Effects of Early-Life Exposure to Topsoil on the Muscle Fiber Characteristics and Gene Expression of Weaned Piglets

Background: The objective of this experiment was to investigate the inuence of early exposure to topsoil on the muscle ber characteristics and transcription related myogenesis, intramuscular fat metabolism, muscle ber types, and mTOR signaling pathway of weaned pigs. Methods: A Total of 180 piglets were separately assigned to No soil, Antibacterial soil, and Normal soil group (each group, n=60), and were fed ad libitum with common antibiotic-free corn-soybean meal diets until day-31. Ten pigs from each group with similar body weight were selected to be slaughtered, and the longissimus dorsi (LD) muscle samples were collected for histological analysis and measurements of genes and proteins expression levels. Result: In the present study, the muscle ber diameter and the area of Normal soil and Antibacterial soil group were signicantly higher than No soil group (P < 0.05). The Normal soil signicantly upregulated the gene expression of MyoG compared to No soil and Antibacterial soil groups (P < 0.05). The gene expression of CD36 and CPT-1 of Normal soil group was signicantly lower than No soil group (P < 0.05), while HSL expression of Normal soil group was signicantly higher than Antibacterial and No soil groups (P < 0.05). The MyHC I of Normal soil group was signicantly higher than No soil group (P < 0.05), but the expression MyHC IIa was lower than No soil group (P < 0.05). The protein expression expressed the similar result with gene expression. In addition, the Normal soil signicantly increased the AMPK and mTOR phosphorylation compared to No soil and Antibacterial soil groups (P < 0.05). Conclusion: These data suggest that early exposure to topsoil regulates the muscle ber growth, modulates the expression pattern related to myogenesis, muscle ber type, intramuscular fat metabolism, and increases the phosphorylation of mTOR and AMPK pathways.


Background
In the modern pig industry, fewer than 6 % of the pig raised in the United States are housed in pasture of dirt pens, and most of them have been moved from outdoors into the slatted-oor indoor systems [1]. This phenomenon was caused by the increased space requirements as well as a need for more complex management practices for outdoor systems compared to indoor systems [2]. The pigs fed indoor rarely have the opportunity to touch the soil, which is a complex system consists of air, water, minerals, organic matter, and biota [3]. The effects of this environmental change are still understudied for the fact that the outdoor system is not a feasible means of swine production for the majority pig industry. However, previous studies show that feeding piglets outdoors during lactation improves growth performance, carcass weight, and feed e ciency [4,5]. In addition, it was reported that early exposure to agricultural soil modulates the gut microbiota maturation of pigs during their early life [6]. Despite these discoveries, many questions remain in regard to the roles of the exposure to outdoor soil in regulating myogenesis, muscle ber type, intramuscular fat metabolism.
A previous investigation con rmed that the muscle ber characteristics could be affected by the animal rearing environment [7]. The muscle ber characteristics (diameter, number, area) and ber type composition are closely related to each other [8], and it is a re ection of animal growth rate [9]. Usually, muscle ber types are de ned by the isoforms of myosin heavy chain (MHC) as MyHC I, IIa, IIb, and IIx, based on their different ATPase types [10]. The gene expression related to the intramuscular fat metabolism, such as PPARγ, CPT1,SREBP1, etc., was affected by rearing systems [11]. Different rearing environments may also impact the lipid, protein, and energy metabolism in skeletal muscle by modulating the mechanistic target of the rapamycin (mTOR) signaling pathway [12].
The objective of this experiment was to explore the effects of exposure to topsoil during pre-weaning on the muscle ber characteristics and expression patterns of genes and proteins related to myogenesis, muscle ber type, intramuscular fat metabolism, and mTOR pathway.

Animals and experimental design
The University of Arkansas's Institutional Animal Care and Use Committee approved all experimental procedures involving animals during the study (ethical approval code: 18059). All 30 sows used in the study were blocked by parity and farrowing body weight. Piglets from each litter were cross-fostered within 24 hours across three sows (within similar parity and body weight). Six piglets with similar body weight from each litter (n = 180) were individually transferred to the same pen in the nursery facility, and kept in their littermates for the entire trial. Piglets were equally allocated to the No soil group, Antibacterial soil group, and Normal soil group (60 piglets in each group). No soil group was exposed to an empty pan, Antibacterial soil group was exposed to a pan with 1 kg of irradiated topsoil (Sterigenics, Fort Worth, TX) to kill bacteria in the soil, and Normal soil group was exposed to a pan with 1 kg of topsoil (Sod Store, Inc., Tontitown, AR). They were fed ad libitum with common antibiotic-free corn-soybean meal diets in littermates for 31 days.

