Reduced growth of calves and its reversal by use of anabolic agents

Domestic Animal Endocrinology 19 (2000) 85–92

Reduced growth of calves and its reversal by use of anabolic agents J.L. Sartina,b,*, M.A. Shoresa, D.D. Schwartza, R.J. Kemppainena, J. Bakera a

Departments of Anatomy, Physiology and Pharmacology, Alabama Agricultural Experiment Station, Auburn University, Auburn, AL 36849-5518, USA b Department of Animal Health Research, Alabama Agricultural Experiment Station, Auburn University, Auburn, AL 36849-5518, USA

Abstract Disease has profound effects on the immune system, endocrine system, and on the growth process. Since diseases are catabolic to the animal, there is current interest in the possible role of anabolic hormones to counter the effects of disease in general and minimize the effects of a disease process on growth and development. A number of anabolic hormones, such as growth hormone (GH) and estradiol ⫹ progesterone (EP), have been studied for their role in enhancing growth and stimulating immune function and are thus candidates for hormonal intervention in disease processes. GH has been shown to be effective in countering some of the deleterious effects of endotoxemia but was ineffective in a parasitic disease model. Studies with EP have shown similar success with both endotoxemia and a parasitic disease model. Moreover, GH and EP do not share a common mechanism of action, suggesting that the effects are not simply due to anabolic actions. While the mechanism of action of GH in endotoxemia has been examined, the effects of EP are via an unknown mechanism, possibly by inhibition of IL-I action or inhibition of nitric oxide overproduction. Keywords: Eimeria bovis; Estrogen; Progesterone; Nitric oxide; Endotoxin

1. Introduction Growth is a complex and well-coordinated physiological process. Any factors that interfere with all or a portion of this mechanism can have a major negative impact on growth. One such detrimental factor is disease. While growth is essentially an anabolic process, disease is by nature a catabolic process. There is, among other factors found with disease, a loss of * Corresponding author. Tel.: ⫹1-334-844-5515; fax: ⫹1-334-844-5388. E-mail address: [email protected] (J.L. Sartin). 0739-7240/00/$ – see front matter © 2000 Elsevier Science Inc. All rights reserved. PII: S 0 7 3 9 - 7 2 4 0 ( 0 0 ) 0 0 0 6 9 - 2

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food intake and a concurrent requirement for energy that exceeds intake. The net result is an increasing dependence on internal sources of energy, beginning with glycogen and fat and progressing to protein. Also associated with critical illness is a prolonged inhibition of plasma growth hormone (GH) and insulin-like growth factor-I (IGF-I) concentrations. The down regulated growth hormone axis may contribute to inhibition of growth rates, reduced immune function and impaired wound healing (for reviews, see 1,2,3,4,5,6,7). Anabolic agents have long been used to increase growth in animals. Estrogen, progesterone and GH are hormones that have been the focus of numerous studies demonstrating anabolic effects and increased growth rates in animals [8]. Anabolic agents (such as estradiol/progesterone, growth hormone, insulin, etc) tend to enhance protein synthesis either directly or through an elegantly orchestrated endocrine response including GH, insulin and insulin-like growth factor-I (IGF-I). The fact that anabolic agents have the opposite effects on metabolism of fat and protein to those seen in catabolic disease, suggests that anabolic hormones may be used as a possible intervention strategy to minimize the impact of disease on growth. Indeed, early studies using GH confirmed that this approach might meet with success. In rats, hypophysectomy reduced survival rates in rats infected with Salmonella typhimuriam while GH treatment was found to increase survival rates [9]. GH treatment of critically ill human patients was found to improve protein synthesis, reduce urea generation, improve wound healing and shorten hospital stay [2]. In addition to GH, there are other anabolic agents that have the potential for counteracting catabolic processes such as disease. For example, clonidine (␣2-receptor agonist) can improve nitrogen balance in patients undergoing esophageal cancer resection [10], insulin and estrogen reduce urinary nitrogen loss in a disease model [11], and IGF-I produced favorable effects on carbohydrate metabolism in E. coli treated piglets [11]. Although all studies have not proved positive, there is an increasing body of evidence to suggest that anabolic agents may have a place in the treatment of various diseases [2].

