Preservation of rooster post-thawed sperm epigenetic modifications, fertility potential and other quality parameters in different extenders using reduced glutathione.

Although rooster semen cryopreservation is an efficient procedure to spread qualified semen samples for reproductive goals, some post-thawed qualified semen samples resulted in poor fertility rate that could be related to epigenetic modifications during the cryopreservation process. This research was conducted to investigate the effect of reduced glutathione (GSH) in different cryopreservation extenders (Lake and Beltsville) on preservation of epigenetic modifications, fertility potential and other quality parameters of rooster sperm after thawing. Semen samples were collected and diluted in Lake and Beltsville extenders as follows: L-0: Lake without GSH, L-G: Lake with GSH, B-0: Beltsville without GSH, and B-G: Beltsville with GSH. After freeze-thawing process, sperm motility, membrane functionality, mitochondrial activity, acrosome integrity, viability, apoptosis status, lipid peroxidation, DNA fragmentation, ROS concentration, epigenetic modifications and fertility potential were evaluated. In results, the type of extender had no effect (P > 0.05) of post-thawed sperm quality. The treatments containing GSH presented higher (P ≤ 0.05) total motility, progressive motility, membrane functionality, mitochondrial activity, acrosome integrity, viability, DNA methylation, fertility as well as lower (P ≤ 0.05) lipid peroxidation, apoptosis, DNA fragmentation and ROS concentration than other treatments. Extender supplementation with GSH had no effect (P > 0.05) on histone methylation, histone acetylation and hatching rate. In conclusion, supplementation of rooster sperm cryopreservation extender with GSH could be an effective strategy to preserve post-thawed sperm DNA methylation, fertility and other quality parameters during reproductive programs.

Preservation of the quality and fertility potential of post-thawed rooster sperm using MitoQ.

Cryopreservation of rooster spermatozoa is an efficient procedure to spread qualified semen samples for reproductive goals in commercial flocks, but the freeze-thawing process reduces the quality and fertility potential of post-thawed sperm cells. This study was aimed to investigate the effect of the mitochondria-targeted antioxidant MitoQ on rooster sperm quality and fertility potential preservation during freeze-thawing process. Semen samples were collected and diluted in the Lake medium, assigned into five equal aliquots, supplemented with 0, 1, 10, 100 and 1000 nM MitoQ, and cryopreserved in liquid nitrogen. After thawing, sperm motility, membrane functionality, abnormal morphology, mitochondria active potential, acrosome integrity, viability, apoptosis status, lipid peroxidation, DNA fragmentation, ROS concentration and fertility potential were evaluated. According to the results, freezing extender supplementation with 10 and 100 nM MitoQ presented higher (P ≤ 0.05) total motility, progressive motility, average path velocity, membrane functionality, mitochondria active potential, acrosome integrity and viability compared to the other groups. On the other hand, lipid peroxidation, apoptosis rate, DNA fragmentation and ROS concentration were lower (P ≤ 0.05) in groups received 10 and 100 nM MitoQ compared to other groups. Moreover, fertility rate was higher in groups received 10 and 100 nM MitoQ compared to control group. Therefore, MitoQ is able to preserve quality parameters and fertility potential of post-thawed spermatozoa in rooster and it could be an effective additive for supplementation of rooster's semen cryopreservation medium during reproductive programs.

Mitochondria-targeted antioxidant "MitoQ" improves rooster's cooled sperm quality indicators and reproductive performance.

