Effects of Diet with "Lianhuang" Additive of Chinese Herb Medicine on Growth Performance and Metabolic Rates of Dietary Nutrients in Donglan Black Chicken.

Jing YAN　Lei QIU　Min CHEN　Fulin WANG　Wenjie LU　Menglei LI　Cui YANG　Jiahuang YANG

Abstract[Objectives] The study was conducted to evaluate the effects of diet with "Lianhuang" additive of Chinese herbal medicine （LACHM） on growth performance and metabolic rates of dietary nutrients in Donglan black chickens （DBCs）. [Methods]80 DBCs aged 1 d were blocked into 4 groups randomly and assigned to 6 replicates in each group with 20 chicks in each replicate. Group A as control group fed basal diet. Groups B， C and D as treatment groups consumed the basal diet added with 0.25%， 0.50% and 1.00% of LACHM， respectively during the test period of 70 d. In the end of feeding test， 24 DBCs with similar body weight from each group were chosen out and used for a metabolism test to determine the metabolic rates of dietary nutrients from each treatment. The metabolism test was conducted as the same design as the feeding test and the DBCs from each group were fed the same diet as previous test. [Results] The ADG of chickens in the test increased by 5.00%-9.15% （P<0.05） except the stage of 50-70 d， and the individual average body weight （IABW） at 70 d in group B also increased by 4.86% （P<0.05）. Compared with group A， the ADG of group C in the stages of 36-49， 1-49 and 1-70 d and the IABW in the end of 70 d increased by 5.90%， 2.66%， 2.68% and 2.57% （P>0.05）， respectively. The ADFI in the stage of 36-49 d from group A was 6.04% more than that from group B （P<0.01）， and 5.12% and 4.13% more than those from groups C and D （P<0.05）， respectively， and the ADFI of group A in the stage of 1-49 d was still 2.86% more than that from group C （P<0.05）. However， no differences in ADFI were found in other stages in the test among the 4 groups （P>0.05）. The F/G in the stages of 22-35 and 1-70 d from group B was 6.32% and 3.41% lower than those from group A （P<0.05）， respectively， and even 11.43% and 7.31% lower in the stages of 36-49 and 1-49 d from group B than those from group A （P<0.01）， respectively. The F/G of group C in the stage of 36-49 d was also 10.15% lower than that of group A （P<0.05）， and still 5.29% lower than that from group A in 1-49 d （P<0.01）， but no differences were found in other stages between the two groups （P>0.05）. The metabolic rates of MD， CP， CF， NFE， CA， TE， Ca and P in the 3 treatment diets for group B， C and D all increased to some extent. Among them， the increasing extents of diets with 0.25% and 0.50% of LACHM for groups B and C were much greater. Therefore， the diet with 0.25% of LACHM could improve the growth performance and metabolic rates of dietary nutrients in DBCs significantly. [Conclusions]This study provides a theoretical reference for the effects of traditional Chinese medicine additives on the growth and metabolic rates of DBCs.

Key words"Lianhuang" additive of Chinese herbal medicine; Donglan black chicken; Growth performance; Nutrient metabolic rate

Feed antibiotics have good disease resistance and growth-pro-moting properties. They have been used in China's livestock and poultry production for a long time， and have played an important role in the development of China's livestock and poultry breeding. However， the side effects and risks caused by the use of antibiotics in a large amount have forced China to adopt a "prohibition of antibiotics"[1-4]. However， in today's "prohibition of antibiotics" era， there is no appropriate substitute. Therefore， finding a corresponding substitute for antibiotics has become a top priority. Many studies have shown that traditional Chinese herbal medicines contain a variety of biologically active substances， which have good antibacterial， disease resistance and growth-promoting effects， and compound Chinese herbal medicine additives contain more types of biologically active substances， so the effect is better[5-7]. Although these studies have preliminarily shown the feasibility of Chinese herbal medicine as a substitute for antibiotics， there are still many problems to be solved， such as the quality of additives， the efficiency of bioactive substances， the stability of the effect and its mechanism[5]. Therefore， further in-depth research is still needed. In this study， according to the pharmacology of traditional Chinese medicine and factors including the types and contents of main bioactive substances contained in various constituents of its formulas， an additive formed with ten Chinese herbal medicines such as Forsythia suspensa， Angelica sinensis， Astragalus memeranaceus and Crataegus pinnatifida were used to carry out feeding and metabolism tests on Guangxi Donglan black chickens （DBCs） to study its effects on the growth performance and feed nutrient use efficiency of black-bone chickens， so as to explore the possibility of its use as an antibiotic substitute. This study will provide a scientific basis for finding antibiotic substitutes after "prohibition of antibiotics".

