The Use of Sodium Hyaluronate in Freezing Media for Bovine and Murine Embryos

Abstract

Four experiments were designed to determine whether sodium hyaluronate (SH) may be used to replace newborn calf serum (NCS) in murine and bovine embryo freezing media. A total of 780 mouse and 178 cattle embryos were frozen. Freezing media were prepared in Dulbecco's phosphate-buffered saline supplemented with 20% NCS and 10% glycerol (v/v) (medium A). In experintents I and II, NCS was replaced by 0.1% SH (w/v) in medium B and by 0.1% polyvinyl alcohol (PVA) (w/v) in medium C. In experiments III and IV, NCS was replaced in mouse embryo freezing medium by three different molecular weights of SH as follows: 0.1 or 0.2% (SH-1; <3 × 105 Da) in medium B. by 0.1 or 0.05% (SH-2; 5-7.5 × 105 Da) in medium C, and by 0.1 or 0.025% (SH-3; >1.2 × 106 Da) in medium D. Embryos were frozen and thawed using standard procedures. Glycerol was diluted from embryos after thawing in a single step with 1.0 M sucrose. After 48 h in culture (Ham's F-10 medium for cattle embryos and BWW medium for mouse embryos), embryos were evaluated for development to expanded or hatched blastocysts. Survival rates were compared by X2 analysis. In experiment I, there were no significant differences in mouse embryo survival or development between NCS, SH, and PVA groups (84.1, 79.2, and 83.3%, respectively). In experiment II, there were no significant differences in bovine embryo survival between NCS and SH groups (67.2 and 67.7%), but both had significantly higher survival than the PVA group (37.9%: P < 0.05). In experiment III, there was a significantly lower (P < 0.05) embryo survival rate in the low-molecular-weight SH-1 group (18.6%) than in medium (SH-2)- or high (SH-3)-molecular-weight groups (78.6 and 92.5%, respectively). Although doubling the concentration of SH-1 in experiment IV resulted in improved embryo survival (63.4%), survival rates were significantly higher (P < 0.05) in the medium-molecular-weight (SH-2; 82.3%) and high-molecular-weight (SH-3; 86.0%) groups despite reduced concentrations of these compounds in the freezing medium. Results suggest that SH can be used to replace biological sera in embryo freezing media.