Esterified glucomannan improves aflatoxin-induced damage of sperm parameters during liquid storage of ram semen at 5°C.

The aim of the present work was to study the effects of aflatoxin (AF) on sperm parameters in rams, and to determine the protective efficiency of esterified glucomannan (EG) co-administered with AF up to 96 h of the liquid storage of ram semen at 5°C. Thirty-two Merino rams (12-14 months old) were used. The animals were examined for their general health status. To ensure their adaptation to the environment and the new feeding regimen, a 15-day acclimatization programme was applied to the animals, prior to the start of the study. Experimental feeding was continued for ninety-two days. The experimental design consisted of four dietary treatments. The control group (C) was fed with commercial feed. The AF group was fed with commercial feed plus 250 µg/day of total AF. The EG group received commercial feed plus 2g/day of EG. The AF + EG group was given commercial feed plus 250 µg/day of total AF and 2g/day of EG. In the study, ejaculates were obtained from rams twice a week for 12 weeks, using an electro-ejaculator. After collected, the ejaculates were diluted with a skimmed milk extender, and stored at 5°C. Sperm motility and rates of abnormal and nonviable spermatozoa were determined for the different treatment groups at 5°C at 0, 24, 48, 72 and 96 h of liquid storage. During the first two weeks of the trial, the groups did not statistically differ from each other for sperm motility or rates of abnormal and nonviable spermatozoa at 0, 24, 48 and 96 h of storage. As from the third week, the short-term storage of semen produced statistically significant differences between the AF group and the other treatment groups for sperm parameters (p<0.05). The administration of aflatoxin was observed to have reduced sperm motility and to have increased the rates of abnormal and nonviable spermatozoa in comparison to the control group (p<0.05), while EG co-administered with AF was determined to have ameliorated the adverse effects of AF on sperm parameters, and this ameliorative effect continued throughout the short-term storage of semen. On the other hand, aflatoxin administration resulted in the deterioration of the sperm parameters in the following weeks, and the combined administration of EG + AF reversed this adverse effect, thus, bringing the sperm parameters closer to the values of the control group. This study demonstrated that, in rams, AF adversely affected sperm, biochemical and testis parameters, and that the combined administration of EG and AF reversibly improved these adverse effects.

Protective effect of esterified glucomannan on aflatoxin-induced changes in testicular function, sperm quality, and seminal plasma biochemistry in rams.

The aim of this study was to determine the effect of aflatoxin (AF) on spermatologic, biochemical, and testis parameters in rams, and the protective efficiency of esterified glucomannan (EG) co-administered with AF. Thirty-two Merino rams (12-14 months old) were used. The experimental design consisted of four dietary treatments. The control group was fed commercial feed. The AF group was fed with commercial feed plus 250 µg/d of total AF. The EG group received commercial feed plus 2 g/d of EG. The AF + EG group was given commercial feed plus 250 µg/d of total AF and 2 g/d of EG. There were treatment, time, and treatment-by-time interaction effects on sperm motility, abnormal spermatozoa, damaged acrosome, and dead spermatozoa (P < 0.01). The percentage of motile sperm was lower and the percentages of abnormal sperm, sperm with damaged acrosomes, and dead sperm were greater in the AF group than in the control, AF+EG, and EG groups, as from week 3 until the end of week 12 (P < 0.05). As from week 3, hyaluronidase activity in the seminal plasma increased significantly in the AF group, compared with the control. The co-administration of AF+EG was found to be effective in preventing the increase in hyaluronidase activity. As week 4, malondialdehyde (MDA) levels were significantly higher in the AF group compared with the control. The combined administration of AF+EG was found to be effective in lowering the MDA levels, increased by AF, to the levels measured in the control (P < 0.05). Although glutathione (GSH) levels were determined to have significantly decreased in the AF group in comparison to the control, it was observed that, in the group co-administered with AF and EG, particularly after week 7, the GSH levels, which had decreased owing to AF, were largely ameliorated (P < 0.05). In conclusion, AF adversely affected spermatologic, biochemical, and testis parameters, and the combined administration of EG with AF reversibly eliminated these adverse effects in rams.

Raffinose and hypotaurine improve the post-thawed Merino ram sperm parameters.

