The basic parameters of ovine protein synthesis were investigated in a series of experiments using variables such as intake, stage of maturity and amino acid addition and deletion. Inter-species comparisons were also made between the ovine and redclaw crayfish (Cherax quadricarinatus).
Protein synthesis was examined in more detail than previous studies by quantifying mRNA translation and specifically ribosome elongation time.
The effect of level of intake on the parameters of protein synthesis in the ovine liver, skin, rumen, duodenum, semimembranosus and longissimus dorsi muscles was investigated (Chapter 3). Protein fractional synthesis rate (FSR) increased in most tissues (liver, duodenum, skin and muscles) in response to increased level of intake. The increase in protein FSR within each tissue was attributed to an increase in mRNA translational efficiency, however there was no effect of increased level of intake on ribosome elongation time leading to the inference that an increase in translational efficiency by level of intake occurred through increased ribosome initiation rates. Ovine ribosome elongation times were documented between 82 (semimembranosus muscle) and 379 seconds (duodenum). A comparison of tissue types found that those tissues with higher total RNA levels had associated higher protein FSR's. Higher protein FSR's and higher translational efficiencies were correlated with longer ribosome elongation times. However the ribosome elongation time per unit of total RNA was lower in tissues with higher translational efficiencies and longer in tissues with lower translational efficiencies.
An increased level of maturity resulted in decreased protein FSR in all tissues. This resulted from a decline in total RNA (liver, skin and muscles) and DNA (except for rumen) and a reduced translational efficiency in liver, duodenum and rumen tissues. In all tissues an increase in ribosome elongation time with increased stage of maturity was documented. Unlike the effect of intake the effects of increased stage of maturity within the tissues are predominantly related to cell capacity for synthesis, but also affected by the ribosome elongation time. The current study also demonstrated that the mechanism of age related decline in protein FSR is not universal for all tissues in the ruminant and not similar to that of intake. Skin and muscle tissue was able to compensate for declines in cell capacity for protein synthesis (RNA) by maintaining or increasing translational efficiency.
Specific nutrient manipulation of the diet through the addition of a complete group of amino acids (arginine, lysine, histidine, threonine, methionine and cysteine) and a limiting group (methionine and cysteine absent) by jugular infusion and the dietary supplementation of fishmeal was undertaken to directly manipulate ribosome elongation times (Chapter 5). The addition of amino acids and fishmeal supplementation increased protein FSR in all tissues through the same parameters as that of increased level of intake (assumed to be at the ribosome initiation level as there were no changes in ribosome elongation time). The deletion of methionine and cysteine from the infusion treatment had no consistent effects in this study.
Redclaw crayfish (Cherax quadricarinatus), a freshwater decapod crustacean display a large variation in weight gain. There was no relationship found between the large variation in weight gain in the redclaw and protein FSR (Chapter 6). Similarly, no relationship was found between protein FSR and any other parameter measured (RNA, DNA, translational efficiency or ribosome elongation rate) in the redclaw. Ribosome elongation rates between the redclaw tail muscle (54.5 to 270.7 seconds) and the ovine muscles (85.7 to 182.9 seconds) appear to be similar with larger variation in the redclaw. The study of redclaw showed that the molecular mechanisms of protein synthesis may be similar across species but the regulation of synthesis and growth is different.
In conclusion, ribosome elongation times have been documented in ovine liver, duodenum, skin, rumen, semimembranosus muscle and longissimus dorsi muscle and the redclaw crayfish for the first time. The effects of increased level of intake, stage of maturity and amino acid and fishmeal supplementation on the ovine tissues with respect to protein FSR, RNA, DNA, translational efficiency and ribosome elongation time are reported. The mechanisms by which level of intake and amino acid and fishmeal supplementation affect protein synthesis were found to be the same, with ribosome initiation postulated to have the predominant effect on protein FSR as there were few treatment effects on ribosome elongation time. Increased stage of maturity effects on protein FSR occur through declining cell capacity for synthesis and reduced translational efficiency. Ribosome elongation time has been shown to be an important influence in this relationship. Practically this study identifies the ability to increase the potential for faster and more efficient growth. Firstly by selecting animals for higher RNA content, shown to have the largest effect on protein synthesis and then for greater translational efficiency based on the ability for higher ribosome initiation rates as more efficient translation leads to higher protein synthesis and growth. This study has also highlighted the potential to increase protein synthesis in specific tissues. Fishmeal has been identified as a supplement with the right balance of nutrients to create higher rates of protein synthesis in the peripheral tissues (specifically muscle).
It was concluded that ribosome elongation time is not the major factor influencing protein FSR but there are differences between tissues and some nutritional treatments related to the size of molecule being synthesized and the velocity of the ribosome along the mRNA.