Endocrinol.
Japon.
1990, 37(2) , 261-273
Plasma
Growth Hormone, Insulin-Like Growth Factor-I, and Milk Production Responses to Exogenous Human Growth Hormone-Releasing Factor Analogs in Dairy Cows KOICHI
HODATE, TETSU JOHKE, AKIHITO AND SHINICHI OHASHI*
OZAWA
National Institute of Animal Industry, Tsukuba Norindanchi P. O. Box 5, Ibaraki 305 *Research Institute for Polymers and Textiles , Tsukuba-shi, Ibaraki 305
Abstract
Responses of plasma growth hormone (GH) and insulin-like growth factor-I (IGF-I), and milk production to subcutaneous (sc) injection(s) of two synthetic human growth hormone-releasing factor (hGRF) analogs were studied in dairy cows. Two mg of each hGRF analog dissolved in 5ml saline per cow were injected into the shoulder area of each experimental animal, and jugular venous blood samples were collected via an indwelling catheter or by venipuncture. Plasma GH and IGF-I concentrations were measured by radioimmnoassay methods. In dry cows, the mean concentration of plasma GH after a single sc injection of hGRF analogs rose to 22.0-28.3ng/ml at about 5h from 1.41.7ng/ml at 0h (just before injection), and returned to the level before injection after 10-12h. On the other hand, the plasma IGF-I began to increase after a lag of 4-6h following a single injection of hGRF analogs, and reached maximum values of 71.1-89.4ng/ml at 20h from 43.7-46.4ng/ml at 0h. The IGF-I concentration at 24h after a single injection of hGRF analogs was still higher than the value for the dry cows given saline. In lactating cows, the plasma concentration of GH at 2h after daily sc injections of hGRF analogs during 14 consecutive days (an injection period) was higher than those for the lactating cows which received saline. Also, during the injection period, the concentration of IGF-I was higher in the lactating cows which received hGRF analog injections than in the cows which received saline injections. During the last 7 days of the injection period, the administration of hGRF analogs increased the mean milk yield by 11-19% in comparison with those for the saline in jected cows. A positive correlation was observed between the mean plasma IGF-I concentration and the mean milk yield in the lactating cows treated with hGRF analogs throughout the injection and a postinjection (11 consecutive days after cessation of hGRF analog injection) periods. The results demonstrate that a single sc injection of hGRF analogs stimulates both GH release and the circulating level of IGF-I in dry cows, and that daily sc injections of hGRF analogs over 14 days enhance milk production, and plasma GH and IGF-I levels in lactating cows. Received
December
19,
1989
262
HODATE
It is well known that exogenous bovine growth hormone (bGH) increases milk production in dairy cows (Peel and Bauman, 1987). On the other hand, exogenous human growth hormone-releasing factor (hGRF) specifically stimulates GH release in cattle (Johke et al., 1984; Moseley et al., 1984). Furthermore, it has been reported that exogenous hGRF (Enright et al., 1986; Pelletier et al., 1987; Lapierre et al., 1988) or bovine GRF (bGRF; Enright et al., 1988) increased milk production in dairy cows, and these increases in milk production are thought to be mediated by increased endogenous GH via hGRF or bGRF. However, the mechanism in which bGH exerts the galactopoietic effect has not been established. Recently, since evidence that bGH does not act directly on mammary tissue is accumulating (Gluckman et al., 1987), it is postulated that at least part of the galactopoietic effect of bGH may be mediated by a secondary mediator, insulin-like growth factor-I (IGF-I). IGF-I mediates some of the endocrine effects of GH, and its concentration is GH-dependent (Gluckman et al., 1987). In cattle, the circulating level of IGF-I is enhanced in response to exogenous bGH (Davis et al., 1987; Breier et al., 1988; Glimm et al ., 1988; Prosser et al., 1989; Cohick et al., 1989). However, to our knowledge, there is only one report (Enright et al., 1989) which demonstrates the increases in somatomedin C after bGRF infusion. In the present experiments, we studied the effects of subcutaneous (sc) injections of two synthetic hGRF analogs with enhanced or prolonged bGH releasing activity (Hodate et al., 1986 ; Mowles et al., 1987), [D-Ala2]-hGRF(1-29)-NH2 (DAhGRF) and [D-Ala2, Ala15]-hGRF(1-29)-NH2 (A 15-DAhGRF), on circulating levels of GH and IGF-I, and milk yield in dairy cows. Some of the preliminary results of these experiments were previously reported (Hodate et al., 1989).
