Cherry laurel fruit extract counters dimethoate-induced reproductive impairment and testicular apoptosis.

Dimethoate is an organophosphorus pesticide used against agricultural insects, which causes oxidative stress and damage in many organs, including the reproductive ones. Cherry laurel (Laurocerasus officinalis Roem.) fruit is rich in vitamins and phenolic compounds with antioxidant effect. The aim of this study was to investigate how effective its extract would be against dimethoate-induced testis and sperm damage in rats. Sixty animals were divided in six groups of 10. Group 1 (control) received only 1 mL of saline (0.9 % NaCl). Group 2 received 7 mg/kg of dimethoate in 1 mL of saline. Group 3 received 4 mg/kg of extract in 1 mL of saline. Group 4 received the extract 30 min before dimethoate administration. Group 5 received vitamin C (positive control, 100 mg/kg in 1 mL of saline) 30 min before dimethoate administration. Group 6 received only dimethoate for the first four weeks and then a combination of dimethoate and extract for another four weeks. All doses were administered daily by oral gavage. After eight weeks of treatment, the rats were euthanised and their reproductive organs removed. We took their body and reproductive organ weights and evaluated testicular oxidative stress, semen characteristics, sperm DNA damage, testicular apoptosis, and histopathological changes. Dimethoate significantly decreased body and reproductive organ weights, sperm motility and concentration, testicular superoxide dismutase, and glutathione-peroxidase activities and significantly increased lipid peroxidation, abnormal sperm rate, sperm DNA damage, testicular apoptosis, and caused histopathological lesions. Cherry laurel extract significantly countered many dimethoate-induced adverse effects, both as pre- and post-treatment, including reproductive organ weight, semen parameters, oxidant-antioxidant balance, sperm DNA integrity, testicular apoptosis, and histological structure. Our findings clearly suggest that the beneficial effects of the extract are associated with countering oxidative stress, lipid peroxidation in particular.

Oxidative stress modulates the expression of apoptosis-associated microRNAs in bovine granulosa cells in vitro.

Despite its essential role in ovulation, oxidative stress (OS) has been found to be cytotoxic to cells, while microRNAs (miRNAs) are known as a major regulator of genes involved in cellular defense against cytotoxicity. However, a functional link between OS and miRNA expression changes in granulosa cells (GCs) remains to be investigated. Here, we investigate the OS modulation of apoptosis-associated miRNAs and their biological relevance in bovine GCs. Following the evaluation of cell viability, accumulation of reactive oxygen species (ROS), cytotoxicity and mitochondrial activity, we used a ready-to-use miRNA PCR array to identify differentially regulated miRNAs. The results showed that exposure to 150 µM H2O2 for 4 h creates remarkable signs of OS in GCs characterized by more than 50% loss of cell viability, higher nuclear factor erythroid 2-related factor 2 (NRF2) nuclear translocation, significantly (p < 0.05) higher abundance of antioxidant genes, significantly (p < 0.001) higher accumulation of ROS, lower mitochondrial activity and a higher (p < 0.001) number of apoptotic nuclei compared to that of the control group. miRNA expression analysis revealed that a total of 69 miRNAs were differentially regulated in which 47 and 22 miRNAs were up- and downregulated, respectively, in stressed GCs. By applying the 2-fold and p < 0.05 criteria, we found 16 miRNAs were upregulated and 10 miRNAs were downregulated. Target prediction revealed that up- and downregulated miRNAs potentially targeted a total of 6210 and 3575 genes, respectively. Pathway analysis showed that upregulated miRNAs are targeting the genes involved mostly in cell survival, intracellular communication and homeostasis, cellular migration and growth control and disease pathways. Our results showed that OS modulates the expression of apoptosis-associated miRNAs that might have effects on cellular or molecular damages.

Gilaburu (Viburnum opulus L.) fruit extract alleviates testis and sperm damages induced by taxane-based chemotherapeutics.

