MEAT SCIENCE AND MUSCLE BIOLOGY SYMPOSIUM: In utero nutrition related to fetal development, postnatal performance, and meat quality of pork 1

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1 Published December 2, 2014 MEAT SCIENCE AND MUSCLE BIOLOGY SYMPOSIUM: In utero nutrition related to fetal development, postnatal performance, and meat quality of pork 1 N. Oksbjerg, 2 P. M. Nissen, M. Therkildsen, H. S. Møller, L. B. Larsen, M. Andersen, and J. F. Young Department of Food Science, Aarhus University, 8830 Tjele, Denmark ABSTRACT: Intrauterine growth restriction (IUGR) occurs naturally in pigs and leads to low birth weight of piglets due to undernutrition caused by placental insufficiency. For 2 main reasons, low birth weight causes economic loss. First, low birth weight pigs have a greater mortality and increasing the litter size causes more low birth weight piglets within litters. Second, surviving low birth weight piglets have reduced performance (i.e., ADG, feed conversion rate, and percentage meat). To develop dietary strategies for preventing IUGR, knowledge of the biological basis of IUGR is required. Muscle fiber number, formed during myogenesis, is correlated positively with performance traits and has been shown in several studies to be reduced in low birth weight pigs. Postnatal muscle hypertrophy is due to satellite cell number per fiber at birth and their rate of proliferation as well as protein deposition (i.e., protein synthesis and degradation). Previous studies and some recent ones indicate that low birth weight littermates in mice are born with fewer satellite cells and studies on pigs show that the rate of satellite cell proliferation may vary within litters. Proteomics studies show that protein synthesis and degradation is downregulated in IUGR pigs and low birth weight pigs also produce meat with less tenderness. Alternative maternal feeding strategies to prevent IUGR have been examined. Increasing maternal global nutrition had no beneficial effect on performance and muscle growth traits in several studies. Feeding excess maternal dietary protein also did not influence muscle growth traits whereas moderately decreased maternal dietary protein may decrease muscle fiber number and performance. On the other hand, addition of L-carnitine to the maternal gestation or lactation diet may increase birth and weaning weights or the muscle fiber number, respectively, in low birth weight pig offspring. Finally, promising data have been obtained on reproductive traits in pigs after addition of functional AA, such as arginine and glutamine, to the gestational diet. Although much is known about the biological basis of IUGR, we still need to learn much more about the mode of action before maternal dietary strategies can be developed to prevent IUGR. Key words: birth weight, myogenesis, protein turnover, satellite cell, tenderness 2013 American Society of Animal Science. All rights reserved. J. Anim. Sci : doi: /jas INTRODUCTION It has been estimated that global meat intake will double from 2000 to 2050 due to increased population and spending rate (FAO, 2006). However, meat production 1 Based on a presentation at the Meat Science and Muscle Biology Symposium titled In utero factors that influence postnatal muscle growth, carcass composition, and meat quality at the Joint Annual Meeting, July 15 19, 2012, Phoenix, AZ, with publication sponsored by the Journal of Animal Science and the American Society of Animal Science. 2 Corresponding author: Niels.Oksbjerg@agrsci.dk Received: September 11, Accepted: November 24, is not sustainable and life cycle assessment analysis indicates that meat production significantly contributes to CO 2 emission in decreasing order: ruminants, pigs, and poultry (Tuomisto and de Mattos, 2011). As a consequence, efficiency of meat production should be improved at all steps from conception to consumption to make meat production more sustainable. In the pig as well as in other meat producing animals in utero nutrition plays a significant role for litter performance. Over- and undernutrition, in particular, as well as nutrient imbalance may lead to intrauterine growth restriction (IUGR), which is defined as impaired growth and development of the

2 1444 Oksbjerg et al. mammalian embryo/fetus or its organs during pregnancy. In pigs, IUGR develops from d 30 to 45 until term (Kim et al., 2009), is more severe than in other meat producing animals, and is caused by placental insufficiency (Wu et al., 2006). Intrauterine growth restriction leads to low birth weight and causes economic loss for 2 main reasons; first, low birth weight pigs have a reduced survival rate and second, low birth weight littermates that survive have decreased performance i.e., reduced daily BW gain, reduced feed intake, increased feed conversion ratio (i.e., kg feed/kg BW gain), and decreased percentage of meat compared with larger littermates (Gondret et al., 2006; Nissen and Oksbjerg, 2011). In addition, nutrients restriction during fetal growth can have permanent effects on physiology and metabolism later on in life, a concept known as fetal programming (Barker, 1998). Many meat quality traits are dependent on the metabolic status at the time of slaughter (Young et al., 2009). As a consequence, IUGR pigs may differ in meat quality traits compared with mean and heavy birth weight littermates. The aim of the present review is to discuss effects of in utero nutrition on survival and muscle traits influencing muscle development and growth as well as meat quality. Previous reviews have been given on the effect of prenatal events on postnatal events (Foxcroft et al., 2006; Rehfeldt and Kuhn, 2006; Nissen and Oksbjerg, 2009b; Rehfeldt et al., 2011a,b; Campos et al., 2012). NEGATIVE CONSEQUENCES OF INTRAUTERINE GROWTH RESTRICTION IN PIG PRODUCTION Survival