1. Introduction
Accumulated evidence supports the favorable effects of acidifiers in nutrient digestion and absorption, immune, antibacteria, and antioxidant aspects by lowering gastrointestinal pH and modulating gut microbiota [
1,
2,
3]. Citric acid (CA) is a weak organic acid which is widely distributed in the fruits of plants and tissues of animals. As an organic acidifier and key component of the tricarboxylic acid (TCA) cycle, CA is more efficient, safe, and environmentally friendly than other acidifiers, such as malic, fumaric [
3], and tartaric acid [
4].
Studies have shown that adding CA to animal diets could stimulate appetite, lower the pH value of the gastrointestinal tract (GIT), activate digestive enzymes, and promote the proliferation of probiotics [
5,
6,
7,
8]. The increased probiotics may elevate the secretion of intestinal lactic acid and short-chain fatty acids (SCFAs), which have been confirmed to be conducive to the renewal and development of epithelial cells [
9]. In addition, as a key component of the TCA cycle, CA could also directly synthesize ATP under stress, reducing the damage caused by stress to the body. Recent reports about CA supplementation in poultry diets have demonstrated that CA could promote the growth, immune status, intestinal structure, and barrier function of broilers [
1,
10], quails [
6], and ducks [
11]; however, reports on the effects on the physical and chemical properties of the diets and gut microbiome of geese are very rare, especially in their growing period.
Poultry is one of the cheapest sources of animal protein, of which goose meat is also an ideal nutritious and healthy food with high protein, low fat, and low cholesterol [
12]. China is the largest producer and consumer of goose meat in the world, with 466 million geese used for meat production in 2022 [
13]. Previous research by our team has demonstrated that the optimal level of CA in the gosling diets should be 1% [
14]. Based on the above research, we further investigated the changes in feeding diets (pH value and acid-binding capacity) and blood hormone levels in growing geese with the increase in CA levels. Moreover, in the present study, we used 16s rDNA sequencing technology instead of the spread plate count method for a more in-depth analysis of the cecum microbiota composition. In conclusion, we hypothesized that the dietary supplementation of CA is also expected to have favorable effects on the growing geese. Hence, this research was performed to further evaluate the effects of dietary CA supplementation at different levels (0.8%, 1.6%, 2.4%, 3.2%, and 4%) on the performance, plasma constituents, small intestinal development, and cecum microbiota of growing Sichuan white geese.
4. Discussion
Supplemental acidifiers for disease prevention and growth promotion are an efficient and price competitive nutritional regulation method. The current research found that 2.4% and 3.2% CA supplementation exerted a favorable growth-promoting function, and this result was in line with Abdel-Fattah et al. [
10], who previously demonstrated that the addition of 2% or 3% CA in broiler diets markedly improved FBW and F/G. A.Nouri et al. [
19] also demonstrated that the ADFI and BW of broilers were improved by 1.5% and 3% CA supplementation. And the improvement in the F/G was also verified by some other studies [
20,
21]. The acidifying effect of CA on diets could have been responsible for the appetite stimulation and activation of digestive enzymes, and eventually led to the improvement of performance data, which was further verified by the decrease in the pH value and acid-binding capacity of treatment diets. Furthermore, Nourmohammadi et al. [
22] illustrated that broilers fed 6% CA diets resulted in adverse effects on ADFI and ADG compared with the control group and 3% CA group, which means that excess citric acid might adversely affect growth performance. Similarly, Fikry et al. [
6] discovered that adding 0.5% to 2% CA to Japanese quails’ diets improved FBW and ADG, but that growth performance increased initially and then decreased with increasing levels of CA, with the best performance achieved at 1%. Esmaeilipour et al. [
23] also found 4% CA supplementation in diets adversely affected the ADG and ADFI of broilers. Our data revealed that the FBW, ADG, and ADFI of the group fed the 4% CA supplemented diet did not improve further or were even repressed, which meant that the inclusion of 4% CA might have been close to the maximum acid-resistant capacity of growing geese, thus severely affecting the growth performance and the efficiency of nutrient absorption. Therefore, the diet should be scientifically supplemented with appropriate levels of CA for the purpose of growth promotion.
