Lactation Response of Dairy Cows Receiving Bovine Somatotropin via Daily Injections or in a Sustained-Release Vehicle R. K. MCGUFFEY, H. B. GREEN, R. P. SASSON, and T. H. FERGUSON Lilly Research Laboratories A Division of Eli Lilly and Company PO Box 708 Greenfield, IN 46140
given by daily injections or in a sustained-release vehicle. (Key words: somatotropin, sustained release, lactation performance)
ABSTRACT
Three experiments were conducted to characterize metabolic and milk production responses of dairy cows receiving recombinantly derived bovine somatotropin administered either by daily injection or in a sustained-release vehicle. In Experiment I, somatotropin (25 mg/d) purified by two methods was given by daily injection for 14 d and resulted in 3.5 and 3.8 kg/d more milk than controls. Percentages of fat and total solids in milk were also increased by somatotropin. Eleven hematology indices and 12 metabolites, minerals, and enzyme activities in serum were unaffected by somatotropin. In Experiments 2 and 3, somatotropin was administered in a sustained-release vehicle during an 84-d treatment period. In Experiment 2, administration of 960 mg of somatotropin at 28-d intervals increased milk and SCM yields by 4.1 and 3.3 kg/d compared with yields of controls. There were no significant differences in other production parameters. In Experiment 3, 320, 640, and 960 mg somatotropin were each administered in the sustained-release vehicle at intervals of 14, 21, and 28 d. An uninjected group served as control. Cows receiving somatotropin averaged 3.5 to 5.9 kg/d more milk than controls across all injection intervals. Among doses, milk yield was greater at 960 mg than at 320 or 640 mg. There were no significant differences in milk or SCM among injection groups. These experiments demonstrate the comparable efficacy of somatotropin when
Received February 27, 1989. Accepted October 2, 1989.
1990 J Dairy Sci 73:763-771
INTRODUCTION
Stricker and Grtiter (21) first described the lactogenic property of crude anterior pituitary extracts in rats. The galactopoietic nature of pituitary extracts derived from cattle was demonstrated in a classical set of experiments with lactating dairy cows by Asimov and Krouze (1). Cotes et al. (8) reported somatotropin (ST) was the protein responsible for galactopoiesis in ruminants, not prolactin or adrenocorticotropin. Following that discovery, the galactopoietic nature of ST was demonstrated under a wide range of experimental conditions. Generally. galactopoiesis occurred when 30 to 40 mg ST were administered daily to dairy animals. Duration of treatment with ST ranged from 4 d to 12 wk. Milk yield prior to treatment ranged from 7 to 35 kg/d (7, 13) and was increased by as much as 7.0 kg/d in one long-term study with ST treatment (7). The experiments used several breeds of dairy cattle in all stages of lactation. Rations consisted of only pasture (17), pasture plus concentrate (7), and forage and concentrates fed separately (5, 13) or as complete diets (11, 16). Somatotropin was injected daily with one exception: Machlin (13) injected every 3rd d. The impact of these early studies was realized when Bauman et al. (3) reported comparable galactopoietic activity of ST from pituitary origin and bovine somatotropin (bST) produced by recombinant DNA technology. Relatively large supplies of bST made longer studies possible. Bauman et al. (4) observed as much as a 41 % increase in milk yield in dairy cows when bST was administered by daily injections for 188 d. 763
764
MCGUFFEY ET AL.