Rate of Intrauterine Growth Restriction Piglets During the last 2 to 3 decades selection for greater number of piglets born per litter has been performed. Under Danish conditions, litter size has increased from 13.3 piglets/litter in 2000 to 16.3 piglets/litter in 2010 (Pedersen et al., 2010). In France, the number of piglets born alive has increased from 10.9 per litter in 1991 to 12.2 per litter in 2001 (Pellois, 2002). However, increasing the litter size increases intralitter variation of birth weight, which has negative consequences for piglet survival. Milligan et al. (2002) examined the relationships between litter size, birth weight, and survival rate of low and high birth weight littermates. They reported that increasing litter size from fewer than 9 to more than 12 reduced birth weight in both low birth weight and high birth weight piglets; however, the survival rate only decreased in small littermates. Quiniou et al. (2002) examined the influence of litter size on litter mean birth weight, within litter variation in birth weight, and the percentage of piglets with a birth weight less than 1 kg. Mean birth weight decreased steadily from 1.59 kg to 1.26 kg when the litter size increased from fewer than 11 to more than 16 piglets per litter. At the same time, the within litter variation in birth weight increased from 0.26 to 0.30 kg, and the percentage of piglets born that weighed <1 kg increased from 7 to 23%. This may be explained by a decreased uterine blood flow for each fetus (Père and Etienne, 2000). Because the within litter variation in birth weight is negatively related with survival (Wolf et al., 2008), it is most likely that genetic improvement in litter size will increase the number of small piglets born followed by increasing their mortality rate. The decreased survival rate of smaller littermates may be related, in part, to less ability to compete for the best teats, lower amounts of colostrum, and decreased milk ingestion leading to low nutritional status (Theil et al., 2011; Campos et al., 2012). Performance of Intrauterine Growth Restriction Piglets Several studies have almost consistently shown that pigs born small have a reduced performance (see Nissen and Oksbjerg, 2009b) compared with piglets born large although the numerical difference may vary depending on feeding strategies, breed, litter size, and growth periods. In our recent study (Nissen and Oksbjerg, 2011), which is representative for the cited studies, we found that ADG was reduced by 30 g/d in low birth weight pigs in the lactation period and that this reduction in daily gain persisted from weaning to slaughter at 150 d of age by 87 g/d compared with heavy birth weight pigs. During the postweaning period, feed intake was reduced by 160 g/d and feed conversion ratio increased by 80 g kg gain in low birth weight pigs compared with heavy birth weight piglets. In that study we did not find any difference in meat percentage of the carcasses between low birth weight pigs and heavy weight pigs, which was in line with Gondret et al. (2005). However, others have found a reduction in meat percentage of low birth weight pigs (Gondret et al., 2006; Rehfeldt et al., 2008) compared with larger littermates. If a fetus has been nutrient restricted during fetal development and fed a diet rich in nutrients during postnatal life, then the offspring is mismatched to the postnatal environment (Gluckman and Hanson, 2006). The smaller birth weight together with the reduced performance of these pigs may indicate a reduced requirement for dietary protein. In our recent study (Nissen and Oksbjerg, 2011), we therefore fed low and high birth weight pigs from weaning to slaughter at d 150 either a diet adequate in protein for growing finishing

3 In utero nutrition and effects on offspring 1445 pigs or a diet containing 30% less protein. However, we found no interactions between birth weight groups and dietary protein level on any performance traits. This indicated that low and heavy birth weight pigs responded to the reduction in dietary protein in a similar way and we suggested that the requirement for dietary protein was similar between the 2 birth weight groups. Changes in Muscle Traits of Intrauterine Growth Restriction Piglets Muscle fiber traits related to growth as a consequence of reduced performance of low birth weight pigs have been studied in several experiments. Muscle tissue develops during gestation (Rehfeldt et al., 2011a,b). The various steps in myogenesis encompass mesenchymal stem cell commitment to myoblasts, myoblast proliferation, alignment, fusion, and differentiation to primitive myotubes. In pigs, 2 populations of muscle fibers develop. The first population, the primary myofibers, develop between d 25 and 50 of gestation. These primary fibers act as a template for the formation of the next population, the secondary fibers. Some researchers (Handel and Stickland, 1987) have suggested that the number of primary fibers is genetically determined and does not vary within litter whereas others have found variation in the number of primary fibers within litter (Nissen and Oksbjerg, 2004; Bérard and Bee, 2010; Rehfeldt et al., 2012a). In pigs, the ratios of secondary to primary fibers vary between 20:1 and 24:1. At d 80 to 90 of gestation, formation of fibers is completed (Stickland et al., 2004) and the number of fibers at birth is directly related to birth weight (r = 0.47; Rehfeldt and Kuhn, 2006), daily BW gain (r = 0.42; Dwyer et al., 1993; Pedersen et al., 2001; Rehfeldt and Kuhn, 2006), feed conversion ratio (r = 042; Dwyer et al., 1993), and meat percentage (r = 0.38; Rehfeldt et al., 2000). These correlations are less than expected, for 2 contributing reasons. First, measuring muscle fiber number in large animals is very tedious and time consuming and is based on