R. B. Simpson2, J. D. A r m s ~ m n g ~ -R.~ , W. Hawef, D. C. h4illefi, E. P. Heimeg and R. M. Campbell6

North Carolina State University, Raleigh 27695-7621 and Hoffmann-La Roche, Inc., Nutley, NJ 07110-1199 ABSTRACT

Angus and Charolais heifers (195 f 7 kg) were actively immunized against growth hormone-releasing factor (GRF) to evaluate. the effect on concentrations of somatotropin (ST), insulin-like growth factor I (IGF-I), insulin (INS), growth, and onset of puberty. primary immunizations were given at 184 f 7 d of age (d 0 of experiment) by injecting (s.c.) 1.5 mg of GRF-(l-29)-Gly-Gly-Cys-NH2 conjugated to 1.5 mg of human serum albumin (GRF, n = 22) or 1.5 mg of human serum albumin (HSAi, n = 21). Booster immunizations of .5 mg of antigen were given on d 62,92,153, and 251.Antibody binding (percentage at 1:2,000dilution) to [lZr]GRF on d 69 was greater (P c .01)in GRFi (53.7f 4.5)than in HSAi (10.1f .6)heifers. Serum concentration (nghnl) and frequency (peakd5 h) of ST release, respectively, on d 78 were lower (P < .Ol) in GRFi than in HSAi heifers (3.3 f .1 vs 5.6 k .2and .9f .3 vs 2.3 f -2). Serum IGF-I (ng/ml) was lower (P < .01)in GRFi than in HSAi heifers on d 69 (41 f 5 vs 112 f 4).Serum INS (pU/ml) on d 78 was lower (P c .05) in GRFi (2.2f .l) than in HSAi (3.8f .2)heifers. Feed intake, AM;,and feed efficiency were lower (P c .05)in GRFi than in HSAi heifers. Hip height was lower (P < -01)and fat thickness was greater (P < .05) in GRFi than in HSAi heifers by d 132 and 167,respectively. Percentage of heifers attaining puberty (progesterone > 1 ng/ml for two consecutive weeks) by d 209 and 379 (12.9 and 18.5 mo of age), respectively, was lower (P < .05) in GRFi (40.9 and 45.5) than in HSAi (81.0and 100). In conclusion, growing heifers were successively immunized against GRF. Active immunization against GRF resulted in decreased serum concentration of ST, IGF-I, and INS. In addition, GRF immunization led to lowered feed intake, AM;,and feed efficiency, increased fat depth, and delayed onset of puberty in heifers. We propose that ST and IGF-I are important metabolic mediators involved in the initiation of puberty in heifers. Key Words: Heifer, F'uberty, GHRF, Somatotropin, IGF-I, Insulin J. Anim. Sci. 1991. 6949144924

'The use of trade names in this publication does not imply endorsement by the North Carolina A R S or criticism of similar products not mentioned. Research reported in lhis publication was funded by Ihe North Carolina A R S . purified ovine somatotropin(ST), ovine ST anti@y A. F. Parlow, Pituitary Hormones and Antisera Center, Torrance, CA), and human Win-like. p w t h factor I antiserum (by L. Undmvood and J. J. Van Wylq Chapel HiU. NC) were obtained through the National Hormone and Pituitary Rograns NJDDK, and Univ. of Maryland, School of Med., Baltimore. MD. FK33-824 was kindly provided by Sandoz pharm. Corp.,

East Hanover, NJ.Appreciation is expressed to D. Askew, L. Cole, B. Huff,E. Jones, C. Marsh, Jr., K. Moore, J. Niemaun, R Stanlro, T.Wagoner, V. Wilson, and T. Wise for their technical assi-. 2Dept. of him. Sci., PhysioL Program, North Carolina Slate Univ. 3T0 whom reprint requests should be sent. 4-t. of ~ n i m .Sei.. NO& carolina state U ~ V . k e p t . of Peptide Res., Hoffmanu-La Roche, Inc. %ept. of him. sci. m.,~ 0 - b ~ocht,~nc. Received April 4, 1991. Accepted June 26, 1991.

4914

Downloaded from https://academic.oup.com/jas/article-abstract/69/12/4914/4704867 by Iowa State University user on 16 January 2019

EFFECT OF ACTIVE IMMUNIZATION AGAINST GROWTH HORMONE-RELEASING FACTOR ON GROWTH AND ONSET OF PUBERTY IN BEEF HEIFERS

ACI’IVE IMMUNIZATION AGAINST GRP IN HEIFERS

4915

Materialsand Methods

Age at puberty is an important factor affecting reproductive performance of beef cattle. Byerley et al. (1987) reported that heifers bred at their third estrus had higher pregnancy rates than heifers bred at first estrus. Heifers that conceive early in their fiist breeding season continue to calve early in subsequent calving seasons and wean heavier calves throughout their lifetimes (Lesmeister et al., 1973). Thus, it is desirable for heifers to attain puberty before, rather than during, their first breeding season. Although the role of the gonadotropins, especially LH, in the attainment of puberty in heifers has been widely studied (Kinder et al., 1987), involvement of somatotropin (ST)and insulin-like growth factor I (IGF-I) in the events preceding the onset of puberty are not clearly understood. Jones et al. (1991) observed an increase in the frequency of release of LH and an increase in serum concentrations of IGF-I before puberty in Angus heifers. Granger et al. (1989) reported lower concentrations of IGF-I and a delay in puberty in heifers fed hay compared with heifers fed hay and supplement. Whether lower serum concentrations of IGF-I mediate the effects of nutritional stress on pubertal onset, or whether IGF-I concentrations merely reflect nutritional stress, has not been determined. Active immunization against growth hormonereleasing factor (GRF-[1-29]-Gly-GlyCys-NH2) abolishes episodic release of ST and lowers serum concentrations of IGF-I in gilts (Armstrong et al., 199Ob) and lactating beef cows (Lloyd et al., 1988). We hypothesized that prepubertal heifers immunized against GRF would provide a useful model for elucidation of the role that ST and IGF-I may play in the events preceding puberty. The objectives of this study were 1) to determine whether growing heifers could be effectively immunized against GRF and to monitor the effect of immunization on ST and IGF-I and 2) to evaluate the effects of decreased concentrations of ST and IGF-I on growth and onset of puberty in beef heifers.

