ESTRUS AND LH RELEASE IN OVARIECTOMIZED HEIFERS FOLLOWING VAGINAL DEVICES CONTAINING OVARIAN STERIODS 1,s

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1 ESTRUS AND LH RELEASE IN OVARIECTOMIZED HEIFERS FOLLOWING VAGINAL DEVICES CONTAINING OVARIAN STERIODS 1,s R. Rajamahendran 2, P. C. Lagu~" and R. D. Baker 3 Macdonald Campus of McGill University 4, Ste. Anne de Bellevue, Quebec HOA 1 CO Summary An experiment was conducted to study the effects of Silastic vaginal devices containing either estradiol-1713, progesterone or progesterone + estradiol-1713 on estrus and luteinizing hormone (LH release in ovariectomized heifers. All heifers treated with a vaginal device that contained estradiol-1713 exhibited estrus and LH release, whereas none of those treated with devices that contained progesterone + estradiol-1713 exhibited estrus or LH release. This experiment demonstrates that progesterone blocks estrogen-induced estrus and LH release in ovariectomized heifers. (Key Words: Ovariectomized Heifers, Estrus, LH, Progesterone, Estradiol-17/3.) I ntroduction Several researchers (Hobson and Hansel, 1972; Short, et al., 1973; Hausler and Malven, 1976) were able to cause a preovulatory-like surge of luteinizing hormone (LH) in plasma of ovariectomized cattle by the administration of estradiol. In addition, 2 mg estradiol can induce estrous behavior and LH release in prepuberal heifers (Gonzalez-Padilla et al., 1975). These reports suggest that estradiol may trigger an acute release of LH in cattle. Consistent with this view is the observation that estrogen concentrations increase in plasma prior to the LH surge in cattle (Echternkamp and 1 This study was supported financially by Quebec Agriculture Research Council Grant No. McA Present address: Lecturer, Department of Animal Husbandry, Univ. of Sri-Lanka, Peradeniya, Sri- Lanka. 3Present address: American Embryos, Inc., 2220 Patterson Road, Middleville, MI Department of Animal Science. s The authors thank Dr. G. D. Niswender (Colorado State Univ.) for the anti-estradiol-6-bsa serum. Hansel, 1971 ; Hendricks et al., 1971 ; Christensen et al., 1971). Hobson and Hansel (1972) observed that LH release did not occur in heifers given estrogen during the luteal phase of an estrous cycle. However, others (Short et al., 1973; Hausler and Malven, 1976) failed to inhibit estrogen-induced LH release with progesterone in ovariectomized heifers. In view of this conflict, further research is needed to define the action of progesterone in control of estrus and LH release in cattle. The objective was to investigate the effects of Silastic vaginal devices containing estradiol- 1713, progesterone or progesterone + estradiol on estrous behavior and progesterone, estradiol-17/3 and LH concentrations in serum of ovariectomized heifers. Materials and Methods Six heifers, 18 to 24 months old that weighed approximately 450 kg, were ovariectomized 21 days before the experiment. The heifers were housed indoors and were fed grain and hay. Heifers were assigned to one of three treatments during treatment periods A, B and C. During period A, two heifers were given progesterone (P4), two estradiol-17/3 (E2) and two progesterone and estradiol-1713 (P4 + E2). Steroids were delivered via pessaries as described below. During periods B and C, the treatments were rotated so that each heifer received all three treatments. Pessaries were in place for 3 days during each treatment period. The interval between treatments was 7 days. The vaginal devices, with a surface of about 160 cm 2, were described by Rajamehendran et al., (1979): One gram progesterone was placed in the lumen of each of two silicone rubber tubes (Dow Coming, Silastic No , I.D.-.79 cm, O. D cm, 15 cm long) and sealed with Silastic medical adhesive (Dow Coming). Pessaries containing estradiol-17r were prepared by coating ends of the silicone 554 JOURNAL OF ANIMAL SCIENCE, Vol. 49, No. 2, 1979

2 ESTRUS AND LH IN HEIFERS 555 [ c. J Figure 1. Diagram of the Silastic vaginal devices: surface area = 160 cm 2. Progesterone (1 g) was placed in each of the 2 tubes. Estradiol-1713 (10 mg) was coated on the four ends of the tubes over an area of about 40 centimeters squared. tubes with 1 ml of medical adhesive containing 10 mg E2. The coating covered a total area of about 40 cm 2 (figure 1). Each device was inserted into the vagina with a plastic speculum and plunger. A string attached to the device and left protruding from the vulva was used to remove the device at the end of treatment. Blood (20 ml) was obtained from a jugular vein before insertion of the device at 0 hr, then at 4-hr intervals for 36 hours. Aliquots of each sample were stored at -20 C until assayed. All heifers were observed for behavioral estrus throughout the experimental period. A vasectomized bull was used to determine time of onset of standing estrus. Progesterone was measured in serum by a radioimmunoassay as described by Rajamahendran et al., (1976) with the following modification. After extraction with petroleum ether, the aqueous phase was frozen at -20 C for 1 hr then the ether extract was decanted into assay tubes. Dr. Gordon D. Niswender of Colorado 50 Sl : 40 P4 E2 35 P4 + E2 25' F- IC HOURS FROM DEVICE INSERTION Figure 2. Mean serum estradiol-1713 in ovariectomized heifers treated with vaginal device containing progesterone (P4), estradiol-1713 (E~) or progesterone + estradiol-17~ (P4 + E2 ). 0 = time of insertion of the devices, n = six observations per treatment. Values for E~-treated heifers were greater than those for P4" (< 01) and P4 + E 2 (P<.05) treated heifers.

