Application of insect freeze tolerant strategies to the freezing of bovine embryos
Hemolymph of Tipula trivittata larvae permits freeze tolerance of this insect due to its content of cryoprotectants and ice nucleating proteins. Spontaneous ice nucleation of the dialyzed hemolymph occurs between -5 C and -11 C up to dilutions of 1:1000. The objectives of this study were to evaluate the effects of seeding temperature (-5 C vs -7 C), and the presence of hemolymph at a low (.1% v/v) and a high (10% v/v) level on the survival of frozen-thawed bovine embryos. In Exp. l, survival rates of 6 and 7 day bovine embryos frozen in medium containing .1% hemolymph and seeded at -5 C or -7 C, were compared to evaluate the effect of seeding temperature. The effect of hemolymph was evaluated by including a control without hemolymph seeded at -7 C. In Exp. 2, survival rates of embryos frozen with and without 10% hemolymph were compared. In Exp. 3 the evaluation of the effect of 10% hemolymph was continued. Also included was a control handled identically to embryos frozen in medium with 10% hemolymph regarding pre and post freeze manipulations but which was not frozen. This allowed evaluation of freezing damage per se. For Exps. l, 2, and 3, survival based on mean final development score and time to advance a developmental stage in vitro did not differ for embryos frozen. However, in Exp.· 3, the control which was not frozen had 30% greater survival than embryos undergoing the same manipulations but which were frozen. Thus, neither seeding temperature nor inclusion of .1% or 10% hemolymph in freezing medium had a significant effect on survival of frozen-thawed bovine embryos.