S e r u m Prolactin and G r o w t h H o r m o n e Responses to T h y r o t r o p i n Releasing H o r m o n e in Postpubertal Cattle I D. T. VINES ~, E. M. CONVEY, and H. A. TUCKER Animal Reproduction Laboratory Department of Dairy Science Michigan State University East Lansing48824 ABSTRACT
INTRODUCTION
Serum prolactin increased 9 to 20 and growth hormone 2.5 to 4 times following administration of thyrotropin releasing hormone in 6 heifers during an estrous cycle, in 26 heifers during pregnancy, and in 16 cows during lactation. Stage of the estrous cycle or pregnancy did not affect quantity of prolactin or growth hormone released after administration of thyrotropin releasing hormone. There was a tendency for the quantity of prolactin released by thyrotropin releasing hormone to be greater at 2 mo than at 4, 6, 8, or 10 mo of lactation. During lactation, growth hormone release after thyrotropin releasing hormone was greatest at 2 too. Prolactin release after thyrotropin releasing hormone was 6 to 16 times greater in summer than in spring or fall in pregnant heifers and approximately 3 times greater in spring, summer, and fall than during the winter in lactating cows. Season did n o t affect release of growth hormone after thyrotropin releasing hormone. Season of year more importantly affects capacity of the anterior pituitary to release prolactin than stages of the estrous cycle, pregnancy, or lactation. On the other hand, season does not affect induced release of growth hormone by thyrotropin releasing hormone. There is, however, an increased capacity of the anterior pituitary to release growth hormone during early lactation, coincident with maximal milk yield.
Prolactin (PRL) and growth hormone (GH) have been implicated as part of a complex of hormones which are essential for mammary growth, lactogenesis, and maintenance of lactation (8). Tashjian et al. (30) observed that thyrotropin releasing hormone (TRH) increased PRL and suppressed GH release from cloned rat pituitary tumor cells. Subsequently, Smith and Convey (29) demonstrated that TRH directly affected bovine pituitary cells cultured in vitro to release PRL and GH. Bowers et al. (6) and Jacobs et al. (13) demonstrated release of PRL in vivo following TRH administration in humans. Release of PRL following TRH also occurred in mature bulls (7, 33), cows (7, 16), prepubertal heifers (35), sheep (11), and rats (4). In addition, GH concentrations in serum increased following TRH in lactating cows (7) and prepubertal heifers (35). The present investigation was to determine if the capacity of the anterior pituitary to release PRL and GH was altered during various physiological states. Accordingly, we determined PRL and GH release following TRH administration to cattle at various stages of the estrous cycle; at 3, 6, and 9 mo of pregnancy; and at various stages of lactation.
MATERIALS AND METHODS Blood Sampling
Received June 27, 1977. 1Published with the approval of the Director of the Michigan Agricultural Experiment Station as Journal Article No. 8125. This research was supported in part
by USPHS Grant AM-15899 and HD-09883. 2Texas Agricultural Extension Service, Texas A & M University System, Overton 75684. 1977 J Dairy Sci 60:1949-1957
A polyvinyl cannula was inserted into a jugular vein of each animal 1 day prior to experimentation. Blood samples were collected at intervals before and after TRH. To condition animals to blood sampling and thereby to establish stable baseline values for PRL, blood samples were collected and discarded (not assayed for hormones) at 15-min intervals for 2 to 3 h prior to start of experimentation. Thereafter, blood samples were collected as in Table 1. Sera were prepared from the blood
1949
1950
VINES ET AL.
TABLE 1. Time (minutes) of blood sampling relative to thyrotropin releasing hormone (TRH) administration at time zero. Experiment
Pre-tre~ment
Post4reatment
1 and2 3
-15,-10,-5,0 -30,-15,-5,0
4,6,8,10,15,20,25,30 4,6,8,10,12,14,16,18,20,25,30
samples and stored at - 2 0 C until assayed for PRL (18, 32) and GH (24).
milk produced. Although five cows were dried off prior to the lO-mo of lactation, their data were included in the analysis.
Experiment 1
Six Holstein heifers were housed in individual tie stalls. Heifers were allowed access to an adjacent lot twice daily and observed for signs of standing estrus. All estrous cycles were between 19 and 22 days long. Heifers received IV injections of 33 /~g TRH/IO0 kg body weight according to a randomized, complete block, repeat measurement design on days 0 (estrus), 2, 4, 7, 15, and 18 of the estrous cycle. Three heifers were bled and given TRH beginning on day 0, and the other three were bled and injected initially on day 15 of an estrous cycle. Experiment 2
Twenty-seven pregnant Holstein heifers received IV injections of 33 gg TRH/100 kg body weight according to a three (stage of pregnancy) by three (month) factorial design. Nine heifers each received TRH during November, April, or July with three heifers at each month in either the first, second, or third trimester of pregnancy. However, only two heifers in the first trimester of pregnancy received TRH during July due to loss of a cannula from one heifer. Each heifer received only one injection of TRH. Experiment 3
Sixteen lactating Holstein cows received IV injections of 33 gg TRH/100 kg body weight at 2, 4, 6, 8, and 10 mo of lactation. Each cow received TRH on 2 consecutive days at each stage of lactation. Cows were started on experiment in groups of four during May, September, October, and November. Cows were maintained in stanchions and fed corn silage, alfalfa hay, and a 14% protein concentrate fed at 1 kg/3 kg Journal of Dairy Science Vol. 60, No. 12, 1977
Analyses of Data
Areas under the curve of hormone response were calculated as in (35). Data were analyzed by analysis of variance in Experiment 1. In Experiment 2, analysis of variance and Tukey's statistic were used to compare means of the areas under individual curves of hormone response (17). Analysis of variance and analysis of covariance were used in Experiment 3. Also there were preselected orthogonal contrasts among means of areas under individual curves of hormone response in Experiment 3. In addition, average hormone concentrations at the post-treatment peak and average time (minutes) required to achieve peak hormone concentrations were calculated for each experiment.
