Insulin-like growth factor-I and insulin-like growth factor binding protein-2 and -5 in equine seminal plasma: association with sperm characteristics and fertility

The objectives of this study were 1) to determine whether insulin-like growth factor-I (IGF-I) and insulin-like growth factor binding proteins (IGFBPs) were present in seminal plasma of stallions; 2) to compare semen parameters (IGF proteins, sperm numbers, morphology, and motility) from stallions at sexual rest (SR) and when sexually active (SA); 3) to compare semen parameters between stallions with high and low seminal plasma IGF-I concentrations; and 4) to examine the relationship between seminal plasma IGF-I concentrations and fertility parameters of stallions. Ejaculates were collected from stallions at SR (n = 51) and SA (n = 46). Concentrations of IGF-I and IGFBP-2 in seminal plasma samples were determined by radioimmunoassay. Presence of IGFBPs in equine seminal plasma was verified using immunoprecipitation and Western ligand blot procedures. IGF-I, IGFBP-2, and IGFBP-5 were present in equine seminal plasma. Concentrations of IGF-I, IGF-I/protein, total IGF-I, IGFBP-2, IGFBP-2/protein, and total IGFBP-2 were not significantly different (P > or = 0.13) in seminal plasma between stallions at either SR or SA. At SR, stallions with higher seminal plasma IGF-I had more total IGFBP-2 per ejaculate (P < 0.01), more morphologically normal sperm (P = 0.05), and higher first-cycle pregnancy rates (P = 0.02). At SA, stallions with higher seminal plasma IGF-I had fewer cycles per pregnancy (P = 0.02). An association of seminal plasma IGF-I concentration with sperm motility, sperm morphology, and pregnancy rates in bred mares suggests that IGF-I may play a role in sperm function.     

Two-dimensional polyacrylamide gel electrophoresis of equine seminal plasma proteins and their correlation with fertility

The objectives of this study were to 1) identify proteins found in stallion seminal plasma utilizing two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) in conjunction with Western blot analysis; and 2) to determine if any of these individual proteins were correlated with stallion fertility utilizing regression analysis. Fertility was quantified by assigning a breeding score for each stallion. Each score was calculated by dividing the number of conceptions by the number of breedings for each stallion for four successive breeding seasons (1992-1995). Ejaculates from stallions of known fertility (n = 6) were collected with a Missouri-style artificial vagina. Immediately after collection, the semen sample was filtered and the gel fraction removed. The resultant sperm-rich fraction was centrifuged in a Beckman Microfuge E at 10,000 x g and the seminal plasma aspirated from the pelleted sperm cells. Two-dimensional PAGE of the seminal plasma was performed under denaturing conditions which revealed that 14 proteins were common in all stallions in the research population. Four of these proteins (SP-1, SP-2, SP-3, and SP-4) were found to be significantly (P < 0.05) correlated with the breeding score assigned for each stallion. Regression analysis of protein optical densities with breeding score indicated that SP-1 (72 kDa, pI 5.6) was positively correlated with fertility (P < 0.05, r2 = 0.706), while SP-2 (75 kDa, pI 6.0), SP-3 (18 kDa, pI 4.3), and SP-4 (16 kDa, pI 6.5) were found to be negatively correlated (P < 0.05, r2 = 0.762, 0.730, 0.775 respectively) with fertility. Western blot analysis of SP-1 indicated there was an antigenic homology with a bovine 55 kDa fertility-associated seminal plasma protein identified in a study by Killian et al. (19). This suggests that the two proteins may have a similar physiological role and therefore common biological properties. These results indicate that analysis of stallion seminal plasma proteins can be used as an indicator of fertilizing capacity. Identification of such proteins in stallion seminal plasma could lead to better insight into the nature of subfertility or infertility in the horse, as well as to indicate better cryopreservation strategies.     

