Dietary Energy Levels from Middle to Late Gestation of Guangdong Small-Ear Spotted Pig: Effects on Body Weight, Muscle Development, and Glucose Metabolism of its Newborn Piglets

Chengjun Hu South China Agricultural University Yunyu Yang South China Agricultural University Minxia Chen South China Agricultural University Xiangyu Hao South China Agricultural University Shuqi Wang South China Agricultural University Linfang Yang Guangdong Yihao Foodstuffs Co., Ltd Shihai Zhang South China Agricultural University Chengquan Tan (  tanchengquan@scau.edu.cn ) South China Agricultural University College of Animal Science https://orcid.org/0000-0003-1686-3778


Introduction
In the last decade, the selection for high proli cacy in modern sow herds has led to a marked increase in litter sizes. One consistent outcome of this strategy was the increased number of low birth weight piglets with less vitality and mature. Maternal nutrition plays a vital role in this performance. For instance, increased maternal feed intake from 1.8 kg/d to 3.5 kg/d during days 90 to 110 of gestation was reported to reduce the occurrence of lightweight piglets [1]. These results suggest that an optimal nutrition level of sows during gestation is important to the improvement of birth weight and its heterogeneity of piglets.
Adequate energy intake during the gestation can improve reproductive performance, litter performance, and colostrum quality [2]. However, excessive energy intake during gestation can reduce feed intake during lactation and pig weight [2,3], increase body weight and backfat thickness loss [4]. In addition, maternal energy intake is closely associated with fetus skeletal muscle development. Study showed that the skeletal muscle differentiation and maturity of fetus were delayed under maternal high energy intake during the gestation [5]. Therefore, the energy intake of gestating sows needs to satisfy both of fetal growth and body condition maintenance.
Guangdong small-ear spotted pigs are Chinese indigenous pig breed that have many unique characteristics, such as desired meat quality and adaptability to crude ber. However, this breed also possesses negative economic features including slow growth rate, low lean-meat percentage, and low feed utilization rate. To our knowledge, no study has elucidated the energy requirement of this breed during pregnancy. Therefore, the aim of this study was to investigate the effects of maternal energy diet feeding from middle to late gestation of Guangdong small-ear spotted pigs on birth weight, muscle development and glucose metabolism of its newborn piglets.

Experimental design and diets
A total of 66 pregnant gilts (Guangdong Small-ear Spotted pig) were assigned to either a control group (CON group, 11.50 MJ/kg digestible energy) or a high-energy diet group (HE group; 13.42 MJ/kg digestible energy) during gestation, with 11 replicate pens per treatment and 3 gilts per pen, respectively.
The nutrient level for the control diet (

Performance measurement of gilts and piglets
After parturition, the birth weight of piglets was measured within 6 h after birth. No difference was observed in the birth weight of the piglets (645 ± 23 g VS 604 ± 8 g) between the two groups. Backfat thickness of gilts was measured at parturition. Backfat thickness was measured at the P2 position using a mode ultrasound scanner (Renco Lean-Meater®, Minneapolis, MN, USA). The numbers of total born, born alive, and stillborn piglets were recorded at farrowing.

Glucose tolerance test (GTT)
After an overnight fast, an intraperitoneal glucose tolerance test (GTT) was performed. Brie y, neonatal piglets with a similar average birth weight (n = 8) were given an intraperitoneal injection of glucose (1 g/kg body weight) over a period of 120 min. Blood glucose levels in tail vein were measured at 10, 20, 30, 45, 60, 90, and 120 min after the initial glucose injection using a glucometer (Sannuo, Changcha, China).

Sample collection
After parturition, piglets (n = 8) with a similar average birth weight were selected for sample collection. Blood samples were collected into 10-mL centrifuge tubes from the jugular vein, followed by centrifugation at 3,000 × g and 4 °C for 15 min to recover the serum. Meanwhile, liver and soleus muscle (about 2 g each) were collected from the carcass and weighed, followed by snap-freezing in liquid nitrogen and storage at -80 °C for further analysis.

