Implications

Improving lamb survival and performance is key to enhancing productivity of sheep farming enterprises worldwide. Pastoral-based production systems often present a challenging environment to manipulate nutrition due to difficult terrain, vast land masses and remote locations. The potential for targeted dietary interventions to influence a range of production phenotypes including survival, growth and meat production offers exciting opportunities to realise the value of increased ewe fecundity. Targeted nutritional interventions in critical developmental time windows may offer potential tools for farmers to improve lamb survival and production performance especially in multiple-born lambs.

Production impact of multiple births

Implementation of nutritional, genetic, management and health strategies by New Zealand sheep farmers has resulted in a 24% increase in the number of lambs born per ewe mated in the last 18 years (increase of 100% to 124% from 1990; Morel et al., Reference Morel, Morris and Kenyon2008). However, lambing percentages >200% have been described (Shorten et al., Reference Shorten, O’Connell, Demmers, Edwards, Cullen and Juengel2013), which are associated with a greater proportion of twin- and triplet-born lambs (Amer et al., Reference Amer, McEwan, Dodds and Davis1999).

It is well accepted that twins and triplets have reduced birth weight which leads to higher mortality rates at lambing compared with singletons (Scales et al., Reference Scales, Burton and Moss1986; Gootwine et al., Reference Gootwine, Spencer and Bazer2007). An average mortality rate of 15% to 20% in twins and 25% to 40% in triplets has been reported in New Zealand (West et al., Reference West, Bruere and Ridler2008; Stafford, Reference Stafford, Kenyon, Morris and West2013), with similar rates observed in Australia (Hinch and Brien, Reference Hinch and Brien2014) and other areas of the globe (Rowland et al., Reference Rowland, Salman, Kimberling, Schweitzer and Keefe1992; Dwyer, Reference Dwyer2007). The first 24 h of life is critical for lamb survival, with nearly 50% of all mortalities occurring during this time frame (Dwyer, Reference Dwyer2007). The negative relationship between lamb survival and number of lambs born highlights the importance of identifying strategies to increase lamb survival in multiple-born lambs to realise the value of the improvements in ewe fecundity.

Intra-uterine growth restriction

Intra-uterine growth restriction (IUGR) is common in multiple-born lambs and is a significant problem for agricultural animal production. The term IUGR is often used to describe a wide range of phenotypic outcomes in offspring that have experienced a restricted intra-uterine environment, and is defined as decreased intra-uterine growth velocity (Ergaz et al., Reference Ergaz, Avgil and Ornoy2005) and thus reduced foetal growth potential. Most studies have focussed on the target outcome of human health where IUGR is often a condition resulting from drastically reduced conceptus nutrient and oxygen supply in late gestation mainly resulting from placental insufficiency. The consequences of IUGR in multiple-born lambs include reduced foetal growth and thus birth weight, higher mortality rates (see review by Kenyon, Reference Kenyon2008), reduced neonatal growth rate and lower muscle mass (McCoard et al., Reference McCoard, McNabb, Birtles, Harris, McCutcheon and Peterson1997). In addition, IUGR can result in permanent negative effects on growth, feed efficiency, body composition and thus poor finishing, meat quality and long-term health, thereby decreasing farmer profits (Wu et al., Reference Wu, Bazer, Wallace and Spencer2006; Kenyon and Blair, Reference Kenyon and Blair2014).

Placental development and nutrient transfer

The primary determinant of foetal growth, and thus lamb birth weight, is the supply of nutrients which depends on placental transport as illustrated by the positive correlation between placental and foetal weight (Mellor, Reference Mellor1983). Sheep have a cotyledonary placentation where the exchange of nutrients and waste products happens at discrete sites called placentomes (Ford, Reference Ford2000). These discrete units of foetal–maternal exchange are composed of foetal (cotyledon) and maternal (caruncle) components (Ford, Reference Ford2000) which can be classified based on their shape (type A to D) and may differ in their maternal–foetal exchange area, oxygen exchange efficiency and glucose transport (Fowden et al., Reference Fowden, Ward, Wooding, Forhead and Constancia2006). Transport of nutrients across the placenta is determined by a range of factors including the concentration gradient between maternal and foetal blood, placental blood flow and metabolism, and specific membrane-bound transporter expression and activity.