Effect of L-carnitine administration on the seminal characteristics of oligoasthenospermic stallions

The effect of orally administered l-carnitine on the quality of semen obtained from stallions with different semen qualities was investigated. Four stallions with proven fertility (high motility group, HM) and with normal seminal characteristics (>50% progressive motility and > 80 x 10(6) spermatozoa/ml), and four questionable breeders (low motility group, LM) with <50% of sperm progressive motility and < 80 x 10(6) spermatozoa/ml, received p.o. 20 g of l-carnitine for 60 days. Blood and semen samples were collected before treatment (T0) and after 30 (T1) and 60 days (T2). Semen evaluation were performed on five consecutive daily ejaculates (n = 120 ejaculates) and conventional semen analysis was carried out on each ejaculate, both at collection and after refrigeration for 24, 48, and 72 h. Furthermore l-carnitine, acetylcarnitine, pyruvate, and lactate concentrations, and carnitine acetyltransferase activity (CAT) were determined both in raw semen and seminal plasma. There were an increase in progressive motile spermatozoa only in the LM group (26.8 +/- 12.9, 39.1 +/- 15.5, and 48.8 +/- 8.6 for T0, T1, and T2, respectively). Free seminal plasma carnitine concentration was higher in the LM group compared to the HM one. Both pyruvate and lactate were higher in the LM group. Raw semen and seminal plasma carnitine and acetylcarnitine levels correlate positively with both sperm concentration and progressive motility; moreover, acetylcarnitine content was positively correlated with total motile morphologically normal spermatozoa. In conclusion, oral administration of l-carnitine to stallions with questionable seminal characteristics may improve spermatozoa kinetics and morphological characteristics; whereas, it seem to be ineffective in normospermic animals.     

Sperm yield after single layer centrifugation with Androcoll-E is related to the potential fertility of the original ejaculate

Many attempts have been made to identify laboratory tests that are predictive of sperm fertility, both to improve the quality of stallion semen doses for artificial insemination (AI) and to identify potential breeding sires if no fertility data are available. Sperm quality at the stud is mostly evaluated by assessing subjective motility, although this parameter can be poorly indicative of fertility. Sperm morphology and chromatin integrity in Swedish stallions are correlated to pregnancy rate after AI. Because single layer centrifugation (SLC) selects for spermatozoa with normal morphology and good chromatin, retrospective analysis was carried out to investigate whether sperm yield after SLC is linked to potential fertility. Commercial semen doses for AI from 24 stallions (five stallions with four ejaculates each, 19 stallions with three ejaculates each; n = 77) obtained during the breeding season were cooled, and sent overnight to the Swedish University of Agricultural Sciences in an insulated box for evaluation, with other doses being sent to studs for commercial AI. On arrival at Swedish University of Agricultural Sciences, the semen was used for SLC and also for evaluation of sperm motility, membrane integrity, chromatin integrity, and morphology. The seasonal pregnancy rates for each stallion were available. The yield of progressively motile spermatozoa after SLC (calculated as a proportion of the initial load) was found to be highly correlated with pregnancy rate (r = 0.75; P < 0.001). Chromatin damage was highly negatively correlated with pregnancy rate (r = −0.69; P < 0.001). Pregnancy rate was also correlated with membrane integrity (r = 0.58; P < 0.01), progressive motility (r = 0.63; P < 0.01), and normal morphology (r = 0.45; P < 0.05). In conclusion, these preliminary results show that sperm yield after SLC is related to the potential fertility of the original ejaculate, and could be an alternative indicator of stallion fertility if breeding data are not available. Single layer centrifugation is fast (30 minutes) and does not require expensive equipment, whereas other assays require a flow cytometer and/or specialist skills. An additional option could be to transport semen doses to a laboratory for SLC if the stud personnel do not want to perform the procedure themselves.     

