1. Introduction
Intensive feeding is becoming more and more common in the broiler industry. Although high stocking density (HSD) can improve the yield of broilers per unit area to some extent, it reduces the health status of individual broilers [
1]. HSD reduced feed intake, body weight gain and carcass weight [
2,
3]; at the same time, HSD reduced the antioxidant capacity of the serum and tissue of broilers [
4,
5,
6], and then caused oxidative stress to produce a large number of stress proteins (such as the heat shock protein) and activate oxygen-induced rate-limiting enzymes (such as HO-1), then accelerated the tissue oxidative damages [
7] and reduced immunity and caused liver damage [
8]. Yarahmadi et al. showed that density stress can increase the proinflammatory cytokine Interleukin-1β (IL-1 β) and tumor necrosis factor-α (TNF-α) contents. The release of proinflammatory factors can mediate inflammation [
9].
Cr(III) has strong antioxidant activity and is considered to be the preferred mineral in poultry diet [
10,
11,
12]. Studies have shown that Cr(III) has pharmacological effects on rodents [
13], and yeast can stimulate digestive enzymes to produce better immune response and higher growth performance [
14]. It is well known that organic chromium has lower toxicity and higher bioavailability than inorganic chromium [
15,
16]. Under conventional conditions, adding organic chromium can promote the growth, fast metabolism and antioxidant capacity of broilers [
17,
18,
19]. Under the condition of heat stress, chromium picolinate (Cr-Pic) supplementation can reduce the level of corticosterone and increase the level of serotonin in broilers, indicating that Cr(III) may have the effect of relieving stress in broilers [
20]. In addition, at high stocking density, chromium methionine (Cr-Met) supplementation can improve production performance and humoral immunity in layers [
21]. Chromium yeast (Cr-yeast) is a yeast preparation enriched with trivalent chromium, which has no toxicity confirmed by animal toxicology tests [
22,
23]. Therefore, Cr-yeast can ensure the safety of feed. It has the advantages of a short production cycle, simple operation and low production cost [
24,
25]. According to the meta-analysis of White and Vincent [
26], in recent years, the research range of chromium in different forms and sources in broiler diets is 200–2000 µg Cr/kg under non-stress, and the research range under heat stress is 200–4 × 10
5 µg Cr/kg. Safwat et al. [
27] reported that adding 1000 and 1500 µg Cr/kg Cr-yeast to the diet of Arbor Acres broilers can reduce the feed conversion ratio of 0–21 days old and 0–35 days old under normal conditions; Sahin et al. [
20] reported that adding 200, 400, 800, and 1200 µg Cr/kg Cr-Pic to the diet of Ross broilers increased feed intake and decreased serum corticosterone content under heat stress. In addition, there have been many studies on the effects of different organic chromium (Cr-Pic, chromium nicotinate (Cr-Nic), chromium propionate (Cr-Pro) and Cr-Met) on broilers, but few studies were reported on the alleviation of high stocking density stress by organic chromium.
Therefore, this experiment aims to explore the effects of different doses of Cr-yeast on the growth performance, serum biochemistry, serum immunity, antioxidant capacity and liver histopathology of broilers under high stocking density stress, in order to obtain the appropriate dosage of chromium yeast and provide a scientific basis for production practice.
4. Discussion
Organic chromium can significantly promote animal growth, which has important practical significance in the production and application of livestock and poultry. In the conventional feeding environment, adding 300 and 400 µg Cr/kg Cr-yeast or 400 µg Cr/kg Cr-Pro to the diet could increase the BWG and reduce the FCR of broilers [
17,
32]. In the heat stress environment, adding 1000 µg Cr/kg Cr-Met or 1500 µg Cr/kg Cr-Nic to the diet can effectively alleviate the heat stress and improve the feed intake and body weight gain of broilers [
33,
34]. However, in the current study, the addition of different levels of Cr-yeast had no significant effect on the growth performance of broilers under HSD. This is similar to previous studies. Rajalekshmi al [
35] found that the addition of 100, 200, 400, 800, 1600, and 3200 µg Cr/kg Cr-Pro had no significant effect on the feed intake of broilers under conventional feeding conditions; the addition of 400 µg Cr/kg organic chromium (Cr-Met, Cr-yeast, Cr-Nic, Cr-Pic) in the diet had no significant effect on ADG, ADFI and FCR [
19]. In addition, Ebrahimzadeh et al. [
36] reported that adding 200, 400 and 800 µg Cr/kg Cr-Met to the diet had no significant effect on the feed intake of broilers under heat stress; their study pointed out that when 500 and 1000 µg Cr/kg Cr-Met were added to the diet, it significantly increased feed intake and body weight gain of broilers under heat stress [
33]. This difference in growth performance results may be caused by differences in stress conditions and chromium levels.