Six-month-old Angus (A) and Charolais (C) heifers (n = 43) were used to examine the effects of active immunization against GRF on concentrations of ST and IGF-I, growth, body composition, and onset of puberty. Heifers were born in the autumn of 1988 from primiparous and multiparous dams; treatments were initiated while calves were nursing their dams. Primary immunizations were administered at 184 f 7 d of age (d 0 of experiment) by injecting 1.5 mg of GRF-(1-29)-Gly-Gly-CysNH2 conjugated to 1.5 mg of human serum albumin (GRFi) or 1.5 mg of human, serum albumin alone (HSAi) (Armstrong et al., 199Ob). Booster immunizations of .5 mg of antigen (approximately 5 ml) were given on d 62, 92, 153, and 251 of the study. Injections were administered (s.c.) at five or six locations between the vulva and udder. Primary and booster doses were emulsified in complete and incomplete Freund’s adjuvant7, respectively. Within each breed, heifers were blocked by sire and age and randomly assigned to GRFi or HSAi (A-GRFi, n = 12; A-HSAi, n =12; CGRFi, n = IO; C-HSAi, n = 9). Heifers were weaned on July 10,1989 (d 47 of experiment). Before weaning, heifers and their dams were allowed to graze grass-legume pasture. At weaning, heifers were assigned to open-sided pens with slotted floors (30 m x 9 m), each equipped with 12 individual, electronic Calan gate feeders6. Heifers were given ad libitum access to a sorghum silageconcentrate diet formulated to support BW gains of .91 kg/d (NRC,1984); individual feed intakes were recorded daily. Heifers were acclimated to the diet from d 47 to 61. Heifers remained in open-sided pens until 1 mo after onset of puberty or termination of the study (d 379 or 18.5 mo of age). Heifer BW was recorded at 2-wk intervals from d 62 until termination of the study. A 126d interval (d 62 to 188) was used for determination of effects of treatment on feed intake, ADG,and feedgain ratio. This time interval was used because a high percentage of HSAi heifers had attained puberty and were removed from the pens with individual feeders by d 188. Hip height was recorded at d 0, 47, 132, 209, and 251. Fat thickness between the 1Ith and 12th ribs was measured on d 56, 111, 167, and 257 using an Aloka Model 210 DX

7~igmachemical CO., st. ~ ~ u i sMO. , *American Calan Inc., Northwood, NH.

Downloaded from https://academic.oup.com/jas/article-abstract/69/12/4914/4704867 by Iowa State University user on 16 January 2019

Introduction

4916

SIMPSON ET A L

Downloaded from https://academic.oup.com/jas/article-abstract/69/12/4914/4704867 by Iowa State University user on 16 January 2019

(ST, NEFA, INS), evacuated glass tubes" (P4, IGF-I), or glass tubes containing sodium fluoride" (GLU). Samples were stored at 4'C until centrifugation at 2,500 x g for 30 min the day of (p4, GLU, IGF-I, T4) or the day after (ST,INS,NEFA) collection. Serum or plasma was stored at -2O'C until it was analyzed. Assays. Antibody binding of GRF was determined by incubating various dilutions of serum (1:lOO to 1:32,000) with [125r]GRF (1-29)-N& as previously described (Armstrong et al., 1990b). Antibody binding failed to exceed 50% at any dilution in several AGRFi heifers; thus, binding was expressed as percentage of binding at a 1:2,000 dilution of serum. Serum concentration of P4 was determined by a solid-phase radioimmunoassay12. Average intra- and interassay CV were 8.3 and 12.7%, respectively, for 11 assays. Sensitivity, defined as 90% of total binding, was .09 nglml. Serum concentration of ST was determined by the procedure described by Armstrong and Spears (1988). The average intra- and interassay CV for seven assays were 11.8 and 9.8%, respectively. Sensitivity of the assay, as described previously, was .85 ns/rnl. Serum concentration of LH was determined by procedures described by Armstrong and Spears (1988). Average intra- and interassay CV for four assays were 3.6 and 5.5%, respectively. Sensitivity of the LH assay was .38 ng/rnl. Serum INS concentration was analyzed via the procedures of Hales and Randle (1963) with modifications described by Jones et al. (1991). Average intra- and interassay CV were 11.7 and 19.1%,respectively, for two assays. Assay sensitivity, as previously described, was 1.1 pSJ/ml. Plasma glucose was analyzed by an automated glucose oxidase methodI3. Serum NEFA were determined via an enzymatic, colorimetric method14. Serum T4 concentrations were determined using a solid-phase radioimmunoassay12. Intraassay CV was 6.9%. Assay sensitivity was 10.0 nglml. Serum concentrations of IGF-I were determined as detailed by Houseknecht et al. (1988) with modifications (Holland et al., 1988; Jones et al., 1991). Average intra- and interassay CV 9Corometrics Medical Systems, Inc., Wallinsford, CT. '%me ~ ~ ~ ~ ~ t CO., u r Denver. i n g CO. were 9.2 and 9.1%. respectively, for two 'lVacutainu Tube,Becton-DicLinson, Rutherford, NJ. assays. Sensitivity of the assay was 5.0 ng/ml. '2Diagnostic Products Corp.. Los Angeles, CA. Statistical Analyses. All analyses were '3Glucose Analyzer 2, Beckman Instruments, Inc.. conducted using GLM procedures (SAS, B r a , CA. 1985). Growth and performance variables were '%AKO Chemicals USA, Inc.. Dallas, TX.

real-time ultrasound9. Internal pelvic area was determined on d 168 using a Rice pelvimeter'o. On d 0, 62, 69, 78, 97, 153, and 160, single blood samples were collected from all heifers (n = 43) via jugular or coccygeal venipuncture for determination of GRF antibody levels. Serum IGF-I and thyroxine (T4) were analyzed in samples collected on d 0, 62, and 69. Beginning on d 62, single blood samples were collected weekly from all heifers for analysis of concentration of progesterone (P4). Puberty was defined as the fist day on which serum P4 exceeded 1 ng/rnl for two consecutive weeks. Samples were obtained at least 4 wk beyond the onset of puberty. On d 78, blood samples were collected from 28 prepubertal heifers (A-GRFi, n = 7; AHSAi, n = 7; C-GRFi, n = 7; C-HSAi, n = 7) at 15-min intervals for 8 h to monitor effects of immunization on hormones and metabolites. All samples from the first five h (-1 to 4 h from feeding) of the collection period were analyzed for ST. Plasma glucose (GLU), serum insulin (INS), and nonesterifid fatty acids (NEFA) were determined in samples collected at -30, 0, 30, 60,120, 240, and 360 min from time of feeding. All samples from the entire 8-h collection period were analyzed for serum LH concentration. At the conclusion of the 8-h period, heifers (n = 28) were administered (i.v.) an endogenous opioid agonist @-Ala2, MePhe4, Met(O)5-ol)-enkephalin (FK33-824; Roemer et al., 1977; 5 p&g of BW) to further evaluate the success of GRF neutralization. Blood samples were obtained at 10-min intervals from 0 to 30 min and at 15-min intervals from 30 to 120 min from time of FK33-824 administration. Re- and postinfusion samples were analyzed for concentration of ST. Cannulas were inserted into the jugular vein approximately 18 h before initiation of sampling. During collection of blood samples, heifers were maintained in individually partitioned, stationary chutes (183 cm x 76 cm). Blood samples were collected in glass tubes

