Environmental factors contributed to circannual rhythm of semen quality

We investigated whether human semen parameters present circannual rhythm or not, and whether environmental factors exert on semen quality. This retrospective study used data of patients mainly from Reproductive Medicine Center and Urology and Andrology Clinic of a general hospital in China. Sperm concentration and motility were measured by computer aided sperm analysis (CASA). Sperm morphology was scored based on the strict criteria (WHO, 2010). The Kruskal–Wallis rank test was used to investigate the relationship between semen parameters and season/month. Partial correlation coefficients were used to analyze the relationship between semen parameters and environmental factors. In this study, we found that sperm concentration and total amount per ejaculate were significantly lower in summer and higher in winter. But, sperm progressive motility and motility were significantly higher in spring and summer (from March to June), lower in autumn and winter (September and October). Unexpectedly, normal sperm morphology and mixed agglutination reaction (MAR) positive rate didn’t vary along with season or month. Furthermore, temperature was negatively related to sperm concentration and total amount per ejaculate. Precipitation was positively associated with progressive motility and normal sperm morphology, but negatively related to sperm head defect percentage. The length of sunlight was positively related to progressive motility. The Air Quality Index (AQI) was positively associated with semen volume and sperm total amount per ejaculate. These suggest seasonal and monthly variation underlying some semen parameters.     

Seasonal changes in semen quality and correlation with plasma hormone profiles in Karan Fries bulls

The aim of the study was to assess the influence of summer and winter seasons on semen quality and plasma hormone concentrations in cross-bred bulls. Semen was collected by an artificial vagina from eight bulls and microscopically evaluated for quality parameters. Semen volume was higher in summer season (p < 0.05) than winter season, whereas nonsignificant variation (p > 0.05) was observed in mass motility, individual motility, sperm viability, sperm concentration and percentage of membrane-intact and acrosome-intact spermatozoa. Plasma prolactin and testosterone concentration were significantly (p < 0.01) higher in summer season than winter season. Plasma testosterone levels were positively correlated with semen volume and negatively correlated with individual motility (p < 0.05). Prolactin showed a significant positive correlation with semen volume. A well-defined seasonal pattern in semen characteristics was not observed and few correlations existed between plasma hormone levels and semen characteristics in Karan Fries bulls.     

Collection method, season and individual variation on seminal characteristics in the llama (Lama glama)

The objective of the present study was to evaluate the effect of semen collection method (electroejaculation "EE" as compared with the artificial vagina "AV"), the season (summer versus winter) and the male used on macroscopic and microscopic characteristics of ejaculates in llamas. A total of 110 ejaculates were collected from six males and 92 of them were analyzed. Ejaculate volume, concentration, total sperm and the following sperm characteristics were studied: motility, membrane function (HOS test), membrane integrity (CFDA/PI fluorochromes) and morphology. A mixed linear model, that considered season and collection method as the fixed variables and the male as the random variable, was used for the statistical analysis. Variability was found between males (p相似文献   

Thiols prevent H2O2-mediated loss of sperm motility in cryopreserved bull semen

We previously showed that cryopreservation of bull spermatozoa in egg yolk Tris extender (EYTG) significantly reduced the intracellular level of thiols. Other studies showed the beneficial effects of adding antioxidants to cryopreserved bull spermatozoa. These studies led us to investigate the effects of various thiols, an important class of antioxidants, on sperm motility of cryopreserved bull semen in a commonly used extender, EYTG. Sperm motility was analyzed by computer-assisted semen analysis (CASA). After thawing, a diluted pool of bull semen was incubated at 38.5 degrees C in airtight tubes with the following thiols for 6 hours: glutathione (GSH/GSSG), cysteine, N-acetyl-L-cysteine (NAC) and 2-mercaptoethanol in the presence or absence of oxidative stress. The oxidative stress was caused by adding H2O2 (100 microM) to diluted semen. Incubation of diluted bull semen in EYTG at 38.5 degrees C over a period of 6 h decreased sperm motility by approximately 9 fold from the start (72 +/- 3, mean +/- SEM, n=4) to the end (9 +/- 4, n=4) of the incubation. We found that all thiols to a concentration above 0.5 mM maintained high sperm motility for 6 h in the absence of an external source of oxidative stress (52 +/- 4, for 4 thiols). However, one mM of each thiol was required to efficiently protect sperm motility in the presence of 100 microM of H2O2 for 6 h. We also found that the GSH concentration in diluted semen was too low (microM) to adequately supply exogenous addition of 72 U/mL of glutathione peroxidase (GPx), an enzyme that detoxifies H2O2 and hydroperoxides using GSH as a cofactor. In fact, a better protection of sperm motility could be achieved with only 5 U/mL of GPx and 0.1 mM of GSH added to diluted semen. Our results also demonstrated that added GSSG (0.5 mM) in diluted semen was not regenerated efficiently to GSH over 6 h. The latter result indicated in the extender that the glutathione redox-cycle was deficient. Therefore, deleterious effects sperm motility after cryopreservation in EYTG can be counteracted by adding various thiols at mM concentration.     