In sheep, uterine capacity is a key factor limiting foetal survival and growth, especially when ewes are carrying multiples (Gootwine et al., Reference Gootwine, Spencer and Bazer2007). Lower birth weights in multiple litters are associated with smaller placentae with reduced placentome number and weight per foetus in twin compared to singleton foetuses (McCoard et al., Reference McCoard, Peterson, McNabb, Harris and McCutcheon2001; Rumball et al., Reference Rumball, Harding, Oliver and Bloomfield2008; van der Linden et al., Reference van der Linden, Sciascia, Sales and McCoard2013) and decreased total placental vascularity (Vonnahme et al., Reference Vonnahme, Evoniuk, Johnson, Borowicz, Luther, Pant, Redmer, Reynolds and Grazul-Bilska2008), suggesting reduced placental nutrient transport. However, the smaller placentas associated with twins have been shown to be more efficient (van der Linden et al., Reference van der Linden, Sciascia, Sales and McCoard2013) which may be a function of compensatory changes in placental structure and function to deliver an adequate nutrient supply to the foetus to support growth (Rumball et al., Reference Rumball, Harding, Oliver and Bloomfield2008). The role of changes in placentome morphology on nutrient transport has not been evaluated directly and warrants further investigation.

Developmental changes occur in maternal and foetal plasma AA concentrations during pregnancy in sheep. Factors that influence maternal and foetal AA profiles include breed (Ashworth et al., Reference Ashworth, Dwyer, McIlvaney, Werkman and Rooke2011), the stage of pregnancy (Kwon et al., Reference Kwon, Ford, Bazer, Spencer, Nathanielsz, Nijland, Hess and Wu2003) and maternal nutrient status (Kwon et al., Reference Kwon, Spencer, Bazer and Wu2004). We have reported that increased foetal growth in response to maternal arginine supplementation from 100 days of pregnancy to term (McCoard et al., Reference McCoard, Sales, Wards, Sciascia, Oliver, Koolaard and van der Linden2013) is associated with improved placental growth, development and function (van der Linden et al., Reference van der Linden, Sciascia, Sales, Wards, Oliver and McCoard2015). Twins have reduced plasma arginine, leucine, histidine and glutamine compared with singletons (van der Linden et al., Reference van der Linden, Sciascia, Sales and McCoard2013), which indicates that AA transport may differ between twin and single placentae and/or differential metabolism of the foetoplacental unit. The mechanism responsible for these differences is unclear however leucine, arginine and glutamine are known activators of the mechanistic target of rapamycin (mTOR) signalling pathway which is a placental nutrient sensor (Roos et al., Reference Roos, Jansson, Palmberg, Säljö, Powell and Jansson2007) that coordinates maternal nutrient availability and foetal nutrient supply (Jansson and Powell, Reference Jansson and Powell2006). In early pregnancy, this pathway plays an important role in the survival and development of the ovine conceptus (Kim et al., Reference Kim, Burghardt, Wu, Johnson, Spencer and Bazer2011) and in humans; placental mTOR signalling is markedly down-regulated during IUGR (Roos et al., Reference Roos, Jansson, Palmberg, Säljö, Powell and Jansson2007). Hyperthermia-induced growth restriction in sheep is also associated with perturbations in placental mTOR signalling (Arroyo et al., Reference Arroyo, Brown and Galan2009). In other species, mTOR signalling controls placental AA transport by regulating the expression of specific AA transporters (Roos et al., Reference Roos, Palmberg, Säljö, Powell and Jansson2005 and Reference Roos, Jansson, Palmberg, Säljö, Powell and Jansson2007). Thus, the activation of placental mTOR signalling and AA transporter expression leading to increased foetal growth is a potential mechanism underpinning the effect of maternal arginine supplementation on ovine foetal growth.

