Liquid Storage Of Ram Semen Research Articles

Liquid Storage of Ram Semen: Associated Damages and Improvement

The successful application of assisted reproductive techniques (ARTs) in ovine as in other mammal species relies on many factors among which the quality of the semen used. After collection, semen samples are generally processed for storage (liquid storage or cryoconservation) before being used for insemination or in vitro embryo production. During the liquid storage process, sperm cells are exposed to artificial conditions which lead to oxidative stress—the imbalance between pro-oxidants and antioxidants (AO), following overproduction of reactive oxygen species (ROS)—resulting in ultrastructural, biochemical and functional damages of spermatozoa. Especially, viability, motility, mitochondrial activity, membrane integrity, and acrosome integrity are reduced while morphological abnormalities, DNA fragmentation, and lipid peroxidation (LPO) are increased, affecting the fertilizing ability and subsequent early embryonic development when using standard extenders. Indeed, an optimal semen extender must not only regulate and support an environment of adequate pH and buffering capacity to protect spermatozoa from osmotic and cooling stresses, but, also prevent the generation and/or scavenge excess ROS. To improve ram semen liquid storage, several methods have been developed with the supplementation of extenders with antioxidants or antioxidant like-compounds (enzymes, amino-acids, vitamins, plant extracts), seminal plasma, sugars, fatty acids, and nanoparticles being a relevant approach. Promising results have been registered with the supplementation of extenders with these compounds, confirming they can be used to preserve ram semen quality and fertility. Therefore, the present review provides an updated overview of the damages and associated mechanisms that ram spermatozoa undergo during liquid storage. Moreover, the supplementation of extenders with different compounds as a tool to improve semen storage is also discussed as well as their efficiency to reduce and/or prevent sperm damages during storage.

Effects of reduced glutathione on ram sperm parameters, antioxidant status, mitochondrial activity and the abundance of hexose transporters during liquid storage at 5 ℃

The objective of this study was to evaluate the effects of reduced glutathione (GSH) supplementation on sperm motility, kinematics, mitochondrial activity and the abundance of sperm hexose transporters during liquid storage of ram semen at 5 ℃. Semen samples from eight rams were pooled and diluted with Tris-egg yolk extender supplemented with 0 (control), 50, 100 and 200 mM GSH, and they were assessed at different processing periods (fresh diluted, stored for 24, 48 and 72 h at 5 ℃). Sperm motility and kinematic parameters were analyzed by computer-assisted sperm analysis (CASA). The hypo-osmotic swelling test (HOST) was used to evaluate the functional integrity of sperm membrane. Eosin-nigrosin and JC-1/PI staining methods were used to assess sperm viability and mitochondrial activity, respectively. The antioxidant status of sperm was examined using biological assays. The changes of sperm hexose transporters (GLUT 3, 5 and 8) were determined using western blot analysis. As compared to the control group, supplementation with 100 and 200 mM GSH improved (P ＜ 0.05) sperm total motility, membrane integrity, motion parameters and mitochondrial activity at 72 h of storage, as well as resulted in the higher sperm antioxidant status after storage from 24 to 72 h. The cooling induced a reduction of GLUT 3 and 8, and GSH treatment at 200 mM retarded the gradual decline of these proteins during the prolonged storage time. Our data suggest that GSH can reduce the decline of semen quality by influencing the antioxidant status and energy metabolism of liquid stored ram spermatozoa. The concentration of 200 mM could be appropriate for ram semen during liquid storage at 5 ℃.

