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Maternal organic selenium supplementation during gestation enhances muscle fiber area and muscle fiber maturation of offspring in porcine model

Background Organic selenium supplementation during gestation improves the antioxidant status and reproductive performance of sows and increases the antioxidative capacity of the intestines of their offspring. This study was conducted to investigate the effect of maternal basel diet (control) supplemented with an organic Se, 2-hydroxy-4-methylselenobutanoic acid (HMSeBA), or inorganic sodium selenite (Na2SeO3) during gestation on the antioxidant status and development of muscle in newborn and weaned piglets. Newborn piglets before colostrum intake and weaned piglets were selected for longissimus dorsi (LD) muscle collection and analysis. Results The results showed that maternal HMSeBA supplementation increased the muscle area and content of Se in the LD muscle of newborn piglets, improved gene expression of selenoproteins, and decreased oxidative status in the LD muscle of both newborn and weaned piglets compared with the control. The expression of muscle development-related genes of newborn piglets in the HMSeBA group was lower than in the control group, whereas the expression of MRF4 in weaned piglets was higher in the HMSeBA group than in the control and Na2SeO3 groups. In addition, HMSeBA supplementation decreased the mRNA expressions of myosin heavy chains (MyHC) IIx and MyHC IIb and the percentage of MyHC IIb; increased the expression of PGC-1α in the LD muscle of newborn piglets; increased the gene expression of MyHC IIa; and decreased the protein expression of slow MyHC and the activity of malate dehydrogenase in the LD muscle of weaned piglets compared with the control group. Conclusions Maternal HMSeBA supplementation during gestation can improve the antioxidative capacity of the muscle of their offspring and promote the maturity of muscle fibres in weaned offspring.

with organic Se improves the amount of Se transferred from sows to their progeny [5,6], thereby enhancing the antioxidative capacity of their offspring [7]. Our previous studies found that maternal supplementation with 2-hydroxy-4-methylselenobutanoic acid (HMSeBA) during gestation increased the plasma concentration of total Se and improved the antioxidative capacities of sows and their offspring [8]. However, little is known about the effects of Se source on muscle development in offspring. Skeletal muscle accounts for 40%-50% of body weight [9]. Muscle development is regulated by several transcription factors. The MyoD family of myogenic regulatory factors (MRFs) are master regulators of myogenic determination and differentiation, while postpartum satellite cells are determined by paired box gene 7 (Pax7) [10]. In addition, myostatin (MSTN) inhibits muscle development [11], while mammalian target of rapamycin (mTOR) promotes muscle hypertrophy [12]. Insulin-like growth factors promote myogenesis and postnatal muscle growth by accelerating protein synthesis and inhibiting protein degradation [13]. Further, Se is involved in the differentiation of chicken embryonic myoblasts [14] and improves the fatty acid composition in poultry muscle tissues [15]. Although Se has an effect on muscle pH and drip loss in growing pigs, the effect of maternal Se supply on offspring muscle development is not clear and is worth exploring.
In most mammals, the number of muscle fibres is determined at birth. Thus, the increase in postnatal skeletal muscle mass results from an increase in muscle fibre size (hypertrophy) [16]. However, the composition of muscle fibre type is continuously changing postnatally [17], wherein days 1 to 14 are the critical periods for transformation. Based on the characteristics of its contraction, skeletal muscle fibre types are classified as slow-twitch (Type I) fibers with MyHC I expression and fast-twitch (Type II) fibers. Fast fibres are divided into type IIa (fast-twitch oxidative type) with MyHC IIa expression, type IIx (fast-twitch oxidative-glycolytic type) with MyHC IIx expression, and type IIb (fast-twitch glycolytic type) with MyHC IIb expression [18]. Muscle fibre types can transform between types I and II. Therefore, the objective of this study was to explore whether maternal addition of HMSeBA during gestation could improve muscle development, selenium deposition, and antioxidant status in the muscles of their offspring.

