1. Introduction
Different species of birds may be categorized as either altricial or precocial, based on the physiology and anatomy of newborn birds and their maturity of behavior at birth [
1]. Pigeon is one of the altricial birds; newly hatched pigeon squabs are characterized by closed eyes, little or no plumage, and undeveloped locomotion organs [
1]. Pigeon squabs are unable to feed independently and require parental care, especially feeding with pigeon milk in a mouth-to-mouth manner, if they are to survive [
2]. Pigeon milk is a curd-like substance regurgitated from the crops of parental pigeons to their squabs [
3]. Pigeon milk, produced by both male and female pigeons, is made up of 60% protein, 32–36% fat, 1–3% carbohydrate, minerals, antibodies, and other nutrients [
2,
4,
5]. Many substances in the pigeon milk of parental pigeons can be transmitted to squabs [
6]. Ding et al. (2020) reported that microbiota play important roles in squabs and can be transmitted from parental pigeons to squabs through pigeon milk [
2]. Xu et al. (2021) reported that supplementing the diets of parental pigeons with 1% linoleic acid improved intestinal morphology and increased intestinal microbial diversity in their squabs [
7]. Researchers have confirmed, then, that supplementation of the diet of parental pigeons may have effects on the growth and health of squabs through its effects on pigeon milk.
Probiotics are beneficial microorganisms that have been shown to inhibit the adherence of pathogenic intestinal bacteria, improve the digestibility of nutrients, and enhance intestinal health [
8,
9,
10]. Researchers have reported that dietary supplementation with probiotic products modulates host-protective immunity against
Clostridium,
Coliform, and
Salmonella infection in both pigs and chickens [
11,
12,
13].
Enterococcus faecium (
E. faecium) is a natural inhabitant of the intestinal tract in poultry; it is known to have positive effects on the growth performance of poultry birds [
14,
15,
16]. Dietary
E. faecium can increase egg weight and serum FSH levels and reduce levels of
Bacteroidetes [
15]. Dietary supplementation with
E. faecium has also been found to reduce ammonia emission in laying hens by increasing the digestibility of nutrients [
14,
17]. Similarly,
Bacillus subtilis (
B. subtilis) has been recognized as safe for animal dietary use [
18]. It can improve growth performance, balance the intestinal microbiota, enhance immunity, and prevent damage to the intestinal mucosa [
19,
20].
Antibiotics are traditionally used as therapeutic agents and growth promoters in animal husbandry around the world, but such usage may cause various toxic side effects such as antibiotic resistance in bacteria, drug residues, and damage to intestinal microorganisms, in addition to having adverse impacts upon environmental sustainability [
8,
9,
21]. Because of this, the use of growth-promoting antibiotics has been forbidden in Europe since 2006, and similar bans were imposed in South Korea and China in 2012 and 2020, respectively [
22,
23]. As a result, studies on such alternative products, such as probiotics, enzymes, and plant extracts, to replace antibiotics have become urgently needed in recent years [
24,
25]. The effects of probiotics on the growth of pigeon squabs have been reported previously. The addition of a mixture of chitosan oligosaccharide and
Clostridium butyricum to the diet of pigeon squabs was shown to enhance intestinal health and prevent loss of body weight [
26]. In another study,
Bacillus velezensis isolated from the feces of pigeons enhanced the expression of immune-regulatory genes against pigeon circovirus by means of dietary supplementation with lyophilized
Bacillus velezensis cells [
27]. However, there have been few reports on the effects on squab growth that may result from supplementing the diets of parental pigeons with
E. faecium and
B. subtilis. The effectiveness of these probiotics is not yet clear. In this study, therefore, we sought to explore the effects of the supplementation with
E. faecium and
B. subtilis, both singly and in combination, in the drinking water of parental pigeons on the growth performance, immunity, intestinal health, and microbiota of their squabs.
4. Discussion
Pigeons are altricial birds. Pigeon squabs are unable to feed by themselves for a period of time after hatching, so they need to be fed with pigeon milk by parental birds [
31]. Nutritional regulation in parental pigeons and the ingredients of crop milk are both important factors affecting the growth and development of squabs [
26,
32]. Probiotics are living microorganisms that beneficially affect host animals by modulating the gut microbiota, reducing disease risk, and improving growth performance [
33,
34].
E. faecium, which has been reported as a probiotic, normally colonizes in the gut. It has been found to exert a positive impact on poultry by improving growth performance, reducing ammonia emission, and preventing
Salmonella infection [
14,
35].
B. subtilis is now established as a prominent probiotic species that is known to promote nutritional digestion and absorption [
36]. In poultry breeding, it is always used as a growth promoter to increase digestibility and growth performance in broilers [
36]. However, there have been few reports on the effects of using
E. faecium or
B. subtilis as supplements in the drinking water of parental pigeons in order to promote performance, immunity, and microorganisms in pigeon milk.