Materials and Methods

Chinese herbal medicine additives

The "Lianhuang" additive of Chinese herbal medicine （hereinafter referred to as LACHM） used in this experiment was composed of ten kinds of Chinese herbal medicines including F. suspensa， A. sinensis， A. memeranaceus， Citrus reticulata Blanco， C. pinnatifida and Glycyrrhiza uralensis. It was provided by Yixin Pharmaceutical Co.， Ltd. in Nanning， Guangxi. Before use， the additive was pulverized， sieved with 100 mesh sieves， and bagged.

Experimental diets and nutrient levels

The feed used in the experiment was provided by Nanning Liyuan Grain and Oil Feed Co.， Ltd. （the formula is confidential; the raw materials are composed of corn， wheat， sorghum and other grains and soybean meal， fish meal， calcium hydrogen phosphate， L-lysine sulfate， methionine hydroxy analogue， vitamin A， vitamin D3， vitamin E， vitamin K3， copper sulfate， ferrous sulfate， zinc sulfate， calcium propionate， etc.）. The main nutrient levels of diets at different stages are shown in Table 1.

Experimental animals， design and feeding management

Feeding management480 one-day-old DBCs （all ♀ chickens） used in the test were provided by Guangxi Liteng Agriculture and Animal Husbandry Development Co.， Ltd. Adopting a single factor completely random design， the test chickens were divided into 4 groups， each of which was set up with 6 replicates， each of which was assigned with 20 chickens. Among them， group A was fed the basal diet as the control group; and groups B， C， and D were supplemented with 0.25%， 0.50% and 1.00% of LACHM in their basal diet， respectively. The experiment was carried out on November 9， 2020 at the chicken farm of the Poultry Research Laboratory of the Guangxi Institute of Animal Science， and the experimental period was 70 d. Before 21 d， they were raised in the brooding room， and the temperature was controlled manually. The initial room temperature was controlled at 33 ℃， and after 1 week， it was lowered by 1 ℃ every 2 d， and finally （before 21 d） maintained at 30 ℃. During the experiment， the chickens had free access to food and water， and the feeding management and immunization procedures were carried out according to the production routine.

Metabolic testAt the end of the 70 d of feeding test， 24 test chickens with similar body weight were selected from each test group， a total of 96 chickens. According to the design of the original feeding test， the chickens were divided into four groups， each of which was set up with 6 replicates， each including 4 chickens. The chickens were fed the diet used in each group in the previous experiment for a 6-day metabolism test. The first 3 d were the pre-trial period， and the collection of feces was carried out in the latter 3 d. During the sampling period， all the chicken feces of the test chickens on the previous day were collected at 8：00 every morning， and the feathers， dander and other sundries were removed， and the materials were mixed uniformly and sampled. The samples were sprayed with 10% sulfuric acid， uniformly bagged and sealed， and stored at -20 ℃. Then， the determination of various nutrients and AIA content was carried out by the Animal Nutrition Laboratory of Guangxi Institute of Animal Science. The metabolic test method was the 4N hydrochloric acid insoluble ash （4NHCl·AIA） method.

