Journal of Integrative Agriculture 2016, 15(0): Available online at ScienceDirect

Journal of Integrative Agriculture 2016, 15(0): 60345-7 Available online at www.sciencedirect.com ScienceDirect RESEARCH ARTICLE Dietary arginine supplementation in multiparous sows during lactation improves the weight gain of suckling piglets ZHU Cui 1, 2, GUO Chang-yi 1, GAO Kai-guo 1, WANG Li 1, CHEN Zhuang 2, MA Xian-yong 1, JIANG Zong-yong 1, 2 1 Key Laboratory of Animal Nutrition and Feed Science (South China), Ministry of Agriculture/Institute of Animal Science, Guangdong Academy of Agricultural Sciences, Guangzhou 510640, P.R.China 2 Agro-Biological Gene Research Center, Guangdong Academy of Agricultural Sciences, Guangzhou 510640, P.R.China Abstract This study investigated the effects of dietary arginine (Arg) supplementation, just during lactation, on sow and litter performance, plasma concentrations of metabolites and hormones, and milk yield and composition in multiparous sows. Thirty-one sows were randomly assigned to 3 dietary treatments supplemented with 0.0 (control, n=10), 0.5% (n=10), or 1.0% (n=11) L-Arg-HCl, respectively. Experimental diets were provided to the sows from d 3 to 21 of lactation. Plasma and milk samples were collected at d 14 and 21 of lactation. The average daily gain (ADG) of piglets from sows fed diets supplemented with 0.5 or 1.0% L-Arg-HCl at d 3 to 14 of lactation, were higher than that of controls (P<0.05). Maternal supplementation with 1.0% L-Arg-HCl also increased ADG of piglets between d 3 and 21 of lactation than that of the controls (P<0.05). There was no significant effect of supplementation on average daily feed intake (ADFI), body weight loss, and backfat thickness loss of lactating sows. Supplementation with 0.5 or 1.0% L-Arg-HCl had a trend towards increasing milk yields and milk fat contents (0.05<P<0.10); milk protein and lactose were unchanged. Supplementation with 1.0% L-Arg- HCl increased plasma concentrations of prolactin and insulin in sows at d 14 and 21 of lactation, and plasma concentrations of non-esterified fatty acid (NEFA), insulin-like growth factor-1 (IGF-1), and nitric oxide (NO) in sows at d 21 of lactation, when compared to the controls (P<0.05). Supplementation with 1.0% L-Arg-HCl increased IGF-1 and spermine in milk at d 14 of lactation, relative to the controls (P<0.05). Plasma Arg concentrations at d 14 and 21 of lactation, as well as plasma NO level and milk IGF-1 at d 21 of lactation, were increased, while plasma urea nitrogen (PUN) concentration at d 14 and 21 of lactation was decreased, by supplementation with 0.5 or 1.0% L-Arg-HCl when compared to the controls (P<0.05). Collectively, dietary supplementation of multiparous sows with Arg, just during lactation, is beneficial for enhancing litter weight gain but the complete mechanism remains to be determined and may involve the maternal endocrine changes and milk polyamines contents. Keywords: arginine, lactating sows, hormone, suckling piglets, milk composition, polyamines Received 25 April, 2016 Accepted 2 June, 2016 ZHU Cui, E-mail: juncy2010@gmail.com; Correspondence JIANG Zong-yong, Tel: +86-20-87596262, Fax: +86-20-87503358, E-mail: jiangz28@qq.com 2016, CAAS. All rights reserved. Published by Elsevier Ltd. doi: 10.1016/S2095-3119(16)61426-0 1. Introduction Arginine (Arg) plays multiple roles in animal metabolism by serving as a substrate for protein synthesis, and as a precursor for the synthesis of important biological molecules