Animal slaughter and sampling
The day prior to harvest, all pigs were weighed, and the pigs of median weight form each pen (10 piglets per group) were selected for sampling on day 31. Following a 12 h period of fasting prior to slaughter with the access of water, the pigs were transported to the University of Arkansas red meat abattoir. Piglets were euthanized by a captive bolt and immediately followed by exsanguination. Then, the muscle samples from longissimus dorsi (LD) of left carcass were removed and subsequently snap frozen at -80℃ in liquid nitrogen for RNA isolation and protein extraction process. Another piece of LD muscle from each pig was cut into 0.5 × 0.5 × 1.0 cm cube, and immediately xed in 10% buffered neutral formalin solution for the histological experiment.

Histological analysis
The LD muscle samples, xed in 10% buffered neutral formalin solution, were dehydrated in alcohol, cleared in xylene, in ltrated, embedded in para n [13], and then were cut in to 3 µm thickness. H&E (hematoxylin and eosin) staining was used to treat the thickness for histological study [14]. Stained cross-sections were viewed and photographed at 175 × by ZOE™ Fluorescent Cell Imager (Bio-rad, Hercules, CA, USA). Five photographs of each cross-section of LD samples were taken, and then they were analyzed using Image-J software (National Institutes of Health, Bethesda, MD, USA). The average muscle ber numbers per area were obtained by counting the total number of bers in ve areas (700,000 µm 2 for each area). The muscle ber diameter (µm) and area (µm 2 ) of each LD muscle was measured by using 300 bers in ve cross-sections of each sample.

RNA isolation and cDNA synthesis
The samples, stored in liquid nitrogen, were homogenized using the Precellys Evolution homogenizer  (Table 1). β-actin was chosen as the house-keeping gene to normalize target gene levels. Real-time qPCR was performed by using iQ™ SYBR® Green Supermix (Bio-rad, Cat. No. 1708890) along with the manufacturer's instructions. The 2 −ΔΔCt method was used to analyze the relative expression, and the relative expression was normalized and expressed as a ratio to the expression in the No soil group.

Statistical analysis
All data were analyzed by one-way ANOVA with SPSS 22.0 statistical software (SPSS Inc., Chicago, IL, USA).
The results were expressed as the Mean ± SEM, and a P < 0.05 was used to determine statistical signi cance.

Muscle ber characteristics
The histochemical section of LD muscle is shown in Fig. 1. The muscle ber characteristics consisted of muscle ber diameter, ber area, and the number of bers are shown in Table 2. The muscle ber diameter of Antibacterial soil group was signi cantly higher than Normal and No soil groups (P < 0.05), and the Normal soil group had a signi cantly higher diameter than the No soil group (P < 0.05). The muscle ber area of Antibacterial soil and Normal soil groups was signi cantly greater than No soil group (P < 0.05). However, there were no signi cant differences in muscle ber number among each group (P > 0.05). Values are the least square mean ± standard error of the mean. Within a row, means with no superscripts or with a common superscript letter are not signi cantly different (P < 0.05).

Myogenesis relative gene expression
The gene expression related to myogenesis (MyoG, Myf5, and MSTN) in the LD muscle was measured by RT-PCR is presented in Fig. 2. The Normal soil signi cantly upregulated the gene expression of MyoG of LD muscle compared to No soil and Antibacterial soil groups (P < 0.05). However, there was no signi cant gene expression differences of Myf5 and MSTN between each group (P > 0.05).

Intramuscular fat relative gene expression
The gene expression related with intramuscular fat deposition and lipolysis are shown in Fig. 3 and Fig. 4. For fat deposition, the gene expression of CD36 in No soil group was signi cantly higher than Normal soil group (P < 0.05), but there was no signi cant difference between No soil group and Antibacterial soil group (P > 0.05). There were no signi cant differences of gene expression of PPARγ, FAS, and SREBP-1 between each group (P > 0.05). The gene expression related with lipolysis such as HSL was signi cantly upregulated in Normal soil group compared to Antibacterial soil and No soil group (P < 0.05). The LPL expression of Normal soil group was signi cantly higher than No soil group (P < 0.05), yet no signi cant difference compared with Antibacterial soil group (P > 0.05) was noticed. However, the CPT1 was down regulated in Normal soil group compared with No soil group (P < 0.05). Additionally, there was no signi cant difference of ATGL expression between each group (P > 0.05).

