Overview

The lower birth weight of twin-born lambs compared to singletons, which has implications for post-natal survival and post-natal growth, is a major constraint for the global sheep industry. However, these limitations are especially relevant for the New Zealand sheep flock due to the increased prolificacy achieved in the last 15 years. Late- gestation and early post-natal skeletal muscle growth is reduced in twins compared to singletons, leading to decreased birth weight and subsequently poorer survival and/or post-natal growth. Therefore, any attempt to improve skeletal muscle growth during pregnancy and/or after birth may have a major impact on sheep production by reducing mortality and increasing growth. Despite the importance of muscle growth in sheep meat production, there is still little understanding of the mechanisms leading to reduced muscle growth in twins. However, studies in monogastrics have shown that dietary supplementation with specific amino acids (AA) significantly increase skeletal muscle growth during pregnancy and after birth (Wu et al., 2007a). This effect is mediated by the signalling role that specific AA have on pathways that regulate protein accretion, such as the mechanistic target of rapamycin (mTOR) pathway. However, it is not fully understood which AA could play a role in the regulation of the pathways involved in muscle growth and the mechanisms associated with specific AA supplementation during pregnancy and post-natally on muscle growth in twin sheep are unclear.

The objectives of this thesis were, firstly, to identify intracellular AA which could act as regulatory factors for muscle growth in late pregnancy and post natally in twin sheep; secondly, to evaluate in vivo the effect of supplementing a potential regulatory AA

on muscle growth, and thirdly to examine the role of mTOR signalling in mediating any observed effects on muscle growth.

The findings of the studies undertaken in this thesis reinforce the observation that twin lambs have reduced muscle growth during late pregnancy and from birth to weaning compared to singletons, regardless of the level of maternal or lamb nutrition. This confirms that factors other than plane of nutrition influence muscle growth. Reduced intracellular concentration of specific AA in muscle, namely arginine (Arg) and glutamine were associated with reduced muscle mass in twin fetuses. This suggested that Arg and glutamine may play a role in fetal muscle growth. In contrast, skeletal muscle weight at weaning was associated with changes in several intracellular AA in muscle,

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with no clear indication of which AA could be driving the difference in muscle growth between singletons and twins. Based on these results and the current literature Arg was selected as a candidate regulator of muscle growth during late pregnancy and post-natally. Two in vivo trials were conducted in order to evaluate, firstly, the effect of maternal Arg

administration (via i.v bolus three times daily), during pregnancy on twin fetal and post-

natal muscle growth, and secondly, the effect of direct Arg supplementation of the lamb (via milk) from birth to weaning on muscle growth.

While maternal supplementation with Arg resulted in no effect on fetal weight and only increased the M. psoas major muscle weight in females at 140 days of

pregnancy, it increased the birth weight of female lambs from supplemented ewes, compared to control females. Results suggested Arg supplementation affected the growth of female fetuses in the last two weeks of pregnancy and this effect was coupled with increased abundance of mTOR and protein synthesis capacity in muscle at 140 days of pregnancy. A developmental programming effect of maternal Arg supplementation was also described for postnatal muscle growth, whereby female lambs from ewes supplemented with Arg during pregnancy had increased muscle growth at market weight compared to control female lambs. Post-natal supplementation of artificially reared lambs with Arg also demonstrated a positive response in muscle growth only in females. This effect was associated with increased weight gain during the first 3-4 weeks of growth, resulting in a 11% to 18% increased muscle weight of lambs at weaning, compared with control females.

This thesis provides a new insight on the role of AA in sheep muscle growth and the effect of Arg supplementation both during pregnancy and post-natally. Similar to what has been observed in monogastrics, Arg supplementation during pregnancy and post- natally influences muscle growth. However, it is intriguing that the effect was observed only in female offspring. The cellular mechanisms explaining the difference in body and muscle growth between female fetuses from supplemented versus control ewes appeared to be associated at least in part with the mTOR pathway. Greater total mTOR concentration in muscle of Arg-supplemented females, leading to an increase in protein synthesis capacity, at least during late pregnancy, could be associated with heavier birth weight, while the role of mTOR during postnatal Arg supplementation was not clear. The causes of the unexpected sexual dimorphism in response to Arg supplementation requires further investigation. However, these results highlight the potential of Arg to improve

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muscle growth in female lambs through supplementation, creating the baseline for future research in ruminants.