Effect of fumigation height and time on cryopreservation of ram semen.

The cooling rate is a crucial factor in the process of freezing semen, influencing the overall freezing effectiveness. The height and time of fumigation can significantly impact the rate of cooling. Appropriate cooling rates can help minimize the formation of ice crystals in spermatozoa and reduce potential damage to them. Therefore, the aim of this study was to evaluate the effect of different fumigation heights and time for the cryopreservation of Hu ram semen. Experiments I-IV assessed the effect of semen cryopreservation by testing the post-thawed spermatozoa total motility (TM), progressive motility (PM) and kinetic parameters fumigated at distances of 2, 4, 6 and 8 cm for durations of 5, 10, 15 and 20 min, respectively. Based on the results of experiments I to IV, experiment V evaluated the effect of semen cryopreservation by testing the post-thawed spermatozoa TM, PM, kinetic parameters, plasma membrane integrity, acrosome integrity and reactive oxygen species (ROS) level fumigated at distances of 2, 4, 6 and 8 cm for duration of 20 min. The results indicated that fumigation at 2 cm for 20 min significantly (P < 0.05) improved spermatozoa TM, PM, mean angular displacement (MAD), plasma membrane integrity and acrosome integrity compared to other groups. Additionally, it significantly (P < 0.05) reduced spermatozoa ROS level compared to the 6 and 8 cm groups. In conclusion, fumigation for 20 min at a distance of 2 cm from the liquid nitrogen surface is the most suitable cooling method for the cryopreservation of Hu ram semen.

Effect of Different Dilution Methods and Ratios of Ram Semen on Sperm Parameters after Cryopreservation.

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.

Astaxanthin Improved the Quality of Hu Ram Semen by Increasing the Antioxidant Capacity and Mitochondrial Potential and Mitigating Free Radicals-Induced Oxidative Damage.

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.

Punicalagin Protects Ram Sperm from Oxidative Stress by Enhancing Antioxidant Capacity and Mitochondrial Potential during Liquid Storage at 4 °C.

The aim of this study was to investigate the effect of punicalagin, an antioxidant, on ram sperm quality. Semen samples were collected and pooled from five rams, then diluted using a Tris-based diluent containing various concentrations (0, 5, 15, 30 and 45 µM) of punicalagin. Sperm motility, plasma membrane integrity, acrosome integrity, total antioxidant capacity (TAC), reactive oxygen species (ROS), malondialdehyde (MDA), mitochondrial membrane potential (MMP), superoxide dismutase (SOD) and catalase (CAT) were measured and analyzed during liquid storage at 4 °C. The results showed that the Tris-based solution containing punicalagin improved sperm motility, plasma membrane integrity, acrosome integrity, TAC, SOD, CAT and MMP, and decreased ROS content and MDA content. At the same time, the semen sample diluted with the Tris-based solution supplemented with 30 µM punicalagin achieved the best effect. The sperm total motility, progressive motility, plasma membrane integrity, acrosome integrity, TAC, SOD, CAT and MMP of the group supplemented with 30 µM punicalagin were significantly (p < 0.05) higher than those of the other groups on the 5th day during the liquid storage at 4 °C. Meanwhile, the ROS content and MDA content were significantly (p < 0.05) lower than those in the other groups. In conclusion, the optimal concentration of punicalagin in the Hu ram semen diluent was determined to be 30 µM. The results indicated that a diluent supplemented with punicalagin could enhance the quality of ram sperm preserved at 4 °C by increasing antioxidant capacity, mitochondrial potential and reducing oxidative stress.

Ovarian Dynamics and Changes in Estradiol-17ß and Progesterone Relationship with Standing Estrus, Preovulatory Follicles, and Ovulation Using Single Prostaglandin F2α Injection in Barbari Goats.