In this study, it was aimed to investigate the possible protective effects of gilaburu (Viburnum opulus L., Glb) fruit extract on testis and sperm damages induced by docetaxel (DTX) and paclitaxel (PTX) chemotherapeutics in rats. Sixty adult male Wistar albino rats were divided into 6 equal groups, 10 animals each. The groups were created as control (weekly i.p. saline injection), Glb (weekly i.p. saline injection and daily oral 100mg/kg Glb), DTX (weekly i.p. injection of 5mg/kg DTX), PTX (weekly i.p. injection of 4mg/kg PTX), DTX+Glb (weekly i.p. injection of 5mg/kg DTX and daily oral 100mg/kg Glb) and PTX+Glb (weekly i.p. injection of 4mg/kg PTX and daily oral 100mg/kg Glb). Following 10-week chronic application, spermatological, biochemical, histopathological, cytopathological and immunohistochemical examinations were performed. DTX and PTX caused significant decreases in absolute and relative weights of all reproductive organs, testosterone level, sperm motility, concentration, Bcl-2 anti-apoptotic immunopositive cell scores of testes and spermatozoa as well as catalase activity in epididymal tissue, superoxide dismutase and glutathione peroxidase activities of testicular and epididymal tissues when compared with the control group. Both drugs also significantly increased abnormal sperm rate, testicular and epididymal malondialdehyde level, Bax pro-apoptotic immunopositive cell scores of testes and spermatozoa, and caused remarkable testicular histological and cytological damages. However, Glb consumption mitigated the PTX-induced decreases in absolute weights of epididimis, seminal vesicles, ventral prostate and both taxanes-induced disturbances in sperm characteristics, imbalances in oxidant/antioxidant system, increments in germ cell apoptosis and testicular histo-and cyto-pathological damages. It was concluded that long-term Glb consumption alleviates the taxanes-induced damages in reproductive system of male rats.

Concentration dependent antioxidative and apoptotic effects of sulforaphane on bovine granulosa cells in vitro.

Sulforaphane (SFN) has received a great deal of research attention because of its ability to induce the production of a battery of antioxidant enzymes in certain concentrations through the activation of the Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) signaling pathway, which may effectively neutralize reactive oxygen species (ROS) induced oxidative stress. This study was conducted to investigate the potential of different concentrations of SFN in inducing antioxidative and apoptotic effects in granulosa cells (GCs). For this purpose, bovine GCs were collected from preovulatory antral follicles and cultured with different concentrations of SFN (0-80 µM) and based on phenotypic evaluation three concentrations were selected: 2 µM (low), 10 µM (medium), and 20 µM (high) for further investigations. The results showed that there was a dramatic loss of cell viability and higher cytotoxic effects of SFN on GCs at higher concentrations (>15 µM). The expression of NRF2 increased significantly (p < 0.05) with fold change ranged 3-8 in SFN treated GCs, whereas Kelch Like ECH Associated Protein 1 (KEAP1) expression was either downregulated or similar as control group under the same conditions. Moreover, the relative expression of the genes (PRDX1, CAT, TXN1and SOD1) downstream to NRF2 activation was found to be highly expressed (fold change ranged from 2 to 5, p < 0.05) in SFN treated GCs compared to the untreated control. In addition, ROS accumulation was higher in GCs treated with 20 µM SFN which in turn results in a higher accumulation of lipid droplets. Compared to control, no changes in the mitochondrial activity was observed at 2 and 10 µM SFN concentrations; however, significantly lower mitochondrial activity was found at high concentration (20 µM). The results of this study clearly showed that 10 µM SFN concentration played a crucial role in activating Nrf2 pathway without inducing apoptotic characteristics and this concentration may have beneficial effects in boosting the production of phase II antioxidant enzymes in GCs. However, at high concentration (20 µM), SFN may generate excessive ROS that causes mitochondrial dysfunction and induces cellular stress and eventually leads to apoptosis. These data strongly suggest a concentration dependent antioxidative and apoptotic effects of SFN on GCs.

