Successful in vitro growth of rat two-cell embryos to blastocysts using a simple chemically defined medium.
The aim of the present study was to formulate a simple chemically defined medium for the in vitro growth of rat two-cell embryos to blastocysts. Embryos from day 2 pregnant rats were retrieved and placed in paraffin oil-covered droplets of "rat two-cell embryo culture medium" (R2ECM) containing combinations of various serum supplements, glucose, L-glutamine, and cultured up to 96 h in a CO(2) incubator. Embryos cultured in the basic medium (R2ECM), as well as those supplemented either with fetal bovine serum (FBS) or male rat serum (MRS) did not develop beyond the two- to four-cell stage. In R2ECM with 0.3% bovine serum albumin (BSA) and 7.5 mM glucose, 44% of embryos reached the blastocyst stage by 96 h in culture, and the blastulation rate increased to about 83% when 1 mM of L-glutamine was added. To evaluate the effects of varying doses of glucose, two-cell embryos were cultured in R2ECM supplemented with 0.3% BSA, 1 mM L-glutamine, and 2.5, 5.0, or 7.5 mM of glucose. The percentage of embryos reaching the blastocyst stage for 2.5, 5.0, and 7.5 mM glucose was 64.6%, 65.3%, and 82.9%, respectively. The present study showed that the modified medium (R2ECM) is a simple chemically defined medium that is capable of supporting in vitro growth of rat two-cell embryos to blastocysts in high proportion (greater than 80%) without the need for change of medium within 96 h of culture.
Goh
V H
VH
Department of Obstetrics and Gynecology, National University Hospital, National University of Singapore, 5 Lower Kent Road, 119074, Singapore, Singapore. obggohhh@nus.edu.sg
Adiga
S K
SK
Tain
C F
CF
Tong
T Y
TY
Ng
S C
SC
D000818:Animals; D001755:Blastocyst / Q000187:drug effects / Q000502:physiology*; D003470:Culture Media; D046508:Culture Techniques; D005314:Embryonic and Fetal Development / Q000187:drug effects / Q000502:physiology*; D005260:Female; D005947:Glucose / Q000494:pharmacology; D005973:Glutamine / Q000494:pharmacology; D011247:Pregnancy; D051381:Rats; D017208:Rats, Wistar
D003470:Culture Media; D005973:Glutamine; D005947:Glucose