Studies of physiological methods for increasing growth rate in beef cattle

The aim of this project was to investigate physiological methods for increasing growth rate in beef cattle. The effect of glucocorticoids with or without clenbuterol on B₂- adrenoceptors (B₂-AR) in skeletal muscle and the effect of fasting on serum leptin were studied.

Muscle growth can be promoted in meat producing animals, humans, and experimental animals by the use of B-agonists. However, these anabolic effects rapidly attenuate due to a decrease in B₂-AR density in skeletal muscle following treatment. Glucocorticoids were demonstrated to increase B₂-AR density in rat lung. Thus, the first section of the project was designed to determine whether glucocorticoids could prevent clenbuterol-induced downregulation of B₂-ARs.

Corticosterone, dexamethasone and clenbuterol were tested for effects on muscle growth and B₂-AR density in male rats. Corticosterone caused a dose-related decrease in total weight gain and gain in gastrocnemius/plantaris bundle (GP muscle), but failed to increase B-AR density in the muscle at any of doses tested (6.25-50mg/kg, sc 5d). GP muscle and the ratio of gain/food intake were increased in the group treated with clenbuterol and decreased in the group treated with dexamethasone when administrated daily for 10d. B-AR density in GP muscle was decreased by both clenbuterol and dexamethasone. In contrast, B-AR density in rat lung was increased by dexamethasone and decreased by clenbuterol. hi concurrent treatment group, dexamethasone prevented clenbuterol-induced downregulation of B₂-ARs in rat lung, but not in GP muscle. Thus, dexamethasone does not enhance the anabolic effect of clenbuterol, but causes a marked catabolic effect instead. The results demonstrate that a B-agonist/glucocorticoid combination would not be useful in animal production, and the discovery that B₂-drenoceptors are regulated differently in different tissues warrants further investigation.

The leptin system is postulated to be a key regulatory hormonal pathway associated with the regulation of body weight through effects on metabolism and appetite both in humans and in rodents. Thus, in the second section of this study, an immunological method for detecting bovine leptin and the effects of fasting on circulating leptin, cortisol, glucose and FFA in beef cattle were investigated.

Polyclonal antibodies against bovine recombinant leptin were produced by immunizations of mice, rats, sheep and cattle. Immunization resulted in a positive response with maximum titres assayed by ELISA of 800 in 6/16 of the C57 BL/6 mice and 4/8 of cattle, 3000 in 7/16 rat, and 102400 in 3/3 sheep. No immune response was observed in BALB/c mice. Spleen lymphocytes derived from immunized rats were fused with the murine myeloma cells. Eight hundred hybridomas were cultured. Unfortunately, no leptin specific monoclonal antibody was found on screening the supernatants from these clones.

Cattle and sheep antibodies were affinity purified and conjugated with horseradish peroxidase or biotin. Two immunoassays, avidin-biotin ELISA and competitive ELISA, were developed for determining bovine leptin. The detection limit for recombinant bovine leptin was 500ng/m1 in the avidin-biotin ELISA and 5ng/m1 in the competitive ELISA. The application of the competitive ELISA developed in this study was tested by measuring leptin concentrations of bovine serum and serum plus standards. The results showed that the leptin concentration of different diluted serum was 45.4 ± 3.31 ng/ml.

However, the concentrations of leptin of 1:2 diluted serum plus each standard were all higher than expected, possibly due to the presence of reserve leptin binding proteins in the serum. Thus, after the problem of putative interference of binding protein in serum is resolved, determination of large numbers of sample from beef cattle, rapidly and economically using the competitive assay, will be possible.

The effect of 24 h fasting on serum leptin levels was studied in beef cattle. Serum leptin 4.17 ± 0.11 ng human equivalent /ml was detected in these cattle using a human leptin RIA kit. The leptin levels of fasting animals decreased as compared to the animals before fasting and re -feeding. Fasting caused a 72.33 ± 28.18 % increase in serum cortisol and 203.01 ± 30.94 % increase in plasma FFA. Plasma glucose did not change. A positive correlation between serum leptin concentrations and body weight were observed in seven cattle (before fasting: r2 = 0.663, P = 0.026; after re-feeding: r2 = 0.902, P = 0.0011), but one other animal was outlying. These results indicated that depriving cattle of food caused a decrease of serum leptin and an increase of stress steroids and plasma FFA, while glucose level maintained normal, which demonstrated that leptin levels are readily influenced by physiological changes and are consistent with a homeostatic signaling function for this hormone.

According to the findings of the fasting study, further work would be needed: (1) to investigate whether there are some relationships between leptin and FFA, glucose, insulin and other hormones in circulation in beef cattle as in rats and mice; and (2) to establish whether immunization of cattle with leptin will decrease serum leptin and whether lower leptin level will stimulate food intake, increase growth and enhance the productivity of beef cattle.