Effects of Cinnamon (C. zeylanicum) Bark Oil Against Taxanes-Induced Damages in Sperm Quality, Testicular and Epididymal Oxidant/Antioxidant Balance, Testicular Apoptosis, and Sperm DNA Integrity.

The aim of this study was to investigate whether cinnamon bark oil (CBO) has protective effect on taxanes-induced adverse changes in sperm quality, testicular and epididymal oxidant/antioxidant balance, testicular apoptosis, and sperm DNA integrity. For this purpose, 88 adult male rats were equally divided into 8 groups: control, CBO, docetaxel (DTX), paclitaxel (PTX), DTX+PTX, DTX+CBO, PTX+CBO, and DTX+PTX+CBO. CBO was given by gavage daily for 10 weeks at the dose of 100 mg/kg. DTX and PTX were administered by intraperitoneal injection at the doses of 5 and 4 mg/kg/week, respectively, for 10 weeks. DTX+PTX and DTX+PTX+CBO groups were treated with DTX during first 5 weeks and PTX during next 5 weeks. DTX, PTX, and their mixed administrations caused significant decreases in absolute and relative weights of all reproductive organs, testosterone level, sperm motility, concentration, glutathione level, and catalase activity in testicular and epididymal tissues. They also significantly increased abnormal sperm rate, testicular and epididymal malondialdehyde level, apoptotic germ cell number, and sperm DNA fragmentation and significantly damaged the histological structure of testes. CBO consumption by DTX-, PTX-, and DTX+PTX-treated rats provided significant ameliorations in decreased relative weights of reproductive organs, decreased testosterone, decreased sperm quality, imbalanced oxidant/antioxidant system, increased apoptotic germ cell number, rate of sperm with fragmented DNA, and severity of testicular histopathological lesions induced by taxanes. In conclusion, taxanes cause impairments in sperm quality, testicular and epididymal oxidant/antioxidant balance, testicular histopathological structure, and sperm DNA integrity, and long-term CBO consumption protects male reproductive system of rats.

Influence of fetuin and hyaluronan on the post-thaw quality and fertilizing ability of Holstein bull semen.

It was determined that fetuin and hyaluronan supplementation did not provide any significant effect on the post-thaw subjective and CASA motility percentages and sperm motion characteristics, in comparison to the controls (P>0.05). Sperm acrosome and total abnormalities were similar in all groups (P>0.05). Groups M (hyaluronan+fetuin) and H (hyaluronan) displayed a higher rate of sperm membrane integrity, compared to that of Group C (control) (P<0.01). According to the results of the comet assay, the lowest percentage of sperm with damaged DNA was achieved in Group H, when compared to all of the experimental groups (P<0.01). Furthermore, all of the additives resulted in a lower rate of sperm with damaged DNA than that of the controls, and thus, reduced DNA damage (P<0.01). For pregnancy rates, there were no significant differences between the extender groups (P>0.05). MDA formation was found to be lower in Groups M and F (P<0.01). In Group M, SOD activity was determined to have significantly increased (23.61±5.62 U/ml) compared to the other groups (P<0.01). All experimental groups had a GSH-Px activity higher than that of the control group (P<0.01).

Cryopreservation of bull sperm: effects of extender supplemented with different cryoprotectants and antioxidants on sperm motility, antioxidant capacity and fertility results.