R ESU LTS Experiment 1
Baseline concentrations of PRL in serum 15 rain before TRH averaged 6 ng/mt in heifers during the estrous cycle. Maximum concentrations of serum PRL after TRH ranged from 52 to 81 ng/ml, and time required to attain these maximums ranged from 10 to 17 min (Table 2). Days of estrous cycle did not affect (P'>.05) the area of the response curve of PRL after TRH. However, maximum concentrations of PRL after TRH, pre-TRH concentrations and areas under the PRL response curve at days 2 and 4 tended to be less than at other stages of the estrous cycle. Concentrations of GH in serum before TRH averaged 6 ng/ml and increased to maximums of 18 to 24 ng/ml after TRH (Table 3). These maximal concentrations were attained 11 to 15 min after TRH. Days of estrous cycle did not
HORMONE RESPONSE TO THYROTROPIN RELEASING HORMONE
1951
TABLE 2. Change in serum prolactin (PRL) concentrations after injection of thyrotropin releasing hormone (TRH) during the estrous cycle. PRL release after TRH a (ng mF l min)
Day of estrous cycle
Pre-TRH PRL (ng/ml)
Peak PRL after TRH (ng/ml)
Time to peak (min)
X
SE
0 (estrus) 2 4 7 15 18
8 3 3 9 5 6
73 61 52 77 74 81
13 12 10 12 11 17
1191 812 820 1166 1368 1560
408 301 179 169 225 247
alntegrated area of plot of PRL concentrations for 30 min after TRH minus baselines before TRH. Values are means -+ standard errors (n = 6).
a f f e c t ( P > . 0 5 ) m a g n i t u d e o f T R H - i n d u c e d release o f GH. Experiment 2 C o n c e n t r a t i o n s o f P R L in s e r u m p r i o r to T R H averaged 14 n g / m l in p r e g n a n t heifers. A f t e r T R H , m a x i m u m P R L c o n c e n t r a t i o n s in s e r u m ranged f r o m 125 to 184 ng/ml, and t i m e r e q u i r e d to attain these m a x i m u m s was 11 t o 12 min (Table 4). Stage o f p r e g n a n c y did n o t a f f e c t ( P ) . 0 5 ) area u n d e r t h e T R H - i n d u c e d P R L r e s p o n s e curve. However, p r e - T R H conc e n t r a t i o n s , m a x i m u m c o n c e n t r a t i o n s after T R H , and r e s p o n s e - c u r v e areas for P R L following T R H a d m i n i s t r a t i o n t e n d e d to be greater w i t h advancing p r e g n a n c y . R e a r r a n g e m e n t o f these data a c c o r d i n g to m o n t h w h e n samples w e r e c o l l e c t e d r e s u l t e d in areas o f P R L re-
s p o n s e curves w h i c h were greater ( P < . 0 1 ) in July t h a n in N o v e m b e r and April. However, areas in N o v e m b e r a n d April were n o t significantly d i f f e r e n t ( P > . 0 5 ) f r o m each o t h e r (Table 5). Overall, baseline c o n c e n t r a t i o n s o f GH in serum b e f o r e T R H averaged 4 ng/ml. M a x i m u m GH c o n c e n t r a t i o n s in serum after T R H administ r a t i o n were 10 to 12 ng/ml, and t i m e s r e q u i r e d to attain t h e s e values were 9 to 13 min (Table 6). N e i t h e r stage o f p r e g n a n c y (Table 6) n o r m o n t h o f year (Table 5) a f f e c t e d ( P > . 0 5 ) t h e q u a n t i t y o f GH released t o T R H as m e a s u r e d by area u n d e r the r e s p o n s e curve. Experiment 3 Each c o w received T R H on 2 c o n s e c u t i v e days at each stage o f lactation to d e t e r m i n e
TABLE 3. Change in serum growth hormone (GH) concentrations after injections of thyrotropin releasing hormone (TRH) during the estrous cycle. GH release after TRH a (ng m1-1 min)
Day of estrous cycle
Pre-TRH GH (ng/ml)
Peak GH after TRH (ng/ml)
Time to peak (min)
R
SE
0 (estrus) 2 4 7 15 18
5 6 6 7 7 5
24 21 18 20 20 24
12 11 11 11 14 15
283 20O 187 206 193 270
43 96 56 46 44 40
alntegrated area of plot of GH concentrations for 30 rain after TRH, minus baseline before TRH. Values are means ± standard errors (n = 6). Journal of Dairy Science Vol. 60, No. 12, 1977
1952
VINES ET AL.
