Plasma G r o w t h H o r m o n e a n d I n s u l i n D u r i n g E a r l y L a c t a t i o n in C o w s F e d Silage Based Diets R. D. SMITH 1, W. HANSEL, and C. E. COPPOCK Department of Animal Science Cornell University Ithaca, NY 14853 ABSTRACT
meet requirements for maintenance and for the rapid increase in milk production (10, 23). There results a period of negative energy balance during which tissue energy stores are mobilized rapidly (10). Later in lactation, the nutritional status of the animal improves as nutrient intake more closely meets the requirements for maintenance and lactation. Although growth hormone (GH) and insulin play important roles in regulating metabolism during reduced nutrient intake and energy imbalance (32), alterations in the plasma concentrations of these hormones in dairy cows during early lactation have not been studied extensively. Therefore, this study was conducted to monitor changes in blood concentrations of insulin and GH during early lactation and to attempt to relate these changes to the nutritional status of the animal. A second objective was to determine the effect on these hormones of feeding diets in which forage was provided only in silage form and supplemented with liberal amounts of concentrate feeds. Earlier studies suggested that the feeding of corn silage as the sole source of forage together with large amounts of concentrate feeds increased the incidence of ketosis (15, 26, 29) and increased animal mortality (30) as compared with diets containing both dry hay and corn silage and comparable amounts of concentrates.
Peripheral plasma insulin, growth hormone, and blood glucose were determined in 24 dairy cows during early lactation. Blood samples were obtained at 4-day intervals during the first 56 days of experimental lactations 1 and 3 of a 3-lactation experiment. The cows were divided equally among three groups and fed one of the following forage rations for three consecutive lactations: corn silage (18.2 kg/day) plus alfalfa-timothy hay ad libitum; corn silage (18.2 kg/day) plus alfalfa-timothy silage ad Iibitum; or corn silage ad libitum. These diets were continued throughout the intervening dry periods except in the latter group where corn silage intake was limited if individual animals exhibited excessive body weight gain. Four animals in each group received prepartum supplemental concentrates. The remaining four animals received no prepartum concentrate supplementation. Following parturition, concentrates were fed in increasing amounts to a maximum of 14.6 kg/day during the first 56 days of lactation. Plasma insulin, growth hormone, and blood glucose were not affected by forage diet or prepartum supplemental concentrate feeding. Plasma insulin and blood glucose increased and plasma growth hormone decreased with time after parturition. These changes were correlated with nutrient intake which increased during the experiment. Changes in plasma insulin and growth hormone are related to metabolic adjustments during early lactation.
MATERIALS AND METHODS
INTRODUCTION
The high producing dairy cow cannot consume enough nutrients during early lactation to Received April 21, 1975. 1Department of Biochemistry, University of Rochester Medical Center, Rochester, NY 14642.