Another potential mechanism mediating the effect of maternal arginine supplementation on foetal growth is placental metabolism of arginine into nitric oxide (NO) which is a major mediator of ovine placental–foetal blood flow during pregnancy (Rosenfeld et al., Reference Rosenfeld, Cox, Roy and Magness1996). Arginine is the major substrate used for NO production and the two enzymes responsible for this process are inducible (iNOS) and constitutive (cNOS) nitric oxide synthase. Kwon et al. (Reference Kwon, Spencer, Bazer and Wu2004) report that the activities of iNOS and cNOS and levels of NO peak in the intercotyledonary placenta, placentome and intercaruncular endometrium during mid-to-late gestation of Columbia crossbred ewes – a period of rapid foetal growth. In addition, it has been reported that treatment of Suffolk ewes, restricted to 50% NRC requirements with sildenafil citrate, dose-dependently increased total AA’s and polyamines in amniotic fluid, allantoic fluid and foetal serum without affecting values in maternal serum, and foetal weight in nutrient-restricted ewes (Satterfield et al., Reference Satterfield, Bazer, Spencer and Wu2010). Sildenafil citrate works by inhibiting the enzyme that breaks down cyclic guanosine monophosphate, the metabolite produced by NO stimulation of guanylate cyclase, and which is responsible for tissue vasodilation. Interestingly, it has recently been shown that NO synthesis stimulates mTOR activation (Ito et al., Reference Ito, Ruegg, Kudo, Miyagoe-Suzuki and Takeda2013; Capobianco et al., Reference Capobianco, Ramirez, Fornes, Powell, Jansson and Jawerbaum2015), suggesting that either of these pathways maybe involved in regulating ovine placental AA transport, and potentially contribute to the differences in placental efficiency between twins and singletons. A greater functional understanding of the foetoplacental unit in relation to nutrient transfer in multiple-born lambs is required to develop nutritional intervention strategies to improve foetal outcome in sheep.

Birth weight

Birth weight is a key contributing factor to lamb mortality with low birth weight increasing a lamb’s risk for starvation and exposure (Dwyer and Morgan, Reference Dwyer and Morgan2006). The birth weight of triplet-born lambs was reported to be 19% to 24% lower than twin-lamb birth weights (Morris and Kenyon, Reference Morris and Kenyon2004; Everett-Hincks et al., Reference Everett-Hincks, Lopez-Villalobos, Blair and Stafford2005; Everett-Hincks and Dodds, Reference Everett-Hincks and Dodds2008), and 36% to 40% lower than singles (Scales et al., Reference Scales, Burton and Moss1986). Lower birth weight is associated with increased surface area to body–mass ratio and lower body energy reserves (Alexander, Reference Alexander1978) which can increase mortality when exposured to cold conditions (Dwyer and Morgan, Reference Dwyer and Morgan2006). The optimal birth weight range for lamb survival is 4 to 6 kg (Dalton et al., Reference Dalton, Knight and Johnson1980; Morel et al., Reference Morel, Morris and Kenyon2008).

Specific AA supplementation during pregnancy has been shown to enhance foetal growth, and thus birth weight in sheep. Notably, intravenous bolus injection with 155 µmol arginine–HCl/kg BW three times daily between 60 days gestation and birth increases birth weight in single and twin lambs from under-fed ewes (Lassala et al., Reference Lassala, Bazer, Cudd, Datta, Keisler, Satterfield, Spencer and Wu2010). However, while the birth weight of quadruplet lambs was increased when well-fed Booroola Rambouillet ewes were injected with an intravenous bolus of 345 µmol arginine–HCl/kg BW, three times daily, from 100- to 21 days of gestation, the birth weight of triplets, twins or singletons was unchanged (Lassala et al., Reference Lassala, Bazer, Cudd, Datta, Keisler, Satterfield, Spencer and Wu2011). In contrast, when twin-bearing Romney ewes were given an intravenous bolus injection of 345 μmol arginine–HCl/kg bodyweight, three times a day, from 100 days gestation to birth, the birth weight of female but not male lambs was increased (McCoard et al., Reference McCoard, Sales, Wards, Sciascia, Oliver, Koolaard and van der Linden2013) suggesting supplementation during the last 2 weeks of gestation may have the potential to influence the birth weight of twin-born lambs. Alternatively, the differences between these studies may reflect breed differences in their response to AA supplementation, or potentially an influence of the nutritional value of the basal diet despite both being formulated to meet or exceed National Research Council (NRC) requirements. Overall, these studies highlight the potential for specific AA supplementation during key developmental time windows (late gestation) to influence lamb birth weight which may have important consequences for survival, especially in lower birth weight lambs. The effect of supplementation with other AA beyond arginine on lamb birth weight has yet to be evaluated. Furthermore, delivery methods that enable delivery of AA via the diet to avoid rumen degradation such as rumen-protected formulations or AA analogues (McCoard et al., Reference McCoard, Sales and Sciascia2016) are required before practical evaluation of the impact of AA supplementation can be evaluated in pasture-fed multiple-bearing ewes on farm.

