C L E A R A N C E OF G O N A D O T R O P I N - R E L E A S I N G H O R M O N E IN BEEF H E I F E R S A F T E R I N T R A M U S C U L A R OR INTRAVENOUS ADMINISTRATION l
Colorado State University, Fort Collins 8 0 5 2 3
SUMMARY Ten heifers (170 to 200 kg) were administered 2.5 mg synthetic gonadotropin-releasing hormone (GnRH) intramuscularly (im) into the left semitendinosus muscle. Muscle biopsies from the injection site were taken from each of two heifers at 2, 8, 24, 48 and 72 hr after injection. Four heifers were administered 100 /~Ci (sp.act.~-l,600 /~Ci/#g)GnRH -12Sl im and muscle biopsies were taken from each of two heifers at 2 hr and 8 hours. An additional four heifers received an im injection of saline and muscle biopsies were taken from each of two heifers at 2 hr and 8 hours. Four heifers were given intravenous (iv) injections of 2.5 mg gonadotropin-releasing hormone. Samples of jugular blood were collected at - 3 0 , - 1 5 , 0, 5, 10, 15, 30, 45, 60, 90, 120, 150, 180, 240, 300, 360, 420 and 480 rain relative to the time of injection from all heifers. In addition, samples were collected at 4-hr intervals until 72 hr from those heifers in which biopsies were performed at 24, 48 and 72 hr after injection. Samples from all heifers receiving 2.5 mg GnRIt or 5 ml saline were assayed for luteinizing hormone (LH), follicle-stimulating hormone (FSH) and gonadotropin-releasing hormone. Levels of GnRH were maximal at 5 rain (186 -+ 43 pg/ml) after iv injection and at 15 min (82 -+ 37 pg/ml) after im administration. The GnRH A 2Sl also reached a maximal level in serum 15 min after the im injection. Levels of GnRH remained above baseline for 1.5 hr after iv administration compared to 4 to 8 hr after im administration. Concentrations of GnRH were elevated in muscle tissue obtained at 2 and 8 hr after injection but had decreased to baseline by 24 hours. Serum levels of LH and FSH were maximal at 2.5 hr after either iv or im injection of gonadotropin-releasing hor-
1Supported in part by NIH grant HD-O7841 and a contract from Parke-Davis Co., Ann Arbor, Mi.
mone. Increases of LH, FSH or GnRH were not observed in heifers administered saline. (Key Words: GnRH, Clearance, Intramuscular Injection, Heifers, LH.)
INTRODUCTION That GnRH induces release of LH and FSH in the bovine has been well documented (Golter et al., 1973; Zolman et al., 1973; Kaltenbach et al., 1974; Schams et al., 1974). Furthermore, GnRH has been used pharmacologically in heifers to induce ovulation after synchronization of estrus (Mauer and Rippel, 1972), to induce ovulation during the early postpartum period (Britt et al., 1974), and to treat ovarian follicular cysts (Kittok et al., 1973; Cantley et a2., 1975). Although GnRH has been used for these therapeutic purposes very little is known about the time required for GnRH to enter the circulation after im injection or the length of time residues of GnRH remain at the site of injection. The objectives of this study were to: 1) compare the patterns of release of LH and FSH after iv or im injection of GnRH; 2) determine the time required for GnRH to enter the circulation after im injection; and 3) determine the concentration of residues of GnRH in muscle at various times after im administration.
EXPERIMENTAL PROCEDURES Animals. A total of 22 Hereford heifers weighing from 170 to 200 kg were divided into four groups. A polyvinyl cannula with a teflon obturator (Longdwell, Becton-Dickinson, Co., Rutherford, N.J.) was inserted into the jugular vein of each animal to facilitate collection of blood samples. Group 1. Ten heifers were given 2.5 mg synthetic GnRH (Parke-Davis Co.) into a semitendinosus muscle in 5.0 ml sterile physiological saline. Blood samples (20 ml) were collected at - 3 0 , - 1 5 , 0, 5, 10, 15, 30, 45, 60, 90, 120,
1264
JOURNAL OF ANIMAL SCIENCE, Vol. 43, No. 6 (1976)
Downloaded from https://academic.oup.com/jas/article-abstract/43/6/1264/4700440 by Iowa State University user on 19 January 2019
J. E. Peterson and T. M. Nett
CLEARANCE OF GnRH IN HEIFERS
the levels of radioactivity. The association of 1251 with GnRH was confirmed by immunoprecipitation.