The cell membrane of rooster sperm is sensitive to cold due to the high content of polyunsaturated fatty acids, which are very susceptible to lipid peroxidation. The present study was conducted to determine the effect of different concentrations of the mitochondrial-targeting antioxidant "MitoQ" on sperm quality and fertility potential of chilled semen in roosters. Semen samples were collected from 10 roosters, diluted in Lake extender, assigned into 5 groups according to MitoQ concentrations (0, 1, 10, 100 and 1000 nM MitoQ) and stored at 5 °C up to 48 h. Motility, mitochondrial activity, viability, membrane integrity, and lipid peroxidation were assessed at 0, 24, and 48 h of cold storage periods. In addition, the fertility potential was assessed using 24 h-cooled semen samples. Our results showed that extender supplementation with MitoQ had no effect (P > 0.05) on chilled semen samples quality parameters at time 0, while at times 24 and 48 h storage, samples contained 100 nM MitoQ presented higher (P ≤ 0.05) total motility, progressive motility, viability and membrane integrity compared to the other groups. In addition, semen samples containing 10 and 100 nM MitoQ showed higher (P ≤ 0.05) mitochondrial activity and lower (P ≤ 0.05) lipid peroxidation than other groups at 24 and 48 h storage. Fertility rate was higher (P ≤ 0.05) when the hens were artificially inseminated with 24 h-chilled semen samples containing 100 nM MitoQ. In conclusion, supplementing Lake Extender with 100 nM MitoQ could be a helpful strategy to preserve chilled semen quality and fertility potential in the rooster.

Flow cytometry study of post-thawed buck spermatozoa: Mito-TEMPO improves cryopreservation performance by controlling apoptosis rate, DNA fragmentation and ROS production.

Sperm cryopreservation is used to spread qualified semen for artificial insemination, but the freezing process reduces sperm quality. This study assessed the efficacy of Mito-TEMPO on post-thawed goat sperm quality. Semen samples divided to five equal groups and after dilution, received different doses of Mito-TEMPO (0, 1, 10, 100 and 1000 µM), and cryopreserved in liquid nitrogen. After thawing, flow cytometry analysis was performed to evaluate sperm mitochondria membrane potential, viability, apoptotic-like changes, DNA fragmentation and ROS concentration. According to the results, Mito-TEMPO (10 and 100 µM) improved (P ≤ 0.05) sperm viability and decreased (P ≤ 0.05) apoptotic-like changes and ROS concentration compared to the other groups. Mitochondria membrane potential was higher (P ≤ 0.05) in groups received 1, 10 and 100 µM Mito-TEMPO. The lowest (P ≤ 0.05) DNA fragmentation was observed in group received 10 µM Mito-TEMPO. In conclusion, mitochondria-targeted antioxidant Mito-TEMPO could be an efficient cryo-additive to enhance flowcytometric quality parameters of post-thawed buck semen.

Supplementation of sperm cooling medium with Zinc and Zinc oxide nanoparticles preserves rooster sperm quality and fertility potential.

Supplementation of sperm cooling medium with helpful additives is a reasonable method to conserve sperm fertility potential during cooling storage process. This study was aimed to determine the effect of sperm cooling medium supplementation with Zinc and Zinc oxide nanoparticles (NZn and NZnO) on rooster semen quality and fertility efficiency during storage periods. Semen samples were diluted in the Lake medium and assigned into five equal aliquots. The first was Control group and the other groups received 50 µg/ml NZn, 50 µg/ml NZnO, 100 µg/ml NZn and 100 µg/ml NZnO. Then, the samples were cooled at 5 °C and conserved up to 45 h. Total motility, progressive motility, mitochondrial activity, viability, membrane integrity and lipid peroxidation of samples were analyzed during 0, 22 and 45 h post-cooling. Artificial insemination was also performed using 22- hrs cooled semen. No difference was found among groups during quality evaluations at 0 h storage. Extender supplementation with 100 µg/ml NZn and NZnO presented higher (P ≤ 0.05) total motility, progressive motility, mitochondrial activity, viability, membrane integrity and lower lipid peroxidation compared to other groups during 22 and 45 h cooling storage. Fertility rate of 22-h cooled-stored semen samples was higher (P ≤ 0.05) in groups contained 100 µg/ml NZn and NZnO compared to the Control group. In conclusion, addition of 100 µg/ml NZn and NZnO to the sperm storage medium could be introduced as an effective method to preserve rooster semen quality during cooling storage period.

Cysteamine enhances quality and fertility potential of rooster semen in cooled storage.