ZHU Cui et al. Journal of Integrative Agriculture 2016, 15(0): 60345-7 3 such as nitric oxide (NO) and polyamines (Wu et al. 2004). Previous study has shown that neonatal pigs have a high requirement for arginine for growth and development (Wu et al. 2004). The low concentration of Arg in sow s milk and declining endogenous Arg synthesis from neonatal small intestine, are insufficient for supporting the potential growth of neonatal and sucking piglets (Wu and Knabe 1994; Kim and Wu 2004; Wu et al. 2004; Frank et al. 2007). Dietary supplementation of sows with Arg provides an effective way to improve arginine provision for sow-reared piglets. Previous studies showed that increasing dietary Arg supplementation in primiparous sows, starting from pregnancy to lactation until weaning, led to an increased litter weight gain of piglets (Mateo et al. 2008). Other studies indicated that dietary supplementation with 1% L-Arg-HCl continuously from d 22 after breeding enhanced pregnancy outcomes in primiparous and multiparous sows under practical production conditions (Gao et al. 2012). This long-term supplementation of sows with Arg was marginally cost-effective. It is of great interest to determine if a similar beneficial effect could be obtained if Arg supplementation was provided for a shorter interval, just during lactation, in multiparous sows. Due to the ethical concerns over lactation research with women and children, swine have been considered to be an ideal animal model to study nutrition influence on mammary gland development and health of offspring (Kim and Wu 2009). The present study, therefore, has tested the hypothesis that dietary Arg supplementation in multiparous lactating sows, exclusively during lactation, might improve the weight gain of sow-reared piglets. Sow and litter performance, plasma concentrations of hormones and metabolites, and milk composition of sows were determined herein. 2. Materials and methods 2.1. Animals, housing and diets The experiment was carried out in a commercial farrow-towean operation (INVA Co. Ltd., Enping, Guangdong, China), and all procedures were approved by the Animal Care and Use Committee of Guangdong Academy of Agricultural Sciences, China. A total of 31 Landrace Large White multiparous sows (average parity=2.5) were moved into the farrowing room with individual slatted-floor farrowing crates on approximately d 110 of pregnancy. Each crate had a single feeder and a nipple drinker, and was equipped with a heat lamp for newborn piglets. The temperature of the farrowing room was maintained at 18 to 20 C. After farrowing, sows with similar body weight and parity were randomly allotted into 3 dietary treatments where the basal diet (Table 1) was supplemented with 0 (control, n=10), 0.5% (n=10) or 1.0% (n=11) L-Arg-HCl. Diets were formulated to meet or exceed the nutrient requirements for lactating sows recommended by the NRC (2012). The 3 experiment diets contained total amounts of 0.82, 1.23, and 1.64% of Arg, respectively and alanine was used for the isonitrogenous control. The L-Arg-HCl and L-alanine were purchased from the Qianjiang Siwei Amino Acids Co. Ltd., Hubei, China, with purities of 99.7 and 99.2%, respectively. All sows were fed 2.5 kg d 1 per sow of the common basal diet (control diet) from d 110 of pregnancy until the start of the experiment on d 3 of lactation. Then the experimental diets were provided until d 21 of lactation. Feed was gradually increased from farrowing to d 5 of lactation, then provided ad libitum. Piglets had no access to creep feed but water was freely available. 2.2. Sow and litter performance The daily feed intake (ADFI) of sows was recorded accord- Table 1 Ingredient and composition of experimental diets (as fed basis) Item Supplemental L-Arg-HCl in diets (%) 0.0 (control) 0.5 1.0 Ingredients (%) Corn 65.12 65.46 65.82 Wheat 12.30 12.30 12.30 Soybean meal 12.00 12.00 12.00 Spray-dried blood cells 2.00 2.00 2.00 Soybean oil 1.80 1.80 1.80 Dicalcium phosphate 1.85 1.85 1.85 Limestone 1.10 1.10 1.10 Salt 0.40 0.40 0.40 Trace mineral premix 1) 0.44 0.44 0.44 Vitamin premix 2) 0.52 0.52 0.52 Amino acid premix 3) 0.77 0.77 0.77 L-Arg-HCl 0.00 0.50 1.00 