Effect of three commercial extenders on sperm motility and fertility in liquid ram semen stored at 15 °C or 5 °C

The effect of different extenders on sperm motility and fertility was evaluated during liquid storage of ram semen at 5 °C and 15 °C. The semen was collected, pooled and diluted in three commercial extenders: Inra 96® (INRA) based on skimmed milk, Biladyl® A fraction (BIL) based on egg yolk, and Ovixcell® (OVIX) based on soybean lecithin. Then, sperm motility was evaluated at 0, 6, 24, 48, 72 and 96 h. In order to evaluate fertility, samples stored at 15 °C were used after dilution in INRA and OVIX. Results showed that progressive motility was significantly higher up to 72 h of storage in sperm samples maintained at 5 °C in comparison with 15 °C, similarly for each tested diluent. When samples were stored at 5 °C in OVIX, kinematic parameters such as velocity (except curvilinear velocity, VCL), trajectory [linearity (LIN), straightness (STR), wobble (WOB)], amplitude of lateral head displacement (ALH) and beat/cross frequency (BCF) were higher than in INRA and BIL. No significant differences in pregnancy rate were detected between INRA (62.6%) and OVIX (58.9%). In conclusion, liquid storage at 5 °C with OVIX extender is an interesting option since non-animal components are used, and this extender offers similar in vitro and in vivo efficacy as other extenders containing animal components.

195 Sperm motion characteristics of ram semen liquid-stored in a milk egg yolk extender at four temperatures

Abstract Varying temperatures have been used for liquid storage of ram semen and that of other species. This study evaluated motility characteristics of ram semen stored at 5, 10, 15, and 20°C for up to 96 h. Two ejaculates were collected and pooled from each of 6 rams using an artificial vagina and evaluated for motility and concentration. Samples were extended in ultra-high temperature pasteurized skim milk and egg yolk (10% v/v) containing penicillin and streptomycin, diluted to 250 million sperm/mL, and packaged in 0.5 mL straws. Semen was held at 32°C during processing, and straws placed in 500 mL jars for storage at 5°C (refrigerator), 10 and 15°C (refrigerated water bath) and 20°C (air conditioned room temperature). Cooling rates were 0.04, 0.25, 0.44, and 0.02 °C/min, and final temperatures reached after 672, 83, 36, and 567 min in the four storage environments, respectively, with cooling rates faster in a liquid than air environment. Straws from individual rams were removed at 6, 24, 48, 72 and 96 h of storage and analyzed with a computer-assisted sperm analyzer after warming to 36°C. Data were analyzed for the effect of storage time, temperature, and their interaction on sperm motion characteristics. Sperm motion characteristics were not affected over time during storage at 5 and 10°C. At 15°C motility parameters decreased in a curvilinear (P < 0.05) relationship with time (progressive motility: 52.6 to 29.7%; rapid motility: 36.5 to 16.5%), and at 20°C in linear (P < 0.001) relationship (progressive motility: 51.9 to 13.1%; rapid motility: 36.1 to 6.3%). Circular motility decreased (P < 0.05) with increasing temperatures after 72 and 96 h of storage, while local motility was not affected by storage temperature and time. Results suggest storage at 10°C may be a viable alternative to storage at 5°C as retention of motility was similar.

Effect of addition of melatonin on liquid storage of ram semen at 4°C.

Adding a certain amount of antioxidants to semen extender has been shown to improve semen quality. The aim of present study was to elucidate whether the supplementation of melatonin to the Tris-based extender (CTR) could enhance the quality of ram spermatozoa during storage at 4°C. Ram semen samples were collected and diluted with CTR extender containing different concentrations (0, 0.05 (M 0.05), 0.1 (M 0.1), 0.2 (M 0.2) or 0.4 (M 0.4) mM) of melatonin. Sperm routine indicators, mitochondrial activity, total antioxidant capacity (T-AOC) and malondialdehyde (MDA) content were analysed in control and melatonin treatment groups. The higher per cent of motility, plasma membrane integrity, mitochondrial activity and T-AOC activity was observed in M 0.05, M 0.1 and M 0.2 groups compared to control group at 5days of storage (p<0.05), while lower percentage of MDA content was observed among these groups (p<0.05). In addition, there were no significant differences in acrosome integrity among the control and M 0.05, M 0.1 and M 0.2 groups during the experiment. The above results show that the addition of 0.05, 0.1, 0.2mM of melatonin is beneficial to the preservation of ram semen during liquid storage at 4°C mainly through antioxidative stress.