Many studies have confirmed that probiotics can improve growth performance by promoting enzymatic digestion through the provision of nutrients, vitamins, enzymes, and necessary growth factors to the host [
8,
37]. However, in this study, supplementation of the drinking water of parental pigeons with
E. faecium and
B. subtilis had no impact on the growth performance of their squabs. Similarly, Wen et al. (2022) also reported that a diet with
C. butyricum had no impact on growth performance [
26]. Other studies have found that supplementation with
C. butyricum had no impact on growth ability in broilers and piglets [
38,
39]. Pigeon milk contains a large number of proteins, including active enzymes and immunoglobulins [
31]. In one previous study, broilers fed a diet supplemented with
Bacillus exhibited significantly higher enzyme activities (lipase, trypsin, and amylase) in their intestinal content, suggesting that supplementation with probiotics promoted digestion and absorption of nutrients; in addition, the serum concentration of IgA was significantly higher in the
Bacillus-treated group [
40]. The results of the present study indicated that supplementation of the water of parental pigeons with
E. faecium and
B. subtilis did not significantly promote the activities of lipase, trypsin, or amylase in the pigeon milk, while concentrations of IgA and IgG in the pigeon milk were significantly higher in the treated group. As previously reported, probiotics have a beneficial impact on the immune system by increasing production of different cytokines, activating immune cells, and increasing systemic immune response [
41]. The IgA antibody plays a crucial role in immunity, especially with respect to humoral adaptive immune response. It can bind to pathogens and prevent them from invading by means of a noninflammatory process [
41]. Probiotics can also activate IgA cycles and maintain the health of mucosal sites. In addition, probiotics may promote the production of IgG antibodies and the activation of IFNγ [
42]. In the present study, we found that supplementation of the drinking water of parental pigeons with
E. faecium and
B. subtilis significantly increased the concentrations of IgA and IgG in pigeon milk, but the impact on the enzyme activities in pigeon milk and growth performance in squabs was found to be not significant.
Microbiota often exist in the intestines and milk of animals in a symbiotic relationship with their host [
43,
44,
45]. Pigeon milk contains many microorganisms in a complex environment that could promote initial microorganism colonization and have an important impact on squab growth [
2,
46]. Microbiota diversity was found to be higher in the crop than in the small intestine and rectum [
47].
Firmicutes and
Proteobacteria were found to be the dominant phyla in the crops of chickens, while the dominant phyla in the crops of pigeons were reported as
Proteobacteria and
Bacteroidetes [
47,
48]. The microbial characteristics of pigeon milk acquired from squabs include eight phyla; amongst these,
Firmicutes,
Actinobacteria, and
Bacteroidetes have the most microbiota [
2]. In the present study, the dominant phyla in pigeon milk were found to be
Firmicutes,
Actinobacteria, and
Bacteroidetes. In humans,
Firmicutes was associated with energy and the absorption of nutrients [
49].
Actinobacteria could produce substances which function as antibiotics [
50]. Supplementation with
E. faecium and
B. subtilis might therefore improve digestion and immunity in squabs that consume pigeon milk from parental birds whose diet is so supplemented.
The main genera were
Lactobacillus,
Bifidobacterium,
Veillonella, and
Enterococcus. Compared with the control group, the group supplemented with
E. faecium and
B. subtilis had higher proportions of
Lactobacillus and
Bifidobacterium. Ding et al. (2020) reported that
Lactobacillus,
Enterococcus,
Veillonella, and
Bifidobacterium were the main microbiota in pigeon milk at the genus level [
2].
Lactobacillus has been shown to stimulate both innate and acquired immune response in many species. Du et al. (2022) showed that
Lactobacillus improved disease resistance in shrimp by regulating the nutritional immune response [
51]. In humans, administration of
Lactobacillus isolated from breast milk improved host immunity in adults through increased levels of immunoglobulins and numbers of immune cells [
52,
53]. In addition,
Lactobacillus and
Bifidobacterium have been shown to have beneficial affects on intestinal immunity by increasing the levels of IgA levels and other immunoglobulin-secreting cells [
54].
Lactobacillus and
Bifidobacteria have also been detected in the breast milk of human mothers in the first year of their child’s life, indicating roles in infant growth [
55,
56]. In mice,
Lactobacillus has been found to protect epithelial cells not exposed to viruses [
57]. In the present study, the groups of pigeons with higher proportions of
Lactobacillus and
Bifidobacterium also presented higher immunoglobulin levels in their pigeon milk. In brief, immunity may be promoted through supplementation with
E. faecium and
B. subtilis.