1. Introduction
Rabbit farming is gaining popularity as an alternative agricultural enterprise that can be adopted in both rural and urban areas due to rabbits’ small body size, rapid growth rate, short gestation interval, high prolificacy, and ability to utilise forages and by-products as major dietary ingredients [
1]. Rabbit meat has a high commercial value and is a popular, nutritious, and a healthful protein source [
2]. Rabbits, on the other hand, are susceptible to pathogenic microorganisms, especially when raised in stressful conditions [
3]. In the rabbit sector, there is an increasing interest in using natural feed additives for consumer safety [
4]. A variety of nutritional treatments, including commercial amino acid and enzyme supplementation, have been employed to promote nutrient utilisation while maintaining economic efficiency with low-protein diets [
5]. Exogenous enzymes have been introduced into the animal’s feed as natural alternative products to complement the endogenous enzymatic capability, which increases feed nutritional value [
6], and can promote caecal fermentation and vary concentrations of volatile fatty acid, affecting the colonization of beneficial bacteria in the cecum, thus contributing to the maintenance of the animal’s good health. It is common practice to add enzyme preparations to conventional diets to improve feed digestibility, efficiency, and performance in animals [
7,
8]. Lysozyme extracted from avian egg whites has been applied in rabbit and broiler production as an effective natural growth promoter and antibacterial agent [
9,
10,
11], and it plays an important defense role in the innate immune system in most mammals [
12]. Lysozyme is a 1,4-β-N-acetylmuramidase with antimicrobial effects due to its ability to break down the peptidoglycan found in bacterial cell walls, which results in the loss of cellular membrane integrity, causing cell death [
13]. Lysozyme is one of the most promising techniques for enhancing rabbit and broiler health and growth by increasing the diversity of gut microbial balance and antioxidative responses [
11,
14], making it an excellent candidate to replace antibiotics in the animal production industry [
15]. Treatment with lysozyme created an abundance of beneficial bacteria and decreased harmful populations in animal guts [
10,
16,
17,
18]. Dietary lysozyme also increased rabbit growth rate, blood health, and antibacterial capacity [
10,
19]. Furthermore, weaned pigs given dietary lysozyme had better growth performance, diversity of beneficial gut microbiota, health of intestinal barriers, and immunological response [
12,
16,
17]. Based on the aforementioned studies and related information, it can be hypothesized that lysozyme would improve the performance, caecal fermentation and microbiota, and antioxidant status of growing rabbits. Therefore, this study was carried out to evaluate the growth performance, carcass characteristics, blood constituents, caecal fermentation, and microbial population of growing rabbits fed lysozyme-supplemented diet.
4. Discussion
A healthy gut is necessary to maintain high feed efficiency and growth performance in farm animals. The present study revealed that the supplementation of lysozyme improved the growth performance, caecal fermentation and bacterial populations, antioxidant status, and overall health status in growing rabbits. Dietary LYZ supplementation enhanced FBW and DGR, with improved FCR. The findings are consistent with those of El-Deep et al. [
10], who found that lysozyme at 100–200 mg/kg is efficient for improving growing rabbit performance. Similar studies on rabbits [
28,
29], poultry [
11], and pigs [
12,
30,
31] observed that feeding them lysozyme increased growth performance while improving the gut barrier function and modulated intestinal microbes, resulting in the activation of intestinal immunity with high absorption capacity. The positive effect of LYZ on FBW, DGR, and FCR in this study may be due to the improvement in antioxidants, caecal fermentation, and beneficial microbial abundance in
L. acidophilus,
L. cellobiosus, and
Enterococcus Sp., as well as enhanced nutrient digestibility, which can be attributed to increased nutrient absorption in the rabbit intestine [
12,
31]. Additionally, the improved growth performance may be attributed to the regulation of metabolism and modulation of the immune response by beneficial microorganisms [
32]. Brundig et al. [
33] found that dietary LYZ raised 18 known metabolites in the blood that are responsible for protein synthesis through the binding of methionine, threonine, and hydroxyproline, resulting in increased feed utilisation and growth performance. In the present study, the improvement in FCR and growth performance could be explained by the richness of LYZ with amino acids as well as its antioxidant and antibacterial properties, which could increase bacterial activity and feeding efficiency in rabbits [
10]. The lysozyme additive had antimicrobial properties with increased protection against harmful microorganisms in the gut of animals [
15,
34]. Indeed, a high concentration of beneficial bacteria improved feed digestion and nutrient absorption [
35].
The size of the caecum and the parameters of caecal fermentation were within the ranges described by Garcia et al. [
36]. Caecal size, N-ammonia, and the proportion of butyric and acetic acids were all different among the LYZ-treated and control groups. The VFA produced by fermentation and absorbed in the rabbit’s hindgut is an important source of energy, providing up to 30 to 40% of the energy required for survival [
37]. High relative abundances of the beneficial microbiota could indicate more active caecum fermentation, which leads to higher butyrate proportion and improved growth performance [
38]. The caecum occupies 40% of the whole-tract content size, and the rabbit ecosystem contains a highly active microbiota that plays an important role in their digestive physiology [
39,
40]. A variety of factors, including the volume and composition of the rabbits’ diet, influence their pH. Changes in organic acid accumulated in the ingesta could be causing the pH fluctuations.
The present study showed that the caecal microbiota of
Lactobacillus acidophilus,
Lactobacillus cellobiosus, and
Enterococcus were significantly increased when LYZ was added to the rabbit diet, while the count of
E. coli decreased. The increased
Lactobacillus count generated by LYZ feeding could be related to LYZ’s antimicrobial, antioxidant, and immunomodulatory properties, which acted as a health indicator for the rabbits [
10,
11].
