All experimental procedures complied with current animal protection laws (Ethics Approval Code: HNUAHE ER 2223101) and were approved by the Animal Care and Use Committee of Henan University of Animal Husbandry and Economy, in accordance with the Guide for the Care and Use of Laboratory Animals.

This study was conducted at Pingdu Huayu Pig Breeding Technology R&D Co., Ltd., Qingdao City, Shandong Province. Forty healthy sows (Large White, on day 85 of gestation) with a parity level of 1–2 were used in this experiment. At 0–85 days of gestation, all sows were fed the same commercial diet with a digestive energy level of 3.1 Mcal/kg and a protein level of 12.8%. On day 85 of pregnancy, the sows were assigned to two treatment groups based on the principle of similar body weight and backfat thickness in a completely randomized design, with 20 replicates in each treatment group and one sow in each replicate. The sows in the control group (Con) were fed a basal diet (dietary formulations and nutritional compositions are shown in Table 1), and the sows in the treatment group (3-HIB) were supplemented with 15 mg/kg body weight of 3-HIB daily based on either the initial or post-parturition body weight in the basal diet. Sows were fed a diet mixed with 3-HIB at a dose corresponding to their body weight at each meal. 3-HIB, a valine metabolite, has not been reported in pig experiments. Therefore, the amount of 3-HIB in the diet was measured according to the addition of leucine metabolite β-hydroxy β-methylbutyrate (HMB) in sows, according to previous studies (18, 19). Each sow was fed twice daily (07:00 and 18:00 h), with a total daily intake of 2.8 kg/day until farrowing. The basal diet during late gestation was formulated to meet nutrient requirements as recommended by the National Research Council in 2012 (NRC 2012) (20), with all nutrient levels meeting or exceeding the NRC (2012) recommendations. All sows were housed in individual crates with an autoloading feeding system, and water was available ad libitum.

During the delivery of sows, the birth weight of each piglet was recorded to calculate litter weight. Litter size, number of live births, and number of stillbirths were recorded, and the weight coefficient of variation (CV) of each litter was calculated. Approximately 5 mL of blood was collected from the auricular marginal vein of sows (n = 12) and centrifuged (3,000 g for 15 min at 4°C) after standing for 15 min to obtain plasma samples; all samples were stored at −20°C for further examination. Three placental samples were collected from six sows in each treatment group, corresponding to the average piglet weight. The three collected placental tissue samples were analyzed for lipid metabolism-related indicators, lipid metabolism-related proteins, morphology, and immunofluorescence.

When sows farrowed, marks were made on the umbilical cords at both the sow and piglet ends to determine the placental sites corresponding to the piglets scheduled for slaughter. Placental samples corresponding to the average piglet weight were collected from six sows in each treatment group. These placental tissue samples were analyzed for lipid metabolism-related indicators—including triglycerides (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), glucose (Glu)—as well as lipid metabolism-related proteins such as sterol regulatory element binding protein 1 (SREBP1), fatty acid synthase (FASN), acetyl-CoA carboxylase (ACC), solute carrier family 27a (SLC27A1), and fatty acid binding protein 3 (FABP3). Additionally, tissue morphology and immunofluorescence (FABP3) were examined. Six female piglets with weights close to the average weight per treatment group were selected from different gilts for blood sample collection and slaughter at farrowing. Blood was collected via the anterior vena cava and separated to obtain plasma samples that were then stored at −20°C for biochemical and fatty acid analysis. The piglets were euthanized after being anesthetized with an intravenous injection of Zoletil 50 (0.1 mg/kg body weight). The weights of the heart, liver, spleen, intestine, and other organs of the piglets were recorded to calculate the organ index. The organ index is calculated as the ratio of the organ weight to the body weight of piglets. Longissimus dorsi muscle samples were collected corresponding to the last rib and stored at −80°C for determining the expression levels of proteins related to lipid metabolism.