Liquid storage of ram semen for 96 h: Effects on kinematic parameters, membranes and DNA integrity, and ROS production

Abstract A complete assessment of morphological and functional characteristics of ram semen during refrigeration is necessary to optimize the process of semen manipulation and storage. The aim of this study was to describe changes in main predictive parameters of ram semen diluted in a commercial soy lecithin-based extender (OVIXcell), during long term liquid storage at 4 °C. Ejaculates of 5 Sarda rams were collected, pooled and diluted in OVIXcell. Samples were cooled at 4 °C and stored at this temperature until 96 h. At 0-24-48-72-96 h semen samples were analysed for the following parameters: motility [computer assisted sperm analysis (CASA)]; integrity of cytoplasm membrane and acrosome (PI/PSA staining); DNA fragmentation index [DFI(%); sperm chromatin structure assay (SCSA)]; levels of reactive oxygen species (ROS; H2DCFDA staining). Effect of time of storage on main parameters and correlations among them were assessed respectively by ANOVA and Pearson's correlation coefficient. Primary and secondary motility parameters were significantly affected by time of storage (P 0.05). Long term storage did not affect the levels of DFI(%) (P > 0.05) while ROS production significantly increased from 48 to 96 h (P

Positive effect of BSA supplemented soybean lecithin based extender on liquid storage of ram semen at 5C

The aim of the study was to evaluate different BSA doses in soybean-lecithin based extenders for liquid storage of ram semen. Pooled semen was divided into four equal volumes and diluted in a single-step dilution method with BSA supplemented (BSA5, BSA7.5, BSA10 groups supplemented 5mg/mL, 7.5mg/mL, 10mg/mL BSA respectively) and BSA-free (control) extenders. Semen samples were assessed for sperm motility, plasma membrane functional integrity using hypo-osmotic swelling test (HOST), damaged acrosome using FITC-Pisum sativum agglutinin (PSA-FITC) at 0, 24, 48 and 72h of storage at 5 °C. Antioxidant activities of groups at 0 and 72h were measured with glutathionine (GSH) concentration, membrane lipid peroxidation status was analyzed using the malondialdehyde (MDA) concentration. Antioxidant capacity of extender also was measured with 2,2-Diphenyl-1-picrylhydrazyl (DPPH) and Dichloro-dihydro-fluorescein diacetate (DCFH-DA) assays. In the study, BSA (5mg/mL) supplementation has beneficial effect on ram sperm parameters at all evaluation times. In addition, the increasing doses of BSA supplementation has a negative effect on sperm parameters especially 48 and 72h of storage

Semen supplementation with palmitoleic acid promotes kinematics, microscopic and antioxidative parameters of ram spermatozoa during liquid storage.

The purpose of the present experiment was to investigate the protective effects of palmitoleate on the quality of ram semen during low temperature liquid storage. Ejaculates were collected using the artificial vagina from four Qezel rams twice a week. Ejaculates were pooled, diluted with Tris-egg yolk extender without palmitoleate (control) or supplemented with 0.125 (P 0.125), 0.25 (P 0.25), 0.5 (P 0.5) and 1 (P 1) mM palmitoleate at a final concentration of 500×106 spermatozoa/ml. Total motility and forward progressive motility (FPM) as well as other spermatozoa kinematics were evaluated by computer-assisted sperm analysis. Moreover, viability and membrane functionality were determined in the spermatozoa. Additionally, amounts of malondialdehyde (MDA), total antioxidant activity (AOA), nitric oxide (NO) and superoxide dismutase (SOD) activities were evaluated in the medium and spermatozoa at 0, 24, 48 and 72hr of storage. The palmitoleate supplementation resulted in a significant (p<.05) increase in total motility and FPM with the highest increase at 0.5 mM concentration for 72hr. P 0.5 group also resulted in the highest percentage of membrane-intact spermatozoa (76.60±1.95%) and viability (75.81±1.34%) at 72hr (p<.05). The amounts of MDA and NO were lower in P 0.125, P 0.25 and P 0.5 groups compared to control at 48hr and 72hr (p<.05). Higher amounts of AOA were obtained in palmitoleate-treated groups in medium and spermatozoa during storage time (p<.05). Furthermore, palmitoleate supplementation increased the SOD activities in spermatozoa compared to the control (p<.05). The results of the present experiment reveal that supplementation with 0.5 mM palmitoleate improves ram spermatozoa motion characteristics, AOA levels and SOD activities during liquid storage. Then, palmitoleate could be used as an antioxidant source during liquid storage of ram semen.

