Antioxidant effect of quercetin in an extender containing DMA or glycerol on freezing capacity of goat semen

This study was performed to evaluate the effectiveness of quercetin as a non-enzymatic antioxidant in combination with glycerol or Dimethylacetamide (DMA), on freezability of goat semen. Ejaculates from four healthy mature Mahabadi goats were collected using an artificial vagina. After primary processing, semen was pooled and extended by egg yolk based extender supplemented with different concentrations of quercetin (10 or 20 μM) along with 5% glycerol or DMA. The extended semen was frozen and sperm motility parameters, viability, abnormality, membrane integrity and lipid peroxidation were assessed after thawing. Results showed that sperm viability, total motility, progressive motility, straightness (STR) and linearity (LIN) were higher (P < 0.05), and abnormality percentage and MDA concentration were lower (P < 0.05) in extender containing DMA. Similarly, higher (P < 0.05) total motility, progressive motility, viability and membrane integrity along with lower (P < 0.05) MDA level were noted in Q10 group. The lowest (P < 0.05) MDA level was observed in DMA extender containing moderate level of quercetin (Q10D). Also the STR was higher (P < 0.05) in Q10D compared to Q10G and Q20G groups. In conclusion, supplementation of extender with 10 μM quercetin in combination with DMA improves the goat sperm motion kinetics and suppresses lipid peroxidation after freezing and thawing. Furthermore, DMA is more effective cryoprotectant for the freezing of goat sperm.     

Addition of superoxide dismutase mimics during cooling process prevents oxidative stress and improves semen quality parameters in frozen/thawed ram spermatozoa

High levels of reactive oxygen species (ROS), which may be related to reduced semen quality, are detected during semen cryopreservation in some species. The objectives of this study were to measure the oxidative stress during ram semen cryopreservation and to evaluate the effect of adding 2 antioxidant mimics of superoxide dismutase (Tempo and Tempol) during the cooling process on sperm motility, viability, acrosomal integrity, capacitation status, ROS levels, and lipid peroxidation in frozen and/or thawed ram spermatozoa. Measuring of ROS levels during the cooling process at 35, 25, 15, and 5 °C and after freezing and/or thawing showed a directly proportional increase (P < 0.05) when temperatures were lowering. Adding antioxidants at 10 °C confered a higher motility and sperm viability after cryopreservation in comparison with adding at 35 °C or at 35 °C/5 °C. After freezing and/or thawing, sperm motility was significantly higher (P < 0.05) in Tempo and Tempol 1 mM than that in control group. Percentage of capacitated spermatozoa was lower (P < 0.05) in Tempo and Tempol 1 mM in comparison with that in control group. In addition, ROS levels and lipid peroxidation in group Tempo 1 mM were lower (P < 0.05) than those in control group. These results demonstrate that ram spermatozoa are exposed to oxidative stress during the cooling process, specifically when maintained at 5 °C and that lipid peroxidation induced by high levels of ROS decreases sperm motility and induces premature sperm capacitation. In contrast, the addition of Tempo or Tempol at 0.5 to 1 mM during the cooling process (10 °C) protects ram spermatozoa from oxidative stress.     

Effects of different extenders on DNA integrity of boar spermatozoa following freezing–thawing

The sperm-rich fraction, collected from eight mature Yorkshire boars, was frozen in an extender containing 9% LDL (w/v), 100 mM trehalose, or 20% yolk (v/v), respectively. Sperm DNA integrity was assessed using the single-cell gel electrophoresis (SCGE). Other sperm quality characteristics such as motility, acrosome and membrane integrity were also monitored. The results showed that freezing–thawing caused an increase in sperm DNA fragmentation, and extender containing 9% LDL could significantly protect sperm DNA integrity (P < 0.05) from the damage caused by cryopreservation and decrease DNA damages compared with extender containing 100 mM trehalose and 20% yolk (v/v). No significant difference in damaged DNA was detected between frozen and unfrozen semen samples for extender of 9% LDL and 100 mM trehalose, but cryopreservation could increase the degree of DNA damage (P < 0.05), the percentage of damaged DNA degree of grade 2 and 3 was significantly increased. The deterioration in post-thaw sperm DNA integrity was concurrent with reduced sperm characteristics. The data here demonstrated that the cryoprotectant played a fundamental role in reducing boar sperm DNA damage and protecting DNA integrity. It can be suggested that evaluation of sperm DNA integrity, coupled with correlative and basic characteristics such as motility, acrosome integrity and membrane integrity, may aid in determining the quality of frozen boar semen.     