measurement of the muscle cross-sectional area divided by the mean fiber area. In slaughter pigs, it is impossible to count all fibers in the cross-sectional area, but a number of subsamplings are performed. However, the number of subsamplings is critical for the accuracy with which the muscle fiber number is determined. In experiments with slaughter pigs, the number of subsamples varies from 1 to 5. These problems were addressed by Cerisuelo et al. (2007), who reported that for muscle fiber number, 4 to 5 subsamples were required to obtain a high repeatability. This is seldom done and may increase the error in the estimation of muscle fiber number and consequently reduce the correlation between the number of fibers and performance traits. Second, the correlation should be calculated in relation to muscle growth rate instead of daily gain because the muscle growth rate can be altered without changing daily gain. Although the biological basis for reduced growth rate of small littermates is unknown, 1 hypothesis proposed is that it is related to a reduction in muscle fiber number because several studies have shown that the reduced performance of low birth weight piglets compared with heavy birth weight piglets is associated with a reduced number of muscle fibers in indicator muscles, semitendinosus or LM (Handel and Stickland, 1988; Bee, 2004; Gondret et al., 2005, 2006; Rehfeldt and Kuhn, 2006; Lösel et al., 2009; Bérard and Bee, 2010). Rehfeldt and Kuhn (2006) reported on the total DNA in semitendinosus of newborn piglets and found that low birth weight piglets had less total DNA content than mean and heavy birth weight piglets. These authors therefore suggested that the reduced number of muscle fibers in low birth weight piglets was caused by decreased myoblast proliferation during myogenesis. However, some studies indicate that the decrease in muscle growth by reduced number of muscle fibers is partially compensated by increased cross-sectional area of muscle fibers (Gondret et al., 2005, 2006; Rehfeldt and Kuhn, 2006). The reason for this is discussed by Rehfeldt and Kuhn (2006). However, the pigs in the studies by Gondret et al. (2005, 2006) were slaughtered and analyzed at the same market weight. Consequently, the low birth weight pigs were 10 to 12 d older and if the cross-sectional area of muscle fibers reflects the growth rate of muscle, the comparison should be made at the same age. In addition, some low birth weight piglets may develop as many muscle fibers as their heavy birth weight littermates; Handel and Stickland (1988) showed that several small littermates exhibited catch-up growth because they had as many muscle fibers as their larger littermates. In the study by Nissen and Oksbjerg (2004), pigs were grouped within litter by carcass weight at slaughter (i.e., d 150) as opposed to birth weight to obtain the greatest variation in muscle fiber number and cross-sectional area of muscle fibers within litter. This way of grouping allows some low weight piglets to appear in the mean and heavy carcass weight groups. It was found that heavy carcass weight pigs had a greater number of muscle fibers compared with mean and low carcass weight pigs, the latter 2 groups having similar numbers of muscle fibers. The mean cross-sectional fiber area was similar for heavy and mean carcass weight pigs but greater than low carcass weight pigs. The lower cross-sectional fiber area may be related with changes in postnatal muscle growth. Besides muscle fiber number, postnatal muscle growth is related to protein deposition and satellite cell

4 1446 Oksbjerg et al. proliferation (Allen et al., 1979). Protein deposition (i.e., hypertrophy and growth in length) is the difference between 2 dynamic processes, that is, the rates of protein synthesis and protein degradation. Satellite cells are mononucleated cells residing between the basal lamina and the sarcolemma. When satellite cells proliferate, 1 or both daughter cells may fuse with the adjacent fibers and add additional DNA to the fibers. Satellite cell proliferation is important for postnatal growth. To exemplify this, irradiation (25 Gy), which inhibits mitotic activity, reduced muscle hypertrophy of pectoralis muscle of tom turkeys (Mozdziak et al., 1997). In a study by Nissen and Oksbjerg (2004), the content of muscle DNA per fiber was estimated as total DNA per muscle fiber in the semitendinosus. The results showed that the total content of DNA per fiber was less in the low carcass weight pigs compared with high carcass weight pigs at slaughter. Because the satellite cell is the main source of DNA present in muscle at slaughter, we speculated that the decreased total content of DNA per fiber was a consequence of a decreased satellite cell proliferation and incorporation into the muscle fibers of low carcass weight pigs. To investigate this in more detail, satellite cells were isolated from semimembranosus of low, mean, and heavy carcass weight pigs originating from 8 litters at 6 wk of age (i.e., at approximately 12 kg BW). These studies showed that satellite cells from low carcass weight pigs proliferated at a slower rate than did satellite cells from either mean or heavy carcass weight pigs, which were similar to each other. Also their ability to differentiate into myotubes was slower than satellite cells from mean and heavy carcass weight pigs (Nissen and Oksbjerg, 2009a). In a study by Brown and Stickland (1993), satellite cells were compared in the biceps brachii muscle of mice divergently selected for growth and in small and large littermates within each selection group. Electron microscopy of muscle sections revealed that the number of satellite cells per fiber was similar between selection groups but less in the smaller littermate compared with the larger littermates. In mice, satellite cells have