L-Alanine 1.70 0.85 0.00 Total 100.00 100.00 100.00 Calculated nutrition composition DE (kcal kg 1 ) 3 418 3 413 3 407 CP (%) 16.30 16.30 16.30 Ca (%) 0.90 0.90 0.90 Available P 0.46 0.46 0.46 Arginine (%) 0.82 1.23 1.64 Lysine (%) 0.97 0.97 0.97 Methionine+Cystine (%) 0.47 0.47 0.47 Threonine (%) 0.63 0.63 0.63 Tryptophan (%) 0.18 0.18 0.18 1) Supplied per kilogram of final diet: 60 mg Zn; 60 mg Fe; 35 mg Mn; 8 mg Cu; 0.35 mg I, and 0.30 mg Se. 2) Supplied per kilogram of final diet: 25 000 IU vitamin A; 3 000 IU vitamin D 3 ; 65 mg vitamin E; 5 mg vitamin K; 12.5 mg riboflavin; 50 mg niacin; 25 mg D-pantothenic acid; 37.5 μg vitamin B 12 ; 5 mg pyridoxine; 2.15 mg folic acid; 0.1 mg biotin; 5 mg thiamin, and 0.75 g choline choline. 3) Supplied per kilogram of final diet: 3.82 g L-lysine-HCl; 0.20 g DL-metioninne; 1.07 g L-threonine; 0.30 g L-tryptophan; 1.60 g L-valine; and 0.67 g L-isoleucine.

4 ZHU Cui et al. Journal of Integrative Agriculture 2016, 15(0): 60345-7 ingly. Sows were weighed at d 3 and 21 of lactation, and body weight loss at d 3 to 21 of lactation was calculated accordingly. The backfat thickness of each sow was measured at the second rib using a B-mode ultrasound scanner (ULTRASOUND WED-2000, Beijing, China) at d 3 and 21 of lactation and the difference was used to determine the backfat loss. Cross-fostering within treatment groups was performed within 48 h after farrowing to standardize litters to 10 piglets per sow. Piglets were weighed at d 3, 14, and 21 of age, and average daily gain (ADG) between d 3 to 14, and d 3 to 21 was calculated accordingly. 2.3. Blood collection and analyses Heparinized blood samples (5 ml) were collected by venipuncture of an ear vein before the morning feeding from 18 sows (6 randomly selected sows in each group) on both d 14 and 21 of lactation. Blood was held on ice and centrifuged (1 400 g for 10 min at 4 C) within 30 min; plasma samples were frozen at 20 C until analyses. Commercial Elisa kits were used for the quantification of plasma prolactin, insulin, and insulin-like growth factor-1 (IGF-1) concentrations, according to the manufacturer s instructions (Adlitteram Diagnostic Laboratories Inc., America). Plasma urea nitrogen (PUN), non-esterified fatty acid (NEFA) and NO concentrations were analyzed using commercial colorimetric kits (Nanjing Jiancheng Bioengineering Institute, Jiangsu, China). Free Arg content in the plasma and milk of sows was measured with an amino acid analyzer (L-8900, HITACHI, Japan), as described in previous studies (Ma et al. 2010; Ren et al. 2014). was analyzed by the Kjeldahl method (AOAC 2000; Kjeldahl 2300 Analyzer, FOSS TECATOR, Sweden). Milk fat analysis (AOAC 2000; Fat 2055 Analyzer, SOX TEX, Sweden) was based on ether extraction of samples and lactose was determined as described by Auldist et al. (2000). The contents of polyamines (spermine, putrescine, and spermidine) were determined by fluorescence-coupled HPLC as described by Wu et al. (2000). 2.6. Statistical analysis The effects of supplementation were analyzed, on a within-day basis, by one-way ANOVA (SAS, ver. 8.0, Cary, NC). Differences among treatments, presented as means±se, were compared with Duncan s tests. P<0.05 was considered to be statistically significant. 3. Results 3.1. Sow and litter performance There were no differences due to treatment (P>0.05) in ADFI, body weight loss, and backfat thickness loss of lactating sows (Table 2). There was a marginal increase (approximately 10% relative to the controls) in milk yield of supplemented sows, at d 14 of lactation (0.05<P<0.10). The ADG of piglets between d 3 and 14 of lactation was higher than in the controls (P<0.05) in sows fed diets supplemented with either 0.5 or 1.0% L-Arg-HCl as was ADG between d 3 and d 21 in piglets of sows supplemented with 1.0% L-Arg-HCl. 2.4. Milk yield determination Milk yield for each sow was calculated from milk intakes of 3 individual piglets within litters by the body weight increase before and after suckling, accordingly to the calculation described by King et al. (1993). 