Effect of culture media and CO2 concentration on embryo development in a bovine embryo in vitro production system

Theriogenology EFFECT OF CULTURE MEDIA AND CO, CONCENTRATION ON EMBRYO DEVELOPMENT IN A BOVINE EMBRYO IN VITRO PRODUCTION SYSTEM J.S. Merton’, J.A.W. van Dillen’, J.F. Hasler’ and J.H.G. den Daas’. ’ Holland Genetics, IVP Laboratory, 5437 BG Beers, The Netherlands. ’ Emtran Inc, 197 Bossler Road, Elizabethtown, PA 17022, Pennsylvania, USA. The objective of the present work was to investigate the effect of two different culture media, TCM 199 and Menezo-B2 with different CO, cqncentrations, on the rates of cleavage and blastocyst formation of IVM and IVF oocytes. Groups of 35 cumulus-oocyte-complexes (COCs), recovered from slaughterhouse ovaries, were matured for 24 h in 0.5 ml TCMl99 supplemented with 10% Fetal Calf Serum, 4 ug FSH/ml and 6 ug LH/ml (bovine, pituitary glands). After IVM, groups of 35 COCs were transferred to 0.5 ml fertilization medium (modified Tyrode-Lactate medium) containing 0.6% fatty acid-free BSA fraction V, 10 uglml heparin, 20uM penicillamine, 10 uM hypotaurine and 1 uM epinephrine. Frozen/thawed semen was Percoll separated and added at a concentration of 0.25 x 106/ml. After 20 to 22 h (Day 1). COCs were vortexed to remove the surrounding cumulus cells and placed in groups of 35 in wells containing a monolayer of BRL cells and either 0.5 ml TCMl99 or Menezo-B2 supplemented with 10% FCS. On Days 4 and 8, embryos were placed on fresh monolayers. Cleavage was evaluated on Day 4 and embryo development on Days 7, 8, 9 and 11. In experiment I, embryos were incubated at 38.5 “C in 5% CO,. In experiment II and Ill embryos cultured in Menezo-B2 were incubated in 4% and 3% CO,, respectively. Table: Rates of cleavage and blastocyst formation of In Vitro matured and fertilized cultured either in TCM199 or Menezo-BP with different levels of CO,. 5xP

Group

CO,

Oocytes

Day8

Day1 1

I

Ml99 B2

5% 5%

1085 702(65)a 1120 700(63)a

157114)a 197(18)b

257124)a 276(24)a

243(95)a 203(74)b

II

Ml99 62

5% 4%

1061 602(57)a 1082 674(62)b

146(14)a 219(20)b

2 17(20)a 254(23)a

23Of 1OO)a 242(95)a

Ill

Ml99 82

5% 3%

1578 940(60)a 1491 993(67)b

210(13)a 355(24)b

340(22)a 45gf31)b

335fg9)a 418fgl)a

Mat

a,b: Values within each experiment analysis (PC 0.05).

(%)Hatching

Blastocysts

Cleaved(%)

Day7

oocytes

and column with different

superscripts

differ

by Chi Square

In experiment I significantly more (PC 0.05) embryos developed to the blastocyst stage on Day 7 in MenezoB2 than in TCM199. However, an equal number of embryos reached the blastocyst stage on Day 8. When Menezo-B2 was used in 5% CO,, the pH of the medium (6.5 - 6.8) affected the quality of the embryos (hatching percentage; P-Z 0.001). By lowering the CO, level, this effect was deleted. In 3% CO, (experiment Ill) Menezo-BP caused a sianificant increase in rates of cleavage -(P-Z’ 0.0001) and blastocyst formation it-r both Day 7 and 8 (P-Z 0.001; Day 7). It is concluded that embryo culture in Menezo62 compared to TCMl99 reauires a lowerino of the CO, concentration to cause an enhanced embryo development rate and an increased number of embryos produced.