Influence of lycopene and cysteamine on sperm and oxidative stress parameters during liquid storage of ram semen at 5 °C

Ejaculates were collected from six Merino rams with the aid of an artificial vagina twice a week. The ejaculates containing spermatozoa with >80% forward progressive motility and concentrations higher than 2×109 spermatozoa/ml were pooled. The present study included two experiments. In experiment 1, each pooled ejaculate was divided into four equal aliquots and diluted (37°C) with the Tris based extender, containing 0 (control), 0.5, 1 and 2mM lycopene, at a final concentration of approximately 400×106 sperms/ml (single step dilution), In experiment 2, cysteamine at concentrations of 0 (control), 0.5, 1 and 2mM, 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 and oxidative stress parameters were evaluated after 0, 24, 48 and 72h of storage at 5°C. The extender supplemented with 0.5mM lycopene resulted in higher mitochondrial activity rate (p<0.05) in comparison to the control group at 72h of storage. Lycopene at 0.5mM dose led to higher sperm motility rate (p<0.05) when compared to 2mM lycopene group at 72h of liquid storage. As regards oxidative stress parameters, only 2mM lycopene increased total glutathione levels (p<0.05) at 0h of storage. The extender supplemented with 1mM cysteamine gave higher motility (p<0.05) at 48h compared to control. As regards oxidative stress parameters, 1 and 2mM cysteamine at 48h and 1mM cysteamine at 72h increased total glutathione levels (p<0.05) compared to control groups. Cysteamine at 1 and 2mM doses decreased lipid peroxidation (p<0.05) at 0h of liquid storage compared to control. Our data suggest that lycopene at 0.5 and 2mM and cysteamine at 1 and 2mM doses can be added to Tris based extender for improving the ram sperm motility, viability, mitochondrial activity and oxidative stress parameters during the liquid storage.

Effects of the Tris, Tes, or skim milk based extender on in vitro parameters of ram spermatozoa during liquid storage

Cryopreservation can seriously damage structure and physiological function of mammalian spermatozoa. However, liquid storage at reduced temperature may be an alternative of cryopreservation if artificial insemination is performed in relatively short time. In this study, effects of the Tris-hydroxymethyl aminomethane (Tris), N-Tris (hydroxymethyl) -methylaminoethane-sulfonic acid (Tes), or skim milk based extender on ram spermatozoa during liquid storage at 4°C were assessed and compared. Semen samples from 6 adult Yunnan semi-fine wool rams with proven fertility were pooled, equally divided into 3 groups according to extenders, and preserved at 4°C for various durations (0, 1, 3, 5, and 7 days). The motility, moving velocity, and hypo-osmotic swelling capability of spermatozoa were evaluated using a computer assisted sperm analyzer system. The acrosome status, membrane integrity, distribution of phosphatidylserine (PS), and mitochondrial membrane potential (MMP) were analyzed with flow cytometry. The results indicated that following liquid storage in the Tris or Tes based extender for 3 days, the motility, hypo-osmotic swelling capability, and MMP of spermatozoa did not show a significant decrease. Additionally, liquid storage in the Tris or Tes based extender for 24h did not deleteriously affect the moving velocity, acrosome status, and membrane integrity of spermatozoa. However, after liquid storage for 24h, the percentage of spermatozoa with membrane exposed PS was significantly increased. On the contrary, the negative effects of skim milk on the above parameters of ram spermatozoa became more serious compared to the Tris or Tes based extender after liquid storage for 24h (P<0.05). There was no significant difference existing between the Tris and Tes based extender as concerning the parameters evaluated in this study. In conclusion, the Tris or Tes based extender may be more suitable for liquid storage of ram spermatozoa at reduced temperature compared to the skim milk based extender. Tris can be substituted by Tes for liquid storage of ram semen. The future study should focus on assessment of molecular changes and fertility of ram spermatozoa after liquid storage.