Endoczinol. April 1990
et al.
Materials
and
Japon.
Methods
Experimental animals Four Holstein dry cows and 16 Holstein lactating cows from the institute herd were used. The dry cows fed at 0830 and 1630h daily. The lactating cows were individually fed corn silage, alfalfa hay cube, and concentrate based on milk production prior to the experiment at 0930 and 1930 h daily in order to meet the nutrient requirements of the Japanese Feeding Standard (Research Council Secretariat of Agriculture, Forestry and Fisheries, 1987). The lactating cows were milked twice daily at 0830 and 1830h.
Preparation of hGRF analogs for injection The hGRF analogs; DAhGRF, an analog with D-Ala substitution in position-2 Ala of hGRF(1-29)-NH2, and A15-DAhGRF, an analog with D-Ala and Ala substitutions in position-2 Ala and -15 Gly of hGRF(1-29)-NH2 respectively, were synthesized by the solid-phase method and purified by HPLC. Two mg of DAhGRF or A15-DAhGRF was dissolved in 5ml sterile saline before use and injected subcutaneously into the shoulder area of each experimental animal at 1130h. Experiment
1:
jection of and circulating Four mean
dry
6 years
weight
either
or
2mg
DAhGRF
per
cow.
for
each
of
early
October.
were
collected
heparin
via
to
after
24h
ples before
were and
and
at
min
intervals
intervals during
during
with
tained
after
until
after
These
chilled
assayed
for
ice,
blood and
for
GH
plasma and IGF-I.
sam-
for 0.5h injection,
before,
at
at at
30-min or
samples
or
blood
intervals saline
hour,
and
centrifugation
samples
from -0.5h
0.5h
DAhGRF
at and
containing
5ml
the first
used out
blood
catheter
1-12h,
12-24h
were
tubes
15-min 24h after
(as A15-
September
venous
intervals during
735kg subcu-
carried
Serial
in-
saline
cows
late
indwelling
at
age,
and/or
centrifuge
injection.
sc
release dry cows
sterile
between
drawn during
in
DAhGRF
Jugular
15-min
injection.
5ml
These
into
GH
injected
treatment
1 week
an
single
mean
were
of
repeatedly intervals
a
of
(bw),
with
control)
of
analogs on level of IGF-I
cows,
body
taneously a
Effects
hGRF
10-
15-min intervals
A15-DAhGRF
were
immediately
samples
were
stored
at -60•Ž
ob-
Vol. 37, No.
2
GH,
IGF-I
AND
LACTATION
Experiment 2: Effects of daily sc injections of DAhGRF on plasma GH and IGF-I levels, and milk production in lactating cows The experiment was carried out over 36 days, which were divided into 3 periods: preinjection (days 0 to 10), injection (days 11 to 24), and postinjection periods (days 25 to 35), between late April and late May. Eight lactating cows (5 years mean age and 687kg mean bw, 182 days after parturition) were divided into two equal groups, control and analog groups. During 14 consecutive days of the injection period, the cows in the control and analog groups received a daily sc injection of 5ml sterile saline and 2mg of DAhGRF, respectively. Jugular venous blood samples from these cows were collected into evacuated tubes containing heparin by venipuncture at 1130h (just before injection) on days 0, 7, 10, 11, 12, 14, 16, 18, 20, 22, 24, 25, 26, 29, 32, and 35, and at 1330h (2h after injection) on days 11, 12, 14, 16, 18, 20, 22, and 24. These blood samples were immediately chilled with ice, and plasma samples were obtained as described for Experiment 1. Milk samples from the cows were collected on days 0, 3, 6, 10, 13, 17, 20, 24, 27, 31, and 35 for composition analysis. Experiment 3: Effects of daily sc injections of A15-DAhGRF on plasma GH and IGF-I levels, and milk production in lactating cows The experiment was carried out as described for Experiment 2 between mid-October and midNovember. Eight lactating cows (4 years mean age, 589 kg mean bw, 185 days after parturition) were divided in the same manner as in Experiment 2. During 14 consecutive days of the injection period, the cows in the control and analog groups received a daily sc injection of 5 ml sterile saline and 2mg of A15-DAhGRF, respectively. Jugular venous blood samples were collected as described for Experiment 2 at 1130h (just before injection) on days 0, 6, 11, 14, 17, 20, 22, 24, 25, 28, 31, and 35, and at 1330h (2h after injection) on days 11, 14, 17, 20, 22, and 24. Milk samples from the cows were collected on days 0, 6, 10, 13, 16, 20, 24, 27, and 34 for composition analysis. Measurements milk components Concentrations
