Local versus systemic effects of exogenous estradiol-17β on ovarian follicular dynamics in heifers...

Ž .Animal Reproduction Science 59 2000 141–157www.elsevier.comrlocateranireprosci

Local versus systemic effects of exogenousestradiol-17b on ovarian follicular dynamics in

heifers with progestogen implants

G.A. Bo a,1, D.R. Bergfelt b, G.M. Brogliatti b, R.A. Pierson c,G.P. Adams b, R.J. Mapletoft a,)

a Departments of Herd Medicine and Theriogenology, UniÕersity of Saskatchewan, 52 Campus DriÕe,Saskatoon, Saskatchewan, Canada S7N 5B4

b Department of Veterinary Anatomy, Western College of Veterinary Medicine, UniÕersity of Saskatchewan,Saskatoon, Saskatchewan, Canada S7N 5B4

c Department of Obstetrics and Gynecology, College of Medicine, UniÕersity of Saskatchewan, Saskatoon,Saskatchewan, Canada S7N 5B4

Received 19 August 1999; received in revised form 24 February 2000; accepted 3 March 2000

Abstract

Two experiments were designed to determine if the suppressive effect of estradiol treatment onovarian follicles in progestogen-implanted heifers is mediated directly at the ovary or systemically,at a higher level. The purpose of Experiment 1 was to determine a minimal effective dose of

Ž .estradiol-17b E-17b that would induce follicle regression in progestogen-implanted heifers. BeefŽ .heifers were implanted with progestogen on Day 2 Day 0sovulation and were assigned

Ž . Ž .randomly to five groups: control sesame seed oil, ns9 ; 0.1 mg of E-17b ns8 ; 0.5 mg ofŽ . Ž . Ž . Ž .E-17b ns8 ; 1 mg of E-17b ns8 ; or 5 mg of E-17b ns8 by intramuscular im injection

Ž .on Day 3. Treatment with 5 and 1 mg of E-17b resulted in smaller P-0.05 day-to-daydiameter profiles of the dominant follicle compared with controls, whereas 0.1 mg of E-17b didnot have an apparent effect on follicle growth. The effect of a dose of 0.5 mg was intermediate

Ž .and tended P-0.06 to result in a smaller diameter profile of the dominant follicle comparedŽ .with control heifers. Experiment 2 was designed to utilize a subminimal dose of E-17b 0.1 mg ,

locally, to determine whether estradiol treatment induces follicle regression through a direct action

) Corresponding author. Tel.: q1-306-966-7149; fax: q1-306-966-7159.Ž . Ž .E-mail addresses: [email protected] G.A. Bo , [email protected] R.J. Mapletoft .

1 Ž . Ž .Present address: Instituto de Reproduccion Animal Cordoba IRAC , J.L. de Cabrera 106 5000 Cordoba,Argentina.

0378-4320r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved.Ž .PII: S0378-4320 00 00140-8

( )G.A. Bo et al.rAnimal Reproduction Science 59 2000 141–157142

on the ovary. Beef heifers received a progestogen ear implant on Day 2 and were assignedŽ . Ž .randomly to five groups on Day 3: control sesame seed oil, ns8 ; 5 mg of E-17b im ns8 ;

Ž .0.1 mg of E-17b im ns8 ; 0.1 mg of E-17b given into the wall of the uterus, near the tip of theŽ Ž . .horn ipsilateral to the dominant follicle intrauterine iu , ns8 ; or 0.1 mg of E-17b given into

Ž Ž . .the stroma of the ovary, immediately adjacent to the dominant follicle intraovarian io , ns6 .Ž .Local iu and io treatments were given via a transvaginal ultrasound-guided needle injection.

Treatment with 5 mg of E-17b im resulted in suppression of the dominant follicle of the firstŽ .follicular wave and early emergence of the second follicular wave P-0.05 . Diameter profiles

of the dominant follicle in heifers treated with 0.1 mg im or 0.1 mg iu differed from those ofcontrol heifers on Day 5, whereas diameter profiles of the dominant follicle in heifers treated with0.1 mg io did not differ from the controls. Daily changes in diameter of the dominant follicle did

Ž .not differ among the three groups treated with 0.1 mg of E-17b im, iu and io . Hourly changes incirculating concentrations of FSH and LH were not detected following estradiol treatment eitherbefore or after the results were combined for all estradiol-treated groups. Results are supportive ofthe hypothesis that the suppressive effect of estradiol in cattle is exerted indirectly through asystemic route rather than directly at the ovary. Although low plasma concentrations of FSH andLH were not detected, systemic treatments with high E-17b dosages resulted in follicularsuppression whereas local treatments with subminimal dosages, within the ovary bearing thedominant follicle, were without effect. q 2000 Elsevier Science B.V. All rights reserved.

