Soybean meal (SBM), an important protein source in the livestock industry, is highly recognized for its significant protein content and widespread availability (Azarm and Lee, 2014; Kim, 2014). However, the price of SBM has increased dramatically and the supply of high-quality protein feeding ingredients (such as SBM and fish meal) has been deficient in recent years. This leads to a higher livestock breeding expense and restricts the development of animal husbandry. Therefore, there is an urgent need for more alternative high-quality protein feeding ingredients to solve the protein source crisis in animal husbandry.

Cottonseed meal (CSM), a by-product obtained from the process of extracting the oil from cotton seed, is an attractive alternative protein source for livestock diets (Nagalakshmi et al., 2007). However, compared to SBM, the application of CSM as a feed ingredient in animal husbandry is limited due to the presence of anti-nutritional factors (Lordelo et al., 2007; Tang et al., 2012) such as free gossypol, cycloproponoic fatty acids, and crude fiber, which may cause negative effects on growth performance and organ functionality (Nie et al., 2015; Świątkiewicz, 2016). Fermented cottonseed meal (FCSM), a product produced by mixing solid CSM with liquid phases and then inoculating the mixture with beneficial microorganisms (Sun et al., 2014), can reduce free gossypol and improve the protein quality of CSM with solid state fermentation (Zhang et al., 2007). It has been suggested that the FCSM partial replacing SBM (about 6–8%) not only can improve the growth performance, immune and antioxidant capacity, and digestibility in broiler chickens (Sun et al., 2013; Nie et al., 2015; Wang et al., 2017; Niu et al., 2020), but also can reduce the F:G (the ratio of feed and gain weight) and diarrhea rate in nursery pigs, growing pigs and finishing pigs (Guan et al., 2017), which indicated that the FCSM has the potential to be a high-quality protein source.

Weaned piglet is a critical group in whole pig production, which consumes a large amount of high-quality protein ingredients. However, there is a lack of relevant research on the application of FCSM in weaned piglets. Therefore, the present study aimed to compare the effects of dietary SBM and FCSM on growth performance, immunity and antioxidant properties, nutrient digestibility, and gut microbiota of weaned piglets.

The animal handling and all procedures of this study received approval from the Animal Care and Use Ethics Committee of the Hunan Agricultural University (Changsha). The FCSM used in this experiment was provided by the Tycoon Group (Xinjiang, China). The SBM used in this experiment was provided by Hunan Lifeng Biological Technology Co., Ltd. (Hunan, China). Table 1 shows the nutrient composition of SBM and FCSM.

Cottonseed meal has not been widely used because it contains a large number of anti-nutritional factors, which result negative effects on growth performance, immune and antioxidant capacity and nutrient digestibility in animals (Nagalakshmi et al., 2007; Nie et al., 2015; Świątkiewicz, 2016). After going through solid state fermentation, FCSM has much less free gossypol and other anti-nutritional factors, and definitively improved protein quality (Zhang et al., 2006, 2007; Sun et al., 2014; Wang et al., 2017). Sun et al. (2013) reported that the appropriate inclusion of FCSM replacing SBM improved the growth of yellow-feathered broiler chickens. Similarly, FCSM supplementation improved the ADG and G:F ratio of yellow-feathered broilers from the 43rd to 64th and the 21st to 64th day, respectively (Nie et al., 2015), which indicated that FCSM is beneficial for broilers as it enhanced their growth performance and digestion. Consistently, the current study showed that the piglets in the FCSM group presented greater growth performance and a lower diarrhea rate than those in the SBM group. The fermentation process of CSM might be one of the reasons, which effectively decreases free gossypol level, and increases acid-soluble protein level in CSM, and therefore further improves the digestive enzyme activity and nutrient digestibility in weaned piglets (Sun et al., 2014; Wang et al., 2017). This explanation has been confirmed by the nutrient digestibility of weaned piglets in FCSM and SBM treatments. The present results demonstrated that the apparent total tract digestibility and ileal digestibility of nutrients, essential and nonessential AA have been enhanced in piglets within the FCSM group.

Moreover, the microbial fermentation process can produce many beneficial substances, such as small-size peptides, exoenzymes, vitamins, organic acids, which can promote the immunity of animals (Johnson, 2013; Zhao et al., 2016). A study have reported that fermentation with Bacillus subtilis BJ-1 can reduce free gossypol level in CSM and dietary inclusion of 12% FCSM can improve immunity (Tang et al., 2012). Wang et al. (2017) reported that the dietary supplementation of FCSM increased serum IgM and IgG levels compared with SBM groups in broilers. In the present study, the concentration of serumal IgG was increased in piglets in the FCSM group in comparison with those in the SBM group, which might be due to the live microbes in FCSM acting as probiotics to enhance the humoral immune response of the animals (Latesh et al., 2013; Stašová et al., 2015).