Effects of different cryoprotective agents on ram sperm morphology and DNAintegrity

This study investigates the effects of glycerol, 1,2 propanediol, sucrose, and trehalose on post-thaw motility, morphology, and genome integrity of Awassi ram semen. Ejaculates of thick consistency with rapid wave motion (>+++) and >70% initial motility were pooled. Sperm were diluted to a final concentration of 1/5 (semen/extender) in 0% cryoprotectant, 6% glycerol, 6% 1,2 propanediol, 62.5 mM sucrose or 62.5 mM trehalose using a two-step dilution method. The equilibrated semen was frozen in 0.25-ml straws. Semen samples were examined for sperm motility, defective acrosomes (FITC-Pisum sativum agglutinin (FITC PSA)), DNA integrity (acridine orange staining (AO)) and apoptotic activity (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) and Caspase-3 activity) at four time points: after dilution with extender A, after cooling to 5 °C, after equilibration and post-thaw. Freezing and thawing procedures (cooling at 5 °C, dilution, equilibration, and thawing) had negative effects on motility (P < 0.001), acrosome integrity (P < 0.001), and DNA integrity as determined by AO (P < 0.001) and TUNEL (P < 0.001) assays. There were positive correlations between sperm with defective acrosomes and apoptotic (AO- and TUNEL-positive) spermatozoa. In contrast, a significant negative correlation was found between sperm motility and defective acrosomes and AO- and TUNEL positivity (P < 0.01). The cryopreservation process acts as an apoptotic inducer in ram semen; all cryoprotectants used in the present study allowed apoptosis to some extent, with negative effects on sperm morphology and DNA integrity. The glycerol group performed better than the propanediol, sucrose, trehalose, and control groups in terms of post-thaw sperm motility but not DNA integrity.     

Effects of different extenders on DNA integrity of boar spermatozoa following freezing-thawing

The sperm-rich fraction, collected from eight mature Yorkshire boars, was frozen in an extender containing 9% LDL (w/v), 100 mM trehalose, or 20% yolk (v/v), respectively. Sperm DNA integrity was assessed using the single-cell gel electrophoresis (SCGE). Other sperm quality characteristics such as motility, acrosome and membrane integrity were also monitored. The results showed that freezing–thawing caused an increase in sperm DNA fragmentation, and extender containing 9% LDL could significantly protect sperm DNA integrity (P < 0.05) from the damage caused by cryopreservation and decrease DNA damages compared with extender containing 100 mM trehalose and 20% yolk (v/v). No significant difference in damaged DNA was detected between frozen and unfrozen semen samples for extender of 9% LDL and 100 mM trehalose, but cryopreservation could increase the degree of DNA damage (P < 0.05), the percentage of damaged DNA degree of grade 2 and 3 was significantly increased. The deterioration in post-thaw sperm DNA integrity was concurrent with reduced sperm characteristics. The data here demonstrated that the cryoprotectant played a fundamental role in reducing boar sperm DNA damage and protecting DNA integrity. It can be suggested that evaluation of sperm DNA integrity, coupled with correlative and basic characteristics such as motility, acrosome integrity and membrane integrity, may aid in determining the quality of frozen boar semen.     