Effect of argan oil on liquid storage of ram semen in Tris or skim milk based extenders

Due to its high antioxidant content, the argan oil could play a beneficial role in liquid storage of ram semen. The aim of this study was to investigate effects of different concentration of argan oil (ARO) on spermatologic parameters, lipid peroxidation and DNA fragmentation during liquid storage of ram semen until 48h. Also effects of extenders and temperature on same parameters were assessed. For these aims, semen samples were collected from Boujaâd rams, extended with Tris egg yolk or skim milk extenders without (control) or supplemented with different concentrations of ARO (1%, 2%, 5% and 10% v/v) at a final concentration of 0.8×109 sperm/mL and stored until 48h at 5°C or 15°C. The sperm quality assessments were performed at different intervals during storage (0, 8, 24 and 48h). Sperm progressive motility started to decrease after 8h of storage in all temperatures – extenders combinations and dropped steadily during the 8–48h interval. However, sperm viability, progressive motility and membrane integrity were markedly higher in ARO groups (especially in 1% in Tris and 5% in skim milk) until 24h and 48h storage at both temperatures compared to controls. The argan oil also decreased the level of spontaneous and induced malondialdehyde (MDA) and the sperm DNA fragmentation until 48h storage. In conclusion, it was determined that addition of argan oil to conventional extenders may improve the quality of ram semen during liquid storage in different temperatures.

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 96h of the liquid storage of ram semen at 5°C. Thirty-two Merino rams (12–14months 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 250μg/day of total AF. The EG group received commercial feed plus 2g/day of EG. The AF + EG group was given commercial feed plus 250μg/day of total AF and 2g/day of EG. In the study, ejaculates were obtained from rams twice a week for 12weeks, 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 96h 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 96h 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.

Royal Jelly improves the sperm parameters of ram semen during liquid storage and serves as an antioxidant source

The current study was carried out to investigate the protective effects of the Royal Jelly (RJ) supplementation on the sperm kinematics, plasma membrane functionality, the level of produced nitric oxide (NO) and total antioxidant capacity during the liquid storage of ram semen at 4°C, for various periods of time (0, 24, 48, 72, 96 and 120h). Semen samples were collected from four rams and pooled, diluted with Tris–egg yolk extender without RJ (control) or supplemented with different concentrations of RJ (0, 0.5, 1, 1.5 and 2%), at a final concentration of 200×106sperm/mL. Sperm viability, kinematics and membrane functionality were determined by nigrosin–eosin staining, computer-assisted sperm analysis (CASA), and by using the hypo-osmotic swelling test (HOST), respectively. Additionally, the oxidative and nitrosative status were evaluated after the RJ supplementation. The RJ supplementation resulted in a significant (P<0.05) increase of sperm viability with the highest increase at 1% RJ concentration for 120h. A significant protective effect of RJ on sperm membrane functionality was obtained at lower concentrations (0.5 and 1%) and in all incubated time points. The most prominent protective effect of RJ on sperm motility parameters was found on the progressive velocity (VSL) as after 72h storage, no significant reduction was found in comparison to the 50% reduction in the control group. The produced NO level during storage time was reduced by addition of RJ at low concentrations (0.5 and 1%). Our data suggest that the RJ supplementation at lower concentrations (0.5 and 1%) improves the ram sperm kinetics and plasma membrane functionality during the liquid and cold storage. Moreover, the protective effect of RJ might be attributed to its antioxidative/antinitrosative capacities.