Three experiments are described that had objectives of: 1) demonstrating comparable galactopoietic activity of bST purified by methods suitable for laboratory purification and for large-scale production; 2) determining the response of dairy cows to bST administered in a sustained-release vehicle; and 3) determining the dose response of dairy cows to bST administered in a sustained-release vehicle at intervals of 14, 21, and 28 d. MATERIALS AND METHODS
Bovine somatotropin (Somidobove 1) was produced by recombinant DNA technology using Escherichia coli as the host vector. After a suitable growth time, bacteria were isolated and heat-killed. A liquid slurry was made of the bST-containing microorganisms. A bST-enriched fraction was isolated from the slurry and was chromatographed using either DEAESephadex (Method A) or DEAE tris-acryl (Method B) as the solid phase. Method A had been used previously in the laboratory to purify ST (24). Method B allowed chromatography of large quantities of bST-enriched fractions. The first experiment compared the galactopoietic activity of bST isolated by each method to ascertain that the methodology of large scale purification did not alter biological activity. Experiment 2 and 3 used bST derived from Method B. Experiment 1
Twenty-four lactating Holstein cows were assigned to this experiment which consisted of 14 d of pretreatment, 14 d of treatment, and 14 d of posttreatment. Milk production during pretreatment (range 19.6 to 33.7 kg/d), days in milk (DIM) (range 44 to 138), and parity were used to block the cows into eight groups of three. Within each group, cows were randomly assigned to one of three treatments: 1) control, 2) bovine somatotropin purified using DEAESephadex (Method A), and 3) bovine somatotropin purified using DEAE tris-acryl (Method B). Each bST treatment was given by subcuta-
lSomidobove is the proprietary name for rccombinantly derived bovine S0matolropin produced by Eli Lilly and Company (12). Journal of Dairy Science Vol. 73,
No.3. 1990
neous injection in the scapular area at an estimated dose of 25 mg/d monomeric bST. Monomeric purity was estimated from peak height using HPLC and from protein content of the purified bST. Solutions were prepared daily on the day before injection, except for Sunday and Monday injections, which were prepared on Friday. All prepared solutions were refrigerated until used. Cows in the control group received a subcutaneous injection daily of 10 ml of .1 M NH4HC0 3, pH 7.75 (vehicle). Bovine somatotropin purified by Method A was delivered in 30 ml of vehicle, and bST purified by Method B was delivered in 20 ml of vehicle. Based upon HPLC analysis and protein content, the average daily dose of bST was 27.6 ± 3.0 mg (Method A) and 26.8 ± 1.6 mg (Method B). Cows were maintained in individual tie stalls with free access to water. They were exercised for at least 2 h daily. Cows were milked twice daily and production was recorded. Milk was collected for composition determinations on Monday p.m. and Tuesday a.m. before treatments were administered, and on Monday p.m., Tuesday a.m., Thursday p.m., and Friday a.m. during both treatment and posttreatment periods. Fat, protein, lactose, and total solids were determined on a percentage basis using a Berwind infrared milk analyzer (Berwind Instruments, Ltd., Osbaldwick Industrial Estate, Osbaldwick, York, YO BUS. England). Solids-corrected milk was calculated according to Tyrrell and Reid (23). A Foss MilkO-Tester (Foss Electric, Hillerod, Denmark) was used to determine SCc. Cows were fed for ad libitum access twice daily a total mixed ration consisting of 60% forage (three parts com silage, one part chopped alfalfa hay on a DM basis) and 40% concentrate on a DM basis (Table 1). Feed issued and feed refused were recorded daily throughout the experiment. Body weight was determined at the beginning and end of the pretreatment period, at the end of the treatment period, at the end of the treatment period. and at the end of the experiment. Two blood samples (one for whole blood, one for serum) were collected by jugular venipuncture 2 d prior to starting treatment. on the final day of treatment, and on the final day of the experiment. Whole blood was analyzed for leukocytes, erythrocytes, erythrocyte morpholo-
SOMATOTROPIN IN A SUSTAINED-RELEASE VEHICLE TABLE 1. Composition of concentrate and estimates of nutrient and energy content. Ingredient
(%)
Corn, cracked yellow Soybean meal Bet pulp, dried Molasses, cane Limestone Sodium phosphate Trace-mineral salt Sodium bicarbonate Vitamin A + D premix' Vitamin E premix I Selenium premix'
48.50 33.10 10.00 3.50 1.65 1.05 1.00 .75 .30 .05 .05
Dry matter, % Crude protein, % NEI, Mcal/kg Calcium, % Phosphorus, %
89.10 24.00 1.74 .85 .69
'Vitamin A .lJld D premix contained 907,000 IU A, 102,000 IU Dylkg; vitamin E premix contained 9070 IV EI kg; selenium premix contained 200 mg of selenium as sodium selenite.