Approximately 100 mg of placental tissue was weighed, 1 mL of tissue lysis solution and iron beads were added, and the mixture was ground by a grinder (JXFSTPRP-64, Jingxin, Shanghai, China). After centrifugation at 12,000 g for 15 min at 4°C, the supernatant was collected to determine lipid metabolism indices. The concentrations of Glu, TG, TC, HDL-C, LDL-C, and total protein in the placenta and plasma were measured using commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), following the manufacturer’s instructions.

The fatty acid composition of piglet plasma was determined by gas chromatography. First, fatty acids in the plasma were extracted using an organic solvent. The samples were then mixed and centrifuged to obtain an organic, aqueous, and protein phase. The organic phase was collected for the subsequent experiments. A methylating reagent was then added to the organic phase to convert the fatty acids into fatty acid methyl esters. After cooling to room temperature, the solution was treated with a saturated NaCl solution and n-hexane to obtain the n-hexane phase, which was used for instrument detection. The flow rate of the gas chromatograph was set to 1.5 mL/min. The capillary column dimensions were 30 m × 320 μm × 0.25 μm. The temperature of the injection port was gradually raised to 230°C within 2 min. Finally, because different fatty acid methyl esters formed different chromatographic peaks, a normalization method was used to calculate the area of each chromatographic peak, thereby reflecting the fatty acid content.

After fixing the tissue with 4% paraformaldehyde, the sample was cut into 5-μm sections using a microtome and placed on slides for drying. Subsequently, the slides were fixed using 4% paraformaldehyde for 30 min. The glass slides were washed 3 times for 5 min with phosphate-buffered saline (PBS). The slices were treated with a 0.1% Triton X-100 solution for 15 min. After washing with PBS 3 times, the slices were blocked with 5% bovine serum albumin (BSA) for 60 min. Subsequently, the slides were incubated with the FABP3 antibody solution and placed in a humidified chamber for incubation overnight at 4°C. Thereafter, the slides were incubated with a fluorescent-labeled secondary antibody at room temperature (25°C) for 60 min. The slides were overlaid with 4′,6-diamidino-2-phenylindole (DAPI) reagent, incubated in the dark for 10 min, and washed 3 times with PBS. Images were acquired using a fluorescence microscope (NIS-Elements, Nikon, Japan).

The expression of lipid metabolism-related proteins in the sow placenta (n = 6) and piglet muscle (n = 6) was detected. Samples from two sows or piglets were randomly combined into one sample, and three samples were obtained from each treatment group for Western blot analysis. The tissue samples were treated with lysates and then lysed using a grinder. The sample was then centrifuged at 12,000 g for 15 min at 4°C. The supernatant was collected, and the protein concentration of each sample was determined using a bicinchoninic acid (BCA) protein concentration assay kit (Thermo Scientific, MA, United States). For electrophoresis, 50 μg of sample was loaded per well/well. The electrophoresis time, membrane transfer time, antibody incubation time, and other parameters involved in the follow-up test process were as described in a previous study. Anti-phosphor-mammalian target of rapamycin (mTOR), anti-phospho-P70, anti-phospho-4EBP1, anti-SREBP1, anti-long-chain acyl-CoA synthetase (ACSL), anti-carnitine palmitoyltransferase (anti-CPT), and anti-carbamoyl-phosphate synthetase (anti-CAD) antibodies were purchased from Proteintech (Proteintech Group, Wuhan, China). Anti-CD36 and anti-ACC antibodies were purchased from Cell Signaling Technology (Danvers, MA, United States). Anti-FASN, anti-SLC27A1, and anti-FABP3 antibodies were purchased from Abcam (Cambridge, UK). Anti-rabbit IgG, anti-mouse IgG, and anti-β-actin antibodies were purchased from AmyJet Scientific (Wuhan, China). Protein expression was measured using the ImageJ software and normalized to β-actin expression levels.

