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The effect of insulin on maturation and development of in vitro -fertilized bovine oocytes
THE EFFECT OF INSULIN ON MATURATION AND DEVELOPMENT OF IN VITRQ-FERTILIZED BOVINE OOCYTE-S L. Zhang, E.G. Blakewood, R.S. Denniston and R.A. Godke Department of Animal Science, LAES, LSU Agricultural Center Louisiana State University, Baton Rouge, Louisiana 70803 USA
The objective of this study was to evaluate the effect of bovine insulin on maturation and development of in vitrqfertilized bovine oocytes. Cumulus-intact oocytes were aspirated from abattoir ovaries (3-8 mm follicles) of mixed-breed dairy and beef cows. The cumulus-intact oocytes were incubated in 0.5 ml of Tissue Culture Medium-199 (TCM-199) with 5% heat-treated fetal calf serum (FCS) at 39°C for 24 h. Oocytes were randomly allotted to either Treatment (Trt) A (n=400), TCM-199 with 5% FCS or Trt B (n=400), TCM-199 with 5% FCS and 1 /.&g/mlof bovine insulin (Sigma: St. Louis, MO) as the oocyte maturation medium. Frozen-thawed sperm cells were washed with Brackett-Oliphant (B-O) medium with 10 mM of caffeine and adjusted to a concentration of 3 x 106 sperm/ml and then exposed to Ca2’ ionophore A23 187 to aid in capacitation. Sperm and oocytes were incubated (39°C) in 100 ~1 drops of B-O medium with 10 mg/ml of BSA (Fraction-V) (Sigma: St. Louis, MO) in an atmosphere of 5% CO, in air. After 4 h of incubation, the oocytes (with their cumulus cells) were moved to TCM-199 with 5% FCS and cultured at 39°C. After 43 h in culture, the cumulus cells attached to the ova were removed and the remaining cells attached to the culture dish were maintained in 4-well culture plates for co-culture. Medium was changed at 48-h intervals during the culture interval (I10 days). In summary, the cumulus complex expansion (CCE) score (1 =poor to 5 =excellent score) was greater (P > 0.01) for the insulin-treated oocytes when compared with the CCE score of oocytes not exposed to supplemental insulin (table). In addition, the % fertilization rate was greater (P < 0.05) for the insulintreated oocytes when compared with the rate of those not exposed to insulin. The % of IVF-embryos developing to morula and blastocyst stages were not different with or without insulin supplementation, however, the number of cells/embryo was greater at the morula stage when insulin was used in the maturation medium than that of the control Bovine Oocvtes IVM With and Without Insulin Treatment Oocyted Group TCM-199 TCM-199 + Insulin
CCE
MATR
FERT
CLEV
MORL
EXBLST
HBLST
g Ap
score
96
%
96
46 (cells)
96 (cells)
46 (cells)
400
3.20’
92.8
66.2’
53.5
40.7 (34)’
26.6 (75)
18.9 (509)
3.61b
93.9
77.0b
54.8
42.3 (49)b
23.3 (93)
17.4 (888)
P
-
-
- P
400 800
P
-
-
lsbMeanswith different superscripts in the same column are significantly different. medium without insulin. In this study, bovine cumulus-intact oocytes had significantly improved cumulus expansion scores and fertilization rates following exposure to insulinsupplemented TCM-199 maturation medium. Also, the number of cells per embryo tended to be higher when bovine insulin was added in the maturation medium. The use of insulin-supplemented maturation medium should not be overlooked in IVF procedures for cattle and other farm animal species.
JANUARY
1991 VOL. 35 NO. 1
301
The objective of this study was to evaluate the effect of bovine insulin on maturation and development of in vitrqfertilized bovine oocytes. Cumulus-intact oocytes were aspirated from abattoir ovaries (3-8 mm follicles) of mixed-breed dairy and beef cows. The cumulus-intact oocytes were incubated in 0.5 ml of Tissue Culture Medium-199 (TCM-199) with 5% heat-treated fetal calf serum (FCS) at 39°C for 24 h. Oocytes were randomly allotted to either Treatment (Trt) A (n=400), TCM-199 with 5% FCS or Trt B (n=400), TCM-199 with 5% FCS and 1 /.&g/mlof bovine insulin (Sigma: St. Louis, MO) as the oocyte maturation medium. Frozen-thawed sperm cells were washed with Brackett-Oliphant (B-O) medium with 10 mM of caffeine and adjusted to a concentration of 3 x 106 sperm/ml and then exposed to Ca2’ ionophore A23 187 to aid in capacitation. Sperm and oocytes were incubated (39°C) in 100 ~1 drops of B-O medium with 10 mg/ml of BSA (Fraction-V) (Sigma: St. Louis, MO) in an atmosphere of 5% CO, in air. After 4 h of incubation, the oocytes (with their cumulus cells) were moved to TCM-199 with 5% FCS and cultured at 39°C. After 43 h in culture, the cumulus cells attached to the ova were removed and the remaining cells attached to the culture dish were maintained in 4-well culture plates for co-culture. Medium was changed at 48-h intervals during the culture interval (I10 days). In summary, the cumulus complex expansion (CCE) score (1 =poor to 5 =excellent score) was greater (P > 0.01) for the insulin-treated oocytes when compared with the CCE score of oocytes not exposed to supplemental insulin (table). In addition, the % fertilization rate was greater (P < 0.05) for the insulintreated oocytes when compared with the rate of those not exposed to insulin. The % of IVF-embryos developing to morula and blastocyst stages were not different with or without insulin supplementation, however, the number of cells/embryo was greater at the morula stage when insulin was used in the maturation medium than that of the control Bovine Oocvtes IVM With and Without Insulin Treatment Oocyted Group TCM-199 TCM-199 + Insulin
CCE
MATR
FERT
CLEV
MORL
EXBLST
HBLST
g Ap
score
96
%
96
46 (cells)
96 (cells)
46 (cells)
400
3.20’
92.8
66.2’
53.5
40.7 (34)’
26.6 (75)
18.9 (509)
3.61b
93.9
77.0b
54.8
42.3 (49)b
23.3 (93)
17.4 (888)
P
-
-
- P
400 800
P
-
-
lsbMeanswith different superscripts in the same column are significantly different. medium without insulin. In this study, bovine cumulus-intact oocytes had significantly improved cumulus expansion scores and fertilization rates following exposure to insulinsupplemented TCM-199 maturation medium. Also, the number of cells per embryo tended to be higher when bovine insulin was added in the maturation medium. The use of insulin-supplemented maturation medium should not be overlooked in IVF procedures for cattle and other farm animal species.
JANUARY
1991 VOL. 35 NO. 1
301