
%0 Journal Article
%A Ideta, A. 
%A Hayama, K. 
%A Urakawa, M. 
%A Ohwada, N. 
%A Aoyagi, Y. 
%T 121 CRYOPRESERVATION OF BOVINE BIOPSIED EMBRYOS UNDER A MAGNETIC FIELD
%J Reproduction, Fertility and Development
%D 2006/12/12
%V 19
%N 1
%P 178-178
%K 
%R https://doi.org/10.1071/RDV19N1AB121
%U https://www.publish.csiro.au/paper/RDV19N1AB121 
%X 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&#8211;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&apos;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. 