4917

ACTIVE IMFvWNEA'TION AGAINST GRP IN HEIFERS

Results

Antibody binding against GRF was higher (P < .Ol) in GRFi than in HSAi heifers on each day in which GRF antibodies were measured, except d 0 (Table 1). Antibodies against GRF were present on d 62 before the first booster, percentage of binding increased (P < .05) in response to the first booster (d 62 vs 69). Percentage of binding (averaged across d 62, 69, 78, 97, 153, and 160) was greater (P c .05) in C-GRFi (58.5 f 5.4) than in A-GRFi (36.6 f 5.6) heifers; percentage of binding was similar in C- and A-HSAi heifers. Mean concentrations of serum ST on d 78 were lower (P < .01) in GRFi than in HSAi heifers (Figure 1). Across treatments, concentrations of ST were lower (P < .05) during the 2nd h after feeding than during the hour before feeding (treatment x time, P > .2). Concentrations of IGF-I were lower ( P < .01) in GRFi than in HSAi heifers on d 62 and 69 but not on d 0 (Table 2). On d 62 and 69, the main effect of breed, but not the treatment x breed interaction, contributed (P < .01) to variation in concentrations of IGF-I (data not shown). Mean concentration and basal ST (ng/ ml) and frquency (peaksL5 h) of ST release from -1 to 4 h from feeding on d 78 were lower (P < -05) in GRFi than in HSAi heifers (Table 2). Amplitude of ST episodes tended to be lower (P = .08) in GRFi than in HSAi heifers.

TABLE 1. PPRCENTAGE OF BINDING OF GROWTH HORMONE-RELEASING FACTOR (GRp>(1-29)NH2 IN S E R W FROM ANGUS AND CHAROLAIS HEIFERS AGAINST GRP-(l-29)-GLY-GLY-CYSNH2-HUMAN SERUM ALBUMIN (GRFi) OR HUMAN SERUM ALBUMIN ( H S A i ) Treatment

Day of exuerimentb 0

62 69 78 97 153 160

GRFi

HSAi

3.2 22.7 53.7 48.5 51.5 42.0 60.9

2.9 f .4 4.9 f .5 10.1 f .6 1.0 f .8 .8 f .2 .6 f .2 .3 f .1

f .6' f 4.3d f 4.9 f 4.0d f 4.9d f 5.1d f4 9

at 1:2,000 dilution. b a y 0 = 184 f 7 d of age. Runary immunizations were administered on d 4 booster immunizations were given on d 62, 92, 153, and 251. 'Mean f SE. kRFI different than HSAi heifers (P < .01).

Downloaded from https://academic.oup.com/jas/article-abstract/69/12/4914/4704867 by Iowa State University user on 16 January 2019

analyzed using the model of treatment, breed, and treatment x breed Age and BW on d 0 were used as covariates; covariates not contributing to variation (P > .05) were deleted from subsequent analyses. The effects of treatment on the percentage of heifers attaining puberty were analyzed using CATMOD (SAS, 1985). The model included treatment, breed, and treatment x breed. Hormone and metabolite data from d 78 were analyzed using a model containing treatment (GRFi vs HSAi), breed (A vs C), treatment x breed, heifer within treatment x breed, time, treatment x time, breed x time, and treatment x breed x time. The effects of treatment, breed, and treatment x breed were tested using the heifer within treatment x breed mean square as the error term. Frequency (pulsesi5 h) and amplitude (ng/ ml) of release of ST were determined for individual heifers during a 5-h period. Frequency (pulses/8 h) and amplitude (ng/ml) of release of LH were determined for individual heifers during an 8-h period. The following criteria were used to define a ST or LH pulse were: 1) the pulse had to occur within 30 min of the previous nadir, 2) the pulse had to be at least 50% greater than the previous nadir, and 3) the amplitude of the pulse had to be greater than the sensitivity of the assay. Basal ST was determined as the mean of all samples after those associated with an episode of release of ST had been deleted. Because approximately 55% of GRFi heifers failed to attain puberty during the study, retrospective analyses were conducted on several variables of interest within the GRFi group. Angus- and C-GRFi heifers that attained puberty by 12.9 mo of age (d 209 of experiment, n = 9) were compared to A-GRFi and C-GRFi heifers that failed to attain puberty by 18 mo of age (d 364 of experiment, n = 13). The model for the retrospective analyses consisted of pubertal status at 12.9 mo of age, breed, and pubertal status x breed. Age and BW on d 0 were used as covariates; covariates not contributing to variation (P > .OS) were deleted from subsequent analyses. Performance variables, antibody binding, and concentrations of hormones and metabolites from d 0 to 78 were used in initial analyses; however, results will be reported only for variables in which pubertal status or the breed x pubertal status interaction was found to be significant (P < .05).

4918

SIMPSON ET AL.

was smaller (P < .01) in GRFi than in HSAi heifers (148 f 4 vs 168 f 5, respectively). Charolais heifers had greater (P < .01) hip height and pelvic area and lower (P < .05) backfat thickness than A heifers on each day measured, C had greater (P < .03) BW than A on d 104 and thereafter (data not shown). The breed x treatment interaction was not significant for hip height, pelvic area, backfat thickness, or BW. During the 126-d interval in which feed intake and gain variables were quantified, feed intake (kg of DWd) and ADG (kg/d), respectively, were lower (P< .05) in GRFi (7.47 f .3 and .71 f .03) than in HSAi (8.44 f .4 and .95 f .02)heifers. Feed:gain ratios were higher (P < .01) in GRFi (10.63 f .3) than in HSAi (8.85 f .4) heifers. The percentage of heifers reaching puberty by 12.9 mo of age (d 209) was lower (Pe .OS) in GRFi (40.9, 9/22) than in HSAi (81.0. 17/ 21) heifers. At 18.5 mo of age (d 379) only 45.5% (10/22) of the GRFi heifers had reached puberty, compared with 100% (21121) of the HSAi group (P < .05) (Figure 4). Neither breed nor breed x treatment contributed (P > .2) to the percentage of heifers attaining puberty on any day analyzed.

