Fig. 1. A healthy, morphologically normal bovine preantral follicle with circular granulosa cell layers visible Ž
. after 10 days of in vitro culture after staining with Hoechst 33342 =400 .
. Concepts, Berkeley, CA, USA for the diameter gain by follicles on different days of
culture. The analysis for maintenance of viability and morphology was done by Chi-square test.
3. Results
Follicles of all initial sizes, and on all control and culture treatments increased in diameters during the period of in vitro culture with the greatest increases in absolute and
proportional size occurring between days 1 and 5 of culture. The culture medium supplements caused the increase in size to be greater than control at both days 5 and 11
Ž .
of culture for all initial size categorizes p - 0.01; Table 1 . For follicles with an initial diameter of 40 mm, the FSH alone and FSH plus EGF supplements were more effective
Ž .
at both days 5 and 11 of culture than the other two treatments p - 0.01 . The same two Ž
. treatments were most effective for 60-mm follicles Table 2
at day 5 of culture Ž
. p - 0.01
and, of the two poorer supplements, FSH plus ITS was significantly Ž
. p - 0.01
better than FSH plus hypoxanthine. By day 11 of culture, there were Ž
. differences between all the treatments with FSH q EGF FSH p - 0.01 FSH q ITS
Ž .
Ž .
p - 0.01 FSH plus hypoxanthine p - 0.05 . This pattern of treatment differences
was established by day 5 of culture when the initial diameter of the follicles was 80 mm Ž
. Ž
. Ž
. Table 3 , FSH q EGF FSH
p - 0.05 FSH q ITS p - 0.01 FSH plus hypox-
Ž .
anthine p - 0.01 and the pattern was maintained at day 11 of culture altogether the
Table 1
q
Ž Effect of FSH, FSHqEGF, FSHqITS , FSHqhypoxanthine in culture medium on follicle diameter 40
. mm , viability and morphology, in vitro cultured for 10 days
Ž .
Treatments Days in culture
At the end of culture follicular Ž
. Ž
. meansSEM; ns 20
intactrnormal 1
5 11
Viability Morphology
Ž .
Ž .
ns10 ns10
A A
Ž .
Ž .
Ž .
FSH 100 ngrml 40.60.2
58.30.6 63.90.4
9 90 8 80
A B
Ž .
Ž .
FSHqEGF 40.40.1
57.40.5 69.70.6
8 80 8 80
Ž .
100 ngrml each
B aC
Ž .
Ž .
Ž .
FSH 100 ngrml q 40.60.1
53.30.7 59.90.4
8 80 9 90
q
Ž .
ITS 1
B bC
Ž .
Ž .
FSH 100 ngrmlq 40.50.1
53.00.5 61.60.8
8 80 8 80
hypoxanthine Ž
. 4 mM
C D
Ž .
Ž .
Control 40.30.1
45.00.2 48.90.2
7 70 6 60
Ž
ab ABCD
Values with different superscripts in the same columns are significantly different p- 0.05,
p- 0.01, .
ANOVA; p 0.05, Chi-square test .
difference between FSH and FSH q EGF was not significant. The four media supple- Ž
. ments did not affect the size of 100-mm follicles Table 4 by day 5, and there was little
difference between them at day 11 although the difference in size at day 11 was Ž
. significant for the comparison between the FSH and FSH q ITS
p - 0.05 culture supplements. None of the culture media caused the follicle diameter to increase in size
to that of the initial diameter of the next larger category of follicle during the 10 days of
Table 2
q
Ž Effect of FSH, FSHqEGF, FSHqITS , FSHqhypoxanthine in culture medium on follicle diameter 60
. mm , viability and morphology, in vitro cultured for 10 days
Ž .
Treatments Days in culture
At the end of culture follicular Ž
. Ž .
Ž .
meansSEM ns 20
intactrnormal 1
5 11
Viability Morphology
Ž .
Ž .
ns10 ns10
A A
Ž .