Caffeic acid improves microscopic sperm parameters and antioxidant status of buffalo (Bubalus bubalis) bull semen following freeze-thawing process

This study investigates the effects of cryopreservation and supplementation of buffalo's semen with Caffeic acid. It studies the effects of different Caffeic acid concentrations on cryopreservation capacity of the buffalo and evaluates their influence on various sperm parameters like motility, viability, progressive motility, sperm plasma membrane integrity, and antioxidant status. Twenty-four semen samples were collected with an artificial vaginal from three adult water buffalos. The semen samples were evaluated and the qualified ejaculates were separated and were diluted in a Tris-based extender. The resulting samples were classified into 5 groups: No antioxidant (control), Control sham (NaOH), Caffeic acid 50 μM, Caffeic acid 100 μM, and Caffeic acid 200 μM. The semen samples encountered cryodamage and the quality was deteriorating during the cryopreservation (P < 0.05). The semen evaluation after thawing showed that the groups of samples receiving 100 μM Caffeic acid had higher viability, total motility, and lower abnormal sperm and better linearity (LIN), curvilinear velocity (VCL), straight-line velocity (VSL) and path velocity and higher intact plasma membrane (P < 0.05) compared to other groups. It is notable that adding 100 μM Caffeic acid to freezing extenders enhances the CAT, GPx, SOD, and GSH and also ameliorates total antioxidant capacity of spermatozoa after thawing. It is notable that the addition of 100 μM Caffeic acid decreases the amount of Malondialdehyde. These reactions lead us to conclude that 100 μM Caffeic acid enhances the semen quality of water buffalo after thawing.     

The effects of antioxidants on semen traits and in vitro fertilizing ability of sperm from the flat-headed cat (Prionailurus planiceps)

Since antioxidants can overcome the negative effects of reactive oxygen species (ROS) during sperm cryopreservation, post-thaw sperm quality in flat-headed cats (Prionailurus planiceps), an endangered species, might benefit from the addition of antioxidants to semen extender. The objectives of this study were to: 1) investigate semen traits; and 2) evaluate effects of the vitamin E analogue Trolox (vitamin E) and glutathione peroxidase (GPx) on the quality of frozen sperm from captive flat-headed cats in Thailand. Eight ejaculates were collected by electroejaculation from four flat-headed cats. Each semen sample was divided into three aliquots and re-suspended in a semen extender as follows: 1) without antioxidant supplementation (control); 2) supplemented with 5 mM vitamin E; or 3) supplemented with 10 U/mL GPx. All samples were cryopreserved and thawed. Subjective sperm motility, progressive motility, and the integrity of the sperm membrane, acrosome and DNA were evaluated at semen collection, after 1 h cold storage, and at 0 and 6 h after thawing. Mitochondrial membrane potential, early apoptotic cells, and embryo development by heterologous in vitro fertilization were evaluated after thawing. Captive flat-headed cats were affected by teratozoospermia. After 1 h cold storage, sperm membrane integrity in samples supplemented with GPx was higher than the control group (54.5 ± 13.7 vs 51.3 ± 13.9; P < 0.05; mean ± SD). Sperm frozen in extender with GPx had higher motility at 6 h and greater mitochondrial membrane potential at 0 and 6 h post-thaw incubation than the other groups (P < 0.05). In conclusion, GPx improved the quality of frozen-thawed sperm in flat-headed cats.     

Effects of mint,thyme, and curcumin extract nanoformulations on the sperm quality,apoptosis, chromatin decondensation,enzyme activity,and oxidative status of cryopreserved goat semen

Goat semen cryopreservation is a challenging process as it results in reduced motility, vitality, and fertility of spermatozoa after freezing. In this study, we evaluated the effects of different herbal extract nanoformulations (NFs) [mint (MENFs), thyme (TENFs), and curcumin (CENFs)], supplemented at either 50 or 100 μg into Tris-extender on the cryopreserved goat semen quality. The hydrothermal squeezing method was used for the preparation of the NFs extracts. The morphological evaluation of the NFs extracts was conducted by transmission electron microscopy. All NFs supplements improved (p < 0.05) the progressive motility, vitality, and plasma membrane integrity of sperm compared with the control extender after equilibration (5 °C for 2 h) and thawing (37 °C for 30 s), but had no effect on sperm abnormality and acrosome integrity. All NFs supplements decreased (p < 0.05) the apoptosis, malondialdehyde level, and chromatin decondensation of sperm cells, while increased (p < 0.05) the total antioxidant capacity and catalase activity in the frozen/thawed extender. Particularly, CENFs at a level of 100 μg showed improvement of sperm parameters and antioxidant status during cryopreservation of goat semen more than TENFs and MENFs. The CENFs improved the quality of goat spermatozoa in post-thawed semen in terms of preventing cryodamage and promoting the cryotolerance of spermatozoa when compared with TENFs and MENFs. Therefore, supplementation of Tris-extender with CENFs could enhance goat semen processing during cryopreservation.     

