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.     

Differential ubiquitination of stallion sperm proteins: possible implications for infertility and reproductive seasonality

Antibodies against ubiquitin, a universal proteolytic marker, show increased cross-reactivity with defective spermatozoa in men and bulls. We investigated sperm ubiquitination in the stallion, a seasonally polyestrous mammal. Immunofluorescence and immunoelectron microscopy demonstrated that anti-ubiquitin antibodies bind to the surface of both membrane-intact and aldehyde-fixed spermatozoa. Cross-reactivity to the ubiquitin-conjugating enzyme E2 was also detected in sperm. Immunohistochemistry showed that ubiquitinated spermatozoa were first detected in the caput epididymis, coincident with a strong accumulation of ubiquitin and ubiquitin C-terminal hydrolase, protein gene product 9.5, in the apical stereocilia of the epididymal epithelium. Testicular spermatozoa did not display significant ubiquitin cross-reactivity. Similarly, lesser accumulation of ubiquitin cross-reactive substrates was identified in the accessory sex glands. Semen samples were collected from three fertile stallions and one subfertile stallion between December and February and probed for ubiquitin by flow cytometry and immunoblotting. Flow cytometric analysis showed that sperm from the subfertile stallion had higher ubiquitin levels than sperm from the other three stallions. In addition, immunoblot analysis of sperm proteins from the subfertile stallion showed two unique ubiquitin cross-reactive bands that were not present in sperm extracts from the three fertile stallions. To screen for a possible role for ubiquitin in seasonal changes in sperm production, semen samples from two fertile stallions were collected in March, June, September, and December and subjected to a flow cytometric ubiquitin assay. The lowest levels of ubiquitin-labeled sperm were found in March, approximately coincident with the onset of the natural horse breeding season. A progressive increase in sperm ubiquitin levels was found during summer and fall, with a peak in December. These data suggest that stallion sperm are differentially ubiquitinated during epididymal maturation and that this ubiquitination may reflect changes in sperm numbers and semen quality. The association between changes in sperm ubiquitination and seasonal changes in sperm production will be subjected to further studies in a larger cohort of animals.     

Effect of storage time and temperature on stallion sperm DNA and fertility

We used the sperm chromatin structure assay (SCSA) to study the change in stallion sperm DNA susceptibility to denaturation after exposure of extended semen to three different storage temperatures (5, 20, or 37 degrees C) at 7, 20, 31, and 46 h. In addition, we compared the rates of sperm DNA denaturation in fertile and subfertile stallions. Among fertile stallions, spermatozoa stored at 20 and 37 degrees C showed a significant (P < 0.05) rise in the SCSA measures (Mean(alpha1), S.D.(alpha(t)), and percent cells outside the main population-COMP(alpha(t))) overtime, with the degree of rise being more dramatic at 37 degrees C. Over all stallions, samples stored at 5 degrees C showed no significant (P > 0.05) changes in the SCSA values measured over time, indicating maintenance of chromatin quality for up to 46 h. The COMP(alpha(t)) from stallions classified as subfertile showed an increased susceptibility to denaturation or decline in chromatin quality between 20 and 31 h when stored at 5 degrees C; however, spermatozoa from fertile stallions did not change during the time intervals analyzed. These data suggest that sperm DNA from some subfertile stallions may decline at a greater rate than spermatozoa from fertile stallions when exposed to similar storage conditions.     