TABLE 2. CONCENTRATIONS OF INSULIN-LIKE GROWTH FACTOR I (IGF-I) ON DAYS 0, 62, AND 69 AND CHARACTERISTICS OF SOMATOTROPIN (ST) ON DAY 78 OF EXPERIMENT IN ANGUS AND CHAROLAIS HEIFERS IMMUNIZED AGAINST GROWTH HORMONE-RELEASING FACTOR ( G W (1-29)-GLY-GLY-CYS-NH2-HuMAN SERUM ALBUMIN (GRFi) OR HUMAN SERUM ALBUMIN ( H S A i ) ~~~

Treatment

HSAi

Item

GRFi

IGF-I, ng/ml na Day Ob Day 62 Day 69

104 f 5 f

22 46

f 7

41

f 5

106 101 112

P>F 21 f 5 f 4 f 4

S P

na Mean, ng/ml Basal,n@ld Amplitude, ngimle Frequency,peaksi.5 h

14 3.3 f .1

5.6 f

3.0 f

4.3

-1 3.7 f 1.1 .9 f .3

.6 .01

.01

-

14 f

-

.2 .2

.o 1 .05

5.9 f .5

.os

f

.01

2.3

.2

'Number of heifers from which blood samples were collected (IGF-I, 1 sample/heifeq ST, 21 sampleaeifer). k a y of experiment, day 0 = 184 It 7 d of age. cSamples collected on d 78. dSamples associated with episodes of ST release were omitted. %Difference between peak and previous nadir. fMean ? SE.

Downloaded from https://academic.oup.com/jas/article-abstract/69/12/4914/4704867 by Iowa State University user on 16 January 2019

Concentrations of ST before and after FK33-824 administration on d 78 were lower (P< .01) in GRFi than in HSAi heifers (Figure 2). A treatment x time interaction (P < .01) was detected; ST increased after FK33-824 in HSAi but not in GRFi heifers. Plasma GLU concentrations (mg/dl) on d 78 were similar (P > .2) in GRFi and HSAi heifers (64 f .9 vs 68 f .4, respectively). Concentrations of INS (pU/ml) on d 78 were lower (P < .05) in GRFi (2.2 f .l) than in HSAi (3.8 f ,2) heifers. Serum NEFA (pEq/ liter) levels on d 78 were not different (P> .8) between GRFi (133 f 5) and HSAi (138 f 6) heifers. Concentrations of T4 (ndml) in GRFi heifers were similar (P> .l) to those in HSAi heifers on d 0, 62, and 69 (88 f 4, 80 f 4, and 74 f 4 vs 82 f 4, 86 3~ 4, and 81 f 2, respectively). Mean BW, hip height, and fat thickness are depicted in Figure 3. By d 84, BW was lower (P < .OS) in GRFi than in HSAi heifers; hip height was lower (P e .01) in GRFi than in HSAi heifers by d 132. Fat depth was greater (P < .05) in GRFi than in HSAi heifers by d 167. Initial BW and hip height (d 0) and backfat thickness (d 56) were similar between treatments (P > .1). Pelvic area (cm2) on d 168

4919

AcIlvE IMMuNIzAnON AGAINST GRF IN HEIFERS

'I

10

-*

I

:

0

- GRFi -~HSAi

were greater (P < .05) in A- and C-GRFi heifers that reached puberty by 12.9 mo of age than in those that did not reach p u b e q before 18 mo of age. A breed x pubertal status interaction was detected because serum IGF-I on d 62 and 69 was greater in pubertal than in nonpukrtal A-GRFi heifers but not in C-GRFi heifers. Serum IGF-I on d 0, 62, and 69 was similar (P > .l)in pubertal and nonpubertal HSAi heifers (data not shown). Discussion

Active immunization against GRF allows a specific lesion in the hypothalamo-pituitary axis without surgery and without removal of other pituitary hormones. Immunization apparently neutralizes GRF before it binds to hypophyseal GRF receptors and stimulates release of ST. orowing A n g u s and Charolais heifers were effectively immunized against GRF, as evidenced by elevated antibody binding of GRF (Table l), suppressed serum concentrations of ST and IGF-I (Table 2 and Figure l), reduced rate of gain and skeletal growth, decreased feed efficiency, increased fat thickness (Figure 3), and failure of an opioid agonist to increase ST (Figure 2). Effects of GRFi on the aforementioned vari-

"1

60

:

I

I

10

0

20

40

60

80

100

120

Minutes from FK33-824 Minutes from Feeding

Figure 2. Concenhations of somatotropin (ST) (mean f

SE)from 0 to 120 min from i.v. injection (5 pg/kg of BW) Figure 1. Mean (*SE) CoIlCentratiom of somaiobnpin (ST)from -60 to 240 min from feeding in Angus aed Charolais heifers. ' XI against growth hormonereleasing factor (GRF)-(l-29)-Gly-Gly-Qs-NHz-haman serum aIbumin(GRFi n = 14) or human serum albumin ( H S A i , n = 14) on d 78. Serum ST was lower (P c .OS) in GRFi than in HSAi heifers across times. Concentrations of ST from 60 to 120 min were lowcr (P c .M)than ST at -60 to 0 min from feeding in HSAi but not in GWi heifers.

of an exfogemus opioid peptide agonist, @AQ,M.SJIX ,~et(o)bi. pK33-824) in Angos and Charolais h e i f e r s imtmmmd * againstgrowth h o n n o n s r e l ~ factor (GRF)-(l-29)-Gly-Gly-CysNH2-human sawn albumin (GWb n = 14) or human serum albumin (HSAi, n = 14) on d 78. Serum ST was lower (P c .OS) in GRFi than in HSAi heifers at all *~CS following the injection O f -3-824. mea^ ST increased (P< .05) following thc injection of PK33-824in HSAi but not in GRFi heifers.