Effect of season on fertility of frozen buffalo semen

Twenty double ejaculates from each of ten water-buffalo bulls were collected in June (non-breeding season) and again in November (breeding season). Fresh semen was screened for sperm quantity, motility, eosin uptake, and sperm morphology and was frozen using lactose, skim-milk, and Tris extenders. Thawed semen was checked for motility and Sephadex filtration. Half of each semen batch was used for artificial insemination in the breeding season and the other half during the non-breeding season.Laboratory screening revealed that June semen had a significantly lower Sephadex filtration rate and a higher percentage of abnormal sperm cells, and three June ejaculates were excluded from further processing due to poor sperm motility. In the remaining ejaculates the motility before freezing and the sperm cell quantity were higher in June semen than in November semen. Eosin uptake, mass motility, and post-freeze-motility did not vary with season. November semen produced significantly higher pregnancy rates than June semen over a total of 3220 inseminations in both seasons. Forty percent of the observed seasonality of buffalo fertility was attributable to the male. No fertility differences appeared between extenders used. When November semen was used, the fertility in adult buffaloes in both seasons was higher than in heifers.     

The contribution of bulls and cows to the seasonal differences in the fertility of dairy cattle in Israel

Semen collected from dairy Israeli-Friesian bulls in the winter and summer was pelleted-frozen and used for inseminations in the collection season, in the other season, and after a 12-month storage period.The semen quality, as assessed by the percentage of ejaculates with initial poor motility and low sperm density and the percentage of spermatozoa surviving the freezing-thawing process, and those revealing acrosomal and tail abnormalities after thawing, was slightly and non-significantly lower in summer than in winter.The fertility of semen collected in both seasons was not affected by a 12-month storage period. The use of winter semen in summer did not improve the low fertility of cows in that season compared with those inseminated with summer semen.It appears from the results of this study that the summer decrease in the fertility of the inseminations is due to the adverse effect of this season on the fertility of the cows.     

Dynamics of exogenous kallikrein-stimulated bovine sperm motility in the presence of seminal plasma kallikrein

Sperm motility is known to be activated and maintained by kallikrein contained within the seminal plasma. We studied the relationship between the levels of seminal plasma kallikrein activity and in vitro exogenous kallikrein-induced sperm motility enhancement. Semen samples were collected from Holstein-Friesian bulls and grouped on the basis of the initial total sperm motility into Group I with > 60 % (mean 75.3 +/- 1.8 %, n = 25), and Group II with < 60 % (mean 51.2 +/- 1.7%, n = 25). Seminal plasma kallikrein activity was measured with the aid of the specific chromogenic substrate S-2266. In Group I the mean activity was 0.983 +/- 0.042 microkat/L, and in Group II it was 0.805 +/- 0.063 microkat/L (P < 0.05). Then each semen sample was divided into a control and an experimental subgroup treated with 16.7 microkat/L of hog pancreatic kallikrein. Total sperm motility was monitored at 1-h intervals. It was found that the addition of exogenous kallikrein stimulated the sperm motility in both groups but in the 4th h after treatment the difference in sperm motility between the experimental and control subgroups of Group I was 12.4 % whereas in Group II it was 21.7 %. We concluded that adding exogenous kallikrein in vitro to semen samples with lower kallikrein activity in the seminal plasma enhanced total sperm motility more than adding it to ejaculates with higher levels of endogenous kallikrein activity.     

