2.5. Statistical analyses Ž
Single-point measurements e.g., day of wave emergence, duration of growing, static .
and regressing phases, onset of regressing phase, maximum follicle diameter were compared among groups by analysis of variance. End points involving repeated mea-
Ž .
surements over time e.g., follicle diameter and hormonal profiles were compared among groups by split-plot analysis of variance to examine the main effects of group
Ž .
Ž .
and time hour or day , and their interactions Gill and Hafs, 1971 . Variation due to sequential data was accounted for by using heifer within group as the error term to test
the effect of group. If main effects or interactions were significant, multiple comparisons Ž
. among groups were made by the method of protected least significant difference LSD .
3. Results
3.1. Experiment 1 Plasma concentrations of estradiol in the respective treatment groups are shown in
Fig. 1. Changes in mean concentration of estradiol in all groups given E-17b were
Ž .
Fig. 1. Mean plasma E-17b concentrations in heifers implanted with progestogen on Day 2 Day 0 s ovulation Ž
and treated intramuscularly with different dosages of E-17b on Day 3. Mean concentrations for control no .
Ž .
E-17b heifers were different P - 0.05 from those of E-17b-treated heifers between 2 and 10 h after 0.1 mg of E-17b, 2 to 12 h after 0.5 mg or 1 mg of E-17b, and between 2 and 24 h after 5 mg of E-17b, respectively.
Bars showing standard error of the means have been omitted for clarity. Experiment 1.
Table 1 Ž
. Mean S.E.M.
follicular characteristics in heifers implanted with progestogen on Day 2 and treated Ž
. intramuscularly with different doses of E-17b on Day 3 ovulation s Day 0 . Experiment 1
End points Control
E-17b Ž
. no E-17b
0.1 mg 0.5 mg
1.0 mg 5.0 mg
n 9
8 8
8 8
First waÕe Ž
. Emergence day
y0.20.1 y0.20.2
0.00.0 y0.10.2
y0.50.8 Dominant follicle
Ž .
Maximum diameter mm 12.50.7
12.40.5 11.10.4
11.20.4 11.20.5
Ž .
Cessation of growth day 6.10.4
5.60.4 5.10.6
5.50.6 5.30.7
ab b
ab ab
a
Ž .
Onset of regression day 10.40.5
12.00.5 11.00.7
10.40.4 9.80.9
Largest subordinate follicle Ž
. Maximum diameter mm
8.10.3 7.80.4
8.60.4 8.10.3
8.40.5 Ž
. Cessation of growth day
3.00.2 2.60.4
3.40.5 2.90.4
3.10.4 Ž
. Onset of regression day
4.30.2 5.10.5
6.40.3 5.00.6
5.10.4 Second waÕe
ab b
ab a
a
Ž .
Emergence day 7.50.3
8.60.4 7.90.3
7.40.4 7.20.4
a,b
Ž .
Means within a row with superscripts not in common are different P - 0.05 .
Ž .
characterized by a dramatic increase P - 0.05 2 h after treatment and a gradual
Ž .
decrease thereafter. Peak concentrations mean S.E.M.
were 118 8, 559 76,
Fig. 2. Mean diameter profiles of the dominant follicles of the first follicular wave in heifers implanted with Ž
. progestogen on Day 2 Day 0 s ovulation and treated intramuscularly with different dosages of E-17b on Day
3. Diameter profiles of the dominant follicles in heifers treated with 5 and 1 mg of E-17b were smaller Ž
. Ž
. P - 0.05 compared with heifers in the 0.1-mg and control no E-17b groups. Diameter profiles of the
Ž .
dominant follicles in heifers treated with 0.5 mg of E-17b tended P - 0.06 to be smaller compared with those heifers treated with 0.1 mg or in the control group, and were not different from those heifers treated with
1 and 5 mg of E-17b. Bars showing standard error of the means have been omitted for clarity. Experiment 1.
903 184 and 3471 595 pgrml for heifers treated with 0.1, 0.5, 1 or 5 mg of E-17b, Ž
. respectively. Mean concentrations in control heifers were lower P - 0.05 than those of
estradiol-treated heifers between 2 and 10 h after 0.1 mg of E-17b, 2 to 12 h after 0.5 mg, or 1 mg of E-17b, and between 2 and 24 h after 5 mg of E-17b.
The effects of E-17b on follicular characteristics and wave emergence in progesto- gen-implanted heifers are shown in Table 1. In the first follicular wave, mean day of
emergence, cessation of growth and maximum diameter of the dominant follicle were not different among groups. However, the mean day of onset of regression of the
Ž .
dominant follicle was earlier P - 0.05 in heifers treated with 5 mg of E-17b than in heifers treated with 0.1 mg of E-17b. Mean day of emergence of the second follicular
Ž .
wave was earlier P - 0.05 in heifers treated with 1 or 5 mg than in those treated with 0.1 mg of E-17b. The day of emergence of the second wave in the control group and the
0.5 mg of E-17b group was intermediate and not different from other E-17b treated groups.
