In the last decades, dietary manipulation with innovative feed supplements, such as medicinal plant and its derivatives, are among the common advanced strategies [5, 24–26]. Medicinal plants are reflected a cornerstone of medicine since earliest times, and have been employed as promoters of antioxidant capacity, immunity and growth in animals [26]. Lasia spinosa Thw. (LST) has been used medicinally since ancient times for its diverse biological activities such as anti-bacterial and anti-septic properties [13–16].
In the present study, dietary LST powder supplementation significantly boosted the growth performance of the chickens involved, produced a remarkable increase in ADG and final body weight, and caused a statistical decrease in FCR when compared to the control diet, in accordance with the findings that the inclusion of LST in fish diets significantly improved the growth performance and feed utilization [20]. In addition, the current study displayed no significant difference in mortality rate was noticed in groups that received LST compared with the control, which agreed with the described by Munglue et al. [20] who perceived no significant alterations concerning survival rate in fish as a response to the dietary LST. In parallel, the supplementation of LST in buffalo had a positive impact on the growth indices [21]. However, the underlying mechanisms of the growth-promoting effect of LST are still not understood. Phesatcha et al. [2] confirmed that the inferior FCR and boosted growth may relate to enhanced appetite and digestion in beef-cattle fed diets enriched with LST (60–90 g/day/head). The authors suggest that this could be related to the constructive role of LST in improving the FI, apparent digestibility, and efficiency of microbial N synthesis in cattle [2], which may be one of the motives that boosted poultry growth in our study.
As natural additives in animal feed, several phytohormones found in medicinal plants are proven to promote growth and feed utilization. Suthikrai et al. [21] found LST contains considerable levels of natural phytoestrogens (10.76-14.55pg/g DM) and phytoandrogens (0.15-0.92pg/g DM). Therefore, it is reasonable to hypothesize that the production of growth hormone and insulin-like growth factor-I may be modulated by phytohormones, resulting in the improvement of growth. Phytoandrogens may also enhance protein synthesis by activating the androgen receptor (AR)-mediated anabolism [28]. In addition, combined with the findings of Buszczak et al. [29], it is possible that the growth-promoting effect of LST may be due to modulating related genes expression related to protein synthesis and cell growth. Moreover, LST is excessively rich in alkaloids, flavonoids, terpenoids, phenolic compounds, steroids, phytosterols, saponins, coumarins, tannins, glycosides and anthraquinone [30]. Kikusato et al. [31] found that plant-derived isoquinoline alkaloids supplementation significantly improved the growth performance of chickens under heat stress conditions. Starčević et al. [32] reported that phenolic compounds boosted the growth performance and antioxidant profile of chickens. It has been reported that phytosterols could promote animal production and antioxidant capacity. Thus, these phytochemical constituents may collectively contribute to the growth-promoting property of LST.
Blood variables are commonly reliable indicators of general health profile, as responses to external and internal stressors and stimuli. The study notices LST supplements did not significantly affect the levels of blood metabolites (except for TBIL, TG, HDL-C and LDL-C), showing that LST had no detrimental impacts or toxicity in the broilers. In the present study, dietary inclusion of LST reduced the triglyceride and LDL-C and boosted TBIL and HDL-C levels in broiler serum. The findings were consistent with prior studies in rats where the levels of triglyceride and LDL-C were significantly decreased with the methanolic leaves extract of LST (200, 400, 800 mg/kg BW) administration in compared to the control groups [12]. Moreover, Kaewamatawong et al. [33] studied the acute to subchronic toxicity of LST in mice and found that LST significantly decreased the triglyceride values but did not induce any toxicological effects in the acute and subchronic term. The presence of anti-hypertensive compounds such as flavonoids and alkaloids present in LST could be responsible to reduce serum triglycerides and LDL-C and enhancing the HDL-C levels and thus avoiding cardiovascular ailment [24]. Further studies should be conducted to validate the underlined mechanism of LST as an anti-hypertensive agent for drug treatments in poultry. The present study has shown that LST significantly improved the levels of Ca, Fe and P in broiler serum as a response to 4% addition of LST in broiler diets. This enhancement could be due to the rich trace element content in LST [11]. In addition, TBIL was significantly reduced only in the LST1 groups and other biochemical parameters were similar among the groups, thus indicating that the LST powder consumption did not have a negative influence on the health status of the animals.
The immunity of poultry is usually affected by the animal’s oxidative status (OS) [8]. An excess amount of OS can cause serious damage to DNA, lipids and proteins. The elimination of extra OS depends on the action of several enzymes such as SOD and CAT [10]. Moreover, chicken meat is relatively rich in polyunsaturated fatty acids, making it vulnerable to oxidation [11, 24]. Therefore, diet-derived antioxidants may be vital in reducing cumulative oxidative damage. Natural antioxidants are broadly found in medicinal herbs. The LST and its extracts have antioxidant property due to its high content of flavonoids and phenolic compounds [19]. It has been reported that the extracts of LST could scavenge the stable radical 1,1-diphenyl-2-pecrylhydrazyl (DPPH) [14], elucidated those polyphenols and ascorbic acid contributed mainly to the total antioxidant activity of rhizomes. Besides, phytochemical constituents in LST such as phytosterols [34] and flavonoids [35], were proven to improve the antioxidant enzyme activity.
In the present study, we measure the levels of antioxidant enzymes to determine the antioxidant status in serum, liver and breast muscle. We found that GSH-Px, SOD and CAT were significantly increased in LST supplemented groups, while the levels of ROS and MDA, as indicators of OS, were significantly reduced. The increased antioxidant enzyme content is parallel with the decreased level of MDA and ROS as response to dietary LST inclusion, indicating that dietary LST supplement can promote the antioxidant capability of birds by enhancing the enzymatic antioxidant scheme. As the improvement of the antioxidant enzyme would exert a positive influence on meat quality [34], so it is logical to speculate that the addition of LST can enhance the meat quality of the chickens involved in this experiment.