In the present study, the improvement in performance due to the addition of 3.2% CA was further verified by the carcass traits, the increase in the TMP, and the decrease in the AFP and SFP, indicating that CA might be beneficial to the increase in net available energy for protein synthesis by improving the oxidation of fatty acids or restraining the biosynthesis of fatty acids [
24]. Consistent with our findings, Haq et al. [
25] found that with the increase in CA addition to 1.5%, the DP significantly increased, while the ABP significantly decreased in ducklings. Moreover, a significant reduction in ABP was also found in 2% or 3% CA supplemented diets by Elnagar et al. [
11]. The proventriculus–gizzard indices and liver indices of broilers were significantly decreased by 0.25% CA [
26]; however, some results reported that CA supplementation in broiler and quail diets did not significantly affect carcass traits [
6,
27]. This divergence may be because of the differences in animal species, genders, age, supplemental dosages, and the composition of ingredients.
Several blood metabolites can be used to determine the animals’ physiological status and overall health. Our results found that plasma ALB remained unchanged, whereas GLO and TP first increased and then decreased as dietary CA increased. The geese fed a 4% CA diet had significantly lower GLO and TP than the group fed a 1.6% or 2.4% CA diet. Similarly, previous studies found that broilers fed a diet containing 3% acidifier showed higher blood GLO and immune organ weight than the control group [
10]. Moreover, Ghazalah et al. [
28] found that the TP and GLO levels of broilers were significantly increased by the addition of 1% to 2% CA, whereas no significant difference was observed in broilers who received an addition of 3% CA. Furthermore, consistent with some previous studies [
6,
11,
29], the decreased plasma Urea and UA levels, which are the main end products of N metabolism, implied that the CA supplementation in geese diets may be beneficial to the efficiency of amino acid utilization and renal function. In addition, previous studies have shown that high plasma UA levels were positively correlated with long-term purine metabolism disorder, which eventually led to gout [
30]. Furthermore, in line with our findings, Elnagar et al. [
11] reported that ducklings fed a diet containing 2% or 3% CA showed lower CREA and Urea levels. Fikry et al. [
6] found that the increase in the amount of CA added, from 0.5% to 1.5%, results in a reduction in plasma Urea levels. Reda et al. [
29] demonstrated that 0.5% to 2% CA supplemented diet reduced quails’ Urea levels, while no significant difference was observed for their CREA levels. Overall, these results imply that the immune status and amino acid utilization efficiency of growing geese could be enhanced by CA supplementation.
IgA, IgG, and IgM are the main antibodies mediating humoral immunity, and increased immunoglobulins have been associated with improved immune function [
31]. T-AOC, GSH-Px, SOD, and CAT are all considered vital indicators of antioxidant status in the body, which can prevent oxidative stress by scavenging free radicals such as reactive oxygen species. MDA has been recognized as a biomarker of lipid peroxidation, which indirectly reflects the degree of cellular damage [
32]. Our results found the levels of plasma GSH-Px were increased in the 2.4% and 3.2 CA groups, suggesting that the antioxidant system was improved to sustain oxidative stability. Our findings are partly consistent with those of Elnagar et al. [
11], who found that CA increased the plasma T-AOC, GSH-Px, and SOD activities of ducklings; however, Mustafa et al. [
33] demonstrated that broilers fed a diet containing organic acids had higher plasma IgG levels. Fikry et al. [
6] demonstrated that quails treated with 5 or 10 g/kg CA supplementation showed significantly higher IgG levels, while IgA levels were significantly reduced with CA addition (up to 20 g/kg). This difference was probably caused by different species or ages. Furthermore, some other organic acids also showed an antioxidant capacity in poultry diets [
34,
35], but their mechanism needs to be further researched.
The secretion and metabolism of hormones are essential for birds’ growth, development, and reproduction. GHs are peptide hormones secreted by the pituitary gland, which plays a vital role in promoting metabolism and maintaining normal development. IGF-1 is a polypeptide similar in structure and function to insulin, which exerts control over the cell growth cycle, maturation, differentiation, proliferation, and protein anabolism [
36,
37]. In this study, supplementing diets with 3.2% CA noticeably increased the level of IGF-1, which might account for the improvement of growth performance observed in the 3.2% CA group.