The effects of E-17b treatment on diameter profiles of the dominant follicle in Ž
progestogen-implanted heifers are shown in Fig. 2. A group-by-day interaction P -
. Ž
. 0.05 in the mean profiles of the dominant follicles was attributed to larger P - 0.05
Ž .
Fig. 3. Mean plasma E-17b concentrations in heifers implanted with progestogen on Day 2 Day 0 s ovulation Ž
. and treated with 5 or 0.1 mg of E-17b intramuscularly im , or 0.1 mg of E-17b into the myometrium near the
Ž . tip of the uterine horn ipsilateral to the dominant follicle iu , or 0.1 mg of E-17b into the ovarian stroma,
Ž . Ž
. immediately adjacent to the dominant follicle io on Day 3. Mean concentrations for control no E-17b
Ž .
heifers were different P - 0.05 from those heifers treated with 5 mg of E-17b between 2 and 24 h and from Ž
. those treated with 0.1 mg im, iu and io at 2 and 6 h after treatment. Concentrations in heifers treated with 0.1
Ž .
mg of E-17b io were lower P - 0.05 than in those heifers treated with 0.1 mg, im and iu, at 2 h after
treatment. Bars showing standard error of the means have been omitted for clarity. Experiment 2.
follicle diameters in control heifers or those treated with 0.1 mg of E-17b compared Ž
. Ž
with heifers treated with 5 mg of E-17b Days 4 to 10 or 1 mg of E-17b Days 8 to .
10 . The mean diameter profile of the dominant follicle in heifers treated with 0.5 mg of E-17b was intermediate and did not differ from those of the 1- and 5-mg groups, but
Ž .
tended P - 0.06 to be smaller than those of the 0.1 mg of E-17b and control groups. Mean diameter profiles of the largest subordinate follicle did not differ among groups.
3.2. Experiment 2 Plasma concentrations of E-17b in each of the experimental groups are shown in Fig.
3. Changes in mean concentrations in all treated groups were characterized by a Ž
. dramatic increase P - 0.01 2 h after treatment and a gradual decrease thereafter. Peak
Ž .
concentrations mean S.E.M. were highest in heifers treated with 5 mg of E-17b im Ž
. 3228.3 305.6 pgrml . Mean peak concentrations in heifers treated with 0.1 mg, im
Ž .
and 0.1 mg, iu were not different 197.2 38.3 and 203.3 39.1 , but both were higher Ž
. Ž
. P - 0.05 compared with heifers treated with 0.1 mg, io 102.6 8.9 . Mean concen-
Ž .
Ž trations in the control group were lower P - 0.05 than in the 0.1 mg groups im, io or
. iu from 2 to 6 h and the 5 mg group from 2 to 24 h after E-17b treatment.
The effects of E-17b treatment on follicular characteristics and wave emergence in progestogen-implanted heifers are shown in Table 2. Mean day of emergence of the first
follicular wave did not differ among groups. The dominant follicle of the first follicular wave in heifers treated with 5 mg of E-17b had a smaller maximum diameter, ceased
Table 2 Ž
. Mean S.E.M. follicular characteristics in heifers implanted with progestogen on Day 2 and treated with 5.0
Ž . or 0.1 mg of E-17b im, or 0.1 mg into the wall of the uterine horn ipsilateral to the dominant follicle iu , or
Ž . Ž
. 0.1 mg into the ovary, adjacent to the dominant follicle io on Day 3 Day 0 s ovulation . Experiment 2
End points Control
E-17b Ž
. no E-17b
Ž .
Ž .
Ž . Ž .
5.0 mg im 0.1 mg im
0.1 mg iu 0.1 mg io
n 8
8 8
8 6
First waÕe Ž
. Emergence day
y0.10.1 y0.30.2
0.00.2 y0.10.2
0.20.4 Dominant follicle
b a
ab ab
b
Ž .
Maximum diameter mm 12.90.6
10.70.4 11.80.5
12.10.3 12.50.6
b a
ab b
ab
Ž .
Cessation of growth day 6.30.8
4.40.7 6.00.5
6.30.4 5.90.3
b a
b b
b
Ž .
Onset of regression day 11.00.8
8.40.8 11.00.6
11.80.6 12.21.1
Largest subordinate follicle Ž
. Maximum diameter mm
8.10.4 7.60.3
8.30.6 7.50.4
7.80.4 Ž
. Cessation of growth day
2.90.3 2.50.4
2.90.5 2.60.5
3.50.6 Ž
. Onset of regression day
5.00.5 5.10.4
5.50.7 5.40.5
5.70.6 Second waÕe
bc a
ab bc
c
Ž .