Quercetin supplemented casein-based extender improves the post-thaw quality of rooster semen

The advantageous influence of quercetin (Q) supplementation in an extender has not yet been evaluated for rooster semen cryopreservation. This research was purposely conducted in order to assess the effect of different quercetin concentrations added into an extender on the sperm quality of the rooster subsequent to a freezing-thawing process. After the freezing-thawing process, spermatozoa quality parameters (membrane functionality, acrosome integrity, motility, viability, and abnormal morphology), endogenous enzymes (SOD, CAT, and GPx), mitochondrial activity, DNA fragmentation index, lipid peroxidation (MDA), and ROS were all evaluated. A total of 75 neat pooled ejaculates (3 ejaculates/rooster) were collected from 25 arbor acres roosters (24 wks) twice a week using abdominal massage technique, then divided into five equal aliquots and diluted with an extender containing different doses of Q (CS-Q) as follows: casein extender without Q (control only), casein extender containing 0.040 mg/mL quercetin (CS-Q 0.040), 0.020 mg/mL quercetin (CS-Q 0.020), 0.010 mg/mL quercetin (CS-Q 0.010), and 0.005 mg/mL quercetin (CS-Q 0.005). Our results depicted that adding to the extender with a 0.010 mg/mL Q enhanced (P < 0.01) sperm motility, membrane function, viability, mitochondrial activity, intact acrosome (P < 0.05), SOD (P < 0.001), CAT, and GPx (P < 0.01) compared to the control group at post-thaw. Compared to the control group and other treatment groups after the freeze-thawing process, the addition of 0.005 mg/mL Q into the extender also showed higher (P < 0.05) improvement in the quality of sperm parameters and a higher (P < 0.01) SOD and CAT but did not improve mitochondrial activity and sperm viability. In addition, there was a lower degree of DNA fragmentation index, lower (P < 0.05) lipid peroxidation and ROS in frozen-thawed sperm treated with 0.010 mg/mL and 0.005 mg/mL Q than in control and the other treatment groups. In addition, 0.020 mg/mL Q supplementation into the extender also reduced DNA fragmentation and improved GPx activity compared to the control group at post-thaw. Different concentrations of Q 0.010 and 0.005 mg/mL added to the extender reduced the percentage of abnormal spermatozoa compared to the other groups. The results of this study showed for the first time that the inclusion of an extender with a suitable quercetin concentration of 0.010 mg/mL improved the post-thawed quality of rooster semen.     

Comparison of TNC and standard extender on post-thaw quality and in vivo fertility of Thai native chicken sperm

Semen extender has a vital role in preservation of sperm cells properties in terms of sperm viability, motility, acrosome integrity, and mitochondrial membrane potential. The objective of the present study was to evaluate a new extender, known as Thai native chicken (TNC) extender compared to BHSV-based and modified Sasaki extenders for freezing chicken semen. Semen from Thai native roosters was collected, pooled, and randomly divided into three groups. Semen was frozen with a simple freezing method using nitrogen vapor and dimethylformamide. In the first experiment, post-thaw motion parameters, viability, acrosome integrity, mitochondrial function, and lipid peroxidation levels were analyzed using computer-assisted sperm analysis, propidium iodide, fluorescein isothiocyanate-conjugate peanut agglutinin, JC-1, and the thiobarbituric acid reaction. Results showed that the type of extender had no effect on the percentage of total motile and curvilinear velocity. The percentage of progressive motile, straight-line velocity, and average path velocity of post-thawed semen were significantly lower in TNC compared to the modified Sasaki extender. However, the percentages of post-thawed acrosome integrity and active mitochondria were significantly higher in TNC extender (P < 0.05). For the second experiment, semen was thawed by using each of extenders thereafter, was inseminated to 48-layer breeder hens to determine the fertility rate. Among the three extenders used, the highest fertility rate was found in TNC extender. In conclusion, TNC extender can be recommended as an appropriate and useful cryopreservation media for native chicken semen since it maintains the quality of rooster semen and fertility after freezing and thawing process.     