Seasonal variation in serum testosterone, testicular volume and semen characteristics in coatis (Nasua nasua)

The objective was to characterize seasonal changes in serum testosterone concentration, testicular volume and sperm quantity and quality in captive coatis (Nasua nasua) from Pantanal, MT, Brazil. Sampling was done once monthly for 1 y. Mean (± SEM) serum testosterone concentrations (767.37 ± 216.2 ng/ml) and total and progressive sperm motility (79.6 ± 3.9%; 3.8 ± 0.3, on a scale of 0 to 5) peaked in July. The highest combined testis volume (10.3 ± 0.4 cm³) and sperm concentration (403 million ± 102 sperm/ml) occurred in August, at the peak of the winter breeding season. No seasonal effects on percentages of morphologically normal sperm, acrosome integrity, or live sperm were detected; however, the percentage of secondary sperm defects was higher in the winter. In conclusion, intricate relationships between testosterone concentration, testis volume, semen concentration and total and progressive sperm motility with high levels of breeding activity were observed during the dry season in the winter (June, July, August), followed by a subsequent decline in these activities during the wet season (i.e., summer: December, January, February). There was no seasonal pattern for production of functionally intact and morphologically normal sperm.     

Quantitative characteristics of Atlantic halibut, Hippoglossus hippoglossus L., semen throughout the reproductive season

The overall objective of the study was to investigate changes in quantitative parameters of Atlantic halibut (Hippoglossus hippoglossus L.) semen throughout the reproductive season in order to systematize the knowledge about biology of Atlantic halibut spermatozoa. Semen samples were collected from February to May from broodstock males kept under either a natural or 3-month advanced photoperiod regime. Spermatozoa concentration, semen pH and osmolality, as well as spermatozoa motility parameters were investigated. The use of catheterization of sperm was examined. Also, fertilization tests were performed. We found that spermatozoa concentration increases in a linear-like mode towards the end of the spawning season, which correlated with a decrease in a number of spermatozoa motility parameters, including actual percentage of motile spermatozoa (MOT), curvilinear velocity (VCL) and straight-line velocity (VSL) of spermatozoa. A breakpoint in MOT occurred when spermatozoa reached a concentration in the range of 17-20 x 10(9) spermatozoa/mL. The fertilization ability of sperm from males kept under natural photoperiod decreased in April. Survival of embryos at 80 degrees days produced by fertilizing eggs of single female with sperm from natural photoperiod males was 88, 76 and 41% on April 09 and 17, and May 01, respectively, whereas using sperm from 3-month delayed photoperiod males for fertilizing eggs from the same female on April 27 resulted in 80% of surviving embryos, not differing significantly from the data from April 09. Physical decomposition of spermatozoa was observed towards the end of the season and it was related to an increase in the whole semen osmolality. Catheterization of semen did not improve spermatozoa motility parameters, however, it reduced the variation in recorded values, especially in the case of pH, caused by contamination with feces or urine. Post-seasonal decrease in spermatozoa concentration was likely related to intensive ageing processes. Based on the present study and available data by other researchers, a model of changes of quantitative parameters in Atlantic halibut semen throughout the reproductive season is proposed.     