2.5. Milk collection and analyses Milk samples (10 ml) were collected from the same 18 sows after blood collection on d 14 and 21 of lactation. For each sow, the anterior, middle, and posterior mammary glands were chosen for manual milk sample collection. Before collection, piglets were separated from their dams for about 1 h. The udder and teats were then cleaned with warm water, gently massaged and the sows were given oxytocin (20 IU, i.m.). Milk samples from the glands in each sow were pooled and frozen at 20 C until analyzed for milk composition and metabolites. Milk protein content 3.2. Plasma and milk free arginine concentrations in sows Plasma concentrations of free Arg in sows at d 14 and 21 of lactation were increased over the controls (P<0.05) by dietary supplementation with 0.5 or 1.0% L-Arg-HCl (Fig. 1); concentrations of other plasma free amino acids did not differ (P>0.05, data not shown), nor did milk free Arg concentrations (P>0.05, Fig. 1). 3.3. Plasma metabolites and hormones Dietary supplementation with 1.0% L-Arg-HCl increased plasma concentrations of NEFA and NO over those in the controls (P<0.05) in sows at d 21 of lactation (Table 3). Concentrations of PUN in sows both at d 14 and 21 of lactation, when compared to the control, were decreased (P<0.05) by dietary supplementation with either 0.5 or 1.0% L-Arg-HCl. Sows receiving 1.0% L-Arg-HCl had increased

ZHU Cui et al. Journal of Integrative Agriculture 2016, 15(0): 60345-7 5 Table 2 Effect of dietary L-Arg-HCl supplementation during lactation on sow performance, milk yield, and litter weight gain Supplemental L-Arg-HCl in diets (%) Item 1) 0.0 (control) 0.5 1.0 Observation (n) 11 10 11 Sow performance ADFI (kg d 1 ) 5.58±0.12 5.32±0.15 5.52±0.14 Body weight loss (kg) 0.80±1.44 4.98±1.61 3.98±1.26 Backfat thickness loss (mm) 0.25±0.56 1.13±0.47 0.78±0.36 Milk yield (kg d 1 ) 2) d 14 10.44±0.43 11.47±0.51 11.55±0.37 d 21 9.49±0.51 10.24±0.46 10.47±0.55 ADG of piglets (g d 1 ) d 3 to 14 203.48±5.46 b 222.93±4.88 a 228.12±6.70 a d 3 to 21 208.61±2.29 b 222.36±2.21 ab 227.59±7.01 a 1) ADFI, average daily feed intake; ADG, average daily gain. 2) Values are means±se (n=3). Data within a row without a common letter are significantly different (P<0.05). The same as below. Control 0.5% L-Arg-HCl 1.0% L-Arg-HCl Milk Arg concentration (μmoll 1 ) 240 210 180 150 120 90 60 30 0 c Plasma a b b c 14 21 a Milk Arg concentration (μmoll 1 ) 15 12 9 6 3 0 Milk 14 21 Lactaion period (d) Lactaion period (d) Fig. 1 Effect of dietary L-Arg-HCl supplementation on plasma and milk Arg concentrations in lactating sows. Values are means±se, n=6. Data within days, without common letters are significantly different (P<0.05). plasma concentrations of prolactin and insulin at d 14 and 21 of lactation. Plasma concentrations of IGF-1 were strikingly increased by supplementation (2 to 3 folds) at day 14 and increased with the higher level of supplementation at d 21 (P<0.05). 3.4. Milk composition and metabolites There was no significant effect of supplementation (P>0.05) in lactose and protein contents of sows milk (Table 4) but there was a slight increase (12%, 0.05<P<0.10) in fat content at d 14 in sows given 1.0% L-Arg-HCl. Dietary supplementation with either 0.5 or 1% L-Arg-HCl increased the concentrations of IGF-1 in milk at d 21, as did the higher level at d 14 (P<0.05). Putrescine concentration in milk decreased and spermine increased (P<0.05) at d 14 with 1.0% L-Arg-HCl. The milk content of spermidine was the highest in sows supplemented with 0.5% L-Arg-HCl and the lowest in those given 1.0 % L-Arg-HCl (P<0.05). 