Endocrine and superovulatory responses in heifers pretreated with FSH or bovine follicular fluid

THERIOGENOLOGY ~1~ AND S~~~~RY RESPONSES IN HEIFERS PRETREATEDWITH FSH OR BOVINE FOLLICULARFLUID J.G. Lussier' and T.D. Carruthers* 'AgricultureCanada Research Station,C.P.90 Lennoxville,Qugbec, Canada, JlM 123 2Departmentof VeterinaryPhysiologicalSciences W.C.V.M., Universityof Saskatchewan Saskatoon,Saskatchewan,Canada, S7N OWO Received for publication: Juzy 6, 1.988 Accepted: February 13, 1989 ABSTRACT

This study examined the effects of altered serum FSH concentration on subsequentovarian response to superovulation. Synchronizedheifers were assigned randomlyon Day 1 of the cycle (estrus= Day 0) to three pretreatmentgroups that consistedof 6-d of saline (7 ml, s.c., b.i.d.; Group I), FSH-P (0.5 mg, i.m., b.i,d.; Group II) or charcoal-extracted bovine follicularfluid (BFF; 7 ml, s.c., b.i.d.; Group III) injections. Superovulationwas initiatedon Day 7 and consistedof FSH-P in decreasing dosages over 4 d (4,3,2,1mg; i.m., b.i.d.),with cloprostenol(500 ~g) on the morning of the third day. A second replicatewith 14 heifers was conductedusing the same protocol but twice the pretreatmentdosage of FSH-P (1 mg) and BFF (14 ml). Endogenousplasma FSH decreasedduring BFF and FSH-P pretreatmentscompared to controls (P < 0.02). Endogenous FSH concentrationsin both primed groups (II and III) were similar to control values (Group I) 12 h after the start of superovulation. Basal LH concentrationswere not differentbetween pretreatmentgroups. The interval from cloprostenoltreatmentto the preovulatoryLH surge in Group III was 21.3 and 23.9 h longer (P < 0.0001) than it was in Groups I and II. The postovulationprogesteronerise was delayed in Group III. The number of corpora lutea (CL) was lowest in the BFF-primedgroup (4.2 + 0.8) comparedwith the FSH-primed(7.4 + 1.3) and the control 412.0 ? 1.8; P < 0.003) groups. In the FSH-primedgroup (0.68 + 0.06 cm ), CL volumes were lar er than in the control group (0.45 + 0.03 cm'), whereas in the BFF-primez group (0.27 + 0.02 cm") CL volumes were smaller compared with the control group (P < 0.0001). Mean FSH concentrationsfor 48 h preceding superovulationand the number of CL per cow were positively correlated (1:= 0.55; P < 0.004; n = 26). We concludedthat both FSH-P and BFF pretreatmentsdecreased the superovulatoryresponseof heifers to FSH-P. The mechanism for this would appear to be associated with reduced endogenousFSH prior to the start of superovulation, Key words: superovulation,follicularfluid, FSH, ovary, cattle. Acknowledgments Presented in part at the 13th Annual Conferenceof the International Embryo Transfer Society,Dublin, Ireland,1987. Funded in part by grants from the National Sciencesand Energy Research Council and the SaskatchewanHealth Research Board. The authors acknowledgethe technicalsupport of C. Zielke.