Biomarkers of in vivo fertility in sperm and seminal plasma of fertile stallions

The global proteome of sperm and seminal plasma of fertile stallions was investigated to determine whether associations with relative in vivo fertility exist. Seven stallions at stud in a commercial breeding station were collected throughout the breeding season and bred to a total of 164 mares to determine conception rates. On three occasions during the breeding season, raw semen was obtained from a regular collection for proteomic analysis using two-dimensional electrophoresis and also assessed for routine semen quality end points. First cycle conception rate was negatively related to ejaculate volume (r = −0.43, P = 0.05) and total IGF1 content (ng) per ejaculate (r = −0.58, P = 0.006), whereas overall pregnancy rate was positively related to sperm concentration (r = 0.56, P = 0.01). The abundance of three proteins known to be involved in carbohydrate metabolism in sperm was positively related to fertility. Furthermore, the abundance of four seminal plasma proteins were identified as being negatively related to fertility; these were identified as kallikrein-1E2 (KLK2), clusterin, and seminal plasma proteins 1 (SP1) and 2 (SP2). Abundance of cysteine-rich secretory protein 3 (CRISP3) was positively related to first cycle conception rate (r = 0.495, P = 0.027) and may provide a good marker of fertility. Based on stepwise regression analysis, clusterin and SP1 in seminal plasma together with sperm citrate synthase were predictive of fertility (r = 0.77, P < 0.0001). This study identified proteins within sperm and seminal plasma that could serve as biomarkers of semen quality and fertility in stallions.     

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

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

Morphological and functional changes of stallion spermatozoa after cryopreservation during breeding and non-breeding season

The study compared quality and freezability of stallion semen during breeding and non-breeding seasons. Ejaculates were collected twice per week from four stallions during May (n = 24) and December (n = 24). The semen was mixed with skim milk extender, centrifuged and resuspended in fresh extender. Aliquots of this sperm suspension were separated from extender and diluted in TALP medium for sperm evaluation or with cryoextender (type "Gent" or a combination of Triladyl and skim milk). Samples of 0.5ml were cryopreserved in straws using a programmed freezer. Parameters of sperm quality were evaluated before and after freezing/thawing. These included percentages of motile spermatozoa and of morphological intact sperm. Typical injuries were demonstrated by scanning electron microscopy (S.E.M.). The acrosomal status was visualised using FITC-conjugated peanut agglutinin, and the acrosome reaction was induced by calcium ionophore A 23187. The chromatin stability was estimated by acridine orange test.In winter, the average percentages of motile and morphologically normal sperm (67 and 74.3%, respectively) were higher than during the breeding season in May (59 and 65.9%; P < 0.05). After freezing/thawing the proportions of vital and intact sperm decreased significantly. The number of motile sperm declined to 15 and 18% in May and December (range 5-40%), and of morphologically intact sperm to 51% in both seasons. Results of S.E.M. showed typical membrane ruptures in the acrosomal region and some sperm with abnormal necks. The proportion of frozen sperm with spontaneous acrosome reaction was higher during winter (86.5 versus 77.0%), suggesting a higher degree of membrane reactivity. Percentages of spermatozoa with denaturated chromatin were minimal and showed minimal differences between fresh and frozen state, stallions or seasons. An additional decondensation treatment with papain and DTE revealed a slightly enhanced number of spermatozoa with denaturable DNA after cryopreservation, especially in December (5.4 +/- 1.3%). The influence of cryoextenders was not significant for most sperm parameters, but there was a high variability between the stallions. Altogether, the influence of factors on the quality of spermatozoa has the following rank order: cryopreservation > stallion > season. Different cellular structures seem to have different susceptibilities to physicochemical stress. The cryopreservation of sperm during December results in survival rates similar to those measured during the breeding season, even more important for successful preservation is the selection of suitable semen donors.     

Advanced fertility diagnosis in stallion semen using transmission electron microscopy