Biochemical parameter
The levels of serum insulin (CSB-E06829p, Cusabio Biotech, Wuhan, China) and non-esteri ed fatty acid (NEFA) (A042-2-1, Nanjing Jiancheng Bioengineering Institute, Nanjing, China) were determined using Malic dehydrogenase (MDH) and succinate dehydrogenase (SDH) enzyme activity assay The MDH and SDH activities in muscle and liver were measured using the commercial assay kits (Solarbio, China) according to the manufacturer's instructions.
Mitochondrial DNA (mtDNA) copy number Total genomic DNA was isolated from muscle and liver using the QIAamp DNA Mini Kit (Qiagen, USA) according to the manufacturer's instructions. Mitochondrial DNA copy number was determined as previously described [6]. The primers were presented in supplemental Table 1.
Real-time quantitative RT-PCR.
Total RNA was isolated from the muscle and liver tissues using the TRIzol reagent (Invitrogen, Carlsbad, USA). cDNA synthesis was performed using 1000 ng RNA according to the manufacturer's instructions of the PrimeScript RT reagent kit (Takara, Dalian, China). Primers used are listed in supplemental Table 1.

Statistical analysis
Data are presented as mean ± SEM. Statistical analyses were performed using SPSS 20.0 (SPPS Inc., Chicago, IL) software. The differences between the two groups were analyzed by the Student's t test. P < 0.05 was considered as statistically signi cant.

Performance of gilts and piglets
The litter weight, total piglets born, piglets born alive and stillbirth are not difference (P > 0.05) between the CON and HE groups. The HE group showed an increase trend (P = 0.07) in the birth weight variation when compared to the CON group (Table 2). Interestingly, the HE diet was shown to decrease (P < 0.05) the piglet birth weight of gilts with total or alive litter size of 12 to 13 (Fig. 1A, B). No difference (P > 0.05) was observed in the muscle weight of newborn piglets in the two groups (Fig. 2). Value are mean ± SEM. The differences between the two groups were analyzed by the Student's t test. 1 There were 11 replicate pens (3 gilts /pens) per group.
Gilts from the HE group showed a higher (P < 0.05) backfat thickness at parturition day (Table 2). Maternal high-energy diet feeding impairs glucose tolerance in offspring We performed a GTT (glucose tolerance test) in neonatal piglets and the results are shown in Fig. 3. The two groups showed no difference (P > 005) in the fasting glucose level at baseline (Fig. 3A). However, the HE piglets were higher (P < 0.05) than the CON piglets in the area under the curve (AUC) analysis of GTT (Fig. 3B). Additionally, the HE piglets were shown to be higher (P < 0.05) than the CON piglets in HOMA-IR value, fasting serum insulin level, and triglyceride concentration (Fig. 3C, D, F). However, no difference (P > 005) was observed in the serum NEFA concentration between the two groups (Fig. 3E).
Maternal high-energy diet feeding induces TG accumulation, but prevents glycogen deposition in the muscle and liver of offspring Compared with the CON group, the HE piglets displayed a higher (P < 0.05) concentration of TG, while a lower (P < 0.05) content of glycogen and glycogen synthase in the muscle (Fig. 4A-D).
Maternal high-energy diet feeding inhibits the activation of insulin signaling pathway in the muscle and liver of offspring In Fig. 5, when compared to the CON group, the HE piglets showed a reduced activation of insulin signaling pathway in the liver and muscle, as evidenced by the decreased protein levels of p-IRS-1 (P < 0.05) and p-AKT (P < 0.05).
Maternal high-energy diet feeding induces the transition of skeletal muscle ber types in offspring Here, we investigated the effects of maternal HE diet on the skeletal muscle ber type in offspring. In soleus muscle, we found that maternal HE diet downregulated the slow-twitch ber-associated genes of MyHC I (P < 0.05) and MyHC IIa (P < 0.05), with a trend (P = 0.08) to upregulate the fast-twitch ber associated gene of MyHC IIb (Fig. 6A). Accordingly, the protein level of MyHC I (P < 0.05) was downregulated, in contrast to the upregulation in the protein levels of MyHC IIb (P < 0.05) and MyHC IIx (P < 0.05) in the soleus muscle of HE piglets versus the CON piglets (Fig. 6B).
Maternal high-energy diet feeding impairs mitochondrial biogenesis in the muscle and liver of offspring In Fig. 7, maternal HE diet was shown to reduce (P < 0.05) the soleus muscle ATP levels (Fig. 7D), with a trend (P = 0.08) to decrease the mtDNA content (Fig. 7E). When compared with the CON group, the HE piglets showed a downregulation (P < 0.05) in the protein levels of peroxisome proliferator-activated receptor gamma coactivator (PGC-1α) (P < 0.05) and nuclear respiratory factor-1 (NRF-1) (P < 0.05) in the muscle (Fig. 7A-C). Additionally, maternal HE diet decreased the SDH (P < 0.05) and MDH (P < 0.05) activities in the muscle of offspring (Fig. 7F).
In Fig. 8, maternal HE diet was seen to reduce (P < 0.05) the hepatic ATP levels (Fig. 8E). Additionally, the protein levels of PGC-1α (P < 0.05) and NRF-1(P < 0.05) were also downregulated in the liver of the HE piglets versus the CON piglets ( Fig. 8A-C). Furthermore, the HE piglets also showed a decrease (P < 0.05) in the SDH activity in the liver due to maternal HE diet treatment (Fig. 8F).