3 556 RAJAMAHENDRAN ET AL. State University, Fort Collings, CO, kindly analyzed the serum samples for LH. Estradiol- 17~3 was quantitated by procedures described in the appendix. Serum hormone data were analyzed statistically as a split-split plot experiment in a Latinsquare design. Periods were considered as main plot units, blood sampling times as subplot units and treatments as sub-sub plot units. The means were analyzed using least square difference (Steel and Torrie, 1960). Results Mean estradiol-17/3 concentrations in serum at 0 hr were not different among treatment groups (figure 2). Estradiol-17fl remained relatively constant throughout the sampfing period in P4-treated heifers (5.1 pg/ml). However at 4 hr, it increased to 40.9 and 32.1 pg/ml for E 2 - and P4 + E2 -treated heifers, respectively. These concentrations of estradiol were greater than (P<.01) pretreatment concentrations for these heifers and all concentrations for P4-treated heifers. After the estradiol in serum of E2-treated heifers reached a maximum, it declined steadily to 12 pg/m] at 16 hr; thereafter, it remained constant until the end of the sampling period. In two of the E2-treated heifers, the serum estradiol concentration declined to the pretreatment level at 28 hours. In P4 + E2-treated heifers, estradiol peaked at 4 hr, declined (P<.01) to 7.4 pg/ml at 20 hr and thereafter remained constant. Overall, estradiol concentrations were greater (P<.01) in E~-treated heifers than that in P4- or P4 + E2-treated heifers. Also, a significant (P<.O1) treatment time interaction was singled out for the serum estradiol concentrations. Progesterone concentrations in serum collected at 0 hr was not different among treatment groups (figure 3). In E2-treated heifers, it remained at about.5 ng/ml throughout the sampling period. In P4-treated heifers, it was higher (P<.O1) at 4 hr than that for E2-treated heifers (3.9 vs.5 ng/ml) and peaked at 8 hours. Thereafter, progesterone decreased gradually and reached 2.5 ng/ml at 36 hours. Changes in progesterone in serum of P4 + E2-treated heifers were not different (P>.05) from those in P4-treated heifers, except that the maximum concentration was at 4 hours. Pretreatment, LH in serum ranged from 5 to 6 ng/milligrams. In heifers treated with P4 and P4 + E2, no change was observed in LH concentrations during the sampling period (figure 4). However, a slight (P<.05) decline in LH concentration was observed at 4 or 8 hr in heifers 7, P4 _.E 5, 4 ~ 3, R~ 2, HOURS FROM DEVICE INSERTION Figure 3. Mean serum progesterone in ovariectomized neiters treated with vaginal device containing progesterone (P4), estradiol-17/3 (E 2 ) or progesterone + estradiol-17fl (P4 + E2 )- 0 = time of insertion of the devices, n = six observations per treatment. Values for P4- and P4 + E2 -treated heifers did not differ (P>.05) than those for E 2 -treated heifers.