gy, leukocyte differential. thrombocytes, fibrinogen. hemoglobin, packed cell volume, mean corpuscular volume. mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration. Serum was analyzed for glucose, urea nitrogen, creatinine. potassium, calcium, cholesterol. triglycerides, sodium, chloride, sorbitol dehydrogenase. total bilirubin. and alkaline phosphatase. Data from the treatment and the posttreatment periods were analyzed statistically using the General Linear Model of SAS (18). The model used for each analysis was:
where Yijk is the observed value of a cow during the treatment period, Il is the population mean value, ti is the treatment i (i = 1,2,3), rj is the block j, b is the regression coefficient. (Xijk - X) is the preliminary period deviation for a cow from the mean. and Eijk is the error deviation. Data from whole blood and serum collected in the treatment and posttreatment periods were subjected to one-way analysis of variance. Significance of treatment effects was compared by Dunnett's T·test (20).
765
Experiment 2
Fourteen lactating Holstein cows were assigned to this experiment, which consisted of 14 d pretreatment and 84 d of treatment. Milk production (range 19.5 to 34.5 kg/d) during pretreatment and DIM (range 63 to 246 d) were used to pair cows for random assignment to one of two treatments. Treatment" were either control (no injection) or 960 mg bST in a sustained-release vehicle (SRV; TD-85; Eli Lilly and Company) given at 28-d intervals. The SRV was administered on d 1. 29, and 57 of the treatment period by subcutaneous injection. Injections were given on alternating sides in the scapular region of the animal. Management, housing, milk recording, and feeding were as described for Experiment 1. Cows were weighed on the day before pretreatment and at l4-d intervals thereafter. Production measures were analyzed statistically by analysis of covariance as in Experiment 1 with the exception that block was excluded from the model. Experiment 3
Seventy-two Holsteins (52 multiparous; 20 primiparous) were used to determine lactation response of cows to a dose range of bST (320, 640. 960 mg) given in the SRV at three injection intervals (14, 21, 28 d). Treatments were arranged in a 3 x 3 factorial and included an uninjected group as control. Each treatment combination included five multiparous and two primiparous cows; the control group contained two additional multiparous cows. The experiment consisted of 14 d of pretreatment and 84 d of treatment. Management, housing, milk recording, and feeding were as described for Experiments 1 and 2. Production parameters were analyzed statistically by analysis of covariance for a 3 x 3 factorial experiment having an additional control group. RESULTS AND DISCUSSION Experiment 1
Three cows were removed from the experiment because of mastitis (Table 2). The onset of mastitis was sudden and severe with daily milk production decreasing from more than 28 Journal of Dairy Science Vol. 73.
No.3, 1990
766
MCGUFFEY ET AL.
TABLE 2. Cow removals. Reason for removal
Cow number
Treatment
26596 26525 29031
Method A Method B Method B
Mastitis Mastitis
2
26581 29304 26584 27248
Control Control Somatotropin Somatotropin
Hardware Hardware Mastitis Dry period
3
29063 29653 2%12 29651 2%50
Control Control 310-321 1 310-328 960-328
Teat injury Mastitis Teat injury Teat injury Mastitis
Experiment
Ma~titis
1Dose of somatotropin (mg) minus interval between injections (days).