Iodine deficiency can lead to lamb mortality (Sargison et al., Reference Sargison, West and Clark1998). Lamb birth weight is negatively affected by grazing ewes on kale crops during gestation, an effect which is reversed with maternal iodine supplementation during pregnancy (i.m.: iodised arachis oil; high=400 mg, medium=300 mg; Knowles and Grace, Reference Knowles and Grace2015). Kale is a complementary forage crop that has low iodine concentrations and contains glucosinolates that inhibit thyroid utilisation of iodine through releasing thiocyanate goitrogens (Stoewsand, Reference Stoewsand1995), therefore in this case, maternal iodine supplementation was correcting for iodine deficiency. However, provided dietary intake of >0.2 to 0.30 mg I/kg dry matter is obtained (Grace and Knowles, Reference Grace and Knowles2010), dietary intake of iodine is usually adequate. Consistent with this notion, birth weight is unaffected by maternal iodine supplementation (26.6 mg/day in the diet) from 119 day gestation to term in ewes fed fresh silage (McGovern et al., Reference McGovern, Magee, Browne, MacHugh and Boland2015). Similarly, lamb birth weight was unaffected in twin- or triplet-born lambs from pasture-fed ewes supplemented with iodine (i.m. injection of 1.5 ml iodised peanut oil 35 days postpartum) despite elevated maternal iodine levels throughout gestation (Kerslake et al., Reference Kerslake, Kenyon, Stafford, Morris and Morel2010). These studies suggest that provided ewes are not iodine deficient, supplementation with iodine during mid-to-late gestation is likely to have limited impact on survival of multiple-born lambs.

Maternal supplementation with polyunsaturated fatty acid (PUFA) increases gestation length in several species (reviewed by Capper et al., Reference Capper, Wilkinson, Mackenzie and Sinclair2006) resulting in a more physiologically mature foetus at birth. However, supplementation with 12 g/ewe per day algae-derived PUFA DHA in twin-bearing Targhee ewes in the last 30 days of gestation and early lactation had no effect on lamb birth weight (Keithly et al., Reference Keithly, Kott, Berardinelli, Moreaux and Hatfield2011). Other studies have also demonstrated variable effects of trace elements and vitamins throughout gestation on lamb birth weight (reviewed by Rooke et al., Reference Rooke, Dwyer and Ashworth2008). However, studies where ewes are supplemented in the last trimester of gestation are scarce. Capper et al. (Reference Capper, Wildinson, Kasapidou, Pattinson, Mackenzie and Sinclair2005) reported that vitamin E supplementation (500 mg/kg; 6 weeks prepartum) of twin- and triplet-bearing ewes increased lamb birth weight. Supplementation of pasture grazing ewes deficient in cobalt with 0.03 or 0.06 mg cobalt/day via weekly drenching throughout gestation also increased lamb birth weight (Quirk and Norton, Reference Quirk and Norton1987). However, maternal supplementation with selenium, an antioxidant, had inconsistent effects (Hammer et al., Reference Hammer, Thorson, Meyer, Redmer, Luther, Neville, Reed, Reynolds, Caton and Vonnahme2011). More research is required to establish whether maternal supplementation strategies in mid-to-late gestation can benefit lamb birth weight in pasture-fed ewes where trace element and mineral status of the ewes is adequate. The practical considerations for maternal trace element supplementation to ewes in pasture-based systems has been reviewed elsewhere (Grace and Knowles, Reference Grace and Knowles2012).

