Prolonged post cooling but not pre-cooling equilibrium length improves the viability of ram sperm cryopreserved in an extender containing low-density lipoproteins

Abstract

The objective was to evaluate: (1) the influence of the length of an equilibrium period done at room temperature on cooled semen diluted in extender containing different low density lipoproteins (LDL) concentration in replacement of whole egg yolk, (2) the effect of different concentration of LDL in replacement of egg yolk in extenders for freezing ram sperm and (3) the influence of the length of an equilibrium period after cooling to 5°C on maintenance of post-freezing sperm viability. In Experiment I, semen samples were diluted in extender containing 15% egg yolk or 2, 4 or 8% LDL (v/v) in replacement of the egg yolk. Diluted semen was subjected to 0, 30, 60, or 120min of equilibrium at room temperature and thereafter cooled in the refrigerator (−0.13°C/min) to reach 5°C in 2.5h. Motility assessments were performed on re-warmed samples (38°C) at 1h interval for 3h; the percentage of sperm with functional integrity of the plasma membrane was determined based on a hypoosmotic swelling test (HOST). There was no interaction (P>0.05) between extenders and duration of pre-cooling equilibrium for the evaluated characteristics. The whole yolk extender preserved motility similar to 8% LDL (P>0.05) and better preserved the motility than 2 or 4% LDL extenders (P<0.05). The plasma membrane integrity was evenly preserved among extenders (P>0.05). In Experiment II, extenders of Experiment I were used to freeze ram semen immediately after final dilution, with neither pre nor post cooling equilibrium period. The following parameters were evaluated after thawing (38°C/30s): sperm longevity (incubation 38°C/3h), functional and structural integrity of sperm membranes (HOST and fluorescent probes) and kinetics of sperm by computer analysis (CASA). Extenders with 4% LDL or 8% LDL had similar (P>0.05) responses than that obtained with whole egg yolk for all evaluated parameters. The 2% LDL extender was less efficacious (P<0.05). In Experiment III, semen samples diluted in whole yolk, 4% LDL or 8% LDL were allowed to equilibrate after cooling to 5°C for 1 or 2h before freezing. After thawing (38°C/30s), samples were subjected to the same tests as in Experiment II. Equilibration for 2h increased (P<0.05) post thaw sperm motility (regardless of the extender). In conclusion, using ram semen, LDL (4% or 8%) replaced effectively whole egg yolk (15%), maximizing its effectiveness when equilibration length at 5°C before freezing was of 2h.