Effect of sperm concentration on characteristics and fertilization capacity of rooster sperm frozen in the presence of the antioxidants catalase and vitamin E.

The objective of this study conducted was to determine the influence of different levels of sperm concentration, including catalase (CAT) and vitamin E (VitE) in rooster semen extender on postthawed quality and fertility of rooster semen. Semen was collected twice a week from six roosters (Arian) and diluted according to experimental treatments consisting of sperm suspensions containing different sperm concentrations (200, 400, and 600 × 106 sperm/mL) without antioxidant supplementation as control (Con) groups (Con200, Con400, and Con600, respectively), sperm suspensions containing different sperm concentrations (200, 400, and 600 × 106 sperm/mL) supplemented with 5-µg/mL VitE (VitE200, VitE400, and VitE600, respectively) and different sperm concentrations (200, 400, and 600 × 106 sperm/mL) supplementation with 100 IU/mL CAT (CAT200, CAT400, and CAT600, respectively). After thawing; sperm motility, membrane integrity, and mitochondrial function were assessed. Fertility and hatchability rates were determined by using 100 artificially inseminated hens. The percentage of total motility (TM) and activity of mitochondria decreased (P < 0.05) as the sperm concentration increased in control groups. So, the lowest percentage of the TM and activity of mitochondria were observed in the Con600 as compared with other treatment groups. Extenders containing 100 IU/mL CAT and 5-µg/mL VitE resulted in higher (P < 0.05) TM, progressive motility, membrane integrity, and activity of mitochondria compared with control groups. Adding VitE and CAT in different sperm concentrations, the percentage of TM, membrane integrity, and activity of mitochondria decreased (P < 0.05) as the sperm concentration decreased. The highest (P < 0.05) membrane integrity, TM, and progressive motility were recorded at VitE400 and CAT400. Including VitE and CAT in rooster extender with different level sperm concentrations had no effect (P > 0.05) on fertility and hatchability rates. In conclusion, although adding VitE and CAT in extender with different levels of sperm concentration improved postthawed quality of rooster semen, but adding VitE and CAT in the extender have no effect on fertility rate.

Are the optimum levels of the catalase and vitamin E in rooster semen extender after freezing-thawing influenced by sperm concentration?

To date, there has no report to evaluate the interaction effects of antioxidant and sperm concentration in rooster semen cryopreservation. This study was aimed to investigate the effects of vitamin E (VitE) and catalase (CAT) at different sperm concentrations on the rooster post-thawed sperm quality. Semen samples were collected twice a week from ten roosters (ROS 308) and diluted according to experimental treatments. The treatments consist of different sperm concentrations (200, 400 and 600 × 106 sperm/mL) with supplementation VitE (5 µg/mL; VitE200, VitE400, and VitE600, respectively) or CAT (100 IU/mL; CAT200, CAT400, CAT600, respectively) and without antioxidants [Control (Con); Con200, Con400, Con600, respectively]. After thawing, motion characteristics were assessed using a CASA system. Plasma membrane integrity and malondialdehyde (MDA) level were evaluated with Hypoosmotic swelling test (HOST) and Thiobarbituric acid (TBA), respectively. The higher percentage of total motility, progressive motility, viability and membrane integrity were obtained in VitE400 (81.16 ± 1.21, 18.44 ± 1.19, 85.47 ± 1.07, 86.91 ± 1.16, respectively) and CAT400 (79.38 ± 1.21, 17.19 ± 1.19, 83.42 ± 1.07, 85.73 ± 1.16, respectively) compared to control groups. Moreover, the lowest percentage of MDA was measured in VitE400, VitE600 and CAT400 rather than other groups (1.489, 1.500, 1.510 ± 0.06, respectively). In conclusion, the results of the present study demonstrate that VitE (5 µg/mL) and CAT (100 IU/mL) independently at sperm concentration, 400 million sperm/mL could beneficial effect for preservation of rooster semen during cryopreservation.

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.

The effects of different levels of vitamin E and vitamin C in modified Beltsville extender on rooster post-thawed sperm quality.

Oxidative damage of sperm by means of reactive oxygen species generated by the cellular components of semen is one of the main reason of declined motility and fertility of sperm during the freeze-thawing process. This study was conducted to determine the influence of vitamin C and vitamin E on rooster post-thawed sperm motility, viability and malondialdehyde (MDA) level. Semen samples from 10 sexually-mature Ross 308 breeder roosters were collected and pooled, divided into nine equal parts and diluted with modified Beltsville extender containing with no antioxidants (control), or containing 100 (C100), 200 (C200), 400 (C400), 800 (C800) µg/mL vitamin C, and 2 (E2), 5 (E5), 10 (E10) and 15 (E15) µg/mL vitamin E. After thawing, total and progressive sperm motility, sperm viability and semen MDA level were assessed. The results shown that C200 and E5 extenders resulted in higher total motility (p < 0.05) compared to other extenders, with exception of E10 extender. Progressive motility was higher in E5 extender (p < 0.05) compared to other extenders, with exception of C200 and E10 extenders. Also, C200 and E5 extenders resulted in higher viability of post-thawed spermatozoa (p < 0.05) compared to other extenders. Finally, the results showed that MDA level was lower in C100 and C200 extenders compared to other extenders (p < 0.05), with exception of E5 extender. In conclusion, the results of the present study demonstrate that C200 and E5 can improve the function of post-thawed rooster spermatozoa.

The effects of different levels of catalase and superoxide dismutase in modified Beltsville extender on rooster post-thawed sperm quality.

Oxidative damage of sperm by means of reactive oxygen species generated by the cellular components of semen is one of the main reasons for decreased sperm motility and fertility during the freeze-thawing process. This study was conducted to determine the influence of catalase (CAT) and superoxide dismutase (SOD) on rooster sperm motility, viability and MDA level after freezing and thawing. Semen samples from 10 sexually-mature Ross 308 breeder roosters were collected and pooled, divided into nine equal parts and diluted with modified Beltsville extender containing no antioxidants (control), or supplemented with 50, 100, 200 and 300 µg/mL CAT, or 50, 100, 200 and 300 U/mL SOD. After thawing, sperm motility and motion parameters were assessed using a CASA system. Sperm viability and MDA level were assessed by eosin-nigrosin and MDA test, respectively. The results of this experiment showed that the extender supplemented with 100 and 200 µg CAT, and 50 U SOD had the highest sperm motility (P<0.05) in sperm motility. Also, addition 100, 200 and 300 µg CAT, and 50 U SOD can improve significantly viability after freeze-thaw. Extender supplemented with 100 µg CAT had significantly lower MDA level compared to control and 300 µg CAT. In conclusion, the results of the present study demonstrate that addition of CAT (100 µg/mL) and SOD (50 U/mL) independently have beneficial effect on quality of post-thawed rooster semen.