The importance of NaCl concentration in a chemically defined medium for the development of bovine oocytes matured and fertilized in vitro. J.M. Lim, J.H. Kim, K. Okuda and K. Niwa

Theriogenology 42:421-432, 1994

The importance of NaCl concentration in a chemically defined

medium for the development of bovine oocytes matured

and fertilized in vitro

J.M. Lim, J.H Kim, K1. Okuda and K. Niwa2

Division of Animal Science and Technology, Faculty jof Agriculture

Okayama University, Okayama 700, Japan

Abstract

Bovine oocytes matured and fertilized in vitro were cultured in a 

chemically defined medium(modified Tyrode's solution) without glucose. 

When different concentrations of NaCl were added to the medium, the 

proportions of embryos developed to the ≥8-cell, morula and blastocyst 

stages 96, 144 and 192h post insemination, respectively, were 

significantly higher at 89 to 114mM than 64 to 76 and 126 to 139mM NaCl. A 

high proportion(28%) of blastocyst stage embryos 192h post insemination 

was obtained at 89mM NaCl. When calculated osmolarity in the medium with 

64mM NaCl was varied by adding D-sorbitol, significantly higher 

proportions of morula stage embryos were obtained at 265 to 315mOsm(27 to 

38%) than 215(9%) and 365(2%)mOsm, but the development to the blastocyst 

stage was difficult at any osmolarities(215 to 365mOsm) tested. In the 

medium with a fixed osmolarity(315mOsm) but with different concentrations

(64 to 114mM) of NaCl, there were no differences in the proportions(29 to 

33%) of morula stage embryos among different NaCl concentrations. However, 

significantly higher proportions of embryos developed to the blastocyst 

stage at 89 to 101mM(22 to 23%) than 64 to 76(0 to 9%) and 114(11%)mM 

NaCl. When Cl- concentration in the medium with 64mM NaCl was adjusted by 

adding choline chloride, significantly higher proportions of embryos 

developed to the morula stage at 97 to 122mM(32 to 40%) than 72(6%) and 147

(2%)mM Cl-, but few embryos developed to the blastocyst stage at any Cl- 

concentrations(72 to 147mM) tested. In the medium with 64 or 114mM NaCl 

and each with 2 different Na+/K+ ratios, there were no differences in the 

proportions of morula and blastocyste stage embryos between different 

Na+/K+ ratios(31 and 39 at 64mM NaCl, and 39 and 47 at 114mM NaCl) at each 

NaCl concentration. When glucose was added to the medium with 89mM NaCl 

120h post insemination, there were no significant differences in the 

proportions(40 to 48%) fo morula stage embryos 144h post insemination 

among different concentrations(0 to 6.95mM) of glucose. The proportion

(33%) of blastocysts 192h post insemination at 2.78mM glucose was 

significantly higher than the values at 0(22%), 5.56(19%) and 6.95(15%)mM 

but not different compared with the values at 1.39(23%) and 4.17(28%)mM. 

In conclusion, NaCl concentration in a defined medium is one of the most 

important factors for the development of bovine embryo to the blastocyst 

stage, but the development of embryos up to the morula stage is also 

regulated by osmolarity and/or Cl- concentration.

key words: bovine, embryos, preimplantation development, defined medium, 

NaCl requirement