Experimental design and animal management
Forty-five Landrace Yorkshire sows after insemination were randomly divided into three groups according to their body weight (239.25 ± 8.54 kg) and backfat thickness (13.90 ± 1.28 mm), and received one of the following diets during gestation: basal diet (Control, n = 15), a basal diet supplemented with sodium selenite (Na 2 SeO 3 ) at 0.3 mg Se per kg (Na 2 SeO 3 , n = 15), and HMSeBA at 0.3 mg Se per kg (HMSeBA, n = 15). The experimental diets were formulated to meet the nutrient requirements of gestating sows as recommended by NRC [19] (Table 1), except for that of selenium. All sows were fed the same lactation diet. HMSeBA (hydroxy-analogue of selenomethionine, Selisseo ® , 2% Se) was provided by Adisseo France S.A.S. and Na 2 SeO 3 (1% Se) was obtained from Chengdu Shuxing Feed Co. Ltd (Chengdu, Sichuan, China).

Sample collection
On the day of birth, 10 piglets from each group (male) were anaesthetised and sacrificed before suckling. Samples of the longissimus dorsi (LD) muscle from the eighth to tenth rib were collected. The remaining piglets were breastfed until they were weaned. On the day of weaning, six piglets from each group (male) were slaughtered and the LD muscle was collected and stored at -80 °C.

Muscle fibre histological analysis
Muscle fibre morphology in pigs was determined by staining the muscle fibres using the classical ATPase method of Guth et al. [20]. All sections were photographed using a digital microscope (Nikon) based on five consecutive random areas. At least 150 muscle fibers were randomly selected by Image-Pro Plus 6.0 Image analysis software (Media Cybernetics Inc., Bethesda, MD, USA), and the diameter and area of muscle fibers in the collected images were measured [21]. The number and cross-sectional area of the muscle fibres were calculated using the software programme Image-Plus 6.0, and muscle density was calculated based on the number of muscle fibres/muscle areas.

Measurement of selenium concentration
The Se level in the muscle was analysed according to the method of Chao et al. [3]. Briefly, approximately 0.5 g of muscle sample was digested with 10 mL HNO 3 and 2 mL H 2 O 2 in a microwave. The solution was then heated and treated with 6 mol/L HCl. A reagent blank test was simultaneously performed. The total Se content was determined using hydride atomic fluorescence spectrometry (AFS-9230, Beijing Auspicious Day Instrument Co., LTD, Beijing, China) [3].

Gene expression and muscle type
Muscle tissue powder was homogenised in TRIzol reagent (Invitrogen, Shanghai, China), then RNA was extracted according to the manufacturer's instructions and RNA concentration was determined. The expression changes of genes were validated by a SYBR-based High-Specificity miRNA qRT-PCR Detection kit (TaKaRa Biotechnology Co., Ltd., Dalian, China) on the Applied Biosystems 7900HT Real-Time PCR Detection System (Applied Biosystems, Carlsbad, USA). Real-time PCR data were analysed using the 2 -∆∆Ct method, with GAPDH as the reference. The primer sequences are listed in Table 2.
According to the ratio between the mRNA expression of each myosin heavy chain subtype and type IIx mRNA (referred to as 1), the proportion of each gene in the total was calculated to obtain the proportion of muscle fibre type. The proportion of MyHC I, MyHC IIa, MyHC IIb, and MyHC IIx mRNA (%) was calculated to represent the proportion of slow oxidation, fast oxidation, fast fermentation, and intermediate type muscle fibres, respectively.

Analysis of metabolic enzyme activities
The activities of succinic dehydrogenase (SDH), malate dehydrogenase (MDH), and lactate dehydrogenase (LDH) in the LD muscle were measured using the assay Table 2 Primer sequences for the target and reference genes

Genes
Primer Sequence (5' to 3') Accession no. kits purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China) and the protocol followed the manufacturer's instructions.