of plasma
GH
and
IGF-I,
and
RESPONSE
plasma
GH
and
IGF-I
263
hGRF
were determined by radioimmunoassay methods (Johke, 1978 ; Ozawa et al., 1989). The IGF-I assay was performed after acid-ethanol extraction of the plasma. The intra- and interassay coefficients of variation for the IGF-I assay were 4.4 and 11.1% respectively. In Experiment 1, as an index of the amount of GH released in response to saline or hGRF analogs, area under the GH response curves (AUC) for 24h after injection were determined. Milk fat, protein, and lactose were measured with an infrared milk analyzer. The t-test was used to assess the significance of the differences in the data (Snedecor and Cochran, 1980).
Results Experiment
1
Effects
of
analogs
on
of
IGF-I
sc
injection
the
dry
mean
are
of
and
from
1.4
and
for and 3-4
to
10-12
saline
rose
0h,
4.75h
respectively. declined, injection
(ng•Emin•Em1-1) mean•}SE)
(10,549.0•}1,636.0) as
were
great
as
The
was
larger
though
the
that
for
AUC
than difference
for
that
for
was
not
significant.
Fig.
1-b
shows
the
concentration
which
saline
the injec-
and
before
(2,540.9•}284.2).
statistically
the
the
5.25
AUC
(P
Japon.
1990, 37(2) , 261-273
Plasma
Growth Hormone, Insulin-Like Growth Factor-I, and Milk Production Responses to Exogenous Human Growth Hormone-Releasing Factor Analogs in Dairy Cows KOICHI
HODATE, TETSU JOHKE, AKIHITO AND SHINICHI OHASHI*
OZAWA
National Institute of Animal Industry, Tsukuba Norindanchi P. O. Box 5, Ibaraki 305 *Research Institute for Polymers and Textiles , Tsukuba-shi, Ibaraki 305
Abstract
Responses of plasma growth hormone (GH) and insulin-like growth factor-I (IGF-I), and milk production to subcutaneous (sc) injection(s) of two synthetic human growth hormone-releasing factor (hGRF) analogs were studied in dairy cows. Two mg of each hGRF analog dissolved in 5ml saline per cow were injected into the shoulder area of each experimental animal, and jugular venous blood samples were collected via an indwelling catheter or by venipuncture. Plasma GH and IGF-I concentrations were measured by radioimmnoassay methods. In dry cows, the mean concentration of plasma GH after a single sc injection of hGRF analogs rose to 22.0-28.3ng/ml at about 5h from 1.41.7ng/ml at 0h (just before injection), and returned to the level before injection after 10-12h. On the other hand, the plasma IGF-I began to increase after a lag of 4-6h following a single injection of hGRF analogs, and reached maximum values of 71.1-89.4ng/ml at 20h from 43.7-46.4ng/ml at 0h. The IGF-I concentration at 24h after a single injection of hGRF analogs was still higher than the value for the dry cows given saline. In lactating cows, the plasma concentration of GH at 2h after daily sc injections of hGRF analogs during 14 consecutive days (an injection period) was higher than those for the lactating cows which received saline. Also, during the injection period, the concentration of IGF-I was higher in the lactating cows which received hGRF analog injections than in the cows which received saline injections. During the last 7 days of the injection period, the administration of hGRF analogs increased the mean milk yield by 11-19% in comparison with those for the saline in jected cows. A positive correlation was observed between the mean plasma IGF-I concentration and the mean milk yield in the lactating cows treated with hGRF analogs throughout the injection and a postinjection (11 consecutive days after cessation of hGRF analog injection) periods. The results demonstrate that a single sc injection of hGRF analogs stimulates both GH release and the circulating level of IGF-I in dry cows, and that daily sc injections of hGRF analogs over 14 days enhance milk production, and plasma GH and IGF-I levels in lactating cows. Received