Discussion
Maternal dietary energy level during pregnancy plays an important role in reproductive performance.
Guangdong Small-ear Spotted pigs are Chinese indigenous pig breed that have many advantages, which are highly valuable to the local pig industry. However, few studies have focused on the energy requirement of this breed during pregnancy. Therefore, the present study was to investigate the effects of dietary energy levels from middle to late gestation on reproductive performance of Guangdong Small-ear Spotted pig. In the present study, Maternal HE diet had no effect on litter weight, total piglets born, piglets born alive and stillbirth, which was in line with previous study [8]. Interestingly, maternal HE diet was shown not effect on average birth weight of piglets, but was shown to increase birth weight variation and decrease the average birth weight of piglets that from gilts with total or alive litter size of 12 to 13. This is a novel nding which suggests that increased maternal dietary energy levels from 11.50 MJ/kg to 13.42 MJ/kg decreased the reproductive performance of gilts with total or alive litter size of 12 to 13.
However, the muscle weight of neonatal piglets was not affected by dietary energy treatment. Similarly, the increased backfat thickness in late gestation was observed in gilts by increasing dietary energy levels [3]. In addition, the glucose tolerance in neonatal offspring was investigated, and maternal HE diet was shown to impair the glucose tolerance of neonatal offspring, as evidenced by GTT test, fasting serum insulin, and HOMA-IR index, which was in line with previous studies reporting that maternal high-fat diet impaired glucose metabolism in offspring [9].
Skeletal muscle consumes nearly 80% of glucose [10], and liver regulates glycogen storage, glucose uptake and output [11], indicating skeletal muscle and liver play an important role in glucose homeostasis. Glucose homeostasis is disrupted when the insulin signal pathway is suppressed in liver and muscle [12,13]. In the present study, the insulin signaling pathway in the liver and muscle of the HE offspring was examined to explore the molecular mechanism underlying their impaired glucose tolerance.
In their liver and muscle, the protein levels of p-IRS1 and p-AKT were seen to be decreased, implying the insulin signaling pathway was impaired. Lipotoxicity is characterized by excessive accumulation of lipid in non-adipose tissues, including liver and muscle, leading to cellular dysfunction. Excessive triglyceride accumulation in muscle is an important contributor to obesity and insulin resistance, and it is also known as a marker of insulin resistance [14]. The increased triglyceride content in the liver and muscle of the HE offspring further con rmed that maternal low-energy diet is bene cial to the glucose homeostasis of the offspring. Additionally, we observed a decrease in the glycogen content in liver and muscle, as well as a decrease in the content of glycogen synthase in muscle. Glycogen synthesis is a major fate for glucose uptake in the body, and glycogen synthase is a key enzyme related to whole-body insulin sensitivity. Previous studies have shown impaired glucose tolerance and metabolism in liver or muscle glycogen synthase knock-out mice [15,16]. Therefore, we speculate that suppressing the insulin signal pathway in liver and muscle may lead to a reduction in glucose utilization, thus impairing the glucose tolerance in the offspring under maternal HE diet treatment.
Skeletal muscle ber types, including MyHC I, MyHC IIa, MyHC IIb, and MyHC IIx, play a role in glucose homeostasis. Slow-twitch muscle bers are more sensitive than fast-twitch muscle bers to insulin stimulation [17]. Additionally, type I and IIa bers are more abundant than type IIb and IIx bers in the protein levels of insulin receptor, glucose transport-4 (GLUT4), and glycogen synthase [18], and the former two bers play a more important role in maintaining glucose homeostasis in response to insulin [19]. Furthermore, MyHC IIx ber exerts a negative effect on glucose uptake [20]. To explore the mechanism underlying glucose homeostasis, we determined the protein levels of MyHC I, MyHC IIa, MyHC IIb, and MyHC IIx in soleus muscle. Under maternal HE diet treatment, the protein levels of type I bers (MyHC I) and MyHC IIa were downregulated in the offspring, in contrast to up-regulation in the protein levels of type II bers (MyHC IIb and MyHC IIx). Based on the results in previous and present studies, impairment of glucose tolerance in offspring under HE diet treatment may be attributed to the decreased protein levels of slow-twitch muscle bers.
Mitochondrial function plays an important role in energy metabolism. Previous studies have shown that mitochondrial function and biogenesis are compromised in liver and muscle in type 2 diabetes, obesity, and insulin resistance [21][22][23][24][25]. PGC1α is a transcription factor coactivator that improves mitochondrial biogenesis via activation of various transcription factors including NRF1 [26]. Type I muscle bers have more mitochondria and higher oxidative metabolism than type II bers [27]. Increased type I muscle ber was observed in PGC-1α overexpressing mice [28]. Consist with the increase of type I muscle ber, the protein levels of PGC-1α and NRF1 were downregulated in the muscle and liver of the HE offspring. Furthermore, ATP content was also observed to decrease in the liver and muscle of the HE offspring. It has been reported that impaired mitochondrial biogenesis and function result in decreased ATP production [29]. The above ndings indicate that maternal HE diet feeding impaired mitochondrial biogenesis in the liver and muscle of the offspring, which can be supported by previous studies reporting that maternal high-energy diet impaired mitochondrial biogenesis in the skeletal muscle of the offspring in pigs [30]. SDH and MDH are respiratory enzymes and exert an important role in mitochondrial function, and the decreased SDH and MDH activities in the HE group further support that maternal HE diet impairs mitochondrial biogenesis in the liver and muscle of the offspring.
In conclusion, maternal high-energy diet from middle to late pregnancy was shown to decrease the birth weight of piglet that from the gilts with total or alive litter size of 12 to 13. In addition, maternal high-energy diet impairs the glucose tolerance in the neonatal offspring, with the mechanisms involved in the downregulation of slow-twitch muscle bers in soleus muscle and the decrease in hepatic and muscular mitochondrial biogenesis. These results contribute to a better understanding of the role of maternal energy diet in the reproductive performance of Guangdong Small-ear Spotted pig, and glucose metabolism and muscle ber transformation in offspring.  Figure 1 The birth weight of piglets of sows with different total (A) or alive (B) litter size. Values are mean ± SEM. The difference between two means was analyzed using the Student's test. * indicates P < 0.05.