Downloaded from https://academic.oup.com/jas/article-abstract/69/12/4914/4704867 by Iowa State University user on 16 January 2019

Serum concentrations of LH on d 78 were similar (P > .2) in GRFi and HSAi heifers (1.16 f .04 vs .93 f .02, respectively). Frequency (pulsesB h) and amplitude (ng/ml) of LH release were also similar between GRFi and HSAi heifers (1.2 f .26 vs 1.7 f .37,P > .2 and 1.6 f .24 vs 1.4 f .21, P > .8, respectively). Mean concentrations of LH on d 78 were higher (P < .01) in C (1.36 f .04)than in A (.73 f .02)heifers; however, no breed differences (P > .7)were found regarding the fresuency and amplitude of LH release. Of the variables considered, antibody binding, ADG from d 0 to 62, and IGF-I concentrations on d 0. 62, and 69 were related (P < .05) to whether GRFi heifers had reached puberty by 12.9 mo of age (pubertal) or had not reached puberty by 18 mo of age (nonpubertal; Table 3). Angus GRFi heifers that attained pubew by 12.9 mo of age had a lower (P < .05) percentage of binding on d 69 than nonpubertal A-GRFi heifers. In contrast, percentage of binding was similar in pubertal and nonpubertal C-GRFG heifers (breed x pubertal status interaction, P < .05). In both breeds, GRFi heifers that attained puberty gained at a higher (P < .01) rate from d 0 to 62 than those not attaining puberty. On d 0 (before immunization), concentrations of IGF-I

4920

SIMPSON ET AL.

130i

0

1004 0

1'

.8

,7. ,7

r,"

0

.6-

120

60

'

, 60

A

GRFi

.--t

'

-

180

'

'

120

180

300

240

240

-

(percentage of binding) to active immunization. Reduced overall and basal concentrations of ST and lowered concentrations of IGF-I were likely the effect of GRFi on pulsatile release of ST. Pulsatile release of ST was severely reduced in GRFi heifers; episodic release of ST was absent in 6 of 14 heifers. In heifers with ST episodes detected. amplitude of ST release tended to be lower in GRFi than in HSA heifers. Active immunization against GRF has been previously shown to suppress episodic release of ST and lower serum IGF-I concentrations in growing steers (Trout and Schanbacher, 1990), lactating cows (Lloyd et al., 1988), and cyclic gilts (Armstrong et al., 199Ob). The observation that GRFi decreased basal ST is in contrast to the absence of a decrease in basal ST in GRFi gilts (Armstrong et al., 1990b). The effects of GRFi on overall and basal concentrations of ST, frequency of ST release (Plouzek et al., 1988), and growth rate (Anderson, 1977) were similar to those after hypophyseal stalk-transection in beef calves. This experiment demonstrated that active immunization against GRF delayed the onset of puberty in Angus and Charolais heifers Figure 4). The effect of GRFi on puberty was similar in Angus and Charolais heifers; a breed x treatment interaction was not detected. As would be expected with decreased concentrations of ST. GRFi heifers deposited more fat

' 300

GRFi GRFi HSAi HSAi

.5-

Q)

0 M

m

.4-

.3-

0..1

1

0

60

120

180

240

300

Day of Experiment

Figure 3. Mean (rt SE) of hip height (upper panel), BW (middle panel), and fat thickness measured between 11th and 12th ribs (lower panel) in Angus and Charolais heifers immunized against growth hormone-releasing factor (GRF)-(1-29)-Gly-Gly-Cy~-NHz-h~ albumin (GRFi, n = 22) or human serum albumin ( H S A i , n = 21). Body weight and hip height were lower (P < .05) and fat thickness was greater (P < .05) in GRFi than in HSAi heifers by d 84, 132. and 167, respectively.

80

140

200

260

320

380

Day of Experiment

Figure 4. Cumulative percentage of Angus and Charolais heifers immunized against growth hormonereleasing factor (GRF)-(1-29)-Gly-Gly-Cys-NH~h~ sem albumin (GRF, n = 22) or human serum albumin (HSAi n = 21) reaching puberty from d 80 to 379 of the experiment. A lower percentage (P< .OS) of GWi than of HSAi heifers reached puberty on d 124 through d 379 (10.2 through 18.5 mo of age). Neither breed nor the breed x treatment interaction contributed (P> .l) to variation in the cumulative percentage attaining puberty.

Downloaded from https://academic.oup.com/jas/article-abstract/69/12/4914/4704867 by Iowa State University user on 16 January 2019

ables were similar in Angus and Charolais heifers (breed x treatment, P > .1). Increased antibody binding and decreased IGF-I concentrations, as well as reduced ADG were detectable before the first booster immunization (administered on d 62 of experiment). In the present study, percentage binding of [1254GRF was higher than that reported for ruminants (Lloyd et al., 1988; Trout and Schanbacher, 1990) but lower than that reported for pigs (Armstrong et al., 1990b). These data also provide evidence for breed differences in the magnitude of response

492 1

ACTIVE IMMUNIZATION AGAINST GRF IN HEIFERS

Breed and pubertal status

~~

n

Percenta e o f b e

a.k3gd

IGF-I, @de Day 0 Day 62 Dav 69

P>Fb

Charolais

Angus

Pubertal

Item

No~ubertal

Nonoubertal

Pubertal

PUB BRD P x B

~

5 27 f Sfg .87 f .Mi 130 94 75

f 9' f I@ f I@

52 98 32 31

7 f gh .66 f .07

f4 f 7h f gh

4 70 f 68 .86 f .05 111

35 32

f8 rt

9

f 5g

67

6 f 3g .64 f 05

83 31 33

f9 f Sg f 48

.1 .01

-01

.8

.04 .9

.01 .01 .01

.03

.4

.01

.03 .01

.02

Weifers not pubertal by 12.9 mo did not reach puberty before 18 mo of age. %ode1 included PUB = pubertal status, BRD = breed, and PUB x BRD. POI IGF-I covariates weight and age at d 0 contributed to variation (P < .05).

on d 0 of experiment only, the

'Percentage binding of ['251]GRF-(1-29)NH~ in serum at 1:2,000 dilution. dADG from d 0 to 62 of experiment; d 0 = 184 f 7 d of age. 1GF-I = insulin-like growth factor I. fMeaa f SE. gh4eans with different superscripts within breed differ (P < .05). 'Pubertal vs nonpubertal across breeds (P < .05).

than HSAi heifers (Figure 3). Based on observational data in humans, increased fat deposition would be expected to be associated with a decrease in age at puberty (Frisch, 1984). However, Brooks et al. (1985) and Grass et al. (1982) concluded that a minimum percentage of body fat was not a prerequisite for puberty in the heifer. The present data clearly demonstrate that factors independent of body fat triggered the onset of puberty in GRFi heifers. Previous studies have shown that pubertal onset is delayed in heifers when ADG is decreased (Wiltbank et al., 1966; Arije and Wiltbank, 1971; Grass et al., 1982; Granger et al., 1989; McShane et al., 1989a). In the present experiment, the possibility cannot be ruled out that pubertal onset in GRFi heifers was delayed because ADG and feed intake were decreased (critical BW was not reached). However, the delay in onset of puberty observed in GRFi heifers was much more prolonged (> 18 mo of age) than that reported in the aforementioned studies. Furthermore, Short and Bellows (1971) reported that heifers fed to achieve different rates of gain attained puberty at different weights. This would imply that a critical BW for onset of puberty is not applicable to heifers.