Muscle ber type relative gene expression
The expression levels of MyHC I, MyHC IIa, MyHC IIb and MyHC IIx were illustrated in Fig. 5. The MyHC I of Normal soil group was signi cantly higher than No soil group (P < 0.05), but no signi cant difference compared to Antibacterial soil group (P > 0.05). However, the MyHC IIa of Normal soil group was signi cantly down regulated compared with No soil group (P < 0.05). There were no signi cant differences of MyHC IIb and IIx between each group (P > 0.05).

Protein expression related with intramuscular fat and myogenesis
HSL, PPARγ, Myf5, and MSTN expressions were further measured using Western blot, and the results were displayed in Fig. 6. The protein abundance of HSL in Normal soil group was signi cantly higher than Antibacterial soil and No soil group (P < 0.05). In addition, PPARγ and MSTN protein abundances were signi cantly decreased in Normal soil group compared with the other two groups (P < 0.05). These protein expressions were consistent with the PCR results. However, there was no signi cant difference of Mfy5 abundance between each group (P > 0.05).

Muscle ber type protein expression
The protein abundances of MyHC I, MyHC IIa, MyHC IIb and MyHC IIx were determined via Western blot (Fig. 7). The protein expression of MyHC I and MyHC IIa was comparable with the PCR results. The MyHC I of Normal soil group was signi cantly greater than No soil and Antibacterial soil group (P < 0.05), and the MyHC IIa of Normal soil group was signi cantly lessened (P < 0.05). The MyHC IIx protein abundance in Normal soil group was also signi cantly decreased compared with other two groups (P < 0.05). Consistently, the MyHC IIb protein abundance was not signi cantly different among all groups (P > 0.05).
Protein expression related with mTOR pathway AMPK and mTOR signaling pathways were determined using Western blotting analysis, and the outcomes were demonstrated in Fig. 8. The results showed that the Normal soil signi cantly increased the AMPK and mTOR phosphorylation compared to No soil and Antibacterial soil groups (P < 0.05), and no signi cant differences were obvious between No soil and Antibacterial soil groups (P > 0.05).