Determined indexes and methods

Determination of body weight， feed intake and feed rewardAt the beginning of the experiment， 21， 35， 49 and 70 d， the fasting body weight of the experimental chickens in each group was weighed before feeding （8：00）， and the average daily gain （ADG） was calculated correspondingly. Meanwhile， the feed intake of the test chickens during the period was measured and the corresponding average daily feed intake （ADFI） was calculated. Finally， according to the ADG and AAFI of each stage， the feed-to-grain ratio （F/G） （that is， the feed reward） of the test chickens was calculated， and the calculation formula was： （F/G）=ADFI（g）/ADG （g）.

Determination of dietary nutrient metabolic ratesThe samples collected during the metabolism test were thawed uniformly， dried at 600 ℃， crushed and bagged before the start of the measurement. Finally， the nutrient and AIA contents in the samples were determined. According to the formula used in the 4NHCl·AIA method， the metabolic rates of various nutrients in the diet were calculated.

DC （%） （nutrient metabolic rate）=100-100[（A₁/A）×（F/F）]

In the formula， A： AIA content in feed （%）， A： AIA content in manure （%）， F： Nutrient content in feed （%）， and F： Nutrient content in manure （%）.

Processing of experimental data

The experimental data were preliminarily processed with Excel， and one-way ANOVA was performed with SPSS16.0 statistical software. The Duncan's method was used to compare the means between groups. The results were expressed as： mean ± standard deviation.

Experimental Results

Effects of LACHM on growth performance of experimental chickens

ADG resultsThe ADG results of the test chickens are shown in Table 2.

The data in Table 2 showed that during the whole test period， except 50-70 d， the ADG of group B in other stages increased by 5.00%-9.15% （P<0.05） compared with that of group A， and the average body weight at 70 d also increased by 4.86% （P<0.05）. The ADG of group C in 36-49， 1-49 and 1-70 d and the average body weight at 70 d also increased by 5.90%， 2.66%， 2.68% and 2.57% compared with group A， respectively， but the differences did not reach the statistical significant level （P>0.05）， and there were no significant differences in other stages between the two groups （P>0.05）. The ADG of group D in 36-49 and 1-70 d and the average body weight at 70 d also tended to increase compared with group A （P>0.05）， but there were no significant differences in other stages between the two groups （P>0.05）. It could be seen that the addition of 0.25% Chinese herbal medicine additive in the diet could significantly improve the weight gain performance of the test chickens， and the weight gain effect of group B was the best among the three test groups.

Effects on feed intakeThe ADFI of the test chickens in each stage is shown in Table 3.

From the data in Table 3， although the ADFI of group A was 6.04% higher than that of group B （P<0.01） and also 5.12% and 4.13% higher than that of groups C and D （P<0.05）， respectively， in 36-49 d， and group A was also 2.86% higher than group C in 1-49 d （P<0.05）， in other stages， especially in 1-70 d， there were no significant differences between the four groups of chickens （P>0.05）. It could be seen that none of the three groups with the addition of LACHM could increase the dietary intake of the experimental chickens， but there were no adverse effects.

Impact on feed rewardThe F/G of the test chickens in each stage is shown in Table 4.

The data in Table 4 show that in 1-21 and 50-70 d， there were no significant differences in F/G between groups A and B （P>0.05）， but in 22-35 and 1-70 d， group B was lower than Group A by 6.32% and 3.41% （P<0.05）， respectively， and in 36-49 and 1-49 d， it was lower than group A by 11.43% and 7.31% （P<0.01）， respectively. In 36-49 d， the F/G of group C was 10.15% lower than that of group A （P<0.05）， and it was also lower than that of group A by 5.29% in 1-49 d （P<0.01）， but in other stages， there were no significant differences between the two groups （P>0.05）. Although the F/G of group D was 7.27% lower than that of group A in 36-49 d （P<0.05）， but the F/G of group A was lower than that of group D by 5.43% in 1-21 d （P<0.05）， and there were no significant differences in other stages between the two groups （P>0.05）. It could be seen that among the three test groups， group B with an addition amount of 0.25% could significantly reduce the feed-to-gain ratio， that is， it significantly improved the feed conversion efficiency （or feed reward）.

Effects on metabolic efficiency of dietary nutrients

The metabolic rates of dietary nutrients in the experimental chickens are shown in Table 5.