Keywords: Heifer; Follicle; Progestogen; Estradiol; Gonadotropins

1. Introduction

The dominant follicle of a follicular wave suppresses the growth of other follicles ofŽ .the wave subordinates and prevents emergence of the next follicular wave until the

Ždominant follicle of the preceding wave regresses or ovulates Ginther et al., 1989c;.Adams et al., 1992a, 1993 . The mechanism by which follicular dominance occurs is not

understood. However, it has been shown that the dominant follicle produces largeŽ . Žamounts of steroidal i.e., estrogen, androgens and non-steroidal factors i.e., inhibin,

.follistatin, growth factors of which some have been shown to suppress follicular growthŽby altering systemic concentrations of FSH Ginther et al., 1989c; Adams et al., 1992a;

.Findlay et al., 1992; Fortune, 1994; Bergfelt et al., 1994 . Intraovarian products mayŽalso act directly or locally on the ovary to alter follicular development Lobb and

.Dorrington 1992; Findlay, 1993 .Experiments in which exogenous estradiol was administered to determine its effect on

Ž .follicular development Hutz et al., 1988; Engelhart et al., 1989 have indicated follicleregression after treatment. Similarly, we have documented suppression of growth of the

Ždominant follicle after estradiol treatment to progestogen-implanted cattle Bo et al.,.1991, 1995a,b . In this regard, the dominant follicle of a follicular wave gains the

Žcapacity to produce larger quantities of estradiol than subordinate follicles Badinga et.al., 1992; Bodensteiner et al., 1996 . Perhaps, the degree of estrogen production by the

dominant follicle is part of a mechanism that facilitates dominance by suppressingŽ . Ždevelopment of other follicles of the wave either directly i.e., locally or indirectly i.e.,

.systemically .

( )G.A. Bo et al.rAnimal Reproduction Science 59 2000 141–157 143

ŽExogenous 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 sucha hypothesis; estradiol treatment to heifers with progestogen-ear implants or during theluteal 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 wasdetected prior to the estradiol-induced LH release in cows treated on Day 16 of the cycleŽ .Engelhart et al., 1989 . Alternatively, estrogen may induce follicular atresia by altering

Ž .tonic LH secretion andror through FSH suppression Price and Webb, 1988 . EstradiolŽ .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 havebeen reported to decrease LH pulse frequency and suppress maximal diameter of the

Ždominant 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, andestradiol 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 inŽ .combination with progestogen Barnes et al., 1981 has been shown to suppress FSH;

Žhowever, 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 circulatingŽ . 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 withinthe ovary rather than through gonadotropin suppression. To our knowledge, there are noreports of a local effect of estrogen on follicular dynamics in cattle.

We hypothesized that the suppressive effect of exogenous estradiol, in combinationwith progestogen, on ovarian follicles in cattle is mediated systemically rather thanlocally. Experiment 1 was designed to determine a minimal effective systemic dose ofestradiol for inducing follicular regression in progestogen-implanted heifers. Experiment2 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 estradioltreatment induces suppression of follicles through a direct action at the level of theovary.

2. Materials and methods

2.1. Experiment 1

Ž .Cross-bred beef heifers ns41 , 15 to 18 months of age and weighing 320 to 420 kgŽ . Žwere treated with 500 mg of cloprostenol intramuscularly im Estrumate, Coopers

.Agropharm, Ajax, ON, Canada and monitored daily by ultrasonography to detectŽ . Žovulation 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, ns9 ; 0.1 mg of E-17b ns8 ; 0.5 mg of E-17b ns8 ; 1

Ž . Ž . Žmg of E-17b ns8 ; or 5 mg of E-17b ns8 . 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 Day3. Day 3 was selected as the day of E-17b treatment because it corresponded to themid-growing phase of the dominant follicle of the first follicular wave and the earliestday the dominant follicle could be consistently differentiated from subordinate folliclesŽ .Ginther et al., 1989a .