The quantification of lipid and protein oxidation in stallion spermatozoa and seminal plasma: seasonal distinctions and correlations with DNA strand breaks, classical seminal parameters and stallion fertility

The goal of this work was to correlate oxidative stress caused by reactive oxygen species (ROS) and DNA damage with classic semen parameters in spermatozoa and seminal plasma of fertile and subfertile stallions. Oxidation was measured in both lipids and proteins, using the thiobarbituric acid reactive species (TBARS) assay and the DNPH carbonyl groups assay, respectively. Sperm DNA damage was monitored using the TUNEL assay. These parameters were monitored in samples obtained during the breeding and the non-breeding seasons. In general, fertile stallions showed better classical semen parameters, and those parameters improved from the non-breeding to the breeding season, although an increase in sperm production was accompanied by a decrease in the semen quality from subfertile stallions in the breeding season. In terms of oxidation levels we found that there were clear differences whether lipids or proteins were considered. In the breeding season there seemed to be a tendency towards normalizing lipid oxidation in spermatozoa and seminal plasma, and protein oxidation in the seminal plasma, of both fertile and subfertile animals. Thus, differences monitored in the non-breeding season were no longer visible. Interestingly, a higher level of protein oxidation was found in the sperm of fertile animals in the breeding season. Considering that there were positive correlations between sperm protein oxidation and sperm motility and vitality, these results suggests that the oxidation of semen proteins may be important for sperm function. On the other hand, lipid oxidation in the seminal plasma seemed to be a general indicator for sperm damage. In the non-breeding season positive correlations between lipid and protein oxidation levels in both sperm and seminal plasma and several defects in sperm function were found, but only for subfertile animals, thus suggesting that lipid and protein oxidation may aid in the identification of subfertile stallions during the non-breeding season. Levels of ROS production never seemed to result in compromised sperm DNA integrity, indicating that measurements were within physiological levels and/or that there is an efficient antioxidant activity in stallion sperm cells.     

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.     

Seminal traits, suitability for semen preservation and fertility in the native Portuguese horse breeds Puro Sangue Lusitano and Sorraia: Implications for stallion classification and assisted reproduction

The Puro Sangue Lusitano (PSL) is the major national breed of horse in Portugal, but no studies exist on its seminal characteristics, or on the possibility of conserving semen for future use. The aim of this study was to evaluate semen parameters, fertility and the aptness to semen preservation in Lusitano Stallions. In order to compare characteristics defined by a single or by multiple semen collections per stallion 152 ejaculates obtained from 152 Lusitano stallions presented at an annual breeding soundness examination as well as data related to 371 ejaculates obtained from 9 PSL were analyzed. These latter samples were also evaluated in terms of their possible use in assisted reproduction and were compared with 113 ejaculates obtained from 4 Sorraia horses, a rare and endangered Portuguese breed. The percentage of motile spermatozoa (PMS) was assessed after collection (AC), after semen dilution (AD) and at 24h of cool-storage. Mean values obtained for sperm motility and morphology and semen pH observed after semen collection differ significantly (P<0.05) between single collection/multiple stallions and multiple collections/limited stallions, and no age related effects were detected. Overall, Lusitano semen quality was comparable to that of related breeds, while Sorraia stallions had very poor semen quality. The response to cool-storage of diluted semen samples differed among stallions and breeds, and the best results for progressive motile sperm cells at 24h were in a range of 35-53% for PSL stallions and were lower for Sorraia stallions. Fertility rates obtained with artificial insemination (AI) averaged at 85% for PSL. With the exception of PMS AC, sperm vitality and semen pH no other seminal trait seemed to influence fertility rates in the Lusitano breed.     

Density gradient centrifugation of sperm from a subfertile stallion and effect of seminal plasma addition on fertility

Stallions are not selected for fertility but for other criteria (pedigree, conformation, performances, progeny), therefore valuable but subfertile stallions with poor semen quality are frequently used in commercial breeding programs. The object of this study was to evaluate whether sperm selection through a silane-coated silica colloid gradient centrifugation, with or without the addition of seminal plasma of a high fertile stallion, could improve the pregnancy rates of an oligospermic valuable stallion in a commercial breeding program. In 2008 breeding season (experiment 1, n=104 mares), simple centrifugation and density gradient centrifugation of the sperm were compared. In 2009 and 2010 breeding seasons (experiment 2, n=125 mares), the effect of the addition of 5% seminal plasma to the extender after sperm selection was evaluated. In all mares deep horn uterine insemination was performed with 1 ml containing 50×10(6) morphologically normal progressive motile spermatozoa, 24-30 h after induction of ovulation with hCG. Pregnancy diagnosis by ultrasonography was performed 14 days following ovulation. Results showed a higher per cycle pregnancy rate (P>0.05) when sperm selection through a density gradient was used (62% vs. 42.3%, exp 1), while the addition of 5% seminal plasma did not influence the outcome (45.9% vs. 47.6%, exp 2) (P>0.05). An age-related decrease in the fertility of the stallion was observed when comparing the results from the different breeding seasons (P<0.05). In conclusion, sperm selection through a discontinuous density gradient enabled a normal per cycle pregnancy rate to be achieved from an oligospermic-subfertile stallion in a commercial breeding program, and no differences were observed regarding the addition of seminal plasma.     