The synergistic effect of trehalose and low concentrations of cryoprotectants can improve post-thaw ram sperm parameters

The objective of this study was to compare the effects of different concentrations of two different cryoprotectants (glycerol, G and ethylene glycol, EG) and trehalose (T), added to the semen extender, on post-thaw ram sperm parameters. Ejaculates, collected from 6 Merino rams, were pooled and evaluated at 37 °C. The pooled samples were divided into six equal aliquots, and diluted in Tris-based extenders containing 5% G, 3% G + 60 Mm T, 1.5% G + 100 Mm T, 5% EG, 3% EG + 60 mM T, and 1.5% EG + 100 Mm T. Subsequently, the samples were cooled to 5 °C, frozen in 0.25-ml French straws, and stored in liquid nitrogen (LN₂). Frozen samples were thawed individually, at 37 °C for 25 s in a water bath, for evaluation. Sperm motility was assessed using a phase-contrast microscope with a warm stage maintained at 37 °C. Acrosome integrity (FITC/PNA-PI), sperm viability (SYBR-14/PI), mitochondrial activity (JC-1/PI), DNA damage (COMET assay) and DNA fragmentation (TUNEL test) were determined. The group of samples diluted in an extender containing 5% of glycerol (Group 5% G) displayed higher percentages of subjective motility, viability and mitochondrial activity of sperm, compared to the other groups (P < 0.05). On the other hand, Group 3% G + 60 mM T yielded the second-best results for subjective motility, viability and mitochondrial activity of sperm, when compared to the other groups. The post-thaw sperm parameters of Group 3% G + 60 Mm T did not show any statistically significant difference from those of Group 5% G. There were no statistically significant differences between the groups for acrosome integrity (P > 0.05).The results of the COMET assay showed that the use of low concentrations of cryoprotectants in combination with trehalose decreased sperm DNA damage. Accordingly, Group 1.5% G + 100 mM T and Group 3% EG + 60 mM T benefited from a significantly stronger cryoprotective effect on DNA integrity, in comparison to Group 5% G (P < 0.05). According to the results of the TUNEL test, the combined use of low concentrations of cryoprotectants with trehalose decreased sperm DNA damage, when compared to the use of 5% glycerol, but this difference was statistically insignificant (P > 0.05).In conclusion, G and EG concentrations can be reduced by adding various amounts of T (60 mM, 100 mM) to the semen extender. The addition of 5% of glycerol and 3% G + 60 mM T to the semen extender did not yield statistically different post-thaw sperm parameters, when compared for protection against cryoinjury. Post-thaw sperm parameters can be improved by the supplementation of the semen extender with 3% G + 60 mM T. Thus, we recommend the use of freezing extenders containing low cryoprotectant concentrations (3% G) combined with trehalose to avoid the high level of toxic and osmotic damage caused by 5% G.     

Use of commercial extenders and alternatives to prevent sperm agglutination for cryopreservation of brown bear semen

The objective of this study was to evaluate different bovine and canine commercial semen extenders for cryopreservation of brown bear ejaculates and the effect of semen collection directly into extender on sperm agglutination. Semen samples were obtained by electroejaculation from 13 adult males. In experiment 1, eleven ejaculates from eight bears were used to evaluate Bioxcell and Andromed as extenders, whereas in experiment 2, nine ejaculates from six bears were used to evaluate Triladyl canine, CaniPro, and Extender 2 as extenders. An extender specifically developed for brown bears (Test-Tris-fructose-egg yolk-glycerol, TTF-ULE/bear) served as a control extender in both experiments. After thawing, total and progressive sperm motility and sperm viability were greater (P < 0.05) for TTF-ULE/bear and Andromed extenders than for Bioxcell in experiment 1 and greater (P < 0.05) for TTF-ULE/bear extender than for Triladyl Canine, CaniPro, and Extender 2 in experiment 2. In experiment 3, addition of handling extender (TTF-H) to the semen collection tube for eight ejaculates from seven bears resulted in less (P < 0.05) sperm agglutination in fresh samples (score 0.5 ± 0.2 vs. 1.8 ± 0.4 in diluted and control samples, respectively) with no effect on pre-freeze and post-thawing semen quality. In conclusion, TTF-ULE/bear is the most suitable extender for brown bear semen cryopreservation, but comparable results can be obtained with the commercial extender Andromed. In addition, collection of ejaculates directly in TTF-H extender decreases sperm agglutination in fresh samples.     