2.2. Experiment 2

Ž .Cross-bred beef heifers ns38 from the same population as those in Experiment 1were treated with cloprostenol, monitored for ovulation and received a progestogen earimplant on Day 2. On Day 3, heifers were assigned randomly to one of five treatment

Ž . Ž .groups: control sesame seed oil, im; ns8 ; 5 mg of E-17b im ns8 ; 0.1 mg ofŽ .E-17b im ns8 ; 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; ns8 ; or 0.1 mg of E-17b into the ovarianŽ .stroma, immediately adjacent to the dominant follicle io; ns6 .

Ž .Local treatments iu and io were administered with a 21-gauge needle soldered to aŽ17-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 themyometrium, near the ovarian end of the uterine horn. A local relationship between

Žvenous 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 wellŽ .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. IntraovarianŽ .io treatment was administered in 0.2 ml of sesame seed oil into the ovarian stromaimmediately 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 theobservational period, 4 days after detection of the emergence of the second follicularwave.

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 theŽ .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 andcontinued until 4 days after the emergence of the second wave. Ultrasound data werecollected without knowledge of treatment groups.

The dominant follicle of a wave was defined as the follicle that reached the largestdiameter. 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 afollicular 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 thedominant 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 follicleappeared 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 weredetermined by collecting blood samples via jugular venipuncture into heparinized tubes.

Ž .Plasma was harvested within 30 min of collection and stored frozen y208C untilassays were done. In Experiment 1, samples were taken every 2 h during the first 12 hafter E-17b treatment and every 6 h for the next 36 h and, in Experiment 2, bloodsamples were collected at 0, 2, 6, 12, 24 and 48 h after E-17b treatment. Plasmaconcentrations of estradiol were measured in all samples using a validated radioim-

Ž .munoassay Joseph et al., 1992 . Standards were prepared in charcoal-stripped bovineserum and the standard curve ranged from 6.25 to 1600 pgrml. Values greater than thestandard curve were diluted in charcoal-stripped serum and re-analyzed. Sensitivity ofthe 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 wereŽdetermined 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 hlater at 0800 h and ended 10 h later at 1800 h. The second serial bleeding period began16 h after E-17b treatment at 2200 h and ended 10 h later at 0800 h. Blood sampleswere taken once every hour for each of the serial bleeding periods. An initial 1-mlsample was withdrawn and discarded, a second 4-ml sample was withdrawn andtransferred to a centrifuge tube. All samples were allowed to clot at room temperatureŽ .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. Serumconcentrations of FSH and LH were determined using a validated radioimmunoassayŽ .Bolt and Rollins, 1983; Bolt et al., 1990 which has been modified and describedŽ .Adams et al., 1992a . Reagents were obtained from the USDA Animal HormoneProgram. 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 LHassay, 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 0.06ngrml 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 were12% for within-assay and 11% for between-assays.


2.5. Statistical analyses

ŽSingle-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 groupŽ . 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 testthe 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 inFig. 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 0sovulationŽand 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 mgof 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 1Ž .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 ovulationsDay 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 waÕeŽ .Emergence day y0.2"0.1 y0.2"0.2 0.0"0.0 y0.1"0.2 y0.5"0.8

Dominant follicleŽ .Maximum diameter mm 12.5"0.7 12.4"0.5 11.1"0.4 11.2"0.4 11.2"0.5Ž .Cessation of growth day 6.1"0.4 5.6"0.4 5.1"0.6 5.5"0.6 5.3"0.7

ab b ab ab aŽ .Onset of regression day 10.4"0.5 12.0"0.5 11.0"0.7 10.4"0.4 9.8"0.9

Largest subordinate follicleŽ .Maximum diameter mm 8.1"0.3 7.8"0.4 8.6"0.4 8.1"0.3 8.4"0.5Ž .Cessation of growth day 3.0"0.2 2.6"0.4 3.4"0.5 2.9"0.4 3.1"0.4Ž .Onset of regression day 4.3"0.2 5.1"0.5 6.4"0.3 5.0"0.6 5.1"0.4

Second waÕeab b ab a aŽ .Emergence day 7.5"0.3 8.6"0.4 7.9"0.3 7.4"0.4 7.2"0.4

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 gradualŽ .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 withŽ .progestogen on Day 2 Day 0sovulation 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 smallerŽ . 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 withthose heifers treated with 0.1 mg or in the control group, and were not different from those heifers treated with1 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 0.5mg, 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 ofemergence, cessation of growth and maximum diameter of the dominant follicle werenot 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 inheifers 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 with0.1 mg of E-17b. The day of emergence of the second wave in the control group and the0.5 mg of E-17b group was intermediate and not different from other E-17b treatedgroups.