2. Effect of anabolic agents on immune function In addition to metabolic and growth effects, these anabolic hormones may have potent effects on immune function in humans and animals. Indeed, GH, GH-releasing hormone (GRH), somatostatin and IGF-I are all synthesized by cells of the immune system (including macrophages, B-cells, T-cells, NK cells; [1]). The secretion of GH by selected immune cells can be stimulated by GH, GRH or thyrotropin releasing hormone (TRH), though there appears to be heterogeneity between cell types as to how GH is regulated and the amount of GH secreted [1]. Of equal importance are the data indicating the receptors for these hormones reside on immune cells and can mediate the activation of immune cells. For example, GH stimulates proliferative responses in human IM-9 lymphocytes and can induce antibody production. Injection of GH into mice will increase DNA production in the spleen and thymus while GH deficient mice have reduced thymic weights. GH can also increase the differentiation of stem cells to erythrocytes and granulocytes [1]. The initial interest in estrogen and disease was stimulated by the observations that the prevalence of some diseases is not the same between the sexes [13]. Subsequent studies have

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Table 1 CD2⫹, CD4⫹ and CD8⫹ PBMC in Control and EP groups Treatment

% CD2

% CD4

% CD8

Con EP

29.5 ⫾ 2.8 42.5 ⫾ 3.8*

9.6 ⫾ 1.1 16.0 ⫾ 2.3*

8.5 ⫾ 0.7 15.5 ⫾ 1.8*

* P ⬍ 0.05. Heath et al. [22]

shown that estrogen may alter the course of an infection by acting on the host, the pathogen, or the host-pathogen system. The effects of estrogens may be either positive or negative depending on the disease and the health status of the host [13]. There are data to indicate estrogen may depress cell-mediated immunity, suppress natural killer cell activity, and enhance antibody formation in human and animal models. Estrogen has been shown to increase plasma TNF␣ concentrations [14]. Low doses of estrogen increased interleukin-I (IL-I) concentrations while high doses appear to reduce plasma IL-I. Finally, both estrogen and progesterone have been shown to block nitric oxide generation in immune cells and in the hypothalamus [15,16].

3. Effect of anabolic agents on immune function in cattle Burton et al [17,18] examined the effects of GH on immune function in cattle. These experiments determined that lymphocytes from cows treated with GH responded to mitogen with an increased proliferation response. In addition, GH was found to produce modest increases in plasma IgG titres. Furthermore, Elsasser et al. [19] found that GH did not influence the CD2⫹, CD4⫹, and CD8⫹ T-lymphocyte populations in calves. However, GH did reduce the secretion of tumor necrosis factor (TNF; a cytokine product of lymphocytes that causes fever, reduced appetite, etc.), in response to endotoxemia in cattle [20]. Burton et al. [21] have also examined the effects of an estrogen-progesterone combination (EP; Synovex S威, 20 mg estradiol benzoate/200 mg progesterone; Ft Dodge Animal Health) and insulin on immune responses in cattle. Insulin caused elevations in mitogen stimulated responses of peripheral blood mononuclear cells, increases in the maximum blastogenic responses to ovalbumin (OVA) immunization, increased total leukocyte counts and an increased ratio of circulating cells expressing CD4⫹/CD8⫹ antigens; all an expression of improved immune responses. Insulin was also associated with a reduced antibody production to injected OVA. On the other hand, EP treatment increased mitogen-stimulated effects on spontaneous blastogenic activity but did not have consistent effects on other immune parameters measured. Heath et al. [22] examined the effects of EP (Synovex C威; 10 mg estradiol benzoate/100 mg progesterone; Ft Dodge Animal Health) on immune parameters in younger Holstein calves and found that estrogen/progesterone implants increase the number of CD2⫹, CD4⫹, and CD8⫹ expressing cells in circulation (Table 1). These studies suggest there is some benefit to the use of anabolic agents, not only for improved growth and feed efficiency, but also for non-specific benefits to the immune system.

