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.

Masoudi R ，Hatami M ，Esmaeilkhanian S ，Zarei F ，Sharafi M ，Hatefi A ... -  《-》

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.

Alipour-Jenaghard P ，Daghigh-Kia H ，Masoudi R 《-》

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 ± 2.4% and 60.8 ± 2.4%), membrane functionality (62.3 ± 2.6% and 64.7 ± 2.6%), mitochondrial activity (49.4 ± 1.7% and 49.8 ± 1.7%), total motility (57.1 ± 1.9% and 58.8 ± 1.9%, respectively), progressive motility (28.9 ± 1.3% and 29.6 ± 1.3%), and lower lipid peroxidation (2.4 ± 0.09 nmol/ml and 2.3 ± 0.09 nmol/ml) compared to control group. Acrosome integrity was higher (P ≤ 0.05) in GSH 4 (91.4 ± 1.8%) compared to other groups. DNA fragmentation and MDA concentrations were higher (P ≤ 0.05) in GSH 8 (12 ± 1.2% and 3.4 ± 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.

Masoudi R ，Sharafi M ，Shahneh AZ ，Khodaei-Motlagh M ... -  《-》

Cryopreservation of rooster semen: Evidence for the epigenetic modifications of thawed sperm.

Although semen cryopreservation is an important, widely used technique for long-term sperm storage, it not only induces partially irreversible damages to sperm but might also deteriorate anatomical, biochemical, and structural organelles. These cellular and epigenetic modifications are the main reasons underlying the decline in sperm motility and fertility during the freeze-thaw process. Using the two Lake and Beltsville semen extenders, the present study aims to evaluate the epigenetic patterns (DNA methylation and histone modification), cellular parameters (e.g., membrane integrity, viability, DNA stability, mitochondria activity, and apoptosis status), and fertility potential of rooster semen collected from six mature roosters before and after cryopreservation according to a standard protocol. The results show that cryopreservation leads to significantly (P < 0.05) reduced values of the parameters examined when compared with those of fresh sperms. While the extenders used exhibit no difference with respect to DNA methylation (DNMT), the Lake extender leads to significant reductions (P < 0.05) in H3K9 acetylation (17.4 ± 1.8) and H3K4 methylation (42 ± 2.3) compared to the Beltsville (9.2 ± 1.8 and 23 ± 2.3, respectively). Compared to the Beltsville extender, the Lake one is also observed to yield a significantly (P < 0.05) superior sperm quality in terms of total motility (TM; 77.2 ± 1.6 vs. 68.3 ± 1.6), average path velocity (VAP; 71 ± 1.4 vs. 53 ± 1.4), and straight-line velocity (VSL; 52 ± 1.5 vs. 34 ± 1.5) as well as significantly (P < 0.05) higher viability (60 ± 1.69 vs. 51 ± 1.69) and membrane functionality (55 ± 3.2 vs. 46 ± 3.2). The Lake extender is also found to outperform the Beltsville one due to its significantly (P < 0.05) higher fertility rate (59.5% vs. 47.2%). The two extenders, however, exhibit no differences in DNA fragmentation, mitochondrial activity, or hatchability rate. The Beltsville extender showed to be superior to the Lake one due to its significantly greater reactive oxygen species percentage (ROS; 45.9 ±.3.2 vs. 28.5 ± 3.2) and apoptosis (29 ± 2.3 vs. 27 ± 2.3). It may be concluded that the Lake extender is capable of improving the cellular and epigenetic parameters of rooster sperms during cryopreservation due to the crucial roles it plays in the protection of sperms against cryo-damages.

Salehi M ，Mahdavi AH ，Sharafi M ，Shahverdi A ... -  《-》

Supplementation of Beltsville extender with quercetin improves the quality of frozen-thawed rooster semen.

Siari S ，Mehri M ，Sharafi M 《-》