The aim of this study was to determine the effects of raffinose and hypotaurine on sperm parameters after the freeze-thawing of Merino ram sperm. Totally 40 ejaculates of five Merino ram were used in the study. Semen samples, which were diluted with a Tris-based extender containing 10mM raffinose, 5mM hypotaurine, 5mM raffinose +2.5mM hypotaurine (H+R) and no antioxidant (control), were cooled to 5 °C and frozen in 0.25 ml French straws and stored in liquid nitrogen. Frozen straws were then thawed individually at 37 °C for 25s in a water bath for evaluation. The addition of raffinose led to higher percentages of subjective and CASA motilities (47.5 ± 12.2%, 46.3 ± 13.6%) compared to controls (38.8 ± 13.8%, 30.5 ± 11.7%, P<0.05). For the CASA progressive motility, 5mM raffinose (20.12 ± 8.82%) had increasing effect in comparison to control (10 ± 7.94%, P<0.05) following the freeze-thawing process. Raffinose and hypotaurine led to higher viability (40.8 ± 4.68%, 40.8 ± 4.7%), high sperm mitochondrial activity (29.5 ± 5.4%, 27.3 ± 4.9%) and acrosome integrity (50.8 ± 8.1, 50.7 ± 4.4) percentages, compared to control groups (31.5 ± 3.5%, 9.5 ± 8.2%, 42.8 ± 7.3%, P<0.05). H+R group only led to high sperm mitochondrial activity when compared to control group. In the comet test, raffinose and hypotaurine resulted in lower sperm with damaged DNA (6.2% and 3.9%) than that of control (9.1%), reducing the DNA damage. For TUNEL assay, The TUNEL-positive cell was distinguished by distinct nuclear staining. Raffinose and H+R groups resulted in lower sperm with TUNEL-positive cell (1.5 ± 1.2% and 2.1 ± 0.9%) than that of control (4.9 ± 2.5%) (P<0.05). In conclusion, findings of this study showed that raffinose and hypotaurine supplementation in semen extenders provided a better protection of sperm parameters against cryopreservation injury, in comparison to the control groups.

Methionine supplementation improves ram sperm parameters during liquid storage at 5°C.

The aim of this study was to investigate the effects of methionine and lipoic acid on ram sperm parameters during liquid storage (5 °C). Ejaculates collected from five Merino rams were pooled at 37 °C. Each pooled ejaculate was divided into five equal aliquots and diluted (37 °C) with five extenders, one of which was without additives, two of which contained methionine at two different doses, and the other two of which contained lipoic acid at two different doses. Sperm parameters were determined at 0, 24, 48, 72 and 96 h of liquid storage at 5 °C. The extenders containing 2 and 4mM of methionine resulted in higher motility percentages, in comparison to the control, up to 96 h of storage. Methionine at doses of 2 and 4mM led to higher viability and sperm mitochondrial activity percentages, when compared to the controls during 48, 72 and 96 h of liquid storage (P<0.05). The findings of this study showed that methionine was of greater benefit to ram sperm parameters during liquid storage.

Effects of dithioerythritol on ram semen after the freeze-thawing process.

The aim of this study was to evaluate the effects of dithioerythritol added to cryopreservation extender on the post-thawed sperm parameters, lipid peroxidation and antioxidant activities of Merino ram sperm. Semen samples from 5 mature Merino rams (1 and 2 years of age) were used in the study. Semen samples, which were diluted with a Tris-based extender containing 0.5, 1, and 2mM dithiothreitol and no antioxidant (control), were cooled to 5°C and frozen in 0.25 ml French straws. Frozen straws were then thawed individually at 37°C for 20s in a water bath for evaluation. The addition of dithioerythritol at 0.5 and 2mM doses led to higher percentages of subjective motility (62.9±4.2% and 63.6±1.8%) compared to control (52.0±4.9%, P<0.05). As regards CASA motility, dithioerythritol 0.25 and 2 mM (60.2±4.5% and 59.6±1.2%) groups were higher from that of control (44.2±8.7%, P<0.05). For the CASA progressive motility, 0.25, 0.5 and 2 mM doses of dithioerythritol (22.0±2.1%, 21.7±2.5% and 24.0±1.2%) had increasing effect in comparison to control (15.0±2.5%). Dithioerythritol at 1 and 2 mM doses for ALH provided higher values compared to the control (P<0.001) following the freeze-thawing process. Supplementation with dithiothreitol did not significantly affect the integrities of sperm membrane and acrosome, and mitochondrial activities. No significant differences were observed in biochemical parameters among the groups (P>0.05). Findings of this study showed that dithioerythritol supplementation in semen extenders, was of greater benefit to sperm motility of frozen-thawed ram sperm.