The immune organ index can be used to evaluate the immune function of broilers. In our study, the HSD significantly reduced the liver index of broilers. Qaid et al. [
37] also found that the relative weight of liver in broilers fed with high density was significantly lower than that in broilers fed with low density. During stress, the body produces a large number of glucocorticoids to activate glucocorticoid receptors to play an anti-inflammatory function, so as to start the resistance response of the internal environment to stimulating factors. When stress is excessive, the expression and function of glucocorticoid receptors are often down regulated, resulting in glucocorticoid resistance, leading to systemic inflammatory response and immune organ dysfunction. [
38,
39,
40]. When we added 200, 400 and 1600 µg Cr/kg Cr-yeast to the diet, the significant decrease in broiler liver index in HSD was alleviated. Sahin et al. [
41] reported that the addition of 250 µg Cr/kg Cr-Pic under heat stress increased the liver and spleen index of broilers; Jahanian et al. [
34] found that the addition of Cr-Met under heat stress increased the thymus and bursa index of broilers. Part of the positive effect of Cr (III) on immune organ index may be due to its anti-inflammatory and antioxidant functions [
36].
The stress response is mainly realized through the hypothalamus pituitary adrenocortical axis (HPA), and the increase in CORT content could be used as a marker of stress response [
38]. In the current study, the HSD group had much higher CORT contents in serum than the LSD group, which means the high stocking density induced the stress situation of broilers. In addition, Ma et al. [
8] reported that the CORT level was significantly higher in the HSD group than the NSD group. In our study, supplementing 400 µg Cr/kg Cr-yeast to the diet of broilers under high stocking density had the lowest contents of serum CORT, which indicated that Cr-yeast could alleviate stress for broilers. The liver is an important metabolic and immune organ. When hepatocytes are damaged, ALT and AST mainly existing in hepatocytes will escape from the cells due to the destruction of the cell membrane and mitochondrial structure, and then enter the blood, so as to increase the activities of ALT and AST in the serum [
42]. Most of the ALP in the serum comes from the liver and bone, and mainly exists in the capillary bile duct cell membrane of the liver. When the hepatocytes are damaged, the capillary bile duct cell membrane in the liver is damaged, causing the capillary bile duct cell membrane to move to the basal side, and then ALP leaks into the serum, raising the concentration of ALP in the serum [
43]. TBA are synthesized by the liver and secreted into the intestine, part of which is reabsorbed by the intestine, then through the portal vein, and it is finally absorbed back by the liver [
44]. When hepatocytes are damaged, bile acids that are reabsorbed from the intestine cannot be effectively absorbed, and the concentration of bile acids in the serum will be increased, so that the level of TBA in the serum can reflect the injury of liver parenchyma [
44,
45]; PA is a glycoprotein synthesized by the liver and is secreted into the blood. When the liver is damaged, the synthesis and secretion function will be reduced, and the concentration of PA in the blood will be reduced [
46]; TRF is mainly synthesized by hepatocytes. The occurrence of liver disease is often accompanied by the decrease in TRF [
47]. In this study, HSD increased the activities of ALT, ALP and the contents of TBA, and decreased the levels of PA and TRF in the serum, indicating that the HSD caused damage to the liver. When we added different levels of Cr-yeast to the diet of broilers in the HSD, the activities of ALT and AST in the serum decreased; when Cr-yeast was added at 800 µg Cr/kg level, the ALP activities and CG contents decreased significantly; when Cr-yeast was added at 400, 800 and 1600 μg Cr/kg level, the TBA contents decreased significantly and PA contents increased significantly; when Cr-yeast was added at 200 and 400 µg Cr/kg levels to the diet, the contents of TRF increased significantly. The results showed that the addition of Cr-yeast alleviated the liver damage of broilers under HSD.