Results of the current study indicated that the dietary LST powder addition affected the structure of the intestine in broilers. It is well known that intestinal morphology can objectively reflect gut health, and villus height as well as crypt depth can partially reflect functional status of the intestine [22]. A previous study demonstrated that the villus height was increased and the intestinal function was improved in fish after supplementation of LST extract in diet [20]. Similarly, in this study, a statistical decrease in crypt depth and increase in villus height and in the ratio of the two was observed in the intestine of chickens fed LST powder compared with the control groups. The increased villus height and villus height to crypt depth ratio for various gut fractions of chickens fed LST powder were in accordance with appropriate growth performance and increased metabolizability of nutrients. In fact, the increase of villus height (Vh) usually results in higher total luminal villus absorptive area and subsequently lead to higher transport of nutrients at the villus surface [36]. Moreover, the decrease in crypt depth (Cd) usually indicates a slower turnover and healthier condition of the gut, which can reduce maintenance requirement and ultimately be beneficial to promote the growth of the animals [37]. It is possible that the richness of fiber fractions in LST rhizome sustain the intestinal integrity through increasing the Vh and Vh/Cd, decreasing the Cd feature [4].
The gut microbiome is closely related to intestinal health, normal physiological functions and poultry production, and the composition of the microbial community could be altered upon the diet and over time [39]. This study is setup necessarily to evaluate the effect of LST powder as a feed additive on the intestinal flora of Chinese yellow-feathered broilers, which will contribute to developing nutrition intervention for optimal health, growth and productivity in poultry. We found that the dominant phyla in the chicken are Firmicutes, Bacteroidetes, Proteobacteria. Furthermore, the primary genera were Bacteroides, Faecalibacterium, Oscillospira in the current study, which was consistent with earlier works recognizing Bacteroidetes as the fundamental genus in the cecum (about 40% of sequences) [40].
Dietary LST powder addition also caused a significant alteration of the gut microbiota diversity in broilers. According to the α-diversity results, the 1% supplementation LST powder significantly increased the abundance of caecal microbiota (PD whole tree index, Chao1 index, observed species and unique OTUs) compared with the other groups, while 2% and 4% supplementation LST powder decreased the abundance (chao1 index and observed species) of caecal micorbiota. In addition, regarding the β-diversity analysis, a clear separation of microbial community due to dietary LST powder inclusion was observed, which means microbial community of each group were significantly different. Elevated levels of diversity mostly contribute to maintaining the stability of intestinal microbiota after exposing to environmental stress, as well as resistance against potential invading pathogens [41]. For these reasons, it is considered that a reduction of α and β diversity should be negative. This suggests that the structure of intestinal flora in the LST1 groups was improved and, thus, better intestinal health. However, LST2 and LST4 groups got a lower diversity, and this may be resulted from the anti-bacterial activity of higher LST powder addition [13, 14].
LEfSe analysis revealed that the relative abundance of Anaerofilum was significantly increased in the LST1 groups compared with other groups, which has been proposed to boost the antioxidant capability and absorption of energy as previously reported by Guo et al. [42]. Interestingly, Odoribacter [43] and Sutterella [44], known as SCFA-producing bacteria, were also overrepresented in the LST1 groups. SCFAs, as the main energy source of colorectal cells, play an essential role in regulating the absorption of several nutrients, digestive and hormonal secretions in the intestine, and participating in energy metabolism widely [43, 44]. In addition, Bacillus has been widely proven to promote growth performance [43, 45]. In this study, we found the enrichment of Bacillus in the LST1 groups, while the best growth performance was found in the LST1 groups. Therefore, the increase of relative abundance in Anaerofilum, Odoribacter, Sutterella and Bacillus in LST1 groups in the present study, suggests the efficacy of 1% LST powder in promoting beneficial bacteria, which would eventually contribute to improved performance and health. These findings suggest that there is close association between intestinal flora and physiological performance.
However, no reports have verified the associations of growth performance, serum metabolites, antioxidant status and intestinal morphology with changed gut microbiome structure in Chinese yellow-feathered broilers fed with LST powder. By using Spearman’s correlation analysis, we revealed that the profiles of taxonomic composition at the genus level in cecal microbiota were significantly associated with growth performance, serum metabolites, antioxidant status and intestinal morphology. Consistently, the relative abundance of Odoribacter and Bacillus was positively correlated with growth performance, which showed that 1% addition level of LST powder could improve ADG and decrease FCR potentially by increasing the abundance of Odoribacter and Bacillus as detected at day 42. In the present study, we also demonstrated that Bacillus, which has been reported to improve intestinal morphology [46, 47], was positively related to the Vh and Vh/Cd of duodenum. Furthermore, the abundance of Bacillus was closely correlated to the concentrations of CAT, SOD and GSH-Px, and many studies have reported Bacillus could participate in boosting of antioxidant scheme [48, 49]. Actually, intestinal flora can regulate the host well-being, immunity and disease by determining the biological value of the diet [48]. Here, we found that serum metabolites including triglyceride, TBIL, Ca, Mg and LDL-C affected by dietary treatment of LST powder, were faithfully connected with the dynamic fluctuations of broiler gut microbiome structure.
Overall, the gut microbiota is reshaped by LST supplements and further contributes to improvement of growth performance, intestinal morphology, antioxidant capacity, Ca and Mg, and reduction of triglyceride, LDL-C and TBIL.