183 EFFECT OF THE ACIDIC ORGANIC BUFFER MES ON BOVINE IN VITRO EMBRYO PRODUCTION

Abstract

Transferred embryos carry the risk of being vehicles of organisms causing diseases. Currently, the risk of in vitro-produced (IVP) embryos is more difficult to assess than the risk of in vivo-derived embryos, since less research has been published on the former. Foot and mouth disease virus (FMDV) is extremely sensitive to a low pH and is likely to be destroyed if embryos are exposed to a low pH for a short time. 2-(N-Morphalino)-ethanesulfonic acid (MES); an organic buffer with pKa 6.1; Sigma, South Africa, M2933) as been shown to destroy FMDV at a rate of 90% per minute at pH 6 and at a rate of 90% per second at pH 5 (Acharya et al. 1990 Vet. Microbiol. 23, 21–34; Thomson “Foot-and-mouth disease,” in Infectious Diseases of Livestock with Special Reference to Southern Africa, ed. Coetzer JAW, Thomson GR, and Tustin RC, Oxford University Press, Cape Town, 825–852). The aim of this study was to test whether exposing bovine oocytes and IVP zygotes to the organic buffer MES, buffered at pH 5.5, is detrimental to the development of bovine IVP embryos. IVM, IVF, and IVC was carried out with 1367 oocytes as described earlier [Jooste et al. 2003 Theriogenology 59, 443]. Oocytes were divided into three groups: 484 were used as controls (no MES exposure); 437 were in a maximal exposure group (MAX), i.e. MES treatment after washing of oocytes, after IVM and after IVF, and 446 had a minimal exposure (MIN), i.e. MES treatment after IVF only. To treat the oocytes with MES, 100 oocytes (from ten droplets) were drawn into a pipette in a maximal volume of 100 μL, and placed in 3 mL of MES, swirled around for 10 s, drawn up again in a maximal volume of 100 μL, and placed in 3 mL of culture medium. Oocytes or zygotes were then washed five times in culture medium before being processed through IVM, IVF, or IVC depending on their stage. Exposure of oocytes to MES varied from 30 to 60 s (10 s swirling and a variable time thereafter to pick up). A chi-square test was used to test for differences in cleavage and Day 7 blastocyst yield between control and treatment groups (P < 0.05). Cleavage (70%; 340/484) and blastocyst yield (32%; 156/484) in the control group were not different from those in MIN (68%; 304/446, and 29%; 131/446, respectively), but were significantly higher than for MAX (57%; 249/437, and 18%; 79/437, respectively). In MAX the MES had a harsh effect on the cumulus cells, making them granular and clumpy in appearance. Oocytes treated in MES solution adhered to the bottom of the dish, which made their handling difficult. Exposure time in MES was therefore variable and longer than initially planned. It is concluded that bovine IVP embryos can be exposed to MES without detrimental effect. Treatment with MAX still resulted in blastocysts but it did not yield good numbers. In future trials, treated dishes should be used to prevent oocyte and zygote adherence. Further research is needed to test whether FMDV can be removed from bovine IVP embryos with the described method.