Conserved ram seminal plasma proteins bind to the sperm membrane and repair cryopreservation damage

Whole seminal plasma (SP) enhances the function and fertility of frozen/thawed ram sperm. The objective of the current study was to investigate whether SP proteins capable of binding to molecules from the sperm plasma membrane were conserved among ram breeds, and whether these proteins were sufficient to overcome cryopreservation-induced reductions in sperm quality. Whole ram SP, obtained from rams of various breeds, improved progressive motility of frozen/thawed sperm at all times evaluated (P < 0.05); however, it did not improve total motility (15 min, P = 0.480; 30 min, P = 0.764; and 45 min, P = 0.795). To identify SP proteins responsible for this effect, a new method was developed to retain SP proteins that bound specifically to the sperm membrane by immobilization of sperm membrane proteins. These proteins specifically bound to the sperm surface, especially the acrosomal region. Lactotransferrin, epididymal secretory protein E1, Synaptosomal-associated protein 29, and RSVP-20 were identified (mass spectrometry) in this fraction. The retained SP proteins fraction repaired ultrastructural damage of frozen/thawed sperm and, with the addition of fructose, significantly improved motility of frozen/thawed sperm. We concluded that SP proteins that bound to the sperm membrane were conserved among ram breeds, and that when added to frozen/thawed semen (along with an energy source), they repaired ram sperm damage and enhanced sperm motility.     

Exposure to the seminal plasma of different portions of the boar ejaculate modulates the survival of spermatozoa cryopreserved in MiniFlatPacks

Spermatozoa present in the first collectable 10 mL of the sperm-rich fraction (SRF) of the boar ejaculate (portion 1, P1) have higher documented viability during and after cryopreservation than spermatozoa in the rest of the ejaculate (portion 2, P2), probably in relation to different features of the surrounding seminal plasma (SP). In the present study, we investigated whether the SP from these ejaculate portions (SP1 or SP2) was able to differently influence sperm viability and chromatin structure of the P1- or P2-contained spermatozoa from individual boars primarily or secondarily exposed (e.g., following cleansing and re-exposure) to pooled SP1 or SP2 from the same males during 60 min. Spermatozoa were subjected to controlled cooling and thawing in MiniFlatPacks (MFPs) and examined for motility (using computer-assisted sperm analysis, CASA) at selected stages of processing. Moreover, sperm plasma membrane intactness (investigated using SYBR-14/propidium iodide, PI), plasma membrane architecture (examined using Annexin-V-PI staining), and chromatin (deoxyribonucleic acid, DNA) integrity (tested using sperm chromatin structure assay, SCSA) were assessed post-thaw (PT). A higher proportion of P1 spermatozoa than of P2 spermatozoa incubated in their native SP portion were confirmed to be motile from collection to PT. When P1 spermatozoa were cleansed from their original SP and re-exposed to pooled P2-SP, sperm kinematics deteriorated from extension to PT. By contrast, cleansed P2 spermatozoa increased motility to P1 levels, especially PT when re-exposed to pooled P1-SP. Such differential effects on motility were not clearly accompanied by biologically related modifications of sperm membrane or chromatin structure. This influence of the SP on sperm kinematics was not sire-dependent and it was presumably related to different concentrations or either SP proteins or bicarbonate in the different ejaculate portions.     