Effects of cysteine and ergothioneine on post-thawed Merino ram sperm and biochemical parameters.

The aim of the current study was to evaluate the effects of cysteine and ergothioneine on the post-thawed sperm parameters, lipid peroxidation and antioxidant activities. Semen samples from 5 mature Merino rams were used in the study. Semen samples, which were diluted with a Tris-based extender containing l-Cysteine and l-(+)-Ergothioneine and no antioxidant (control), were cooled to 5°C and frozen in 0.25 ml French straws. Frozen straws were then thawed individually at 37°C for 20s in a water bath for evaluation. Ergothioneine at doses of 2 and 4mM increased percentages of subjective motility, VSL and VCL, compared to controls following the freeze-thawing (P<0.001). Ergothioneine at three different doses led to higher rates of progressive motility and VAP, compared to control groups (P<0.001). Cysteine and ergothioneine at three doses provided the higher rates of ALH, in comparison to no antioxidant group (P<0.001). As regards CASA motility, supplementation with antioxidants did not provide any significant difference on the percentage of post-thaw sperm CASA motilities, in comparison to the control. In regards of sperm membrane integrity, only cysteine 1mM provided a greater protective effect, compared to control (P<0.001). Percentages of sperm with high mitochondrial activity were dramatically increased with cysteine at doses of 1 and 2mM, compared to control (P<0.05). No significant differences were observed in sperm acrosome integrities among groups. CAT activity was increased significantly only in cysteine1mM compared to control group (P<0.001). Cysteine at doses of 2 and 4mM showed a tendency of increased activities of CAT when compared to control. But these increases were not statistically significant. Supplementation with antioxidants did not significantly affect activities of SOD and GPx. Findings of this study showed that ergothioneine supplementation in semen extenders, was of greater benefit to motility and motion characteristics of frozen-thawed ram sperm.

Effects of antioxidants on post-thawed bovine sperm and oxidative stress parameters: antioxidants protect DNA integrity against cryodamage.

This study was conducted to determine the effects of methionine, inositol and carnitine on sperm (motility, abnormality, DNA integrity and in vivo fertility) and oxidative stress parameters (lipid peroxidation, total glutathione and antioxidant potential levels) of bovine semen after the freeze-thawing process. Nine ejaculates, collected with the aid of an artificial vagina twice a week from each Simmental bovine, were included in the study. Each ejaculate, splitted into seven equal groups and diluted in Tris-based extender containing methionine (2.5 and 7.5 mM), carnitine (2.5 and 7.5 mM), inositol (2.5 and 7.5 mM) and no additive (control), was cooled to 5 °C and then frozen in 0.25 ml straws. Frozen straws were then thawed individually at 37 °C for 20s in a water bath for the evaluation. The extender supplemented with 7.5 mM doses of carnitine and inositol led to higher subjective motility percentages (61.9±1.3% and 51.3±1.6%) compared to the other groups. The addition of methionine and carnitine at doses of 2.5 and 7.5 mM and inositol at doses of 7.5mM provided a greater protective effect in the percentages of total abnormality in comparison to the control and inositol 2.5 mM (P < 0.001). As regards CASA motility, 7.5 mM carnitine (41.6±2.9% and 54.2±4.9%) and inositol (34.9±2.0% and 47.3±2.2%) caused insignificant increases in CASA and total motility in comparison to the other groups. All of the antioxidants at 2.5 and 7.5 mM resulted in lower sperm with damaged DNA than that of control, thus reducing the DNA damage (P < 0.05). No significant differences were observed in CASA progressive motility and sperm motion characteristics among the groups. In fertility results based on 59-day non-returns, no significant differences were observed in non-return rates among groups. As regards biochemical parameters, supplementation with antioxidants did not significantly affect LPO and total GSH levels in comparison to the control group (P > 0.05). The maintenance of AOP level in methionine 2.5 mM was demonstrated to be higher (5.06±0.38 mM) than that of control (0.96±0.29 mM) following the freeze-thawing (P < 0.001). Supplementation with these antioxidants prior to the cryopreservation process protected the DNA integrity against the cryodamage. Furthermore, future research should focus on the molecular mechanisms of the antioxidative effects of the antioxidants methionine, carnitine and inositol during cryopreservation.

