Effect of a Prolonged Release Formulation of Recombinant Bovine Somatotropin on Plasma Concentrations of Hormones and Metabolites, and Milk Production in Dairy Cows KOICHI HODATE, AKIHITO OZAWA AND TETSU JOHKE National Institute of Animal Industry, Ibaraki 305, Japan Abstract. The effect of a prolonged release formulation of recombinant DNA derived bovine somatotropin (rbst) on the plasma concentrations of growth hormone (GH), insulin-like growth factor-i (IGF-I), insulin, glucose, blood urea nitrogen (BUN) and nonesterified fatty acids (NEFA), and milk production in lactating dairy cows was studied. Eight cows were divided into two equal groups. One group was the noninjected control, and cows in the other group received a single subcutaneous injection of 640 mg rbst. Plasma GH levels in the rbst-treated cows were higher than in the control cows for 10 days after the injection. Plasma IGF-I concentrations were significantly higher in the rbst-treated cows than in the control for 14 days after the treatment. In the rbst-treated cows, the plasma concentrations of insulin and glucose tended to be higher than those in the control until 7 days after the injection. Also, plasma NEFA levels were higher in the rbst-treated cows for 10 days. In contrast, plasma BUN levels were significantly lower in the rbst-treated cows for 17 days after the treatment. For 28 days after the injection, the mean daily milk yield in rbst-treated cows was 4.5 kg (21.2%) more than that in the control cows. In the rbst-treated cows, a highly positive correlation was observed between the mean daily milk yield and the mean plasma concentration of IGF-I throughout the postinjection period. These results indicate that the rbst treatment induces the increase in the circulating concentrations of GH, IGF-I, insulin, glucose and NEFA and the decrease in BUN, and suggests that a part of the galactopoietic effect of rbst may be mediated by IGF-I. Key words: Plasma hormones, Metabolites, Milk production, Recombinant bst, Lactating cows. (Endocrinol Japon 38: 527-532, 1991) IN 1937, Asimov and Krouze [1] reported for the hormone derived from the anterior pituitary. In 1985, Bauman et al. [3] demonstrated that longterm daily injections of rbst were as effective as bovine anterior pituitary increased milk production in cows. Subsequent studies by Young and his pituitary-derived bovine GH in enhancing milk colleagues showed that the galactopoietic response production in cows. Thereafter the prolonged was due to growth hormone (GH) [2]. Recent release formulations of rbst, which have a long advances in recombinant DNA technology have lasting activity and can be injected every 14 or 28 made recombinant bovine GH or somatotropin days, were developed, and the galactopoietic action of these formulations was examined in dairy (rbst) available in large quantities. It has a similar structure and biological activity to the natural Received: July 4, 1991 Accepted: September 20, 1991 Correspondence to: Dr. Koichi HODATE, Department of Animal Physiology, National Institute of Animal Industry, Tsukuba Norindanchi, P. O. Box 5, Ibaraki 305, Japan. cows [4, 5]. However, as far as we know, there are few reports referring to the influence of these formulations on circulating levels of hormones and metabolites [6, 7]. In the present series of experiments, we studied the influence of a single injection of rbst in a prolonged release formula-
HODATE et al. tion on plasma concentrations of GH, insulin-like growth factor-i (IGF-I), insulin, glucose, blood urea nitrogen (BUN) and nonesterified fatty acids (NEFA), and milk production in lactating cows. (Beckman), respectively. Concentrations of plasma NEFA were estimated with a NEFA assay kit (Iatron). 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 [11]. Materials and Methods Results Changes in concentrations of plasma hormones in the cows before and after treatment with rbst are shown in Fig. 1. In the rbst-treated cows, the concentrations of GH were increased by the rbst injection, and were maintained at a level which was higher than that of the control cows until day 10 after the treatment. The concentrations of IGF-I in the rbst-treated cows rose to 259.7ng/ml The experiments were carried out over 57 days between mid January and early March. Eight multiparous lactating Holstein dairy cows averaging 188 days postpartum from the institute herd were used. The 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 [8], and supplemented with Italian ryegrass hay at 1200 h daily during 28 consecutive days after rbst injection. The cows were milked twice daily at 0830 and 1830 h, and the daily milk yield was recorded. The 8 cows were divided into two equal groups. One group was the noninjected control. Each cow in the other group received a single subcutaneous injection of a prolonged release formulation of 640 mg rbst which was prepared for every 28 day injection (provided by Eli Lilly) at 1130 h on day 0 of a 57-day experimental period. Blood samples (10ml) from the