PARENT SESSION
Cryopreservation

ABILITY OF CRYOPRESERVED BOAR SPERM TO REGULATE INTERNAL CALCIUM.

Buhr, Mary¹, Pettitt, Murray², ^{1 2}

ABSTRACT- Sperm regulate internal calcium (Ca_i²⁺) to control capacitation and the acrosome reaction. We hypothesized that the poor fertilizing ability of cryopreserved boar sperm was related to their ability to regulate Ca_i²⁺. Split ejaculates (n=4) were cryopreserved in 4 extenders: minimal [MIN: BF5 extender minus egg yolk, glycerol, Orvus ES© Paste (OEP)]; MIN + 20% egg yolk, 3% glycerol (EYGLY), 0.33% (w/v) OEP (EYGLY:OEP); EYGLY + 0.1 or 0.01% of detergent Pluronic© F-127 (EYGLY:0.1P; EYGLY:0.01P). During 30 min after thawing into Ca²⁺-free buffer, Ca_i²⁺ was stable in sperm cryopreserved in EYGLY:0.01P extender (P>0.05) but changed in sperm cryopreserved in other extenders (P<0.05). Sperm were then provided with 0 or 1mM Ca²⁺. In the first 60 sec after adding 1 mM Ca²⁺, Ca_i²⁺ increased in sperm cryopreserved in EYGLY:0.1P (5.01 ± 2.04 x 10^-4 Ca²⁺ units/min; P<0.05) but not in the other extenders nor in any sperm receiving 0 mM Ca²⁺ (P>0.05). Presumably membrane damage during cryopreservation in EYGLY:0.1P allowed an immediate influx of exogenous Ca²⁺. During the next 30 min, Ca_i²⁺ increased in sperm cryopreserved in MIN, EYGLY:0.1P and EYGLY:0.01P extenders challenged with Ca²⁺ (P<0.05) while there was no change in the sperm cryopreserved in EYGLY:OEP (P>0.05), suggesting that these sperm are less damaged and can exclude exogenous Ca²⁺ for at least 30 min. Ca_i²⁺ levels following 0 mM Ca²⁺ increased only in EYGLY:0.1P (P<0.05); this harsher treatment may make Ca²⁺ available from intracellular stores or moribund cells. Pregnancy rates (animals with embryos/animals inseminated) and fertilization rates (oocytes fertilized/total oocytes in pregnant animals) confirmed the superiority of sperm cryopreserved in EYGLY:OEP extender [5/6 pregnant, 50/95 fertilized, P<0.05 vs EYGLY:0.1P (2/6 and 25/67) and EYGLY:0.01P (1/6 and 8/10)], MIN had 0% pregnant. Cryopreservation extender affects boar sperms' Ca_i²⁺ regulation and fertility.

KEY WORDS: freezing, fertility, calcium, sperm