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Fig. 1. Effect of GH treatment on plasma IGF-I in Sarcocystis infected calves. C ⫽ control; I ⫽ infected; PF ⫽ pair-fed; GH ⫽ growth hormone. *P ⬍ 0.05. Elsasser et al., 1998 (ref. 23).

4. Effect of anabolic agents in disease models of cattle Based on the hypothesis that anabolic agents can counter catabolic events during diseases and since anabolic agents enhance immune system function, several studies have tested the hypothesis that these agents may be effective in experimental models of disease. Elsasser et al. [20] used E. coli endotoxin injection to model bacterial endotoxemia. Cattle treated with GH and injected with a low dose of endotoxin (0.2 ␮g/kg BW) had diminished plasma concentrations of TNF, thromboxane and cortisol compared to cattle treated with endotoxin alone. Encouraged by these positive findings, Elsasser et al. [23] examined the effects of GH treatment on the animal’s responses to a parasitic disease, Sarcocystis cruzi. In this instance, food intake was not improved by the GH treatment in the infected cattle. Sarcocystis infection produced a prolonged reduction in plasma IGF-I, as is typical for critical illness [23]. However, GH treatments could not maintain plasma IGF-I concentrations in the infected calves (Fig. 1). Moreover, there was no evidence of anabolic effects on muscle protein accretion in the GH treated, infected cattle. In addition, average daily fat accretion was negative in the GH-infected animals compared to the infected calves without GH. Thus while there were some benefits provided from GH treatments in the endotoxemic model, GH effects in the parasitic disease model were unfavorable. Heath et al. [22] used Eimeria bovis (coccidiosis) infection as a model of a catabolic circumstance. In this study, Holstein calves were provided EP (Synovex C威) or sham implants and three weeks later given 250,000 oocysts of E. bovis or saline orally. At 15 days after inoculation, symptoms of infection began to be apparent. Calves treated with EP prior to inoculation with E. bovis developed diarrhea, fever, reduced food intake, but in all cases the onset of symptoms associated with this disease were either delayed in onset or reduced in duration (but not magnitude) when compared to infected cattle not receiving EP. For

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Fig. 2. Effect of estrogen/progesterone on body weight in E. bovis infected calves. Con ⫽ control; EP ⫽ Synovex; PF ⫽ pair-fed; C ⫽ coccidiosis. *P ⬍ 0.05. Heath et al., 1997 (ref. 22).

example, calves infected with E. bovis had a reduction in food intake that began after day 16 postinfection and reached a maximum 60% reduction in food intake at day 20 through day 24 postinfection. Food intake in infected cattle was still reduced by 40 –50% at day 28 postinfection. By contrast, calves treated with EP and infected with E. bovis had a maximum 25–30% reduction in food intake that had normalized by day 24 postinfection. Body weight from day 14 to 28 postinfection decreased about 3 kg in infected calves compared to a positive weight gain of 12 kg during the same time period in EP-treated, infected calves (Fig. 2). The reduced weight gain observed in infected calves was not exclusively due to reduced food intake as calves pair-fed to infected calves had a positive weight gain during the same time period. Diarrhea was present in all infected calves not receiving EP by day 16 postinfection, but was absent in all EP-treated, infected calves until day 18 postinfection. Moreover, feces had returned to normal by day 23 postinfection in EP-treated, infected calves versus day 26 for infected calves not treated with EP. Although there were hormonal and immunological data collected with this study, there were no compelling explanations for the positive effects of EP on the physiological responses of these calves. At this point there is no specific explanation for the ability of EP to improve an animals response to a catabolic disease model. One approach to find a mechanism for the beneficial effects of EP would be to compare the effects of EP to an anabolic agent with a known mechanism of action in a catabolic disease model. In endotoxemia, GH reduces tumor necrosis factor, cortisol and thromboxane concentrations in plasma, all indicators of a mechanism for GH effects [20]. We therefore sought to use this model and compare the effects of EP treatment on the progression of this catabolic insult to the calf. Endotoxin administration produces an initial hyperglycemia that is reduced in EP treated calves [24]. The hyperglycemic effect of endotoxin was not affected in cattle given GH prior to the endotoxin [19]. After the initial hyperglycemia, there is a prolonged hyperglycemia in endotoxin-treated calves given EP, suggestive of insulin resistance that persisted for 24 h [24]. Interestingly, GH treated calves also had a prolonged hyperglycemia following endo-