The objectives of this study were to compare glycerol (G) and ethylene glycol (EG) at different concentrations and trehalose (T) or cysteine (C; with/without) in Tris extender for cryopreservation of bull semen. Twenty-four ejaculates obtained from three bulls were included in the study. Each ejaculate was divided into four equal aliquots and diluted using both of the Tris extenders with G (5% or 7%) or EG (3% or 5%). After that, each extenders were divided into three equal aliquots and diluted using both of the 5 mM C or 25 mM T, and control (without additives) was cooled to 4 ° C and frozen in 0.25 ml French straws. The addition of 3% and 5% EG without antioxidants resulted in the least Computer-Assisted Sperm motility Analysis (CASA) motility as compared with the other groups. Treatment with 25 mM T in 3% EG beneficially effected acrosome morphology as compared with the other groups. Also, treatment with 3% EG with 25 mM T and 5% EG resulted in a greater rate of total abnormalities. Treatment with 3% G yielded a slightly greater percentage of membrane integrity by Hypo-Osmotic Swelling Test (HOST) assessment than that of the other groups. Treatment with 3% EG with 5 mM C resulted in the greatest concentration of malondialdehyde (MDA). The glutathione peroxidase (GPx) antioxidant activity was increased in the C-treatment groups when compared to the other groups. Treatment with 5% EG and 5 mM C resulted in less chromatin damage and detrimental impacts on tail moment. Treatment with 5% EG led to greater non-return rates of inseminated cows. However, this result was not considered to be statistically important.

Comparing ethylene glycol with glycerol and with or without dithiothreitol and sucrose for cryopreservation of bull semen in egg-yolk containing extenders.

There are few studies performed for investigating the roles of different ratio and cryoprotectants with dithiothreitol or sucrose on sperm motility characteristics and antioxidant capacities of post-thawed bull spermatozoa. The objectives of this study were to compare glycerol (G) and ethylene glycol (EG) at different concentrations as cryoprotectants and dithiothreitol (D) or sucrose (S) (with/without) as antioxidants in Tris extender for cryopreservation of bull semen. Twenty-four ejaculates obtained from three bulls were included in the study. Each ejaculate was split into four equal aliquots and diluted using both of the Tris extenders with glycerol (5% or 7%) or ethylene glycol (3% or 5%). After that, each extenders were split into three equal aliquots and diluted using both of the dithiothreitol 5mM or sucrose 25 mM, and control (without additives) was cooled to 4 °C and frozen in 0.25-ml French straws. when compared to control, different doses cryoprotectants and antioxidants addition no significantly increased the percentages of post-thaw sperm progressive and motitilities, acrosome abnormality and plasma membrane integrity (P>0.05). However, EG3+S yielded the greatest percentages of the total abnormality (P<0.05). As regard to antioxidant activities G7 and EG5 led to lowest MDA activity with or without D or S but, these results were not supported to the GPx activity (P<0.01). The sperm motion characteristics such as VAP, VCL, ALH and BCF gave significantly different results (P<0.05). When compared the DNA integrity, different doses cryoprotectants without antioxidants addition significantly increased the percentages of the tail intensity and tail moment (P<0.05). There were no significant differences observed in non-return rates among all treatment groups (P>0.05).

In vitro effects of l-carnitine and glutamine on motility, acrosomal abnormality, and plasma membrane integrity of rabbit sperm during liquid-storage.

This study was designed to evaluate the in vitro effects of l-carnitine and glutamine (Gln) on the sperm quality parameters of liquid-stored rabbit semen maintained up to 24 h at 5°C. Pooled and extended ejaculates were divided into two equal portions. l-Carnitine doses of 0.5, 1 and 2mM were added to the first portion, and glutamine was added at the same doses to the second portion. All samples were cooled to 5°C and examined at 0, 6, 12 and 24 h of liquid storage. Supplementation of the semen extender with three different doses of l-carnitine provided significant increases in the percentage of motile sperm at 12 h (P<0.01), and 24h (P<0.001) and enabled significant protection of the sperm plasma membrane (P<0.01) at 12 and 24h of cool-storage, in comparison to the control samples. Only the 2mM dose of l-carnitine significantly (P<0.01) decreased the rate of acrosomal damage when compared to the control group. Furthermore, all doses of Gln caused a significant (P<0.01) decrease in acrosomal damage at 6h, and provided significant improvement (P<0.01) in sperm motility, acrosomal and plasma membrane integrities at 12 and 24h of liquid storage, when compared to the controls. In conclusion, the supplementation of liquid-stored rabbit semen with l-carnitine and Gln provided a protection for sperm against cool storage-induced functional and structural damages.