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THERIOGENOLOGY INTRODUCTION The superovulatory response in cattle is extremely variable and is influenced by 1) the genetic composition (1,2); 2) the number of antral follicles capable of responding to gonadotropins (3,4); 3) the stimulatory agent used, its biological activity and mode of injection (5-11); and 4) the time of the estrous cycle at which superovulatory treatment is begun (3,12,13). The role of endogenous folliclestimulating hormone (FSH) secretion in the superovulatory response of cattle is not well defined. Therefore, the objectives of our experiment were to determine the effects of altered serum FSH concentrations on subsequent ovarian response to superovulation. Charcoal-extracted bovine follicular fluid (BFF), a rich source of inhibin (14-16), was administered to depress endogenous FSH concentrations (17-20), thereby decreasing a low dosage of exogenous ovarian exposure to FSH. Alternatively, porcine FSH (FSH-P, Schering, Pointe-Claire, Quebec, Canada) was administered to increase ovarian exposure to FSH. These pretreatments were given for a period of 6 d prior to the start of superovulation to allow time for alterations to occur in the population of follicles greater than 2 nun in diameter , a size believed to be capable of responding to a superovulatory treatment (4). Antral follicle growth from 1.5 to 8.5 mm in diameter has been shown to require approximatively 6 d in cattle (21). We hypothesized that pretreatment with a low dosage of FSH-P would stimulate the growth of small and medium size follicles, thereby increasing the number of follicles capable of responding to the superovulatory treatment, and that decreased endogenous FSH in BFFpretreated heifers would result in a reduction of follicular development and maturation, thereby decreasing the superovulatory response. MATERIALSAND METHODS Animal Treatments Twenty-eight Hereford heifers (297 f 21 kg) ,were randomly placed into two replicate groups and held in separate pens at the University of Saskatchewan Goodale Research Farm, from August to November, 1985. The heifers in the first replicate (n = 14) were synchronized with two injections of cloprostenol (500 ,ug; Estrumate, Coopers Agropharm, Ajax, Ontario, Canada) 11 d apart. On Day 1 of the synchronized cycle (estrus = Day 0), they were assigned randomly to three pretreatment groups: I) control, II) FSH-P, or III) charcoal-extracted bovine follicular fluid Pretreatments consisted of a 6-d period of saline (7 ml, s.c., (BFF). b.i.d.; Group II) or BFF (7 ml, b.i.d.; Group I), FSH-P (0.5 mg, i.m., s.c., b.i.d.; Group III) injections. Superovulation was initiated on the morning of Day 7 and consisted of FSH-P in decreasing dosages over 4 d with cloprostenol (500 pg) on the morning of (4,3,2,1 mg, i.m., b.i.d.) Heifers exhibited estrus the morning of the fifth day the third day. (Day 11). Jugular blood samples were taken at 12-h intervals (0800 and 2000 h), and both ovaries were removed approximately 2 d postovulation 120 h after cloprostenol injection) by flank laparotomy. The (i.e., second replicate (n = 14) was begun 10 d later, using twice the pretreatment dosages of FSH-P (1 mg) and BFF (14 ml).

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FollicularFluid Preparation Bovine ovaries were collectedat an abattoir and put on ice until follicularfluid was aspirated from all folliclesless than 20 mm in diameter. Cyst-like follicleslarger than 20 ~~l~ll in diameterwere discarded. The pooled follicularfluid was centrifuged(2,500 rpm) for 15 min at 4'C and the supernatantwas stored at -2O'C. When the follicularfluid was thawed, activatedcharcoal (NoritA; BDH Chemicals, Toronto, Canada) was added (5mg/ml)and the solution stirred for 4 h at 4Oc. The charcoalwas removedby centrifugation(3,000 rpm) for 30 min at 4°C followedby filtration(WhatmanNo. 1). Crystallinepenicillin was added (100 IV/ml) and the BFF was aliquottedinto injectiondoses and stored at -2O'C until immediatelyprior to injection. EndocrineAnalysis To assess the endocrineprofile, jugular blood sampleswere taken at 12-h intervals,allowed to clot at room temperature(22'C) for 6 h, centrifuged(3,000 rpm) for 15 min at 4'C and the serum was frozen (-2O'C)until assayed. Endogenousgonadotropinhormone concentrations (FSH, LH) were determinedby double antibody radioimmunoassays (RIA). Serum LH concentrationswere determinedin a single assay using a previouslydescribedmethod (22). The assay used bovine LH as tracer (LEB-17116-2;L.E. Reichert,Union University,Albany, NY), NIH-bLH-BlO as standard, rabbit anti-ovineLH as first antibody (GDN #15; G.D. Niswender,Colorado State University,Ft. Collins, CO), and a sheep anti-rabbitgammaglobulinas a precipitatingsecond antibody. The sensitivityof the assay was 0.025 ng per assay tube and the intraassay coefficientof variationwas 10.4%. The endogenousFSH concentrationswere determinedin a single assay by a previouslydescribedmethod (23) which used ovine FSH as tracer (NIAMDD-oFSH-I-l), ovine FSH (NIAMDD-oFSH-BP-l) as standard,rabbit anti-ovineFSH (NIAMDD-anti-OFSH-1) as the first,antibody,and sheep anti-rabbitgammaglobulinas the precipitatingsecond antibody. The sensitivityof the assay was 0.06 ng per assay tube and the intraassay coefficientof variationwas 9.4%. Follicularfluid and serum (200 ~1) were extractedwith petroleum ether and the concentrationof progesterone(P4) was determined (24). We used a first antibody raised against 4-pregnen-11u-01-3, 20 dione hemisuccinater)$nked to bovine serum albumin. Progesterone-llw glucuronide-[ I-iodotyraminel(Amersham,Oakville,Ontario, Canada) was used as tracer, and the complex was precipitatedwith a sheep anti-rabbit second antibody.The sensitivityof the assay was 30 pg per assay tube and the intraassaycoefficientsof variationwere 5.4% and 11.0% for the low and high referencesamples, respectively. Based on the LH surge and the pretreatmentperiod, serum samples within heifer and day (0800 and 2000 h) were pooled to obtain sufficient volume to allow determinationof estradiol-17S(E, ) concentrations. Concentrationsof E, were determinedas previouslydescribed (25) and validated for cow serum and follicularfluid samples (26). Briefly,