4 ESTRUS AND LH IN HEIFERS 557 treated with P4 + E2 or P4, respectively. In E2-treated heifers, a slight decline (P<.05) in serum LH was also observed at 4 hr, but serum LH increased and reached a maximum of 32.7 ng/ml at 16 hr followed by a decline (P<.O1) at 24 hours. Serum LH concentrations in E2- treated heifers were higher (P<.OS) than those for P4- and P4 + E2-treated heifers. No difference (P<.05) in LH concentrations was observed between P4- and P4 + E2-treated heifers. All heifers treated with E2 displayed standing estrus. Onset of estrus ranged from 18 to 26 hr from the time of insertion of the device. Two heifers treated during period A had shorter intervals to estrus than the other heifers. Two of the P4 + E2-treated heifers also exhibited slight signs of estrus at 24 hr during period A. Discussion From extensive studies in the ovariectomized ewe (Robinson, 1959), it is clear that both progesterone and estradiol-17/3 play an important role in induction of estrous behavior. In the present study, all ovariectomized heifers treated with E 2 exhibited estrus within approxi- mately 24 hr, whereas none of the P4- or P4 + E2-treated heifers exhibited standing estrus. The results suggest that progesterone blocks estrogen induced estrous behavior. This observation agrees with other reports in ovariectomized heifers (Short et al., 1973) and ewes (Scaramuzzi et al., 1971). Maximum concentrations of serum estradiol obtained with E2 and P4 + E2 treatments were approximately four times that reported for intact heifers at proestrous (Wettemann et al., 1972). In the present study, serum estradiol was greater in ovariectomized heifers treated with E2 than in those treated with P4 + E2. The reason for the difference is not clear but it is possible that progesterone either affected the release of estradiol from the device or the absorption of estradiol through the vaginal wall. The observation that E2-treated heifers released LH is in agreement with other reports in ovariectomized heifers (Swanson, 1974; Gonzalez-Padilla et al., 1975). These observations and the fact that blood estradiol levels increase before estrus (Echternkamp and Hansel, 1971; Christensen et al, 1971) strongly P4 E2 P4 + E2 30, 25' v = 20, 15' lo, 5. o o HOURS FROM DEVICE INSERTION Figure 4. Mean serum LH in ovariectomized heifers treated with vaginal device containing progesterone (P4) estradiol-17# (E 2 ) or progesterone + estradiol-17/3 (P4 + E2 ). 0 = time of insertion of the devices, n = six observations per treatment. Values for E~-treated heifers were higher (P<.01) than those for P4- or P4 + E2-treated heifers.

5 558 RAJAMAHENDRAN ET AL. suggest that estrogen causes the preovulatory surge of LH release in cattle. The fact that P4 treatment did not influence the concentration of Serum LH is also in agreement with data of others for ovariectomized (Hobson and Hansel, 1972; Short et al., 1973; Hausler and Malven, 1976) and cycling cows (Hobson and Hansel, 1972). Maximum concentrations of serum progesterone obtained with P4 and P4 + E2 treatment were comparable to levels in cycling heifers during the luteal phase of an estrous cycle (Hendricks et al., 1971; Echternkamp et al., 1971; Rajamahendran et al., 1976). In contrast to the results of Beck et al. (1976), no difference was observed in the concentration of serum progesterone in heifers treated with P4 or P4 + E2. The estrogen-induced LH release during the luteal phase is lower than during the estrous phase of the cycle (Hobson and Hansel, 1972). Similarly, in the present study, progesterone was able to block estrogen induced LH release in ovariectomized heifers. This study confirms the findings of others in ovariectomized (Scaramuzzi et al, 1971) and intact ewes (Bolt et al, 1971). However, with similar experimental models, Short et al. (1973) and Hausler and Malven (1976) failed to inhibit estrogen-induced LH release by injecting progesterone into ovariectomized heifers. Perhaps, Silastic intravaginal devices may provide a more physiological release of steroid hormones. The present study clearly indicates that, in the ovariectomized heifer, progesterone can block estrogeninduced LH release. Appendix Estradiol antiserum was used at a dilution of 1:8000 in.1% gelatin buffer (.1 M sodium phosphate, ph 7.4). The same buffer was used for preparing dilute solutions of 2,4,6,7-3 H-estradiol-17/3 (New England Nuclear; 20,000 DPM/.05 ml). Estradiol-17/3 standards were prepared in benzene: methanol (9:1 v/v). All the above solutions were stored at 4 C. The detailed procedure consisted of the following steps. The serum sample (lml) was extracted with 6 ml of