Neonatal vitality

Lighter birth weight, newborn lambs or lambs with lower rectal temperatures exhibit reduced vigour (Dwyer and Morgan, Reference Dwyer and Morgan2006), and less drive to suckle (Alexander and Williams, Reference Alexander and Williams1968), which increased their risk of hypothermia (Dalton et al., Reference Dalton, Knight and Johnson1980). Increased mortality and morbidity of multiple-born lambs has been linked to compromised immune function (Dønnema et al., Reference Dønnema, Randbya, Hektoenb, Avdemc, Meling, Våge, Ådnøya, Steinheima and Waage2015). Vitamin E is one of the micronutrients that may have an impact on immune functions and health. It protects biological membranes from oxidative damage by acting as scavengers of reactive oxygen species and is linked to IgG production (Huber, Reference Huber1988). A number of vitamin E supplementation studies have been conducted during mid-to-late pregnancy to assess the production performance of multiple-born lambs. Dønnema et al. (Reference Dønnema, Randbya, Hektoenb, Avdemc, Meling, Våge, Ådnøya, Steinheima and Waage2015) have shown that oral vitamin E supplemented (360 IU/ewe per day; 6 to 7 weeks prepartum) Norwegian White Sheep with ⩾3 lambs have a significantly lower rate of stillbirths compared with control ewes. However, this was not observed in ewes with ⩽2 lambs, which is in agreement with a previous study conducted in twin-bearing Hardy Speckled Faces ewes orally supplemented with 200 IU vitamin E per ewe per day for the last 8 weeks of gestation (Merrell, Reference Merrell1998). The mechanism of action is currently not known, however it could be linked to reduced oxidative stress or lack of IgG stimulation in twin-bearing ewes (Daniels et al., Reference Daniels, Hatfield, Burgess, Kott and Bowman2000).

Long-chain PUFAs have also been used to assess their effect on lamb viability, as they are known to influence neuronal division, synaptic transmission and retinal development potentially improving early neonatal behaviour. Several studies have shown that supplementation of twin- and triplet-bearing ewes with PUFAs in the last 4 to 9 weeks of gestation improved lamb vigour (Capper et al., Reference Capper, Wildinson, Kasapidou, Pattinson, Mackenzie and Sinclair2005 and Reference Capper, Wilkinson, Mackenzie and Sinclair2006; Pickard et al., Reference Pickard, Beard, Seal and Edwards2005 and Reference Pickard, Beard, Seal and Edwards2008). For example, inclusion of 6 or 12 g of DHA from 9 weeks before lambing improved measures of lamb vigour including time to suckle and time to stand (Pickard et al., Reference Pickard, Beard, Seal and Edwards2005 and Reference Pickard, Beard, Seal and Edwards2008).

Thermoregulation

Brown adipose tissue (BAT) is a specialised fat store that is used by the newborn lamb to generate about 50% of the total heat produced (Symonds and Lomax, Reference Symonds1992; Satterfield and Wu, Reference Satterfield and Wu2011), facilitating an effective adaptation to the cold challenge of the extra-uterine environment and preventing hypothermia (Alexander and Williams, Reference Alexander and Williams1968). Hypothermia is a major cause of on-farm lamb losses in the first few days of life (Everett-Hincks and Dodds, Reference Everett-Hincks and Dodds2008). Low birth weight lambs exhibited lower rectal temperatures (Dwyer and Morgan, Reference Dwyer and Morgan2006), greater lactate concentrations (Stafford et al., Reference Stafford2007) and lower plasma thyroid hormone concentrations (Kerslake et al., Reference Kerslake, Kenyon, Stafford, Morris and Morel2010). These factors are known to negatively impact on the ability of a newborn lamb to maintain body temperature after birth and likely contribute to mortality (Kerslake et al., Reference Kerslake, Kenyon, Stafford, Morris and Morel2010). We have shown that during cold exposure there was a rapid decrease in heat loss in the newborn lamb (McCoard et al., Reference McCoard, Henderson, Knol, Dowling and Webster2014b). Therefore, increasing BAT stores and/or the activity of BAT has the potential to improve survival.

Rooke et al. (Reference Rooke, Dwyer and Ashworth2008) reviewed the role of trace elements and vitamin supplementation of the ewe on various traits in the lamb including thermoregulatory capacity. Of the micronutrients evaluated (Cobalt, Copper, Iodine, Iron, Manganese, Selenium (Se), Zinc, vitamins A and E and n-3 fatty acids), Se, vitamin E and fatty acids were identified as the most likely candidates to improve lamb survival. Many of the studies undertaken have evaluated supplementation throughout pregnancy and/or have studied the responses in ewes fed a concentrate or a conserved forage-based diet, rather than within a pasture-based feeding system. As some diets are deficient in some micronutrients, for example lower vitamin E levels in dry stored feeds compared with spring fresh forage (Kivimae and Carpena, Reference Kivimae and Carpena1973), many of the studies reported in the literature may have limited application to a pasture-based system. Trace element supplementation in pasture-fed ewes can improve lamb performance (Grace and Knowles, Reference Grace and Knowles2012) however specific evaluation of thermoregulatory capacity of the neonates following maternal supplementation has not been directly evaluated.