to 4 kg or less in 2 d for two cows receiving bST purified by Method B. The third cow, which received bST from Method A, had clinical indications of mastitis for the first 3 d of pretreatment. During treatment, milk yield decreased by approximately 8 kg/d 2 d after onset of mastitis. Milk production by this cow returned to premastitis yields following antibiotic therapy. Lactation responses of cows to daily injection of bST are shown in Table 3. Cows receiv-
ing bST purified by methods A and B averaged 3.8 and 3.5 kg/d more (P
given by daily injections or in a sustained-release vehicle. (Key words: somatotropin, sustained release, lactation performance)
ABSTRACT
Three experiments were conducted to characterize metabolic and milk production responses of dairy cows receiving recombinantly derived bovine somatotropin administered either by daily injection or in a sustained-release vehicle. In Experiment I, somatotropin (25 mg/d) purified by two methods was given by daily injection for 14 d and resulted in 3.5 and 3.8 kg/d more milk than controls. Percentages of fat and total solids in milk were also increased by somatotropin. Eleven hematology indices and 12 metabolites, minerals, and enzyme activities in serum were unaffected by somatotropin. In Experiments 2 and 3, somatotropin was administered in a sustained-release vehicle during an 84-d treatment period. In Experiment 2, administration of 960 mg of somatotropin at 28-d intervals increased milk and SCM yields by 4.1 and 3.3 kg/d compared with yields of controls. There were no significant differences in other production parameters. In Experiment 3, 320, 640, and 960 mg somatotropin were each administered in the sustained-release vehicle at intervals of 14, 21, and 28 d. An uninjected group served as control. Cows receiving somatotropin averaged 3.5 to 5.9 kg/d more milk than controls across all injection intervals. Among doses, milk yield was greater at 960 mg than at 320 or 640 mg. There were no significant differences in milk or SCM among injection groups. These experiments demonstrate the comparable efficacy of somatotropin when
Received February 27, 1989. Accepted October 2, 1989.
1990 J Dairy Sci 73:763-771
INTRODUCTION
Stricker and Grtiter (21) first described the lactogenic property of crude anterior pituitary extracts in rats. The galactopoietic nature of pituitary extracts derived from cattle was demonstrated in a classical set of experiments with lactating dairy cows by Asimov and Krouze (1). Cotes et al. (8) reported somatotropin (ST) was the protein responsible for galactopoiesis in ruminants, not prolactin or adrenocorticotropin. Following that discovery, the galactopoietic nature of ST was demonstrated under a wide range of experimental conditions. Generally. galactopoiesis occurred when 30 to 40 mg ST were administered daily to dairy animals. Duration of treatment with ST ranged from 4 d to 12 wk. Milk yield prior to treatment ranged from 7 to 35 kg/d (7, 13) and was increased by as much as 7.0 kg/d in one long-term study with ST treatment (7). The experiments used several breeds of dairy cattle in all stages of lactation. Rations consisted of only pasture (17), pasture plus concentrate (7), and forage and concentrates fed separately (5, 13) or as complete diets (11, 16). Somatotropin was injected daily with one exception: Machlin (13) injected every 3rd d. The impact of these early studies was realized when Bauman et al. (3) reported comparable galactopoietic activity of ST from pituitary origin and bovine somatotropin (bST) produced by recombinant DNA technology. Relatively large supplies of bST made longer studies possible. Bauman et al. (4) observed as much as a 41 % increase in milk yield in dairy cows when bST was administered by daily injections for 188 d. 763
764
MCGUFFEY ET AL.