The relationship between ADG and puberty, however, is complicated by the observation that, across breeds, GRFi heifers that reached puberty by d 209 (12.9 mo of age) had greater ADG from d 0 to 62 than GRFi heifers that did not reach puberty by d 364 (18 mo of age, Table 3). This 6 2 d time span coincided with approximately 7 to 8 mo of age; this interval included the last 47 d before weaning and the 1 5 4 acclimation period to the silageconcentrate diet. Preweaning rate of gain has been shown to have a significant effect on age of puberty in heifers (Wiltbank et al., 1966; Arije and Wiltbank, 1971). Conceivably, an aberration in metabolism early in life (7 or 8 mo of age) could affect follicular development in a manner that results in a delay in puberty. In A-GRFi heifers, pubertal status was related to a lower response to GRFi as evidenced by percentage of binding, ADG !?om d 0 to 62, and serum IGF-I concentrations on d 62 and 69 (Table 3). Thus, A-GRFi heifers that reached puberty by 12.9 mo of age may not have had antibody levels sufficient to delay puberty. In contrast, in C-GRFi heifers, antibody binding or IGF-I on d 62 and 69 were not related to pubertal status. This breed difference cannot be explained from the present experiment; however, Jones et al.

Downloaded from https://academic.oup.com/jas/article-abstract/69/12/4914/4704867 by Iowa State University user on 16 January 2019

TABLE 3. RETROSPEcIlVE COMPARISONS BASED ON PUBERTAL STATUS AT 12.9 MONTHS OF AGE" IN ANGUS AND CHAROLAIS HEIFERS IMMUNIZED AGAINST GROWTH HORMONE-RELEASING FACTOR (GRF)-( 1-29)-GLY-GLY-CYS-NH2-HUMAN SERUM ALBUMIN

4922

SIMesON ET AL..

higher serum IGF-I concentrations when 6 mo of age (d 0 of experiment). In A-GRFi heifers only, serum IGF-I was greater on d 62 and 69 in heifers that were to reach puberty by 12.9 mo of age than in heifers that did not. This observation may be directly related to antibody concentrations in A-GRFi heifers, because binding followed an inverse pattern to that of IGF-I (Table 3). Jones et al. (1991) reported an increase in fi-equency of pulsatile LH release and an increase in serum IGF-I before puberty in A, but not in C heifers. Possible effects of ST and(or) IGF-I (endocrine or paracrine) on the hypothalamo-pituitary axis are unknown. However, GRF immunization did not affect the mean concentration or release pattern of LH on d 78 of this study. The observation that serum INS levels were lower in GRFi heifers is in agreement with reports that INS increased in response to ST treatment in heifers (EXsemann et al., 1986; McShane et ai., 1989b), steers (Eisemann et al., 1989), daj.cows (McDowell et al., 1987), and ewes (Hart et al., 1985). Lipogenesis and glucose oxidation were elevated in adipose tissue from sows that were actively immunized against GRF (Coffey et al., 1989). The decreased levels of ST and INS and increased fat depth in GRFi heifers likely reflected an increase in sensitivity of adipose tissue to the effects of INS. The lower concentrations of INS measured in GRFi heifers should not be overlooked as a possible factor in the delayed onset of puberty in those heifers. Armstrong and Britt (1987) reported a decrease in fresuency of LH release and lowered concentrations of serum INS in nutritionally anestrous gilts. Administration of INS increased ovulation rate in cyclic gilts when given at a dose sufficient to cause a decrease in GLU (Cox et al., 1987). Matamoros et al. (1990) reported that exogenous INS reduced the rate of atresia in medium-sized follicles from d 17 to 19 of the estrous cycle in gilts. Flowers et al. (1989) found that gilts that received excess dietary energy before estrus had increased serum INS concentrations, increased fresuency of LH release, and increased ovulation rates. Also, ovulation rate in energydeprived beef heifers was increased by exogenous INS (Harrison and Randel, 1986). Collectively, these data indicate that INS may be a mediator through which alterations in metabolism affect activity of the

hypothalamic-hypophyseal-ovarian axis.

Downloaded from https://academic.oup.com/jas/article-abstract/69/12/4914/4704867 by Iowa State University user on 16 January 2019

(1991) reported that IGF-I increased before puberty in Angus but not in Charolais heifers. Precise mechanisms through which GRF, ST, and(or) IGF-I, acting via an endocrine, paracine. or autocrine avenue, may influence the onset of puberty have not been fully elucidated. Puberty is delayed in ST-deficient children and this is corrected with ST replacement (Goodman et d., 1968). Administration of recombinant human ST hastened age at first ovulation in rhesus monkeys (Wilson et al., 1989); however, administration of ST did not hasten the onset of puberty in A n g u s heifers (McShane et al., 1989a). Arsenijevic et al. (1989) reported a transient delay in sexual maturation in male rats that were passively immunized against GRF, which was associated with decreased synthesis and release of FSH. Advis et al. (1981) demonstrated that reduced serum ST delayed the onset of puberty in female rats, mediated in part through a decrease in the number of ovarian LH receptors. Thus, circulating levels of ST may play an important role, likely permissive in nature, in the endocrine events that precede puberty. The possibility exists that GRF itself may exert an effect on ovarian function and thus onset of puberty. Plasma GRF levels increase during puberty in humans (Argente et al., 1986). Moretti et al. (1990) demonstrated that the addition of GRF enhanced FSH-induced cyclic adenosine 5'-monophosphate production, progesterone biosynthesis, ammame activity, and LH receptor formation in rat granulosa cells. Any effect of ST on the Occurrence of puberty may be mediated through IGF-I. The observation that immunization against GRF resulted in decreased serum IGF-I and a delay in the onset of puberty gives rise to speculation that serum, and probably ovarian, IGF-I concentrations may be a factor in the eventual onset of puberty. Echtemkamp et al. (1990) reported a positive correlation (R = .69) between IGF-I concentrations in serum and follicular fluid. Insulin-like growth factor I may exert an autocrine or paracrine effect on granulosa cell differentiation and function (Hammond et al., 1988; Schams et al., 1988). Cara et ai. (1990) found that IGF-I enhances LH binding to rat theca-interstitial cells by increasing the LH binding capacity of those cells. In the present study, A- and C-GRFi heifers that attained puberty by 12.9 mo of age had

