![]() ![]() All the tubes were cooled to 4☌ for 90 min before assessing for the motility, morphology, and sperm membrane integrity. The final semen concentration after dilution was 200×10 6 sperm/ml. The mixture was centrifuged at 800 g for 10 min to remove seminal plasma, divided into four aliquots, and resuspended with FH-20, Tris, INRA Freeze, or EquiPlus extenders ( Table-1). Sample with minimum concentration of 200×10 6/ml and motility >50% was selected for the study.įiltered semen of each ejaculate was diluted (1:1) with centrifugation medium containing 6.0 g glucose, 0.37 g ethylenediaminetetraacetic acid, 0.37 g sodium citrate, 0.12 g sodium bicarbonate, 100,000 IU penicillin, and 0.08 g streptomycin in 100 mL distilled water. Sperm concentration and motility were determined using CASA system (ISAS ® program, Prosser R+D, Paterna, Valencia, Spain). After that, the ejaculate was evaluated for general and progressive motility and sperm concentration. The semen volume was measured in a graduated cylinder. The gel was removed immediately from the semen sample after semen collection using sterile gauze and then transferred to a water bath at 37☌. Missouri model AV was used for the semen collection, in this trial. Therefore, the current study aims to determine the best extender which contained different types of buffers, Tris buffer in Tris-based extender and raffinose, glucose, and lactose buffers in HF-20 extender, that were locally manufactured compared with commercial extenders like INRA Freeze and EquiPlus. Besides the ability of sugars to dissolve the cryoprotectant after cryopreservation, it also reduces the osmotic shock. Thus, sugars decrease the concentration of cryoprotectant required for cryopreservation. As a result of the high molecular weight of sugars such as glucose, fructose, and raffinose, therefore, these buffers will not be able to penetrate the sperm membrane. The variation of buffer capacity may have an effect on the sperm biological system and enzymes. Different types of buffers could be supplemented to the semen extender to balance the pH during semen preservation. Therefore, more consideration is required for semen extender to improve the frozen semen quality for horses. Lactic acid is produced by the glycolytic metabolism of the sperm that leads to diminished pH in the semen sample. Semen pH is one of the essential factors that affect the semen quality due to its effect on sperm motility and metabolism. Besides, horses showed high variations between stallions and ejaculates on sperm parameters and pregnancy rate of frozen semen. Unfortunately, a considerable number of stallions have poor post-thawed semen quality and fertility. However, most of the Arabian mares in the Kingdom of Saudi Arabia were bred naturally. ![]() Furthermore, cryopreserved semen is a basic approach in the modern assisted reproductive technology such as in vitro fertilization and intracytoplasmic sperm injection. The use of frozen semen could preserve horse’s genetics for unbounded time and enables to maintain the semen for the future in any event such as death. Frozen semen has more benefit than fresh or chilled semen therefore, this makes it popular in the horse industry. Furthermore, the number of mares that can be impregnated by one stallion during the breeding season will increase due to the possibility of dividing the ejaculate into several insemination doses. ![]() Therefore, the use of AI becomes the basic technique in the modern horse industry. Artificial insemination (AI) has quickened the genetic improvement due to the comfort involved in transporting the semen from the insemination center to the place where the mare is inseminated which offers a broader choice to breeders outside the country.
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