Routine semen analysis of stallions is based on light microscopy (LM). However, there are still a number of animals that are subfertile or even infertile not being identified with conventional semen analysis. The objective of this study was to investigate the suitability of transmission electron microscopy (TEM) for advanced fertility diagnosis in stallion. We examined ejaculates of 46 stallions with known fertility. Animals were divided into three different groups: group 1, fertile stallions (pregnant mares> or =70%, n=29); group 2, subfertile stallions (pregnant mares 10-69%, n=14); group 3, infertile stallions (pregnant mares<10%, n=3). Ejaculates were collected in spring 2002. Conventional semen analysis (volume, sperm concentration, motility, live:dead ratio and percentage of morphologically normal sperm) was immediately performed after semen collection. Ultrastructural analysis included the evaluation of 200 acrosomes, heads, midpieces and cross-sections of tails as well as 100 longitudinal sections of tails from every ejaculate. Using LM, we found a significant increase of morphological deviations from 24.5% (x ) in group 1 to 34.5% in group 2 and 73.5% in group 3. Using TEM, we found a significant increase of detached acrosomes from 6.1% in group 1 to 7.6% in group 2 and 21.4% in group 3. Deviations in tubule pattern were also increased (but not significant) from 2.7% in fertile and 2.8% in subfertile to 11.4% in infertile stallions as well as multiple tails from 1.9% in fertile to 2.0% in subfertile and 8.9% in infertile. Our data indicate that TEM is suitable for advanced fertility diagnostic in stallions, giving a connection between fertility and morphology. It suggests that the most likely reason for sub- and infertility in stallion in case of increased LM pathomorphology of semen are acrosomal alterations, especially detached acrosomes.     

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.     

Sperm transport and survival in the mare

Following the deposition of semen in the mares uterus, spermatozoa must be transported to the site of fertilization, be maintained in the female tract until ovulation occurs, and be prepared to fertilize the released ovum. Sperm motility, myometrial contractions, and a spontaneous post-mating uterine inflammation are important factors for the transport and survival of spermatozoa in the mares reproductive tract. Fertilizable sperm are present in the oviduct within 4 hours after insemination. At this time, the uterus is the site of a hostile inflammatory environment. Our data suggest that spermatozoa trigger an influx of polymorphonuclear neutrophils (PMNs) into the uterine lumen via activation of complement. Furthermore, seminal plasma appears to have a modulatory effect on the post-mating inflammation through its suppressive effect on PMN chemotaxis and migration. Spermatozoa that safely have reached the oviduct can be stored in a functional state for several days, but prolonged sperm storage in the female tract is not required for capacitation and fertilization in the horse. The caudal isthmus has been proposed as a sperm reservoir in the mare. The pattern of sperm transport and survival of spermatozoa in the mares reproductive tract are different between fertile and subfertile stallions, between fertile and some infertile mares, and between fresh and frozen-thawed semen. Possible explanations for these differences include a selective phagocytosis of damaged or dead spermatozoa, impaired myometrial activity in subfertile mares, bio-physiological changes of spermatozoa during cryopreservation, and the removal of seminal plasma during cryopreservation of equine semen.     

Effect of heterologous and homologous seminal plasma on stallion sperm quality

Removing most of the seminal plasma (SP) from stallion semen has been shown to improve survival during cooled storage, yet adding small quantities of SP may improve pregnancy rates or cryosurvival. Furthermore, there is considerable controversy about whether the stallion's own SP or heterologous SP produces the best effect, possibly because of the variation between stallions in SP proteins or because some homologous SP remained in the sperm preparation. The SP is removed completely from stallion spermatozoa prepared by colloid centrifugation. Thus, the aim of the present study was (1) to investigate the effect of adding back SP to colloid centrifuged spermatozoa to determine its effect on spermatozoa; and (2) to investigate whether the stallion's own SP had a greater or lesser effect than heterologous SP. Conventional semen doses were sent from a stud overnight to the laboratory using standard transport conditions. Once at the laboratory, the semen samples were used for single layer centrifugation with Androcoll-E, and the resulting sperm preparations were treated with heterologous SP. Adding SP had a small but significant effect on sperm motility but no effect on the proportion of spermatozoa that had acrosome reacted. There were significant increases in hydrogen peroxide production and chromatin damage (P < 0.001). When homologous and heterologous SP were compared, considerable variation was observed between stallions, so that it was not possible to predict whether homologous or heterologous SP, or no SP, will produce the best motility for spermatozoa from any given stallion. Therefore, it is necessary to test different combinations of spermatozoa and SP to find the optimal effect on motility. The SP from most stallions increased reactive oxygen species and chromatin damage. In conclusion, the interaction between SP and spermatozoa depends on the origin of both SP and spermatozoa. If it is desirable to add SP to stallion sperm samples, it should be done directly before insemination rather than before storage, because of increased hydrogen peroxide production and sperm chromatin damage.     