TNF-α is secreted by activated T cells; it is an effective pro-inflammatory and immunomodulatory cytokine, which is related to the inflammatory state [
48]. As a key pro-inflammatory cytokine, IL-1-β participated in a variety of autoimmune inflammatory reactions and cell activities [
49], and IL-10 can stimulate B cell maturation and antibody production to inhibit inflammation [
50]. The decrease in T3 represents less thyroid hormone secretion and hypothyroidism, and the occurrence of hypothyroidism is related to the decrease in self-immunity. A large number of studies have proved that heat stress can reduce the contents of T3 and T4 in the serum of broilers [
51,
52]. Excessive thyroid hormone secretion in this study may lead to the increase in the thyroid hormone [
53]. Compared with the LSD, the T3 content of the serum in broilers decreased significantly and T4 increased significantly in the HSD. This phenomenon proved that HSD was the reason for the decrease in broiler immunity. We found that the addition of 800 µg Cr/kg Cr-yeast to the diet significantly reduced the TNF-α and IL-1-β contents of serum in broilers at HSD, and significantly increased the IL-10 level of serum; when we added 400, 800, 1600 µg Cr/kg Cr-yeast to the broiler diet at HSD, the decrease in T3 and the increase in T4 were alleviated, so that the addition of Cr-yeast can inhibit the occurrence of inflammation and enhance the immunity to a certain extent.
The body produces excessive free radicals in the oxidation state, which has strong oxidation and can damage the body’s tissues and cells. SOD and GSH-Px can prevent cell oxidative damage by scavenging free radicals, SOD can catalyze the disproportionation of free radicals, remove excessive superoxide anion (O
2−) in the body and inhibit the cascade reaction of oxygen free radicals [
54]; GSH-Px is an important peroxidase, which can improve the ability of the body to decompose peroxide products [
55]. In this study, HSD decreased T-AOC contents and GSH-Px activities in the serum, and T-AOC contents and T-SOD activities in the breast. This is consistent with the research results of Simitzis et al. [
56] and Simsek et al. [
57]. It showed that the HSD has produced oxidative stress in broilers. In the HSD, the addition of 200 and 1600 μg Cr/kg Cr-yeast in the diet increased the content of T-AOC, and adding 400 μg Cr/kg Cr-yeast increased the content of GSH-Px in serum. Adding 400 μg Cr/kg Cr-yeast increased the activities of T-SOD and the content of T-AOC in the breast. In addition, although HSD did not change the antioxidant index of broiler liver, 400 μg Cr/kg Cr-yeast can increase the T-AOC contents and the activities of T-SOD and GSH-Px in the broiler liver under HSD. Therefore, Cr-yeast may have strong antioxidant activity and could improve the antioxidant capacity of broilers.
Through histopathological analysis, we found that HSD increased inflammatory factors in hepatocytes and serious tissue cavitation. It is known that hepatocytes contain a large number of mitochondria, which are the main place for biological redox reactions. Therefore, the liver not only produces a large amount of ROS, but also is the main organ attacked by ROS. Oxidative stress in livestock and poultry can cause liver structural damage and functional disorder. It is also an important pathological feature of hepatitis, fatty liver, liver fibrosis and other diseases [
58]. The results showed that HSD made broilers produce oxidative stress, resulting in liver inflammation, which is consistent with the research results of others [
58,
59]. When 800 μg Cr/kg Cr-yeast was added to the diet of high-density broilers, there were almost no liver inflammatory factors, which also showed that yeast chromium had a certain anti-inflammatory effect and alleviated liver injury.
In recent years, the research dose range of chromium in different forms and sources in broiler diets is 200–2000 µg Cr/kg under non-stress, and the research range under heat stress is 200–4 × 10
5 µg Cr/kg [
26]. However, there are few studies on the recommended dose. The role of chromium in different body functions may be different. The range obtained in this study is wide, and further research may be needed to narrow the supplementation range.