been identified at d 16 of gestation, that is, during the period when fetuses experience most variation in nutrition due to variation in blood supply. All together, the results indicate that satellite cells may contribute to the variation in muscle growth within litter. However, more research is needed for clarifying the role of the satellite cell in IUGR animals. Changes in protein turnover (i.e., synthesis and degradation), independent of satellite cells, also may be involved in IUGR. Therefore, Wang et al. (2008) used proteomics to explore changes in protein expression in new born piglets and found 12 differentially expressed proteins in muscle tissue from newborn low birth weight piglets compared with mean birth weight piglets. Their results indicated that protein synthesis and degradation may be elevated in mean birth weight piglets compared with low birth weight piglets. In support of this, preliminary data from our laboratory using proteomic technology indicates a greater protein turnover in muscle of high birth weight female fetuses at d 116 of gestation by upregulating proteins important for protein synthesis and degradation (e.g., elongation factor TY and proteasome subunit α type I; N. Oksbjerg, M. Andersen, H. S. Møller, L. B. Larsen, and J. F. Young, unpublished data). In growing and finishing pigs, the protein turnover is also increased in female pigs having free access to the diet compared with female pigs on restricted feeding (Therkildsen et al., 2004). With the use of explorative high field nuclear magnetic resonance metabolomics, we found and confirmed with gas chromatography-mass spectrometry that myo-inositol and D-chiro-inositol were increased in plasma of low weight fetuses at d 110 of gestation compared with heavy weight fetuses (Nissen et al., 2011). Because these inositols have been implicated in glucose intolerance and type 2 diabetes, these metabolites may be interesting as early predictors for fetal programming of metabolic diseases in adult life in humans and pigs. To better understand the mode of action of stunted growth, omics technologies may be valuable (Wang et al., 2008; Nissen et al., 2004; Oster et al., 2012). The IGF Axis in Intrauterine Growth Restriction Piglets Muscle tissue expresses various components of the IGF system (Theil et al., 2006), which regulates proliferation and differentiation of myoblasts and satellite cells (Oksbjerg et al., 2004; Rehfeldt et al., 2011a). Because both proliferation and differentiation of satellite cells in vitro were affected by birth weight (Nissen and Oksbjerg, 2009a), the IGF system may at least partly play a role in IUGR piglets during development. In 1 study, a greater expression of several components in the IGF system [i.e., type I IGF-receptor, IGF-binding protein (IGFBP)-3 type II IGF-receptor, and IGFBP-5] were observed (Tilley et al., 2007) and another study reported 24% less IGF-I in plasma from IUGR pigs compared with heavy birth weight pigs (Gondret et al., 2005), indicating a role for the IGF system in IUGR. Meat Quality of Intrauterine Growth Restriction Pigs Meat quality can be divided into technological quality traits such as color, water holding capacity and drip loss, protein content and its characteristics, lipid content and

5 In utero nutrition and effects on offspring 1447 its characteristics, content of connective tissue and its characteristics, antioxidant status, shear force, and sensory quality such as appearance, flavor, tenderness, and juiciness (Andersen et al., 2005). The rate and the extent of the ph decline postmortem, measured as ph measured 45 min. postmortem and 24 hours postmortem (ph24), respectively, have a large impact on important meat quality traits (e.g., water holding capacity and meat color). The postmortem ph decline is dependent on the concentration of high energy phosphate compounds and glycogen at the time of slaughter. For postmortem metabolism, ATP is formed by anaerobic reactions and is due to glycogen breakdown (i.e., glycolysis) to lactate and H +. Initially, metabolism for generating ATP also comes from phosphorus compounds (i.e., creatine phosphate and ADP) present in muscle at the time of slaughter. Creatine phosphate + ADP can be converted to ATP, and the concentrations of these high energy rich compounds significantly influence the onset of glycolysis. If the animal is rested before slaughter, the concentration of phosphorus compounds are high and this will delay the onset of glycolysis. In contrast, if the animal is stressed before slaughter, resulting in reduced concentrations of creatine phosphate and ATP, and the onset of glycolysis will start immediately (Young et al., 2009). In the latter situation, the carcass will still be hot and in combination with low ph this will cause protein denaturation by which the myosin bridges will shrink and reduce the myofibrillar space and the water will be squeezed out and increase drip loss. Furthermore, this situation will also cause paler meat. Nissen and Oksbjerg, (2011) found a higher ph 45 in low birth weight pigs compared with heavy birth weight pigs whereas others found no difference between birth weight groups (Gondret et al., 2005, 2006). On the other hand, Rehfeldt et al. (2008) reported a decrease in ph 45 in low birth weight pigs. The reasons for these inconsistent results are not clear, but the use of various breeds, stunning methods, and level of stress before slaughter may have been contributing factors. Drip loss of the meat postmortem was either unaffected by birth weight (Gondret et al., 2005, 2006; Nissen and Oksbjerg, 2011) or increased in low birth weight or increased in low birth weight pigs compared with mean birth weight pigs (Rehfeldt and Kuhn, 2006; Rehfeldt et al., 2008). The only color determinant that has been reported to be affected by birth weight