Influence of methionine and dithioerythritol on sperm motility, lipid peroxidation and antioxidant capacities during liquid storage of ram semen.

The aim of this study was to investigate the effects of methionine and dithioerythritol, added to the Tris extender, on ram sperm motility and LPO (lipid peroxidation) and antioxidant capacities during liquid storage up to 72 h at 5°C. Ejaculates collected from five Merino rams, were evaluated and pooled at 37°C. This study included two experiments. In experiment 1, each pooled ejaculate was divided into four equal aliquots and diluted (37°C) with the base extender, containing 0 (control), 1, 2 and 4 mM methionine, at a final concentration of approximately 4×10(8)sperms/ml (single step dilution), in a 15-ml plastic centrifuge tube. In experiment 2, dithioerythritol, at concentrations of 0 (control), 0.5, 1 and 2 mM, was used as an additive in the extender, and the procedure explained above was applied for the division of aliquots and the dilution of semen. Diluted semen samples were kept in glass tubes and cooled from 37 to 5°C in a cold cabinet, and maintained at 5°C. Sperm motility and LPO and total glutathione (GSH) and glutathione peroxidase (GPx) capacities were determined at 5°C for periods of 0, 24, 48 and 72 h of liquid storage. The extender supplemented with 1 mM methionine led to higher motility percentages (77.0±1.2%), in comparison to the control group (66.0±4.9%), during 72 h of liquid storage (P<0.05). As regards dithioerythritol, it did not statistically improve the motility rates for any of the storage times at 5°C. In biochemical assays, differences in LPO levels between the groups with antioxidants and the control groups were not statistically significant. Compared to the control group, no significant difference was observed in GSH and GPx activities following the addition of methionine, during 72 h of storage. Total GSH and GPx activities did not increase significantly upon supplementation with 0.5 and 1 mM of dithioerythritol, compared to the control group, at any of the time points (P>0.05). Dithioerythritol at 2 mM led (P<0.01) to elevating GSH activity, compared to the control group, during 72 h of liquid storage. GPx activity was approximately 10 times higher for 2 mM of dithioerythritol (P<0.001), compared to that of the control group at all time points. The question regarding the sustainability of sperm survival, LPO and antioxidant capacities following liquid storage of semen remains unanswered. Further studies are required for a better understanding of the biochemical changes and to obtain more information on the determination of lipid peroxidation and antioxidant capacities during cooled storage of ram semen.

Effects of hypotaurine, cysteamine and aminoacids solution on post-thaw microscopic and oxidative stress parameters of Angora goat semen.

This study was conducted to determine the effects of cysteamine, hypotaurine and aminoacids solution (BME) on standard semen parameters, lipid peroxidation and antioxidant activities of Angora goat semen after the freeze-thawing process. Ejaculates collected from four Angora goats were evaluated and pooled at 37 degrees C. Semen samples, which were diluted with a Tris-based extender containing the antioxidants hypotaurine (5 mM) and cysteamine (5 mM), and an aminoacid solution (13%), and an extender containing no antioxidants (control), were cooled to 5 degrees C and frozen in 0.25-ml French straws in liquid nitrogen. Frozen straws were thawed individually at 37 degrees C for 20s in a water bath for evaluation. Supplementation with cysteamine, hypotaurine and BME caused significant (P<0.05) increases in sperm motility, and significant (P<0.05) decreases in total abnormality rates in comparison to the control group. While all in vitro treatments did not affect the acrosomal abnormality rates, hypotaurine and BME but not cysteamine significantly (P<0.05) increased the HOST results as compared to the control group. Supplementation with antioxidants and BME did not significantly affect MDA levels and CAT activity in comparison to the control group (P>0.05). The antioxidants hypotaurine and cysteamine decreased SOD activity when compared to the BME group and controls (P<0.001).