TABLE 4. Change in serum prolactin (PRL) concentrations after injection of thyrotropin releasing hormone (TRH) during pregnancy. PRL release after TRH a (ng ml -~ min)
Month of pregnancy
Pre-TRH PRL (ng/ml)
Peak P RL after TRH (ng/ml)
Time to peak (min)
X
SE
3 6 9
12 14 15
125 148 184
12 12 11
2205 2831 3155
1309 1037 1210
alntegrated area of plot of PRL concentrations for 30 min after TRH, minus baselines before TRH. Values are means ± standard errors (n = 8 for heifers at 3-too stage of pregnancy ; n = 9 for heifers at 6- and 9-mo of pregnancy).
possible carry-over effects on s e r u m P R L a n d / or G H c o n c e n t r a t i o n s . However, t h e q u a n t i t y o f P R L released on the 2 c o n s e c u t i v e days did n o t differ ( P > . 0 5 ) ; t h e r e f o r e , data for days 1 a n d 2 were p o o l e d f o r f u r t h e r analysis. Prior t o T R H , P R L c o n c e n t r a t i o n s in s e r u m averaged 16 ng/ml. M a x i m u m P R L c o n c e n t r a t i o n s in s e r u m a f t e r T R H r a n g e d f r o m 202 to 2 8 2 n g / m l at t h e five stages o f l a c t a t i o n , a n d t i m e r e q u i r e d t o a t t a i n t h e s e m a x i m a l values was 12 t o 16 rain (Table 7). Stage o f l a c t a t i o n did n o t a f f e c t ( P > . 0 5 ) q u a n t i t y of P R L released a f t e r T R H . However, areas u n d e r P R L r e s p o n s e curves f o l l o w i n g T R H t e n d e d to be g r e a t e s t a t 2 m o of l a c t a t i o n (Table 7). Stage of p r e g n a n c y did n o t a f f e c t ( P > . 0 5 ) q u a n t i t y o f P R L released
TABLE 5. Change in serum prolactin (PRL) and growth hormone (GH) concentrations after injection of thyrotropin releasing hormone (TRH) into pregnant heifers during various seasons (months).
Month of year November April July
PRL release after TRH a (ng ml -~ min) X SE 455 1193 7097
115 270 1010 b
GH release after TRH a (ng m1-1 min) X SE 121 132 134
24 27 21
alntegtated area of plot of hormone concentrations for 30 rain after TRH, minus baselines before TRH. Values are means -+ standard errors (n -- 9) for heifers during November and April; n = 8 for heifers during July). bGreater (P . 0 5 ) q u a n t i t y o f P R L released f o l l o w i n g T R H . However, m o n t h of y e a r a f f e c t e d ( P < . 0 1 ) q u a n t i t y of P R L released f o l l o w i n g T R H (Table 9). C o n c e n t r a t i o n s o f G H in s e r u m b e f o r e T R H averaged 5 ng/ml. M a x i m u m c o n c e n t r a t i o n s o f GH in s e r u m were 13 t o 24 n g / m l a t t h e five stages of l a c t a t i o n (Table 10). T i m e s r e q u i r e d t o a t t a i n t h e s e m a x i m u m s were 9 to 13 rain. Areas o f r e s p o n s e o f G H a f t e r T R H were n o t d i f f e r e n t ( P > . 0 5 ) o n t h e 2 c o n s e c u t i v e days at each stage o f l a c t a t i o n ; t h e r e f o r e , d a t a were p o o l e d f o r f u r t h e r analysis. One-way analysis o f variance s h o w e d t h a t stage of l a c t a t i o n a f f e c t e d ( P < . 0 5 ) q u a n t i t y of G H released. R e s p o n s e areas f o r GH were greater ( P < . 0 5 ) a t 2-too o f l a c t a t i o n as c o m p a r e d with 4, 6, 8, or 10 m o . Stage o f p r e g n a n c y did n o t a f f e c t ( P > . 0 5 ) q u a n t i t y of G H released; h o w e v e r , stage o f l a c t a t i o n a n d m o n t h o f p r e g n a n c y were correl a t e d closely (r=.83). T h e r e f o r e , a least squares analysis o f covariance was used to a n a l y z e t h e i n d e p e n d e n t effects o f stage of l a c t a t i o n , m o n t h o f p r e g n a n c y , a n d season of year using in t u r n o n e o f these variables as a m a i n e f f e c t a n d t h e o t h e r t w o as covariates (Table 11). All possible c o m b i n a t i o n s were analyzed. However, n e i t h e r t h e m a i n effects o f stage o f l a c t a t i o n nor month of p r e g n a n c y was significant
HORMONE RESPONSE TO THYROTROPIN RELEASING HORMONE
1953
TABLE 6. Change in serum growth hormone (GH) concentrations after injection of thyrotropin releasing hormone (TRH) during pregnancy. PRL release after TRH a (ng m1-1 min)
Month of pregnancy
Pre-TRH GH (ng/ml)
Peak PRL after TRH (ng/ml)
Time to peak (min)
Y~
SE
3 6 9
4 4 4
11 10 12
13 12 9
132 112 142
19 24 28
alntegrated area of plot of GH concentrations for 30 min after TRH, minus baselines before TRH. Values are means -+ standard errors (n = 8 for heifers at 3-mo of pregnancy ; n = 9 for heifers at 6- and 9-mo of pregnancy).