cows were collected into evacuated tubes containing heparin by venipuncture of the jugular vein at 1130 h 14, 7, 3, 1 and 0 days before the injection and 1, 3, 5, 7, 10, 14, 17, 21, 24, 28, 35 and 42 days after the injection. Blood samples on day 0 were taken just before the rbst injection. These blood samples were immediately chilled with ice, and the plasma was obtained after centrifugation and stored at-60 Ž until assayed for hormones and metabolites. Milk samples from cows were collected 14, 7 and 3 days before the treatment, on the day of the treatment and 3, 7, 10, 14, 17, 21, 24, 28, 35 and 42 days after the treatment for composition analysis. Concentrations of plasma GH and IGF-I were determined by radioimmunoassay methods [9, 10]. The IGF-I assay was performed on the acid-ethanol extraction of the plasma. Plasma concentrations of insulin were estimated with an Eiken ICL RIA kit. Concentrations of plasma glucose and BUN were measured with a Glucose Analyzer 2 (Beckman) and BUN Analyzer 2
RBST ON HORMONE, METABOLITE AND LACTATION Figure 2 shows the changes in the concentrations of plasma metabolites before and after the treatment with rbst. In the rbst-treated cows, the concentrations of glucose tended to be higher than those for the control during the 7 days after the treatment. And the concentrations of NEFA were higher in the rbst-treated cows than in the control until day 10 after the injection. In contrast, the concentrations of BUN in the rbst-treated cows decreased after the rbst injection, and the values were significantly lower than the control until day 17. Milk yields for each group were similar throughout the preinjection period (days-14 to 0)(Fig.3 and Table 1). However, during 28 days after the treatment, the mean daily milk yield in the rbst-treated cows was 4.5kg (21.2%) more than that in the control cows. In the rbst-treated cows, Days Fig.2. Plasma concentrations of glucose, BUN and NEFA before and after treatment with rbst. Arrows indicate time of rbst injection (day 0). Asterisks indicate the statistical difference;*p<0.05, **P<0.01 as compared with the corresponding values for controls. Each point with a bar represents the mean }SE for 4 cows., control; œ, rbst. (Mean) 7 days after the injection from 55.9ng/ml on day 0. Thereafter, the concentrations for the rbst-treated cows were significantly higher than the control until day 14, and then returned to the level of the control by day 17. The concentrations of insulin tended to be higher in the rbst-treated cows than in the control for 7 days after the injection. Days Fig.3. Daily milk yield before and after treatment with rbst. Arrow indicates time of rbst injection (day 0). Asterisks indicate the statistical difference;*p<0.05 as compared with the corresponding values for controls. Each point represents the mean for 4 cows., control; œ, rbst. Table 1. Effect of a single injection of a prolonged release formulation of recombinant bovine somatotropin (rbst) on milk yield (kg/day) Values are expressed as the mean }S. 1) Control=noninjected; rbst=640mg rbst injected on day 0. a; Indicates significant difference from control within each period (P<0.001).
HODATE et al. Table 2. Effect of rbst on protein concentration (%) in milk Values are expressed as the mean }SE for 4 cows. 1) Control=noninjected; rbst=640mg rbst injected on day 0. a; Indicates significant difference from control within each day (P<0.05). Table 3. Effect of rbst on dry matter intake and feed efficiency Values are expressed as the mean. 1) Control=noninjected; rbst=640mg rbst injected on day 0. 2) DMI=dry matter intake; kg/day. 3) FE =feed efficiency; kg milk yield/kg DMI. a; Indicates significant difference from control within each period (P<0.001). a highly positive correlation (r=0.93, n=13; P<0.01) was observed between the mean daily milk yield and the mean concentration of IGF-I throughout the postinjection period (days 0 to 42). In contrast to this, the correlation between the mean daily milk yield and the mean concentration of GH was not statistically significant (r=0.33, n=13). Concentrations of fat and lactose in the milk for each group were not significantly different throughout the experimental period, but protein concentrations in the milk for the rbst-treated cows were noticeably lower than the control on days 3 and 7 after the injeciton (Table 2). Prior to the treatment, daily feed intakes in each group were similar (Table 3). However, for 28 days after the treatment, the average daily feed intake decreased in the rbst-treated cows (-7.4%). Gross efficiency of milk production (kg milk yield/kg feed intake) was improved up to 30% in the rbst-treated cows compared with the control cows. Discussion The present results indicate that an injection of rbst in a prolonged release formulation induces an increase in circulating concentrations of GH, IGF-I, insulin, glucose and NEFA and a decrease in BUN in lactating cows. These results extended the previous results obtained by Lanza et al.