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 ... -  《-》

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 ... -  《-》

Comparative evaluation of Nabi and Beltsville extenders for cryopreservation of rooster semen.

Two experiments were conducted to evaluate the new rooster semen freezing extender which is containing a low level of glycerol and soybean lecithin as an alternative protective agent in the extender. The aim of the first experiment was to evaluate a new extender for freeze-thawing rooster semen known as "Nabi" extender compared to Beltsville. Second experiment was also performed to determine whether the Nabi extender has negative reactions on fertilization after artificial insemination (AI) or no. In the first experiment, post-thaw motion parameters, mitochondrial function and sperm apoptosis were analyzed using Sperm Class Analyzer (SCA), rhodamine-123 and Annexin-V, respectively for frozen-thawed semen in Nabi and Beltsville extender. Results showed that total motility, progressive motility, velocity parameters (VCL, VSL, VAP, LIN and STR) and live spermatozoa with active mitochondria were significantly higher in Nabi compare to Beltsville extender (P < 0.01). Also, the percentages of post-thawed live and early apoptotic spermatozoa were significantly higher in Nabi compared to Beltsville extender (14.46 ± 0.95 vs. 19.27 ± 0.95 and 14.83 ± 4.51 vs. 39.27 ± 4.51, respectively). For apoptotic spermatozoa, the percentages of post-thawed late apoptotic spermatozoa were significantly lower in Nabi (29.66 ± 3.11) compared to Beltsville extender (69.07 ± 3.11), but the type of extender had no effect on the percentages of post-thawed necrotic spermatozoa. In the second experiment, 20 broiler breeder hens (Ross 308) were inseminated with thawed semen using the new freezing diluents or fresh semen for determination of fertility rate. Fertility rate with thawed semen (with Nabi extender) was lower compared to fresh semen (by approximately 8% points). It can be concluded that Nabi extender would improve post-thawed rooster sperm in vitro quality compared to Beltsville extender. The fertility rates of insemination in hens with freeze-thaw sperm were comparable with fresh sperm.

Nabi MM ，Kohram H ，Zhandi M ，Mehrabani-Yeganeh H ，Sharideh H ，Zare-Shahaneh A ，Esmaili V ... -  《-》

Fertility and flow cytometric evaluations of frozen-thawed rooster semen in cryopreservation medium containing low-density lipoprotein.

Shahverdi A ，Sharafi M ，Gourabi H ，Yekta AA ，Esmaeili V ，Sharbatoghli M ，Janzamin E ，Hajnasrollahi M ，Mostafayi F ... -  《-》

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

Siari S ，Mehri M ，Sharafi M 《-》