is lightness (L*). Therefore, a marginally greater L* of low birth weight pigs in the LM was reported by Rehfeldt and Kuhn (2006) whereas Nissen and Oksbjerg (2011) reported a slightly lower L* of low birth weight pigs, in agreement with a higher ph 45, compared with heavy birth weight pigs. Here it is important to emphasize that consumers cannot distinguish these small differences in meat color. Table 1. Dietary composition and calculated chemical composition of the gestation and lactation diets Dietary level of protein 1 Ingredients, g/kg Gestation diets Adequate protein Low protein Lactation diets Adequate protein Low protein Barley Wheat 860 Soybean meal Wheat bran 60.7 Corn starch Green flour Animal fat Molasses L-lysine (chalk) Threonine (chalk) Monocalcium phosphate Chalk Salt Vitamin premix Calculated chemical composition, g/kg CP Crude fat Crude fiber Crude ash Starch Sugars MJ net energy/kg diet (FUp) 4 NE KJ/kg Adequate protein = 136 g CP/kg diet; Low protein = 92 g CP/kg diet. Gilts of purebred Danish Landrace mated to purebred Danish Landrace boars were fed during pregnancy as well as during lactation diets containing either an adequate level of CP (n = 8) or diets containing a 30% less CP (n = 8). The diets were isoenergetic. Low birth weight and heavy birth weight piglets of both genders (female and entire male pigs) were penned individually from weaning at d 28 until slaughter at d 150 of age. During rearing the pigs were fed either a diet adequate in protein or a diet where the protein level was reduced by 30%. The study was conducted as a 2 (dietary protein during gestation and lactation) 2 (birth weight) 2 (gender) 2 (postnatal dietary protein level) factorial design. Feed uptake was recorded and the pigs were weighed at birth, weaning, and slaughter. Here we report the effect of maternal dietary protein during pregnancy as well as during the lactation period on the performance of the offspring (Table 2) and the interactions between gender and maternal dietary proteins on meat quality traits (Table 3). Statistical analyses were conducted using SAS (SAS Inst. Inc., Cary, NC) using the MIXED procedure. The model included the fixed effects of maternal dietary protein, birth weight, gender, and postnatal dietary protein levels as well as their interactions when significant. Gilts within maternal dietary protein level were included as a random factor. Slaughter weight was included as a covariate in the model for meat quality and muscle enzyme when significant. The effect of birth weight and postnatal dietary protein level has been published recently (Nissen and Oksbjerg, 2011). 2 In the form of 40% lysine and 50% chalk. 3 In the form of 50% threonine and 40% chalk. 4 One FUp = 7.72 MJ NE.

6 1448 Oksbjerg et al. Tenderness (i.e., sensory score) and instrumental shear force are dependent on proteolysis by the calpain system, the concentration of intramuscular fat, and the content and characteristics of connective tissues. Thus, meat tenderness increases when intramuscular fat increases up to 3%. Intramuscular fat has been reported to be increased (Karunaratne et al., 2005; Rehfeldt et al., 2008) or unchanged in LM (Gondret et al., 2005, 2006), increased in semitendinosus (Gondret et al., 2005, 2006), and unchanged in rhomboideus (Gondret et al., 2005, 2006) in muscles of low birth weight pigs compared with heavy birth weight pigs. In LM, increased collagen (Gondret et al., 2006) and type II collagen (Karunaratne et al., 2005) were observed in low birth weight pigs. Degradation of myofibrillar proteins, causing tenderization of meat during aging, is dependent on proteolytic enzymes. In addition to being a determining enzyme system for protein degradation in vivo, the calpain system is believed to also limit the tenderization of the meat. Calpain exists in various isoforms, including micromolar calcium-dependent calpain and millimolar calcium-dependent calpain. Furthermore, the endogenous inhibitor of the calpains, calpastatin, regulates the activities of the calpains. Because the calpains are determining for protein degradation in vivo and also in postmortem tenderization (Koohmaraie et al., 2002), the muscle protein degradation rate in vivo is related to tenderness. In support of this, it was found that increased feeding level and compensatory growth response increases in vivo protein degradation and tenderness (Therkildsen et al., 2004). Thus, it was demonstrated that the tenderness decreased (Gondret et al., 2006) and the shear force increased (Nissen and Oksbjerg, 2011) in loin samples from low birth weight pigs compared with heavy birth weight pigs. In the study by Nissen and Oksbjerg (2011), it was found that the calpastatin was upregulated, meaning that the activity of the calpains may have been reduced in low birth weight pigs. In support of this, an increased level of calpastatin in LM from fetal calves at d 125 of gestation in nutrientrestricted cows was increased whereas the activity of micromolar and millimolar calpain were unchanged (Du et al., 2004). Thus, the net effect of changes in calpain activity, content of heat stable and total collagen, and intramuscular fat may explain the less tender meat in low birth weight pigs compared with heavy birth weight pigs. PREVENTION OF INTRAUTERINE GROWTH RESTRICTION Several studies have been conducted with the purpose of preventing IUGR in piglets. These studies have focused first on effects of maternal dietary global feed intake and under- or overnutrition on performance and muscle traits and second on dietary addition of L-carnitine and functional AA to the gestational diet on reproductive traits and muscle traits. Global Feeding Level during Gestation Provision of maternal nutrients directed to the fetus is critical for fetal growth. In general, pregnant sows are fed restrictively