This study investigated the effects of supplementing Lake extender with cysteamine (CYS) on rooster semen quality in cold storage and it's fertility performance. Semen samples were diluted with Lake extender supplemented with different concentrations of CYS (0, 1, 2, 4 and 8 mM) and were cooled and stored at 5 °C for a period of 46 h. Motility, membrane functionality, viability, lipid peroxidation, and mitochondria membrane potential were evaluated at 0, 23 and 46 h of storage. Fertility was assessed at 23 h of storage. Although at the beginning time (0 h), parameters were not affected, 1 mM of CYS improved (P ≤ 0.05) total motility, progressive motility and mitochondria membrane potential during 23 and 46 h storage. Moreover, 1 and 2 mM CYS improved (P ≤ 0.05) membrane functionality and viability compared to other groups. Lipid peroxidation was lower (P ≤ 0.05) in samples diluted with 1 and 2 mM CYS compared to the others. Artificial insemination with 23-hrs cooled-stored semen produced the higher (P ≤ 0.05) fertility rate in groups received 1 and 2 mM CYS compared to the control group. In conclusion, addition of 1 and 2 mM CYS to the extender could be helpful to protect rooster semen against structural and functional damages of cooling storage process.

Rooster frozen-thawed semen quality following sublethal xanthine oxidase treatments.

Reactive oxygen species are associated with cryodamage and may be a factor causing or exacerbating cellular cryodamage during freezing and thawing processes. Induction of sublethal oxidative stress as a new approach for preconditioning of sperm improves the cryo-resistance of sperm. The aim of this study was to investigate effects of sublethal concentrations of xanthine oxidase (XO), which induces oxidative stress before cryopreservation on values for semen quality variables of rooster sperm post-thawing. Semen samples were collected from 15 roosters and treated with different concentrations of XO [XO-0, XO-0.005, XO-0.05, XO-0.5, XO-5, and XO-50 U/ml]; then, the effects of treatments with XO as sublethal stressors, were examined. Results indicated the XO-0.5 and XO-5 treatments resulted in a greater percentage of sperm total motility, progressive motility, viability, and membrane functionality compared to other groups. There was no difference after treatments with XO-0, XO-0.005, and XO-0.05 on sperm total motility, membrane functionality, apoptosis, mitochondria activity, and viability. There was a greater percentage of mitochondria activity in sperm of the XO-0.05, XO-0.5, and XO-5 groups. Furthermore, there was the greatest concentration of malondialdehyde (MDA) in samples of the XO-50 group. Values for sperm abnormal morphology, acrosome integrity, and DNA fragmentation were not different among samples post-thawing. Sperm treated with XO-0.5 and XO-5 had a greater fertilization capacity than those of the control group. In conclusion, treatment of sperm with 0.5 and 5 U/ml XO as inducers of mild oxidative stress before cryopreservation, improved several function quality indices of sperm post-thawing.

Effect of Dietary Fish Oil on Semen Quality and Reproductive Performance of Iranian Zandi Rams.

The current study was conducted to evaluate the effect of dietary fish oil on the semen quality and fertility potential of Zandi rams. For this purpose, a total of 15 Iranian Zandi rams were randomly assigned into three equal groups. The first group was a negative control and were fed without oil supplement. The second group was a positive control, and their diet contained palm oil, and the last group had a diet containing fish oil. All the diets were isocaloric and isonitrogenous. The rams were fed for 70 days, and the semen smaples were collected every 10 days. In experiment I, the evaluated parameters included semen volume, sperm concentration, motility, membrane integrity, and viability. In experiment II, 210 Iranian Zandi rams received CIDR for 12 days and 400 IU of equine chorionic gonadotropin at the time of CIDR removal. Then, they were assigned into three equal groups and artificially inseminated with semen samples. According to the obtained results, the supplementation of ram diet with fish oil as a source of omega-3 fatty acids improved the semen volume, sperm concentration, total motility, progressive motility, viability, membrane integrity, pregnancy rate, parturition rate, and lambing rate of the rams (P≤0.05). In conclusion, fish oil as a dietary supplement for rams could be an effective strategy to improve the semen quality of rams for artificial insemination and other goals.