Analysis of antioxidant enzyme activity and malondialdehyde content
The enzyme activities of total superoxide dismutase (T-SOD), glutathione peroxidase (GSH-Px) and catalase (CAT), total antioxidant capability (T-AOC), and malondialdehyde (MDA) level in the LD muscle were determined according to the manufacturer's instructions (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

Western blot
Western blotting was performed as previously reported [22]. LD muscle samples were homogenised in RIPA lysis buffer (Beyotime biotechnology, Shanghai, China) containing a protease inhibitor (Roche, Shanghai, China

Statistical analysis
Data were analysed using one-way ANOVA procedure of the SPSS software (version 21.0; SPSS Inc., Chicago, IL, USA). Duncan's multiple range test was used to compare the differences between the groups with normally distributed data, while the data without a normal distribution were analysed using non-parametric analysis. Results are presented as mean ± standard error (SE). Differences were recognised as significant when P < 0.05, and a tendency was considered when 0.05 ≤ P < 0.10.

Maternal organic Se supplementation increased the muscle area in LD muscle of weaning piglets
In this study, maternal organic Se supplementation increased the muscle area of the offspring, while there was no effect on muscle density (Fig. 1).

Maternal organic Se supplementation increased the content of Se in LD muscle of newborn piglets
Compared to that in the control and Na 2 SeO 3 groups, maternal HMSeBA supplementation during gestation increased Se content in the LD muscle of newborn piglets (Table 3). Compared with the control group, maternal HMSeBA supplementation significantly reduced birth weight (P < 0.05) but had no effect on the weight or weight of LD as a percentage of body weight in newborn and weaned piglets. The body weights of the piglets were similar between the three groups at weaning (Table 3).

Maternal organic Se supplementation changed the expression of muscle development-related genes in offspring
Compared to the control group, maternal HMSeBA and Na 2 SeO 3 supplementation decreased the mRNA levels of Myf5, MyoD, MyoG, and Pax7 (P < 0.05), whereas only maternal Na 2 SeO 3 supplementation reduced the expression of MRF4 in newborn piglets (P < 0.05) ( Fig. 2A). Maternal organic Se supplementation during gestation decreased the expression of mTOR compared to that in the Na 2 SeO 3 group (P < 0.05) ( Fig. 2A). Moreover, in weaned piglets, maternal HMSeBA supplementation increased the expression of MRF4 compared with that in the Na 2 SeO 3 and control groups (P < 0.05) (Fig. 2B).

Maternal organic Se supplementation during gestation changed muscle fibre type in offspring
The mRNA levels of MyHC I, MyHC IIa, MyHC IIb, and MyHC IIx in the LD muscle of newborn piglets were analysed. The results showed that, compared with the control group, maternal HMSeBA supplementation decreased the mRNA expression of MyHC IIb and MyHC IIx (P < 0.05) and increased the expression of PGC-1α, while maternal Na 2 SeO 3 supplementation only decreased the mRNA expression of MyHC IIb (Fig. 3A). In addition, the percentage of MyHC IIb fibres was reduced in both the Na 2 SeO 3 and HMSeBA groups compared with the control group (P < 0.05) (Fig. 3B). Additionally, the protein level of PGC-1α was increased in the HMSeBA group when compared with the control and Na 2 SeO 3 groups (P < 0.05), whereas there was no difference in the protein expression of slow MyHC and fast MyHC (Fig. 3C, D).
In weaned piglets, maternal HMSeBA supplementation increased the mRNA level of MyHC IIa compared with that in the control group (P < 0.05) (Fig. 3E). However, maternal HMSeBA supplementation did not change the percentage of MyHC I, MyHC IIa, MyHC IIb, or MyHC IIx (Fig. 3F). Piglets from the HMSeBA group had lower slow MyHC and PGC-1α levels than piglets from the control group (P < 0.05), while piglets from the Na 2 SeO 3 group had higher slow MyHC and fast MyHC and lower PGC-1α levels than piglets from the control group (Fig. 3G, H).

Effects of maternal organic Se supplementation during gestation on the activities of metabolic enzymes in the LD muscle of the offspring
The activities of LDH and MDH in the LD muscle of newborn piglets were lower (P < 0.05) in the HMSeBA group than  in the Na 2 SeO 3 group (Fig. 4A). In addition, MDH activity in weaned piglets was lower (P < 0.05) in both the HMSeBA group and Na 2 SeO 3 group than in the control group (Fig. 4B).