and energy requirements are between 20 and 35 MJ ME, corresponding to 2 to3 kg concentrate/d. In studies where gilts or sows have been offered a low-energy intake, birth weight was reduced (Atinmo et al., 1974; Buitrago et al., 1974). In other animal species, such as rodents, a decreased number of muscle fibers were reported when rats were born to dams that were undernourished during gestation and lactation (Bedi et al., 1982). Feeding sows beyond the requirements has been suggested as a mean for preventing natural occurring IUGR. Thus, Dwyer et al. (1994) found an increased number of secondary fibers in semitendinosus of especially the low birth weight pigs followed by increased daily BW gain and improved feed conversion ratio of offspring born to sows fed twice the amount of feed of that required from d 25 to d 50 of gestation. However, other studies, where muscle fiber number and performance of offspring born to sows fed increased feed in specific windows (Nissen et al., 2003; Bee, 2004; Cerisuelo et al., 2009; McNamara et al., 2011), were unable to confirm the results by Dwyer et al. (1994). McNamara et al. (2011) found a decrease in daily BW gain of offspring born to sows fed twice the requirement and Nissen et al. (2003) found that low carcass weight pigs born to sows fed ad libitum from d 25 to 50 of gestation had a decreased estimated muscle deposition rate compared with those born to control fed sows. Finally, Cerisuelo et al. (2009) found that a decreased number of both primary and secondary muscle fibers were compensated with slightly larger cross-sectional areas of mainly type IIb fibers, without affecting performance, in response to increased maternal feeding level during d 45 to 85 of gestation. In agreement with these studies, Nissen et al. (2005) did not find any changes in the IGF-I, IGF-II, and IGFBP-1 to -4 in umbilical cord blood of fetuses collected at d 50 or 70 in response to increased maternal feed intake. Meat Quality and Global Feeding Changes in meat quality traits of pigs born to sows given increased amount of feed during gestation are minimal. No changes in ph24, drip loss, color traits L*, redness (a*), and yellowness (b*), or pigment content was reported (Nissen et al., 2003). Also Cerisuelo et al.

7 In utero nutrition and effects on offspring 1449 (2009) found no effect on ph 45 in semimembranosus and LM; however, ph 24 was slightly increased and L* slightly reduced in offspring born to sows fed increased energy level. McNamara et al. (2011) found an increase in ph 45 in semimembranosus of pigs born to sows fed increased amount of the diet in various windows during gestation. Color traits and drip loss also was affected by increased maternal feed intake. Dietary Protein during Gestation Severe maternal protein restriction either in selected periods or throughout gestation causes stunted fetal growth and a decrease in postnatal growth and mature size of the offspring (Atinmo et al., 1974; Pond et al., 1990). However, recent studies with more moderate gestational protein restriction (Table 1) have been performed. As shown in Table 2, we found no severe effects on postnatal growth performance in offspring after feeding pregnant gilts a 30% reduced dietary protein level during gestation and during lactation. However, there was a tendency (P < 0.08) for ADG to be reduced by 40 g/d, but feed intake, feed conversion ratio, and meat content of the carcasses were changed by maternal protein feeding. In addition, recent experiments on the effect of various maternal dietary protein levels throughout gestation on muscle traits of offspring were published recently (Rehfeldt et al., 2012a,b). In those experiments, gilts were fed isoenergetic diets during gestation with varying protein levels, including a low (6.5%), adequate (12%), or high (30%) level of CP. Although pigs born to gilts fed either excess or reduced dietary protein had decreased birth weight, pigs born to gilts fed excess protein had similar number of primary and secondary fibers, total muscle fiber number, and total content of muscle DNA compared with neonates born to gilts fed adequate CP (Rehfeldt et al., 2012a). These similarities persisted to slaughter at 188 d of age (Rehfeldt et al., 2012b). In contrast, feeding gilts a low protein diet resulted in a decreased number of primary and secondary fibers, total muscle fiber number, and total content of DNA in neonatal offspring (Rehfeldt et al., 2012a) compared with pigs born to gilts fed control diet. These differences persisted until slaughter and indicated less potential (i.e., number of muscle fibers) for muscle growth. In line with this, a slightly decreased carcass weight at slaughter together with reduced meat content in offspring born to low protein gilts indicates that the muscle growth rate had been reduced. As discussed earlier, satellite cells may also be affected by the maternal nutrition and the total DNA content of the semitendinosus was measured at d 83 and 188, and the accumulation of DNA during this period was 465, 449, and 379 mg in offspring born to gilts fed adequate, excess, or limited protein, respectively, indicating decreased satellite cell proliferation in offspring born to low protein gilts, at least from d 83 to 188. Meat Quality and Dietary Protein Level during Gestation Meat quality traits have also been addressed in response to the protein level of the gestational diet. Thus, in the study by Rehfeldt et al. (2012b) it was found that the shear force of the loin was reduced (i.e., indicative of increased tenderness) in the offspring born to low and high protein fed gilts whereas the maternal dietary protein level did not affect ph 45, ph 24, water-holding capacity, color, and intramuscular fat. Our results (Table 3) did not show any main effects on meat quality traits in offspring related