Effect of Carrier and Administration on Luteinizing Hormone Release By Gonadotropin Releasing Hormone

Effect of Carrier and Administration on Luteinizing Hormone Release By Gonadotropin Releasing Hormone

TECHNICAL NOTES Effect of Carrier and Administration on Luteinizing H o r m o n e Release By Gonadotropin Releasing H o r m o n e D. J. KESLER and D...

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TECHNICAL

NOTES

Effect of Carrier and Administration on Luteinizing H o r m o n e Release By Gonadotropin Releasing H o r m o n e D. J. KESLER and D. L. VINCENT Department of Animal Science University of Illinois Urbana 61801

ABSTRACT

duced release of LH is considerably shorter than a preovulatory LH surge, which is about 8 to 10 hr (5, 10). Golter et al. (6), however, reported an LH surge of about 10 h when GnRH was dissolved in 2% carboxymethylcellulose and injected subcutaneously in bulls. Furthermore, in postpartum dairy cows Britt et al. (1) reported that mean peak LH concentrations were 4 h following subcutaneous implantation of a gelatin capsule containing GnRH. Although a variety of carriers and methods of administration have been used, the effect of carrier and method of administration on GnRHinduced LH release has not been examined. This study was to evaluate the effect of various carriers and methods of administration on GnRHinduced LH release in ovariectomized ewes.

Ovariectomized ewes in groups of four were administered either saline (I) or 50 ~tg of gonadotropin releasing hormone (II to VII). Methods of administration and carriers were: II, intramuscular saline; III, intravenous saline; IV, subcutaneous saline; V, subcutaneous gelatin; VI, subcutaneous carboxymethylcellulose; and VII, subcutaneous gelatin capsule. Only subcutaneous administration of gonadotropin releasing hormone in the gelatin capsule (VII) prolonged the interval from treatment to luteinizing hormone peak; however, there were fewer ewes with peak luteinizing hormone concentations equal to or greater than 100 ng/ml in VII than II, IV, V, and VI. Ewes in V, VI, and VII had luteinizing hormone surges of longer duration than 11 and III. Ewes in VII had the longest luteinizing hormone surge, which was 7.5 h , even though luteinizing hormone concentrations did not return to pretreatment concentrations by 8 h posttreatment in three of the four ewes.

MATERIALS AND METHODS

INTRODUCTION Synthetic gonadotropin releasing hormone (GnRH) has induced release of luteinizing hormone (LH) in domestic animals during various physiological states (14, 15). In most studies, GnRH has been dissolved in saline and administered by intramuscular or intravenous injection. Following GnRH treatment in ewes, concentrations of LH in plasma increase within 15 rain, peak LH is .5 to 2.0 h posttreatment, and concentrations of LH in plasma return to pretreatment concentrations by 4 to 6 hr following treatment (13, 15). This GnRH-in-

Received February 27, 1980. 1980 J Dairy Sci 63:2121-2125

Twenty-eight mature ewes ovariectomized in September, 1977, were evenly assigned to one of seven groups. In November, 1977, ewes in II to VII were administered 50/ag of GnRH, whereas ewes in Group I received an injection of saline (.9% with .9% benzyl alcohol). The methods of administration and carriers of GnRH were: II, intramuscular saline; III, intravenous saline; IV, subcutaneous saline; V, subcutaneous 2% gelatin; VI, subcutaneous 2% carboxymethylcellulose; and VII, subcutaneous gelatin capsule. F o r II, III, and IV, GnRH was dissolved in .9% saline with .9% benzyl alchol and stored at 4°C until used. For GnRH in the 2% gelatin carrier, gelatin powder for a 2% solution was added to saline (.9% with .9% benzyl alcohol) and stirred continuously over heat until the gelatin powder was dissolved. The mixture was allowed to cool to approximately 32°C at which time the GnRH was added. After thorough mixing, 1 ml of the mixture was aspirated into syringes and stored at 4°C until used. The GnRH in the 2% carboxymethylcellulose carrier was prepared the same as described for 2% gelatin except carboxymethyl-