Radioimmunoassay o f Lll, FSII and GnRtt. Validated double-antibody radioimmunoassays were used for the quantification of bovine LH (Niswender et al., 1969) and bovine FSH (Akbar et al., 1974b). The radioimmunoassay procedure for GnRH was similar to that reported previously (Nett et al., 1973) with the following modifications. In brief, 200 /al of serum and 200 /al of anti-GnRH sera (R-42 diluted 1:100,000 in 1:400 normal rabbit serum) were incubated for 24 hr at 4 C, GnRII) 2Sl (15,000 cpm) was then added and the tubes were incubated at 4 C for an additional 24 hours. At this time, 200 /al of appropriately diluted sheep anti-rabbit gamma globulin were added and incubation was continued for 72 hours. At the end of the 72-hr incubation 2 ml of cold PBS were added to each tube, the tubes were centrifuged at 2,300 x g for 30 rain, the supernatants were decanted and the precipitates were counted. Immunoprecipitation of GnRH-12SL lmmunoprecipitation was performed as described for LH, FSH and prolactin (Akbar et al., 1974a). In brief, duplicate 200 /al aliquots of muscle extracts or serum samples from heifers in group 2 were incubated at 4 C with excess (1:400) rabbit anti-GnRH sera for 24 hours. Two-hundred microliters of appropriated diluted sheep anti-rabbit gamma globulin was added to each tube and incubation was continued for 72 hours. Three milliliters of cold PBS were added to each tube, the tubes were centrifuged at 2,300 x g for 30 min, the supernatants were decanted and the precipitates (immunoprecipitable G n R H I 2 S l ) were counted in a Nuclear Chicago automatic gamma spectrometer. R ESU LTS
Levels of GnRH in serum after iv or im administration of 2.5 mg GnRH are depicted in figure 1. Levels of GnRH were maximal 5 min after iv administration (186 + 43 pg/ml) but had decreased to baseline (
Colorado State University, Fort Collins 8 0 5 2 3
SUMMARY Ten heifers (170 to 200 kg) were administered 2.5 mg synthetic gonadotropin-releasing hormone (GnRH) intramuscularly (im) into the left semitendinosus muscle. Muscle biopsies from the injection site were taken from each of two heifers at 2, 8, 24, 48 and 72 hr after injection. Four heifers were administered 100 /~Ci (sp.act.~-l,600 /~Ci/#g)GnRH -12Sl im and muscle biopsies were taken from each of two heifers at 2 hr and 8 hours. An additional four heifers received an im injection of saline and muscle biopsies were taken from each of two heifers at 2 hr and 8 hours. Four heifers were given intravenous (iv) injections of 2.5 mg gonadotropin-releasing hormone. Samples of jugular blood were collected at - 3 0 , - 1 5 , 0, 5, 10, 15, 30, 45, 60, 90, 120, 150, 180, 240, 300, 360, 420 and 480 rain relative to the time of injection from all heifers. In addition, samples were collected at 4-hr intervals until 72 hr from those heifers in which biopsies were performed at 24, 48 and 72 hr after injection. Samples from all heifers receiving 2.5 mg GnRIt or 5 ml saline were assayed for luteinizing hormone (LH), follicle-stimulating hormone (FSH) and gonadotropin-releasing hormone. Levels of GnRH were maximal at 5 rain (186 -+ 43 pg/ml) after iv injection and at 15 min (82 -+ 37 pg/ml) after im administration. The GnRH A 2Sl also reached a maximal level in serum 15 min after the im injection. Levels of GnRH remained above baseline for 1.5 hr after iv administration compared to 4 to 8 hr after im administration. Concentrations of GnRH were elevated in muscle tissue obtained at 2 and 8 hr after injection but had decreased to baseline by 24 hours. Serum levels of LH and FSH were maximal at 2.5 hr after either iv or im injection of gonadotropin-releasing hor-
1Supported in part by NIH grant HD-O7841 and a contract from Parke-Davis Co., Ann Arbor, Mi.
mone. Increases of LH, FSH or GnRH were not observed in heifers administered saline. (Key Words: GnRH, Clearance, Intramuscular Injection, Heifers, LH.)