Season of ejaculate collection influences the freezability of boar spermatozoa

The aim of this retrospective study was to evaluate whether the season of ejaculate collection influences the freezability of porcine sperm. A total of 434 ejaculates were collected from boars of six different breeds over three years (2008–2011) and throughout the four seasons of the year identified in the northern hemisphere (winter, spring, summer and autumn). The ejaculates were cryopreserved using a standard 0.5 mL straw freezing protocol. Sperm quality was assessed before (fresh semen samples kept 24 h at 17 °C) and after freezing and thawing (at 30 and 150 min post-thawing in semen samples kept in a water bath at 37 °C), according to the percentages of total motility, as assessed by the CASA system, and viability, as assessed by flow cytometry after staining with SYBR-14, PI and PE-PNA. The data, in percentages, on sperm motility and viability after freezing and thawing were obtained at each evaluation time (recovered) and were normalized to the values before freezing (normalized). The season of ejaculate collection influenced (P < 0.01) sperm quality before freezing and after thawing (recovered and normalized), irrespective of the breed of boar. Sperm quality was lower in summer, both in terms of motility and viability, and in autumn, in terms of motility, than in winter and spring. Seasonality in the normalized data indicates that the season of ejaculate collection influences sperm freezability, regardless of the season’s influence on sperm quality before freezing. Consequently, the spermatozoa from ejaculates collected during summer and, to a lesser extent, also in autumn, are more sensitive to cryopreservation than those from ejaculates collected during winter and spring.     

Seasonal changes of semen quality and freezability in Franches-Montagnes stallions

The objective of this study was to investigate seasonal changes of semen quality parameters in Franches-Montagnes stallions and to compare the freezability of ejaculates collected in autumn and winter. Experiments were performed using 15 stallions from the National Stud Farm in Avenches (Switzerland). Ejaculates were collected and evaluated every month during 1 year as well as cryopreserved in autumn and winter (September to February). In fresh semen the gel-free volume, concentration, motility and morphology (normal sperm, major defects, vacuoles and acrosome defects) were evaluated and in frozen-thawed semen the motility as well as the viability (SYBR-14/PI) were performed. To analyse seasonal differences four periods of 3 months each were defined as autumn (September, October, November), winter (December, January, February), spring (March, April, May) and summer (June, July, August). During the 1-year experiment all fresh semen quality parameters demonstrated a clear seasonal and individual pattern. The gel-free volume was significantly (P<0.05) higher in spring and summer compared to autumn and winter while sperm concentration was significantly (P<0.05) lower in spring than at any other time of the year. Total sperm number was significantly (P<0.05) higher and sperm motility significantly (P<0.05) lower in summer than in other seasons. Regarding sperm morphology, normal sperm was significantly (P<0.05) higher in autumn than in winter and summer and major defects were lowest (P<0.05) in autumn. In frozen-thawed semen motility was significantly (P<0.05) improved in the ejaculates collected in autumn compared to winter, while viability showed no obvious differences. Our results clearly demonstrate that individual and seasonal differences occurred in semen quality of Franches-Montagnes stallions. Ejaculates collected in autumn (September, October, November) demonstrated good quality, especially regarding sperm morphology, and were more suitable for cryopreservation because of better motility in frozen-thawed semen collected during autumn than in winter.     

Decrease in glutathione content in boar sperm after cryopreservation. Effect of the addition of reduced glutathione to the freezing and thawing extenders

Although glutathione content in boar spermatozoa has been previously reported, the effect of reduced glutathione (GSH) on semen parameters and the fertilizing ability of boar spermatozoa after cryopreservation has never been evaluated. In this study, GSH content was determined in ejaculated boar spermatozoa before and after cryopreservation. Semen samples were centrifuged and GSH content in the resulting pellet monitored spectrophotometrically. The fertilizing ability of frozen-thawed boar sperm was also tested in vitro by incubating sperm with in vitro matured oocytes obtained from gilts. GSH content in fresh semen was 3.84 +/- 0.21 nM GSH/10(8) sperm. Following semen cryopreservation, there was a 32% decrease in GSH content (P < 0.0001). There were significant differences in sperm GSH content between different boars and after various preservation protocols (P = 0.0102 ). The effect of addition of GSH to the freezing and thawing extenders was also evaluated. Addition of 5 mM GSH to the freezing extender did not have a significant effect on standard semen parameters or sperm fertilizing ability after thawing. In contrast, when GSH was added to the thawing extender, a dose-dependent tendency to increase in sperm fertilizing ability was observed, although no differences were observed in standard semen parameters. In summary, (i) there was a loss in GSH content after cryopreservation of boar semen; (ii) addition of GSH to the freezing extender did not result in any improvement in either standard semen parameters or sperm fertilizing ability; and (iii) addition of GSH to the thawing extender resulted in a significant increase in sperm fertilizing ability. Nevertheless, future studies must conclude if this is the case for all boars. Furthermore, since addition of GSH to the thawing extender did not result in an improvement in standard semen parameters, this suggests that during the thawing process, GSH prevents damage of a sperm property that is critical in the fertilization process but that is not measured in the routine semen analysis.     

