Probiotics, defined as living microorganisms with proven health benefits upon proper administration, have been extensively studied for their capacity to enhance host immunity, optimize gut health, and improve overall animal growth performance (1, 2). Commonly employed probiotic strains encompass Lactobacillus acidophilus, Lactobacillus plantarum, Bacillus subtilis, and Saccharomyces cerevisiae (3–5). However, the widespread utilization of live probiotics has unveiled associated risks, including variable colonization patterns, instability of live bacteria, and potential strain transfer across the intestinal barrier (6). Consequently, concerns regarding these risks have led to an increased adoption of paraprobiotics, specifically inactivated probiotics (7). Various methods, including heat, high pressure, and ultraviolet light, have been explored for probiotic deactivation, with heat treatment often preferred. This choice is due to its effectiveness, cost-efficiency, and ability to preserve probiotic integrity, ensuring prolonged functionality and stability. Heat treatment stands as a widely adopted approach for preserving probiotic attributes across different contexts (8). Recent studies have substantiated that non-viable bacteria, notably inactivated probiotics, can confer health benefits akin to their living counterparts. These benefits encompass the modulation of immunity, pathogen suppression, and the enhancement of gut microbes to uphold overall animal health. Notably, these functional attributes of inactivated probiotics are attributed to their release of lipophosphatidic acid (LTA), peptidoglycan, and extracellular polysaccharide (EPS) components (9, 10). Additionally, the ease of storage and transportation of inactivated probiotics, compared to live bacteria, further underscores their practical advantages (7, 11, 12).

The weaning period poses a critical juncture for rabbits, characterized by an underdeveloped digestive tract and exposure to multiple stressors, including dietary transitions and relocation to new enclosures, potentially impacting their overall growth and developmental trajectories (13). Post-weaning, the abrupt cessation of breast milk antigen supply results in a rapid decline in intestinal immunity (14). Consequently, rabbits become more susceptible to environmental variations, eliciting stress responses that can manifest as diarrhea and, in severe cases, culminate in death (15).

Prior research has demonstrated the positive impact of dietary supplementation with alive LA on enhancing the dietary conversion rate, promoting growth performance, and augmenting beneficial intestinal flora in rabbits (4). Moreover, LA has been shown to fortify immunity, improve the integrity of the intestinal barrier, diminish the occurrence of diarrhea, and reduce mortality rates (4, 5, 16). Despite these established benefits, there remains a scarcity of literature addressing the utilization of heat-killed LA in rabbit production. This study aims to bridge this knowledge gap by investigating the repercussions of incorporating heat-killed LA into rabbit diets. The investigation also encompasses an in-depth analysis of its effects on growth performance, digestibility, digestive enzymes, antioxidant status, and immune capacity in rabbits. The results of this research will provide valuable scientific insights and provide a powerful reference for the rational application of heat-killed LA in rabbit production.

The weaning stage poses a critical period for rabbits, susceptible to external stimuli such as dietary changes and environmental shifts, potentially leading to appetite loss, and even an increase in mortality and diarrhea rates (19). Zhu et al. (20) observed that the supplementation of 500 mg/kg of inactivated complex probiotics in the diet positively influenced cecum microbiota, consequently improving diet conversion and growth performance in yellow-finned broilers. Noteworthy findings by Tartrakoon et al. (21) demonstrated enhanced feed efficiency and ADG in pigs through the utilization of 20 mg/kg of heat-killed L. plantarum. Consistent with prior studies, the current research underscores that the addition of 600 mg/kg and 800 mg/kg of heat-killed LA significantly reduces F/G and enhances the growth performance of rabbits. This improvement in feed efficiency correlates with the observed elevation in the activity of digestive enzymes (lipase, protease, cellulase) within the intestinal tract of rabbits (22). Notably, the potential role of heat-killed LA in regulating gut microflora and diminishing the incidence of diarrhea aligns with the findings of Moal et al., who observed a reduction in harmful bacterial adhesion in the intestine through the use of inactivated probiotics (23), resulting in a decreased frequency of diarrhea in piglets (24). In summary, we hypothesize that the improved growth performance may also be related to heat-killed LA by modulating gut microflora and mitigating the occurrence of diarrhea.

