Preliminary studies found that progressive motility of ram sperm declined ~75% when stored at 4°C for 24 h, and continued to decline over time when using extenders supplemented with 5% egg yolk. The current study evaluated the effects of different combinations of extenders, ethylene glycol (EG), egg yolk, and penicillamine, hypotaurine, and epinephrine on ram sperm progressive motility during storage. Semen collected from 3 Katahdin and 2 Suffolk rams by electroejaculation was distributed across treatment combinations consisting of either TRIS citrate or milk extender supplemented with 5 or 20% (v/v) egg yolk, ± 1% ethylene glycol (EG) and ± 20 µM penicillamine, 10 µM hypotaurine and 2 µM epinephrine (PHE). For each semen collection, TRIS citrate extender was prepared from a 4× solution so that the TRIS, citric acid and fructose concentration were constant at 300, 94.7, 27.8 mM, respectively, regardless of semen dilution factor. A 4× milk extender was also used so that the extender contained 10% (w/v) milk powder, regardless of semen dilution factor. Both extenders were supplemented with 50 µg mL−1 of gentamicin. Semen was diluted in extender to a final concentration of 300 million sperm/mL in 1.5-mL tubes, and cooled to 4°C over a 2- to 3-h period. Semen was evaluated initially and daily for 3 days, using computer-assisted sperm analysis. Repeated-measures data were analysed using the mixed model (JMP 12.0 software; SAS Institute Inc., Cary, NC, USA) for main effects of extender, supplements, and their interactions. Nonsignificant interactions were removed from the model before reanalysis. Data are presented as LSMeans ± standard errors. Initially, sperm progressive motility averaged 41 ± 6.2% across treatments. After an initial decline, overall progressive motility did not change (P > 0.05) significantly (mean of 22.3 ± 1.6 and 23.05 ± 1.3% at 48 and 72 h, respectively). Over time and across treatment combinations, mean progressive motility was maintained to a greater extent (P < 0.01) by milk than TRIS-based extender (28.2 ± 1.1 v. 18.9 ± 1.1%, respectively). Across extenders, progressive motility of sperm was similar (P = 0.50) for 5 and 20% egg yolk (22.2 ± 1.4 v. 24.4 ± 1.4). Addition of 1% EG increased (P < 0.01) progressive motility (25.8 ± 1.05 v. 21.3 ± 1.05). Addition of PHE also increased (P < 0.01) progressive motility from 20.9 ± 1.04 to 26.3 ± 1.04%. There was an interaction between EG and % egg yolk, primarily due to an effect on sperm stored in TRIS citrate extender. Addition of 1% EG to extender containing 5% egg yolk improved (P < 0.01) progressive motility from 18.5 ± 1.5 to 26.9 ± 1.5%). Addition of 1% EG to TRIS citrate extender also increased (P < 0.05) progressive motility, from 14.6 ± 1.5 to 23.2 ± 1.5%. Results indicate that milk extender supplemented with 1% EG, PHE, and either 5 or 20% egg yolk is capable of maintaining progressive motility of ram semen at ~60% of its initial value when stored at 4°C for up to 72 h. Additional studies are needed to evaluate pregnancy rate after insemination of ewes with stored semen.
Cryopreservation of Indonesian native chicken semen by using dimethyl sulfoxide and various level of ethylene glycol as cryoprotectants