Three experiments are described that had objectives of: 1) demonstrating comparable galactopoietic activity of bST purified by methods suitable for laboratory purification and for large-scale production; 2) determining the response of dairy cows to bST administered in a sustained-release vehicle; and 3) determining the dose response of dairy cows to bST administered in a sustained-release vehicle at intervals of 14, 21, and 28 d. MATERIALS AND METHODS
Bovine somatotropin (Somidobove 1) was produced by recombinant DNA technology using Escherichia coli as the host vector. After a suitable growth time, bacteria were isolated and heat-killed. A liquid slurry was made of the bST-containing microorganisms. A bST-enriched fraction was isolated from the slurry and was chromatographed using either DEAESephadex (Method A) or DEAE tris-acryl (Method B) as the solid phase. Method A had been used previously in the laboratory to purify ST (24). Method B allowed chromatography of large quantities of bST-enriched fractions. The first experiment compared the galactopoietic activity of bST isolated by each method to ascertain that the methodology of large scale purification did not alter biological activity. Experiment 2 and 3 used bST derived from Method B. Experiment 1
Twenty-four lactating Holstein cows were assigned to this experiment which consisted of 14 d of pretreatment, 14 d of treatment, and 14 d of posttreatment. Milk production during pretreatment (range 19.6 to 33.7 kg/d), days in milk (DIM) (range 44 to 138), and parity were used to block the cows into eight groups of three. Within each group, cows were randomly assigned to one of three treatments: 1) control, 2) bovine somatotropin purified using DEAESephadex (Method A), and 3) bovine somatotropin purified using DEAE tris-acryl (Method B). Each bST treatment was given by subcuta-
lSomidobove is the proprietary name for rccombinantly derived bovine S0matolropin produced by Eli Lilly and Company (12). Journal of Dairy Science Vol. 73,
No.3. 1990
neous injection in the scapular area at an estimated dose of 25 mg/d monomeric bST. Monomeric purity was estimated from peak height using HPLC and from protein content of the purified bST. Solutions were prepared daily on the day before injection, except for Sunday and Monday injections, which were prepared on Friday. All prepared solutions were refrigerated until used. Cows in the control group received a subcutaneous injection daily of 10 ml of .1 M NH4HC0 3, pH 7.75 (vehicle). Bovine somatotropin purified by Method A was delivered in 30 ml of vehicle, and bST purified by Method B was delivered in 20 ml of vehicle. Based upon HPLC analysis and protein content, the average daily dose of bST was 27.6 ± 3.0 mg (Method A) and 26.8 ± 1.6 mg (Method B). Cows were maintained in individual tie stalls with free access to water. They were exercised for at least 2 h daily. Cows were milked twice daily and production was recorded. Milk was collected for composition determinations on Monday p.m. and Tuesday a.m. before treatments were administered, and on Monday p.m., Tuesday a.m., Thursday p.m., and Friday a.m. during both treatment and posttreatment periods. Fat, protein, lactose, and total solids were determined on a percentage basis using a Berwind infrared milk analyzer (Berwind Instruments, Ltd., Osbaldwick Industrial Estate, Osbaldwick, York, YO BUS. England). Solids-corrected milk was calculated according to Tyrrell and Reid (23). A Foss MilkO-Tester (Foss Electric, Hillerod, Denmark) was used to determine SCc. Cows were fed for ad libitum access twice daily a total mixed ration consisting of 60% forage (three parts com silage, one part chopped alfalfa hay on a DM basis) and 40% concentrate on a DM basis (Table 1). Feed issued and feed refused were recorded daily throughout the experiment. Body weight was determined at the beginning and end of the pretreatment period, at the end of the treatment period, at the end of the treatment period. and at the end of the experiment. Two blood samples (one for whole blood, one for serum) were collected by jugular venipuncture 2 d prior to starting treatment. on the final day of treatment, and on the final day of the experiment. Whole blood was analyzed for leukocytes, erythrocytes, erythrocyte morpholo-
SOMATOTROPIN IN A SUSTAINED-RELEASE VEHICLE TABLE 1. Composition of concentrate and estimates of nutrient and energy content. Ingredient
(%)
Corn, cracked yellow Soybean meal Bet pulp, dried Molasses, cane Limestone Sodium phosphate Trace-mineral salt Sodium bicarbonate Vitamin A + D premix' Vitamin E premix I Selenium premix'
48.50 33.10 10.00 3.50 1.65 1.05 1.00 .75 .30 .05 .05
Dry matter, % Crude protein, % NEI, Mcal/kg Calcium, % Phosphorus, %
89.10 24.00 1.74 .85 .69
'Vitamin A .lJld D premix contained 907,000 IU A, 102,000 IU Dylkg; vitamin E premix contained 9070 IV EI kg; selenium premix contained 200 mg of selenium as sodium selenite.