Restoration of seminal plasma to stallion spermatozoa selected by colloid centrifugation increases sperm progressive motility but is detrimental to chromatin integrity

There is controversy about whether the presence of some seminal plasma (SP) in an equine insemination dose is necessary for promoting fertility. A new technique for improving stallion sperm quality, single layer centrifugation (SLC) using a species-specific colloid, Androcoll-E, selects a sperm subpopulation that is highly motile with normal morphology, intact membranes and good chromatin integrity from the rest of the ejaculate and removes SP. The present study was designed to investigate the effect of restoring homologous SP (5% and 10%) on the progressive motility, velocity, and chromatin integrity of SLC-selected stallion spermatozoa in 44 semen samples over time. Sperm progressive motility (P < 0.01) and the proportion with class A velocity (>50 μm/sec) were increased in samples where SP was restored, whereas the proportion with class B velocity (10 to 50 μm/sec) was decreased compared with SLC samples. However, after 24 h cold storage of treated samples, progressive motility was not different for the SP-treated groups compared with SLC, whereas chromatin damage DNA fragmentation index (%DFI) was higher. In contrast, adding SP to untreated 24 h-stored SLC samples did not affect progressive motility although it did increase the proportion of spermatozoa with class A velocity. There was individual variation between stallions whether 5% or 10% SP produced a greater increase in progressive motility. In conclusion, 5% to 10% SP can be added back to SLC-selected samples if considered necessary to optimize fertility. However, it should be added immediately before insemination rather than before storage of the sperm dose, to benefit from the transient increase in sperm progressive motility and avoid increased chromatin damage.     

Effect of dialysis or centrifugation on post-thaw motility and fertility of Santa Gertrudis bull semen collected by electroejaculation

Electroejaculated semen from Santa Gertrudis bulls was used to study the effect of centrifugation (600 x g for 5 min) or dialysis [molecular weight cutoff <14,000 Daltons (Da)] on post-thaw motility and on fertility in beef cattle. Analysis of post-thaw motility showed that the main effects (bulls and semen treatment) were significant (P<0.05). Dialysis significantly improved post-thaw motility in four of seven bulls. Initial volume of seminal plasma in the ejaculate was negatively correlated to post-thaw motility (r = -0.73). No significant improvement in post-thaw motility was observed for bulls with high volumes of ejaculate. In a fertility trial, calving rates of heifers synchronized with PGF(2)alpha and inseminated at 72 and 96 h after the second PGF(2)alpha injection with dialyzed or commercial semen were not statistically different (P>0.05; 54.4% vs 55.4%). These results show that dialysis could be used to improve post-thaw motility of electroejaculated bull semen without altering its fertilizing capacity. However, a high initial volume of seminal plasma seems to have a deleterious effect on sperm freezability that cannot be reversed by dialysis.     

The importance of porcine sperm parameters on fertility in vivo

It would be desirable to use semen parameters to predict the in vivo fertilizing capacity of a particular ejaculate. In animal production, an ejaculate is divided into multiple doses for artificial insemination (AI); therefore, it would be economically beneficial to know the functional quality (i.e., fertility) of the semen before it is inseminated. To identify a predictive assay of the fertilizing capacity of a porcine ejaculate, we performed 4 rapid assays of sperm quality (motility, viability, physiological status as assessed by chlortetracycline fluorescence, and ATP content) on samples from 9 ejaculates, before and after a thermal stress test (42.5 degrees C, 45 min). These parameters were subsequently correlated with in vivo fertility resulting from AI with 2 sperm doses, 3 x 10(9) or 0.3 x 10(9) motile cells in 70 mL (optimal or suboptimal sperm number per insemination, respectively) from these same ejaculates. No parameter was correlated to the fertility rates obtained after inseminating with the optimal semen doses, either before or after the thermal stress test (P > 0.05). However, with respect to the animals inseminated with the suboptimal semen dose, sperm motility (the percentage of motile spermatozoa as assessed visually by microscopy) prior to thermal stress was well-correlated to fertility rates (r = 0.783, P = 0.01). The percentage of spermatozoa displaying the chlortetracycline Pattern AR (acrosome reaction) was also statistically related to fertility (r = 0.05, P = 0.04), but the biological importance of this relationship is questionable given the small variation among ejaculates (range: 0 to 2%). No other sperm parameter was significantly related to fertility rates in this group (P > 0.05). These data, therefore, indicate that sperm motility is a useful indicator of sperm fertilizing capacity in vivo. Moreover, to identify a predictor of semen fertility it is critical that the number of spermatozoa used during insemination is sufficiently low to detect differences in sperm fertilizing efficiency.     