Figure 2
Effects of maternal dietary energy levels on muscle weight of newborn piglets. Values are mean ± SEM, n=7-8/group. The difference between two means was analyzed using the Student's test.  between two means was analyzed using the Student's test. * indicates P < 0.05; ** indicates P < 0.01; ns, no signi cant difference.

Figure 5
Page 20/22 Effects of maternal dietary energy levels on the inulin signaling pathway activation in the muscle and liver of piglets. Western blotting analysis was used to determine the protein levels of p-IRS-1, IRS-1, p-AKT, and AKT in the liver (A) and soleus muscle (C) of piglets. (B, D) Summarized data. Values are mean ± SEM, n=7-8/group. The difference between two means was analyzed using the Student's test. * indicates P < 0.05; ** indicates P < 0.01; *** indicates P < 0.001.

Figure 6
Effects of maternal dietary energy levels on the transition of skeletal muscle ber types in piglets. qPCR and Western blotting analyses were employed to measure the mRNA (A) and protein (B, C) levels of MyHCI, IIa, IIb, and IIx in soleus muscle, respectively. Values are mean ± SEM, n=7-8/group. The difference between two means was analyzed using the Student's test. * indicates P < 0.05.

Figure 7
Effects of maternal dietary energy levels on the muscle mitochondrial biogenesis of piglets. Western blotting analysis was used to determine the protein levels of NRF-1 and PGC1-α in soleus muscle (A). The ATP level (B), mtDNA content (C), and activities of SDH and MDH (D) in soleus muscle. Values are mean ± SEM, n=7-8/group. The difference between two means was analyzed using the Student's test. * indicates P < 0.05.