to maternal dietary protein; however, there were some interesting gender maternal dietary protein level interactions in meat quality traits, and our data indicate that the meat quality traits Table 2. Performance of pigs born to gilts feed either an adequate protein or a diet with reduced (Low) protein amount 1 Dietary maternal protein content 2 Adequate Low Variable protein protein SEM P-value Birth wt, kg Weaning wt, kg Gain (birth to weaning), g/d Performance from weaning to slaughter at d 150 BW gain, g/d Feed intake, kg/d Kg feed/kg BW gain Meat, % Gilts of purebred Danish Landrace mated to purebred Danish Landrace boars were fed during pregnancy as well as during lactation diets containing either an adequate level of CP (n = 8) or diets containing a 30% less CP (n = 8). The diets were isoenergetic. Low birth weight and heavy birth weight piglets of both genders (female and entire male pigs) were penned individually from weaning at d 28 until slaughter at d 150 of age. During rearing the pigs were fed either a diet adequate in protein or a diet where the protein level was reduced by 30%. The study was conducted as a 2 (dietary protein during gestation and lactation) 2 (birth weight) 2 (gender) 2 (postnatal dietary protein level) factorial design. Feed uptake was recorded and the pigs were weighed at birth, weaning, and slaughter. Here we report the effect of maternal dietary protein during pregnancy as well as during the lactation period on the performance of the offspring. Statistical analyses were conducted using SAS (SAS Inst. Inc., Cary, NC) using the MIXED procedure. The model included the fixed effects of maternal dietary protein, birth weight, gender, and postnatal dietary protein levels as well as their interactions when significant. Gilts within maternal dietary protein level were included as a random factor. Slaughter weight was included as a covariate in the model for meat quality and muscle enzyme when significant. The effect of birth weight and postnatal dietary protein level has been published recently (Nissen and Oksbjerg, 2011). 2 Adequate protein = 136 g CP/kg diet; Low protein = 92 g CP/kg diet.

8 1450 Oksbjerg et al. are improved in male pigs when born to gilts fed a low protein diet. Thus, the ph 45 was elevated and thawing loss and shear force decreased in male pigs born to gilts fed a low protein diet compared with male offspring born to gilts fed an adequate dietary protein level. The increase in ph 45 in LM may be due to a different ability to cope with stress before slaughter. The better muscle oxidative capacity, the lesser the depletion of the phosphorus compounds. Because the muscle citrate synthase activity was greater in the male pigs, this may contribute to explain the higher ph 45 in male pigs born to gilts fed a low protein diet and this, in turn, may increase the water-holding capacity. In addition, the decreased activity of lactate dehydrogenase may likewise reduce the rate of glycolysis in male offspring born to gilts fed the low protein diet. The reduction in shear force in the loin of the male pigs born to gilts fed the low protein diet could not be explained by the calpastatin because it was unchanged. However, micromolar calcium-dependent calpain was not measured in that study. Regarding female pigs, our data are in agreement with those of Rehfeldt et al. (2012b) except for shear force, which was decreased. Again the discrepancies may be due to the gender, aging time, and stunning methods. As described earlier no simple means to prevent IUGR can be derived from the results on maternal global nutrition and maternal dietary protein level on fetal growth and subsequent postnatal growth of the small littermates. However, studies with bioactive compounds have recently emerged. These encompass dietary L-carnitine, L-arginine, and glutamine and porcine growth hormone. L-carnitine L-carnitine is active in the transport of longchain fatty acids into the mitochondrial matrix for gestational diet have been studied and birth weight as well as weaning weight have been reported to be reported that feeding 400 mg/d L-carnitine by syringe at weaning dependent on birth BW group. Thus, the control of low birth weight piglets had, as expected, a weight littermates. However, when small piglets were equaled that of the mean birth weight piglets (Lösel et al., 2009). It remains to be studied whether increased until market weight and, likewise, if it is possible to increase piglet uptake of carnitine by feeding the sow L-carnitine during gestation and suckling. These need to be studied to explore the positive effect of L-carnitine. L-arginine Besides being used for protein synthesis, L-arginine is also a precursor for compounds with biological activity, such as nitric oxide ( ) and polyamines. The conversion epithelial nitric oxide synthase. In addition, arginine can also be converted to ornithine and further to polyamines (i.e., putrescine, spermidine, and spermine) catalyzed by arginase and ornithine decarboxylase, respectively (Wu et al., 2006). Nitric oxide is a potent vasodilator and stimulates angiogenesis. Because both dilation per se and increased angiogenesis reduce resistance toward protein levels and offspring gender on meat quality traits 1 Dietary maternal protein 2 Adequate protein Low protein P- Variable Male Female Male Female SEM value 3 ph b 6.25 a 6.20 a 6.19 ab Thawing loss, % 11.6 a 9.7 b 10.6 ab 10.3 ab Shear force, N 55.0 a 49.5 ab 45.5 b 50.3 ab CS activity a 4.02 b 3.56 ab 3.32 ab LDH activity 6 1,602 a 1,607 a 1,445 b 1,737 c Calpastatin 7 1,733 2,076 1,880 2, NS 8 Means bearing different superscripts differ (P < 0.05). 