ACTIVE IMMUNIZATION AGAINST GRF IN HEDFERS

4923

Body composition of beef heifers at puberty. Theriogelmplicatlons

Active immunization against growth hormone-releasing factor and the concomitant decrease in serum somatotropin, insulin-like growth factor I, and insulin concentrations resulted in a delay in puberty of growing heifers. Feed intake, feed efficiency, rate of gain, and skeletal growth were also decreased in heifers that were immunized against growth hormone-releasing factor. Immunization against growth hormone-releasing factor resulted in greater fat thickness, yet puberty was delayed. Literature Cited Advis. J. P., S. Smith White and S. R. Ojeda. 1981. Activation of growth hormone short loop negative feedback delays puberty in the female rat. EndocMology 108:1343. Anderson. L. L. 1977. Development in calves and heifers after hypophysial stalk transection or hypophysectomy. Am. J. Physiol. 232FA97. Argente, J., D. Evain-Brion. A. MunoliVia, P.Garnier, M. Hernandez and M. Donuadieu. 1986. Relationship of plasma growth hormone-releasing hormone levels to pubertal changes. J. Clin. Endocrinol. & Metab. 63: 680. Arije, G. F. and J. N. Wiltbank. 1971. Age and weight at puberty in Hereford heifers. J. Anim. Sci. 33:401. Armstrong, J. D. and J. H. Britt. 1987. Nutritionally-induced anestrus in gilts: Metabolic and endocrine changes associated with cessation and resumption of estrous cycles. J. Anim. Sci. 65508. Armstrong, J. D., K. L. Esbenshade, M. T. Coffey, E. Heimer, R Campbell, T. Mowles and A. Felix. 1990a. Opioid control of growth hormone in the suckled sow is primarily mediated through growth hormone releasing factor. Domest. Anim. Endocrinol. 7191. Armstrong,J. D., K. L. Esbenshade, J. L. Johnson, M. T. Coffey. E. Heimer, R. M. Campbell, T. Mowles and A. Felix. 1990b. Active immunization of pigs against growth hormone-releasingfactor: Effect on concentrations of growth hormone and insulin-like growth factor. J. Anim. Sci. 68:427. Armstrong, J. D. and B. H. Johnson. 1989. Agonists of endogenous opioid peptides suppress LH,and stimu-

nology 24:235. Byerley. D. J., R B.Staigmiller, J. G. Baardinelli and R. E. Short. 1987. Pregnancyratesof beef heifers bred either on puberal or third estrus. J. Anim. Sci. 65545. Cara, I. F., J. Fan,J. Azzarello and R. L. Rosesield. 1990. Insulin-like growth factor-1 enhances luteinizing hormone binto rat ovarian theca-interstitial cells. I. Clin. Invest. 86560. Coffey, M. T., J. D. Armstrong and K.L. Esbenshade. 1989. Effect of active immunization against growth hormone releasing factor (GRF') on metabolism of sows. J. Anim. Sci. 67(Suppl. 1):197 (Abstr.). Cox, N. U, M.J. Stuart, T. G. Nthen, W.A. Bennett and H. W. Miller. 1987. Enhancement of ovulationrateingilts by increasing d i e m energy and administering insulin during follicular growth. J. Anim. Sci. 64507. Echternkamp. S. E., L. J. Spicer, K. E. Gregory, S. F. Canning and J. M. Hammond. 1990. Concentrations of insulin-like growth factor-1 in blood and ovarian follicular fluid of cattle selected for twins. Biol. Reprod. 43% E i s e m a ~ ,J. H.. A. C. Hammond, D. E. Bauman, P. J. Reynolds, S. N. McCutcheon, H. F. Tyrrell and G. L. Haaland. 1986. Effect of bovine growth hormone administration 011 metabolism of growing Hereford heifers: protein and lipid metabolism and plasma concentrations of metabolites and hormones. J. Nu&. 116:2504. Eisemann, J. H..A. C. Hammond, T. S. Rumsey and D. E. Bauman. 1989. Nitrogen and protein metabolism and metabolites in plasma and urine of beef steers treated with somatotropin. J. Anim. Sci. 67:105. Flowers, B., M.J. Martin. T. C. Cantley and B. N. Day. 1989. Endocrine changes associated with a dietary-induced inmase in ovulation rate (flushing) in gilts. I. Anim. Sci. 67771. Frisch,R E. 1984.Body fat, puberty and fertility. Biol. Rev. 59:161. Goodman,H. G., M. M. Grumbach and S.L. Kaplae 1968. Growth and growth hormone. II. A comparison of isolated growth-hormone deficiency and multiple pituitary-hormone deficiencies in 35 patients with idiopathic hypopituitary d w a r f i i . N. Engl. J. Med. 27857. Granger, A. L., W.E.Wyatt, W. M. Craig, D. L. Thompson, Jr. and F. G. Hembry. 1989. Effects of breed and wintering diet on growth, puberty and plasma concentrations of growth hormone and insulin-like growth factor-1 in heifers. Domest. Anim.Endocrinol. 6253. Grass, J. A., P.J. Hansen, J. J. Rutledge and E. R Hnuser.

Downloaded from https://academic.oup.com/jas/article-abstract/69/12/4914/4704867 by Iowa State University user on 16 January 2019

late cortisol and growth hormone during the follicular The lack of ST response to FK33-824 in phase in heifers. J. Endocrinol. 121:ll. GRFi heifers provides further evidence that Armstrong, J. D. and J. W. Spears. 1988. Intravenous endogenous opioid peptide stimulation of ST is administration of ionophores in ruminants: Effects on mediated via GRF. Others have reported a lack metabolism independent of the m e n . J. Anim. Sci. 66:1807. of ST response to FK33-824 in GRFi cows (Lloyd et al., 1988) and gilts (Armstrong et al. Armstrong, J. D. and J. W. Spears. 1991. Changes in growth hormone and luteinizing hormone following acute or (1990b). Infusion of FK33-824 increased ST in chronic administrationof an opioid agonist, FK33-824, heifers during the follicular phase of the in wethers. J. Anim. Sci. 69:774. estrous cycle (Armstrong and Johnson, 1989) Arsenijevic, Y.. W. B.Wehenberg, A. Conz, A. Eshkol, P. C. Sizonenko and M. L. Aubert. 1989. Growth and in wethers (Armstrong and Spears, 1991). hormone (GH)deprivation induced by passive immuConversely, administration of an opioid antagnization against rat GH-releasingfactor delays sexual onist decreased ST in the pig (Schneller et al., mahvdton in the male rat. Endocrinology 124:3050. 1987; Armstrong et al., 1990a). Brooks, A. L., R.E. Morrow and R. S. Youngquist. 1985.