Seasonal differences in ram seminal plasma revealed by partition in an aqueous two-phase system

Seminal plasma plays an important role in maturation of spermatozoa through hormonal, enzymatic and surface-modifying events. We have previously shown that adsorption of seminal plasma proteins (SPPs) to the sperm cell surface partially restores the functional characteristics of damaged spermatozoa, reproducing those of live cells. In the present report, we investigate the hypothesis that seasonal differences in seminal plasma could affect its ability to recover membrane integrity of cold-shocked sperm. The effect of seminal plasma proteins, obtained in breeding (bsSPPs) and non-breeding (nbsSPPs) season, on cold-shocked ram spermatozoa previously freed from seminal plasma, was analysed by centrifugal counter-current distribution (CCCD) in an aqueous two-phase system as well as membrane integrity determination by fluorescence markers. Cold-shock treatment greatly lowered cell viability in both breeding and non-breeding season spermatozoa. The cold-shocked sperm viability obtained was approximately 20%. The loss of heterogeneity and the decrease in viability revealed by CCCD analysis was reversed by the addition of increasing amounts of bsSPP, which induced restoration of the surface characteristics of viable-like spermatozoa, as well as an increase in the number of recovered viable sperm. However, this restoring effect was much lower when nbsSPPs were added, even in a sixfold higher concentration than used with bsSPPs. Incubation of cold-shocked cells with both kinds of proteins performed in both seasonal periods, showed that the recovering effect was related to the season when the plasma sample was obtained rather than to the semen season. The addition of bsSPPs to cold-shocked sperm accounted for a nearly 50% reversion for both studied breeding seasons. However, the reversion percentages obtained with nbsSPPs were significantly lower (P<0.05) than those found with bsSPPs in both studied seasonal periods. This different reversion capacity of bsSPPs and nbsSPPs was related to a different protein composition, as revealed by comparative sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis. The bands of 20, 21, 24, 36 and 67 kDa of the bsSP sample profile decreased in winter–spring SP, and were even less intensely stained in summer SP. Densitometric analysis of the stained gel patterns allows automatic comparison among the separated bands, and revealed an important decrease in the content of several bands. The 21.5 kDa band showed the highest decrease, lowering to 14% in June–August plasma with respect to the value obtained in September–December plasma.     

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.     

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.     

Influence of prostaglandin F2 alpha on sperm production and seminal characteristics of the stallion

Six mature stallions were used to test the effect of prostaglandin F2 alpha (PGF2 alpha ) on sperm production and seminal characteristics. Semen was collected from each stallion twice weekly 1 hr following a 10 mg intramuscular injection of PGF2 alpha or a sham injection. A switchback design was used so that three stallions received PGF2 alpha and three served as controls during the first 9 weeks (period 1). Treatment regimens were reversed during the second 9 weeks (period 2). Treatment of stallions with PGF2 alpha resulted in an increase (P less than .05) in gel free seminal volume and a decrease in sperm cell concentration. Total spermatozoa, sperm cell motility, and percentage of primary and secondary sperm abnormalities of ejaculates were not significantly affected by treatment of stallions with PGF2 alpha before semen collection. All treated stallions exhibited a pronounced sweating response to the drug. During the experiment, two of the six stallions masturbated within 20 to 30 minutes after PGF2 alpha treatment without achieving an erection.     

Components of stallion seminal plasma and the effects of seminal plasma on sperm longevity

Seminal plasma is a mixture of secretions produced in the testes, epididymides and accessory sex glands, and ejaculated as several consecutive fluid fractions. The composition of seminal plasma and the effects on sperm longevity vary between fractions and individual stallions. This review focuses on the sequence of ejaculation, constituents of seminal plasma and their potential use as fertility markers as well as the influence of seminal plasma on spermatozoa during storage.     

The effect of vesiculectomy on seminal characteristics in the stallion.