Apparent digestibility serves as a key indicator of an animal’s proficiency in nutrient digestion within the diet, and this is intricately linked to digestive enzyme activity, contributing positively to growth performance (25, 26). Mammalian food nutrients primarily undergo digestion orchestrated by various digestive enzymes, with a minor portion being subject to digestion by intestinal bacteria (27, 28). Palamidi et al. (29) reported an elevation in lipase activity and improved digestibility in broilers when fed heat-killed complex probiotics. Similarly, Dawood et al. (30) observed a significant increase in amylase, protease, and lipase activities following the inactivation of L. plantarum, leading to enhanced nutrient digestion. In concordance with these findings, the current study demonstrated a substantial increase in trypsin, lipase, and cellulase activities upon supplementing diets with 600 mg/kg and 800 mg/kg of heat-killed LA. This trend aligned with the concurrent increase in the apparent digestibility of CP and CF in rabbits. Furthermore, the inactivation of LA was found to enhance the intestinal microbial composition, favoring an increased relative abundance of beneficial bacteria such as Lactobacillus and Bacillus in the intestine (31, 32). This augmentation in beneficial microbial communities within the gut can potentiate their ability to induce the production of endogenous enzymes (33, 34). Consequently, the inactivation of LA appears to contribute to the enhancement of digestive enzyme activity, ultimately improving digestibility and, consequently, the growth performance of rabbits.

Oxidation reactions, yielding ROS, are constant processes within organisms. Under normal physiological conditions, animal cells maintain a delicate equilibrium between ROS production and clearance (35). Excessive accumulation of free radicals poses a significant threat to tissues, leading to intestinal damage, nutrient malabsorption, and severe diarrhea (36, 37). Antioxidant defense enzymes, including SOD and GSH-Px, play pivotal roles in counteracting oxidative stress (38). SOD, functioning as the primary defense against free radicals, not only exhibits superb antioxidant effects but also inhibits the synthesis and release of inflammatory factors (39). T-AOC, encompassing both enzymatic and non-enzymatic antioxidant activities, provides a comprehensive assessment of the organism’s antioxidant status (40). Previous research, such as Liu et al. (41) and Dawood et al. (30) has highlighted the antioxidant properties of heat-killed LA and heat-killed L. plantarum, respectively. In this experiment, supplementation with heat-killed LA demonstrated a notable increase in SOD activity and T-AOC capacity in rabbits, with the most pronounced effects observed at supplementation doses of 600 mg/kg and 800 mg/kg. This augmentation in antioxidant-related enzyme activity may be attributed to the ability of heat-killed Lactobacillus to activate the Nrf2 signaling pathway (42, 43). Activation of the Nrf2 signaling pathway triggers the transcription and translation of downstream antioxidant genes (such as SOD and T-AOC), bolstering antioxidant status in response to environmental stress and sustaining normal intestinal function (44).

Immunoglobulin (IgA, IgM, and SigA etc.) is one of the components of animal immune system and an important parameter reflecting the immune capacity of animal body (45). IgA plays a crucial role in protecting the mucosal surface by preventing the entry, binding, and colonization of toxins and pathogens (46). IgM is the immunoglobulin with the strongest initial immune effect, and is an important barrier against pathogenic invasion (47). In this study, supplementation with heat-killed LA significantly increased the levels of IgA and IgM in serum, which reflected that heat-killed LA could improve the immune capacity of rabbits. As the main immune defense line of intestinal mucosa, SigA has the function of neutralizing pathogens and bacterial exotoxins in intestinal mucosa and maintaining the stability of intestinal flora (48). Danladi et al. found that supplementation of feed with inactivated Lactobacillus plantarum significantly increased the intestinal SigA content of broiler chickens and improved immunity (49). Consistent with the results of previous studies, we found that feed supplementation with 600 mg/kg and 800 mg/kg of heat-killed LA significantly enhanced the secretion of intestinal SigA, protected the intestinal immune barrier, and reduced the occurrence of intestinal diseases. TNF-α is a pro-inflammatory cytokine produced mainly by macrophages and monocytes, which is involved in the inflammatory response, and when its level is too high, it increases intestinal permeability and causes an enteritis response (50). It was found that LTA isolated from Lactobacillus can block the phosphorylation of NF-κB and the phosphorylation degradation of IkB, thereby inhibiting the expression of TNF-α (51). In this experiment, we found that supplementation with heat-killed LA reduced the serum levels of TNF-α. This may be related to the blocking of NF-κB phosphorylation by heat-killed LA. It was also shown that heat inactivation of LA does not cause an inflammatory response in the meat rabbit organism and has no adverse effect on its health.