Specific PUFA such as linoleic acid are a key energy source for BAT in lambs (Lammoglia et al., Reference Lammoglia, Bellows, Grings and Bergman1999). However, twin-bearing Taghee ewes supplemented with 12 g/ewe per day of algae-derived DHA during the last 30 days of gestation had no effect on lamb thermogenesis (Keithly et al., Reference Keithly, Kott, Berardinelli, Moreaux and Hatfield2011). In contrast, twin-bearing ewes fed rumen-protected fat which was high in saturated and monounsaturated fatty acids or high in n-6- and n-3-PUFAs at a level of 2% or 4% for the last 40 days of gestation may improve cold tolerance in newborn lambs (Chen et al., Reference Chen, Carstens, Gilbert, Theis, Archibeque, Kurz, Slay and Smith2007). It is important to note however, that when fed at 8% of the ewe diet, cold tolerance was markedly reduced coupled with reduced palmitate oxidation from BAT indicating decreased ability to oxidise fatty acids, independently of cytochrome c oxidase activity, GDP binding or uncoupling protein 1 (UCP-1) gene expression. These observations highlight the potential for PUFAs to increase thermogenesis but in a dose-dependent manner.

Iodine supplementation of the ewe can elevate thyroid hormone level in the ewe and newborn lamb (Andrewartha et al., Reference Andrewartha, Caple, Davies and MacDonald1980; Rose et al., Reference Rose, Wolf and Haresign2007) which may increase rectal temperatures in the lamb compared with supplemented lambs (Donald et al., Reference Donald, Langlands, Bowles and Smith1994). Negative relationship between maternal iodine supplementation in late gestation and immunoglobulin G (IgG) levels in the newborn lamb have been reported (Boland et al., Reference Boland, Callan, Brophy, Quinn and Crosby2006; Rose et al., Reference Rose, Wolf and Haresign2007; Boland et al., Reference Boland, Hayes, Sweeney, Callan, Baird, Keely and Crosby2008). Recently, McGovern et al. (Reference McGovern, Magee, Browne, MacHugh and Boland2015) reported that ewes supplemented with 26.6 mg/day iodine (either as calcium iodate or potassium iodide) mixed in concentrate feed as a carrier, from 119 days gestation to term, was linked to a failure of IgG absorption and thus passive transfer which may have been the result of suppressed thyroid hormone status. These results imply there are negative effects of maternal iodine supplementation in late gestation, however, the direct effect on lamb survival and subsequent impact on lamb survival and immune function later in life remain to be established. In ewes fed a 100% pasture diet, Kerslake et al. (Reference Kerslake, Kenyon, Stafford, Morris and Morel2010) reported no difference in lamb heat production following maternal iodine supplementation (i.m. injection of 1.5 ml iodised peanut oil; Flexidine 26% w/w iodine bound to ethyl esters of unsaturated fatty acids in oil 35 days before mating) despite elevating maternal iodine levels throughout gestation.

Parenteral arginine supplementation of well-fed twin-bearing ewes from 100 to 140 days of gestation has been shown to increase brown fat stores in the foetuses at 140 days gestation by about 15% (McCoard et al., Reference McCoard, Sales, Wards, Sciascia, Oliver, Koolaard and van der Linden2013) through increased fat cell hypertrophy, resulting in a 0.6°C increase in core-body temperature of twin-born lambs within 2 h of birth (McCoard et al., Reference McCoard, Wards, Koolaard and Salerno2014a). Increased BAT deposition in foetuses from under-fed ewes and diet-induced obese sheep at 125 days of gestation has also been reported in response to maternal arginine supplementation (Satterfield and Wu, Reference Satterfield and Wu2011). These studies highlight the benefits of maternal arginine supplementation to increase thermoregulatory capacity and potential survival. However, validation of these findings in the field, and quantification of the impacts on lamb survival are required.