Cryopreservation of Indian red jungle fowl (Gallus gallus murghi) semen with polyvinylpyrrolidone

The Indian red jungle fowl is a sub-species of the genus Gallus native to South Asia; facing high risk of extinction in its native habitat. During cryopreservation, permeable cryoprotectants like glycerol are usually employed and we previously showed encouraging results with 20% glycerol. Because bird spermatozoa contain very little intracellular water, the possibility of replacing an internal cryoprotectant by an external one is opened. In the present study, we tested the replacement of internal cryoprotectant glycerol by the external cryoprotectant Polyvinylpyrrolidone (PVP). PVP is a non-permeable cryoprotectant and keeps the sperm in glassy state both in cooling and warming stages without making ice crystallization within the sperm cell. We evaluated the effect of various levels of polyvinylpyrrolidone (PVP) on Indian red jungle fowl semen quality and fertility outcomes. The qualifying semen ejaculates collected from eight mature cocks were pooled, divided into five aliquots, diluted (37 °C) with red fowl semen extender having PVP [0% (control) 4% (w/v), 6% (w/v), 8% (w/v) and 10% (w/v)]. Diluted semen was cryopreserved and stored in liquid nitrogen. The whole experiment was repeated/replicated for five times independently. Sperm motility, plasma membrane integrity, viability and acrosome integrity were recorded highest (P < 0.05) with 6% PVP at post-dilution, cooling, equilibration and freeze-thawing. Higher (P < 0.05) no. of fertile eggs, fertility, no. of hatched chicks, percent hatch and hatchability was recorded with 6% PVP compared to control. It is concluded that 6% PVP maintained better post-taw quality and fertility of Indian red jungle fowl spermatozoa than glycerol and can be used in routine practice avoiding the contraceptive effects of glycerol.     

Effect of different concentrations of l-carnitine in extender for semen cryopreservation in sheep

This study assessed the effect of l-carnitine (LC) in sheep semen extenders containing or not egg yolk for cryopreservation in sheep. Two extenders (TRIS-egg yolk or the commercial optiXcell™ IMV medium) were used, totaling six groups: IMV – (0, 5 and 10 mM LC) and TRIS – (0, 5 and 10 mM LC). After the freezing-thawing process and throughout incubation at 38 °C for up to 3 h, several parameters were evaluated: sperm kinetics, hypoosmotic, plasma membrane integrity, capacitation status and lipid peroxidation level. The supplementation of either 5 or 10 mM LC randomly affected some parameters and, overall, TRIS was superior (P < 0.05) than IMV extender. In the LC-groups, IMV had greater (P < 0.05) oxidative stress than TRIS. In conclusion, although LC affected isolated parameters, its supplementation in semen extender had no consistently beneficial effect on freezing-thawing ram sperm.     

Quality and fertilizing ability of electroejaculated cat spermatozoa frozen with or without Equex STM Paste