The metabolic rates of DM in groups B and C increased by 5.75% and 6.41% compared with group A， respectively （P<0.01）， and group D was also higher than group A by 5.20% （P<0.05）. Compared with group A， the metabolic rates of CP in groups B， C and D increased by 4.26%， 4.20% and 3.97% respectively （P<0.05）. However， there were no significant differences in the metabolic rate of EE among the four groups （P>0.05）. The metabolic rates of CF in groups B and C were significantly higher than that in group A （P<0.01）， and group D was also significantly higher than group A （P<0.05）. The metabolic rates of TE in groups B， C and D increased by 2.85%， 2.78 and 2.81%， respectively， compared with group A （P<0.05）. The metabolic rate of NFE in group B was significantly higher than that in group A （P<0.01）， and groups C and D were also significantly higher than group A （P<0.05）. The metabolic rate of CA in group C was significantly higher than that in group A （P<0.01）， and the two groups B and D were also significantly higher than group A （P<0.05）. The metabolic rates of Ca in groups B and C were 20.24% and 22.84% higher than that in group A （P<0.01）， respectively， and group D was also 13.59% higher than group A （P<0.05）. The metabolic rates of P in groups B and C were 24.86% and 24.03% higher than that in group A， respectively （P<0.01）， and group D was also 19.04% higher than group A （P<0.05）. It could be seen that the three groups added with different amounts of LACHM had different degrees of improvement in the metabolic efficiency of main nutrients in the diet， and the improvement with the addition of 0.25% and 0.50% was more significant.

Conclusions and Discussion

Discussion

DBCs are an excellent local chicken breed in Guangxi， which is mainly distributed in 14 townships such as Donglan， Aidong and Changjiang in Donglan County， Guangxi and surrounding counties and towns with an annual output of more than 1 million chickens. The whole body of DBC is black， and the skin， meat， bones and internal organs are light black[8]. The size of the chickens is medium and small. The adult （150 d） roosters are 1.3-2.5 kg， and the hens are 1.1-2.0 kg; and at 120 d， the average weight and daily weight gain of the roosters are 1 336.6 and 11.1 g， respectively， and those of the hens are 913.9 and 8.4 g， respectively[9]. DBCs have the advantages of good rough feed tolerance， strong disease resistance， tender and delicious meat and high medicinal value， and are widely favored by people[10].

In this study， when 0.25%， 0.50% and 1.00% of LACHM were added to the diet of the three experimental groups B， C and D in the four groups of DBCs for 70 d， the ADG of group B was significantly higher than that of the control group A， and it increased in 1-49 or 1-70 d， respectively， by 6.76% and 5.00%， which were significant; and although group C had a certain improvement trend compared with group A， it did not reach the level of statistical difference. It could be seen that the addition of 0.25% of LACHM in the diet had a good effect on the growth of the test chickens， and it also showed that this addition level of the diet was suitable.