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cellulose powder was used. All injections (11 to VI) had 50 ~tg G n R H / m l solution. F o r VII, 50 #g of GnRH was dissolved in 10/al of saline (.9% with .9% benzyl alcohol), p i p e t t e d into one end of a Number 00 gelatin capsule, quick frozen, the remaining end of the capsule replaced and stored at - 2 0 ° C until used. Blood was collected via jugular venipuncture into heparinized vacutainers immediately prior to treatment, at .5-h intervals for 4 h and at 5.0, 6.0, 7.0, and 8.0 h posttreatment. Following collection, blood was chilled on ice water until centrifugation at 10,000 x g for 10 min at 4°C. Plasma was decanted and stored at - 2 0 ° C until assayed. LH Assay

The LH in plasma was quantified by double antibody radioimmunoassay as reported by Niswender et al. (12) except 12si was the radioactive label. The anti-ovine LH antiserum (GDN #15), diluted 1/40,000, exhibited a mean binding of 44%. Plasma samples were assayed in duplicate 100 gtl volumes. The NIH-LH-S 16 ovine LH was used as the ovine standard at a range of .1 to 10 ng per tube (92.9% bound to 14.5% bound, respectively). The .1 ng/tube (92.9% bound) point o f the standard curve was greater (P<.05) than 0 ng/tube (100% bound). When various volumes (50, 100, and 200 gtl) of pooled sheep plasma were assayed, LH's were 1.5, 1.7, and 1.3 ng/ml, respectively. The interassay coefficient of variation was 6.2%. Standard curves showed intraasaay coefficients of variation of 2.1% at the lowest concentration of the curve to 5.3% at the highest concentration of the curve. Data Analysis

Following treatment (0 to 8 h), LH data from all groups were analyzed by split plot analysis of variance (4). The interval from treatment to LH peak and the duration of LH surge were analyzed by analysis of variance (17). The duration of the LH surge was defined as the interval LH concentrations remained above pretreatment concentrations. A multiple range test was used for indentification of differences between means (9). Concentrations o f LH in plasma from the peak to the return to pretreatment content were analyzed also by linear regression and analysis of covariance Journal of Dairy Science Vol. 63, No. 12, 1980

(16). A Chi-square test described by Fisher and Yates (2) was used to test differences in enumerative data. R ESU LTS

Although concentrations o f LH in plasma fluctuated in the control ewes, increase in plasma LH was not sustained. The GnRH induced release of LH in all treated ewes. Even though the mean concentration of LH in plasma in III was less (P<.05) than in II, IV, V, and VI, all groups administered GnRH had higher (P<.05) concentrations of LH than I (control) (Table 1). Concentrations of LH in plasma increased by .5 h posttreatment in all but two ewes which were in VII. In those two ewes, concentrations o f plasma LH were higher than pretreatment concentrations by 1 h posttreatment. The mean interval from GnRH treatment to peak LH concentrations was .8 to 1.6 h for II to VI (P>.05) (Table 1). The interval from GnRH treatment to LH peak in VII, however, was 2.8 -+ .5 h and was longer (P<.05) than for II t¢ VI. In II, IV, V, and VI, at least three of the four ewes in each group had an LH peak equal to or greater than 100 ng/ml, whereas only two ewes in III and one ewe in VII had peak LH concentrations equal to or greater than 100 ng/ml (Table 1). There were fewer (P<.05) ewes with peak LH concentrations equal to or greater than 100 ng/ml in VII than II, IV, V, and VI. Both carrier and m e t h o d of administration affected duration of GnRH induced LH release (Table 1). Although subcutaneous injection of GnRH in saline (IV) induced an LH surge 1.7 h longer (P<.05) than the LH surge following intravenous GnRH treatment in saline (III), neither was different (P<.05) from the LH surge induced by intramuscular injection of GnRH in saline (II). Dissolving GnRH in gelatin (V) or carboxymethylceUulose (VI) and injecting subcutaneously did not prolong (P>.05) the LH surge as compared to dissolving in saline and injecting subcutaneously (IV). However, the LH surge induced by GnRH dissolved in gelatin or carboxymethylcellulose and injected subcutaneously was longer (P<.05) than the LH surge induced by GnRH in saline and injected intravensouly or intramuscularly. Subcutaneous administration of GnRH in a

"FABLE 1. Number of ewes with peak LH concentrations ~ I 0 0 ng/ml, interval from treatment to LH peak, duration o f the LH surge, and mean LH release in ovariectomized ewes administered GnRH.