Sperm mediated gene transfer in pig: Selection of donor boars and optimization of DNA uptake

Transgenic animals are produced primarily by microinjecting exogenous DNA into the male pronuclei of a zygote. Microinjection is successful in mice but not efficient in farm animals, limiting its general utility. We have pursued an alternative technology for producing transgenic animals: Sperm Mediated Gene Transfer (SMGT). Based on our finding that sperm cells bind and internalize exogenous DNA, we used sperm as a vector for transmitting, not only their own DNA, but also, the exogenously-introduced gene of interest to the zygote. SMGT is highly efficient (up to greater than 80%) and relatively inexpensive; it can be used in species refractory to microinjection, whenever reproduction is mediated by gametes. In this report, we describe the procedure for selection of sperm donors and optimization of DNA uptake that are the key steps for the successful outcome of SMGT. We found that the nominal parameters that boar sperm should possess to serve as a good vector for exogenous DNA are the quality of semen based on standard parameters used in conventional animal breeding programs (volume, concentration, presence of abnormal sperm cells, motility at time of collection, and high progressive motility after 2 hr) and the ability of the sperm cells to take up and internalize exogenous DNA. The results described provide significant advances in SMGT technology applied to pigs, so that transgenic pigs can be efficiently obtained. Mol.     

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

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

Seasonal variation in freezability of buffalo semen

Fifty-six buffalo semen ejaculates of mass activity greater than plus three on a five-point scale (28 each in summer and winter seasons) were frozen in Tris yolk glycerol (TY-G) extender with four hours equilibration time. Motility of semen was checked after first extension (initial motility), after equilibration time, 15 minutes after freezing, and 30 days after freezing. Motility increased during winter season. Significant differences (P<0.01) in motility after freezing were observed between summer and winter seasons.     

Semen collection,cryopreservation and artificial insemination in the dromedary camel

Collection of semen with a bovine artificial vagina (AV) was attempted with each of 14 camels over a period of 2 years. Semen samples were evaluated, extended and cryopreserved. Frozen thawed semen, diluted cooled semen or whole semen was used to inseminate some female camels which were induced to ovulate with hCG. Males ejaculated semen into the AV in 74.6% collection attempts. The male copulated for at least 200s in 62.9% attempts. The remaining copulations were of shorter duration. Similarly, 49.3% ejaculates were at least 3ml of semen. Libido and donation of semen improved from December onwards and reached a peak after mid January with peak performance persisting until April. It declined during May. The majority of camels had lost libido and refuse to donate semen by the end of May. Camel semen is in gel form. While 35.9% of 203 semen samples exhibited no individual sperm motility, 28.5% exhibited low to fair grade individual sperm motility and only 35.4% exhibited >50% sperm motility. Differences existed between animals (P<0.01) and months (P<0.05) of collection, while effect of copulation time was not significant. Mass motility was not observed in camel semen. Individual sperm motility develops after liquefaction of semen. Addition of caffeine but not chymotrypsin improved the individual motility. The mean live percent sperm count and normal acrosome were 73.3+/-1.0 and 92.0+/-0.5, respectively. Only 51.1% of 45 semen samples with pre-freeze motility of >50% and 25% of 16 semen samples from low pre-freeze motility group with an overall success of 44.2% of 61 semen samples were successfully preserved. Wide variation was observed in the freezability of semen from different males. Attempts to impregnate female camels with liquid semen, frozen thawed semen and whole semen after hCG induced ovulation resulted in 0/10, 1/13 and 4/10 pregnancies.     