The purpose of the present research was to define ovarian follicular dynamics and plasma endocrine profiles in response to a single PGF2α injection, administered indiscriminately during the breeding season of Barbari goats. Ovarian dynamics were observed at every 12 h interval by using B mode ultrasonography, blood samples for hormonal analysis such as estradiol 17ß and progesterone were collected at every 12 h interval, and bucks with aprons were used to identify standing estrus at every 6 h interval. Relative to PGF2α, the start of standing estrus and ovulation differ (p < 0.05) between early- (n = 7), intermediate- (n = 6), and late-responding (n = 6) goats. The highest plasma level of estradiol 17ß was detected 12 h prior to ovulation. The average diameter of the ovulatory follicle and length of standing estrus were comparable (p > 0.05) between the goats. The corpus luteum degenerated more quickly (p < 0.05) in early- than intermediate- and late-responding goats. Dominant follicle diameter and estradiol 17ß concentration also differ (p < 0.05) among groups. Although the plasma level of progesterone did not vary (p = 0.065), the variation in progesterone concentration with time differed (p < 0.05) amongst the goats. As a result, this research indirectly reveals that the beginning of standing estrus, end of estrus, and ovulation after PGF2α might fluctuate in Barbari goats because of follicular and hormonal dynamics during the luteal phase.

Effects of Different Diluents on Semen Quality of Hu Ram Stored at 4 °C.

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.

Alpha-lipoic acid improves the quality of ram spermatozoa stored at 4°C by reducing oxidative stress and increasing mitochondrial potential.

Introduction: Ram spermatozoa inevitably produce a large number of reactive oxygen species (ROS) during liquid storage, leading to oxidative stress and a decline of spermatozoa quality. Therefore, it is particularly important to add exogenous antioxidants during the process of semen liquid preservation. The purpose of this study is to investigate whether adding alpha-lipoic acid (ALA) to ram semen can reduce oxidative stress and enhance spermatozoa quality during the liquid storage at 4°C. Methods: Different concentrations of ALA (0, 0.025, 0.05, 0.1, 0.5, 1 mM) were added to semen and stored at 4°C. During storage at 4°C, spermatozoa motility, kinetic parameters, membrane integrity, acrosome integrity, energy metabolism parameters (mitochondrial membrane potential (ΔΨM) and adenosine triphosphate (ATP)) and oxidative stress parameters [ROS, malondialdehyde (MDA), total antioxidant capacity (TAC), superoxide dismutase (SOD)] were assessed. Results and discussion: The results indicated that 0.1 mM ALA significantly (p<0.05) improved spermatozoa total motility (TM) and progressive motility (PM), plasma membrane integrity, acrosome integrity, ΔΨM, ATP, TAC, and SOD, while significantly (p<0.05) reducing spermatozoa ROS and MDA content compared to the control group. In conclusion, ALA can reduce damage caused by oxidative stress in spermatozoa and effectively improve the quality of semen preserved at 4°C. And the optimal concentration is 0.1 mM.

Chlorogenic Acid Improves Quality of Chilled Ram Sperm by Mitigating Oxidative Stress.

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.

Effects of Taurine on Sperm Quality during Room Temperature Storage in Hu Sheep.

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.

Follicular dynamics and changes in oestradiol-17ß, progesterone and LH profiles following PGF2α induced oestrus in early and late ovulating Beetal goats.

This study compares the factors associated with variable interval to oestrus and ovulation between early versus late ovulating goats following PGF2α administration. The time of ovulation in Beetal goats (n = 38) was monitored through transrectal ultrasound at every 6 hr following a single dose of PGF2α (experiment 1). Variations in oestrus and ovulation times were further explored through the changes in follicular dynamics, endocrine profiles and behaviour in another set of goats (n = 13) following single PGF2α given randomly during the luteal phase (experiment 2). The ovulation time varied between 60 and 96 hr, and 57% of ovulations occurred by 72 hr following PGF2α (experiment 1). Accordingly, the goats (n = 13) in the second experiment were retrospectively divided either into early and/or late ovulating, that is, ≤72 and/or ≥84 hr following PGF2α . The onset of oestrus, peak estradiol-17ß concentration and LH surge after PGF2α was first observed in early than late ovulating goats (p < 0.05). The goats ovulating early had larger follicle and smaller CL in diameter at the time of PGF2α administration than those ovulating late (5.4 ± 0.2 vs. 4.3 ± 0.2 mm and 10 ± 0.6 vs. 11.8 ± 0.3 mm, respectively; p < 0.05). Likewise, plasma progesterone concentration tended to be lower (p = 0.087) in early than late ovulating goats. In conclusion, the size of dominant follicle and CL at the time of PGF2a determines the interval to ovulation following a single dose of PGF2a during the luteal phase.