4. Discussion The current study demonstrated for the first time that dietary supplementation of multiparous sows between d 3 and 21 of lactation with 1.0% L-Arg-HCl improved the weight gain of piglets. This improvement of growth performance of offspring may have been mediated by increased maternal plasma concentrations of hormones, arginine and its metabolite NO and the increased amounts of IGF-1 and spermine in milk. Traditionally, Arg has not been considered to be a limiting AA for milk synthesis because of its relatively high content in conventional corn-soybean diets for sows (Wu et al. 2014). The quantity of Arg taken up by the porcine mammary gland exceeds the amount secreted in milk (Trottier et al. 1997), indicating that the total mammary requirement for Arg is higher than that estimated from the amino acid profile of milk. Insufficient delivery of Arg to the mammary glands, therefore is a major constraint to maximal weight gain of

6 ZHU Cui et al. Journal of Integrative Agriculture 2016, 15(0): 60345-7 Table 3 Effects of dietary L-Arg-HCl supplementation on plasma concentrations of metabolites and hormones in lactating sows Supplemental L-Arg-HCl in diet (%) Item 1) 0.0 (control) 0.5 1.0 NEFA (mmol L 1 ) d 14 0.39±0.02 0.41±0.01 0.43±0.01 d 21 0.37±0.02 b 0.40±0.01 ab 0.41±0.01 a PUN (mmol L 1 ) d 14 4.84±0.10 a 4.53±0.08 a 4.28±0.10 b d 21 4.88±0.09 a 4.50±0.09 a 4.36±0.11 b NO (μmol L 1 ) d 14 82.74±5.13 87.50±4.81 95.24±4.72 d 21 74.70±3.61 b 79.76±2.98 ab 87.80±3.79 a Prolactin (ng ml 1 ) d 14 2.55±0.17 b 3.17±0.36 ab 4.09±0.41 a d 21 2.10±0.07 b 3.55±0.48 ab 4.11±0.59 a Insulin (ng ml 1 ) d 14 1.89±0.10 b 2.19±0.08 ab 2.38±0.13 a d 21 1.81±0.08 b 2.10±0.10 ab 2.19±0.09 a IGF-1 (ng ml 1 ) d 14 17.82±1.81 b 35.55±10.44 a 48.07±5.00 a d 21 19.27±0.98 b 24.99±1.43 ab 25.89±1.69 a 1) NEFA, non-esterified fatty acid; PUN, plasma urea nitrogen; NO, nitric oxide; IGF-1, insulin-like growth factor-1. Values are means±se, n=6. Table 4 Effects of dietary L-Arg-HCl supplementation on milk composition, concentrations of IGF-1 and polyamines in milk of lactating sows Supplemental L-Arg-HCl in diets (%) Item 1) 0.0 (control) 0.5 1.0 Milk lactose (%) d 14 4.52±0.03 4.49±0.10 4.59±0.13 d 21 4.72±0.06 4.67±0.09 4.89±0.13 Milk fat (%) d 14 6.36±0.51 6.63±0.22 7.13±0.29 d 21 6.67±0.49 6.81±0.56 6.67±0.49 Milk protein (%) d 14 4.90±0.04 4.92±0.05 4.93±0.06 d 21 4.91±0.06 4.92±0.06 4.95±0.05 IGF-1 (ng ml 1 ) d 14 21.43±1.53 b 22.25±1.81 b 44.29±9.10 a d 21 21.79±1.71 c 29.12±2.10 b 38.70±1.65 a Spermine (μg ml 1 ) d 14 1.18±0.09 b 1.28±0.11 ab 1.60±0.14 a d 21 2.39±0.27 2.34±0.36 2.47±0.54 Putrescine (ng ml 1 ) d 14 42.86±8.03 a 23.76±6.44 ab 13.59±3.40 b d 21 10.15±0.83 6.96±0.98 7.98±1.83 Spermidine (μg ml 1 ) d 14 4.00±0.18 6.81±1.96 7.90±1.14 d 21 13.53±2.05 ab 17.60±1.70 a 11.36±0.47 b 1) IGF-1, insulin-like growth factor-1. Values are means±se (n=6). Arg on growth performance of artificially-reared piglets (Kim and Wu 2004; Yao et al. 2008). The present study demonstrated that the benefits, 12% increase in piglet gain, can be obtained by supplementing Arg to the diet of sows during lactation. Dietary Arg supplementation during both pregnancy and lactation improved the reproductive performance of sows (Mateo et al. 2007; Gao et al. 2012). Moreover, the within-litter variation of piglet birth weight was reduced by supplementing L-Arg in gestation diet of sows during the last third of pregnancy (Quesnel et al. 2014). The final nursery body weight of piglets was also increased by Arg and ractopamine supplementation to sows from d 25 to 53 of gestation (Garbossa et al. 2015). There were no observed changes here in indices of sow performance (body weight, backfat thickness, and ADFI) by dietary supplementation with Arg, just during lactation, consistent with an earlier finding in first-parity sows