Discussion
In relation to previous studies, muscle ber characteristics (muscle ber diameter, number, cross section area, and muscle ber type) were affected by intrinsic factors (breed, sex, and age) and extrinsic factors such as nutrition value [16][17][18]. Based on the results, our study indicated that different topsoil treatments also had effects on the muscle ber characteristics and the transcription related with myogenesis and muscle ber type. C Larzul, L Lefaucheur, P Ecolan, J Gogue, A Talmant, P Sellier, P Le Roy and GJJoas Monin [9] pointed out that the muscle ber cross section area had a positive relationship with the pig growth rate. Relevantly, the muscle ber area of Normal soil group and Antibacterial soil group was signi cantly higher than No soil group and indicated a better muscle growth rate. In addition, the muscle ber diameter is positively related to the cross-section area [16], and our results were consistent with this theory.
Hyperplasia (increasing muscle ber number) and hypertrophy (increased ber size) are two main processes that regulate muscle growth [19]. MRFs are a family of helix-loop-helix transcription aspects, including MyoG, Myf5, etc., that modify muscle hyperplasia and hypertrophy [20]. Myf5 is the primary MRFs which directly proliferates myogenic progenitor cells towards a myogenic lineage, and MyoG is the secondary MRFs which regulates the differentiation and fusion of myoblasts to form myo bers [21,22]. The expression of MSTN is mainly in skeletal muscle and its expression has a negative relationship with muscle growth [23]. It was also reported that the mutation of MSTN increased the muscle growth and muscle mass in many animals including pigs, sheep, rabbits, and cattle [24][25][26][27]. This experiment con rmed that exposure to topsoil during the pre-weaning stage changed the transcription related with myogenesis. Exposure to Normal soil upregulated the gene expression of Myf5, while downregulated the MSTN transporter. The results indicated that exposure to topsoil had a positive impact on the myogenesis of piglets.
According to our results, the transcription of different muscle ber types of weaned pigs was also affected by the topsoil. In general, there are four different muscle ber types (type I, type IIa, type IIb, and type IIx) which can be detected in pig skeletal muscle, and they are distinguished by different types of myosin heavy chain (MyHC I, IIa, IIb, and IIx) [10]. Type I is slow-oxidative ber, type IIa is fast oxideglycolytic ber, and both type IIb and IIx are fast glycolytic bers [28]. Different muscle ber types represent different ATPase characteristics of bers [29], for example, type I ber is rich in mitochondria which provides ATP by its fatty acid oxidation, and type II bers, which are classi ed as IIa, IIb, or IIx by its myosin heavy chain (MyHC) isoforms expression, utilize glucose to supply energy [10,30]. The muscle ber types are impacted by complicated intrinsic and extrinsic factors such as breed, gender, age, nutrient level, and physical activity [31]. Based on this theory and our transcription results, exposure to the topsoil, as an extrinsic factor, also altered the muscle ber type composition and the energy utilization forms of weaned pigs, and contacting with Normal soil increased the proportion of type I ber which relies on the oxidative activity while decreased the type II bers which have more glycolytic metabolism. The changes of muscle ber characteristics of weaned pigs may also affect the postmortem metabolism or even the consequential meat quality.
In addition, the gene and protein expression related with intramuscular fat deposition and fat removal ofpiglets were in uenced by the exposure to topsoil. For the intramuscular fat deposition, exposure to Normal soil downregulated the CD36 gene and PPARγ protein expression. CD36 is a primary fatty acid transporter expressed in animal skeletal muscle [32,33], and its abundance on the plasma membrane of obesity animals has a positive correlation with the rate of fatty acid uptake [34]. PPARγ is a ligandactivated transcription factor, expressed in many tissues (skeletal muscle and adipocytes) which accelerates adipogenesis and insulin sensitivity [35,36], and its expression regulates the stimulation of adipocyte differentiation and fat deposition [37]. In terms of lipolysis, Normal soil unregulated the HSL, LPL expression, while downregulated the CPT1 gene expression. HSL detached fatty acids from intracellular triacylglycerol for oxidation and exportation [38]. LPL is a rate-limiting enzyme for the hydrolysis of triacylglycerol, and its catalyzed reaction products, fatty and monoacylglycerol, are used by adipose tissue and skeletal muscle as element of neutral lipids [39]. CPT1 was reported to be correlated with mitochondrial fatty acid oxidation [40].
The mTOR pathway plays an important role in modulating amino acid metabolism and re ecting the availability of amino acid [41,42]. In addition, AMPK is a key energy sensor which regulates the cellular energy [43], and it can also modulates oxidative stress and mitochondrial function [44]. In this investigation, we found that exposure to topsoil increased the mTOR and AMPK signaling pathway in weaned pigs, and it activated their phosphorylation. The results indicated that the amino acid and cellular energy metabolism of piglets were modulated by the exposure to topsoil during weaning period.

Conclusions
The current experiment proved that exposure to topsoil promotes the myogenesis, modulates the transcription related with muscle ber types, intramuscular fat deposition and lipolysis, regulates the AMPK and mTOR signaling pathway of weaned piglets.

Not applicable
Availability of data and material The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors claim no competing interests   Effect of topsoil on gene expression related with myogenesis of weaned piglets. Values are the least square mean ± standard error of the mean. Within a row, means with no superscripts or with a common superscript letter are not signi cantly different (P < 0.05).

Figure 3
Effect of topsoil on gene expression related with intramuscular fat deposition in weaned piglets. Values are the least square mean ± standard error of the mean. Within a row, means with no superscripts or with a common superscript letter are not signi cantly different (P < 0.05).

Figure 4
Effect of topsoil on gene expression related with intramuscular fat lipolysis in weaned piglets. Values are the least square mean ± standard error of the mean. Within a row, means with no superscripts or with a common superscript letter are not signi cantly different (P < 0.05).

Figure 5
Effect of topsoil on gene expression related with muscle ber type of weaned piglets. Values are the least square mean ± standard error of the mean. Within a row, means with no superscripts or with a common superscript letter are not signi cantly different (P < 0.05).

Figure 6
Effect of topsoil on protein expression related with intramuscular fat and myogenesis of weaned piglets.
Values are the least square mean ± standard error of the mean. Within a row, means with no superscripts or with a common superscript letter are not signi cantly different (P < 0.05).

Figure 7
Effect of topsoil on muscle ber type protein expression of weaned piglets. Values are the least square mean ± standard error of the mean. Within a row, means with no superscripts or with a common superscript letter are not signi cantly different (P < 0.05).