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THERIOGENOLOGY serum samples (0.5 to 2.0 ml) were extracted with a mixture of petroleum ether and ethyl acetate (5.5:1; v/v). Tritium-labelled E (NET-517: estradiol [2,4,6,7,16,17-3H(N)]; New England Nuclear, Do&al, Qugbec, Canada) was used as tracer and the first antibody was developed in a rabbit against estradiol-7a-butyrate:BSA. Bound E, was separated from free E, with activated charcoal. The sensitivity of the assay was 1.0 pg coefficients of variation were 6.7% per assay tube; intra- and interassay and 13.6%, respectively. Testosterone was extracted from BFF using diethyl ether and was measured with a tritiurr-based assay using sheep antisera against testosterone 3-(o-carboxymethyl)oxime:BSA as described (27). The sensitivity of the assay was 5 pg per assay tube. Ovarian Analysis The ovaries from the superovulated heifers were placed in Bouin’s fixative solution immediately after removal, left at room temperature for 5 d then transfered to 70% alcohol. Each ovary was serially sectioned into approximately l-mm thick slices with a disposable microtome blade. Perpendicular measurements were taken at the largest cross section of each follicle and CL, and their mean diameters and approximate volumes Follicles were classified according to their diameter: were calculated. Class 1 = 2 to 3 mm, Class 2 = 4 to 6 mm, Class 3 = 7 to 10 mm and Class 4 = larger than 10 mm. Statistical

Analysis

Endocrine profiles were analyzed by ANOVAusing a split plot design with repeated measures (28). The General Linear Models procedure of SAS (29) was performed with pretreatment as the main effect. Comparison between groups for each day was done using orthogonal polynomial contrasts with degrees of freedom adjusted by the Greenhouse-Geisser The orthogonal contrasts were 1) Epsilon correction factor (29,30). control compared with FSH-P and BFF pretreated groups and 2) the FSH-P compared with BFF group. Prior to statistical analysis, CL numbers were transformed (Y-l’*) to reduce the heterogeneity of variance. Numbers and volumes of follicles and CL were analyzed by ANOVA, with pretreatment as the main effect (31). Group means were compared by the Student-NewmanKeuls test. Regression and Pearson’s correlation analysis of mean FSH concentrations and CL numbers per heifer were done according to SPSS’ (31). RESULTS Endocrine

Analysis

One BFF heifer was discarded from the results when based on the it failed to synchronize, and one control heifer was serum P, profile, Endocrine removed when it failed to superovulate, only 2 corpora lutea. analysis was first performed on data from Day -3 to Day 10 relative to the start of pretreatment, then the hormone concentrations were realigned on the preovulatory LH surge on a within-pretreatment group basis, and a