supplemented with 1% L-Arg-HCl (Mateo et al. 2008). Likewise, the improved litter weight gains (both d 3 to 14 and d 3 to 21 of lactation) in multiparous sows given 1% dietary L-Arg-HCl supplementation were consistent with performance of piglets from similarly-treated primiparous sows (Mateo et al. 2008). In contrast, no significant effect on the growth of suckling piglets was obtained from Arg supplemented multiparous sows in a thermoneutal or hot environment (Laspiur and Trottier 2001). Litter weight gain obviously reflects milk production or nutrient concentrations in milk (King et al. 1993). Stimulating mammary development and increasing milk yield, are potential ways for increasing milk Arg intake for suckling piglets. It was logical to first consider milk yield and milk nutrient contents to account for this faster growth of piglets. Neither milk protein nor milk lactose contents were affected by dietary L-Arg-HCl supplementation of the sows but there were comparable, though not-significant, increases in milk yield and milk fat content. The latter may reflect similar trends towards greater fat mobilization in supplemented sows, likely following their net energy balance. Similarly, no significant effect of changing the supply of Arg on milk yield or milk protein synthesis was observed in dairy cows (Doepel and Lapierre 2011; Haque et al. 2013). Supplementation with Arg significantly increased the Arg concentration in the sows plasma, but not in their milk, as in the case of primiparous sows (Mateo et al. 2008), and is likely due to the extensive catabolism of Arg in lactating porcine mammary tissue (O Quinn et al. 2002). Urea, the major end product of amino acid oxidation in mammals (Meijer et al. 1990), reflected by PUN concentrations indicates the efficiency of whole-body nitrogen utilization in lactating sows, with greater PUN indicating less efficient protein utilization (Soltwedel et al. 2006). Our finding that dietary supplementation with Arg reduced PUN concentrations, which may suggest an improvement in the efficiency of utilization of dietary amino acid for milk production. The plasma concentrations of NEFA reflect postpartum energy status and fat catabolism of sows (Revell et al. 1998)

ZHU Cui et al. Journal of Integrative Agriculture 2016, 15(0): 60345-7 7 when negative energy balance is partially compensated by mobilization of body fat. This net catabolism of fat during lactation produces sufficient energy for the mammary gland, even without high protein mobilization (Hultén et al. 1993). The increased concentrations of NEFA found here with Arg supplementation, especially on d 21, are consistent with greater demands accompanying enhanced export of energy in milk. The metabolic state can be partially assessed by measurement of circulating metabolites and metabolic hormones (Quesnel and Prunier 1995). Arg is an essential amino acid for lactating sows for optimal mammary gland growth. Mammary uptake of nutrients is dependent on their availability from the circulation, but is influenced by hormones such as insulin, prolactin and IGF-1 (Farmer et al. 2008). Prolactin is vital for the initiation and maintenance of lactation in sows and dietary supplementation with 1.0% L-Arg-HCl here significantly increased plasma concentrations of prolactin at d 14 and 21 of lactation. Insulin increases cell division in mammary tissue from lactating pigs (Buttle and Lin 1991) and has an obvious extra-mammary role in regulating fat and protein metabolism and hence provision of nutrients to mammary tissue, thus influencing milk constituents and yield in sows (Schams et al. 1994; Breier 1999). The present study showed that dietary supplementation with 1.0% L-Arg-HCl increased plasma insulin concentrations at d 14 and 21 of lactation, consistent with observations at d 7 and 21 of lactation in