(P~>.05) w h e n t h e o t h e r was used as a covariate. However, GH released to T R H d u r i n g t h e 2 n d m o o f l a c t a t i o n was again n u m e r i c a l l y greater t h a n all o t h e r values. Season o f y e a r did n o t a f f e c t (P2>.05) t h e q u a n t i t y of G H released as m e a s u r e d b y area u n d e r t h e r e s p o n s e curve b y e i t h e r o n e - w a y analysis o f v a r i a n c e or analysis of covariance. DISCUSSION
T h e p r e s e n t s t u d y agrees w i t h n u m e r o u s o t h e r studies (7, 16, 26, 33, 34, 35) d e m o n s t r a t i n g release of P R L a n d GH f o l l o w i n g a d m i n i s t r a t i o n of T R H in cattle. Changes in s e r u m c o n c e n t r a t i o n s o f ovarian steroids t h a t n o r m a l l y o c c u r d u r i n g t h e estrous cycle (36) d o n o t i m p o r t a n t l y a f f e c t basal or T R H - i n d u c e d P R L secretion. T h e s e results agree w i t h t h o s e o f Beck e t al. (3), w h o f o u n d n o d i f f e r e n c e s in s e r u m P R L c o n c e n t r a t i o n s in o v a r i e c t o m i z e d heifers given n o h o r m o n e , estradiol-17/3, proges-
t e r o n e , or a c o m b i n a t i o n of estradiol-17/3 a n d progesterone. In a d d i t i o n , K o p r o w s k i a n d T u c k e r (19) f o u n d n o differences in s e r u m P R L c o n c e n t r a t i o n s d u r i n g t h e n o r m a l e s t r o u s cycle in l a c t a t i n g cows. In total, these d a t a suggest t h a t e s t r o g e n a n d p r o g e s t e r o n e are o f m i n o r i m p o r t a n c e in regulating c o n c e n t r a t i o n s of P R L in s e r u m d u r i n g t h e e s t r o u s cycle. Basal P R L c o n c e n t r a t i o n s in s e r u m increased w i t h a d v a n c i n g p r e g n a n c y in heifers (23), cows (15, 27), a n d ewes (22). In a d d i t i o n , Bates et al. (2) a n d Reece a n d T u r n e r (25) r e p o r t e d t h a t p i t u i t a r y p r o l a c t i n c o n t e n t increased d u r i n g late p r e g n a n c y in cattle. These o b s e r v a t i o n s agree with t h e t e n d e n c y we f o u n d f o r t h e a n t e r i o r p i t u i t a r y to release greater q u a n t i t i e s o f s e r u m p r o l a c t i n a f t e r T R H as p r e g n a n c y a d v a n c e d . Arai a n d Lee (1) r e p o r t e d a gradual decline of P R L c o n c e n t r a t i o n s in s e r u m w i t h a d v a n c i n g p r e g n a n c y in ewes. Davis et al. (10) also f o u n d decreasing or l o w s e r u m P R L c o n c e n t r a t i o n s in ewes d u r i n g a d v a n c i n g p r e g n a n c y , b u t P R L
TABLE 7. Change in serum prolactin (PRL) concentrations after injection of thyrotropin releasing hormone (TRH) during lactation.
Month of lactation
Pre-TRH PRL (ng/ml)
Peak PRL after TRH (ng/ml)
Time to peak (rain)
2 4 6 8 10
22 18 13 12 16
282 202 210 234 241
16 15 12 12 13
PRL release after TRH a (ng ml -~ rain) SE 5199 3521 3743 3790 4724
792 521 575 361 606
alntegrated area of plot of PRL concentrations for 30 min after TRH, minus baselines before TRH. Values are means + standard errors (n = 16). Journal of Dairy Science Vol. 60, No. 12, 1977
1954
VINES ET AL.
TABLE 8. Change in serum prolactin (PRL) and growth hormone (GH) concentrations after injections of thyrotropin releasing hormone (TRH) during seasons. PRL release after TRH a (ng m1-1 mira) Seasons Ma~May June-Aug Sept-Nov Dec-Feb
4274 5836 5009 1782
GH release after TRH a !ng m1-1 rain)
SE
R
SE
368 525 516 244 b
156 139 213 131
29 36 31 29
alntegrated area of plot of hormone concentrations for 30 rain after TRH, minus baselines before TRH. Values are means -+standard errors. bLower (P.05) q u a n t i t y of P R L released following T R H administration, areas under P R L response curves t e n d e d to be greatest at 2-too lactation. This increased pituitary responsiveness during early lactation m a y be associated with lactational p e r f o r m a n c e since milk yields are maxim u m at this time. However, the correlation coefficient b e t w e e n T R H - i n d u c e d release of P R L and average daily milk yield for the 5 days preceding T R H administration within stage of lactation was --.01. Schams and Karg (27) also r e p o r t e d no significant correlation b e t w e e n
TABLE 9. Change in serum prolactin (PRL) concentrations after injection of thyrotropin releasing hormone (TRH) during concurrent lactation, pregnancy, or month of year a. Lactation
Month
PRL release after TRH b (ng m1-1 rain)
2
4622
4
4177
6
4234
8
3304
10
4193
Pregnancy
Month
PRL release after TRH b (ng mt -t rain)
0 1 2 3 4 5 6 7 8
3771 5275 4623 4287 4822 4011 4065 1602 3410
Month of year
Month
PRL release after TRH b (ng ml -l rain)
Jan Feb Mar Apr May June July Sept Oct Nov Dec
1559 1883 5031 3602 4122 6558 5916 7467 4229 3184 2423
aEach variable used as main effect with other two variables serving as covariates. Three separate analyses were performed because of high correlation (r = .83) between stage of lactation and month of pregnancy. bIntegrated area of plot of PRL concentrations for 30 rain after TRH, minus baselines before TRH. Values are means. Journal of Dairy Science Vol. 60, No, 12, 197q
HORMONE RESPONSE TO THYROTROPIN RELEASING HORMONE
1955
TABLE 10. Change in serum growth hormone (GH) concentration after injection of thyrotropin releasing hormone (TRH) during lactation. GH release after TRH a (ng m1-1 rain)
Month of lactation
Pre-TRH GH (ng/ml)
Peak GH after TRH (ng/ml)
Time to peak (min)
X
SE
2 4 6 8 10
6 4 5 4 4
24 13 16 15 14
13 13 11 12 9
264 134 144 147 136
40 b 30 28 37 34
alntegrated area of plot of GH concentrations for 30 rain after TRH, minus baselines before TRH. Values are means +-standard errors (n = 16). bGreater (P
INTRODUCTION
Serum prolactin increased 9 to 20 and growth hormone 2.5 to 4 times following administration of thyrotropin releasing hormone in 6 heifers during an estrous cycle, in 26 heifers during pregnancy, and in 16 cows during lactation. Stage of the estrous cycle or pregnancy did not affect quantity of prolactin or growth hormone released after administration of thyrotropin releasing hormone. There was a tendency for the quantity of prolactin released by thyrotropin releasing hormone to be greater at 2 mo than at 4, 6, 8, or 10 mo of lactation. During lactation, growth hormone release after thyrotropin releasing hormone was greatest at 2 too. Prolactin release after thyrotropin releasing hormone was 6 to 16 times greater in summer than in spring or fall in pregnant heifers and approximately 3 times greater in spring, summer, and fall than during the winter in lactating cows. Season did n o t affect release of growth hormone after thyrotropin releasing hormone. Season of year more importantly affects capacity of the anterior pituitary to release prolactin than stages of the estrous cycle, pregnancy, or lactation. On the other hand, season does not affect induced release of growth hormone by thyrotropin releasing hormone. There is, however, an increased capacity of the anterior pituitary to release growth hormone during early lactation, coincident with maximal milk yield.