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THERIOGENOLOGY Preliminary analysis showed no differsecond analysis was performed. the results ences between replicates for FSH, LH, P, and E, and therefore were pooled (Figure 1). The overall FSH mean from Day -3 to Day 10 differed between groups It was higher in the control group than in the FSH-P and (P < 0.03). BFF-primed heifers (P < 0.02). Endogenous FSH appeared to decline more slowly following pretreatment with exogenous FSH-P as compared with the abrupt reduction following BFF pretreatment. Mlve hours after the start of the superovulatory treatment (Day 8), concentrations of endogenous FSH were not different between pretreatment groups (FSH-P and BFF), as compared by orthogonal contrasts (day comparison). Statistical analysis demonstrated a day effect (P < 0.0001) and a day by treatment Analysis of basal LH concentrations during the interaction (P < 0.05). pretreatment and superovulation period (Days -4 to 10) showed a day effect (P < 0.02) but no treatment or day by treatment effects (P > 0.3). The analysis of P concentrations for the pretreatment and superovulatory periods (Figure 23 indicated a day effect (P < 0.0001) but no treatment (P > 0.5) or day by treatment interaction effects (P > 0.9). After realignment on the preowlatory LH surge (Figure 1; Days 10.5 to 14), overall mean FSH concentration did not differ among treatment groups (P > 0.3) but did vary by day (P < O.OOOl), and there was a day by treatment interaction (P < 0.03). The FSH profile shows a delayed preovulatory FSH surge for the BFF-pretreated heifers compared with FSH-P and control heifers. Analysis of basal LH from Days 10.5 to 14 indicated no treatment effect (P > 0.5); however, a day effect (P < 0.0001) and a day by treatment effect (P < 0.0001) were present. The interval from cloprostenol injection to the LH surge was longer (P < 0.0001) in the BFF-pretreated group (76.0 f 3.9 h) than the FSH-P (52.1 + 3.2 h) or the control (54.7 + 2.2 h) groups. The analysis of P concentrations indicated a treatment effect (P < 0.003), a day elfect (P < 0.0001), and a day by treatment effect (P < 0.002), reflecting a delayed increase in luteal P, secretion (Figure 2). From the beginning to the end of the experiment (Days 1 to 14), the analysis of the pooled serum samples for the estradiol-178 (Figure 2) indicated no replicate or treatment effects (P > 0.15). There was a significant day effect (P < 0.0001) but no day by treatment interaction Steroid concentrations in the charcoal-extracted BFF used (P > 0.20). for pretreatments were 0.66 ng/ml for estradiol-178, 1.28 ng/ml for progesterone and 0.08 ng/ml for testosterone. Ovarian Analysis There were no significant differences between replicates for CL or follicle numbers and volumes; therefore, the results were pooled. The number and volume of postovulatory Class 3 follicles (7 to 10 mm in diameter) was higher in the FSH-P pretreated group than in the control group (Table 1). CL numbers differed between groups (P < 0.003); the BFF group was lower than the FSH or control group and the FSH group tended to be lower than the control group (Table 1). The calculated CL volumes differed between pretreatment groups (P < 0.0001). The mean CL volume of

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Figure 1. Mean concentrations(2 S!SM)of FSH and LH for heifers treated with saline (control;n = 9; -R-), FSH-P (n = 8;--O-- ) or charcoal-extractedbovine follicularfluid (BFF; n = 9; ---m--- ) during the pretreatments(Days 0 to 6), superovulation(Days 7 to 10) and the period following superovulationafter realignmenton the preovulatoryLH surge (Days 10.5 to 14).

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Mean concentrations (+ SEX) of P and E, for heifers treated with saline (conkrol; n = 9; --p), FSH-P (n = 8; --O-) or charcoalextracted bovine follicular fluid (BFF; n = 9; ---I--) during the pretreatments (Days 0 to 61, superovulation (Days 7 to 10) and the period following superovulation (Days 11 to 14).

