Ž Exogenous estrogens have been shown to induce an LH surge Short et al., 1979;
. Kinder et al., 1991 and it was suggested that LH release may be associated with
induction of follicular atresia. However, results from our laboratory do not support such a hypothesis; estradiol treatment to heifers with progestogen-ear implants or during the
luteal phase of the cycle did not induce an LH surge yet growth of the dominant follicle
Ž .
was suppressed Bo et al., 1993, 1994 . In another laboratory, follicular atresia was detected prior to the estradiol-induced LH release in cows treated on Day 16 of the cycle
Ž .
Engelhart et al., 1989 . Alternatively, estrogen may induce follicular atresia by altering Ž
. tonic LH secretion andror through FSH suppression Price and Webb, 1988 . Estradiol
Ž .
has been reported to decrease LH pulse amplitude in sheep Rawlings et al., 1984 and Ž
. cattle Price and Webb, 1988 and, progesterone or progestogen implants in cattle have
been reported to decrease LH pulse frequency and suppress maximal diameter of the Ž
dominant follicle in a dose-dependent manner Ireland and Roche, 1982; Adams et al., .
1992a; Savio et al., 1993a,b; Stock and Fortune, 1993; Sanchez et al., 1995 . Estrogen- induced suppression of LH may be apparent only during a progestational phase, and
estradiol and progesterone may have a synergistic effect on altering circulating concen- Ž
. trations of FSH. Estradiol alone
Butler et al., 1983; Wolfe et al., 1992 or in
Ž .
combination with progestogen Barnes et al., 1981 has been shown to suppress FSH; Ž
however, suppression was more prolonged in the progestogen-implanted heifers Bolt et .
al., 1990 . It has been suggested that estradiol and progesterone given in combination Ž
. have an additive suppressive effect on both LH and FSH Price and Webb, 1988 .
Treatment of rhesus monkeys with estradiol into the ovary bearing the largest Ž
. dominant
follicle caused follicular atresia without apparently altering circulating Ž
. Ž
. concentrations of LH or FSH Hutz et al., 1988 . Dierschke et al. 1994 also reported
that estrogen induced follicular atresia in monkeys and rats by an action directly within the ovary rather than through gonadotropin suppression. To our knowledge, there are no
reports of a local effect of estrogen on follicular dynamics in cattle.
We hypothesized that the suppressive effect of exogenous estradiol, in combination with progestogen, on ovarian follicles in cattle is mediated systemically rather than
locally. Experiment 1 was designed to determine a minimal effective systemic dose of estradiol for inducing follicular regression in progestogen-implanted heifers. Experiment
2 was designed to utilize a dose just below the minimal effective systemic dose of
Ž . Ž
. estradiol-17b E-17b
subminimal dose; 0.1 mg locally, to determine whether estradiol treatment induces suppression of follicles through a direct action at the level of the
ovary.
2. Materials and methods
2.1. Experiment 1 Ž
. Cross-bred beef heifers n s 41 , 15 to 18 months of age and weighing 320 to 420 kg
Ž . Ž
were treated with 500 mg of cloprostenol intramuscularly im Estrumate, Coopers
. Agropharm, Ajax, ON, Canada and monitored daily by ultrasonography to detect
Ž .
Ž ovulation Day 0 . On Day 2, heifers received a progestogen ear implant Syncro-Mate-B,
. Sanofi, Overland Park, KS, USA and were assigned randomly to one of five groups:
Ž .
Ž .
Ž .
control sesame seed oil, n s 9 ; 0.1 mg of E-17b n s 8 ; 0.5 mg of E-17b n s 8 ; 1 Ž
. Ž
. Ž
mg of E-17b n s 8 ; or 5 mg of E-17b n s 8 . Each dose of E-17b Sigma, St. Louis, .
MO, USA was dissolved in 1 ml of sesame seed oil. Administration was done by a single im injection into the hind leg in the region of the semitendinosus muscle on Day
3. Day 3 was selected as the day of E-17b treatment because it corresponded to the mid-growing phase of the dominant follicle of the first follicular wave and the earliest
day the dominant follicle could be consistently differentiated from subordinate follicles Ž
. Ginther et al., 1989a .
2.2. Experiment 2 Ž
. Cross-bred beef heifers n s 38 from the same population as those in Experiment 1
were treated with cloprostenol, monitored for ovulation and received a progestogen ear implant on Day 2. On Day 3, heifers were assigned randomly to one of five treatment
Ž .
Ž .
groups: control sesame seed oil, im; n s 8 ; 5 mg of E-17b im n s 8 ; 0.1 mg of Ž
. E-17b im n s 8 ; 0.1 mg of E-17b given into the wall of the uterus, near the tip of the
Ž .
horn ipsilateral to the dominant follicle iu; n s 8 ; or 0.1 mg of E-17b into the ovarian Ž
. stroma, immediately adjacent to the dominant follicle io; n s 6 .
Ž .
Local treatments iu and io were administered with a 21-gauge needle soldered to a Ž
17-gauge, 60-cm-long barrel using transvaginal ultrasound-guided needle puncture 5.0 .