Protective effect of melatonin on sperm motility parameters on liquid storage of ram semen at 5°C

Oxidative damage to sperm resulting from reactive oxygen species generated by the cellular components of semen during liquid storage is possibly one of the main causes of the decline in motility during storage. The aim of this study was to determine the effects of melatonin as an antioxidant on sperm motility parameters in liquid storage of ram semen at 5°C. A total number of 24 ejaculates were collected using the artificial vagina from four chal rams and three replicates of the ejaculates were diluted with a Tris-based extender with melatonin at the doses of 0.1, 1 and 3 mM and an extender containing no melatonin (control). Diluted semen samples were cooled from 37 to 5°C. A computerassisted sperm motility analysis was used to analyze sperm motion characteristics. The sperm motility parameters such as progressive motility, straight linear velocity (VSL), curvilinear velocity (VCL), average path velocity (VAP), head displacement (ALH), sperm track straightness, (STR) and linearity (LIN) were determined in the storage of semen at 5°C for a period of 48 h. The extender supplemented with 1 mM melatonin led to higher percentages of progressive motility and LIN (46.4 and 58.9, respectively) and higher value of VSL and VAP (78.4 and 91.1 µm/s, respectively) in comparison to the control (P 0.05) effect in ALH and STR of the whole semen compared to the control sample. The results indicated that supplementation of melatonin to the semen extender can improve the semen motility.

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 24h. Semen samples from four rams were pooled, diluted with Tris-egg yolk extender without antioxidants (control) or supplemented with either CAT (100, 200, and 400U/mL) or GSH (100, 200, and 400mM) at a final concentration of 50×106sperm/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 24h, 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 200U/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 400mM 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 1.5h of incubation. Based on the results of the present study, the addition of CAT (100 and 200U/mL) reduced the deleterious effects of cooling on total motility in ram sperm maintained at 5°C for 24h, although it did not affect the functionality of the sperm membranes. However, the addition of 400mM GSH caused negative effects on the velocity parameters of the sperm.

Influence of sperm concentration on the motility, morphology, membrane and DNA integrity along with oxidative stress parameters of ram sperm during liquid storage

The aim of this study was to determine the influences of two different concentrations in terms of motility, morphology, membrane integrity (viability and HOST response: HE-test; modified hypoosmotic swelling test (HOST) associated with supravital eosin staining test), DNA integrity (COMET assay) and oxidative stress parameters (MDA, malondialdehyde; AOA, total antioxidant activity; GSH, reduced glutathione; NOx, nitric oxide) of liquid stored ram sperm for 5 days. Two different concentrations suitable for laparoscopic and cervical inseminations were employed. A total of 5 Pirlak rams (Daglic × Kivircik, local breed) with satisfactory breeding potential were selected. Semen samples were collected by artificial vagina. Ejaculates were extended to 25 and 100 million sperm per ml with Tris-based egg-yolk (T) extender at room temperature and stored at 4 °C. The concentration significantly had effects on motility, morphology, membrane and DNA integrity along with oxidative stress parameters ( P < 0.05). Overall changes in the motility, morphology, membrane and DNA integrity along with oxidative stress parameters of ram sperm diluted with T in the 100 × 10 6 ml −1 concentration and preserved at 4 °C for a short term was found to be better preservation than that of diluted with T in the 25 × 10 6 ml −1 concentration. Significant concentration × days of storage interactions were found for all parameters except the NOx. Further studies are required to elucidate the oxidative stress changes on sperm quality parameters in different concentrations during the liquid storage of ram semen.

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.