Treatment groupa

Carrier vehicle

Method of administration

Number with and LH peak >i 100 ng/ml

Time to LH peak (h)

Duration of LH surge c (h)

intramuscular Intravenous Subcutaneous Subcutaneous

0 4e 2 ef 3e 4e

"18 e .8 e 1.6 e 1.4 e

:2'

Subcutaneous Subcutaneous

3e 1f

1.5 e 2.8 f

SE b 1d II III IV V VI

Saline Saline Saline Gelatin Carboxymethylcellulose Gelatin capsule

VII

Mean LH release (ng/ml)

,X

SE b

X'

SE b

.2 •5 .4

511 ef 4.1e 5.8fg 6.5g h

... .6 .3 .5 .2

8.6 d 45.7 f 27.0 e 45.3 f 54.2 f

3.9 6.5 4-. 5 4.0 3.0

.2 .5

7.0gh 7.5 h

.6(1 )i .20)i

48.9 f 41.0ef

2.2 7.3

Z >

2: 0

aFour ewes per group. bstandard error. Clnterval that LH concentrations remained above pretreatment concentrations. d c o n t r o l ewes. e'f'g'hMeans with a different superscript are significant (P<.05). iFignres in parentheses are the number of ewes in which LH concentrations did not return to pretreatment concentrations, <

g t~

Z 9

b~

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KESLER AND VINCENT

gelatin capsule (VII) induced the longest LH surge. Even though the duration LH surge for ewes in VII was n o t different (P>.05) f r o m V and VI, in three of the f o u r ewes in VII, LH concentrations did n o t return to pretreatm e n t concentrations. The linear decrease in LH from peak c o n c e n t r a t i o n s to the return to p r e t r e a t m e n t c o n c e n t r a t i o n s is in Figure 1. Only VI and VII had different regression slopes (P<.O1) f r o m II and III. DISCUSSION

The preovulatory LH surge in the ewe is characterized by an LH release of 8 to 10 h (5, 11). Although peak concentrations of LH induced by e x o g e n o u s G n R H are similar to the preovulatory LH surge, the d u r a t i o n of the LH release is only about half the duration during the preovulatory LH surge (13, 15). R o u t e of administration and carrier as in this study and dosage as r e p o r t e d by Kinder et al. (8) appear to affect duration of LH release induced by exogenous GnRH. F u r t h e r m o r e , the dosage of G n R H and the total a m o u n t o f LH released by exogenous G n R H appear to be associated with G n R H induced ovulation in anestrous ewes (3, 8, 13). In cattle, Britt et al. (1) showed that subcutaneous administration of G n R H in a

0

-20

~j-4C O.

z -6C Lfl ne ~t~ -8C

~

GROUP SLOPE 'mT

MEAN PEAK

-tO.6

-20.1 -21.6

trIM

-25.0 -26.3

.J

HOURS

Figure 1. Linear decrease in plasma LH from peak concentrations to the return to pretreatment concentrations. Journal of Dairy Science Vol. 63, No. 12, 1980

gelatin capsule induced ovulation in all postpart u m dairy cows treated whereas others (7, 18) showed a 50% ovulation response w h e n postp a r t u m dairy cows were administered intramuscularly the same dosage of G n R H in saline. Subcutaneous administration of G n R H in carboxymethylcellulose or in the gelatin capsule appears to prolong effectively LH release. Therefore, future e x p e r i m e n t s are warranted to c o m p a r e carrier and m e t h o d of administration on ovulation responses to G n R H in anestrous ewes and p o s t p a r t u m cows. ACKNOWLEDGMENTS

The G n R H was supplied generously by R. H. Ripple ( A b b o t t Laboratories). Antisera for r a d i o i m m u n o a s s a y of LH ( G D N # 1 5 ) was supplied generously by G. D. Niswender. Purified ovine LH ( L E R - 1 0 5 6 - C 2 ) was supplied by L. E. Richert, and the NIH-LH-S16 standard was supplied by the National Institute of Arthritis, Metabolism, and Digestive Diseases. REFERENCES