Lactobacillus, a defensive bacteria, provides a variety of health benefits to the host, including improving the digestion of nutrients [
41] and promoting the response of the gut-associated immune system [
42]. The antibacterial activities of dietary LYZ suppressed the proliferation of pathogenic bacteria such as
E. coli in the digestive tract, resulting in improved pig health [
12,
17]. The current findings are consistent with those of El-Deep [
10], who found that using LYZ reduced the count of
E. coli in growing rabbits. Furthermore, lysozyme acts as an antibacterial agent by hydrolyzing the peptidoglycan in pathogenic microbial cell walls [
43].
The present results showed that total VFA concentrations were higher in the LYZ groups than in the control group. According to García et al. [
36], total VFA concentrations could reach 99.8 mmoll
−1 depending on the rabbit’s age, physiological health status, and feed ingredients. Fermentation within the caecum appears to have proceeded normally following LYZ administration, as VFA concentrations amounted to 65.86 mmoll
−1. The concentration of total VFA in the caecum has been used to estimate microbial activity indirectly since total VFA is the principal product of microbial fermentation. The VFA is rapidly absorbed in rabbits’ hindgut and provides a regular supply of energy for herbivorous animals that use bacterial fermentation as part of their digestion [
44]. Lowering total VFA would be nutritionally detrimental to the animal because it stimulates colon mucosal growth [
45], a protective factor against pathogenic microbiota [
46,
47]. Acetate dominates in the rabbit’s caecum, followed by butyrate and then propionate [
48]. The molar proportion of butyrate exceeded that of propionate in the rabbit VFA profile, which is in contrast to most herbivorous and omnivorous mammals, which produce more propionate than butyrate in their digestive tracts [
49]. The present study revealed that production of acetic and butyric acids increased with LYZ supplementation in the diet at the expense of propionic acid. In contrast, the increased acetogenesis associated with LYZ supplementation may result in the higher production of acetates, at the expense of propionates. Reduced acetogenesis (microbial synthesis of acetate from CO
2 and H
2) characterises rabbit caecal fermentation, which is gradually replaced by methanogenesis with age [
50]. Acetates contribute to lipogenesis and cholesterologenesis and activate gluconeogenesis from lactate and pyruvate [
51]. Lactate is produced by bacterial fermentation in the caecotroph of the stomach, and it is then subsequently consumed during the caecotroph’s digestion in the small intestine [
44]. Supplementing with LYZ was also associated with an increase in butyrate, an essential precursor for lipogenesis [
51].
Concerning NH
3-N concentration, LYZ supplementation induced a decremental effect compared to control. According to Macfarlane and Gibson [
52], NH
3-N concentration in the caecum could be influenced by a series of factors, such as H
2 pressure, chyme reaction, and carbohydrate availability. Proteolytic activity is relatively higher in the rabbit caecum than in ruminants [
53], and ammonia concentrations fluctuate between 1.86–23.9 mmoll
−1, as reported by Garcia et al. [
36]. Additionally, LYZ supplementation reduced the pH of the caecum. When VFA concentration rises and ammonia concentration falls in rabbit caecal chyme, the pH value lowers [
36], and so the pH drop associated with LYZ groups coincided well with VFA and NH
3-N concentrations. Lipid profiles (TL, PL, TG, TC, HDL, LDL, and vLDL), and the LDL: TC ratio, LDL: TC ratio, HDL: LDL ratio, and LDL: HDL ratio in LYZ-fed rabbits were considerably lower than those in the control group, indicating a correlation between lysozyme consumption and lipid profiles. The PL plays an important role in transporting cholesterol and excess TG from the body to the liver, where it is released into the bile juice rather than being deposited on arterial walls or accumulating fat [
54]. The decreased TC, TG, TL, and carcass fat deposition in the LYZ-treated rabbits could be explained by the higher PL levels in these rabbits. The capacity of PL to reduce intestinal cholesterol absorption, improve biliary cholesterol excretion, and modify the expression and activity of transcriptional factors and enzymes involved in lipoprotein metabolism could explain these positive effects [
55]. HDL is known as “good” cholesterol because it aids in the removal of other, more dangerous types of cholesterol from the circulation. This finding also supports LYZ’s role in rabbits as a health-promoting supplement [
55]. Lysozyme reduced fat percentage in carcass components and lowered serum lipid profile compared to the control group, indicating that dietary LYZ had a beneficial effect on lipid-reduction in rabbits.
The TAC is a biomarker for reducing agents in the blood and their ability to scavenge oxidative free radicals [
56]. SOD levels were higher in rabbits fed a diet supplemented with lysozyme, indicating that lysozyme improves antioxidant status efficiency. Despite the fact that GST is essential for xenobiotic detoxification within cells, excessive amounts in the blood indicate cell damage [
57]. Higher blood total antioxidant activity in LYZ-treated groups may have resulted in improved health as well as higher total antioxidant activity or a reduction in other forms of free radical stress. In both cases, enhanced antioxidant activity reduces oxidative stress exposure. The levels of TAC, SOD, and GST in the serum of rabbits fed dietary LYZ were considerably greater, indicating that these rabbits’ antioxidant defence systems’ ability to scavenge oxidative stress processes had improved. Lysozyme is a good source of antioxidants that can protect cells from free radicals, minimise toxicity, and potentially protect the liver [
58]. According to Lin and Yen [
59], beneficial gut bacteria create a variety of substances that suppress cytotoxicity, eliminate free radicals, and capture reactive oxygen species. El-Deep et al. [
10] and Fritz et al. [
60] revealed that lysozyme supplementation enhanced immunity due to changes in immunological and antioxidant status, which is consistent with our findings. Oliver et al. [
31] reported that supplementing lysozyme to pig diets resulted in a similar rise in immune responses.