The development of BAT and onset of BAT thermogenesis is mediated by rapid up-regulation of genes including UCP-1 around birth (Symonds et al., Reference Symonds, Sebert and Budge2011). Expression of UCP-1 is a marker of BAT thermogenesis and several factors and cofactors influence UCP-1 expression including PPARγ-co-activator-1α (PGC-1α) which regulates mitochondrial biogenesis and oxidative metabolism and PRD1-BF-1-RIZ1 homologous domain containing protein-16 (PRDM-16) which is responsible for BAT lineage determination (Kajimura et al., Reference Kajimura, Seale and Spiegelman2010). We have shown that increased BAT mass in late gestation foetuses in response to maternal arginine supplementation is associated with increased expression of UCP-1 and PRDM-16, and that plasma cortisol may up-regulate UCP-1 expression in the near-term ovine foetus (McCoard et al., Reference McCoard, Wards, Koolaard and Salerno2014a). Up-regulation of PRDM16 indicates that arginine may signal the commitment of precursor cells to the BAT lineage which in turn may have important implications maintaining neonatal core-body temperature, as well as mediating whole body metabolism, adipocyte-muscle cross-talk and energy partitioning (Satterfield and Wu, Reference Satterfield and Wu2011, Tan et al., Reference Tan, Yin, Wu, Liu, Tekwe and Wu2012). Nitrous oxide and mTOR signalling have been implicated in the arginine-induced changes in mitochondrial biogenesis and thus BAT (Tan et al., Reference Tan, Yin, Wu, Liu, Tekwe and Wu2012). In the ovine neonate mTOR signalling may play a greater role (McCoard et al., Reference McCoard, Wards, Koolaard and Salerno2014a); however, this remains to be evaluated directly.

Although non-shivering thermogenesis is the first line of defence against cold exposure in the newborn lamb, the second line of defence is shivering thermogenesis which is initiated only after body temperatures fall significantly (Alexander and Williams, Reference Alexander and Williams1968). Shivering thermogenesis can provide up to 50% of maximal heat production during cold exposure in the newborn lamb. Shivering and non-shivering thermogenesis to facilitate heat production during cold exposure in the newborn lamb are equally important, with shivering thermogenesis becoming the primary source of heat production after the first few days of life (Alexander and Williams, Reference Alexander and Williams1968). It has been postulated that adaptation to the extra-uterine environment post-birth may involve cross-talk between different muscle and fat deposits and their interaction with other organs involved in BAT function (Symonds, Reference Symonds, Sebert and Budge2013), however these interactions remain to be elucidated. Skeletal muscle and BAT may have a common origin (Seale et al., Reference Seale, Bjork, Yang, Kajimura, Chin, Kuang, Scimè, Devarakonda, Conroe, Erdjument-Bromage, Tempst, Rudnicki, Beier and Spiegelman2008) and in humans a link between muscle volume and functional BAT has been suggested in children and adolescents (Gilsanz et al., Reference Gilsanz, Chung, Jackson, Dorey and Hu2011), highlighting the potential importance of skeletal muscle growth during gestation and its contribution to BW thermoregulatory capacity and thus survivability at birth.

Twin-born lambs have reduced muscle mass compared with singletons (McCoard et al., Reference McCoard, McNabb, Birtles, Harris, McCutcheon and Peterson1997), with the divergence in muscle mass appearing after 100 days gestation (McCoard et al., Reference McCoard, Peterson, McNabb, Harris and McCutcheon2001) contributing to the lower birth weight of twins. During foetal development, skeletal muscle has lower priority, in terms of nutrient partitioning, compared with other tissues such as brain, heart and liver, resulting in muscle being more vulnerable to nutrient deficiency (Zhu et al., Reference Zhu, Ford, Means, Hess, Nathanielsz and Du2006). Newborn lambs also exhibit high rates of AA oxidation supporting the notion that low-birth weight lambs at birth are less mature compared with high birth weight lambs in some aspects of metabolic and endocrine development (Greenwood et al., Reference Greenwood, Hunt, Slepetis, Finnerty, Alston, Beermann and Bell2002).