Flow cytometry study of post-thawed bulk spermatozoa: Mito-TEMPO improves cryopreservation performance by controlling apoptosis rate, DNA fragmentation and ROS production.

Sperm cryopreservation is used to spread qualified semen for artificial insemination, but the freezing process reduces sperm quality. This study assessed the efficacy of Mito-TEMPO on post-thawed goat sperm quality. Semen samples divided to five equal groups and after dilution, received different doses of Mito-TEMPO (0, 1, 10, 100 and 1000 µM), and cryopreserved in liquid nitrogen. After thawing, flow cytometry analysis was performed to evaluate sperm mitochondria membrane potential, viability, apoptotic-like changes, DNA fragmentation and ROS concentration. According to the results, Mito-TEMPO (10 and 100 µM) improved (P ≤ 0.05) sperm viability and decreased (P ≤ 0.05) apoptotic-like changes and ROS concentration compared to the other groups. Mitochondria membrane potential was higher (P ≤ 0.05) in groups received 1, 10 and 100 µM Mito-TEMPO. The lowest (P ≤ 0.05) DNA fragmentation was observed in group received 10 µM Mito-TEMPO. In conclusion, mitochondria-targeted antioxidant Mito-TEMPO could be an efficient cryo-additive to enhance flowcytometric quality parameters of post-thawed bulk semen.

Conservation of Buck's Spermatozoa during Cooling Storage Period through Cooling Medium Supplementation with L-Carnitine.

This research examined the influence of the addition of L-carnitine (LC) to cooling medium on buck's semen quality during cooling storage periods at 4oC. Semen samples were collected, diluted, assigned into four groups, and received LC (0, 1, 5, and 10 mM LC). The samples were then chilled to 4oC and stores for 48 h. Sperm total motility, progressive motility, viability, lipid peroxidation, membrane integrity, and mitochondrial activity were examined at 0, 24, and 48 h of cooling storage. At time 0 of cooling storage, different treatments showed no impact on the quality of sperm samples (P>0.05). During 24 and 48 h of chilling periods, the supplementation of cooling medium with 5 mM LC presented greater motility, viability, membrane integrity, and mitochondrial activity (P≤0.05), compared to the other groups. Moreover, the treatment of 5 mM LC caused lower lipid peroxidation (P≤0.05) than the other treatments at 24 and 48 h storage times. In conclusion, the supplementation of buck's cooling storage medium with 5 mM LC is a suitable way to protect buck spermatozoa during 24 and 48 h storage against cold-induced structural and functional damages.

Modulation of Negative Effects of Physiological Stress on Frozen-Thawed Semen with Nutrition of Organic Selenium in Ross 308 Rooster.

Current experiment was carried out in factorial 2×2 arrangement to study the effects of stress (with or without dexamethasone administration) and addition of dietary selenium (with or without selenium supplementation in the diet) in male broiler breeder on the quality of frozen-thawed sperm under oxidative stress induced by dexamethasone. A total of 24 broiler breeder roosters with the age of 28 weeks were used based on a completely randomized design with four therapeutic approaches (factorial 2×2) and six birds in each approach. The experimental treatments were: 1) basal diet without selenium supplementation and injection of saline (CON), 2) basal diet with dexamethasone injection (4 mg/kg BW, three times every other day for one week), (DEX), 3) without dexamethasone injection and supplementation with 0.3 mg/kg selenium (Sel-Plex), and 4) dexamethasone injection and basal diet supplemented with 0.3 mg/kg of diet selenium (Sel-Plex+Dex). Sperm samples were collected from roosters. Motility, progressive motility, plasma membrane integrity, viability, malondialdehyde concentration and antioxidant parameters were evaluated in fresh and frozen-thawed semen. In spite of non-significant interaction effects, factorial analysis indicated the significant effect of every factor on different experimental parameters in fresh and frozen-thawed semen (P<0.05); The results revealed that total and progressive motility, plasma membrane integrity and viability were lower in DEX group when compared with other treatments (P<0.05). On the other hand, malondialdehyde concentration was higher in DEX group in comparison with Con, Sel-Plex and Sel-Plex+DEX groups (P<0.05). Moreover, total antioxidant capacity, level of glutathione peroxidase and superoxide dismutase were lower in DEX group as compared with other treatments (P<0.05). Our findings indicated that administration of selenium in dexamethasone-receiving roosters (Sel-Plex+DEX) improved the parameters of fresh and frozen-thawed sperm; but the best results were observed in Sel-Plex treatment. Therefore, selenium supplementation in the diet of roosters without dexamethasone injection improved total motility, progressive motility, membrane integrity, viability, malondialdehyde, total antioxidant capacity, glutathione peroxidase and superoxide dismutase pre- and post-freezing. It can be concluded, selenium in organic forms in stressed and non-stressed rooster's diet might improve all motility and antioxidant parameters in fresh and frozen-thawed sperm.