An optimal protocol for cat semen cryopreservation has not yet been defined. Addition of Equex STM Paste has been tested for epididymal cat spermatozoa but not for ejaculated cat spermatozoa. Furthermore, the effect of Equex STM Paste on fertilizing ability of cryopreserved semen has never been evaluated in that species. Therefore, the aims of the current study were to investigate if addition of Equex STM Paste to a freezing extender for electroejaculated cat (Felis catus) semen would improve postthaw sperm quality and if sperm fertilizing ability after cryopreservation with or without Equex STM Paste was preserved. Semen was collected by electroejaculation and frozen in a Tris-glucose-citrate egg yolk extender supplemented with (0.5% vol/vol) or without Equex STM Paste. In Experiment 1, sperm motility, membrane integrity, and acrosomal status were determined immediately after collection and at 0, 3, and 6 h postthaw. In Experiment 2, frozen semen from the two groups was used for in vitro fertilization (IVF) of in vitro-matured cat oocytes. Cleavage rate was recorded 30 h after IVF, and embryo development was evaluated on Days 6 and 7 of culture. In Experiment 1, the rate of motile spermatozoa after freezing-thawing was higher when Equex STM Paste was added to the freezing extender, but progressive motility score was not influenced (P > 0.05). Sperm membrane integrity was positively affected (P < 0.05) by the addition of the detergent. Intact acrosomes after thawing were similar (P > 0.05) between groups. Even if the decreasing rates of motility and membrane integrity were more rapid in presence of Equex than those in controls, total motility and sperm viability were similar at 3 and 6 h after thawing (P > 0.05). In Experiment 2, there was no difference in fertilizing ability and embryo development between the two groups (P > 0.05). The results of this study demonstrate that the addition of Equex STM Paste in the freezing extender avoids the loss of motile spermatozoa and maintains fertilizing ability of frozen-thawed spermatozoa.     

Short-term storage sperm of coho salmon (Oncorhynchus kisutch) at 4 °C: Effect of sperm: Extender dilution ratios and antioxidant butyl-hydroxytoluene (BHT) on sperm function

Short-term storage of semen is a necessary key procedure in fish; it allows maximizing the use of gametes. Nevertheless, sperm quality decreases during storage has been associated with oxidative stress damage due to an increase in reactive oxygen species (ROS) during storage. This study was designed to optimize a short-term storage protocol for Coho salmon (Oncorhynchus kisutch) spermatozoa, evaluating the effect of extender dilution and the addition of butyl-hydroxytoluene (BHT) antioxidant on sperm function parameters. In the first experiment, fresh semen was diluted in Storfish®: extender dilution (1:2 and 1:3) and a control sample undiluted and stored at 4 °C for 7-days. In both experiments motility (MO), viability and integrity of plasma membrane, mitochondrial membrane potential (MMP) and superoxide anion level (O₂⁻) were evaluated at 0, 3 and 7 days. Result shows that, 1:3 dilution maintained a higher sperm function for a longer period time. In the second experiment, spermatozoa were suspended in Storfish® (1:3) supplemented with two different concentrations of BHT (1.0 mM and 2.0 mM) and a control sample without antioxidant and stored at 4 °C for 7 days. The results demonstrated that, antioxidant-supplemented samples greater MO than control samples (P < 0.05). The viability remained >75% during storage in all groups. MMP was higher in 2.0 mM BHT compared to 1.0 mM and control (P < 0.05), in addition, this concentration reduced O₂⁻ level (P < 0.05). In conclusion, sperm: extender dilution 1:3 and adding of 2.0 mM BHT in sperm storage extender may enhance protection sperm function in Oncorhynchus kisutch against effects harmful of the oxidative stress during the in vitro storage.     

Freeze–thaw induced genotoxicity in buffalo (<Emphasis Type="Italic">Bubalus bubalis</Emphasis>) spermatozoa in relation to total antioxidant status

Use of cryopreserved semen has become an important tool in assisted reproduction but freezing and thawing cause sub-lethal damage to spermatozoa. This is detrimental to sperm because of the membrane damage including permeability and integrity. An excess generation of reactive oxygen species (ROS) creates oxidative stress due to reduced antioxidant status of the cryopreserved spermatozoa. In the present study fresh buffalo semen was collected and divided into two aliquots. One aliquot was used for fresh semen analysis and the other was cryopreserved in Tris-egg yolk-citrate extender. The semen samples were used to study different sperm quality parameters like motility, viability, membrane integrity and total antioxidant status. The DNA integrity in fresh and cryopreserved spermatozoa was also studied using comet assay. The sperm quality parameters like post-thaw sperm motility, viability, membrane integrity and total antioxidant status of cryopreserved spermatozoa were significantly lowered (P < 0.05) compared to fresh spermatozoa. The DNA fragmentation in cryopreserved spermatozoa was significantly higher (P < 0.01) as compared to fresh spermatozoa. The results show that the irreversible DNA damage occurs in spermatozoa during cryopreservation.     