Appropriate Chinese herbal medicine additives can promote the growth of chickens， and the reason may be related to the bioactive substances contained in various Chinese herbal medicines in the additives. A. sinensis， A. memeranaceus， C. reticulata Blanco， C. pinnatifida and G. uralensis used in this study contain active substances such as polysaccharides， flavonoids， volatile oils， and alkaloids to varying degrees[11-16]. Studies have shown that polysaccharides can promote the proliferation of beneficial bacteria in the digestive tract and inhibit the reproduction of harmful bacteria， so they can improve the intestinal micro-ecosystem and maintain intestinal health. Meanwhile， they can also promote the growth of duodenal and jejunum villi and increase the surface area of the intestine for nutrient absorption. Moreover， polysaccharides can also up-regulate the expression of carbohydrate-active enzyme （CAZymes） genes and improve the activity of CAZymes[17-18]. Therefore， they can improve the digestion and absorption of feed nutrients， thereby promoting animal growth. Fang et al.[19] and Shi et al.[20] added compound Chinese herbal medicine polysaccharides to chicken diets for research， and the results showed that the growth performance of chickens could be significantly improved. Flavonoids have antioxidant， immune-regulating and growth-promoting functions. Jiang et al.[21] added 750 mg/kg of flavonoids from Artemisia argyi to the diet of AA roosters， which significantly increased the ADG in the period of 22-42 d and significantly decreased F/G. Yang et al.[22] added hesperidin （also called bioflavonoids） to the diet of AA male chicks， which also significantly increased the ADG in 22-42 or 1-42 d， and significantly reduced F/G. It is believed that flavonoids can increase villus height and crypt depth of the small intestine， and promote intestinal development by increasing the activity of intracellular alkaline phosphatase and the synthesis of DNA in intestinal epithelial cells.  Meanwhile， flavonoids can also promote gastrointestinal motility， speed up the digestion of chyme， and improve the utilization efficiency of feed. Volatile oils are also known as essential oils. Studies have shown that plant essential oils （PEOs） can increase the palatability of feed and stimulate the secretion of digestive juices and mucus in the gastrointestinal tract. They can inhibit the reproduction of harmful bacteria in the digestive tract， and adjust and stabilize the microflora of the digestive tract. PEOs can also increase villus height and crypt depth of the intestinal tract， increase the surface area of small intestinal villi， and improve the digestion and absorption of the intestinal tract[23-24]. The study adding PEOs to broiler diets also showed that PEOs could significantly increase the activity of trypsin， α-amylase and intestinal maltase[25]， so they can improve the digestibility of feed nutrients， and promote the absorption and utilization of nutrients. Therefore， adding an appropriate amount of PEOs to the diet can significantly improve the growth performance and feed use efficiency of chickens[26-27]. The research also shows that the effect of adding composite PEOs is better than that of single PEO[28]. In addition， the above-mentioned bioactive substances also have antibacterial， antiviral and antioxidant effects[29-31]. Through these effects， the influences of certain negative factors on the growth of chickens can be eliminated， and the growth and development of chickens can be further promoted. Studies have shown that Chinese herbal medicine additives composed of A. memeranaceus， A. sinensis， C. reticulata Blanco， C. pinnatifida and G. uralensis as the main components have a good effect in promoting the growth performance of chickens[7，32-34]. The results of this study are also very similar， which once again proves the growth-promoting effect of such additives on chickens.

During the experiment， because the ADG of group B was significantly higher than that of group A， and there was no significant difference in ADFI between the two groups， the F/G of group B was lower than that of group A， indicating that the addition of 0.25% of LACHM in the diet could significantly reduce the F/G of chickens， that is， the feed conversion efficiency was improved. It showed that the improvement of the growth performance of the experimental chickens in group B was achieved by the improvement of the feed conversion efficiency， which is very meaningful for production practice， because the production benefits can be improved without increasing feed costs.

From the results of the metabolism test， it could be seen that the three different addition amounts of the additive could improve the metabolic rates of main nutrients in the diet to different degrees， and the improvement effects of the addition amounts of 0.25% and 0.50% were the best.  It showed once again that the improvement of the growth performance in group B with the addition of the additive at 0.25% to the diet was achieved through the improvement of feed conversion efficiency. However， why did the improved feed conversion efficiency of other two groups， especially group C， not lead to improved growth performance？ However， why did the improved feed conversion efficiency of other two groups， especially group C， not lead to improved growth performance？ The reason is not clear at present. Superficially， it might be related to the addition amount of additives， because the addition amount of group C was twice as high as that of group B， and the amount of group D was three times higher than that of group B. What was its mechanism？ And was there any other reason besides this？ These are not yet known， and further research is required.

Conclusions

① The addition of 0.25% of LACHM in the diet could significantly improve the growth performance of DBCs， and significantly reduce the feed/weight ratio of black-bone chickens.

② None of the three addition amounts of LACHM could increase the dietary feed intake of DBCs， but they did not negatively affect the feed intake of the experimental chickens.

③ The three addition amounts of LACHM could improve the metabolic rates of the main nutrients in the diet to different degrees， among which the increases by the addition amounts of 0.25% and 0.50% were significant.

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