[6] and Gallo and Block [7]. Gallo and Block took blood samples every 2 weeks, whereas in our trial the intervals between blood samplings were shortened in order to investigate in detail the changes in circulating concentrations of hormones and metabolites. Our results also indicate that rbst is a potent stimulator of milk production in lactating cows. This confirms the previous results [4, 5, 12]. The increase in the circulating concentrations of IGF-I in the rbst-treated cows (Fig.1) may be due to the stimulation of de novo synthesis in the liver and other tissues by the increased circulating levels of GH due to the exogenous administration of rbst. It has been observed that the circulating concentration of IGF-I in the cattle began to increase following a time lag a several hours after injections of GH or human GH-releasing factor
RBST ON HORMONE, METABOLITE AND LACTATION analogs [13-16]. A highly positive correlation was observed between the mean plasma IGF-I concentration and the mean daily milk yield in the rbst-treated cows throughout the postinjection period. This may mean that IGF-I plays a role in mediating a part of the galactopoietic action of rbst. It has been reported that specific binding sites to bovine GH in mammary membranes from lactating ewes or cows have not been demonstrated, and that the arterial infusion of GH into the udder of lactating ewes did not increase milk production [ 17]. These observations lead us to postulate that the effects of GH on the mammary tissue are indirect and are mediated by IGF-I. Indeed, IGF-I stimulated milk production in the lactating bovine mammary tissue (acini) cultured in vitro [ 18], and the infusion of free IGF-I into the pudic artery supplying the mammary gland of lactating goats caused an increase in milk secretion [ 19]. Furthermore, the bovine GH-induced galactopoiesis in goats was associated with increased concentrations of IGF-I in the mammary tissue [20]. However, since conflicting results have also been obtained [21, 22], the physiological role of IGF-I in mediating the galactopoietic effect of bovine GH still remains to be elucidated. Interestingly, in 1990, Glimm et al. [23] reported that GH receptor messenger RNA was identified in mammary tissue from lactating cows and that the GH receptor gene is primarily expressed in the alveolar epithelial cells, and suggested that the lactating bovine mammary gland is a GH target tissue. However, further investigations are required to confirm their results. The increase in plasma glucose (Fig.2) could be due to the increased hepatic gluconeogenesis and the reduced glucose oxidation by the rbst treatment, and then the secretion of insulin may be stimulated in response to the higher glucose level (Fig.1). It has been reported that liver slices from lactating cows receiving GH had significantly higher rates of propionate conversion to both glucose and carbon dioxide [24], and that glucose oxidation declined 19% during GH administration in dairy cows [2]. Glucose thus provided would be used as the additional glucose required for the lactose synthesis accelerated by the rbst treatment. Generally, GH induces NEFA release from adipose tissue, and then increases the blood concentration of NEFA. If cows have a negative energy balance following the treatment with bovine GH, lipid reserves are mobilized and oxidation of NEFA is increased. Thereby the other nutrients such as glucose and amino acids are preserved, and preferentially partitioned to the mammary gland for milk synthesis [2]. The increase in plasma NEFA levels in the rbst-treated cows (Fig.2) may reflect the above. On the other hand, anabolic agents such as GH and sex steroids induce a reduction of BUN. If cows have a negative nitrogen balance, the percentage of milk protein concentration tends to decline [2]. In the present study, the feed intake for the rbst-treated cows for 28 days after the injection did not increase (Table 3), but the milk production was enhanced (Table 1 and Fig.3). Therefore, the rbst-treated cows were probably unable to provide adequate amino acids to the mammary gland to maintain protein yields in proportion to milk yields. The changes in the percentage of the protein concentration in the milk appear to be very sensitive to the amount of dietary protein being consumed [2]. In conclusion, the results obtained in this study indicate that rbst has lipolytic and diabetogenic effects which may be important in the partitioning of nutrients toward the mammary gland, and suggest that a part of the galactopoietic action of rbst may be mediated through IGF-I. Acknowledgements The authors are grateful to NIAMDD, U.S.A. for the generous gift of purified bovine GH. The authors wish to thank Mr. H. Fuse, Dr. Y. Obara and Mr. M. Odai for their valuable assistance. 1. Asimov GJ, Krouze NK (1937) The lactogenic preparations from the anterior pituitary and the increase of milk yield in cows. J Dairy Sci 20: References 289-306. 2. Peel CJ, Bauman DE (1987) Somatotropin and lactation. J Dairy Sci 70: 474-486.