The effects of E-17b treatment on diameter profiles of the dominant follicle inŽprogestogen-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 0sovulationŽ .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 fromŽ .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 aftertreatment. 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 comparedŽ . Žwith 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 ofE-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. PeakŽ .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, imŽ .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 . Žtrations 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 firstfollicular wave did not differ among groups. The dominant follicle of the first follicularwave in heifers treated with 5 mg of E-17b had a smaller maximum diameter, ceased

Table 2Ž .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 , orŽ . Ž .0.1 mg into the ovary, adjacent to the dominant follicle io on Day 3 Day 0sovulation . 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 waÕeŽ .Emergence day y0.1"0.1 y0.3"0.2 0.0"0.2 y0.1"0.2 0.2"0.4

Dominant follicleb a ab ab bŽ .Maximum diameter mm 12.9"0.6 10.7"0.4 11.8"0.5 12.1"0.3 12.5"0.6b a ab b abŽ .Cessation of growth day 6.3"0.8 4.4"0.7 6.0"0.5 6.3"0.4 5.9"0.3b a b b bŽ .Onset of regression day 11.0"0.8 8.4"0.8 11.0"0.6 11.8"0.6 12.2"1.1

Largest subordinate follicleŽ .Maximum diameter mm 8.1"0.4 7.6"0.3 8.3"0.6 7.5"0.4 7.8"0.4Ž .Cessation of growth day 2.9"0.3 2.5"0.4 2.9"0.5 2.6"0.5 3.5"0.6Ž .Onset of regression day 5.0"0.5 5.1"0.4 5.5"0.7 5.4"0.5 5.7"0.6

Second waÕebc a ab bc cŽ .Emergence day 8.0"0.4 6.9"0.2 7.4"0.3 8.1"0.5 9.0"0.4

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 earlierŽ .P-0.05 . Mean characteristics of the dominant follicle of the first follicular wave andthe 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

Žprogestogen-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 5mg im of E-17b, and on Day 5 in those heifers treated with the 0.1 mg im and iu. Meandiameter of the dominant follicle in heifers treated with 5 mg of E-17b was smallerŽ . Ž .P-0.05 compared with heifers in the control no E-17b group or those treated with0.1 mg E-17b io on Days 5 to 12 and those treated with 0.1 mg E-17b im or iu on Days7 to 12. Diameter profiles of heifers treated with 0.1 mg of E-17b im, iu and io were notdifferent. Diameter profiles of the largest subordinate follicles did not differ amonggroups.

The effects of E-17b treatment on systemic concentrations of gonadotropins areshown in Fig. 5. As the mean hourly changes in FSH and LH were not significantlydifferent among groups in the first or second serial bleeding periods, data for theE-17b-treated groups were combined and compared with the control group. Again, therewas 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 withŽ . Ž .progestogen on Day 2 Day 0sovulation 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 ofŽ .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 inŽ .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 0.1Ž .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 havebeen omitted for clarity. Experiment 2.


Ž .Fig. 5. Mean "S.E.M. plasma FSH and LH concentrations in heifers implanted with progestogen on Day 2Ž . Ž .Day 0sovulation 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 theŽ .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 fromall estradiol-treated heifers. Subsequent analyses indicated mean FSH concentrations were lower in the control

Ž . Ž . Žgroup 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 secondserial bleeding period in the combined-treatment group.

serial bleeding period. However, comparison of concentrations of gonadotropin betweenthe 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 LHwere 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 theestradiol-treated group.

4. Discussion

Ž .In Experiment 1, a single systemic im injection of supraphysiologic doses of E-17b

in progestogen-implanted heifers resulted in a dose-dependent increase in circulatingconcentrations of estradiol. Concentrations after treatment were higher than expected.However, the first blood sample was taken 2 h after treatment in the present study,

Žwhereas sampling began 6 h after E-17b treatment in the previous study Bo et al.,.1994 . Values at 6 h in the present study were similar to those reported previously. The

Ž . Ždynamics of E-17b absorption peak at 2 h after treatment and metabolism basal levels.at 24 to 50 h depending on the dosage used are in agreement with reports from other