Successful unilateral extirpation of an inflamed seminal vesicle in a stallion led to systematic trials of the influence of a reduction and absence of the secretion of this gland upon semen characteristics. Operations were performed by the method described for the bull. The volume of ejaculates dropped and sperm concentration per ml increased in each of 2 stallions from which the seminal vesicles had been uni- or bi-laterally removed. Total sperm number and motility remained uninfluenced, but the percentage of eosin-stained spermatozoa increased in the unilaterally operated stallion and the percentage of abnormal spermatozoa increased significantly in both. Concentration of citric acid per ml and per ejaculate was significantly reduced after bilateral vesiculectomy. Ergothioneine concentration per ml increased in the unilaterally and in the bilaterally operated stallion.     

High Levels of Cadmium and Lead in Seminal Fluid and Blood of Smoking Men are Associated with High Oxidative Stress and Damage in Infertile Subjects

We measured the levels of malondialdehyde (MDA), protein carbonyls, glutathione S-transferase (GST) and reducte glutathione (GSH) in seminal plasma and spermatozoa from 95 subjects including 50 infertile patients to evaluate the association between oxidative stress and damage and the components of the anti-oxidant defenses in seminal plasma and spermatozoa of infertile subjects and concentrations of cadmium (Cd) and lead (Pb) in the blood and seminal plasma because of tobacco smoke exposure. The reactive oxygen species (ROS) in spermatozoa were also evaluated by luminol (5-amino-2,3-dihydro-1,4-phthalazinedione)-enhanced chemiluminescence assay. The sperm count, motility, and morphology in the smokers infertile group were found to be lower than those in the fertile male group and nonsmokers infertile group (p < 0.001). Concentrations of Cd, Pb, MDA, protein carbonyls, and ROS levels in the smokers infertile group were significantly higher than those in the fertile male and nonsmokers infertile male groups (p < 0.001). However, GSH levels and GST activities were decreased in the smokers infertile male group than those in the fertile male and nonsmokers infertile male groups (p < 0.001). The results indicate that smoking could affect semen quality and oxidative lipid and protein damage in human spermatozoa. From Pearson correlation analysis, positive correlations were demonstrated between the seminal plasma Cd and seminal plasma protein carbonyls and between seminal plasma Pb and spermatozoa ROS levels in smokers of the subfertile group, while there was a significant positive correlation between blood Cd and ROS levels in smokers of the fertile group. There was also a significant negative correlation of the Cd level of the blood and GSH levels of the sperm and seminal plasma. These findings suggest that cigarette smoking enhances the levels of Cd and Pb in seminal plasma and blood and the extent of oxidative damage associated with a decrease in components of the anti-oxidant defenses in the sperm of infertile males.     

Administration of oxytocin immediately after insemination does not improve pregnancy rates in mares bred by fertile or subfertile stallions

It is probable that reduced pregnancy rates in mares bred to subfertile stallions is attributable, in part, to the reduced number of normal spermatozoa that colonize the oviduct. Administration of oxytocin stimulates both uterine and oviductal contractility. The hypothesis that oxytocin may enhance sperm transport to/into the oviducts, and thereby increase pregnancy rates, was tested in 2 trials. For both trials, fertile estrous mares with follicles > or = 35 mm in diameter were inseminated once at 24 h after administration of 1500 to 2000 U hCG. The inseminate dose was limited to 100 million spermatozoa in order to lower pregnancy rates and thus increase the chance of detecting a treatment effect. Pregnancy status was determined by transrectal ultrasound examination 14 to 16 d after insemination. In Trial 1, 49 mares were inseminated with 4 mL extended semen from 1 of 3 stallions (1 fertile and 2 subfertile males). Immediately after insemination, the mares were administered either 20 U oxytocin or 1 mL saline intravenously. In Trial 2, 51 mares were inseminated with 4 mL extended semen from 1 of 4 stallions (1 fertile and 1 subfertile male used in Trial 1, and 2 additional fertile males). Immediately after insemination, and again 30 min later, mares were administered either 5 U oxytocin or 0.25 mL saline intramuscularly. To test for effects of treatment with oxytocin and for the interaction between semen quality and treatment, a generalized linear mixed regression model was used that accounted for the split-plot design (treatment within stallions), the random effect of stallion, the fixed effect of semen quality, the binary outcome of a single breeding trial, and the varying number of trials per stallion/treatment groups. Three treatment protocols or regimens were used: placebo, 5 U oxytocin injected twice intramuscularly, and 20 units oxytocin injected twice intravenously. Semen was classified as high (fertile stallions) or low (subfertile stallions) quality. No interaction between semen quality and treatment was detected (P > 0.10). The pregnancy rate of mares treated with oxytocin immediately after insemination was 30% (15/50) compared with 50% (25/50) for mares treated with saline immediately after breeding. Administration of oxytocin did not affect pregnancy rates (P > 0.10).     