Impact of seminal and serum zinc on semen quality and hormonal status: A population-based cohort study of Russian young men

BackgroundTrace elements are important factors in human reproductive health. Among them, special attention is paid to zinc, which is an essential trace element and is necessary for the normal functioning of the male reproductive system and the process of spermatogenesis. The aim of the study was to investigate the association between seminal and serum zinc concentrations and semen quality and reproductive hormone levels in population of Russian young men.MethodsThe study population consisted of 626 young Russian men (median age 22.5 years), recruited from the general population, regardless of their fertility status. Each participant provided semen and blood sample, information about his lifestyle and ethnicity. Semen quality (sperm concentration, motility and morphology), reproductive hormone levels (testosterone, estradiol, LH, FSH and inhibin B), and serum and seminal zinc concentrations were evaluated. The semen samples were analyzed according to the WHO laboratory manual (WHO, 2010). Serum hormones were measured by enzyme immunoassay, zinc concentrations were determined using spectrophotometry and direct colorimetry without deproteinization.ResultsZinc was present in the seminal plasma in a significantly higher concentration than in the blood serum (median serum Zn concentration was 23.6 μmol/L vs seminal Zn concentration 1571.8 μmol/L). The seminal zinc concentration was positively related to the total sperm count, sperm concentration, progressive motility and normal morphology (Spearman’s test: 0.221; 0.286; 0.269; 0.183, respectively, p < 0.001), while the serum Zn concentration was negatively related to serum testosterone and estradiol levels (r = −0.249 and r = −0.096, respectively, p < 0.001−0.05). It was found that the seminal Zn content in men with normal semen quality was higher compared to men with lowered semen quality (means: 6.37 and 5.03 μmol/ejaculate, respectively, p < 0.001). Similarly, the semen volume, total sperm count, sperm concentration, progressive motility, normal morphology and the serum testosterone level in men with the seminal Zn deficiency were lower than in men with the normal seminal Zn content.ConclusionBased on the results of our population-based study, seminal Zn levels were closely associated with semen parameters in young men, so Zn deficiency may be an important risk factor for lowered semen quality. Seminal Zn determinations should be considered as a useful tool in addition to other parameters in assessing male fertility.     

Determination of acrosin amidase activity in equine spermatozoa

Acrosin amidase activity of spermatozoa has been been associated with in vitro fertilization success in humans and has been proposed as an additional method for assessing sperm function in vitro. In this study, acrosin amidase activity was determined in equine spermatozoa by the hydrolysis of an arginine amide substrate. This assay includes a detergent to release acrosomal enzymes into a medium of basic pH to activate proacrosin to acrosin, which subsequently hydrolyses N-alpha-benzoyl-DL-arginine para-nitroanilide-HCl (BAPNA) to a chromogenic product. Spermatozoa (n = 3 ejaculates from each of 4 stallions) were washed free from seminal plasma by centrifugation through Ficoll and incubated with a detergent-substrate mixture (BAPNA in triton X-100; pH = 8.0) at room temperature for 3 h in the dark. At the end of the 3-h incubation, benzamidine was added to test samples to stop the reaction, and samples were centrifuged to remove spermatozoa. Absorbance at 410 nm was measured to determine acrosin amidase activity (microIU acrosin/10(6) sperm). Acrosin amidase activity increased with sperm concentration (P < 0.001; r(2) = 0.75), and there were significant effects (P < 0.001) of stallion and ejaculate within stallion on acrosin activity. Acrosin activity detectable in equine seminal plasma was 312 +/- 49 microU/ml (n = 3 ejaculates). Addition of a cryopreservation medium containing egg yolk, skim-milk, glycerol and sucrose to equine spermatozoa and subsequent cryopreservation significantly (P < 0.05) increased acrosin amidase activity compared with spermatozoa from raw semen. This result is in contrast to that previously reported for frozen-thawed human spermatozoa. Determination of acrosin amidase activity in equine spermatozoa may provide an alternative method for assessing sperm function in vitro; however, further studies are needed to determine the relationship between acrosin activity and fertility in the horse.     