Ž .laboratories Rexroad et al., 1977; Randel et al., 1979 . It would seem that increasedŽ .circulating concentrations of E-17b for a period of 12 h 0.5 or 1.0 mg of E-17b to 24

Ž .h 5.0 mg of E-17b is adequate to cause suppression of the dominant follicle at thisstage of development of the first follicular wave. Elevated concentrations of E-17b for10 h, as was seen in the 0.1 mg of E-17b group, did not seem to be sufficient to inducefollicular suppression. We have shown previously that the administration of 5.0 mg of

ŽE-17b to progestogen-implanted heifers causing significantly elevated circulating E-17b

.concentrations for approximately 42 h will induce follicular regression and emergenceof a new follicular wave, regardless of the stage of development of the dominant follicle

Ž .of the first follicular wave Bo et al., 1994, 1995a,b . Therefore, greater doses orlonger-acting forms of estrogen would not seem to be necessary, and may even result in

Ždelayed andror asynchronous emergence of the new follicular wave Bo et al., 1993,.1994 .

Experiment 2 utilized a subminimal dose of E-17b chosen from Experiment 1 toexamine the local versus systemic effects of estradiol on follicular development. The ioŽ .local treatment dose of 0.1 mg of E-17b raised systemic concentrations of estradiolŽ .-125 pgrml for about 6 h and follicle growth was not suppressed. Conversely, the

Ž .systemic treatment iu or im with 0.1 mg of E-17b resulted in a greater increase inŽ .systemic concentrations of estradiol -250 pgrml for a similar period of time

Ž .approximately 6 h and a transient suppression of follicular growth. The intraovariandose of 0.1 mg, E-17b and volume of sesame seed oil used as a vehicle was similar to

Ž .that previously used in monkeys Hutz et al., 1988 , but contrary to findings in monkeys,this treatment did not result in follicular regression in cattle. The dosage used for thelocal treatment is considerably greater than the E-17b present in the fluid in the

Ž .dominant follicle, which has been reported to be about 1500 ngrml Singh et al., 1998 .Therefore, the amount given adjacent to the dominant follicle would have been expectedto induce profound follicular suppression compared with systemic treatments if theeffects of E-17b were mediated locally.

Injection of 0.1 mg of E-17b into the uterine wall also represented a local treatment.With the close association of the vasculature between drainage of the cranial end of the


Ž .uterine horn and supply to the ovary Ginther, 1974 , E-17b treatment was expected toelevate estradiol concentrations in the arterial blood supplying the ovary containing thedominant follicle. Results indicated a slightly greater increase in systemic concentrationsof estradiol compared with the intraovarian injection. Furthermore, results indicated a

Ž .transient suppression of follicular growth for 1 day Day 5 before the diameter of thedominant follicle increased similar to that in the control group. In Experiment 1,systemic treatment with 0.1 mg of E-17b im did not appear to suppress folliculargrowth, whereas, in Experiment 2, there was a transient suppression of follicular growthsimilar to that following local treatment into the uterine wall. Although the differencebetween the two experiments with respect to systemic treatment is unknown, theintrauterine treatment may have been more representative of a systemic than a localtreatment.

Differences in the concentrations of circulating E-17b in the 0.1 mg groups may havebeen due to the volume of sesame seed oil in which E-17b was injected into the ovaryŽ .0.2 ml versus 1.0 ml for im and iu sites or its absorption from the site of injection. Inany case, results support an indirect or systemic route of action. When systemicconcentrations of E-17b were higher, follicular growth was affected, at least transiently,

Ž .and as dose of E-17b and circulating concentrations increased, follicular growth wasmore profoundly affected.

The effects of 0.5 and 1 mg of E-17b on dominant follicle growth in Experiment 1further support the notion that the suppressive effect of estradiol is synergistic withprogestogenrprogesterone. Similar dosages of estradiol benzoate have been used toincrease synchrony of estrus andror ovulation and fertility when given after prosta-

Ž .glandin treatment Peters et al., 1977 or 24 to 48 h after the removal of progesterone-re-Žleasing vaginal devices in cattle McDougall et al., 1992; Day et al., 1997; Fike et al.,

.1997; Hanlon et. al., 1997; Martinez et al., 1998 . If estrogens given alone had a directsuppressive effect on ovarian follicles, such treatments would have been expected toresult in decreased fertility.