1 Britt, J. H., R. J. Kittok, and D. S. Harrison. 1974. Ovulation, estrus and endocrine response after GnRH in early postpartum cows. J. Anita. Sci. 39:915. 2 Fisher, R. A., and F. Yates. 1963. Statistical tables for biological, agricultural and medical research. 6th ed. Hafner Publishing Co., New York, NY. 3 Frandle, K., R. W. Wright, and J. J. Reeves. 1977. Fertility of sheep eggs from LHRH induced ovulations. J. Anita. Sci. 45 (Suppl. 1):159. (Abstr.) 4 Gill, G. J., and H. D. Hafs. 1971. Analysis of repeated measurements of animals. J. Anita. Sci. 33:331. 5 Goding, J. R., K. J. Catt, J. M. Brown, C. C. Kaltenbach, I. A. Cumming, and B. J. Mole. 1969. Radioimmunoassay for ovine LH. Secretion of LH during estrus and following estrogen administration in sheep. Endocrinology 85:133. 6 Goiter, T. D., J. J. Reeves, C. C. O'Mary, A. Arimura, and A. V. Schally. 1973. Serum LH levels in bulls treated with synthetic luteinizing hormonereleasing hormone/foUicle stimulating hormonereleasing hormone (LH-RH/FSH-RH). J. Anita. Sci. 37:123. 7 Kesler, D. J., H. A. Garverick, R. S. Youngquist, R. G. Elmore, and C. J. Bierschwal. 1978. Ovarian and endocrine responses and reproductive performance following GnRH treatment in early postpartum dairy cows. Theriogenology 9 : 363. 8 Kinder, J. E., T. E. Adams, T. M. Nett, D. H. Coy, A. V. Schally, and J. J. Reeves. 1976. Serum gonadotropin concentrations and ovarian response in ewes treated with analogs to LH-RH/FSH-RH. J. Anita. Sci. 42:1220.

TECHNICAL NOTE 9 Kramer, C. Y. 1956. Extension of multiple range test to group means with unequal number of replications. Biometrics 12: 307. 10 Land, R. B., J. Pelletier, J. Thimonier, and P. Maulion. 1973. A quantitative study of genetic differences in the incidence of oestrus, ovulation and plasma luteinizing hormone concentrations in the sheep. J. Endocrinol. 58:305. 11 Legan, S. J., and F. J. Karsch. 1979. Neuroendocrine regulation of the estrous cycle and seasonal breeding in the ewe. Biol. Reprod. 20:74. 12 Niswender, G. D., L. E. Reichert, A. B. Midgley, and A. V. Nalbandov. 1969. Radioimmunoassay for bovine and ovine luteinizing hormone. Endocrinology 84:1166. 13 Reeves, J. J., G. K. Tarnausky, and P. K. Chakraborty. 1974. Serum LH in ewes treated with synthetic luteinizing hormone-releasing hormone/follicle

14 15 16 17 18

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stimulating hormone-releasing hormone (LH-RH/ FSH-RH) at three periods of anestrus. J. Anim. Sci. 38:369. Schally, A. V. 1978. Aspects of hypothalamic regulation of the pituitary gland. Science 202:18. Seeger, K., and R. Humke. 1975. LH/FSH releasing hormone in veterinary medicine. The Blue Book 25:156. Snedecor, G. W., and W. G. Cochran. 1967. Statistical methods. 6th ed. The Iowa State University Press, Ames. Steel, R.G.D., andJ. H. Torrie. 1960. Principles and procedures of statistics. McGraw-Hill, New York. NY. Zaied, A. A., H. A. Garverick, C. J. Bierschwal, R. G. Elmore, R. S. Youngquist, and A. J. Sharp. 1979. Effect of ovarian activity cycles in postpartum cows. J. Anita. Sci. 49 (Suppl. 1):351. (Abstr.)

ERRATA Effects of Selection on Variances and Covariances o f S i m u l a t e d First and Second Lactations. M. F. Rothschild, C. R. Henderson, and R. L. Quaas, D e p a r t m e n t of A n i m a l Science, Cornell University, Ithaca, NY 14853. V o l u m e 62, p. 999, Table 4, last column, row 9. The true value under R P should be .3904. Digestibility and Associative Effects of Protected Tallow. D. S. Kronfeld and S. D o n o g h u e , D e p a r t m e n t of Clinical Studies, School o f Veterinary Medicine, University of Pennsylvania, K e n n e t t Square 19348. V o l u m e 63, p. 642. Senior a u t h o r ' s name is D. S. Kronfeld, n o t D. A. Kronfeld as shown. I m m u n o g l o b u l i n P r o d u c t i o n and T r a n s p o r t by the M a m m a r y Gland. B. L. Larson, H. L. Leary, Jr., and J. E. Devery, D e p a r t m e n t of Dairy Science, University of Illinois, Urbana, IL 61801. V o l u m e 63, p. 665. A u t h o r ' s n a m e " H e a r y " should read " L e a r y " .

Journal of Dairy Science Vol. 63, No. 12, 1980