Supplementation of ram's semen extender with Mito-TEMPO I: Improvement in quality parameters and reproductive performance of cooled-stored semen.

Supplementation of cooling medium with some antioxidants could be a helpful way to improve sperm quality during chilling process. The current study was aimed to assess the influence of using Mito-TEMPO in cooling medium on quality parameters and reproductive performance of sheep semen during chilling process. In this study, diluted semen samples were assigned into 5 parts, and received 0, 0.5, 5, 50 and 500 µM Mito-TEMPO. The prepared samples were stored at 5 °C up to 48 h. Chilled sperm motility, viability, abnormal morphology, mitochondrial membrane potential, membrane functionality and malondialdehyde concentration were assessed during 0, 24 and 48 h. For evaluation of reproductive performance, artificial insemination was performed via 24 h-chilled semen. In results, at time 0, no difference was observed among groups. Using 5 and 50 µM Mito-TEMPO resulted in higher (P ≤ 0.05) cooled sperm total motility, progressive motility, membrane functionality, viability and lower malondialdehyde concentration than the other groups during 24 and 48 h storage. The rate of mitochondrial membrane potential was greater (P ≤ 0.05) in treated groups with 5, 50 and 500 µM Mito-TEMPO. Pregnancy, parturition and lambing rates were higher (P ≤ 0.05) when ewes were inseminated with 24 h-chilled semen samples containing 5 and 50 µM Mito-TEMPO compared to the control group. Therefore, supplementation of cooling medium with Mito-TEMPO (5 and 50 µM) could be an efficient method to improve the quality and reproductive efficiency of ram's cooled semen during storage period.

The mitochondria-targeted antioxidant Mito-TEMPO conserves rooster's cooled semen quality and fertility potential.

The PUFAs content of rooster sperm cells makes them vulnerable to the thermal shocks during chilling storage, which reduces the fertility performance of cooled sperm. Extender supplementation with antioxidants is a reasonable method to conserve sperm fertility potential during cooling storage process. The aim of this study was to determine the effect of Mito-TEMPO addition to the Lake medium on rooster sperm quality and fertility potential during cooling process. Semen samples were diluted in the Lake medium and assigned into five equal aliquots and supplemented with 0, 0.5, 5, 50 and 500 µM Mito-TEMPO. Then, the samples were cooled at 5 °C and conserved up to 50 h. Total motility, progressive motility, morphology, viability, membrane integrity, lipid peroxidation and mitochondrial activity of samples were analyzed during 0, 25 and 50 h of cooling period. Artificial insemination was also conducted using 25 h-cooled semen. No significant difference was observed among different treatments during quality evaluations at 0 h storage. Extender supplementation with 5 and 50 µM Mito-TEMPO presented greater (P ≤ 0.05) total motility, progressive motility, viability, membrane integrity and lower lipid peroxidation compared to other groups during 25 and 50 h cooling storage. Mitochondrial activity was higher (P ≤ 0.05) in groups received 5, 50 and 500 µM Mito-TEMPO than others. Fertility rate of 25 h-cooled-stored samples was higher (P ≤ 0.05) in groups containing 5 and 50 µM Mito-TEMPO compared to control group. In conclusion, addition of 5 and 50 µM Mito-TEMPO as a mitochondria-targeted antioxidant to the storage medium could be a suitable method to conserve rooster semen quality against stressful conditions of cooling storage process.

