Aim: Different types of extenders have a variety of components which show the tolerance effect on sperm protection during freezing procedures. In the present study, we have examined the impact of the extenders HF-20 and Tris, which were locally manufactured, and they are competing with commercial extenders INRA Freeze® (IMV Technologies, France) and EquiPlus Freeze® (Minitube, Germany) on the quality of horses frozen semen. Materials and Methods: A total of 15 ejaculates from three healthy stallions were collected and cryopreserved in the same environment. Each semen sample collected was divided into four equal parts and processed. All samples were analyzed before and after freezing for motility, viability, plasma membrane integrity, and morphology. Furthermore, twenty mares were inseminated using post-thawed semen. Results: There were no differences observed among all extenders in all the parameters before freezing. Sperm cryopreserved using HF-20 showed better motility, viability, and plasma membrane integrity than Tris extender. The Tris extender showed the most inferior quality of post-thawed semen between all the extenders. HF-20, INRA Freeze®, and EquiPlus Freeze® extenders revealed the same capacity of semen preservation in vitro and in vivo. Conclusion: HF-20 extender has the same quality as INRA Freeze® and EquiPlus Freeze® that can be considered as one of the best extenders for the semen cryopreservation in horses. In contrast, Tris extender needs some degree of improvement.

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Present study aimed to investigate the effect of adding antioxidants, cysteine and ascorbic acid on the levels of glutamic oxaloacetic transaminase (GOT), glutamic‐pyruvate (GPT), alkaline phosphatase (ALP), lactate dehydrogenase (LDH) and γ‐glutamyl transpeptidase (GGT) enzymes of post‐thawed stallion sperm. Ten ejaculates were collected each from four healthy stallions and cryopreserved using HF‐20 freezing extender containing either 0 mg/ml cysteine or ascorbic acid, 0.5 mg/ml cysteine and 0.5 mg/ml ascorbic acid. All samples in freezing extender containing cysteine or ascorbic acid or none of them were assessed for sperm motility, viability, plasma membrane integrity, morphology and enzymes concentration. The ALP, LDH and GGT were significantly higher in 0‐group compared with cysteine and ascorbic acid groups. The sperm motility of frozen‐thawed semen with 0‐group was significantly better compared with cysteine and ascorbic acid groups. The variation on viability, sperm membrane integrity and morphology were insignificant between all treated groups. Therefore, these enzymes were reduced when using antioxidants in the freezing extender. Results of the present study suggest that concentration of ALP, LDH and GGT enzymes could be used as parameters for prediction of frozen‐thawed stallion semen.

This study is aimed at investigating effects of supplementation of stallion' semen extender with various concentrations of Gum Arabic (GA) versus egg yolk (EY) on viscosity, sperm motility and survival during cooling and freezing. Physical sperm characteristics; i.e. curvilinear velocity (VCL), straight line velocity (VSL), average path velocity (VAP), linearity (LIN) and straightness index (STR) were evaluated. Based on the sperm velocity (velocity of the average path), individual spermatozoons were classified into two major groups; i.e., progressively motile (>45 µm/sec) and immotile (0-45 µm/sec) spermatozoa. Addition of 3, 9 or 15% of GA to HF-20 extender resulted in linear decreases in VCL, VSL and VAP and a decrease in the percentage of progressively motile spermatozoa. Dilution of horse semen samples with high viscosityextenders (i.e., high percentage of GA) decreased the VCL, VSL and VAP in fresh and chilled semen. Freezing semen in high viscosity-extenders reduced percentage of progressively motile spermatozoa compared with those of low viscosity-extenders. In refrigerated and frozen semen samples, the extender containing 15% GA had detrimental effects on the percentage of progressively motile sperm cells and velocity of progressive motile sperm. Moreover, cooling sperm in extenders containing 9 or 15% of GA for 72 hours resulted in complete motility cessation. In conclusion, GA could replace EY in stallion semen extenders at a level of 3% to maintain the physical and biological characteristics of cold and frozen semen.

Twinning induction of single-bearing Noemi ewes is an important avenue to maximize the economic feasibility of sheep production. Sixty Noemi ewes were used and randomly assigned to six treatment groups (n=10/group).Two sources of FSH [i.e., porcine (P) vs. human (H)] were given as a single dose or in six doses. The control 1 group was given a single dose of saline (C1), while the control 2 group was given six doses of saline (C6). Ewes in group 3 (P1) were given a single dose of p-FSH, in group 4 six doses of p-FSH (P6), in group 5 a single dose of h-FSH (H1), and in group 6 six doses of h-FSH (H6). The ewes were inserted with CIDR for 10 days with FSH given on day 8. A fertile ram was used at the onset of estrus. Blood samples were collected for hormone analyses. The time between CIDR removal and onset of estrus (63, 38 and 26 hrs. in C, P, and H, respectively) was shortened by FSH administration. FSH increased the incidence of twinning, however single dose resulted in more stillbirths and mortalities. The neonatal survival rate decreased in the P1 (40%) compared to the P6 (65%) treatments. Both sources of FSH raised progesterone and estradiol 17-β compared to the controls. Contrariwise, both h- and p-FSH reduced T4; however, h-but not p-FSH raised T3. In conclusion, using rh-FSH at six descending doses of a total 180 IU in Noemi ewes produced two viable neonates. Moreover, the exogenous FSH raised the sex hormones and T3 in the ewes.

Semen cryopreservation is very important in animal agriculture to maximize the number of daughters of genetically superior males and to distribute the cryopreserved semen of good males all over the world. However, the freezing process generates some damage to sperm that reduce their fertilizing ability after thawing. Moreover, egg yolk, which is the most common animal-origin cryoprotectant used in semen dilution, is considered a source of biosecurity risk. In the current study, we aimed to compare the replacement of egg yolk in the extender by gum arabic (5%) along with supplementation with antioxidant cysteine or ascorbic acid on semen quality and freezability in Noemi rams in vitro. Semen from six rams were collected with an artificial vagina two times per week. Semen evaluation parameters such as color, volume, pH, general motility, percentage motility, concentration and cell viability ratio were assessed. Spermatozoa motility and concentration were estimated with the computer-assisted semen analysis system. The semen samples were frozen using a Tris extender containing either 15% egg yolk or 5% gum arabic. For antioxidant-supplemented extenders, cysteine or ascorbic acid was dissolved at concentrations of 0.10, 0.50 or 1.0 mM in egg yolk or gum arabic extender. The semen from each ejaculate of each ram were resuspended with a specific extender with glycerol (5%); the final volume after dilution was 1 mL semen to 4 mL extender. The samples were then cooled to 4 °C for 120 min, loaded into 0.5 mL straws and frozen in liquid nitrogen for 7 days. Supplementation of gum arabic or egg yolk extenders for ram semen with antioxidants such as cysteine or ascorbic acid has beneficial effects on semen quality after cold storage or cryopreservation. However, supplementation of a 5% gum arabic extender with cysteine at 0.5 or 1 mM concentration or ascorbic acid at 0.5 mM concentration improved the quality of spermatozoa postcryopreservation. It could be concluded that gum arabic is a good alternative for egg yolk in Noemi ram semen extenders. Antioxidants are necessary to support the addition of gum arabic to the extender to help the ram spermatozoa to survive freezing–thawing and oxidative stresses.

Authors' Contribution MHS, AZD, NA and AH designed the study plan. NUK collected samples and executed the experimental work. MS, MAKand NR recorded and analyzed the data and wrote the article.

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