MHz convex-array transducer; Corometrics Medical Systems, Wallingford, CT, USA . Ž .
Intrauterine iu treatments were administered in 1 ml of sesame seed oil into the myometrium, near the ovarian end of the uterine horn. A local relationship between
Ž venous drainage of the proximal portion of the uterine horn uterine branch of the
. Ž
. ovarian vein
and the arterial supply of the ovary ovarian artery has been well
Ž .
documented Ginther, 1974 . Treatment into the uterine wall was intended to induce an elevation of E-17b locally in the ovary containing the dominant follicle. Intraovarian
Ž . io treatment was administered in 0.2 ml of sesame seed oil into the ovarian stroma
immediately adjacent to the dominant follicle. The io treatment was similar to that used Ž
. in monkeys Hutz et al., 1988 . Progestogen ear implants were removed at the end of the
observational period, 4 days after detection of the emergence of the second follicular wave.
2.3. Ultrasonography Heifers were examined daily by transrectal ultrasonography using a 7.5-MHz trans-
Ž .
ducer Aloka SSD500, ISM, Edmonton, AB to individually identify and monitor the dominant and largest subordinate follicles of the first follicular wave and to detect the
Ž .
day of emergence of the second follicular wave Knopf et al., 1989 . Accordingly, ultrasound scanning commenced 2 or 3 days prior to the expected time of ovulation and
continued until 4 days after the emergence of the second wave. Ultrasound data were collected without knowledge of treatment groups.
The dominant follicle of a wave was defined as the follicle that reached the largest diameter. Subordinate follicles were defined as those that appeared to originate from the
Ž .
same follicular pool as the dominant follicle Ginther et al., 1989a . Emergence of a follicular wave was defined as the day that the dominant follicle was retrospectively
Ž .
identified at a diameter of 4 to 5 mm Ginther et al., 1989a . Cessation of growth of the dominant follicle was defined as the day that the dominant follicle appeared to cease a
Ž .
progressive increase in diameter Ginther et al., 1989a . The first day that the follicle appeared to begin a progressive decrease in diameter was defined as the onset of
Ž .
regression of the dominant follicle Ginther et al., 1989a,b . 2.4. Blood sampling and hormone assays
In Experiments 1 and 2, changes in circulating concentrations of estradiol were determined by collecting blood samples via jugular venipuncture into heparinized tubes.
Ž .
Plasma was harvested within 30 min of collection and stored frozen y208C until assays were done. In Experiment 1, samples were taken every 2 h during the first 12 h
after E-17b treatment and every 6 h for the next 36 h and, in Experiment 2, blood samples were collected at 0, 2, 6, 12, 24 and 48 h after E-17b treatment. Plasma
concentrations of estradiol were measured in all samples using a validated radioim-
Ž .
munoassay Joseph et al., 1992 . Standards were prepared in charcoal-stripped bovine serum and the standard curve ranged from 6.25 to 1600 pgrml. Values greater than the
standard curve were diluted in charcoal-stripped serum and re-analyzed. Sensitivity of the assay was 5 pgrml and coefficients of variation were 14 within-assay and 16
between-assays.
In Experiment 2, changes in circulating concentrations of FSH and LH were Ž
determined by collecting blood samples via an indwelling jugular catheter SV-70 vinyl tubing, i.d. 1.0 mm and o.d. 1.5 mm; Dural Plastics and Engineering, Dural, NSW,
. Australia . Heifers were fitted with a catheter on Day 2 and held in stalls overnight. On
Day 3, E-17b treatments were given at 0600 h. The first serial bleeding period began 2 h later at 0800 h and ended 10 h later at 1800 h. The second serial bleeding period began
16 h after E-17b treatment at 2200 h and ended 10 h later at 0800 h. Blood samples were taken once every hour for each of the serial bleeding periods. An initial 1-ml
sample was withdrawn and discarded, a second 4-ml sample was withdrawn and transferred to a centrifuge tube. All samples were allowed to clot at room temperature
Ž
. 12–18 h ; the clots were removed, the tubes were centrifuged and the serum was
Ž .
poured-off into storage vials and frozen y208C
until assays were done. Serum concentrations of FSH and LH were determined using a validated radioimmunoassay
Ž .
Bolt and Rollins, 1983; Bolt et al., 1990 which has been modified and described Ž
. Adams et al., 1992a . Reagents were obtained from the USDA Animal Hormone
Program. For the FSH assay, USDA-bFSH-I-2 was used for iodination and reference Ž
. standards, and USDA-5-Pool bFSH-b was used as the primary antiserum. For the LH
assay, USDA-bLH-B-6 was used for iodination and reference standards, and USDA- 309-684p was used as the primary antiserum. Sensitivity of the FSH assay was 0.06
ngrml and coefficients of variation were 12 for within-assay and 10 for between-as- says. Sensitivity of the LH assay was 0.29 ngrml and coefficients of variation were
12 for within-assay and 11 for between-assays.
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