Ž .
Ž .
FSH 100 ngrml 60.70.1
84.91.4 91.01.4
9 90 9 90
A B
Ž .
Ž .
FSHqEGF 60.80.1
85.11.3 95.41.3
9 90 9 90
Ž .
100 ngrml each
B aC
Ž .
Ž .
Ž .
FSH 100 ngrml q 60.50.1
79.70.4 86.10.4
9 90 8 80
q
Ž .
ITS 1
C bC
Ž .
Ž .
FSH 100 ngrmlq 60.80.1
75.90.4 83.10.6
8 80 8 80
hypoxanthine Ž
. 4 mM
D D
Ž .
Ž .
Control 60.40.1
66.80.1 70.70.2
7 70 7 70
Ž
ab ABCD
Values with different superscripts in the same columns are significantly different p- 0.05,
p- 0.01, .
ANOVA; p 0.05, Chi-square test .
Table 3
q
Ž Effect of FSH, FSHqEGF, FSHqITS , FSHqhypoxanthine in culture medium on follicle diameter 80
. mm , viability and morphology, in vitro cultured for 10 days
Ž .
Treatments Days in culture
At the end of culture follicular Ž
. Ž .
Ž .
meanSEM ns 20
intactrnormal 1
5 11
Viability Morphology
Ž .
Ž .
ns10 ns10
aA A
Ž .
Ž .
Ž .
FSH 100 ngrml 80.70.1
99.41.0 111.31.2
9 90 8 80
bA A
Ž .
Ž .
FSHqEGF 80.50.1
101.81.0 113.11.4
9 90 9 90
Ž .
100 ngrml each
B B
Ž .
Ž .
Ž .
FSH 100 ngrml q 80.60.1
96.30.4 105.70.8
9 90 8 80
q
Ž .
ITS 1
C C
Ž .
Ž .
FSH 100 ngrmlq 80.30.1
92.70.3 101.00.4
9 90 8 80
hypoxanthine Ž
. 4 mM
D D
Ž .
Ž .
Control 80.30.1
87.90.2 91.40.2
8 80 6 60
Ž
ab ABCD
Values with different superscripts in the same columns are significantly different p- 0.05,
p- 0.01, .
ANOVA; p 0.05, Chi square test .
culture although follicles, which were 100 mm in diameter at the start of culture had achieved a diameter of 120 mm, the size of an in vitro mature follicle, after 4 days in
culture. The overall follicular viability and morphology were better maintained with the
Ž treatments than the controls in all cases but, were not significantly different
p 0.05, .
Chi-square test .
Table 4
q
Ž Effect of FSH, FSHqEGF, FSHqITS , FSHqhypoxanthine in culture medium on follicle diameter 100
. mm , viability and morphology, in vitro cultured for 10 days
Treatments Days in culture
At the end of culture Ž
. Ž .
Ž . Ž
. meanSEM
ns 20 follicular
intactrnormal 1
5 11
Viability Morphology
Ž .
Ž .
ns10 ns10
A A
Ž .
Ž .
Ž .
FSH 100 ngrml 100.30.1
118.80.9 130.01.0
8 80 8 80
A aA
Ž .
Ž .
FSHqEGF 100.20.0
119.31.2 129.00.9
8 80 8 80
Ž .
100 ngrml each
A bA
Ž .
Ž .
Ž .
FSH 100 ngrml q 100.40.1
120.01.0 132.91.9
9 90 8 80
q
Ž .
ITS 1
A A
Ž .
Ž .
FSH 100 ngrmlq 100.50.1
119.31.1 131.91.5
8 80 8 80
hypoxanthine Ž
. 4 mM
B B
Ž .
Ž .
Control 100.50.1
108.50.2 112.00.6
7 70 7 70
Ž
ab AB
Values with different superscripts in the same columns are significantly different p- 0.05,
p- 0.01, .
ANOVA; p 0.05, Chi-square test .
4. Discussion
