Effect of donor bovine serum fractionation on preimplantation mouse embryos in culture

Theriogenology 41:257, 1994 EFFECT OF DONOR BOVINE SERUM FRACTIONATION ON PREIMPLANTATION MOUSE EMBRYOt.q IN CULTURE C.N. Millham, M.B. Tomesi, &.T. P a l a s z ' a n d J. Archer Department of Veterinary Pathology a n d ' H e r d Medicine and Theriogenology, WCVM, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 0W0 Mammalian serum is considered to be an essential component of culture media for mammalian embryos. Studies were designed to determine the effect of fractionation of steer serum (donor bovine serum, DBS; Bocknek, Rexdale, ON, CAN) on embryo development in culture. DBS was fractionated by dialysis, using cellulose membranes with pore sizes to retain fractions of different molecular weights: 1.0Kd, 3.5Kd, 8.0Kd, 14.0Kd, 25.0Kd (Spectrum Medical Industries Inc, Los Angeles, CA, USA). The DBS fractions were analyzed for total protein, albumin, glucose, and electrolytes and were added to the culture medium in two concentrations: experiment I - 5% and experiment II - 1% . Embryos were collected from superstimulated mice 2.5 days after hCG administration into Dulbeccos phosphate buffered saline (PBS) containing suffactant (0.15% Pluronic, Vetrepharm Inc., London, ON, CAN). Four-cell embryos were cultured in BWW medium under paraffin oil in a humidified atmosphere of 5% CO,~ in air at 37°C. Embryo morphology was examined microscopically every 24h for 4 d~ys. Data within each experiment were subjected to Chi-square analysis. After 48h in culture, embryo development in experiment I was significantly higher in all fractions than in control (serum-free, albumin-free) medium, and in experiment II, embryo development was significantly higher in 1% DBS and all fractions than in the control medium (P<0.05). Results after 96h are shown in the following table.

Embryo development and hatched/hatching rates after 96h culture in different fractions of DBS at concentrations of 5% (experiment I) or 1% (experiment II). Experiment I - (5%) Groups

N

% Embryo Development

Experiment II - (1%)

% Hatched/ Hatching

N

% Embryo Development

% Hatched/ Hatching

Control

126

2.4 c

0.8 d

106

0.9 d

0.9 d

DBS

119

47.1 b

39.5 bc

104

79.8 bc

66.4 ab

1.0 Kd

132

65.2 a

39.4 bc

105

75.2 c

51.4 c

3.5 Kd

130

76.9 a

58.5 a

104

87.5 ab

70.2 a

8.0 Kd

123

68.3 a

49.6 ab

103

79.6 bc

63.0 abc

14.0 Kd

120

65.0 a

45.0 b

104

76.9 bc

53.9 bc

25.0 Kd 122 49.2 b 27.9 c 104 94.2 a 71.2 a abcd - Percentages within a column with superscripts not in common are significantly different (P<0.05).

Results indicate that in vitro development of four-cell mouse embryos to the blastocyst/hatching stage is stimulated by serum or a fraction of serum. After 96h in culture, embryo development in most fractions of DBS at very low concentrations (1%) was equal to or less than in complete DBS, but at 5% DBS, embryo development was higher in the majority of the fractions than in complete serum. These data suggest that DBS contains components that are inhibitory to embryo development at levels as low as 5% and that fractionation by dialysis may remove some of these components.

Copyright © 1994 Butterworth-Heinemann

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