Effect of cryopreservation medium and straw size on post-thaw survival of mouse embryos in vitro

THERIOGENOLOGY EFFECT OF CRYOPRESERVATION NEBIUH AND STRAW SIZE ON PUST-THAW SURVIVAL OF HOUSE EHBRYOS JN VITRO G.D. Corbin, C.E. Pope and B.L. Dresser Center for Reproduction of Endangered Wildlife Cincinnati Zoo and Botanical Garden, Cincinnati, OH 45220 The most widely used medium for embryo cryopreservation has been phosphate buffered saline (PBS). Recent studies have indicated that HEPES buffered media may be more suitable for embryo holding and culture than PO, buffered media. The purpose of this study was to compare &I vitro survival rate of mouse embryos after cryopreservation in a PO, buffered medium (PBS) ye that of a HEPES buffered medium (Tyrodes). We also examined the effect of size of straw (ST) (.25 ml 5 .50 ml), in which embryos were frozen, on in vitro survival post-thaw. Swiss Webster females (Charles River, 3 to 10 wk old) were injected with 10 IU (IP) pregnant mare serum gonadotropin. Human chorionic gonadotropin (hCG, 10 IU, IP) was injected 48 h later (day 0) and females were singly paired with sexually mature Swiss Webster males. A total of 823 embryos (early to mid-morula) were collected on day 3 (64 h post-hCG), pooled and held in modified HEPES (15 mH) Tyrodes (HeTy) medium containing 10% heatinactivated newborn calf serum (NCS) until being randomly assigned into one of four treatment groups as follows: Gp 1 = PBS in -25 ml ST (n=206); Gp 2 = PBS in -50 ml ST (w197); Gp 3 = HeTy in .25 ml ST (n=212); Gp 4 = HeTy in .50 ml ST (n=208). PBS was supplemented with 1.0 mM glutamine, 0.36 mM pyruvate, 5.55 mM glucose, 0.63 g/l penicillin, 0.50 g/l streptomycin and 10% NCS. The supplement for HeTy was modified to contain 0.09 mM pyruvate and 0.78 mM Ca lactate without glucose. Embryos were equilibrated with glycerol in 3 steps at 37'C: 8 min each in .44 M and .88 M and 20 min in the final concentration of 1.4 H during which time embryos were loaded into straws (5 to 37/straw). Embryos were then placed in a programnable freezer (Hoxan) at -6'C and held for 3 min prior to being manually seeded. After an additional 3 min equilibration at -6'C embryos were cooled to -34'C at a rate of 0.5'C/min before plunging into liquid nitrogen. Thawing was accomplished by exposure to room temperature air (24'C) for 10 set, then the straw was plunged into a 37'C water bath for 15 to 20 sec. Embryos were rehydrated for 10 min at 37'C in 0.8 M sucrose in the medium in which they were frozen. After a 3X wash (3 min/wash) in the treatment medium, embryos were cultured for 72 h in modified Ham's F-10 t 10% heat-inactivated fetal calf serum at 38'C in 5% CO /air. At 24 h intervals embryos were evaluated for stage of development and quality. Only embryos developing into morphologically normal expanding or later blastocysts at 72 h post-thaw were considered as surviving. Many of the blastocysts included as survivors were hatching from the zona pellucida and/or attaching to the culture dish at the final evaluation. The in vitro survival rate for each of the four groups was: Gp 1 = 68% (140/206), Gp 2 = 68% (135/197), Gp 3 = 77% (164/212), Gp 4 = 69% (143/208). The survival rate of embryos frozen in HeTy (Gps 3 and 4) was slightly higher (P=O.O7) than those frozen in PBS (Gps 1 and 2). Straw size did not have an effect (P=O.19) on post thaw development nor was there an interaction between media and straw size (P=O.13). From these results, we conclude (1) that mouse embryos can be successfully frozen in either .25 ml or .50 ml straws, and (2) that HeTy is a suitable medium for mouse embryo cryopreservation and further studies with embryos of other species are warranted.

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JANUARY198OVOL.33NO.l