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Fig. 3. EP modulation of endotoxin effects on plasma urea nitrogen in calves. Pooled SEM ⫽ 0.9. Asterisks indicate differences from EP/endotoxin. ␣ vs EP control (P ⬍ 0.05). McMahon et al., 1998 [24].

toxin injection [19]. Endotoxemic calves had a 24 h dry matter intake that was reduced by more than 65% while calves treated with EP and injected with endotoxin had food intake reduced by only about 35%. This was similar to the previous EP study with E. bovis infection, but was not measured in the GH study. Examination of plasma cortisol and TNF [24] suggested that these responses were unaffected by the presence of EP whereas GH had a positive effect to reduce these responses to endotoxin [20]. Thus the primary mechanisms for GH action in endotoxemia were not apparent with EP. Either the GH and EP mechanisms differ or the higher dose of endotoxin used by McMahon et al. [24] overcame any effects of EP. Finally, calves treated with EP and endotoxin had a lower blood urea nitrogen (BUN) concentration than endotoxin treated calves (Fig. 3), indicative of a greater rate of proteins being synthesized rather than broken down [24]. These data indicate the anabolic actions of EP can partially antagonize the catabolic effects of endotoxemia. Elsasser et al. [19] found a similar decrease in BUN in cattle treated with GH and endotoxin, suggesting anabolic agents in cattle partially prevented the catabolic effects of disease. Thus, while we find some differences between EP and GH modifications of endotoxemia in cattle, there are several similarities between these agents in the physiological responses to endotoxemia in cattle related to positive anabolic responses. While the mechanism for EP actions is not known at this point, we had predicted that an anabolic agent would reduce the endotoxin stimulated increase in BUN. Perhaps the mere fact that the animal was sheltered from the extreme catabolic situation in endotoxemia is sufficient to explain the mechanism for the protective effects of the EP in cattle. While we have shown that EP can antagonize the catabolic effects of disease, the mechanism(s) for the protective effects of EP in disease models in cattle is (are) unclear [25]. In an effort to determine the mechanisms for EP actions, we are pursuing several lines of additional research. One hypothesis is that there is an effect of EP to inhibit immune cell production of specific cytokines such as IL-I. The reduction in plasma IL-I concentrations

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would lessen the symptoms associated with disease (such as fever or appetite reduction). A second hypothesis is that EP inhibits the inducible nitric oxide synthase, thus reducing the overproduction of NO observed in pathological circumstances. Since excess NO production is thought to produce cell damage and death and EP can inhibit NO synthase, an inhibition of NO production is an attractive hypothesis to explain the protective effects of EP in cattle.

5. Conclusions Anabolic agents have been proposed for use as adjuncts to disease therapy in humans and animals. EP would appear to be one of those anabolic agents for use in cattle. Indeed, used as a prophylactic, EP can enhance growth rates, stimulate immune function, and reduce the severity of disease effects on an animal. Thus, in addition to directly enhancing growth rates, EP has the added benefit of reducing the impact of factors that limit productivity in cattle.

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