INTRODUCTION That GnRH induces release of LH and FSH in the bovine has been well documented (Golter et al., 1973; Zolman et al., 1973; Kaltenbach et al., 1974; Schams et al., 1974). Furthermore, GnRH has been used pharmacologically in heifers to induce ovulation after synchronization of estrus (Mauer and Rippel, 1972), to induce ovulation during the early postpartum period (Britt et al., 1974), and to treat ovarian follicular cysts (Kittok et al., 1973; Cantley et a2., 1975). Although GnRH has been used for these therapeutic purposes very little is known about the time required for GnRH to enter the circulation after im injection or the length of time residues of GnRH remain at the site of injection. The objectives of this study were to: 1) compare the patterns of release of LH and FSH after iv or im injection of GnRH; 2) determine the time required for GnRH to enter the circulation after im injection; and 3) determine the concentration of residues of GnRH in muscle at various times after im administration.
EXPERIMENTAL PROCEDURES Animals. A total of 22 Hereford heifers weighing from 170 to 200 kg were divided into four groups. A polyvinyl cannula with a teflon obturator (Longdwell, Becton-Dickinson, Co., Rutherford, N.J.) was inserted into the jugular vein of each animal to facilitate collection of blood samples. Group 1. Ten heifers were given 2.5 mg synthetic GnRH (Parke-Davis Co.) into a semitendinosus muscle in 5.0 ml sterile physiological saline. Blood samples (20 ml) were collected at - 3 0 , - 1 5 , 0, 5, 10, 15, 30, 45, 60, 90, 120,
1264
JOURNAL OF ANIMAL SCIENCE, Vol. 43, No. 6 (1976)
Downloaded from https://academic.oup.com/jas/article-abstract/43/6/1264/4700440 by Iowa State University user on 19 January 2019
J. E. Peterson and T. M. Nett
CLEARANCE OF GnRH IN HEIFERS
the levels of radioactivity. The association of 1251 with GnRH was confirmed by immunoprecipitation.
Radioimmunoassay o f Lll, FSII and GnRtt. Validated double-antibody radioimmunoassays were used for the quantification of bovine LH (Niswender et al., 1969) and bovine FSH (Akbar et al., 1974b). The radioimmunoassay procedure for GnRH was similar to that reported previously (Nett et al., 1973) with the following modifications. In brief, 200 /al of serum and 200 /al of anti-GnRH sera (R-42 diluted 1:100,000 in 1:400 normal rabbit serum) were incubated for 24 hr at 4 C, GnRII) 2Sl (15,000 cpm) was then added and the tubes were incubated at 4 C for an additional 24 hours. At this time, 200 /al of appropriately diluted sheep anti-rabbit gamma globulin were added and incubation was continued for 72 hours. At the end of the 72-hr incubation 2 ml of cold PBS were added to each tube, the tubes were centrifuged at 2,300 x g for 30 rain, the supernatants were decanted and the precipitates were counted. Immunoprecipitation of GnRH-12SL lmmunoprecipitation was performed as described for LH, FSH and prolactin (Akbar et al., 1974a). In brief, duplicate 200 /al aliquots of muscle extracts or serum samples from heifers in group 2 were incubated at 4 C with excess (1:400) rabbit anti-GnRH sera for 24 hours. Two-hundred microliters of appropriated diluted sheep anti-rabbit gamma globulin was added to each tube and incubation was continued for 72 hours. Three milliliters of cold PBS were added to each tube, the tubes were centrifuged at 2,300 x g for 30 min, the supernatants were decanted and the precipitates (immunoprecipitable G n R H I 2 S l ) were counted in a Nuclear Chicago automatic gamma spectrometer. R ESU LTS
Levels of GnRH in serum after iv or im administration of 2.5 mg GnRH are depicted in figure 1. Levels of GnRH were maximal 5 min after iv administration (186 + 43 pg/ml) but had decreased to baseline (