Influence of various antioxidants added to TCM-199 on post-thaw bovine sperm parameters, DNA integrity and fertilizing ability.

Supplementation of the semen extender with antioxidants did not produce any significant effect on CASA and progressive motilities and sperm motility characteristics, in comparison to the control group (P > 0.05). For sperm acrosome and total abnormalities, TCM-199 supplemented with cysteine (2.60 ± 0.24% and 4.80 ± 0.20%), glutamine (2.80 ± 0.20% and 6.40 ± 0.40%), carnitine (2.60 ± 0.24% and 6.00 ± 0.63%) and methionine (3.40 ± 0.51% and 9.20 ± 0.86%) at doses of 2 mM provided a better protective effect, compared to that of the controls (8.00 ± 0.44 and 15.60 ± 1.895). As regards sperm membrane integrity, supplementation with 2 mM of glutamine and methionine (56.00 ± 1.70% and 62.40 ± 1.78%, respectively) resulted in higher rates, when compared to the control group (41.40 ± 4.74%). According to the results of the COMET assay, only the use of TCM-199 supplemented with 2 mM of cysteine reduced DNA damage and resulted in percentages of sperm with damaged DNA (2.17 ± 0.18%) lower than those of the control group (3.16 ± 0.32%) (P < 0.001). For pregnancy rates, there were no significant differences among the extender groups (P > 0.05).

The effect of bovine serum albumin and fetal calf serum on sperm quality, DNA fragmentation and lipid peroxidation of the liquid stored rabbit semen.

The aim of the present study was to determine the effects of the bovine serum albumin (BSA) and fetal calf serum (FCS) on sperm quality, DNA fragmentation and lipid peroxidation of liquid stored rabbit semen stored up to 72 h at 5 °C. Ejaculates were collected from five New Zealand male rabbits by artificial vagina and pooled at 37 °C following evaluation. Each pooled ejaculate was split into three equal experimental groups and diluted to a final concentration of approximately 40 × 10(6)sperm/ml (single step dilution), in an Eppendorf tube, with the Tris based extender containing BSA (5mg/ml), FCS (10%) or no additive (control) at 37 °C, cooled to 5 °C and stored for up to 72 h. The extender supplemented with BSA and FCS did not improve the percentages of motility and acrosomal abnormality during 48 h compared to the control. The additives BSA and FCS had a significant effect in the maintaining of plasma membrane integrity between 48 and 72 h storage period, compared to the control (P<0.01). The supplementation of BSA and FCS had a protective effect on motility (P<0.05), plasma membrane integrity (P<0.01) and acrosomal integrity (P<0.01) at 72 h compared to the control. The supplementations with BSA and FCS led to a reduction in DNA damage of rabbit sperm at 48 and 72 h during storage period, compared to the control (P<0.001). Although supplementation of BSA and FCS caused significant (P<0.01) decreases in malondialdehyde (MDA) level at 48 h and 72 h, they significantly (P<0.01) increased the glutathione peroxidase (GPx) antioxidant activity up to 72 h when compared to the control group. In conclusion, BSA and FCS supplementation to liquid stored rabbit semen provide a protection for spermatozoa against cool storage-induced DNA damage and plasma membrane integrity by their antioxidative properties.

The effects of different sugars on motility, morphology and DNA damage during the liquid storage of rat epididymal sperm at 4°C.

This study evaluated the protective effects of supplementation with three different sugars on the motility, morphology and DNA integrity of rat epididymal sperm chilled and stored at 4°C Epididymides were obtained from each donor. Rat epididymal sperm was diluted in Ham's F10 plus raffinose, Ham's F10 plus trehalose, Ham's F10 plus fructose, and Ham's F10 medium for control purposes. Thereafter, the extended sperm were chilled and stored in liquid form at 4°C. Sperm motility, morphological abnormalities and DNA damage were determined at 0 and 12h after chilling. No significant difference was observed in any of the parameters evaluated at 0h, before storage (P>0.05). After 12h of storage, all sugar additives led to statistically higher motility, normal sperm morphology and DNA integrity in comparison to the control group. Raffinose gave the best motility percentages (32.86±1.84%) after 12h of storage at 4°C, compared to the other groups (P<0.001). In conclusion, Raffinose, trehalose and fructose provided a better protection of sperm functional parameters against chilling injury, in comparison to the control group.