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 20 s in a water bath for evaluation.Ergothioneine at doses of 2 and 4 mM 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 1 mM 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 2 mM, compared to control (P < 0.05). No significant differences were observed in sperm acrosome integrities among groups. CAT activity was increased significantly only in cysteine1 mM compared to control group (P < 0.001). Cysteine at doses of 2 and 4 mM 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.     

Effect of Simmental bull seminal plasma protein in egg yolk-citrate extender on Kacang buck semen fertility

The genetic resources of Indonesia's indigenous Kacang goat require preservation. Artificial insemination is expected to accelerate population increases and preserve genetic resources simultaneously. The present study was the maiden attempt for cryopreservation of Kacang buck sperm. The objectives of this study were to determine whether the supplementation of superior Simmental bull seminal plasma protein in egg yolk-citrate extender could improve the quality of post-thawed Kacang buck sperm, increase conceptions rates, and improve kidding rates. Buck semen was diluted without supplementation (T0) and with supplementation of 2.5 mg (T1) and 5 mg (T2) of Simmental bull seminal plasma protein per mL egg yolk-citrate extender. Extended semen was packed in 0.25 mL straw as cryopreserved frozen semen. Post-thawed semen samples were evaluated for viability, motility, intact plasma membranes, malondialdehyde level, and DNA fragmentation. Estrus was synchronized for sixty Kacang does, which were divided randomly into three groups and inseminated using post-thawed semen. The progesterone serum concentration of the does was measured 7 and 22 days post-insemination to detect ovulation and conception. Pregnancy was confirmed using abdominal palpation at 43 days post-insemination and by observing birth. The T1 group showed the highest (P < 0.05) post-thawed viability, motility, and intact plasma membrane. Conception, pregnancy and kidding rates were also higher in T1 than other treatment groups. In conclusion, the 2.5 mg Simmental bull seminal plasma protein supplementation per mL egg yolk-citrate extender provided the best seminal quality and fertility of post-thawed Kacang buck semen.     

The influence of trehalose, taurine, cysteamine and hyaluronan on ram semen Microscopic and oxidative stress parameters after freeze-thawing process

There is a lack of information regarding lipid peroxidation and antioxidant capacity in cryopreserved ram semen, and cryopreservation is associated with the production of reactive oxygen species (ROS) which lead to lipid peroxidation (LPO) of sperm membranes, resulting in a loss of motility, viability and fertility of sperm. The aim of this study was to determine the influence of certain additives and their different doses on standard semen parameters, lipid peroxidation and antioxidant activities after the cryopreservation/thawing of ram semen. Ejaculates collected from four Akkaraman rams, a native breed of sheep, were evaluated and pooled at 33 degrees C. Semen samples which were diluted with a Tris-based extender containing additives including trehalose (50, 100mM), taurine (25, 50mM), cysteamine (5, 10mM), and hyaluronan (0.5, 1mg/ml), and an extender containing no additives (control) were cooled to 5 degrees C and frozen in 0.25ml French straws, being stored in liquid nitrogen. Frozen straws were thawed individually at 37 degrees C for 20s in a water bath for evaluation. The use of a Tris-based extender supplemented with 50mM trehalose, 25mM taurine, and 5 and 10mM cysteamine led to higher percentages of post-thaw motility, in comparison to the control group (P<0.01). No significant differences were observed in the percentages of acrosome and total abnormalities, and the hypoosmotic swelling test upon the supplementation of the freezing extender with antioxidants after the thawing of semen. In biochemical assays, the addition of antioxidants did not cause significant differences in levels of malondialdehyde (MDA), glutathione (GSH), and glutathione peroxidase (GSH-Px), after thawing, when compared to groups with no additives. In this study, catalase (CAT) activities were higher in the group that was applied 25mM taurine as an antioxidant, than in all of the other groups (P<0.001). Compared to the controls, antioxidant treatment with 100mM trehalose, 50mM taurine, 5mM cysteamine and 0.5mg/ml hyaluronan, significantly elevated vitamin E (vit E) levels in samples (P<0.001).