Supplementation of chilling storage medium with glutathione protects rooster sperm quality.

This study was aimed to evaluate the effect of addition of reduced glutathione (GSH) to the extender on the rooster's semen quality parameters and fertility potential. Semen samples were diluted with Lake extender contained 0, 0.5, 1, 2, 4 and 8 mM GSH. Then, were chilled to 5 °C and stored for a period of 48 h. Sperm motion characteristics, viability, membrane integrity, lipid peroxidation, mitochondrial activity and fertility potential were evaluated. At the initiation of the experiment (0 h), GSH did not affect sperm parameters, while 2-4 mM GSH improved (P ≤ 0.05) quality indicators during storage periods. Moreover, the samples treated with 2-4 mM GSH have had a lower lipid peroxidation compared to other groups (P ≤ 0.05). Artificial insemination using the semen samples, which had been stored in groups treated with 2-4 mM GSH for a period of 24 h, led to greater (P ≤ 0.05) fertilizing potential compared to the control group.

Improvement of rooster semen quality using coenzyme Q10 during cooling storage in the Lake extender.

Sensitivity of rooster semen to stressful condition of cooling restricts the semen storage in commercial flocks for artificial insemination. This study was accomplished to investigate the effect of coenzyme Q10 (CoQ10) addition to the Lake extender during chilled-storage on the parameters of sperm quality and fertility performance. Roosters' pooled semen samples were assigned into equal parts and diluted with Lake extender supplemented with different concentrations of CoQ10 (0, 1, 2, 5 and 10 µM CoQ10). Then, semen samples were cooled to 5 °C and stored over 48 h. Total and progressive motilities, abnormal morphology, viability, membrane functionality, lipid peroxidation (LPO) and mitochondria active potential of diluted sperm were evaluated at 0, 24 and 48 h of cooling storage. Fertility performance of cooled stored semen was examined at 24 h of cooling storage. Although CoQ10 did not affect sperm quality at the starting time of cooling storage (0 h), extender supplementation with 5 µM of CoQ10 showed higher (P ≤ 0.05) sperm total and progressive motilities, membrane functionality, viability and mitochondria active potential at 24 h as well as total motility, viability and membrane functionality at 48 h in contrast with other groups. Moreover, lipid peroxidation was lower (P ≤ 0.05) in semen samples diluted with 5 µM CoQ10 at 24 and 48 h compared to others. After artificial insemination with 24 h chilled-stored sperm, fertility efficiency was higher (P ≤ 0.05) in treatments contained 5 µM CoQ10 compared to the control group. According to the results, using optimum dose of CoQ10 could be helpful to save rooster semen against chilled storage structural and functional damages.

Effects of reduced glutathione on the quality of rooster sperm during cryopreservation.