Effect of glutamine and sugars after bull spermatozoa cryopreservation.

The objective of this study was to evaluate the effects of the addition of different sugars (raffinose, sucrose, and trehalose) on bull spermatozoa cryopreserved in a commercial extender (Optidyl) supplemented with glutamine on semen parameters, fertilizing ability and superoxide dismutase (SOD) activity. Nine ejaculates for each bull were used in the study. Semen was frozen in five different extenders: raffinose 25 mM plus glutamine 3 mM (RGO), sucrose 25 mM plus glutamine 3 mM (SGO), trehalose 25 mM plus glutamine 3 mM (TGO), glutamine 3 mM (GO) and control (O). Insemination doses were processed so that each 0.25 mL straw contained 15 x 10(6)sperm. Groups of GO and RGO resulted in the higher rates of subjective (54.0 ± 1.7% and 64.0 ± 1.1%; P < 0.01) and CASA motilities (53.0 ± 2.7% and 61.0 ± 4.4%; P < 0.001), respectively compared to the other groups. The supplementation of additives did not provide an effect on the level of post-thaw sperm CASA progressive motilities, the sperm motion characteristics and pregnancy rates. GO and RGO provided the better protective effect for sperm acrosome (4.0 ± 0.5% and 12.0 ± 0.6%) and total abnormalities (5.0 ± 0.3% and 13.0 ± 0.7%; P < 0.001), respectively. At the HOST values, the additives did not give to result the protective effect in comparison to Optydil extender without additives (P > 0.05). For pregnancy rates, there were no significant differences among the groups. The supplementation of additives did not provide any significant difference on the level of SOD activity (P > 0.05). It can be also thought that these sugars might have worked with glutamine in a synergy. Thereby, sugars such as raffinose and sucrose with glutamine in freezing extender may be recommended to facilitate bull semen freezability.

The effect of cysteine and glutathione on sperm and oxidative stress parameters of post-thawed bull semen.

The aim of this study was to determine the effects of antioxidants such as reduced glutathione (GSH) and cysteine in Laiciphose® extender on semen parameters, fertilizing ability, lipid peroxidation (LPO) level and glutathione peroxidise (GPx) activity of post-thawed bull semen. Totally 54 ejaculates of three bulls were used in the study. Five groups, namely; GSH (0.5 and 2 mM), cysteine (5 and 10 mM) and control group, were conducted to test the antioxidants in Laiciphose®. Insemination doses were processed that each 0.25-mL straw contained 15 x 106 sperm. The addition of antioxidants did not present any significant effect on the percentages of post-thaw sperm morphology (acrosome and total abnormalities), subjective, CASA and progressive motilities, as well as sperm motility characteristics (VAP, VSL, VCL, LIN and ALH), compared to the control groups (P > 0.05). GSH 0.5mM (55.5±7.38%) and cysteine 10 mM (48±5.65%) led to lower rates of DNA damage, compared to control (P < 0.05). As regards to MDA level, cysteine at 10 mM dose gave the highest level (4.99±0.44 nmol/L) (P < 0.001). GPx activity was demonstrated to be higher level upon the addition of 5 mM cysteine when compared to the other groups (P < 0.05). With respect to fertility results based on 60-day non-returns, the supplementation of antioxidants did not present significant differences (P > 0.05). The results of this study may provide an useful information for the future studies in this area. So, further studies could be suggested to achieve better information in terms of the DNA damage and fertilizing capacity of bull sperm frozen with effective antioxidants.

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.

