Development of in-vitro-derived bovine embryos in protein-free media: effects of amino acids, glucose, pyruvate, lactate, phosphate and osmotic pressure

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doi:10.1071/RD03090

Development of in-vitro-derived bovine embryos in protein-free media: effects of amino acids, glucose, pyruvate, lactate, phosphate and osmotic pressure

G. Wirtu ^A ^B ^F, C. E. Pope ^B, P. Damiani ^B, F. Miller ^C, B. L. Dresser ^B ^D, C. R. Short ^A, R. A. Godke ^A ^E, B. D. Bavister ^A ^D

^A Comparative Biomedical Sciences, Louisiana State University (LSU) School of Veterinary Medicine, Baton Rouge, LA 70803, USA.
^B Audubon Nature Institute Center for Research of Endangered Species, New Orleans, LA 70131, USA.
^C Cyagra Inc., Gibbon, NE 68840, USA.
^D Department of Biological Sciences, University of New Orleans, LA 70148, USA.
^E Department of Animal Sciences, LSU Agricultural Center, Baton Rouge, LA 70803, USA.
^F To whom correspondence should be addressed. email: gwirtu1@lsu.edu





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Reproduction, Fertility and Development 15(8) 439–449 http://dx.doi.org/10.1071/RD03090
Submitted: 14 October 2003 Accepted: 9 February 2004 Published online: 12 March 2004

Abstract

In experiment 1, the effects of a group of either 20 (i.e. glutamine + essential + non-essential) or 11 (i.e. hamster embryo culture medium (HECM)-6) amino acids were evaluated in modified potassium simplex optimised medium (mKSOM) or basic medium (BM)-3. In experiment 2, the effects of glucose, pyruvate, lactate, phosphate or all four substrates were evaluated in low- or high-osmotic pressure BM-3 (255 and 275 mOsmol respectively) containing 20 amino acids (BM-3-20aa). In experiment 1, mKSOM containing 20 amino acids (mKSOM-20aa) supported the highest frequency of total, expanded (Days 7, 8 and 9) and hatched blastocysts. In experiment 2, supplement type affected the frequency of development to at least the morula stage (Day 7), expanded (Day 8), hatched (Day 9) or total blastocysts and cell number per blastocyst. Osmotic pressure affected the frequency of expanded blastocysts (Day 7) and blastocyst cell number. Regardless of the osmotic pressure, BM-3-20aa containing glucose (0.2 mm) supported the highest frequency of blastocyst development. The interaction between supplement type and osmotic pressure was not significant; however, treatment mean differences were more marked in high- than in low-osmotic pressure medium. In conclusion, the beneficial effects of amino acids on in vitro embryo development are influenced by the base medium. Moreover, glucose-containing media supported a higher frequency of embryonic development than pyruvate- and/or phosphate-supplemented media, indicating that glucose plays more important roles in non-energy generating pathways.

Keywords: chemically defined, glycolysis, in vitro culture, Krebs cycle.