primiparous sows (Mateo et al. 2008). Notably, IGF-1 contributes to mammary differentiation and lactogenesis (Lee et al. 1993; Schams et al. 1994) and, as in other species, can be expected to be galactopoietic in pigs. The increased concentrations of plasma IGF-1 observed here, particularly at d 14, might be a key factor in mediating the effects of Arg supplementation on performance of the piglets; comparable changes occurred in milk IGF-1 that could directly affect the piglets. The mammary gland is a site of extensive synthesis and degradation of AA including the partial metabolism of Arg to NO and polyamines via the NOS and arginase pathways in sows (O Quinn et al. 2002). Both NO and polyamines play critical roles in mammary angiogenesis, milk production and consequent growth of the litter. Enhanced synthesis of NO from Arg may mediate increased mammary blood flow, thereby increasing nutrient delivery for milk production (Meininger and Wu 2002; Kim and Wu 2009; Cieslar et al. 2014). Increased plasma NO concentration at d 21 of lactation from Arg supplementation at d 21 of lactation was consistent with this but it was impractical to measure blood flow in the present study. Additionally, dietary arginine supplementation ameliorated the intestinal abnormalities in mycotoxin-challenged growing pigs possibly due to its metabolite NO (Yin et al. 2014) since it is critical for the normal physiology of the gastrointestinal tract (Rhoads et al. 2008). Polyamines such as spermine, putrescine, and spermidine, regulate cell proliferation and differentiation of mammary epithelial cells (Johnson 1988) and influence lactogenesis and protein synthesis (Meininger and Wu 2002). They directly stimulate the intestinal development of neonates (Reeds et al. 2000). Supplementation of lactating sows with Arg clearly influenced the polyamines as spermine increased and putrescene decreased in milk; changes in spermidine were complex as the 0.5% level caused greater changes than did 1.0%. It cannot be determined if these changes in milk polyamines exerted any direct effects on the piglets or simply reflected altered processing of Arg within the mammary gland. 5. Conclusion This study demonstrated that dietary supplementation of multiparous sows with 1% L-Arg-HCl, just during lactation, was effective in improving the weight gain of piglets. This briefer period of supplementation, rather than during both pregnancy and lactation, may prove to be a cost-effective means of increasing growth performance of piglets to weaning. The complete mechanism bringing about the response is not clear but may include maternal endocrine changes and more completely satisfying the mammary requirement for Arg. These resulted in a trend towards increased production of milk with a higher fat content, without obvious increases in maternal catabolism. Changes in milk content of polyamines potentially exerted direct effects on the sucking piglets. Acknowledgements The authors gratefully acknowledge the financial supports provided by the China Agriculture Research System (CARS- 36), the Hundred Outstanding Talents Training Program at Guangdong Province, China, the Special Program for Guangdong Research Institutions Innovation and Construction, China (2012B060600005), the Natural Science Foundation of Guangdong Province, China (2015A030310332), and the Science and Technology Program of Guangdong Province, China (2013B050800016). We gratefully acknowledge W B Currie (Cornell University, Ithaca, NY) for suggestions on presentation. References AOAC (Association of Official Analytical Chemists). 2000. Official Methods of Analysis. 17th ed. AOAC International, Arlington, VA. Auldist D E, Carlson D, Morrish L, Wakeford CM, King R H. 2000. The influence of suckling interval on milk production

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