Although results support an indirect or systemic route of follicular suppression byestradiol, hourly changes in circulating concentrations of FSH and LH were not detectedfollowing estradiol treatment. Additional analyses for changes in FSH between serialbleeding periods within each group indicated significantly higher concentrations during

Ž .the second serial bleeding period Hours 16 to 26 compared with the first serialŽ .bleeding period Hours 2 to 12 for both control and treated groups. The change in FSH

between the two serial bleeding periods may be attributable to low amplitude surges ofFSH that have been detected in plasma samples collected at 4-h intervals during the

Ž .early luteal phase in cattle Bergfelt et al., 1997 . Regardless, the present results do notŽ .agree with those reported previously Bo et al., 1994 in which treatment of progesto-

gen-implanted heifers with 5.0 mg of E-17b suppressed plasma FSH for about 24 h andresulted in regression of the dominant follicle. Although comparable results with respectto alterations in development of the dominant follicle following different doses ofestradiol and routes of administration are indicated in the present study, the lack of anydetectable changes in FSH do not allow for a similar conclusion. The difference in FSHresults between the previous and present studies may be attributable to the time of

Ž .treatment Day 1 versus Day 3, respectively andror the FSH assay. In the present


study, samples were collected at a time when systemic FSH would be expected to be atŽ .basal concentrations Adams et al., 1992a,b . Therefore, the FSH values in the control

group and at the time of treatment with E-17b would have been near the sensitivity ofŽ .the assay, an area of great variability Adams et al., 1992a,b . Furthermore, a recent

report indicated that the bovine antiserum, used herein, was inferior to ovine antiserumŽ .when used in radioimmunoassays for measuring FSH Crowe et al., 1997 .

Changes in circulating concentrations of LH were similar between the two serialbleeding periods for the control group; however, LH concentrations were significantly

Ž .higher during the first serial bleeding period Hours 2 to 12 compared with the secondŽ .Hours 16 to 26 in the estradiol-treated groups. Bolus administration of estradiol thatresulted in supraphysiological concentrations in circulating estradiol has been reported to

Ž .exert a biphasic effect on LH secretion Kesner et al., 1981; Butler et al., 1983 ;secretion was initially inhibited and this was followed by a surge release. In a previous

Žstudy, treatment of heifers on Day 1 with 5.0 mg of E-17b without exogenous. Žprogestogenrprogesterone was followed by a surge release of LH 16 to 17 h later Bo

.et al., 1994 . Conversely, when the same treatment was done in progestogen-implantedheifers no surge in LH was detected. The progestogen ear implant may have partiallyblocked the estradiol-induced LH release, resulting in apparent higher concentrations ofLH between 9 and 12 h after treatment. Furthermore, considering that LH wasapparently lower in both estradiol-treated and control groups during the second serialbleeding period, the increasing concentrations of progestogen from the ear implant andprogesterone from the CL may have suppressed LH release. Although increases inprogestogenrprogesterone concentrations have been shown to result in decreased LH

Ž .pulse frequency and follicular suppression Savio et al., 1993b , this effect was notapparent in the present study.

5. Conclusion

The results of the present study confirm those of previous studies that exogenousE-17b induces follicular suppression and results in emergence of a follicular wave 3 to 5

Ž .days later in progestogen-implanted heifers. A systemic im injection of 1 mg E-17b

seemed to be as effective as 5 mg in inducing follicular suppression and possible use ofsmaller dosages requires further investigation. Results from Experiment 2 partiallysupport the hypothesis that the suppressive effect of E-17b on ovarian follicles inprogestogen-implanted cattle is exerted indirectly through a systemic route, rather thandirectly at the level of the ovary. Although decreases in plasma concentrations of FSHand LH were not detected, systemic treatment with high dosages of E-17b resulted infollicular suppression, whereas local treatments of subminimal dosages into the uterinehorn ipsilateral to the ovary bearing the dominant follicle or into the ovary bearing thedominant follicle did not induce follicular regression. Within these experiments, thegreater the circulating concentrations of E-17b the greater was the amount of follicularsuppression; however, it is not clear as to how much or how long concentrations inblood must be elevated.


Acknowledgements

This research was supported by the Natural Sciences and Engineering ResearchCouncil of Canada and the University of Saskatchewan. We thank Sanofi, OverlandPark, KS, USA for Syncro-Mate-B and Coopers Agropharm, Ajax, ON, Canada forEstrumate. We also thank B. Kerr and the Goodale Research Farm staff for animalhandling and feeding.

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