The purpose of this study was to investigate the beneficial effects of reduced glutathione (GSH) for cryopreservation of rooster semen. In experiment 1, semen samples were collected from 15 roosters and diluted in the Lake extender that contained various concentrations of GSH as follows: Lake without GSH (control, GSH 0), Lake containing 0.5 mM (GSH 0.5), 1 mM (GSH 1), 2 mM (GSH 2), 4 mM (GSH 4) and 8 mM (GSH 8) GSH. Viability, membrane functionality, morphology, mitochondrial activity, acrosome integrity, motion parameters, lipid peroxidation and DNA fragmentation were assessed after thawing. In experiment 2, reproductive performance of thawed semen was evaluated via artificial insemination. Supplemented extenders with 2 and 4 mM GSH presented higher (P ≤ 0.05) viability (59.4 ±â€¯2.4% and 60.8 ±â€¯2.4%), membrane functionality (62.3 ±â€¯2.6% and 64.7 ±â€¯2.6%), mitochondrial activity (49.4 ±â€¯1.7% and 49.8 ±â€¯1.7%), total motility (57.1 ±â€¯1.9% and 58.8 ±â€¯1.9%, respectively), progressive motility (28.9 ±â€¯1.3% and 29.6 ±â€¯1.3%), and lower lipid peroxidation (2.4 ±â€¯0.09 nmol/ml and 2.3 ±â€¯0.09 nmol/ml) compared to control group. Acrosome integrity was higher (P ≤ 0.05) in GSH 4 (91.4 ±â€¯1.8%) compared to other groups. DNA fragmentation and MDA concentrations were higher (P ≤ 0.05) in GSH 8 (12 ±â€¯1.2% and 3.4 ±â€¯0.09 nmol/ml). In experiment 2, higher (P ≤ 0.05) fertility rate was observed in GSH 2 and GSH 4 (61.9% and 63.8%, respectively) compared to control (41.4%) group. In conclusion, supplementation of Lake extender with 2 and 4 mM GSH improves the cryo-survival and fertility potential of rooster sperm and it could be an applied method for improvement of reproductive goals.

L-carnitine is a survival factor for chilled storage of rooster semen for a long time.

Rooster sperm is sensitive to cooling, which restricts procedures to store sperms for extended periods of time for artificial insemination of commercial flocks. This study was conducted to evaluate the suitability of adding L-carnitine (LC) to chilled-storage of rooster sperm and its effects on sperm quality parameters and its fertility potential during storage at 5 °C. Pooled semen from roosters were divided into six equal aliquots and diluted with media supplemented with different concentrations of LC (0, 0.5, 1, 2, 4 and 8 mM LC). Diluted semen samples were cooled to 5 °C and stored over 48 h. Motility, viability, membrane functionality, lipid peroxidation and mitochondria activity of the sperm were assessed at 0, 24 and 48 h of storage. Moreover, fertility potential of chilled stored sperm was considered at 24 h of storage. While sperm quality was not affected by LC at the beginning of storage (0 h), supplementation of extender with 1 and 2 mM of LC significantly improved the percentage of sperm motility, viability, membrane integrity and mitochondria activity at 24 h and 48 h compared to other groups. Lipid peroxidation was significantly reduced in sperm samples diluted with 1 and 2 mM LC at 24 h (2.15 ± 0.52 nmol/ml and 2.21 ± 0.52 nmol/ml) and 48 h (3.42 ± 0.49 nmol/ml and 3.38 ± 0.49 nmol/ml) compared to other groups. Furthermore, fertility rates during artificial insemination using sperms cooled for 24 h in the presence of 1 and 2 mM LC were significantly higher (78%) than in the control group (64%). These findings suggest that optimum doses of LC could protect rooster sperm against cool storage-induced functional and structural damages.

L-Carnitine in rooster semen cryopreservation: Flow cytometric, biochemical and motion findings for frozen-thawed sperm.

Rooster semen cryopreservation is not efficient for artificial insemination in breeder flocks. L-Carnitine (LC) has been evaluated for effectiveness in cryopreservation media on the characteristics of rooster sperm after freeze-thawing. Motility characteristics, membrane functionality, abnormal morphology, apoptotic like changes, mitochondria activity and lipid peroxidation of rooster sperms were assessed after freeze-thawing with different concentrations of LC in Beltsville medium. Semen samples were collected from 12 roosters, twice a week, and diluted in the extenders that contained different concentrations of LC. Supplementation of Beltsevile with 1 and 2 mM LC was found to result in higher total motility (68.2± 1.7% and 69.1± 1.7%, respectively), progressive motility (28.4± 1.6%, 29.8± 1.6%), membrane functionality (76.2± 1.9% and 75.9± 1.9%), viability (58.2 ± 1.1%, 59.1 ± 1.1%) and lower significant of lipid peroxidation (2.53 ± 0.08 nmol/ml, 2.49 ± 0.08 nmol/ml) compared to control group containing no LC. Lower motility, progressive motility, and viability were observed in frozen-thawed sperm in extender containing 8 mM LC (35.8± 1.7%, 9.6± 1.2% and 27.1 ± 1.2%, respectively) compared to control. Morphology and mitochondrial activity were not affected by different concentrations of LC. Our results showed that supplementation of Beltsville extender with 1 and 2 mM LC significantly improved the quality of rooster sperm quality after freeze-thawing.

