Effects of Dietary Tongmai Granule Residue Supplementation on Growth Performance, Nutrient Digestibility, Blood Biochemical Parameters and Rumen Fermentation in Sheep

The purpose of this study was to investigate the effects of Tongmai granule residue (TGR) on the growth performance, apparent nutrient digestibility, blood biochemical parameters, and rumen fermentation in Doper x Small Tail Han hybrid sheep. Twenty-four healthy female lambs (6–7 months of age; 29.64 ± 1.80 kg initial body weight) were randomly divided into 3 groups and fed for 75 days. The control group was fed a commercial concentrate and the other groups were fed diets supplemented with 10% and 15% TGR respectively. Dietary 15% TGR supplementation decreased average daily gain, apparent digestibility of neutral detergent ber, but increased the feed conversion ratio, apparent digestibility of crude fat and organic matter in lambs (P < 0.05). The 10% TGR group signicantly increased the apparent digestibility of organic matter (P < 0.05). Dietary supplementation of 10% and 15% TGR signicantly increased serum high density lipoprotein cholesterol (HDL-C) content on day 15, IgG content on day 30, and IgA content on day 60 of the feeding period (P < 0.05). Dietary supplementation of 15% TGR signicantly increased the content of IgA on the 30th day (P < 0.05), and signicantly decreased blood urea nitrogen (BUN) on the 30th day (P < 0.05) of the feeding period. TGR at 15% signicantly decreased the contents of acetic acid, propionic acid, butyric acid, and total volatile fatty acids (TVFA) in the rumen (P < 0.05), and signicantly increased the pH, NH3-N concentration, and acetic acid: propionic acid ratio (P < 0.05). The results suggest that, the addition of TGR to diet can improve the apparent digestibility of crude fat and organic matter, and improve the anti-atherosclerosis ability and immune function of Doper x Small Tail Han hybrid female lambs. The dietary addition of 10% TGR yielded the best feeding effect in this experiment. A; IgG, immunoglobulin G; IgM, immunoglobulin M.


Introduction
Chinese herbal medicine, is a valuable medical resource in China known for its numerous functions in health care such as prevention and treatment of diseases. For over 2000 years, herbal medicines have been used in China, as feed additives in animal husbandry to promote animal production and health.
These herbs provide animals with nutrients such as peptides, proteins, essential amino acids, starch, oligosaccharides, vitamins, fatty acids, and organic trace minerals, and as well, possess many bioactive ingredients with antibacterial activity, immune enhancement, and stress reduction properties (Wang et al., 1998;Guo et al., 2004). Also, previous studies on Chinese herbs as feed additives have shown that these herbs can modulate the immune response, nutritional metabolism, and intestinal health of foodproducing animals (Gong et al., 2014).
In recent years, the Chinese herbal medicine industry has been developing rapidly due to the continuous increase in the usage of Chinese herbal medicine resources. This results in an increased yield in Chinese herbal medicine residue. Moreover, due to poor extraction processes and extraction e ciency, the herbal residue still contains nutrients such as cellulose, hemicellulose and lignin, as well as 30% − 50% medicinally active ingredients (Wang et al., 2010;Meng et al., 2017). For example, the contents of calycosin and formononetin in Radix Astragali residue were 75.16% and 77.06%, respectively (Chen et al., 2011). Furthermore, Salviae Miltiorrhizae Radix residue contains 3.62% tanshinone IIA, 2.56% cryptotanshinone, 2.75% tanshinone , and 1.02% methylenetanshinone (Shi et al., 2010), indicating that Chinese herbal medicine residue could be further utilized. Zhu et al. (2016) suggested that Astragalus residue improved the immunity and digestion ability of white ducks resulting in a corresponding increase in body weight and weight of immune organs. In another study, it was shown that herbal residue fermentation supernatant (Jianweixiaoshi tablets) inhibits urease activity, slows down cell in ltration in the gastric area, reduces the level of gastric in ammatory cytokines IL-6, IL-8 and TNF-α, and helps to improve a disturbed microbiota, indicating strong potential in treating Helicobacter pylori infections (Meng et al., 2017).
Tongmai formula is a popular Chinese herbal medicine formula composed of three traditional Chinese herbal medicines: Chuanxiong rhizoma (rhizomes of Ligusticum chuanxiong), Puerariae lobatae Radix (roots of Pueraria lobata) and Salviae Miltiorrhizae Radix (roots of Salvia miltiorrhiza) in a ratio of 1:1:1. It is widely used in the treatment of cerebrovascular and cardiovascular diseases, such as high blood lipids, myocardial infarction and atherosclerosis (Wu et al., 2017). So far, research on Tongmai formula is mainly focused on its chemical composition. As such, no research has been conducted on the application of TGR in sheep production. Therefore, the main objective of the present study was to determine the effects of TGR on the growth performance, apparent nutrient digestibility, blood biochemical parameters and rumen fermentation in Doper × Small Tail Han hybrid sheep, and to explore the feasibility of TGR as a ruminant feed additive.

Materials And Methods
All experimental protocols involving animals were approved by the Institutional Committee for Animal use and Ethics of the College of Animal Nutrition and Feed Science of Jilin Agricultural University and also in agreement with the provincial rules and regulations.

Tongmai Granules
The fresh Tongmai Granules which mainly include Salvia miltiorrhiza, