In the current intensive production system, a certain number of lambs are growth-hindered for various reasons, resulting in “growth-retarded lambs.” Severe malnutrition during the gestation or neonatal periods (Du et al., 2010), illness followed by pneumonia or coughing, and infection with parasites can restrict the healthy development and growth of animals (Gu et al., 2017; Ma et al., 2020b). The growth-retarded lambs with low body weight and feed conversion rate, high morbidity and mortality could reduce the economic incomes of sheep farming, thus, promoting the compensatory growth of growth-retarded lambs is an urgent issue to be addressed.

The healthy development of the gastrointestinal tract plays an important role in nutrient absorption, immune system development and healthy growth of animals (Celi et al., 2017). Zhang et al. (2018) demonstrated that the intestinal development was delayed and intestinal integrity, immune function, and oxidative status were impaired in intrauterine-growth-retarded suckling lambs. A study on the growth-retarded yaks revealed that they had lower rumen weight compare to the normal yaks (Hu et al., 2016). Ma et al. (2020a) inferred that the gastrointestinal development of growth-retarded yaks was dysplastic, as evidenced by the lower papillae height and surface area of the rumen. Severe malnutrition during the early lives of animals do harm for the structure, functions and microflora of gastrointestinal tract, and subsequently restricts the growth of animals (Meyer and Caton, 2016; Zhang et al., 2018). Looking for an effective additive to promote the gastrointestinal tract development may be a feasible method to improve the compensatory growth of growth-retarded lambs. On the other hand, the intestinal microbiota has a critical effect on regulating gastrointestinal homeostasis and function of host. Parker et al. (2017) reported that the stability of the microbial community is beneficial for the host. Ma et al. (2021a) found that modulating the microbiota (increase the relative abundances of Ruminococcaceae, Treponema, Clostridiaceae, Prevotellaceae, and Lachnospiraceae, which were related to oligosaccharide, starch, and cellulose degradation) in gastrointestinal of growth-retarded yaks could effectively enhance the digestibility, gastrointestinal fermentation and digestive enzyme activity. Considering the complex interplay between gut microbiota and host health, interventions based on the modulation of gut microbiota might be one of the potential strategies (Xie et al., 2022).

Currently, probiotics are supposed to have beneficial effect on the host health (World Health Organization, 2001) when they are supplemented with proper amounts. They are used as a safe additive to rebalance the intestinal microflora, prevent diseases, improve inflammation and digestion, and promote growth of animals (Riggs et al., 2006; Kalil, 2014). Lactobacillus spp., Saccharomyces spp., Enterococcus spp., and Bacillus spp. are widely used probiotic supplements in livestock and poultry production (Markowiak and Śliżewska, 2018). Guo et al. (2020) revealed that Lactobacillus sp. is one of the most important bacterial direct-fed microbial used as a growth promoter for ruminants. They have been identified to compete for nutrients in the gastrointestinal and produce antibacterial compounds, which allowed them to occupy specific niches of the gastrointestinal mucosa and activate the innate immune system of young ruminants (Frizzo et al., 2011). Moreover, Bacillus subtilis species were found to improve intestinal health of livestock by regulating immune function to protect against pathogenic challenge (Rolfe, 2000). A study conducted on the effect of B. subtilis on Duhan lambs demonstrated that B. subtilis altered the rumen fermentation pattern and increased the growth performance of lambs (Gao et al., 2023). Du et al. (2018) found that supplementation with probiotics (especially Bacillus spp.) can stabilize the intestinal microbiota of growth-retarded calves and improve their growth performance. In a review paper of Chapman et al. (2011), it was suggested that multi-strain probiotics appear to show greater efficacy than single strains. And Røssland et al. (2005) found that coculture of Lactobacillus with Bacillus cereus could stimulate the biosynthetic capacities of lactic acid strains.

Consequently, we hypothesized that nutritional intervention used to regulate the gastrointestinal microbiota, especially the rumen microbiota, would promote gastrointestinal health and the compensatory growth of growth-retarded lambs. In this study, a probiotic (co-fermented by Lactobacillus acidophilus and Bacillus subtilis) product was supplemented to the basal diet to detect their effects on the growth performance, plasma indices and ruminal bacterial community of growth-retarded lambs. The main objective of the current study is to provide insights into the effective nutritional intervention and its potential mechanisms for promoting the compensatory growth of growth-retarded lambs.

Lambs with growth retardation have higher morbidity and mortality and lower growth performance. No surprise, 4 growth-retarded lambs died during the feeding trial and the growth rate of growth-retarded lambs was slower than that of normal lambs in the current study. Ma et al. (2021b) reported the same findings in growth-retarded yaks. The positive effects on ADG and DMI indicated that dietary supplementation with probiotic was beneficial to improve growth-retarded lamb growth. Abdel-Wahed et al. (2022) found that both Bacillus sp. and Lactobacillus sp. can be used as probiotics to achieve therapeutic purposes, such as increased the growth performance of ewes and reduced the incidence of diarrhea in their newborn lambs (Khattab et al., 2020). Also, the results of a previous experiment using the same probiotic showed that it could enhance the growth performance of piglets (Xie et al., 2022). For ruminants, Ossimi lambs that received Lactobacillus sp. showed the highest weight gain (El-Ashker et al., 2018). Khattab et al. (2020) reported that probiotic bacteria (B. subtilis and L. casei) supplementation enhanced the weaning weight, average body weight gain and the health status of the lambs. Another report showed that Lactobacillus acidophilus supplementation enhanced the feed intake and daily weight gain of Nubian goats (Azzaz et al., 2015). The positive effect of probiotics supplementation may contribute to the stabilization of gastrointestinal microbiota, which promotes growth and enhances the host resistance to diseases (Xue et al., 2011; Du et al., 2018).

Various stressful conditions, such as malnutrition during the neonatal periods and illness followed by coughing, broken the balance between antioxidant defense system and free radical generation system. The lower concentrations of SOD and GSH-Px and the higher level of MDA in the plasma of growth-retarded lambs than normal lambs indicated that the animals were in a state of oxidative stress, therefore, the growth-retarded lambs had lower growth performance. With probiotic supplementation, the oxidative stress of growth-retarded lambs was alleviated, which was evidenced by the increased SOD and GSH-Px activities and the decreased MDA content. It most likely that probiotics can scavenge excess free radicals and improve the antioxidant capacity of animals (Amaretti et al., 2013). Using Lactobacillus farciminis was also reported to affect the antioxidant status of lambs positively (Aydin et al., 2021).

Immunoglobulin is a nonspecific immune molecule in animals (Chen et al., 2021), and IgA, IgM, and IgG are the three main types of immunoglobulins in blood. In the current study, lower concentrations of plasma IgG in growth-retarded lambs suggested that their immune function was less developed than normal lambs. Low immune function may have made growth-retarded lambs more susceptible to disease, which could lead to higher morbidity and mortality (4 growth-retarded lambs died during the feeding trial). Previous studies reported that growth-retardation animals have higher levels of the proinflammatory cytokines TNF-α and IL-6 in blood than growth-normal animals (Zhang et al., 2018; Ma et al., 2021a). In line with these studies, growth-retarded lambs exhibited a higher inflammatory response compared to normal lambs in this study. However, probiotic supplementation ameliorated the inflammatory response of growth-retarded lambs, which was evidenced by increased IgG and decreased levels of the pro-inflammatory cytokines IL-6 and TNF-α. Chen et al. (2022) found that dietary supplementation with the same probiotic product as our study (L. acidophilus and B. subtilis) could increase IgG and decrease IL-6, TNF-α and IFN-γ in the intestines of laying hens. Some studies reported that probiotics treatment enhanced the concentration of IgG in the serum of lambs (Jia et al., 2018; Chen et al., 2021). Lactobacillus delbruekii supplements could be a useful nutritional support for the immune system in healthy lambs (El-Ashker et al., 2018). Altmeyer et al. (2014) inferred that any factors could modify the gut situation will influence immunity, because there were a large number of immune cells in the gastrointestinal tract.

Volatile fatty acid, an energy source of ruminant, contribute to ruminant health by sustaining the rumen ecosystem. It is well documented that rumen VFA, especially butyrate, are the primary chemical stimulators of rumen development (Plöger et al., 2012; Wang et al., 2016). The present results concerning reducing the total VFA with the GR group indicated that growth-retarded lambs had poorer rumen fermentation than normal lambs. In line with this study, growth-retarded yaks have lower rumen VFA fermentation than normal yaks (Hu et al., 2019). Furthermore, the increased concentrations of total VFA and butyrate in the rumen of growth-retarded lambs through the supplementation with probiotic might have beneficial effects on the rumen development of these lambs, which could then recover the growth rate. A meta-analysis by Frizzo et al. (2011) reported that probiotic supplementation could increase the feed digestion, promote the synthesis of VFA, and enhance the growth performance of cattle. Probiotics were also used to maintain gut health and improve feed efficiency by increasing the absorption rate of volatile fatty acids (Krehbiel et al., 2003). In addition, the rumen NH₃-N concentration decreased in the GRP group, which indicated that the diet supplemented with probiotic product improved NH₃-N utilization. Similar results revealed that the supplementation with probiotic decreased the NH₃-N concentration of lambs (Hassan et al., 2020; Chen et al., 2021). Ammonia-N in the rumen is the main nitrogen source for the synthesis of microbial protein, and the declining of NH₃-N in the GRP group is in line with the increase in MCP concentration. Probiotics product can improve stimulation of ruminal microorganism activity by assimilation of NH₃-N into microbial protein (Gado et al., 2011).

In this study, the rumen bacterial communities of normal lambs and growth-retarded lambs were compared, and the results of PCoA indicated that the composition and structure of rumen bacterial were distinct between the GN and GR groups. In line with our findings, some studies have reported that the gastrointestinal composition differs between growth-retarded and normal animals (Hu et al., 2019; Ma et al., 2020b), suggesting that the gastrointestinal bacteria can affect the growth of animals. It was also found that the probiotic supplementation group seemed to be comingled together and tended to separate from the GR group. As compared to the factor of host species, dietary factors made more markedly effect on the rumen microflora (Henderson et al., 2015). Du et al. (2018) fed a B. amyloliquefaciens/B. subtilis diet to growth-retarded calves aged 3–6 months for 30 days, resulted in more stable of bacterial structure. In the current study, probiotics were used to intervene in the growth of growth-retarded lambs. Chaucheyras-Durand and Durand (2010) found that antibacterial compounds produced by probiotics, such as organic acids, hydrogen peroxide, bacteriocins, and biosurfactants, can inhibit the growth of pathogenic microorganisms. Lactobacillus, which is the main component of probiotic products, is an essential member of the intestinal microbiota. It has positive effect on the ruminal environment by promoting the growth of lactic acid utilizing bacteria, and then provide an optimum condition for ruminal microorganisms (Cherdthong et al., 2021). Moreover, as another main component, Bacillus subtilis is believed to affect the composition and function of microbial communities through the production of antimicrobials, promoting the growth of beneficial microbes and overall gut health (Luise et al., 2019; He et al., 2020).

Bacteroidetes and Firmicutes were the dominant bacteria in the rumen of lambs in this study. Ley et al. (2006) found that Firmicutes performed an important function on the energy absorption. Furthermore, Firmicutes are also reported to play a key role in the degradation of oligosaccharides, starch, and cellulose (Ahmad et al., 2020). Ma et al. (2020b) found that growth-retarded yaks with low feed efficiency exhibited lower relative abundance of Firmicutes in the rumen. In this study, growth-retarded lambs had a lower Firmicutes relative abundance, which was correlated with their lower growth performance. At the genus level, Prevotella, Ruminococcus and Succiniclasticum were the dominant bacteria in the three groups. The members of Ruminococcus, which can produce all organic acids, are cellulolytic, fiber-degrading bacteria (Ezaki, 2015). Li and Guan (2017) found that beef cattle with higher feed efficiency exhibited higher relative abundance of Ruminococcus in the rumen. Compared with the GN group, we observed lower Ruminococcus relative abundance in the GR group, indicating that growth-retarded lambs had a lower ability to degrade fiber. However, Ruminococcus tended to be increased by probiotic supplementation, this result suggested the positive effects of probiotic supplementation on microbial changes and might improve rumen digestion. Van Gylswyk (1995) revealed that Succiniclasticum play an important role on the succinic acid production and can convert succinic acid to propionic acid, a major ruminal short chain fatty acid. The relative abundance of Succiniclasticum was the lowest in the GR group, but supplementation with probiotic increased its abundance. This change was consistent with the findings of VFA concentration. Therefore, it was inferred that dietary supplementation with probiotics could improve rumen fermentation, which was in line with the increases in total VFA concentration and growth performance of growth-retarded lambs.

The VFAs, especially butyrate, could stimulate rumen epithelial metabolism (Baldwin and McLeod, 2000; Górka et al., 2011). The butyrate concentration was closely positively correlated with the relative abundances of Prevotella and Selenomonas. It was also found that Succiniclasticum and Acidaminococcus were positively correlated with total VFA and acetate concentrations, which could explain our previous findings of higher total VFA and acetate in the GRP group. Hence, the ruminal epithelium functional deficiencies of growth-retarded lambs could be repaired by nutritional interventions, which was attribute to the increase of beneficial bacteria abundance and the VFA concentrations (especially butyrate production) in the rumen.

In summary, the results obtained in the present study provide evidence that dietary supplementation with probiotic (co-fermented by Lactobacillus acidophilus and Bacillus subtilis) contributes to a number of ways to improve growth performance of growth-retarded lambs, including improvements in antioxidant ability, a decrease in the inflammatory response, and better regulation of the rumen microbiota. These findings may help sheep farmers use nutritional interventions to promote the compensatory growth of growth-retarded lambs.

The ruminal bacterial 16S rRNA gene sequencing data have been deposited in NCBI Sequence Read Archive database (accession number: PRJNA857331).

The animal study was reviewed and approved by All animal research procedures were approved by the Animal Use and Care Committee, Zhejiang A&F University (ZAFUAC2016002, Hangzhou, China).

HM: conceptualization, methodology, writing–original draft preparation, writing–review, editing, and funding acquisition. WJ: investigation, data collection, sample analysis, and writing–original draft preparation. YY: investigation, data collection, and sample analysis. YZ: investigation and data collection. ZL: conceptualization. CW: conceptualization, methodology, formal statistical analysis, and funding acquisition. All authors read and agreed to the published version of the manuscript.

This research was supported by the fund for the National Natural Science Foundation of China (31902179 and 32172742) and Zhejiang Provincial Cooperative Promotion Plan of Major Agricultural Technologies (2021XTTGXM05-4).

ZL was employed by the company Hangzhou King Techina Feed Co., Ltd.

The remaining 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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