December
19,
1989
262
HODATE
It is well known that exogenous bovine growth hormone (bGH) increases milk production in dairy cows (Peel and Bauman, 1987). On the other hand, exogenous human growth hormone-releasing factor (hGRF) specifically stimulates GH release in cattle (Johke et al., 1984; Moseley et al., 1984). Furthermore, it has been reported that exogenous hGRF (Enright et al., 1986; Pelletier et al., 1987; Lapierre et al., 1988) or bovine GRF (bGRF; Enright et al., 1988) increased milk production in dairy cows, and these increases in milk production are thought to be mediated by increased endogenous GH via hGRF or bGRF. However, the mechanism in which bGH exerts the galactopoietic effect has not been established. Recently, since evidence that bGH does not act directly on mammary tissue is accumulating (Gluckman et al., 1987), it is postulated that at least part of the galactopoietic effect of bGH may be mediated by a secondary mediator, insulin-like growth factor-I (IGF-I). IGF-I mediates some of the endocrine effects of GH, and its concentration is GH-dependent (Gluckman et al., 1987). In cattle, the circulating level of IGF-I is enhanced in response to exogenous bGH (Davis et al., 1987; Breier et al., 1988; Glimm et al ., 1988; Prosser et al., 1989; Cohick et al., 1989). However, to our knowledge, there is only one report (Enright et al., 1989) which demonstrates the increases in somatomedin C after bGRF infusion. In the present experiments, we studied the effects of subcutaneous (sc) injections of two synthetic hGRF analogs with enhanced or prolonged bGH releasing activity (Hodate et al., 1986 ; Mowles et al., 1987), [D-Ala2]-hGRF(1-29)-NH2 (DAhGRF) and [D-Ala2, Ala15]-hGRF(1-29)-NH2 (A 15-DAhGRF), on circulating levels of GH and IGF-I, and milk yield in dairy cows. Some of the preliminary results of these experiments were previously reported (Hodate et al., 1989).
Endoczinol. April 1990
et al.
Materials
and
Japon.
Methods
Experimental animals Four Holstein dry cows and 16 Holstein lactating cows from the institute herd were used. The dry cows fed at 0830 and 1630h daily. The lactating cows were individually fed corn silage, alfalfa hay cube, and concentrate based on milk production prior to the experiment at 0930 and 1930 h daily in order to meet the nutrient requirements of the Japanese Feeding Standard (Research Council Secretariat of Agriculture, Forestry and Fisheries, 1987). The lactating cows were milked twice daily at 0830 and 1830h.
Preparation of hGRF analogs for injection The hGRF analogs; DAhGRF, an analog with D-Ala substitution in position-2 Ala of hGRF(1-29)-NH2, and A15-DAhGRF, an analog with D-Ala and Ala substitutions in position-2 Ala and -15 Gly of hGRF(1-29)-NH2 respectively, were synthesized by the solid-phase method and purified by HPLC. Two mg of DAhGRF or A15-DAhGRF was dissolved in 5ml sterile saline before use and injected subcutaneously into the shoulder area of each experimental animal at 1130h. Experiment
1:
jection of and circulating Four mean
dry
6 years
weight
either
or
2mg
DAhGRF
per
cow.
for
each
of
early
October.
were
collected
heparin
via
to
after
24h
ples before
were and
and
at
min
intervals
intervals during
during
with
tained
after
until
after
These
chilled
assayed
for
ice,
blood and
for
GH
plasma and IGF-I.
sam-
for 0.5h injection,
before,
at
at at
30-min or
samples
or
blood
intervals saline
hour,
and
centrifugation
samples
from -0.5h
0.5h
DAhGRF
at and
containing
5ml
the first
used out
blood
catheter
1-12h,
12-24h
were
tubes
15-min 24h after
(as A15-
September
venous
intervals during
735kg subcu-
carried
Serial
in-
saline
cows
late
indwelling
at
age,
and/or
centrifuge
injection.
sc
release dry cows
sterile
between
drawn during
in
DAhGRF
Jugular
15-min
injection.