Association of classical semen parameters, sperm DNA fragmentation index, lipid peroxidation and antioxidant enzymatic activity of semen in ram-lambs

The objective was to determine relationships among classical semen characteristics, sperm chromatin structure assay (SCSA), lipid peroxidation and antioxidant enzymatic activity in ram-lamb semen. Fifty-seven ram-lambs were electroejaculated, and routine semen evaluation was conducted (as part of a breeding soundness evaluation). The percentage of sperm DNA fragmentation index (%DFI) and the percentage of sperm with abnormally high DNA stainability (HDS; immature spermatozoa) were determined by SCSA using the metachromatic properties of acridine orange. Semen was centrifuged at 800 x g for 15 min to separate spermatozoa and seminal plasma and the aliquots were stored at -70 degrees C until analyzed. Lipid peroxidation, superoxide dismutase (SOD), and glutathione peroxidase (GPx) levels in seminal plasma and spermatozoa were measured by spectrophotometric assays. The classical semen parameters were negatively related to lipid peroxidation and GPx activity in spermatozoa; motility and morphology were negatively related to %DFI (P < 0.05). Based on Kruskal-Wallis pair-wise comparison of median values among breeding soundness outcome groups, %DFI was lower in the satisfactory group compared to other groups (P < 0.05) and the lipid peroxidation and GPx activity in seminal plasma and spermatozoa were lower in satisfactory and questionable groups (P < 0.05). However, the SOD was lower in the unsatisfactory group (P < 0.05). In summary, classical semen parameters were negatively related to % DFI, lipid peroxidation and GPx activity in ram-lamb spermatozoa and seminal plasma. There were indications that SOD and GPx have crucial protective roles against the toxic effect of reactive oxygen species (ROS) in ram-lamb semen.     

The effects of age, season and fertility status on plasma and intratesticular insulin-like growth factor I concentration in stallions

The purposes of this study were to establish the basal plasma and testicular insulin-like growth factor-I (IGF-I) values for stallions ranging in age from 6 months to 23 years and to determine if IGF-I could be used as a marker for declining fertility. Blood and testes were obtained from 28 light horse stallions and colts. Of the 28 stallions, 22 were considered fertile and were categorized by age (<2 y, 5 to 10 y, 11 to 15 y, and 16 to 23 y); 12 age-matched stallions were grouped as to fertility status (fertile, subfertile, infertile); and all 28 stallions were grouped as to season of castration (breeding season vs. non-breeding season). In colts less than 2 years of age, IGF-I concentrations in plasma and testicular extracts were higher (P < 0.01) than in the other age groups and were higher in the breeding season than in the non-breeding season (P < 0.01). No significant differences in plasma or testicular extract concentrations of IGF-I were found among fertility groups. The results of this study demonstrate that plasma and testicular IGF-I levels are high in stallions younger than 2 years of age and then decline and plateau in stallions older than 5 years of age, suggesting that IGF-I may be involved in testicular development. The results allude to a possible seasonal effect on IGF-I production. However, it is difficult to separate true seasonality and the effect of age as only those stallions less than 2 years old exhibited variation between seasons. The IGF-I does not appear to have a direct relationship with declined fertility in the stallions tested, suggesting that IGF-I may not be a reliable biomarker for the diagnosis of subfertility and infertility.