HODATE et al. 3. Bauman DE, Eppard PJ, DeGeeter MJ, Lanza GM (1985) Responses of high-producing dairy cows to long-term treatment with pituitary somatotropin and recombinant somatotropin. J Dairy Sci 68: 1352-1362. 4. Chilliard Y (1988) Long-term effects of recombinant bovine somatotropin (rbst) on dairy cow performances. Ann Zootech 37: 159-180. 5. Bauman DE, Hard DL, Crooker BA, Partridge MS, Garrick K, Sandles LD, Erb HN, Franson SE, Hartnell GF, Hintz RL (1989) Long-term evaluation of a prolonged-release formulation of N- methionyl bovine somatotropin in lactating dairy cows. J Dairy Sci 72: 642-651. 6. Lanza GM, Eppard PJ, Miller MA, Franson SE, Ganguli S, Hintz RL, Hammond BG, Bussen SC, Leak RK, Metzger LE (1988) Response of lactating dairy cows to multiple injections of sometribove, USAN (recombinant methionyl bovine somatotropin) in a prolonged release system. Part III. Changes in circulating analytes. J Dairy Sci 71 (Suppl 1):184 (Abstract). 7. Gallo GF, Block E (1990) Effects of recombinant bovine somatotropin on nutritional status and liver functon of lactating dairy cows. J Dairy Sci 73: 3276-3286. 8. Research Council Secretariat of Agriculture, Forestry and Fisheries (1987) Japanese Feeding Standard for Dairy Cattle. Ministry of Agriculture, Forestry and Fisheries, Tokyo (In Japanese). 9. Johke T (1978) Effects of TRH on circulating growth hormone, prolactin and triiodothyronine levels in the bovine. Endocrinol Japon 25: 19-26. 10. Ozawa A, Hodate K, Miyamoto S, Ohtani F, Tsushima T, Johke T (1991) Plasma profiles of insulin-like growth factor-i for 24hours and after injection of bovine growth hormone in dairy heifers. Anim Sci Technol (Jpn)(submitted). 11. Snedecor GW, Cochran WG (1980) Statistical Methods. 7th ed, The Iowa State University Press, Ames, Iowa, USA, 83-106. 12. McGuffey RK, Green HB, Basson RP (1988) Performance of Holsteins given bovine somatotropin in a sustained delivery vehicle. Effect of dose and frequency of administration. J Dairy Sci 71 (Suppl 1): 177 (Abstract). 13. Breier BH, Gluckman PD, BassJJ (1988) Influence of nutritional status and oestradiol-17Ĉ on plasma growth hormone, insulin-like growth factors-i and -II and the response to exogenous growth hormone in young steers. J Endocr 118: 243-250. 14. Glimm DR, Baracos VE, Kennelly JJ (1988) Effect of bovine somatotropin on the distribution of immunoreactive insulin-like growth factor-i in lactating bovine mammary tissue. J Dairy Sci 71: 2923-2935. 15. Cohick WS, Plaut K, Sechen SJ, Bauman DE (1989) Temporal pattern of insulin-like growth factor-i response to exogenous bovine somatotropin in lactating cows. Domest Anim Endocrinol 6: 263-274. 16. Hodate K, Johke T, Ozawa A, Ohashi S (1990) Plasma growth hormone, insulin-like growth factor-i and milk production response to exogenous human growth hormone-releasing factor analogs in dairy cows. Endocrinol Japon 37: 261-273. 17. Gluckman PD, Breier BH, Davis SR (1987) Physiology of the somatotropic axis with particular reference to the ruminant. J Dairy Sci 70: 442-466. 18. Baumrucker CR (1986) Insulin like growth factor 1 (IGF-1) and insulin stimulates lactating bovine mammary tissue DNA synthesis and milk production in vitro. J Dairy Sci 69 (Suppl 1): 120 (Abstract). 19. Prosser CG, Fleet IR, Corps AN, Froesch ER, Heap RB (1990) Increase in milk secretion and mammary blood flow by intra-arterial infusion of insulin-like growth factor-i into the mammary gland of the goat. J Endocr 126: 437-443. 20. Prosser CG, Royle C, Fleet IR, Mepham TB (1991) The galactopoietic effect of bovine growth hormone in goats is associated with increased concentrations of insulin-like growth factor-i in milk and mammary tissue. J Endocr 128: 457-463. 21. Shamay A, Cohen N, Niwa M, Gertler A (1988) Effect of insulin-like growth factor I on deoxyribonucleic acid synthesis and galactopoiesis in bovine undifferentiated and lactating mammary tissue in vitro. Endocrinology 123: 804-809. 22. Davis SR, Gluckman PD, Hodgkinson SC, Farr VC, Breier BH, Burleigh BD (1989) Comparison of the effects of administration of recombinant bovine growth hormone or N-Met insulin-like growth factor-i to lactating goats. J Endocr 123: 33-39. 23. Glimm DR, Baracos VE, Kennelly JJ (1990) Molecular evidence for the presence of growth hormone receptors in the bovine mammary gland. J Endocr 126: R5-R8. 24. Pocius PA, Herbein JH (1986) Effects of in vivo administration of growth hormone on milk production and in vitro hepatic metabolism in dairy cattle. J Dairy Sci 69: 713-720.