Prolactin (PRL) and growth hormone (GH) have been implicated as part of a complex of hormones which are essential for mammary growth, lactogenesis, and maintenance of lactation (8). Tashjian et al. (30) observed that thyrotropin releasing hormone (TRH) increased PRL and suppressed GH release from cloned rat pituitary tumor cells. Subsequently, Smith and Convey (29) demonstrated that TRH directly affected bovine pituitary cells cultured in vitro to release PRL and GH. Bowers et al. (6) and Jacobs et al. (13) demonstrated release of PRL in vivo following TRH administration in humans. Release of PRL following TRH also occurred in mature bulls (7, 33), cows (7, 16), prepubertal heifers (35), sheep (11), and rats (4). In addition, GH concentrations in serum increased following TRH in lactating cows (7) and prepubertal heifers (35). The present investigation was to determine if the capacity of the anterior pituitary to release PRL and GH was altered during various physiological states. Accordingly, we determined PRL and GH release following TRH administration to cattle at various stages of the estrous cycle; at 3, 6, and 9 mo of pregnancy; and at various stages of lactation.
MATERIALS AND METHODS Blood Sampling
Received June 27, 1977. 1Published with the approval of the Director of the Michigan Agricultural Experiment Station as Journal Article No. 8125. This research was supported in part
by USPHS Grant AM-15899 and HD-09883. 2Texas Agricultural Extension Service, Texas A & M University System, Overton 75684. 1977 J Dairy Sci 60:1949-1957
A polyvinyl cannula was inserted into a jugular vein of each animal 1 day prior to experimentation. Blood samples were collected at intervals before and after TRH. To condition animals to blood sampling and thereby to establish stable baseline values for PRL, blood samples were collected and discarded (not assayed for hormones) at 15-min intervals for 2 to 3 h prior to start of experimentation. Thereafter, blood samples were collected as in Table 1. Sera were prepared from the blood
1949
1950
VINES ET AL.
TABLE 1. Time (minutes) of blood sampling relative to thyrotropin releasing hormone (TRH) administration at time zero. Experiment
Pre-tre~ment
Post4reatment
1 and2 3
-15,-10,-5,0 -30,-15,-5,0
4,6,8,10,15,20,25,30 4,6,8,10,12,14,16,18,20,25,30
samples and stored at - 2 0 C until assayed for PRL (18, 32) and GH (24).
milk produced. Although five cows were dried off prior to the lO-mo of lactation, their data were included in the analysis.
Experiment 1
Six Holstein heifers were housed in individual tie stalls. Heifers were allowed access to an adjacent lot twice daily and observed for signs of standing estrus. All estrous cycles were between 19 and 22 days long. Heifers received IV injections of 33 /~g TRH/IO0 kg body weight according to a randomized, complete block, repeat measurement design on days 0 (estrus), 2, 4, 7, 15, and 18 of the estrous cycle. Three heifers were bled and given TRH beginning on day 0, and the other three were bled and injected initially on day 15 of an estrous cycle. Experiment 2
Twenty-seven pregnant Holstein heifers received IV injections of 33 gg TRH/100 kg body weight according to a three (stage of pregnancy) by three (month) factorial design. Nine heifers each received TRH during November, April, or July with three heifers at each month in either the first, second, or third trimester of pregnancy. However, only two heifers in the first trimester of pregnancy received TRH during July due to loss of a cannula from one heifer. Each heifer received only one injection of TRH. Experiment 3
Sixteen lactating Holstein cows received IV injections of 33 gg TRH/100 kg body weight at 2, 4, 6, 8, and 10 mo of lactation. Each cow received TRH on 2 consecutive days at each stage of lactation. Cows were started on experiment in groups of four during May, September, October, and November. Cows were maintained in stanchions and fed corn silage, alfalfa hay, and a 14% protein concentrate fed at 1 kg/3 kg Journal of Dairy Science Vol. 60, No. 12, 1977
Analyses of Data
Areas under the curve of hormone response were calculated as in (35). Data were analyzed by analysis of variance in Experiment 1. In Experiment 2, analysis of variance and Tukey's statistic were used to compare means of the areas under individual curves of hormone response (17). Analysis of variance and analysis of covariance were used in Experiment 3. Also there were preselected orthogonal contrasts among means of areas under individual curves of hormone response in Experiment 3. In addition, average hormone concentrations at the post-treatment peak and average time (minutes) required to achieve peak hormone concentrations were calculated for each experiment.