Emergence day 8.00.4
6.90.2 7.40.3
8.10.5 9.00.4
a,b,c
Ž .
Means within a row with superscripts not in common are different P - 0.05 .
Ž .
growth and regressed earlier compared with control heifers P - 0.05 . Correspond-
ingly, the mean day of emergence of the second follicular wave was also earlier Ž
. P - 0.05 . Mean characteristics of the dominant follicle of the first follicular wave and
the emergence of the second follicular wave in the three groups treated with 0.1 mg of Ž
. E-17b im, iu and io and the control group were not different.
The effects of E-17b treatment on diameter profiles of the dominant follicle in Ž
progestogen-implanted heifers are shown in Fig. 4. A group-by-day interaction P -
. 0.05 was attributed to a smaller mean follicle diameter in the treated heifers. Differ-
ences from the control group were detected from Days 5 to 12 in heifers treated with 5 mg im of E-17b, and on Day 5 in those heifers treated with the 0.1 mg im and iu. Mean
diameter of the dominant follicle in heifers treated with 5 mg of E-17b was smaller Ž
. Ž
. P - 0.05 compared with heifers in the control no E-17b group or those treated with
0.1 mg E-17b io on Days 5 to 12 and those treated with 0.1 mg E-17b im or iu on Days 7 to 12. Diameter profiles of heifers treated with 0.1 mg of E-17b im, iu and io were not
different. Diameter profiles of the largest subordinate follicles did not differ among groups.
The effects of E-17b treatment on systemic concentrations of gonadotropins are shown in Fig. 5. As the mean hourly changes in FSH and LH were not significantly
different among groups in the first or second serial bleeding periods, data for the E-17b-treated groups were combined and compared with the control group. Again, there
was no significant difference between groups for FSH or LH in either the first or second
Fig. 4. Mean diameter profiles of the dominant follicles of the first follicular wave in heifers implanted with Ž
. Ž
. progestogen on Day 2 Day 0 s ovulation and treated with 5 or 0.1 mg of E-17b intramuscularly im , or 0.1
Ž . mg of E-17b into the wall of the uterine horn near the tip ipsilateral to the dominant follicle iu , or 0.1 mg of
Ž . E-17b into the ovarian stroma, immediately adjacent to the dominant follicle io on Day 3. Mean diameter of
Ž .
the dominant follicle in heifers treated with 5 mg of E-17b was smaller P - 0.05 compared with heifers in Ž
. the control no E-17b group and those heifers treated with 0.1 mg E-17b io on Days 5 to 12 and those treated
with 0.1 mg E-17b im or iu on Days 7 to 12. Mean diameter of the dominant follicle in heifers treated with 0.1 Ž
. mg of E-17b im was smaller P - 0.05 than in the control group only on Day 5. Diameter profiles of heifers
treated with 0.1 mg of E-17b im, iu and io were not fdifferent. Bars showing standard error of the means have been omitted for clarity. Experiment 2.
Ž .
Fig. 5. Mean S.E.M. plasma FSH and LH concentrations in heifers implanted with progestogen on Day 2 Ž
. Ž
. Day 0 s ovulation and treated with 5 or 0.1 mg of E-17b intramuscularly im , or 0.1 mg of E-17b into the
Ž . wall of the uterine horn near the tip ipsilateral to the dominant follicle iu , or 0.1 mg of E-17b into the
Ž . ovarian stroma, immediately adjacent to the dominant follicle io on Day 3. Since there was no significant
difference among the treatment groups for either FSH or LH, the treated group represents combined data from all estradiol-treated heifers. Subsequent analyses indicated mean FSH concentrations were lower in the control
Ž .
Ž .
Ž group P - 0.03 and the combined-treatment group P - 0.0007 in the first serial bleeding period Hours 2
. Ž
. to 12 compared with the second Hours 16 to 26 . Mean LH concentrations were similar in the control group
Ž .
between the two serial bleeding periods but were higher P - 0.0004 for the first compared with the second serial bleeding period in the combined-treatment group.
serial bleeding period. However, comparison of concentrations of gonadotropin between the serial bleeding periods within groups indicated significant changes. The mean
Ž .
concentrations of FSH were lower in the control group P - 0.03 and the estradiol-
Ž .
Ž .
treated group P - 0.0007 in the first serial bleeding period Hours 2 to 12 compared Ž
. with the second serial bleeding period Hours 16 to 26 . Mean concentrations of LH
were similar in the control group between the two serial bleeding periods but were
Ž .
higher P - 0.0004
in the first than in the second serial bleeding period in the estradiol-treated group.
4. Discussion