There are a large number of microorganisms in the intestinal tract of animals, and their species composition and activity play an important role in the immune and inflammatory responses of animals (52, 53). Therefore, the balance of intestinal flora is related to the health of animals. Under normal conditions, the composition of the intestinal flora is relatively stable, but at the same time it can be disturbed by external factors (54). In the current study, probiotic supplements or probiotic products were found to be one of the effective ways to maintain a healthy balance of gut microbiota (55–58). In addition, species-rich communities enhance intestinal microecological stability and reduce susceptibility to bacterial invasion and intestinal inflammation (59). In this study, dietary heat-killed LA can regulate the cecal microflora structure of rabbits and increase the relative abundance of Phascolarctobacterium and Alistipes. Phascolarctobacterium and Alistipes are probiotics that promote intestinal health. Their main function is to produce short chain fatty acid (SCFAs), which play a vital role in maintaining nutrient metabolism and microbial homeostasis (60, 61). Acetic acid is not only the most abundant SCFAs in the gut, but also inhibits the NF-κB signaling pathway by down-regulating the expression of TLR gene, thereby reducing the content of TNF-α in the body and reducing the inflammatory response (62). In addition, SCFAs can also accelerate the activation, differentiation and antibody production of B cells by promoting the synthesis of acetyl-CoA, 5‘-adenosine triphosphate and fatty acids in B cells, thus promoting the secretion of SigA, IgA, and IgM (63, 64). SCFAs can also act as an activator of Keap1-Nrf2 signaling pathway (65, 66), and the activation of Nrf2 is positively correlated with SOD, GSH-Px, and T-AOC (44, 67). The above results showed that heat-killed LA can promote the proliferation of Phascolarctobacterium and Alistipes, thereby reducing serum TNF-α level, and increasing jejunum SigA, serum IgM, serum IgA, ileum GSH-Px, jejunum GSH-Px, ileum SOD and serum T-AOC levels, which may be due to the regulation of SCFAs by Phascolarctobacterium and Alistipes. These results provide a theoretical basis for understanding heat-killed LA-mediated microbial promotion of immunity and antioxidant capacity in rabbits. However, further microbiome-based studies are needed to elucidate the mechanisms by which Phascolarctobacterium and Alistipes promote immunity and antioxidant capacity in rabbits.

In conclusion, the incorporation of heat-killed LA into the diet demonstrates a capacity to enhance intestinal digestive enzyme activity, bolster antioxidant status, modulate the immune capacity, increase the relative abundance of beneficial bacteria in the cecum and foster overall organismal health in rabbits. This ultimately translates into improved growth performance. Upon comprehensive analysis, the recommended additive levels of heat-killed LA in rabbit diets appear to be 600 and 800 mg/kg.

The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding authors.

The animal study was approved by Animal Care and Use Committee of Hebei Agriculture University (Baoding, China). The study was conducted in accordance with the local legislation and institutional requirements.

MX: Writing – original draft, Conceptualization, Funding acquisition, Project administration, Resources, Supervision, Validation, Writing – review & editing. CL: Writing – original draft, Conceptualization, Data curation, Formal analysis, Methodology, Writing – review & editing. DW: Writing – original draft, Data curation, Formal analysis, Investigation, Methodology, Resources, Software. FW: Methodology, Project administration, Software, Writing – review & editing. LK: Writing – original draft, Data curation, Formal analysis, Investigation, Methodology. ZJ: Writing – original draft, Data curation, Formal analysis, Investigation, Methodology. WH: Writing – original draft, Data curation, Formal analysis, Investigation, Methodology. SC: Writing – original draft, Data curation, Formal analysis, Investigation, Methodology. WF: Writing – original draft, Data curation, Formal analysis, Investigation, Methodology. YL: Writing – original draft, Conceptualization, Project administration, Resources, Supervision, Validation, Visualization, Writing – review & editing. BC: Writing – original draft, Funding acquisition, Supervision.