The effect of raffinose and methionine on frozen/thawed Angora buck (Capra hircus ancryrensis) semen quality, lipid peroxidation and antioxidant enzyme activities.

The aim of the present study was to determine the effects of different doses of raffinose and methionine on post-thawed semen quality, lipid peroxidation and antioxidant enzyme activities of Angora buck (Capra hircus ancryrensis) sperm following cryopreservation. Ejaculates collected from three Angora bucks were evaluated and pooled at 37 degrees C. Semen samples, which were diluted with a Tris-based extender containing the additives raffinose (2.5, 5, 10mM) and methionine (2.5, 5, 10mM) and an extender containing no antioxidants (control), were cooled to 5 degrees C and frozen in 0.25 ml French straws. Frozen straws were thawed individually at 37 degrees C for 20s in a water bath for evaluation. The freezing extender supplemented with 2.5 and 5mM methionine led to higher percentages of CASA motility (63.6+/-7.0; 63.4+/-3.1%, respectively), in comparison to the controls (P<0.01) following the freeze-thawing process. The addition of antioxidants did not provide any significant effect on the percentages of post-thaw subjective and CASA progressive motilities as well as sperm motion characteristics (VSL and VCL), compared to the control groups (P>0.05). The freezing extender with raffinose (5 and 10mM) and methionine at three different doses (2.5, 5 and 10mM) led to lower percentages of acrosome abnormalities, in comparison to the controls (P<0.001). In the comet test, raffinose (5 and 10mM) and methionine (10mM) gave scores lower than those of the controls, and thereby reduced DNA damage (P<0.05). Malondialdehyde formation was found to be lower (1.8+/-0.1 nmol/L) in the group of 5mM raffinose, compared to the controls following the freeze-thawing process (P<0.01). The additives did not show any effectiveness on the maintenance of SOD, GSH-PX and GSH activities, when compared to the controls (P>0.05). In conclusion, methionine and raffinose play a cryoprotective role against sperm CASA motility, acrosome abnormality and DNA damage. Raffinose 5mM exhibited antioxidative properties, decreasing MDA levels. Further studies are required to obtain more concrete results on the characterization of microscopic parameters and antioxidant activities in cryopreserved goat sperm with different additives.

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).

The influence of cysteine and taurine on microscopic-oxidative stress parameters and fertilizing ability of bull semen following cryopreservation.

Oxidative stress significantly damages sperm functions such as motility, functional integrity, endogenous antioxidant enzyme activities and fertility due to lipid peroxidation induced by reactive oxygen species (ROS). The aim of this study was to determine the effects of antioxidants such as taurine and cysteine in Bioxcell extender on standard semen parameters, fertilizing ability, lipid peroxidation (LPO) and antioxidant activities comprising reduced glutathione (GSH), glutathione peroxidase (GSH-Px), catalase (CAT) and superoxide dismutase (SOD) after the cryopreservation/thawing of bull semen. Nine ejaculates for each bull were included in the study. Three groups, namely taurine (2mM), cysteine (2mM), and control, were designed to analyze the antioxidants in Bioxcell. Insemination doses were processed so that each 0.25-ml straw contained 15 x 10(6) sperm. The addition of cysteine led to higher motility, compared to the other groups (P<0.001). Cysteine showed a greater protective effect on the percentages of acrosome damage and total abnormalities in comparison to the other groups (P<0.001). No significant differences were observed in hypo-osmotic swelling test (HOST), following supplementation with antioxidants during the freeze-thawing process. No significant difference was observed in non-return rates among groups. In biochemical assays, the additives did not show effectiveness on the elimination of malondialdehyde (MDA) formation and maintenance of GSH and GSH-Px activities, when compared to controls. CAT activity (35.1+/-8.1 kU/g) was demonstrated to be significantly higher upon the addition of 2mM taurine (P<0.001), while the level of MDA increased, indicating oxidative stress in this group. SOD activity (21.4+/-2.9 U/g protein) was significantly elevated in the group with cysteine, compared to the other groups (P<0.001).