1 Gilts of purebred Danish Landrace mated to purebred Danish Landrace boars were fed during pregnancy as well as during lactation diets containing either an adequate level of CP (n = 8) or diets containing a 30% less CP (n = 8). The diets were isoenergetic. Low birth weight and heavy birth weight piglets of both genders (female and entire male pigs) were penned individually from weaning at d 28 until slaughter at d 150 of age. During rearing the pigs were fed either a diet adequate in protein or a diet where the protein level was reduced by 30%. The study was conducted as a 2 (dietary protein during gestation and lactation) 2 (birth weight) 2 (gender) 2 (postnatal dietary protein level) factorial design. Feed uptake was recorded and the pigs were weighed at birth, weaning, and slaughter. Here we report the effect of maternal dietary protein during pregnancy as well as during the lactation period on the performance of the offspring (Table 2) and the interactions between gender and maternal dietary proteins on meat quality traits (Table 3). Statistical analyses were conducted using SAS (SAS Inst. Inc., Cary, NC) using the MIXED procedure. gender, and postnatal dietary protein levels as well as their interactions when random factor. Slaughter weight was included as a covariate in the model for and postnatal dietary protein level has been published recently (Nissen and 2 Adequate protein = 136 g CP/kg diet; Low protein = 92 g CP/kg diet. 3 offspring gender. 4 ph 45 = ph measured 45 min. postmortem. 5 CS = citrate synthase (μmol NAD+/NADH per gram of wet muscle). 6 LDH = lactate dehydrogenase (μmol NAD+/NADH per gram of wet muscle). 7 Florescent units per milligram muscle. 8

9 In utero nutrition and effects on offspring 1451 of L-arginine may increase blood flow across the placenta and, in turn, increase the transfer of nutrients from the mother to the fetus. Polyamines stimulate cell proliferation and differentiation of muscle cells and may, therefore, have a positive impact on myogenesis. Thus, lower levels of arginine and polyamines were found in muscles of IUGR piglets (Wu et al., 2010). Dietary inclusion of 1% of L-arginine from d 30 and throughout gestation increased the reproductive performance of sows by increasing the total number of piglets born alive per litter by 2 as well as increasing the litter weight and reducing the number of piglets born dead by 1.2. However, the variation in birth weight was not affected by dietary inclusion of L-arginine (Mateo et al., 2007). In further studies Bérard and Bee (2010) investigated whether dietary inclusion of L-arginine between d 14 and 28 of gestation had any influence on the muscle tissues development of the offspring. They found that at d 75 of gestation, the number of primary fibers increased and the secondary to primary fiber ratio decreased in the semitendinosus of offspring born to sows treated with L-arginine. Glutamine, like arginine, belongs to the group of functional AA and may stimulate protein synthesis via mammalian target of rapamycin signaling and by stimulating the enzyme ornithine decarboxylase, thereby promoting conversion of ornithine to polyamines that, in turn, stimulate DNA and protein synthesis (Wu et al., 2010). Thus, when a mixture of 8 g L-arginine and 12 g glutamine was added as top dressing to the gestational basal diet of 2 kg/d from d 30 to 114 of gestation, the total number of piglets born alive, total litter weight at birth for all piglets born, and litter weight for piglets born alive were increased. In this experiment, the variation in birth weight among all piglets born and among all piglets born alive was decreased (Wu et al., 2010). It would be interesting to examine whether L-arginine and L-carnitine act additively, antagonistically, or synergistically. However, more research exploring the mode of action of L-arginine, glutamine, and L-carnitine to prevent IUGR would be very valuable. Porcine Growth Hormone Porcine GH has also been used in research to prevent IUGR. However, from the time of the review by Rehfeldt et al. (2004), no additional data has been generated, to our knowledge. Therefore, readers are referred to Rehfeldt et al. (2004). SUMMARY AND CONCLUSIONS Small littermate piglets occurring naturally at birth cause economic loss in pig production for mainly 2 reasons: 1) a decreased survival rate and 2) surviving littermates born small have a poorer postnatal growth potential. The biological basis for the stunted growth, reduced feed intake, poorer feed conversion ratio, and lower meat percentage in surviving piglets born small is often related to reduced number of muscle fibers. Some compensation may occur as the cross-sectional area of muscle fibers may increase in low birth weight pigs. Only a few studies have addressed the contribution of postnatal growth traits (i.e., satellite cell proliferation and protein turnover) to the reduced performance. However, satellite cells may be involved because low birth weight littermates of mice are born with fewer satellite cells and because there is intralitter variation in satellite cell proliferation in growing pigs. Protein turnover (i.e., synthesis and degradation) may also be affected in low birth weight littermates because it has been demonstrated that proteins involved in protein synthesis and protein degradation were downregulated compared with mean birth weight pigs. Meat quality traits of small littermates have shown inconsistent results; however, the tenderness of meat from small littermates may be less, likely due to decreased protein degradation. Effective means to prevent development of small littermates has not yet been successful. Thus, overor undernutrition globally or for dietary protein has not given rise to interventions. In addition, low dietary protein level may program meat quality in male pigs. However, recent data on maternal dietary L-carnitine, L-arginine, and glutamine are promising for enhancing reproductive traits. 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