R ESU LTS Experiment 1
Baseline concentrations of PRL in serum 15 rain before TRH averaged 6 ng/mt in heifers during the estrous cycle. Maximum concentrations of serum PRL after TRH ranged from 52 to 81 ng/ml, and time required to attain these maximums ranged from 10 to 17 min (Table 2). Days of estrous cycle did not affect (P'>.05) the area of the response curve of PRL after TRH. However, maximum concentrations of PRL after TRH, pre-TRH concentrations and areas under the PRL response curve at days 2 and 4 tended to be less than at other stages of the estrous cycle. Concentrations of GH in serum before TRH averaged 6 ng/ml and increased to maximums of 18 to 24 ng/ml after TRH (Table 3). These maximal concentrations were attained 11 to 15 min after TRH. Days of estrous cycle did not
HORMONE RESPONSE TO THYROTROPIN RELEASING HORMONE
1951
TABLE 2. Change in serum prolactin (PRL) concentrations after injection of thyrotropin releasing hormone (TRH) during the estrous cycle. PRL release after TRH a (ng mF l min)
Day of estrous cycle
Pre-TRH PRL (ng/ml)
Peak PRL after TRH (ng/ml)
Time to peak (min)
X
SE
0 (estrus) 2 4 7 15 18
8 3 3 9 5 6
73 61 52 77 74 81
13 12 10 12 11 17
1191 812 820 1166 1368 1560
408 301 179 169 225 247
alntegrated area of plot of PRL concentrations for 30 min after TRH minus baselines before TRH. Values are means -+ standard errors (n = 6).
a f f e c t ( P > . 0 5 ) m a g n i t u d e o f T R H - i n d u c e d release o f GH. Experiment 2 C o n c e n t r a t i o n s o f P R L in s e r u m p r i o r to T R H averaged 14 n g / m l in p r e g n a n t heifers. A f t e r T R H , m a x i m u m P R L c o n c e n t r a t i o n s in s e r u m ranged f r o m 125 to 184 ng/ml, and t i m e r e q u i r e d to attain these m a x i m u m s was 11 t o 12 min (Table 4). Stage o f p r e g n a n c y did n o t a f f e c t ( P ) . 0 5 ) area u n d e r t h e T R H - i n d u c e d P R L r e s p o n s e curve. However, p r e - T R H conc e n t r a t i o n s , m a x i m u m c o n c e n t r a t i o n s after T R H , and r e s p o n s e - c u r v e areas for P R L following T R H a d m i n i s t r a t i o n t e n d e d to be greater w i t h advancing p r e g n a n c y . R e a r r a n g e m e n t o f these data a c c o r d i n g to m o n t h w h e n samples w e r e c o l l e c t e d r e s u l t e d in areas o f P R L re-
s p o n s e curves w h i c h were greater ( P < . 0 1 ) in July t h a n in N o v e m b e r and April. However, areas in N o v e m b e r a n d April were n o t significantly d i f f e r e n t ( P > . 0 5 ) f r o m each o t h e r (Table 5). Overall, baseline c o n c e n t r a t i o n s o f GH in serum b e f o r e T R H averaged 4 ng/ml. M a x i m u m GH c o n c e n t r a t i o n s in serum after T R H administ r a t i o n were 10 to 12 ng/ml, and t i m e s r e q u i r e d to attain t h e s e values were 9 to 13 min (Table 6). N e i t h e r stage o f p r e g n a n c y (Table 6) n o r m o n t h o f year (Table 5) a f f e c t e d ( P > . 0 5 ) t h e q u a n t i t y o f GH released t o T R H as m e a s u r e d by area u n d e r the r e s p o n s e curve. Experiment 3 Each c o w received T R H on 2 c o n s e c u t i v e days at each stage o f lactation to d e t e r m i n e
TABLE 3. Change in serum growth hormone (GH) concentrations after injections of thyrotropin releasing hormone (TRH) during the estrous cycle. GH release after TRH a (ng m1-1 min)
Day of estrous cycle
Pre-TRH GH (ng/ml)
Peak GH after TRH (ng/ml)
Time to peak (min)
R
SE
0 (estrus) 2 4 7 15 18
5 6 6 7 7 5
24 21 18 20 20 24
12 11 11 11 14 15
283 20O 187 206 193 270
43 96 56 46 44 40
alntegrated area of plot of GH concentrations for 30 rain after TRH, minus baseline before TRH. Values are means ± standard errors (n = 6). Journal of Dairy Science Vol. 60, No. 12, 1977
1952
VINES ET AL.