The gut pH value is directly associated with gastric acid secretion and digestive enzyme activity, thus impacting the absorption of nutrients [
38]. In the present study, we found that dietary the addition of CA significantly decreased the pH value and acid-binding capacity of diets; however, only the jejunum content’s pH value observed a significant decrease due to the addition of CA. Similar to our previous findings [
14], the diets containing CA only significantly reduced the jejunal content’s pH value. Nourmohammadi et al. [
22] also reported that the addition of 3% CA only significantly reduced the jejunal content’s pH value, while no significant differences were found in the content pH value of other segments of the GIT. However, some studies found that the addition of an acidifier to water significantly reduced the pH value of all segments of the GIT [
39,
40]. This difference indicates that the acidifier’s effect on gut pH value regulation differs based on species, tract segments, dosages, and the form of the acidifier used.
The small intestine plays an essential role in the digestion and absorption of nutrients, and small intestine morphology indicators (VH, CD, and VH/CD) are directly associated with the digestive capacity and intestinal health of animals. In this study, geese from the 3.2% CA group showed the highest duodenum and jejunum VH/CD, indicating that the intestinal absorption capacity was improved by CA. These results coincide with those of Nourmohammadi et al. [
7], who reported that 3% and 6% CA supplementation in broilers significantly increased duodenum VH/CD. The positive effect of CA on the intestinal development of poultry has also been reported by Khosravinia et al. [
41]. The increase in VH/CD may be related to the antibacterial ability of CA as an organic acidifier, which may be beneficial as it disrupts bacterial layers and interferes with the bacterial metabolism of many pathogenic bacteria, ultimately preventing intestinal damage [
42]. Noteworthily, excess CA (4%) supplementation exerted no further beneficial effects on small intestinal morphology in the VH/CD of the duodenum and jejunum compared with the 3.2% CA group, which may be directly associated with the decreased feed consumption.
The cecum is the main place for microbial fermentation in poultry. As shown by the beta diversity, the dietary supplementation of 3.2% CA had a significant effect on cecum microbiota communities. This can be presumably explained by the reduced pH value in the gut, which is consistent with several findings on CA [
6,
10,
14]. Clustering results showed that Firmicutes and Bacteroidota are the top two dominant bacterial phyla in geese ceca, which is consistent with the findings of Fang et al. [
43]. Differential analysis results showed that 3.2% CA supplementation in diets increased the relative abundance of several probiotics, including
Muribaculaceae,
CHKCI001,
Erysipelotrichaceae_UCG_003, and
UCG_009, which contributes to a reduction in damage caused by intestinal disease.
Muribaculaceae is a beneficial bacterium in the intestine, belonging to the genus Mycobacterium. Increased
Muribaculaceae was considered to be directly associated with a prolonged lifespan and improved anti-inflammatory functions in mammals [
44].
Erysipelotrichaceae_UCG_003 is known as a key butyrate-producing member, which is positively associated with the antioxidant and anti-inflammatory capabilities in birds [
45,
46]. Additionally,
CHKCI001 and
UCG_009 are generally regarded as growth-promoting bacteria. Zheng et al. [
47] reported that
CHKCI001 was positively associated with growth performance in geese.
UCG_009 can promote the hydrolyzation of proteins into polypeptides and amino acids [
48]. Meanwhile, several pathogens such as Proteobacteria,
Atopobiaceae,
Streptococcus,
Acinetobacter,
Pseudomonas, and
Alistipes were observed to be reduced by CA supplementation in our study. Proteobacteria is the major phylum of Gram-negative bacteria, and the decreased abundance of Proteobacteria can alleviate intestinal damage and maintain organ health [
49].
Atopobiaceae,
Streptococcus, and
Pseudomonas are common opportunistic pathogens in poultry, which are highly associated with inflammation [
50].
Acinetobacter is reported to be harmful to broiler’s respiratory system and to increase the mortality rate during broiler breeding [
51]. Cobo et al. [
52] pointed that
Alistipes is highly relevant to chronic intestinal inflammation, and other studies demonstrated that
Alistipes is positively correlated with cancer progression and mental disorders [
53]. Considering the improvement in growth performance, carcass traits, antioxidant status, and small intestinal morphology in 3.2% CA supplemented geese, we can surmise that the addition of CA could stimulate intestinal development and maintain the health of the host’s gut by facilitating the abundance of several probiotics and reducing the abundance of several pathogens.