Comparison of conventional and automated freezing methods on PB2 rooster semen cryopreserved with glycerol and dimethylsulfoxide tris coconut-water extender

Background: Poultry semen cryopreservation remains an easy and promising way of preserving and transferring poultry germplasm. Standardizing and optimizing freezing procedure with natural extender may be a reliable step towards achieving better post-thawed sperm quality. This study was conducted to investigate the effects of four different freezing protocols (FP) on frozen rooster semen extended with tris coconut water extender (TCWE). A total of 20 roosters were used and trained for semen collection. TCWE was prepared by adding coconut water to tris buffer and kept at 37 °C. Semen was collected and pooled from the roosters and was evaluated for motility before dilution procedure. Three different concentrations (8, 10, and 15%) of two intracellular cryoprotectants glycerol and dimethylsulfoxide (DMSO) were supplemented in TCWE. Pooled semen was divided into six equal fractions, and TCWE containing cryoprotectants in different concentrations were diluted with the semen in ratio 1:2 (semen:extender). Diluted semen was manually filled in 0.25ml straws and sealed. Semen straws were equilibrated for 4 h at 4 °C. Each straw fraction was further divided into four parts, and subjected to four FP (slow freezing 1, 2, 3 and fast freezing 4). Each FP was done on samples containing 8, 10, and 15% glycerol and 8, 10, and 15% DMSO. After each protocol, semen straws were finally deep into liquid nitrogen –196 °C. After 48h, the straws were thawed individually to evaluate post/thawed motility, viability, and membrane integrity. The experiment consists of three trials. Results: At 8% glycerol concentration, FP1 has significantly (P > 0.05) the highest percentage motility (73.33%) compared to FPs 2, 3, and 4 (56.68, 50.00, and 23.33% respectively). At 10% glycerol, FP2 had the highest motility (48.33%) and HOST (64.00%). At 15% glycerol, FP4 (fast freezing) had the highest percentage motility (71.67%), viability (76.33%), and HOST (71.67%). At 8%, DMSO concentrations FP4 had the highest significant (P > 0.05) motility, viability, and HOST. Ten percent and 15% DMSO revealed no significant (P > 0.05) difference in most parameters among all the FPS. DMSO performed better than glycerol irrespective of concentrations in all FPS on most parameters evaluated.

Conclusions: Fast freezing performed better with 15% concentration of glycerol, while slow and fast freezing performed better with 10% and 15% DMSO concentrations, which implies that DMSO may serve as better cryoprotectant for poultry semen freezing irrespective of freezing protocols adopted. Page 2 of 8 Keywords: PB2 rooster, Sperm, Cryoprotectants and freezing protocols, Extender