TABLE 4. Change in serum prolactin (PRL) concentrations after injection of thyrotropin releasing hormone (TRH) during pregnancy. PRL release after TRH a (ng ml -~ min)
Month of pregnancy
Pre-TRH PRL (ng/ml)
Peak P RL after TRH (ng/ml)
Time to peak (min)
X
SE
3 6 9
12 14 15
125 148 184
12 12 11
2205 2831 3155
1309 1037 1210
alntegrated area of plot of PRL concentrations for 30 min after TRH, minus baselines before TRH. Values are means ± standard errors (n = 8 for heifers at 3-too stage of pregnancy ; n = 9 for heifers at 6- and 9-mo of pregnancy).
possible carry-over effects on s e r u m P R L a n d / or G H c o n c e n t r a t i o n s . However, t h e q u a n t i t y o f P R L released on the 2 c o n s e c u t i v e days did n o t differ ( P > . 0 5 ) ; t h e r e f o r e , data for days 1 a n d 2 were p o o l e d f o r f u r t h e r analysis. Prior t o T R H , P R L c o n c e n t r a t i o n s in s e r u m averaged 16 ng/ml. M a x i m u m P R L c o n c e n t r a t i o n s in s e r u m a f t e r T R H r a n g e d f r o m 202 to 2 8 2 n g / m l at t h e five stages o f l a c t a t i o n , a n d t i m e r e q u i r e d t o a t t a i n t h e s e m a x i m a l values was 12 t o 16 rain (Table 7). Stage o f l a c t a t i o n did n o t a f f e c t ( P > . 0 5 ) q u a n t i t y of P R L released a f t e r T R H . However, areas u n d e r P R L r e s p o n s e curves f o l l o w i n g T R H t e n d e d to be g r e a t e s t a t 2 m o of l a c t a t i o n (Table 7). Stage of p r e g n a n c y did n o t a f f e c t ( P > . 0 5 ) q u a n t i t y o f P R L released
TABLE 5. Change in serum prolactin (PRL) and growth hormone (GH) concentrations after injection of thyrotropin releasing hormone (TRH) into pregnant heifers during various seasons (months).
Month of year November April July
PRL release after TRH a (ng ml -~ min) X SE 455 1193 7097
115 270 1010 b
GH release after TRH a (ng m1-1 min) X SE 121 132 134
24 27 21
alntegtated area of plot of hormone concentrations for 30 rain after TRH, minus baselines before TRH. Values are means -+ standard errors (n -- 9) for heifers during November and April; n = 8 for heifers during July). bGreater (P . 0 5 ) q u a n t i t y o f P R L released f o l l o w i n g T R H . However, m o n t h of y e a r a f f e c t e d ( P < . 0 1 ) q u a n t i t y of P R L released f o l l o w i n g T R H (Table 9). C o n c e n t r a t i o n s o f G H in s e r u m b e f o r e T R H averaged 5 ng/ml. M a x i m u m c o n c e n t r a t i o n s o f GH in s e r u m were 13 t o 24 n g / m l a t t h e five stages of l a c t a t i o n (Table 10). T i m e s r e q u i r e d t o a t t a i n t h e s e m a x i m u m s were 9 to 13 rain. Areas o f r e s p o n s e o f G H a f t e r T R H were n o t d i f f e r e n t ( P > . 0 5 ) o n t h e 2 c o n s e c u t i v e days at each stage o f l a c t a t i o n ; t h e r e f o r e , d a t a were p o o l e d f o r f u r t h e r analysis. One-way analysis o f variance s h o w e d t h a t stage of l a c t a t i o n a f f e c t e d ( P < . 0 5 ) q u a n t i t y of G H released. R e s p o n s e areas f o r GH were greater ( P < . 0 5 ) a t 2-too o f l a c t a t i o n as c o m p a r e d with 4, 6, 8, or 10 m o . Stage o f p r e g n a n c y did n o t a f f e c t ( P > . 0 5 ) q u a n t i t y of G H released; h o w e v e r , stage o f l a c t a t i o n a n d m o n t h o f p r e g n a n c y were correl a t e d closely (r=.83). T h e r e f o r e , a least squares analysis o f covariance was used to a n a l y z e t h e i n d e p e n d e n t effects o f stage of l a c t a t i o n , m o n t h o f p r e g n a n c y , a n d season of year using in t u r n o n e o f these variables as a m a i n e f f e c t a n d t h e o t h e r t w o as covariates (Table 11). All possible c o m b i n a t i o n s were analyzed. However, n e i t h e r t h e m a i n effects o f stage o f l a c t a t i o n nor month of p r e g n a n c y was significant
HORMONE RESPONSE TO THYROTROPIN RELEASING HORMONE
1953
TABLE 6. Change in serum growth hormone (GH) concentrations after injection of thyrotropin releasing hormone (TRH) during pregnancy. PRL release after TRH a (ng m1-1 min)
Month of pregnancy
Pre-TRH GH (ng/ml)
Peak PRL after TRH (ng/ml)
Time to peak (min)
Y~
SE
3 6 9
4 4 4
11 10 12
13 12 9
132 112 142
19 24 28
alntegrated area of plot of GH concentrations for 30 min after TRH, minus baselines before TRH. Values are means -+ standard errors (n = 8 for heifers at 3-mo of pregnancy ; n = 9 for heifers at 6- and 9-mo of pregnancy).