Eight cows were selected on lactation number from each of three groups of animals previously assigned to one of the following forage diets: corn silage (18.2 kg/day) plus hay ad libitum (H); corn silage (18.2 kg/day)plus hay crop silage ad libitum (HCS); or corn silage ad libitum (CS). The forages were harvested yearly and fed as indicated for three consecutive lactations and intervening dry periods to the H and HCS groups. The CS group was fed corn silage ad libitum during lactation, but silage intake was restricted during the dry
248
GROWTH HORMONE AND INSULIN DURING LACTATION period in individuals exhibiting excessive body weight gain. Corn silage dry matter (DM) ranged from 28 to 36% during the three lactation study. Hay (barn dried) and hay crop silage (37 to 47% DM) were harvested on the same date from alternate strips of the same field of first growth alfalfa (75%)-timothy (25%). One-half (4) of the animals in each group received prepartum supplemental concentrates (16.9% crude protein) while the other half received no supplemental concentrates. Beginning 21 days prior to predicted parturition, concentrates were increased .45 kg per day to a maximum of 1% b o d y weight in the supplemental group. Nonsupplemented animals received 1.8 kg of concentrate feed per day throughout the period. During wk 1 to 8 of lactation, concentrates (16.9% crude protein) were fed in gradually increasing amounts to a maximum of 14.6 kg per day, or less if 10% refusal occurred. In the CS group, .9 kg/day of a soybean meal (50% crude protein)-mineral mixture was substituted for .9 kg of the regular concentrate mix to balance the low protein and mineral content of the corn silage. Additional detailed information concerning rations and feeding procedures is in (4, 25). Blood Sampling
Blood samples were obtained during the first and third lactation on treatment by jugular puncture at 4-day intervals beginning on the day of parturition and continuing until 56 days postpartum. All samples were collected between 1100 and 1230 h (6 to 7 h after feeding) as rapidly as possible to minimize the effects of stress associated with blood sampling. Blood samples were collected in tubes containing sodium citrate and were cooled immediately on ice. The plasma was separated within 1 h by centrifugation at 4 C and stored at - 2 0 C until analyzed. Hormone Assays
Radioimmunoassay techniques were validated and used to determine the plasma concentrations of insulin and growth hormone. Insulin was measured using an insulin immunoassay kit (Amersham/Searle) with bovine insulin (Lot 795372, 24.1 U/rng, Eli Lilly C o . ) a s
249
the standard. Dilutions of bovine plasma produced inhibition curves parallel to those produced by the standard hormone preparation diluted in the assay buffer. Recoveries of known amounts of standard insulin added to plasma ranged from 90 to 104%. In 11 assays, the coefficient of variation within assay for repetitive determinations in the same samples averaged 5.6 -+ .6% (mean -+ SE). The coefficient of variation between assays was less than 6% for pooled plasma samples having insulin concentrations ranging from 9.7 to 40/.tU/ml. Plasma GH concentrations were determined by means of the dextran-coated charcoal technique (28), with anti-sheep GH supplied by A. Trenlde, and bovine growth hormone (B1003B, Wilhelmi) for iodination and the preparation of standards. The potency of this GH preparation was 1.4 IU/mg. GH was iodinated at 2 to 3 wk intervals with the chloramine T method (11). The iodinated preparation was purified by column chromatography (Sephadex G-IO0, 1 x 22 cm) immediately prior to use in the assay. By this method, inhibition curves produced by assaying dilutions of bovine plasma were parallel to those when increasing concentrations of standard GH were assayed. Recovery of known amounts of GH added to plasma ranged from 90 to 100%. Coefficients of variation between assays were 9.2, 9.3, and 10.3% for plasma pools averaging 8.7, 13.1, and 32 ng/GH per ml, respectively. The mean within assay coefficient of variation for 21 assays was 6.1 -+ .9%. Blood glucose determinations were made using" Glucostat reagents purchased from Worthington Biochemicals. Statistical Analysis
The data were grouped into three time periods /rod subjected to analysis of variance (13). Means were compared by Sheffe's procedure (9). Simple linear correlations were determined on ungrouped data. R ESU LTS
The data were collected during experimental lactation 3. Similar plasma hormone concentrations and changes therein were observed during lactation 1. As previously reported (4, 5), forage diet did not affect nutrient intake or milk production. Prepartum concentrate supJournal of Dairy Science Vol. 59, No. 2
250
SMITH ET AL.
