Development of two-in-one cock's semen diluent for storage at freezing temperature

Semen diluter is very important for the exploitation of AI to the maximum and it contributes markedly to the cost incurred in this process. Two-stage diluter for semen storage at freezing temperatures was considered in view of improving the efficacy with minimum complexity by decreasing motility and metabolism during storage at phase-1 and recovery of motility prior to insemination which is required for the entry of spermatozoa into sperm host gland by using phase-2 diluter. At step one, K₂HPO₄ and KH₂PO₄ were used maintaining osmolarity around 300 mOsm for the selection of appropriate buffer pH to reduce the motility for longer storage at freezing temperature and recovery of spermatozoa with greater or equal to 60% motility and sustained less than 20% dead and abnormal spermatozoa. In step2 for further increase of the storage time with out affecting the osmolarity and maintaining the above said criteria of motility and percent dead, sodium glutamate was substituted in three levels on best identified buffer combination at step one. In step three, glucose was added in three levels on the best-selected media at step two for further improvement of storage time. Antibiotics (2ml, out of 300mg of penicillin and 250mg of streptomycin dissolved in 5ml of distilled water) were added per liter of the diluter. Following this the best phosphate buffer for storage at freezing temperature was 6.5pH but the best recovery buffer for freezing temperature was 7.0-pH. The selected sodium glutamate level for storage purpose for freezing temperature levels were 25% substituting of the phosphate buffer with sodium glutamate level. Finally addition of 0.6% glucose yielded the best results. The final osmolarity of the diluents was found to be 346 mOsm. The spermatozoa could sustain the desired motility and normal livability for 72 hours with the final diluents.