5ml
These
into
GH
injected
treatment
1 week
an
single
mean
were
of
repeatedly intervals
a
of
(bw),
with
control)
of
analogs on level of IGF-I
cows,
body
taneously a
Effects
hGRF
10-
15-min intervals
A15-DAhGRF
were
immediately
samples
were
stored
at -60•Ž
ob-
Vol. 37, No.
2
GH,
IGF-I
AND
LACTATION
Experiment 2: Effects of daily sc injections of DAhGRF on plasma GH and IGF-I levels, and milk production in lactating cows The experiment was carried out over 36 days, which were divided into 3 periods: preinjection (days 0 to 10), injection (days 11 to 24), and postinjection periods (days 25 to 35), between late April and late May. Eight lactating cows (5 years mean age and 687kg mean bw, 182 days after parturition) were divided into two equal groups, control and analog groups. During 14 consecutive days of the injection period, the cows in the control and analog groups received a daily sc injection of 5ml sterile saline and 2mg of DAhGRF, respectively. Jugular venous blood samples from these cows were collected into evacuated tubes containing heparin by venipuncture at 1130h (just before injection) on days 0, 7, 10, 11, 12, 14, 16, 18, 20, 22, 24, 25, 26, 29, 32, and 35, and at 1330h (2h after injection) on days 11, 12, 14, 16, 18, 20, 22, and 24. These blood samples were immediately chilled with ice, and plasma samples were obtained as described for Experiment 1. Milk samples from the cows were collected on days 0, 3, 6, 10, 13, 17, 20, 24, 27, 31, and 35 for composition analysis. Experiment 3: Effects of daily sc injections of A15-DAhGRF on plasma GH and IGF-I levels, and milk production in lactating cows The experiment was carried out as described for Experiment 2 between mid-October and midNovember. Eight lactating cows (4 years mean age, 589 kg mean bw, 185 days after parturition) were divided in the same manner as in Experiment 2. During 14 consecutive days of the injection period, the cows in the control and analog groups received a daily sc injection of 5 ml sterile saline and 2mg of A15-DAhGRF, respectively. Jugular venous blood samples were collected as described for Experiment 2 at 1130h (just before injection) on days 0, 6, 11, 14, 17, 20, 22, 24, 25, 28, 31, and 35, and at 1330h (2h after injection) on days 11, 14, 17, 20, 22, and 24. Milk samples from the cows were collected on days 0, 6, 10, 13, 16, 20, 24, 27, and 34 for composition analysis. Measurements milk components Concentrations
of plasma
GH
and
IGF-I,
and
RESPONSE
plasma
GH
and
IGF-I
263
hGRF
were determined by radioimmunoassay methods (Johke, 1978 ; Ozawa et al., 1989). The IGF-I assay was performed after acid-ethanol extraction of the plasma. The intra- and interassay coefficients of variation for the IGF-I assay were 4.4 and 11.1% respectively. In Experiment 1, as an index of the amount of GH released in response to saline or hGRF analogs, area under the GH response curves (AUC) for 24h after injection were determined. Milk fat, protein, and lactose were measured with an infrared milk analyzer. The t-test was used to assess the significance of the differences in the data (Snedecor and Cochran, 1980).
Results Experiment
1
Effects
of
analogs
on
of
IGF-I
sc
injection
the
dry
mean
are
of
and
from
1.4
and
for and 3-4
to
10-12
saline
rose
0h,
4.75h
respectively. declined, injection
(ng•Emin•Em1-1) mean•}SE)
(10,549.0•}1,636.0) as
were
great
as
The
was
larger
though
the
that
for
AUC
than difference
for
that
for
was
not
significant.
Fig.
1-b
shows
the
concentration
which
saline
the injec-
and
before
(2,540.9•}284.2).
statistically
the
the
5.25
AUC
(P