]
Hay + Corn Silage
]
H
Grass Silage + Corn Silage
]
O
CornSilage 22
14
Mean= SEM n=8
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I
18
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plementation did not affect plasma hormones or glucose. Data were, therefore, pooled within forage treatments. Insulin in the cows fed silage as the sole source of forage tended to be lower than that in the cows consuming corn silage plus dry hay (Fig. 1). While these differences were not significant (P>.10), an increase in plasma insulin with time after parturition was noted in all forage groups (Fig. 1). When data from all groups were pooled within times, plasma insulin throughout
t h e first
56 days of lactation (Table 1). Hasma insulin during the first 20 days of lactation was lower (P
meet requirements for maintenance and for the rapid increase in milk production (10, 23). There results a period of negative energy balance during which tissue energy stores are mobilized rapidly (10). Later in lactation, the nutritional status of the animal improves as nutrient intake more closely meets the requirements for maintenance and lactation. Although growth hormone (GH) and insulin play important roles in regulating metabolism during reduced nutrient intake and energy imbalance (32), alterations in the plasma concentrations of these hormones in dairy cows during early lactation have not been studied extensively. Therefore, this study was conducted to monitor changes in blood concentrations of insulin and GH during early lactation and to attempt to relate these changes to the nutritional status of the animal. A second objective was to determine the effect on these hormones of feeding diets in which forage was provided only in silage form and supplemented with liberal amounts of concentrate feeds. Earlier studies suggested that the feeding of corn silage as the sole source of forage together with large amounts of concentrate feeds increased the incidence of ketosis (15, 26, 29) and increased animal mortality (30) as compared with diets containing both dry hay and corn silage and comparable amounts of concentrates.
Peripheral plasma insulin, growth hormone, and blood glucose were determined in 24 dairy cows during early lactation. Blood samples were obtained at 4-day intervals during the first 56 days of experimental lactations 1 and 3 of a 3-lactation experiment. The cows were divided equally among three groups and fed one of the following forage rations for three consecutive lactations: corn silage (18.2 kg/day) plus alfalfa-timothy hay ad libitum; corn silage (18.2 kg/day) plus alfalfa-timothy silage ad Iibitum; or corn silage ad libitum. These diets were continued throughout the intervening dry periods except in the latter group where corn silage intake was limited if individual animals exhibited excessive body weight gain. Four animals in each group received prepartum supplemental concentrates. The remaining four animals received no prepartum concentrate supplementation. Following parturition, concentrates were fed in increasing amounts to a maximum of 14.6 kg/day during the first 56 days of lactation. Plasma insulin, growth hormone, and blood glucose were not affected by forage diet or prepartum supplemental concentrate feeding. Plasma insulin and blood glucose increased and plasma growth hormone decreased with time after parturition. These changes were correlated with nutrient intake which increased during the experiment. Changes in plasma insulin and growth hormone are related to metabolic adjustments during early lactation.
MATERIALS AND METHODS
INTRODUCTION
The high producing dairy cow cannot consume enough nutrients during early lactation to Received April 21, 1975. 1Department of Biochemistry, University of Rochester Medical Center, Rochester, NY 14642.
Eight cows were selected on lactation number from each of three groups of animals previously assigned to one of the following forage diets: corn silage (18.2 kg/day) plus hay ad libitum (H); corn silage (18.2 kg/day)plus hay crop silage ad libitum (HCS); or corn silage ad libitum (CS). The forages were harvested yearly and fed as indicated for three consecutive lactations and intervening dry periods to the H and HCS groups. The CS group was fed corn silage ad libitum during lactation, but silage intake was restricted during the dry
248