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THERIOGENOLOGY the BFF group was smaller than the control or FSH-P groups, whereas mean CL volume for the FSH-P group was the largest (P < 0.05). When each CL for the differentpretreatmentgroups were plotted accordingto their volume, a shift in the frequencydistributionwas observed. The BFFpretreated group had a CL volume distributionwith a shift to smaller CL size, while FSH-P pretreatedheifers had a tendencytowards a larger size CL than the control heifers (data not showed). A mean FSH concentrationfor the 48 h precedingsuperovulatory treatmentwas calculatedand related to the number of CL per heifer present followingsuperovulation;a positive correlationwas found (r = 0.55; P < 0.0036; Y = 0.0283X + 0.5378; n = 26). No correlationswere found between mean FSH concentrationbefore superovulationand the number of folliclesin any size class 2 d followingthe superovulatoryestrus. DISCUSSION The pretreatmentof heifers with charcoal-extracted BFF effectively reduced the endogenousserum FSH concentrationsfor the duration of the treatmentwithout a concomitantreductionof basal LB secretion. However, a higher degree of FSH suppressionwas not found when double the amount of BFF was injected (14 ml comparedwith 7 ml). Maximal suppression of 30 to 45% was achieved, similar to the maximum 30 to 40% specific suppressionof FSH release obtainedwhen bovine pituitary cells were incubated for 48 h with 1.56 ,ulof charcoal-extracted BFF (Lussierand Carruthers,unpublishedobservations). A similardegree of specific suppressionwas obtained when charcoal-extracted BFF was incubatedwith isolated rat pituitarycells (32). We have shown a decrease in endogenous immunoreactiveconcentrationsof FSH in BFF- and FSH-P treated heifers; however,we question whether the remainingcirculatingFSH has an altered bioactivity(33). Heifers pretreatedwith exogenousFSH-P showed a gradual decrease in endogenousFSH during the pretreatmentperiod. Although the FSH-P preparationdoes cross-reactslightly in our FSH radioinununoassay (less than l%), we believe that the FSH concentrationsreported for+the FSH-P group representonly endogenousFSH. This is based on the amount injected (0.5 or 1.0 mg of FSH-P per 12 h), the half-lifeof FSH estimated at 5 h (9,34), the blood sampling regimen (12 h postinjection) and the undetectableconcentrationof exogenouslyadministeredporcine FSH (4 mg) 12 h after injectionin cattle (9). It has been previouslyshown in vivo that BFF decreasescirculating FSH concentrationsin cattle (17-19),our results support these findings. The decrease in endogenousFSH during pretreatmentwith FSH-P was unexpectedand may have resulted from promotingand/or supportingthe growth and endocrineactivity of medium and large follicles,which would in turn releasemore inhibin (32,35,36),follistatin(37) and/or E,, thereby suppressingFSH synthesisand/or release. However, the presence of both FSH and LH in the commercialpreparation(FSH-P)used for FSH pretreatmentdoes not allow the effects obtainedon endogenousFSH concentrationand ovarian responsebe specificallyattributedto either FSH or LB activity. Although the overall mean E, concentrationswere not significantlydifferentbetween groups, the FSH-P heifers tended (P <

