) and membrane integrity (SYBR-14/PI staining): All values given are means+/-S.E.M. No changes in motility, progressive motility and membrane integrity or semen longevity for 24 h were detected. A slight but significant reduction of morphologically abnormal spermatozoa was found (weeks 1-4: 43.7+/-7.1%; weeks 13-16: 39.4+/-7.2%, p<0.05). Results show that a supplementary diet with antioxidants in the given concentration and duration does not result in pronounced effects on semen quality of stallions. It is therefore questionable to support stallions with dietary antioxidants as long as they receive an adequately balanced basal diet.     

Procedures for handling fresh stallion semen

Handling procedures for semen to be used at the stud-farm and for transport are reviewed. Proper handling of semen is required throughout the entire process, from semen collection to the insemination of the mare. Semen shall not be exposed to mechanical damage, light, cold or heat. All equipment that comes in contact with semen must be warm, clean, dry and free from toxic residues. Skim-milk extender appears to be the medium best suited for the preservation of stallion semen during cooling and storage. When used immediately, semen is usually extended 1:1 (v:v), but for transport, concentrations of 25 to 100 x 10(6) spermatozoa/mL are recommended. The proportion of semen plasma should be reduced to < 20%. by centrifuging, by collecting only the first 3 sperm-rich fractions, or by substantially diluting of the ejaculate. The storage temperature can be between 20 to 15 degrees C, if shipment time is no more than 12 h; for longer storage, temperatures < 10 degrees C are recommended. Semen can be cooled rapidly from 35 to 19 degrees C. In the temperature zone between 19 and 8 degrees C, stallion spermatozoa are sensitive to cold shock, and the cooling rate should be slowed to 0.05 degrees C/min. Rapid cooling can be resumed below 8 degrees C. At low temperatures, removal of oxygen-rich air is beneficial for the survival of spermatozoa. The Equitainer transport container keeps a constant temperature of 5 degrees C for 48 h and is therefore recommended for transportation lasting over 24 h.     

Recent advances in cooled-semen technology

The majority of horse registries approve the use of artificial insemination, and horse breeding has widely taken benefit from the use of cooled-stored semen. New insights into cooled-semen technology open possibilities to reduce problems such as impaired semen quality after cooled-storage in individual stallions. The stallion itself has major impacts on quality and fertility of cooled-stored semen. Dietary supplementation of antioxidants and polyunsaturated fatty acids improves semen quality in a variety of species, but only few studies on this topic exist in the horse. Proper semen collection and handling is the main key to the maintenance of semen quality during cooled-storage. Semen collection should be achieved by minimal sexual stimulation with a single mount; this results in high sperm concentration, low content of seminal plasma and minimal contamination with bacteria. Milk-based semen extenders are most popular for semen processing and storage. The development of more defined extenders containing only the beneficial milk ingredients has made extender quality more constant and reliable. Semen is often centrifuged to decrease the seminal plasma content. Centrifugation results in a recovery rate of only 75% of spermatozoa in the semen pellet. Recovery rates after centrifugation may be improved with use of a "cushion technique" allowing higher centrifugation force and duration. However, this is not routinely used in cooled-semen technology. After slow-cooling, semen-storage and shipping is best performed at 5 degrees C, maintaining semen motility, membrane integrity and DNA integrity for up to 40 h after collection. Shipping containers created from Styrofoam boxes provide maintenance of semen quality at low cost.     

Effect of solid storage at 15 degrees C on the subsequent motility and fertility of rabbit semen

We conducted two studies to improve preservation of rabbit semen. The objective of the first study was determine whether a glucose- and fructose-based extender with two different amounts of gelatin would solidify at 15 degrees C, and to evaluate the influence of gelatin supplementation on sperm motility parameters after storing semen up to 10 days at 15 degrees C. The fertility of rabbit semen diluted in the best gelatin-supplemented extender established in Study 1 and stored for up to 5 days was evaluated in the second study. In Study 1, semen was collected with an artificial vagina from 40 bucks. Each ejaculate was diluted to (80-100) x 10(6) spermatozoa/mL (1:3, semen/extender) at 37 degrees C in one of the three following glucose- and fructose-based extenders: control (standard liquid extender), semi-gel or gel (0.7 or 1.4 g gelatin in 100 mL extender, respectively). Pools of semen were allocated among 0.6 mL plastic artificial insemination (AI) guns. Thirty (10 per extender group) AI doses were immediately analyzed (0 h) and the remainder stored in a refrigerator (15 degrees C) for 12, 24, 36, 48, 72, 96, or 240 h. All doses with gelatin extenders solidified at 15 degrees C. Semen samples, prewarmed to 37 degrees C, were evaluated with a computer-assisted sperm analysis (CASA) system. The percentage of motile cells was significantly lower using the liquid compared to the gel extenders during semen storage from 0 to 96 h. Although significance was lost, these differences persisted after 240 h of storage. Motility of spermatozoa in the semi-gel extender was intermediate between that of liquid and gel extender throughout the study. Study 2 was performed on 1250 multiparous lactating does. Five homogeneous groups of 250 does previously synchronized were inseminated using semen previously stored for 120, 96, 72, 48 or 24 h, respectively. Rabbit does receiving 24 h-stored semen (diluted with the control extender used in Study 1) served as controls. The remaining females received seminal doses supplemented with 1.4 g/100mL gelatin (gel extender used in Study 1). Kindling rates for rabbit does inseminated with gelatin-supplemented (solid) semen doses stored for 48 h (88%) or 72 h (83%) were similar to those recorded for liquid controls stored for 24 h (81%), whereas rates significantly decreased when the semen was solid and stored for 96 h (64%) or 120 h (60%) before AI. In conclusion, rabbit spermatozoa were effectively stored in the solid state at 15 degrees C, with fertility preserved for up to 5 days. Solid storage of rabbit semen would facilitate commercial distribution.     

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.