(P~>.05) w h e n t h e o t h e r was used as a covariate. However, GH released to T R H d u r i n g t h e 2 n d m o o f l a c t a t i o n was again n u m e r i c a l l y greater t h a n all o t h e r values. Season o f y e a r did n o t a f f e c t (P2>.05) t h e q u a n t i t y of G H released as m e a s u r e d b y area u n d e r t h e r e s p o n s e curve b y e i t h e r o n e - w a y analysis o f v a r i a n c e or analysis of covariance. DISCUSSION
T h e p r e s e n t s t u d y agrees w i t h n u m e r o u s o t h e r studies (7, 16, 26, 33, 34, 35) d e m o n s t r a t i n g release of P R L a n d GH f o l l o w i n g a d m i n i s t r a t i o n of T R H in cattle. Changes in s e r u m c o n c e n t r a t i o n s o f ovarian steroids t h a t n o r m a l l y o c c u r d u r i n g t h e estrous cycle (36) d o n o t i m p o r t a n t l y a f f e c t basal or T R H - i n d u c e d P R L secretion. T h e s e results agree w i t h t h o s e o f Beck e t al. (3), w h o f o u n d n o d i f f e r e n c e s in s e r u m P R L c o n c e n t r a t i o n s in o v a r i e c t o m i z e d heifers given n o h o r m o n e , estradiol-17/3, proges-
t e r o n e , or a c o m b i n a t i o n of estradiol-17/3 a n d progesterone. In a d d i t i o n , K o p r o w s k i a n d T u c k e r (19) f o u n d n o differences in s e r u m P R L c o n c e n t r a t i o n s d u r i n g t h e n o r m a l e s t r o u s cycle in l a c t a t i n g cows. In total, these d a t a suggest t h a t e s t r o g e n a n d p r o g e s t e r o n e are o f m i n o r i m p o r t a n c e in regulating c o n c e n t r a t i o n s of P R L in s e r u m d u r i n g t h e e s t r o u s cycle. Basal P R L c o n c e n t r a t i o n s in s e r u m increased w i t h a d v a n c i n g p r e g n a n c y in heifers (23), cows (15, 27), a n d ewes (22). In a d d i t i o n , Bates et al. (2) a n d Reece a n d T u r n e r (25) r e p o r t e d t h a t p i t u i t a r y p r o l a c t i n c o n t e n t increased d u r i n g late p r e g n a n c y in cattle. These o b s e r v a t i o n s agree with t h e t e n d e n c y we f o u n d f o r t h e a n t e r i o r p i t u i t a r y to release greater q u a n t i t i e s o f s e r u m p r o l a c t i n a f t e r T R H as p r e g n a n c y a d v a n c e d . Arai a n d Lee (1) r e p o r t e d a gradual decline of P R L c o n c e n t r a t i o n s in s e r u m w i t h a d v a n c i n g p r e g n a n c y in ewes. Davis et al. (10) also f o u n d decreasing or l o w s e r u m P R L c o n c e n t r a t i o n s in ewes d u r i n g a d v a n c i n g p r e g n a n c y , b u t P R L
TABLE 7. Change in serum prolactin (PRL) concentrations after injection of thyrotropin releasing hormone (TRH) during lactation.
Month of lactation
Pre-TRH PRL (ng/ml)
Peak PRL after TRH (ng/ml)
Time to peak (rain)
2 4 6 8 10
22 18 13 12 16
282 202 210 234 241
16 15 12 12 13
PRL release after TRH a (ng ml -~ rain) SE 5199 3521 3743 3790 4724
792 521 575 361 606
alntegrated area of plot of PRL concentrations for 30 min after TRH, minus baselines before TRH. Values are means + standard errors (n = 16). Journal of Dairy Science Vol. 60, No. 12, 1977
1954
VINES ET AL.
TABLE 8. Change in serum prolactin (PRL) and growth hormone (GH) concentrations after injections of thyrotropin releasing hormone (TRH) during seasons. PRL release after TRH a (ng m1-1 mira) Seasons Ma~May June-Aug Sept-Nov Dec-Feb
4274 5836 5009 1782
GH release after TRH a !ng m1-1 rain)
SE
R
SE
368 525 516 244 b
156 139 213 131
29 36 31 29
alntegrated area of plot of hormone concentrations for 30 rain after TRH, minus baselines before TRH. Values are means -+standard errors. bLower (P.05) q u a n t i t y of P R L released following T R H administration, areas under P R L response curves t e n d e d to be greatest at 2-too lactation. This increased pituitary responsiveness during early lactation m a y be associated with lactational p e r f o r m a n c e since milk yields are maxim u m at this time. However, the correlation coefficient b e t w e e n T R H - i n d u c e d release of P R L and average daily milk yield for the 5 days preceding T R H administration within stage of lactation was --.01. Schams and Karg (27) also r e p o r t e d no significant correlation b e t w e e n
TABLE 9. Change in serum prolactin (PRL) concentrations after injection of thyrotropin releasing hormone (TRH) during concurrent lactation, pregnancy, or month of year a. Lactation
Month
PRL release after TRH b (ng m1-1 rain)
2
4622
4
4177
6
4234
8
3304
10
4193
Pregnancy
Month
PRL release after TRH b (ng mt -t rain)
0 1 2 3 4 5 6 7 8
3771 5275 4623 4287 4822 4011 4065 1602 3410
Month of year
Month
PRL release after TRH b (ng ml -l rain)
Jan Feb Mar Apr May June July Sept Oct Nov Dec
1559 1883 5031 3602 4122 6558 5916 7467 4229 3184 2423
aEach variable used as main effect with other two variables serving as covariates. Three separate analyses were performed because of high correlation (r = .83) between stage of lactation and month of pregnancy. bIntegrated area of plot of PRL concentrations for 30 rain after TRH, minus baselines before TRH. Values are means. Journal of Dairy Science Vol. 60, No, 12, 197q
HORMONE RESPONSE TO THYROTROPIN RELEASING HORMONE
1955
TABLE 10. Change in serum growth hormone (GH) concentration after injection of thyrotropin releasing hormone (TRH) during lactation. GH release after TRH a (ng m1-1 rain)
Month of lactation
Pre-TRH GH (ng/ml)
Peak GH after TRH (ng/ml)
Time to peak (min)
X
SE
2 4 6 8 10
6 4 5 4 4
24 13 16 15 14
13 13 11 12 9
264 134 144 147 136
40 b 30 28 37 34
alntegrated area of plot of GH concentrations for 30 rain after TRH, minus baselines before TRH. Values are means +-standard errors (n = 16). bGreater (P