The results of the production performance and related biochemical indices were analyzed for the homogeneity of variance and then tested using Student’s t-test in SPSS version 19.0 (IBM Corporation, Chicago, IL, USA). All data are presented through tables and figures as means and standard error of the mean (SEM). A p-value of < 0.05 was considered statistically significant, whereas values of 0.05 ≤ p < 0.10 were considered a tendency.

Newborn piglets were divided into four groups according to their body weight (<1.0 kg, 1.0–1.2 kg, 1.2–1.4 kg, and >1.4 kg, respectively). The body weights of the pigs of different grades were statistically analyzed (Table 2). The addition of 3-HIB to the diet had no significant effect on the total number of offspring. 3-HIB supplementation significantly reduced the number of piglets below 1 kg compared to the Con group (p < 0.05), but had no significant effect on piglets of other body weight grades. Compared to the Con group, 3-HIB supplementation significantly reduced the number of stillbirths, the rate of weak piglets (p < 0.05), and the variation in birth weight of piglets (p < 0.05). 3-HIB supplementation to the sow diet did not affect the organ indices of newborn piglets (Table 3).

Lipid metabolism-related indicators in the plasma and placental homogenates of sows, including glucose (Glu), triglyceride (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C), were determined (Table 4). Compared to the Con group, the addition of 3-HIB to the diet of sows had no significant effect on the concentration of each index in the plasma, but tended to increase the concentration of TG (p = 0.069). In the placenta of sows, TG concentrations were significantly higher in the 3-HIB supplementation group than in the Con group (p < 0.05). In addition, 3-HIB supplementation tended to increase the Glu concentration (p = 0.079).

The fatty acid composition in the plasma of newborn piglets was detected (Table 5). For saturated fatty acids, higher proportions of C18:0 and total saturated fatty acids were observed in the 3-HIB group than in the Con group (p < 0.05). However, 3-HIB supplementation did not affect the monounsaturated fatty acid content in the plasma of piglets. For polyunsaturated fatty acids, C20:4 n-6, C20:5 n-3, C22:6, and total polyunsaturated fatty acid concentrations were significantly higher in the 3-HIB supplementation group than in the Con group (p < 0.05).

To investigate the reasons for the increase in plasma triglycerides and fatty acids in newborn piglets in the 3-HIB supplementation group, this study further analyzed the expression of lipid metabolism-related proteins in the placenta (Figure 1). Compared to the Con group, the 3-HIB addition group showed significantly increased expression of SREBP1 protein in the placenta (p < 0.05), along with significantly upregulated expression of its downstream fatty acid transport-related proteins, including SLC27A1 and FABP3 (p < 0.05, Figure 1A). The results of placental tissue immunofluorescence analysis were consistent with those of western blotting. The 3-HIB supplementation group showed higher fluorescence intensity of the FABP3 protein (Figure 1C). The expression of de novo fatty acid synthesis-related proteins (FASN and ACC) was not affected by 3-HIB supplementation (Figure 1A). Furthermore, assessment of the expression of mTOR signaling pathway-related proteins revealed that p-mTOR and p-4EBP1 were significantly increased in the 3-HIB addition group compared to the Con group (p < 0.05). These findings suggest that 3-HIB regulates the expression of downstream fatty acid transporters by activating the mTOR signaling pathway.

The present study investigated the expression of lipid metabolism-related proteins in piglet muscle tissues (Figure 2). The expression of FABP3 protein in muscle tissue was significantly higher in the 3-HIB addition group than in the Con group (p < 0.05). Simultaneously, the expression of the rate-limiting enzyme of fatty acid oxidation (carnitine palmitoyltransferase 1 [CPT-1]) in the muscle tissue was significantly increased in the 3-HIB addition group (p < 0.05), indicating that increased fatty acid oxidation may provide more energy for cell proliferation. The expression of mTOR signaling pathway-related proteins in the muscle tissue was consistent with that in the placenta. The expression of p-mTOR and p-4EBP1 proteins was significantly higher in the 3-HIB addition group than in the Con group (p < 0.05).