The immunity of piglets is highly related to antioxidant capacity (Liu et al., 2021). Besides, antioxidase can improve the immunity by promoting the bacterial clearance and regulates phagocyte numbers (Peterman et al., 2015). In addition, a microbial FCSM increased the antioxidant activity in diets for Nile tilapia (Lim and Lee, 2011). Similarly, the present study showed that the antioxidation-related enzymes in serum, jejunum, ileum and liver were improved in piglets in the FCSM group compared with that in the SBM group. Additionally, the serum MDA was reduced in piglets in the FCSM group, which indicated that FCSM improved the antioxidative abilities compared with SBM (Wang et al., 2017). This may be one of the reasons that the improvement of growth performance in piglets has found in the FCSM group (Ma, X. et al., 2019; Ma, X. K. et al., 2019).

Many studies suggest that food or feeds fermented by probiotics may be potentially an effective strategy to regulate the gut microbiota and its metabolites (Azad et al., 2020; Niu et al., 2020; Gu et al., 2021; Li et al., 2021). The VFAs, especially butyric acid, as a microbial metabolite, can not only improve the growth performance, but also boost the immunity of piglets (Fang et al., 2014; Zhao et al., 2018). Zhao et al. (2018) reported that dietary fiber increases the butyrate-producing bacteria and improves growth performance of weaned piglets. Tsai et al. (2019) discovered that feeding sodium butyrate during the nursery phase tended to alter blood cell count and improve growth performance of weaned pigs. In the present study, the VFAs of ileum and cecum were increased in the FCSM group than that in the SBM group. This may explain the improved growth performance of piglets. Moreover, VFAs, as important intermediate products during anaerobic digestion, may influence the fermentation characteristics of hindgut (Jha and Berrocoso, 2016; Seradj et al., 2018). The protein fermentation metabolites in the hindgut are amines, SCFAs and BCFAs, among which amines must be converted from nitrogen-containing groups, while BCFAs are only produced from the fermentation of three branched chain amino acids, leucine, isoleucine and valine (Jha and Berrocoso, 2016; Seradj et al., 2018). Over-fermentation of protein in the hindgut is an important cause of diarrhea in piglets. The current study found that FCSM replacing SBM decreased the isobutyrate and isovalerate of the cecum, suggesting that FCSM reduced hindgut fermentation, which may be responsible for the reduced diarrhea rate in piglets.

In recent years, the interaction and connections between dietary protein, gut microbe and host has received increasing attention. Segmented exogenous microbiota transplantation proved the spatial heterogeneity of bacterial colonization along the gastrointestinal tract, i.e., the microbiota from one specific location selectively colonizes its homologous gut region (Li et al., 2020). The number of microorganisms in the hindgut was higher than that in the foregut (Jamet et al., 2011), which is consistent with the result in the present study. It has been previously shown that FCSM enhanced the Simpson index of ileum in piglets, which has a great contribution towards the improvement of intestinal health and maturation in piglets (Wang, X. et al., 2019). Lower protein concentration or better protein sources in the diets can improve hindgut health by preventing the proliferation of pathogenic bacteria and reduced the risk of colitis (Vidal-Lletjós et al., 2017; Najafabadi et al., 2019). Studies have shown that protein fermentation can change the composition and function of intestinal flora (Lu et al., 2019; Wang, H. et al., 2019). Consistently, in the present study, the analysis of the PCoA has shown that the microbial composition between the SBM and FCSM groups are slightly different.

Firmicutes and Bacteroidetes were the most dominant phyla in the pig (Chen et al., 2017). In our study, Firmicutes is the most dominant phyla in ileum and Firmicutes and Bacteroidetes in cecum. Lactobacillus, as a potential probiotic, possesses the resistance to pathogen, anti-inflammatory and antioxidant capacity, and ability to improve of gut microbiota profile (He et al., 2019; Yang et al., 2020). Wang et al. (2017) has discovered that Lactobacilli and total anaerobic bacteria counts in ceca digesta of birds fed FCSM were improved compared with birds fed CSM on days 21 and 42. Likewise, the FCSM replacing the SBM has also enhanced the relative abundance of Lactobacillus of ileum and cecum in the present study. He et al. (2019) has found that Lactobacillus johnsonii L531 reduced pathogen load and helped maintain SCFA levels in the intestine of pigs challenged with Salmonella enterica Infantis. Therefore, this might be one of the reasons that piglets in the FCSM group had higher level of VFAs than that in the SBM group. On the other hand, proteobacteria is the largest phylum of bacteria, including many pathogenic bacteria, such as Escherichia coli, Salmonella, Vibrio cholerae, Helicobacter pylori and other well-known species. In the current study, the relative abundance of Proteobacteria was decreased in piglets in the FCSM group, suggesting that the FCSM has the potential function to inhibit harmful bacteria, and improves the gut microbiota profile than SBM (Wang et al., 2017). In conclusion, FCSM replacing SBM improved the growth performance, immunity and antioxidant properties, nutrients digestibility possibly via the altering gut microbiota profile and its metabolites in weaned piglets.

The data presented in the study are deposited in the (NCBI SRA) repository, accession number (PRJNA743130).

The animal study was reviewed and approved by Animal Care and Use Ethics Committee of the Hunan Agricultural University.

XG and XM: conceptualization, methodology, and software. ZL, NL, XL, and FZ: literature collection. XG and JW: writing–original draft preparation. JC, QJ, and BT: writing–reviewing and editing. XM: funding acquisition. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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