Organic Se supplementation increased antioxidation indicators in LD muscle of offspring
Newborn piglets from the HMSeBA group had higher muscle GSH-Px activity and lower MDA content (P < 0.05) than newborn piglets from the Na 2 SeO 3 and control groups and had higher T-SOD activity (P < 0.05) than newborn piglets from the control group (Table 4). Besides, the MDA content in the LD muscle of weaned piglets was lower (P < 0.05) in the HMSeBA group than in the control and Na 2 SeO 3 groups (Table 4).

Maternal organic Se supplementation regulated the expression of selenoprotein genes in LD muscle of offspring
We then analysed the mRNA levels of selenoproteins in the LD muscle. Results showed that maternal HMSeBA supplementation during gestation increased the mRNA expression of GPX1 and decreased the mRNA expression of SEPHS2 and DIO2 (P < 0.05) compared to the control group in the LD muscle of newborn piglets (Fig. 5A). Besides, the mRNA expression of GPX1 and GPX3 was higher (P < 0.05) and the mRNA expression of DIO2 was lower (P < 0.05) in the newborn piglets of the Na 2 SeO 3 group than in the control group (Fig. 5A).
In the LD muscle of weaned piglets, maternal HMSeBA supplementation increased the expression of SEPHS2 (P < 0.05) while Na 2 SeO 3 supplementation increased the expression of GPX1 (P < 0.05) compared to the control group. Both HMSeBA supplementation and Na 2 SeO 3 supplementation decreased the expression of GPX3 (P < 0.05) compared to the control group (Fig. 5B). Compared with the Na 2 SeO 3 group, maternal HMSeBA supplementation increased the expression of SELP and SELW (P < 0.05) (Fig. 5B).