Seminal plasma affects membrane integrity and motility of equine spermatozoa after cryopreservation

Effects of seminal plasma on post-thaw motility and membrane integrity of cryopreserved horse spermatozoa were investigated. Carboxyfluorescein diacetate staining was used for the assessment of sperm membrane integrity. Adding 30% of seminal plasma from stallions with high post-thaw sperm motility to ejaculates from stallions with low post-thaw sperm motility increased progressive motility from 24.0 +/- 1.6 to 34.5 +/- 1.9% (P < 0.05) and membrane integrity from 27.0 +/- 2.1 to 34.3 +/- 2.3% membrane-intact spermatozoa (P < 0.05). Conversely, the addition of seminal plasma from stallions with low post-thaw sperm motility to ejaculates from stallions with high post-thaw motility decreased progressive motility from 36.0 +/- 1.6 to 30.0 +/- 2.7% (P < 0.05) but did not induce changes in membrane integrity. Seminal plasma from stallions with opposite post-thaw motility therefore clearly influenced the resistance of spermatozoa to the freezing and thawing process. We conclude that the individual composition of seminal plasma affects the suitability of stallions for semen cryopreservation.     

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.     

Seminal carnitine and acetylcarnitine content and carnitine acetyltransferase activity in young Maremmano stallions

The reproductive characteristics and seminal carnitine and acetylcarnitine content as well as carnitine acetyltransferase activity of young Maremmano stallions (n=25) are reported. The stallions were subjected to semen collection in November and January; in each trial two ejaculates were collected 1h apart. The total motile morphologically normal spermatozoa (TMMNS) and the progressively motile spermatozoa at collection and during storage at +4 degrees C were evaluated. Seminal L-carnitine (LC), acetylcarnitine (AC), pyruvate and lactate were measured using spectrophotometric methods, whereas carnitine acetyltransferase activity was measured by radioenzymatic methods. Since there were no major significant differences in seminal and biochemical characteristics between the November and January trials, data were also pooled for the first and second ejaculates. Significant differences (P<0.001) were observed between the first and second ejaculates for sperm count (0.249+/-0.025 versus 0.133+/-0.014x10(9)/ml), total number spermatozoa by ejaculate (12.81+/-1.23 versus 6.36+/-0.77x10(9)), progressively motile spermatozoa (48.6+/-3.0 versus 52.6+/-3.0%) and TMMNS (3.35+/-0.50 versus 2.02+/-0.37x10(9)). In the raw semen the LC and AC were significantly higher in the first ejaculate than in the second (P<0.001), whereas, pyruvate and pyruvate/lactate ratio were higher in the second ejaculate (P<0.05). Seminal plasma AC and LC concentrations resulted higher in the first ejaculate (P<0.001). The pyruvate/lactate ratio was higher in the second ejaculate (P<0.05). Both raw semen and seminal plasma LC and AC concentrations were positively correlated with spermatozoa concentration (P<0.01); in raw semen AC was also correlated to TMMNS (P<0.01). Lactate levels of raw semen was correlated to progressively motile spermatozoa after storage (P<0.01). In the second ejaculate, significant correlations were also observed among AC/LC ratio in raw semen and progressively motile spermatozoa after 48 and 72h of refrigeration. Furthermore, AC levels were correlated to lactate concentration. The positive correlation between LC, AC and spermatozoa concentration, and between AC and TMMNS indicated carnitine as potential semen quality marker. Moreover, the correlation between AC/LC ratio and progressive spermatozoa motility after refrigeration, suggests that carnitine may contribute towards improving the maintenance of spermatozoa viability during in vitro storage.     