Effect of dietary fish oil supplementation on ram semen freeze ability and fertility using soybean lecithin- and egg yolk-based extenders.

Ram semen cryopreservation is not efficient for artificial insemination in commercial herds. Beneficial effects of dietary fish oil have been evaluated for cryopreservation of ram semen in soybean lecithin (SL) and egg yolk (EY)-based extenders. A factorial study (two diets × two extenders) was used to analyze the effects of two diets supplemented with fish oil (n-3 fatty acid) or palm oil (saturated fatty acids; [SFAs]) to freeze ram semen in two extenders containing SL or EY. Motility characteristics, membrane integrity, abnormal morphology, mitochondria activity, acrosome integrity, apoptotic status, and fertilizing ability were assessed after freeze-thawing. Although diet had significant (P ≤ 0.05) effects on the quality parameters of frozen-thawed sperm, effects of extenders on these traits were not significant (P > 0.05). The higher significant (P ≤ 0.05) percentage of total motility and progressive motility were observed in n-3/SL (44.83 ± 1.56 and 28.33 ± 1.4) and n-3/EY (43.33 ± 1.56 and 28.50 ± 1.4) than SFA/SL (32.16 ± 1.56 and 14.00 ± 1.4) and SFA/EY (31.66 ± 1.56 and 12.66 ± 1.4) groups. Moreover, n-3/SL and n-3/EY produced the higher significant (P ≤ 0.05) percentage of membrane integrity of sperm (39.83 ± 1.4 and 37.33 ± 1.4) than SFA/SL and SFA/EY (29.83 ± 1.4 and 28.5 ± 1.4). For viability results, the higher significant percentage of live sperm was observed in n-3/SL and n-3/EY (43.16 ± 1.38 and 45.66 ± 1.38) than SFA/SL and SFA/EY (28.66 ± 1.38 and 27.5 ± 1.38). For fertility trials, n-3-based diets (n-3/SL and n-3/EY) improved significantly (P ≤ 0.05) pregnancy rate (44% and 46%), parturition rate (42% and 42%), and lambing rate (46% and 44%) compared with the SFA-based diets (SFA/SL and SFA/EY). No interaction effects have been found between diets and extenders (P > 0.05). It seems that dietary fish oil can improve the semen performance after freezing-thawing process and artificial insemination aside from type of extenders.

Fertility and flow cytometry study of frozen-thawed sperm in cryopreservation medium supplemented with soybean lecithin.

Semen cryopreservation can provide genetic resources for a large number of females from a small number of superior males. Optimization of cryopreservation media to achieve the highest quality of post-thaw semen is crucial. Soybean lecithin has evaluated as a plant-based cryoprotectant for substitution of egg yolk in ram semen extender. Flow cytometric and fertility assessments were applied following cryopreservation procedure in two experimental groups (SL group: extender containing 1% w/v soybean lecithin and EY group: extender containing 20% v/v egg yolk). The higher percentage of live sperm and the lower percentage of dead sperm were obtained in SL (47.66 ± 1.38, 52.33 ± 1.69, respectively) extender compared to EY (41.16 ± 1.38, 58.83 ± 1.69). For motion characteristics, plasma membrane integrity, acrosome integrity and mitochondria activity, no significant difference was observed between SL and EY extenders. In artificial insemination experiment, there was no significant difference in pregnancy rate, lambing rate and twining rate between SL and EY extenders. It can be concluded that SL extender can be an efficient alternative extender to preserve ram sperm during cryopreservation procedure without adverse effects.