121 CRYOPRESERVATION OF BOVINE BIOPSIED EMBRYOS UNDER A MAGNETIC FIELD

Abstract

Although fresh biopsied embryos have almost the same ability to develop to term as fresh intact embryos, embryos that are cryopreserved after biopsy have lower developmental competence than non-biopsied cryopreserved embryos. Thus, an improved freezing protocol is needed for the cryopreservation of bovine biopsied embryos. Applying a magnetic field during the cryopreservation of porcine spermatozoa has been shown to improve the survival rate after thawing (Masuda et al. 1995 Jap. J. Swine Sci. 32, 203–205). Therefore, we investigated whether using a magnetic field would improve the survival of frozen bovine biopsied embryos. Seven-day embryos were recovered nonsurgically from superovulated Holstein heifers or cows. Grade 1 (IETS classification) embryos were selected for this study. Embryos were biopsied by taking a few cells with a stainless steel blade at room temperature. A 0.25-mL straw was filled in the following order: PBS containing 0.3 M sucrose, air, cryoprotective agent (PBS containing 4% ethylene glycol (Sigma-Aldrich Japan K.K., Tokyo, Japan), 4% propanediol (Sigma), and 4% BSA (Albumax 1^®; GIBCO, Grand Island, NY, USA)) containing a biopsied embryo, air, and PBS containing 0.3 M sucrose as described (Aoyagi et al. 1996 Theriogenology 45, 165 abst). The biopsied embryos were frozen either in a methanol bath embryo freezer that induced 0.5 to 1.5 mTesla magnetic fields (ABI Co., Ltd., Abiko, Japan) (MF-embryos) or in a conventional methanol bath embryo freezer (Con-embryos) as a control. In experiment 1, 83 frozen MF-embryos and 81 frozen Con-embryos were thawed in air for 7 s, followed by thawing in 30°C water for 20 s, squeezed out of the straw into PBS + 5% fetal calf serum, and cultured in CR1aa + 5% calf serum for 24 h. Apoptosis of the embryos was detected using the TUNEL assay, and cell numbers of embryos were counted under a fluorescence microscope. In experiment 2, frozen MF- and Con-embryos were transferred nonsurgically to recipient females 6 to 8 days after estrus. Pregnancies were determined by ultrasonography on Day 60. Data were analyzed by chi-square analysis or Fisher's PLSD test following ANOVA. In experiment 1, after 24 h of culture, the survivability of MF-embryos (86.7%) was not significantly different from that of Con-embryos (79.0%). However, the average cell number of the MF-embryos cultured 24 h post-thawing (98.5 ± 3.6) was significantly greater than that of the Con-embryos (85.9 ± 4.1; P < 0.05). Furthermore, the rate of apoptosis in the MF-embryos (12.2 ± 1.1%) after 24 h of culture was significantly less than that of Con-embryos (25.6 ± 1.4%; P < 0.05). In experiment 2, the pregnancy rate of females that were implanted with MF-embryos (70.8%, 17/24) was higher than that of females implanted with Con-embryos (56.3%, 9/16), but the sample sizes were not large enough to tell whether the difference was significant (P > 0.05). In conclusion, the present results showed that cryopreservation of biopsied embryos under a magnetic field inhibited apoptosis post-thawing, and may have led to an increased pregnancy rate in recipient females.