4924

SIMPSON ET AL. netics of key metabolites in lactating dairy cows at

peak and mid-lactation. Aust. J. Biol. Sci. 40:191. McShane, T.M.,K. K.Schillo, J. A. Boling, N. W. Bradley and J. B. Hall. 1989a. Effects of recombinant DNAderived somatotropin and dietary energy intake on development of beef heifers: I. Growth and puberty. J. h i m . Sci. 67:2230. McShane, T. M., K.K. Schillo, M. J. Estieme, J. A. Boling, N. W. Bradley and J. B. Hall. 1989b. Effects of recombinant DNA-derived somatotropin and dietary energy intake on development of beef heifers: II. Concentrations of hormones and metabolites in blood sera. J. Anim. Sci. 672237. Moretti, C., A. Bagnato, N. Solan,G. Frajese and K. J. Catt. 1990. Receptor-mediated actions of growth hormone releasing factor on granulosa cell differentiation. Endocrinology 127:2117. NRC.1984.Nutrient Requirementsof Beef Cattle (6th Ed.). National Academy Press. Washington, DC. Plomk, C. A., J. R Molina, D. L. Hard, J. Rivier, A. e c t s of growth Trenkle and L. L. Anderson. 1988. W hormone-releasing factor and somatostatin on growth hormone secretion in hypophyseal stalk-transected beef calves. Proc. Soc. Exp. Biol. Med. 189:158. Roemcr,D., H.H.Buescha. R. C.Hill, J.Pless, W.Bauer, F. Cardinaux, A. Closse, D. Hauser and R Huguenin. 1977. A synthetic enkephalin analogue with prolonged parenteral and oral analgesic activity. Nature 268547. SAS. 1985. SASBTAT Guide for Personal Computers (6th Ed.). SAS Inst., Inc., Cary, NC. Schams. D., R. Koll and C.H. Li. 1988.Insulin-like growth factor-1 stimulatesoxytocinand progesterone production by bovine granulosa cells in culture. J. Endocrinol. 116:97. Schneller, H. E.,N. M. Cox and M. R. Rainey, Jr. 1987. Effect of naloxone administration to estrogen-treated lactating sows on luteinizing hormone, growth hormone and ovulation, J. Anim. Sci. 65(Suppl. 1):409 (Abstr.). Short,R. E. and R. A. Bellows. 1971. Relationships among weight gains, age at puberty and reproductive performance in heifers. J. Anim. Sci. 32:127. Trout, W.E. and B. D. Schanbacher. 1990. Growthhormone and insulin-like growth factor-I responses in steers actively immunized against somatostatin or growth hormone-releasing factor. J. Endocrinol. 1 L .123. Wilson, M. E.. T. P. Gordon, C. G. Rudman and J. M. Tanner.1989.E f f e c t s of growth hormoneon the tempo of sexualmaturation in female rhesus monkeys. J. Clin. Endocrinol. & Metab. 68:29. Wiltbank, I. N., K. E. Gregory, L.A. Swiger, J. E. Ingalls, J. A. Rothliibexger and R. M. Koch. 1966. Wecu of heterosis on age and weight at pubeay in beef heifers. J. Anim. Sci. 25:744.

Downloaded from https://academic.oup.com/jas/article-abstract/69/12/4914/4704867 by Iowa State University user on 16 January 2019

1982. Genotype x environmental interactions on productive traits of bovine females.I. Age at puberty as influenced by breed, breed of sire, dietary regimen season. J. h i m . Sci. 55:1441. Hales, C. N. and P. J. Randle. 1963. Immunoassay of insulin with insulin-antibodyprecipitate. J. Biochem. 88:137. Hammond, J. M., C. Hsu, J. S. Mondschein and S. F. Canning. 1988. Paracrine and autocrine functions of growth factors in the ovarian follicle. J. Anim. Sci. 66(Suppl. 2):21. Hanison,L. M. and R. D. Randel. 1986. Influence of insulin and energy intake on ovulation rate, luteinizing hormone and progesterone in beef heifers. J. Anim. Sci. 63:1228. Hart,1.C., P.M.E. Cbadwick S.James and A. D. Simmonds. 1985.Effect of intravenousbovine growth hormone or human pancreatic growthhormone-releasing factor on milk production and plasma hormones and metabolites in sheep. J. EndocrinOl. 105:189. Holland, M. D.,K. L. Hossner, G. D. Niswender, T. H. Elsasser and K. G. Odde. 1988. Validation of a heterologous radioimmunoassay for insulii-like growth factor-] in bovine seaum. J. Endouinol. 119: 281. Houseknecht, K. L., D. L. Boggs, D. R. Campion, J. L. Sartin, T. E. Kiser, G. B. Rampacek and H.E. Amos. 1988. Effect of dietary energy source and level on serum growth hormone, insulin-like growth factor 1, growth and body composition in beef heifers. J. Anim. Sci. 66:2916. Jones, E. J., J. D. Armstrong and R. W. Harvey. 1991. Changes in metabolites, metabolic hormones and luteinizinghormone before puberty in Angus, Braford, Charolais, and Simmenlal heifers. J. Anim. Sci. 69: 1607. Kinder, J. E.,M. L.Day and R. J. Kittok. 1987. Endocrine regulation of puberty in cows and ewes. J. Reprod. Fertil (Supp1.)34:167. Lesmeister, J. L., P.J. Burfening and R.L. Blackwell. 1973. Date of first calving in beef cows and subsequent calf production. J. Anim. Sci. 36:l. Lloyd, K. E., J. D. Armstrong, K. L. Esbenshade, E. Heimer, R. Campbell, T.Mowles and A. Felix. 1988. Active immunization of cows against growth hormone releasing factor. J. Anim. Sci. 66(Suppl. 1):388 (Abstr.). Matamoros, I. A., N. M. Cox and A. B. Moore. 1990. Exogenous insulin and additional energy & e t follicular distribution, follicular steroid concentrations, and granulosa cell human chorionic gonadotropin binding in swine. Biol. Reprod. 43:l. McDowell, G. H., I. C. Hart,J. A. Bines, D. B. Lindsay and A. C. Kirby. 1987. Wects of pituitary-derived bovine growth hormone on production parameters and bioki-