Inclusion of bovine serum albumin in semen extenders to enhance maintenance of stallion sperm viability

Semen from seven mature stallions was used to test the motility response of sperm cells when 3% bovine serum albumin (BSA) was added to seminal plasma and skim milk diluents. A total of 45 ejaculates was collected by artificial vagina and immediately evaluated for percent motile spermatozoa (PMS), rate of forward movement (RFM) and sperm cell concentration. Aliquots (four from each ejaculate) of raw semen containing 500x10(6) sperm cells were exposed to each of the following treatments: (1) seminal plasma (SP), (2) SP+BSA, (3) skim milk (SKM), (4) SKM+BSA; and incubated in 50-ml tubes at 37 C. The sperm cell characteristics, PMS and RFM, of each treatment suspension were reevaluated at 0, 0.5, 1, 2, 6, 12, 18 and 24 hr post-treatment. Inclusion of BSA and the type of extender, either seminal plasma or skim milk, significantly (P<0.05) affected the PMS and RFM of spermatozoa. Analysis of means within evaluation times showed that PMS maintenance was enhanced (P<0.05) when BSA was included in extenders at all incubation intervals except 24 hr. SKM+BSA maintained the highest (P<0.05) PMS for the first 2 hr with SP+BSA sustaining the highest (P<0.05) PMS from 12 to 24 hr. Skim milk alone sustained higher (P<0.05) PMS than the SP diluent for the first 6 hr of incubation, whereas SP maintained a higher (P<0.05) PMS than SKM from 18 to 24 hr. The RFM of spermatozoa was greatest (P<0.05) for the first 6 hr of incubation when exposed to SKM+BSA. Seminal plasma + BSA sustained a higher (P<0.05) RFM for the first 6 hr of incubation than SP alone, but not higher than SKM at this interval. Skim milk sustained a higher (P<0.05) RFM of spermatozoa for the first 6 hr of incubation than SP. These data support the hypothesis that BSA protects spermatozoa from the harmful effects of lipid peroxidation. Including this substance in semen extenders may prolong maintenance of sperm motility.     

Vasectomy effect on canine seminal plasma biochemical components and their correlation with seminal parameters

Three semen samples were collected at 48 h intervals from 20 mature research dogs previously conditioned to manual semen collection. Vasectomy was performed in all dogs, and 15 days after surgery, another three ejaculates were similarly collected. The semen was evaluated, and centrifuged to obtain seminal plasma for measurement of pH, and concentrations of total proteins (TP), total chlorides (Cl), calcium (Ca), potassium (K), and sodium (Na). The seminal plasma protein profile was evaluated by SDS-PAGE; molecular weights and the integrated optical density (IOD) of each band were estimated. There was a negative correlation between K concentration and progressive motility (r = -0.49, P = 0.027), sperm vigor (r = -0.60, P = 0.0053), and plasma integrity, evaluated by both the hypo-osmotic swelling test (r = -0.50, P = 0.026) and a fluorescent stain (r = -0.45, P = 0.046). Positive correlations between Na and K pre- and post-vasectomy (r = 0.88, P < 0.001; r = 0.56, P < 0.01, respectively) were verified. There were a total of 37 bands pre-vasectomy and 35 post-vasectomy (range, 100.6-3.6 kDa). Bands B9 and B13 (42.6 and 29.2 kDa) were not present post-vasectomy. The IOD of band B3 (73.5 kDa) was higher (P = 0.03) pre-vasectomy, compared to post-vasectomy; conversely, the IODs of bands B29 and B37 (7.8 and 3.6 kDa) increased (P = 0.026 and 0.047). Pre-vasectomy, there was a positive correlation (r = 0.49, P = 0.029) between band B37 band (3.6 kDa) and the Na:K ratio. In conclusion, K appeared to be involved in sperm motility in dogs and could be a tool to evaluate sperm function. The prostate contributed several elements to canine seminal plasma. Vasectomy changed Ca concentrations and the protein profile of the seminal plasma. Further studies must be performed to clarify the function of these elements on the in vivo fertility of dogs.