Seasonal changes in semen quality and freezability in the Warmblood stallion

The objective of this study was to investigate seasonal changes in stallion semen quality and to determine the best time for semen cryopreservation. Experiments were performed using 10 Warmblood stallions from the National Stud Farm in Avenches (Switzerland). Ejaculates were collected and frozen every other week during 1 year from January to December 1999. Volume, concentration, and motility, and the number of morphologically normal sperm and sperm with major defects (abnormal heads, acrosome defects, nuclear vacuoles, proximal droplets, abnormal midpieces) were evaluated. For all frozen-thawed semen samples motility as well as viability (SYBR-14/PI) was tested, and the hypoosmotic swelling test (HOS) was performed. To analyze seasonal differences 4 periods of 3 months each were defined: autumn (September, October, November), winter (December, January, February), spring (March, April, May) and summer (June, July, August). During the 1 year experiment all semen quality parameters showed a clear seasonal pattern. The volume, total sperm count and motility in fresh semen were significantly higher (P<0.05) in summer than in winter, while sperm concentration was significantly lower in summer compared to the other seasons. Regarding morphology, normal sperm was significantly lower (P<0.05) in summer than at any other time of the year and higher values (P<0.05) were found for major defects in summer than in spring and autumn. In frozen-thawed semen motility was significantly (P<0.05) improved in autumn when compared to spring and summer. Viability was lowest in summer and differed significantly (P<0.05) from other seasons. The HOS test revealed significantly more (P<0.05) membrane damaged spermatozoa in winter than in spring, summer and autumn. Our results demonstrate that in our climatic conditions clear seasonal differences occur in semen quality of fresh and frozen-thawed semen and that cryopreservation of stallion semen should preferably be performed in autumn.     

Reduced glutathione content in human sperm is decreased after cryopreservation: Effect of the addition of reduced glutathione to the freezing and thawing extenders

In this study, total glutathione content was determined in human spermatozoa before and after cryopreservation. Total GSH in fresh semen was 4.47 ± 0.46 nmol/10⁸ cells. Following semen cryopreservation, GSH decreased to 1.62 ± 0.13 nmol/10⁸ cells, a 64% reduction (p < 0.01). This decrease in GSH content was associated with a decrease in sperm progressive motility (68% of reduction, p < 0.01). Addition of 1 mM GSH to the freezing extender increased the percentage of total motility and sperm viability. It also modified the motility pattern measured by CASA with changes in the straight-line and average path velocities and wobble of the curvilinear trajectory. Addition of GSH to the freezing media reduced spermatozoa ROS levels and increased the level of sulfhydryl groups on membrane proteins. Nevertheless, no effect of GSH addition on lipid membrane disorder or chromatin condensation was detected. Addition of 1 or 5 mM GSH to the thawing media increased the percentage of motile and progressively motile spermatozoa, but no effect on viability was detected. In conclusion, the antioxidant defensive capacity of the GSH is severely altered by the freeze–thawing process. The addition of GSH to the freezing and thawing extender could be of partial and limited benefit in improving the function of frozen human spermatozoa.     

Association of heat shock protein 70 with semen quality in boars

This study attempted to clarify the relationship between the levels of 70kDa heat shock protein (HSP70) and semen quality in boars. Semen samples from 29 (13 Duroc, 9 Landrace, and 7 Yorkshire) boars (mean age=25.2+/-2.2 months) were examined. Three to four ejaculates per boar, collected during cool and hot seasons, were evaluated in terms of the sperm concentration, sperm motility, percentage of normal and abnormal sperm, as well as percentage of sperm with proximal and distal plasma droplets. Significant seasonal and breed differences in semen quality were observed. Experimental results indicate that the semen quality of Landrace boars was better than those of Yorkshire and Duroc boars (P<0.05) and semen quality declined significantly during the hot season (P<0.05). One-dimensional SDS-PAGE analysis of spermatozoa proteins indicated that protein profiles did not significantly differ between seasons and among breeds. Both constitutive and stress-inducible form of HSP70 were detected in boar spermatozoa by Western blot analysis. The level of HSP70, which revealed no difference among breeds within a season, was significantly lower during the hot season in all the three breeds (P<0.05). Although there appeared to be low correlation coefficients between the level of HSP70 and semen quality traits, the semen quality tended to decline significantly in samples with a lower level of HSP70. Results in this study suggest that the levels of HSP70 in boar spermatozoa are significantly lower during the hot season and might be associated with semen quality.     

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.