gy, leukocyte differential. thrombocytes, fibrinogen. hemoglobin, packed cell volume, mean corpuscular volume. mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration. Serum was analyzed for glucose, urea nitrogen, creatinine. potassium, calcium, cholesterol. triglycerides, sodium, chloride, sorbitol dehydrogenase. total bilirubin. and alkaline phosphatase. Data from the treatment and the posttreatment periods were analyzed statistically using the General Linear Model of SAS (18). The model used for each analysis was:
where Yijk is the observed value of a cow during the treatment period, Il is the population mean value, ti is the treatment i (i = 1,2,3), rj is the block j, b is the regression coefficient. (Xijk - X) is the preliminary period deviation for a cow from the mean. and Eijk is the error deviation. Data from whole blood and serum collected in the treatment and posttreatment periods were subjected to one-way analysis of variance. Significance of treatment effects was compared by Dunnett's T·test (20).
765
Experiment 2
Fourteen lactating Holstein cows were assigned to this experiment, which consisted of 14 d pretreatment and 84 d of treatment. Milk production (range 19.5 to 34.5 kg/d) during pretreatment and DIM (range 63 to 246 d) were used to pair cows for random assignment to one of two treatments. Treatment" were either control (no injection) or 960 mg bST in a sustained-release vehicle (SRV; TD-85; Eli Lilly and Company) given at 28-d intervals. The SRV was administered on d 1. 29, and 57 of the treatment period by subcutaneous injection. Injections were given on alternating sides in the scapular region of the animal. Management, housing, milk recording, and feeding were as described for Experiment 1. Cows were weighed on the day before pretreatment and at l4-d intervals thereafter. Production measures were analyzed statistically by analysis of covariance as in Experiment 1 with the exception that block was excluded from the model. Experiment 3
Seventy-two Holsteins (52 multiparous; 20 primiparous) were used to determine lactation response of cows to a dose range of bST (320, 640. 960 mg) given in the SRV at three injection intervals (14, 21, 28 d). Treatments were arranged in a 3 x 3 factorial and included an uninjected group as control. Each treatment combination included five multiparous and two primiparous cows; the control group contained two additional multiparous cows. The experiment consisted of 14 d of pretreatment and 84 d of treatment. Management, housing, milk recording, and feeding were as described for Experiments 1 and 2. Production parameters were analyzed statistically by analysis of covariance for a 3 x 3 factorial experiment having an additional control group. RESULTS AND DISCUSSION Experiment 1
Three cows were removed from the experiment because of mastitis (Table 2). The onset of mastitis was sudden and severe with daily milk production decreasing from more than 28 Journal of Dairy Science Vol. 73.
No.3, 1990
766
MCGUFFEY ET AL.
TABLE 2. Cow removals. Reason for removal
Cow number
Treatment
26596 26525 29031
Method A Method B Method B
Mastitis Mastitis
2
26581 29304 26584 27248
Control Control Somatotropin Somatotropin
Hardware Hardware Mastitis Dry period
3
29063 29653 2%12 29651 2%50
Control Control 310-321 1 310-328 960-328
Teat injury Mastitis Teat injury Teat injury Mastitis
Experiment
Ma~titis
1Dose of somatotropin (mg) minus interval between injections (days).
to 4 kg or less in 2 d for two cows receiving bST purified by Method B. The third cow, which received bST from Method A, had clinical indications of mastitis for the first 3 d of pretreatment. During treatment, milk yield decreased by approximately 8 kg/d 2 d after onset of mastitis. Milk production by this cow returned to premastitis yields following antibiotic therapy. Lactation responses of cows to daily injection of bST are shown in Table 3. Cows receiv-
ing bST purified by methods A and B averaged 3.8 and 3.5 kg/d more (P