Protective effect of taurine, glutathione and trehalose on the liquid storage of ram semen

Oxidative damage to sperm resulting from reactive oxygen species generated by the cellular components of semen during liquid storage is possibly one of the main causes for the decline in motility and fertility during storage—the other detrimental cause is low temperature on the destabilisation of sperm membrane structure. The aim of this study was to determine the effects of the addition of the anti-oxidants taurine and glutathione (GSH), and the membrane structure stabiliser, trehalose, on sperm viability during low temperature liquid storage. A total number of 36 ejaculates were collected using the artificial vagina from four Chios rams and nine replicates of the ejaculates were diluted with a Tris-based extender containing additives as the control. The sperm motility, percentage abnormal sperm, plasma membrane intact sperm and the hypo-osmotic swelling test (HOST) were determined during storage of semen at 5 °C for a period of 0, 6, 24 and 30 h of liquid storage, respectively. Trehalose at a level of 50 mM provided the best maintenance of motility at 6 and 30 h ( P < 0.05), and gave the highest percentage (69.0 ± 2.0% and 64.6 ± 1.8%, respectively) of viable sperm at 24 and 30 h ( P < 0.01). Trehalose treatment at a concentration of 50 mM also resulted in the highest percentage of membrane-intact sperm (53.7 ± 2.9%) after performing HOST at 30 h. The anti-oxidant treatments GSH 5–10 mM and taurine at 50 mM provided a significant improvement in sperm survival during the 6 h of liquid storage at 5 °C ( P < 0.05). In conclusion, many aspects of sperm protection, e.g. sperm motility, viability and membrane stabilisation of the sperm cells during relative low temperature storage, are the key factors determining the preservation of sperm function. Future efforts toward improving function of ram sperm kept in low temperature storage should concentrate on anti-oxidant additives. The results of this study provide a new approach to the preservation of sperm from rams of the Chios and related breeds, and so contribute to the improvement of these breeds for the world sheep industry.

A comparison between tris and milk diluents for liquid storage of ram semen in Iran

The use of semi-skimmed cow milk diluent for liquid storage of ram semen is more convenient in Iranian sheep farming condition than other solutions. Preliminary tests showed that the motility and live percentages of ram spermatozoa were significantly (P&gt;0.05) lower for semen samples were diluted in citrate buffer than semen samples in either tris buffer or semi-skimmed cow milk (Ahangari, 1996). This study was carried out for liquid storage of ram semen under Iranian farm conditions.

Liquid storage of ram semen in the absence or presence of some antioxidants.

The antioxidants superoxide dismutase (SOD), catalase (CAT), cytochrome c (CHc) and glutathione peroxidase (GP) were added at various concentrations to Tris-glucose-yolk diluent (TGY), and their effects on motility, acrosome integrity and fertility of ram spermatozoa were assessed after extension and liquid storage. All the antioxidants improved the motility and acrosome integrity of spermatozoa, and a combination of SOD and CAT had an additive effect on the survival of spermatozoa stored at 5 degrees C but not at 25 degrees C. There was a linear improvement in survival of spermatozoa with increasing dose of antioxidants except for CAT for which doses higher than 200 U mL-1 were toxic. The proportion of oocytes fertilized in vitro declined with time of semen storage (P < 0.001), and was better for semen diluted with TGY containing SOD or CAT than TGY without antioxidants when stored for 7 days (116/246, 47% v. 25/79, 32%; P < 0.05) but not for 14 days (23/174, 13% v. 8/66, 12%). Fertilization rates were unaffected by the presence or absence in the diluent of CHc or GP. The proportions of ewes with fertilized ova and of recovered ova fertilized were better after insemination with semen diluted in TGY containing SOD and CAT than TGY without antioxidants when stored for 14 days (9/18, 50% and 20/40, 50% v. 2/13, 15% and 5/32, 16%; P < 0.05) but not for 7 days (9/20, 45% and 16/48, 33% v. 8/16, 50% and 24/41, 59%). Pregnancy rates were better after intrauterine insemination of ewes with fresh semen than stored semen (11/18, 61% v. 21/75, 28%; P < 0.01), and with semen stored in TGY containing SOD and CAT than in TGY without antioxidants (15/37, 41% v. 6/38, 16%; P < 0.05).