Amino acid availability regulates skeletal muscle mass by stimulating protein synthesis and reducing protein degradation. Amino acids act as precursors of nitrogenous substances, such as polyamines and NO which likely mediate growth and development of muscle fibres (Wu et al., Reference Wu, Bazer, Burghardt, Johnson, Kim, Li, Satterfield and Spencer2010). In addition, AA exert a signalling effect on the regulating factors controlling myogenesis (Yoon and Chen, Reference Yoon and Chen2013). In the later stages of pregnancy, skeletal muscle growth increases rapidly and the foetus responds to infusion of specific (e.g. arginine) or a mix of AA by increasing protein synthesis (Liechty et al., Reference Liechty, Boyle, Moorehead, Auble and Denne1999; de Boo et al., Reference De Boo, van Sijl, Smith, Kuik, Lafeber and Harding2005). This response during foetal life appears to be associated with the plasma level of insulin in the foetus (Brown and Hay Reference Brown and Hay2006). However, in IUGR sheep models, when AA are infused directly into the foetus, net foetal protein accretion increases independently of insulin changes (Brown et al., Reference Brown, Rozance, Thorn, Friedman and Hay2012).

During late gestation, changes in specific rather than total intracellular muscle AA concentrations are associated with lower muscle mass in twins (Pacheco et al., Reference Pacheco, Treloar, Kenyon, Blair and McCoard2010). Notably, arginine and glutamine appeared to be closely related to foetal mass and the mass of the semitendinosus muscle (Pacheco et al., Reference Pacheco, Treloar, Kenyon, Blair and McCoard2010). Further, a reduction in the concentration of specific intracellular free AA such as arginine, leucine, valine and glutamine which play important roles in muscle growth, may be limiting for skeletal muscle hypertrophy in twins (Sales et al., Reference Sales2013), consistent with the correlation between the weight of the foetal semitendinosus muscle in twins with intracellular concentrations of free arginine (r=0.66, P<0.01) and glutamine (r=0.49, P<0.01) in late gestation (Sales et al., Reference Sales, Pacheco, Blair, Kenyon, Nicholas, Senna Salerno and McCoard2014).

Compared with singletons, twin foetal sheep have down-regulated mTOR signalling in late gestation which may be related to long-term restricted nutrient availability leading to reduced ribosome number and abundance of the translational machinery per ribosome (Sciascia et al., Reference Sciascia, Pacheco, Bracegirdle, Berry, Kenyon, Blair, Senna Salerno, Nicholas and McCoard2010). These results may explain, at least in part, the restricted myofibre hypertrophy and reduced muscle mass observed in twins relative to singletons. Activation of mTOR signalling in skeletal muscle is under the control of the arginine-family of AA (e.g. arginine and glutamine) and leucine (Meijer and Dubbelhuis, Reference Meijer and Dubbelhuis2004). Consistent with this notion, maternal intravenous bolus injection of arginine three times daily from 100 days gestation to birth increased the capacity for protein synthesis in foetal muscle which is associated with increased abundance of mTOR near birth (Sales, Reference Sales, Pacheco, Blair, Kenyon and McCoard2014). In the same experiment, we reported that maternal arginine administration increased core-body temperatures of the lambs within 2 h of birth (McCoard et al., Reference McCoard, Wards, Koolaard and Salerno2014a). Although arginine increased the capacity for skeletal muscle growth in lambs, the potential cross-talk between skeletal muscle growth and thermoregulatory capacity of the lambs remains to be elucidated. Furthermore, the effect of maternal arginine supplementation on lamb survival in pasture-fed ewes in a commercial farm system also remains to be determined.

Future prospects

Improved productivity and profitability of the sheep meat industry has been made possible by increasing lambing percentages. However, multiple-birth lambs suffer from IUGR which negatively impacts early-life development and growth. The application of specific nutritional components such as AA, vitamins, trace elements and PUFAs in mid-to-late gestation (summarised in Table 1) have the potential to influence traits associated with lamb survival including placental nutrient transfer and thus foetal growth, birth weight, lamb vigour and thermoregulatory capacity. Undoubtedly, further research into the utility of macro and micronutrients in pasture-fed ewes on foetal growth, lamb survival and postnatal performance, critical intervention time windows and identification of delivery routes and stages of growth that are both cost-effective and practical to implement in pasture-based grazing systems should be the focus of future research activities. Further, discovery of the role other nutrients play in regulating foetal growth and survival is required to increase our knowledge of the potential for nutraceuticals to decrease lamb mortality and morbidity. We hope the animal field will grasp this line of research and continue to expand this knowledge base and the potential it has in improving sheep production.

Table 1 Summary of the observed effects on foetal-neonatal growth and development when supra-nutritional levels of specific nutrients are supplied to multiple-bearing ewes during mid-to-late pregnancy

IgG=immunoglobulin G; PUFA=polyunsaturated fatty acid.