GROWTH HORMONE AND INSULIN DURING LACTATION period in individuals exhibiting excessive body weight gain. Corn silage dry matter (DM) ranged from 28 to 36% during the three lactation study. Hay (barn dried) and hay crop silage (37 to 47% DM) were harvested on the same date from alternate strips of the same field of first growth alfalfa (75%)-timothy (25%). One-half (4) of the animals in each group received prepartum supplemental concentrates (16.9% crude protein) while the other half received no supplemental concentrates. Beginning 21 days prior to predicted parturition, concentrates were increased .45 kg per day to a maximum of 1% b o d y weight in the supplemental group. Nonsupplemented animals received 1.8 kg of concentrate feed per day throughout the period. During wk 1 to 8 of lactation, concentrates (16.9% crude protein) were fed in gradually increasing amounts to a maximum of 14.6 kg per day, or less if 10% refusal occurred. In the CS group, .9 kg/day of a soybean meal (50% crude protein)-mineral mixture was substituted for .9 kg of the regular concentrate mix to balance the low protein and mineral content of the corn silage. Additional detailed information concerning rations and feeding procedures is in (4, 25). Blood Sampling
Blood samples were obtained during the first and third lactation on treatment by jugular puncture at 4-day intervals beginning on the day of parturition and continuing until 56 days postpartum. All samples were collected between 1100 and 1230 h (6 to 7 h after feeding) as rapidly as possible to minimize the effects of stress associated with blood sampling. Blood samples were collected in tubes containing sodium citrate and were cooled immediately on ice. The plasma was separated within 1 h by centrifugation at 4 C and stored at - 2 0 C until analyzed. Hormone Assays
Radioimmunoassay techniques were validated and used to determine the plasma concentrations of insulin and growth hormone. Insulin was measured using an insulin immunoassay kit (Amersham/Searle) with bovine insulin (Lot 795372, 24.1 U/rng, Eli Lilly C o . ) a s
249
the standard. Dilutions of bovine plasma produced inhibition curves parallel to those produced by the standard hormone preparation diluted in the assay buffer. Recoveries of known amounts of standard insulin added to plasma ranged from 90 to 104%. In 11 assays, the coefficient of variation within assay for repetitive determinations in the same samples averaged 5.6 -+ .6% (mean -+ SE). The coefficient of variation between assays was less than 6% for pooled plasma samples having insulin concentrations ranging from 9.7 to 40/.tU/ml. Plasma GH concentrations were determined by means of the dextran-coated charcoal technique (28), with anti-sheep GH supplied by A. Trenlde, and bovine growth hormone (B1003B, Wilhelmi) for iodination and the preparation of standards. The potency of this GH preparation was 1.4 IU/mg. GH was iodinated at 2 to 3 wk intervals with the chloramine T method (11). The iodinated preparation was purified by column chromatography (Sephadex G-IO0, 1 x 22 cm) immediately prior to use in the assay. By this method, inhibition curves produced by assaying dilutions of bovine plasma were parallel to those when increasing concentrations of standard GH were assayed. Recovery of known amounts of GH added to plasma ranged from 90 to 100%. Coefficients of variation between assays were 9.2, 9.3, and 10.3% for plasma pools averaging 8.7, 13.1, and 32 ng/GH per ml, respectively. The mean within assay coefficient of variation for 21 assays was 6.1 -+ .9%. Blood glucose determinations were made using" Glucostat reagents purchased from Worthington Biochemicals. Statistical Analysis
The data were grouped into three time periods /rod subjected to analysis of variance (13). Means were compared by Sheffe's procedure (9). Simple linear correlations were determined on ungrouped data. R ESU LTS
The data were collected during experimental lactation 3. Similar plasma hormone concentrations and changes therein were observed during lactation 1. As previously reported (4, 5), forage diet did not affect nutrient intake or milk production. Prepartum concentrate supJournal of Dairy Science Vol. 59, No. 2
250
SMITH ET AL.
]
Hay + Corn Silage
]
H
Grass Silage + Corn Silage
]
O
CornSilage 22
14
Mean= SEM n=8
j
I
18
6
a a.
4
fi
1 21 40
1-- 20
:.:.1 12
:-:::1 :.:.~
o
~0
:.:-:1 .:.:-I
8
41--56
K
i!iii
]]!]1
6
plementation did not affect plasma hormones or glucose. Data were, therefore, pooled within forage treatments. Insulin in the cows fed silage as the sole source of forage tended to be lower than that in the cows consuming corn silage plus dry hay (Fig. 1). While these differences were not significant (P>.10), an increase in plasma insulin with time after parturition was noted in all forage groups (Fig. 1). When data from all groups were pooled within times, plasma insulin throughout
t h e first
56 days of lactation (Table 1). Hasma insulin during the first 20 days of lactation was lower (P