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THERIOGENOLOGY 0.15) to have higher concentrations of E, when compared with BFF and Moreover, when the superovulatory treatment was control heifers. started, the mean E, concentration appeared to reach higher levels in the FSH-P group than in the control or BFF group, which suggests the presence However, due to the pooling of of larger and/or more active follicles. serum samples and the dilution effect of jugular blood sampling, these E, Further experimentation should include differences were not significant. ovarian vein catheterization to better characterize the effects of pretreatment on E, secretion in cattle. The preovulatory surge of LH and FSH was delayed by approximately 24 h in the BFF-pretreated heifers, but the precise timing of the LH and FSH surge was not obtained due to the 12-h sampling interval. This observation supports the hypothesis that follicular development was suppressed by BFF treatment, and at the time superovulation was initiated, only less mature follicles were able to respond. Therefore, additional time was required for these follicles to grow and mature sufficiently to trigger the gonadotropin surge. This hypothesis is supported by ultrasonographic observations that injection of BFF (11 ml, for 6 d reduced follicular growth in association with S.C., b.i.d.) All animals in the three treatment groups exhibited suppressed FSH (38). a post-ovulatory FSH increase, similar to that previously reported (39, The delayed postovulatory rise of P, in BFF-primed heifers would 40). also support delayed ovulation and CL formation. The numbers and volumes of CL were lower in BFF-treated compared with control or FSH-P-treated group. The somewhat less numerous but larger CL obtained from the FSH-P-primed heifers compared with control heifers supports the hypothesis that pretreatment with a gonadotropin preparation (FSH-P) stimulated the development of follicles, which then Larger CL presumably resulted responded to superovulatory treatment. from larger follicles at the time of ovulation. However, contrary to our hypothesized increase in superovulatory response following FSH-P pretreatment, we obtained a tendency toward a lower.mean CL number than was found in the control heifers. These results contradict those of others (41,42), who reported an increase in superovulatoy response following pretreatment with FSH-P at the beginning of the cycle. Ware et al. (41) pretreated with 10 mg of FSH-P on Day 2 or 3 of the cycle and initiated superovulation from Day 11 to Day 16, whereas Pajamahendren et al. (42) pretreated with 2.5 mg of FSH-P on Days 3 and 4 and began superovulation In both experiments (41,42) the numbers of CL from Day 9 to Day 13. obtained in their control groups were lower compared with ours, 61 and Their CL numbers were also lower compared with 42.5%, respectively. those reported for superovulatory responses by other research groups The discrepancies between our results and theirs (41,42) (6,8,13,43). may also have resulted from the different age or breed of cattle used, material used, or the time the different priming regimens, superovulatory at which the superovulatory treatment was begun. Meanwhile, our priming results are in agreement with Grass0 et al. (43), who observed a decreased superovulatory response when 10 mg of FSH-P was administered on They also reported that FSH-P-primed heifers Day 3 of the estrous cycle. having more large follicles (2 7 nun) as detected by ultrasonoyraphy at the beginning of superovulation had a reduced superovulatory response. Saumande et al. (3) reported a reduced ovarian response after PHSG

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THERIOGENOLOGY stimulation when large follicles were present in the ovary at the start A reduced superovulatory response was also obtained of superovulation. when superovulatory treatment was inititated following selection of the preovulatory follicle at the end of a normal estrus cycle (44). A negative correlation has been found between the number of healthy follicles larger than 2 mm in diameter and the PMSGinduced ovulation rate in the ewe (45). Staigmiller and England (46) have shown in cattle that ovariectomy ipsilateral to the largest follicle is followed 4 d later by an increase in the size of follicles and the quantity of follicular fluid in the remaining ovary, whereas no changes were observed when the ovariectomy was contralateral to the largest follicle. In addition, no preovulatory follicles developed during human menopausal gonadotropin therapy in women when a dominant follicle was present (47). Cur data support these observations, since FSH-P pretreatment reduced endogenous FSH to concentrations similar to those found in the BFF-primed group. This decrease of endogenous FSH may be related to the sustained growth of one or more follicles which secreted increased amounts of inhibin, E, and/or follistatin, thereby inhibiting growth of small and medium follicles. The presence of large follicles at the start of superovulation may then have resulted in the formation of fewer but larger CL. The positive correlation between endogenous FSH concentrations prior to superovulation and the ovulatory response, suggests that low FSH at the start of superovulation may indicate the presence of large, active follicles on the ovary which suppress the overall ovarian response. Lussier et al. (48) have reported that BFF injections, at the same time as superovulatory treatments with FSH-P, block the superovulatory response. This supports the hypothesis of a direct action by BFF at the ovarian level (49). Therefore, the suppressive action of large follicles could be mediated by negative feedback at the pituitary level suppressing FSH release and/or by direct action at the ovarian level on small follicles (inhibin: 50; Follicle Regulatory Protein: 51). Phillippo and Rowson (12) and Lindsell et al. (13) reported a higher superovulatory response when superovulation was started on Days 8 to 12 of the cycle. Andrieu (52) has observed by ultrasonographic monitoring that the large follicle appearing after ovulation showed signs of regression by Day 10 to 11 of the cycle. This observation has also been recently supported (53-55). Moreover, the period from Day 3 to Day 8 has been characterized by the presence of one large estrogen-active follicle, but by Day 9 all follicles were estrogen-inactive (56-58). The presence of a large active follicle before Day 6 or 7 may explain the lower superovulatory response obtained at this time; the regression of this dominant follicle by Day 9 offers an opportunity to start superovulation before the natural selection of another large dominant follicle around Days 12 to 14 (52-55). Our results and those of others (3,43,44,45,47) support the concept that the presence of large active follicles at the start of superovulation may be detrimental to the superovulatory response and responsible for much of the variability in superovulatory responses. Further work should be directed at stimulating small and medium follicle development before initiating superovulation, altering dominant follicle viability and characterizing the suppressive actions of large follicles on small and medium size follicles.