Discussion
Although there have been many studies on Se nutrition, there is little research on the effect of maternal Se nutrition on the development of offspring muscle fibres in swine models. It is well known that the muscle occupies an important position in animal production, and the foetal period is important for muscle development. Our findings revealed that maternal HMSeBA supplementation during gestation increased the muscle area. Diniz et al. [23] found that maternal organic Se supplementation during late gestation resulted in the upregulation of myosin and actin filament-associated  Table 4 Effect of maternal selenium supplementation during gestation on the oxidative status of offspring MDA Malondialdehyde, T-AOC Total antioxidant capability, CAT Catalase, T-SOD Total superoxide dismutase, GSH-P X Glutathione peroxidase. Data were expressed as the mean ± SE. a,b P < 0.05 between different superscripts within the same line genes in newborn calves, potentially allowing for optimal muscle function and contraction. Excessive oxidative stress may be a key factor in early foetal loss [24], whereas moderation of oxidative stress can promote muscle development during the embryonic period through the Wnt signalling pathway [25]. Wnt proteins are known to be involved in myogenesis as they can regulate the expression of Pax3, Pax7, and MRF genes [26]. Our results showed that compared with control, maternal organic Se supplementation during gestation significantly decreased the gene expression of myf5, MyoD, MyoG, and Pax7 in the LD muscle and the body weight of newborn piglets. However, at weaning, the expression of MRF4 mRNA was significantly increased compared to the control and Na 2 SeO 3 groups, while the body weight and LD muscle weight were similar among the three groups. These data suggest that piglets in the HMSeBA group experienced catch-up growth [27] during the newborn period and will have better muscle development potential because of the higher MRF4 expression [28]. Selenoprotein W (SELW), without a known biological function [29], is the most widely distributed selenoprotein in muscles under normal conditions [30]. Therefore, SELW may be involved in muscle metabolism. Loflin et al. [31] showed that SELW is involved in muscle growth and differentiation. Li et al. [32] found that increased expression of the SELW gene was associated with enhanced water-holding capacity in meat. In our study, we showed an increase in SELW gene expression in weaned piglets in the HMSeBA group compared to the Na 2 SeO 3 group, which suggests that piglets in the HMSeBA group might have better muscle development and meat quality in the future. Further studies with growing pigs are required to confirm this. The perinatal period is critical for muscle development in piglets [16]. If muscle development is restricted during this period, muscle growth is affected, resulting in permanent damage [33]. Lefaucheur et al. [34] found that undernutrition during the first postnatal week could decrease hypertrophy of the future fast-twitch glycolytic fibres, delay contractile and metabolic maturation in later maturation processes, and increase the percentage of MyHC I-containing fibres in the psoas muscle. The activities of SDH, MDH, and LDH are considered indicators of muscle oxidation and glycolysis. Several reports have shown that the activity of SDH and MDH is higher in oxidised fibres than in glycolytic fibres, while the activity of LDH in glycolytic fibres is higher than that in oxidised fibres [21,35,36]. In the present study, our results indicated that more oxidised muscle fibres were transformed into glycolytic muscle fibres in the HMSeBA group during the period between birth and weaning. In addition to the change in PGC-1α expression, adequate Se leads to higher feed intake [37] and improved antioxidant status [38] which may be another reason for this phenomenon.
It is well known that maternal nutrition during pregnancy has a profound impact on foetal development. Se can be added as an antioxidant to sow diets during pregnancy and lactation [39]. Dietary selenium can be used to synthesise selenoprotein P in the liver, which can then be transferred to the foetus through the cord blood, placenta [40], colostrum and milk [41], and other transport systems. The Se in the foetus is then deposited in different tissues, and supplied to some Se-containing proteins according to a hierarchy in selenoprotein expression which play different roles in different tissues [42]. Chao et al. [3] showed that HMSeBA supplementation increased Se content in the muscle compared to Na 2 SeO 3 supplementation. In addition, Se concentrations in neonatal pigs from sows fed yeast Se was higher than those fed Na 2 SeO 3 [43]. Our study also found that maternal HMSeBA supplementation increased Se content in the LD muscle of newborn piglets compared to the control and Na 2 SeO 3 groups. The higher efficiency of organic Se in absorption, tissue accumulation, and antioxidant bioavailability [3,4] may be the reason for this.
Newborn piglets suffer from severe oxidative stress at birth owing to their incomplete antioxidant system [44]. Therefore, the development and growth process of the foetus is easily affected by oxidative stress, and this negative effect may extend to later stages in life. In the current study, we found that the activities of muscle GSH-Px and T-SOD in newborn piglets were significantly increased, while MDA content was decreased in the HMSeBA group compared to the control group and Na 2 SeO 3 group. This result suggests that maternal HMSeBA supplementation during gestation improves the antioxidant capacity of the foetus. Furthermore, TXNRD2 expression was higher in the HMSeBA group than in the control group. These results were similar to the results of Zhan et al. who found that maternal selenomethionine supplementation during gestation and lactation improved the antioxidant status in muscle as compared to the Na 2 SeO 3 supplementation [7]. These results indicate that maternal supplementation of organic selenium during pregnancy can improve not only the redox status in the LD muscle of newborn piglets but also the redox status in weaned piglets. The half-life of Se in muscles is 12 d [45]. Sows fed organic Se had a greater transfer efficiency of Se to the neonate, colostrum, milk, weaned piglets, and sow tissues than sows fed inorganic Se. Our previous study also found that SELP content in milk on day 7 of lactation in the HMSeBA group was higher than that on day 0 of lactation [8]. These data suggest that piglets from the organic Se group take up more Se from sows through maternal milk, and Se can last longer in organic form. This may be one of the reasons for the improvement in the antioxidant status in the muscle of weaned piglets.

Conclusion
The present study showed that maternal HMSeBA supplementation during pregnancy increased muscle Se deposition in newborn piglets and improved the antioxidative capacity and development of offspring muscle. It is very interesting that there are significant changes in muscle fibres during birth and weaning. Current results indicate maternal organic Se supplementation during gestation may be beneficial for muscle development in the offspring.