fresh anhydrous diethylether in a 15 x 125 mm tube (Kimax) fitted with a Teflon-lined screw cap. The mixture was shaken on a horizontal Eberback shaker for 10 min, centrifuged (1,250 x g) for 2 min and then placed at -20 C overnight. The solvent (extract) was decanted into assay tubes (12 x 75 mm) and evaporated under nitrogen at 40 C. The tube was rinsed with.1 ml of methanol. Extracted samples and standards were evaporated to dryness. To the tubes were added.2 ml of phosphate buffer,.05 ml of 3H_estradiol_ 17/3 and.05 ml of diluted estradiol antiserum. The tubes were vortexed for 5 sec and allowed to stand at 4 C overnight. Following this incubation period, fresh dextran-coated charcoal was added to separate the bound and unbound fractions. The tubes were vortexed for 5 sec, allowed to stand at 4 C for 15 rain and centrifuged at 1,250 g for 20 min at 4 C. The supernatant was decanted into vials to which 10 ml of scintillation cocktail (3.8 L toluene, g PPO, mg POPOP and 100 ml Beckman Bio-Solv-3) were added and mixed. Radioactivity was counted ~ 24 hr later in a liquid scintillation spectrometer (Beckman LS-235: counting efficiency for 3H, 70%) set to count a sample for 10 min or to an error rate of 1.5%, whichever occurred first. The radioimmunoassay was validated for the estimation of estradiol-17t3 in bovine serum. Specificity of the antiserum was checked for estrone, progesterone and corticosterone; less than.5% cross-reactivity was observed, confirming the data of England et al (1974) using the same antiserum. Accuracy of the assay examined over the range of 10 to 320 pg estradiol-17/3 showed no systematic error. The estimated quantities of estradiol-17/3 were closely correlated (r =.99, b =.95) with expected values. Results of the analysis of the internal standard (40 pg/sample) included in each assay were consistent. Inter- and intra-assay coefficients of variation (6.8 and 7.2%) were satisfactory for physiological studies. The sensitivity of the method (5 pg/sample) was adequate to measure estradiol-1713 in 1 ml of serum. Literature Cited Beck, T. W., V. G. Smith, B. E. Seguin and E. M. Convey Bovine serum LH, GH and prolactin following chronic implantation of ovarian steroids and subsequent ovariectomy. J. Anirn. Sci. 42:461. Bolt, D. J., H. E. Kelly and H. W. Hawk Release of LH by estradiol in cycling ewes. Biol. Reprod. 4:35. Christensen, D. S., J. N. Wihbank and M. L. Hopwood Blood hormone levels during the bovine estrous cycle. J. Anita. Sci. 33:251. Echternkamp, S. E. and W. Hansel Plasma estrogens, luteinizing hormone and corticoid

6 ESTRUS AND LH 1N HEIFERS 559 levels in postpartum cows. J. Dairy Sci. 54:800. England, B. G., G. D. Niswender and A. R. Midgley, Jr Radioimmunoassay of estradiol-17# without chromatography. J. Clin. Endocrinol. Metab. 38:42. Gonzalez-Padilla, E., G. D. Niswender and J. N. Wiltbank Puberty in beef heifers. II. Effect of injections of progesterone and estradiol- 17~3 on serum LH, FSH and ovarian activity. J. Anita. Sci. 40:1105. Hausler, C. L. and P. V. Malven Interaction of progesterone, GnRH, estradiol in the control of LH release in castrate heifers. J. Anita. Sci. 42:1239. Henricks, D. M., J. F. Dickey and J. R. Hill Plasma estrogen and progesterone levels in cows prior to and during estrus. Endocrinology 89:1350. Hobson, W. C. and W. Hansel Plasma LH levels after ovariectomy, corpus luteum removal and estradiol administration in cattle. Endocrinology 91:185. Rajamahendran, R., P. C. Lagu~ and R. D. Baker Plasma progesterone levels in cycling and gonadotrophin-prostaglandin-treated heifers. Can. J. Anim. Sci. 57:37. Rajamahendran, R., P. C. Lagui~ and R. D. Baker Plasma progesterone levels in cycling and activity in prepuberai heifers treated with progesterone. Can. J. Anita. Sci. (In press). Robinson, T. J In Cole, H. H. and P. T. Cupps (eds.). Reproduction in Domestic Animals. Vol. 1, p Academic Press, New York. Scaramuzzi, R. J., S. A. Tillson, I. H. Thorneycroft and B. V. Caldwell Action of exogenous progesterone and estrogen on behavioral estrus and luteinizing hormone levels in the ovariectomized ewe. Endocrinology 88:1184. Short, R. E., B. E. Howland, R. D. Randel, D. S. Christensen and R. A. Bellows Induced LH release in spayed cows. J. Anita. Sci. 37:551. Steel, R.G.D. and J. H. Torrie Principles and Procedures of Statistics. 481 p. McGraw Hill Book Co., Inc. New York. Swanson, L. V Hormone feedback in prepuberal Holstein heifers. J. Anim. Sci. 39:228. (Abstr.). Wettemann, R. P., H. D. Hafs, L. A. Edgerton and L. V. Swanson Estradiol and progesterone in blood serum during the bovine estrous cycle. J. Anita. Sci. 34:1020.