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REFERENCES 1. Mariana, J.C., Girard, P. and Chupin, D. Differencesintraracesde reponse ovariennea un traitementde superovulationpar PMSG chez des vaches de race charolaise. Frequencedes naissancesg8mellaires. Ann. Zootech.Jj:345-353 (1977). 2.

Bindon, B.M., Piper, L.R., Cahill, L.P., Driancourt,M.A. and O'Shea, T. Genetic and hormonal factors affectingsuperovulation. Theriogenology25:53-70 (1986).

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Saumande,J., Chupin, D., Mariana, J.C., Ortavant,R. and Mauleon, P. Factors affectingthe variabilityof ovulation rates after PMSG stimulation. In: Sreenan,J.M. (ed). Control of Reproductionin the cow. Martinus%ijhoff, The Hague, London, 1978, pp. 195225.

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Schams, D., Menzer, CH., Schallenberger,E., Hoffman,B., Hahn, J. and Hahn, R. Some studieson pregnant mare serum gonadotropin(PMSG) and on endocrine responsesafter applicationfor superovulationin cattle. In: Sreenan,J.M. ted). Control of Reproductionin the Cow. Martinus-ijhoff, The Hague, London, 1978, pp. 122-143.

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Murphy, B.D., Mapletoft,R.J., Manns, J.G. and Humphrey,W.D. Variabilityin gonadotrophinpreparationsas a factor in the superovulatoryresponse. Theriogenology3:117-125 (1984).

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Lerner, S.P., Thayne,W.V., Baker, R.D., Henschen,T., Meredith,S., Inskeep, E.K., Dailey, R.A., Lewis, P.E. and.Butcher,R.L. Age, dose of FSH and other factorsaffectingsuperovulationin Holstein cows. J. Anim. Sci. g:176-183 (1986).

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Chupin, D., Cognie, Y., Combarnous,Y., Procureur,R. and Saumande, J. Effect of purifiedLH and FSH on ovulation in the cow and ewe. In: Roche, J. and O'callaghan,D. feds). FollicularGrowth and &lation Rate in Farm Animals. Martinus Nijhoff, The Hague, 1987, pp. 63-72.

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Demoustier,M., Beckers,J.-FR., Van Der Zwalmen,P., Closset,J., Gillard, J. and Ectors, FR. Determinationof porcine plasma follitropinlevels during superovulationtreatmentin cows. Theriogenology%:379-386 (1988).

D., Mariana, J.C., Gibson, W.R. and Roux, X. Cycles folliculaireset croissanceterminalesdu folliculechez la vache au tours du cycle normal ou apres stimulation. In: Salat-Baroux,J. and Thibault,Ch. teds). Periode Peri-Ovulatoire, Soci6te Francaise pour 1'Etude de la Fertiliti. Masson, Paris, 1984, pp. 67-84.

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Eldsen, R.P., Nelson, L.D., Seidel, G.E.,Jr. Superovulatingcows with folliclestimulatinghormone and pregnantmare's serum gonadotrophin. Theriogenology9:17-26 (1978).

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