The purpose of this study was to investigate whether the addition of chlorogenic acid (CGA) to a sheep semen extender could improve the quality of chilled sheep sperm. Ejaculates (n = 80) were collected from five Hu rams with an artificial vagina. The ejaculates were mixed and divided into five equal parts, diluted with a CGA-free Tris–egg yolk extender (control), or supplemented with 0.2, 0.4, 0.8, and 1.2 mg/mL. The sperm kinematic parameters (viability, progressive motility), functional integrity of plasma membrane and acrosome, adenosine triphosphate (ATP) concentration and antioxidant parameters (Catalase (CAT), Superoxide dismutase (SOD) activity, total antioxidant capacity (T-AOC), ROS level and Malondialdehyde (MDA) content) were evaluated during storage of the semen. The results indicated that: PM, plasmatic membrane integrity and acrosomal integrity in 0.8 mg/mL CGA were higher (p < 0.05) from day 1 to 5. The ROS level in CGA groups was lower than the control (p < 0.05). CAT, SOD, ATP, and T-AOC were highest at 0.8 mg/mL concentration within 1 to 5 days. The above results indicated that the right concentration of CGA improved the quality of Hu ram sperm during chilling storage.

The present study aimed to investigate whether the presence of Tau protected Hu sheep sperm from ROS stress during storage at room temperature. The semen was diluted with extender (Tris-based) at room temperature, supplemented with different concentrations of Tau (0, 10, 20, 40, 80, or 100 mM), and stored at 15 °C. Sperm quality parameters (sperm progressive motility, kinetic parameters, plasma membrane integrity rate, acrosome integrity rate, and MMP) and antioxidant parameters (ROS, MDA, SOD, CAT, and T-AOC) were evaluated during the preservation of semen. The addition of Tau, especially at a concentration of 20 mM, exerted positive effects on sperm quality parameters and antioxidant parameters compared to the sperm without Tau treatment (control group). The addition of Tau, especially at a concentration of 100 mM, exerted negative effects on sperm quality parameters and antioxidant parameters compared to the control group. Interestingly, the results indicated that the sperm acrosome integrity rate did not change during storage time. In conclusion, the addition of Tau to sperm preserved at room temperature can enhance the antioxidant ability of sperm, reduce the LPO on the 5th day, and improve the quality of semen preserved at room temperature. These results implied that Tau had potential to enhance Hu sheep sperm reproductive performance.

The objective of this research was to investigate the effect of astaxanthin supplementations of semen extender on the quality of Hu ram semen after up to five days of preservation at 4 °C. Semen samples were collected from five healthy Hu rams using an artificial vagina during breeding season (April to August 2023) and diluted with a basic extender supplemented with control (0), 1 µM, 2 µM, 3.5 µM, or 4.5 µM of AXT. Overall, 170 semen ejaculate samples (34 repetitions) from five healthy Hu rams were used in our research study. The results revealed that the addition of AXT (3.5 µM) significantly (p ≤ 0.05) increased the sperm kinematic indexes (T.M%, P.M%, MAD%, STR%, and LIN %), sperm viability, plasma membrane integrity, acrosome integrity, total antioxidant content (T-AOC), and mitochondrial membrane potential (MMP) of the Hu rams spermatozoa after up to five days of preservation at 4 °C. Contrary to that, the addition of the best concentration of AXT (3.5 µM) to the semen extender significantly (p ≤ 0.05) reduced the reactive oxygen species (ROS) and malondialdehyde (MDA) concentration of Hu ram semen. In conclusion, the results of the current study indicate that the addition of a semen extender with AXT improves the quality of Hu ram spermatozoa by increasing the total antioxidant capacity (T-AOC) and mitochondrial membrane potential (MMP). On the other hand, reducing free radicals induced oxidative (ROS) and per oxidative (MDA) damage to Hu ram semen.

The dilution method and ratio were tested to assess their effects on the Hu ram semen after cryopreservation. Experiment I aimed to explore the effect of various dilution ratios (1:1, 1:2, 1:3, 1:4) of diluent I (Tris-based and egg yolk) under the condition of 1:1 dilution of diluent II (diluent I and glycerol) on the Hu ram semen preserved in liquid nitrogen regarding spermatozoa motility and kinetic parameters. Experiment II aimed to investigate the effect of various dilution ratios (1:1, 1:2, 1:3, 1:4) of diluent I under the condition of 1:2 dilution of diluent II to the Hu ram semen for cryopreservation on spermatozoa motility and kinetic parameters. The purpose of experiment III is to assess the effect of various dilution methods and ratios on the cryopreservation of Hu ram semen by detecting spermatozoa motility, kinetic parameters, plasma membrane integrity, acrosome integrity and reactive oxygen species (ROS) level. Experiment III includes four groups: one-step dilution method and two-step dilution method. The two-step dilution method includes two groups: 1:2, 1:1 and 1:3, 1:2, and the one-step dilution method includes two groups: 1:5 and 1:11. The results indicated that the post-thawed spermatozoa total motility (TM), progressive motility (PM) and average motion degree (MAD) were highest in the 1:2 group and significantly higher (p < 0.05) than those in the 1:1 and 1:4 groups under the condition of 1:1 dilution of diluent II. The post-thawed spermatozoa TM and PM of the 1:3 group were significantly higher (p < 0.05) than those of the other groups under the condition of 1:2 dilution of diluent II. The post-thawed spermatozoa TM, PM, plasma membrane integrity and acrosome integrity of the two-step group (1:3, 1:2) were the highest and significantly higher (p < 0.05) than those in the other groups. Additionally, the post-thawed spermatozoa ROS level of the two-step group (1:3, 1:2) was significantly lower (p < 0.05) than that in the one-step groups (1:5 and 1:11). Therefore, a two-step dilution (1:3, 1:2) was found to be the most suitable method and ratio for diluting the Hu ram semen after cryopreservation.

This study aimed to investigate the effects of various diluents on the quality of Hu ram sperm stored at 4 °C. Semen samples were collected from three Hu rams and diluted with diluents A (Sodium citrate–Glucose–Egg yolk), B (Sodium citrate–Glucose), C (Fructose–Skimmed milk powder–Soy lecithin), and D (Tris–Fructose–Citric acid–Egg yolk). Total motility (TM), straight-line velocity (VSL), average path velocity (VAP), curvilinear velocity (VCL), average motion degree (MAD), acrosome integrity, membrane integrity, and reactive oxygen species (ROS) were evaluated. The results showed that diluent D had better preservation in terms of the sperm TM, VSL, VCL, VAP, MAD, and membrane and acrosome integrity. On the third day of the storage, the sperm PM of diluent D was higher than that of other diluents (p < 0.05). The ROS level of diluent D was lower than that of other diluents on the fifth day (p < 0.05). On the seventh day of the storage, the sperm TM in diluent D reached 50%, which was the highest in all diluent groups. On the seventh day of the storage, the integrity of the sperm membrane and the integrity of the acrosome of the sperm in diluent D were the highest in all diluent groups (p < 0.05). In conclusion, these results indicated that diluent D improved the semen quality during storage at 4 °C. In this study, diluent D was the best diluent formula for Hu ram semen stored at 4 °C.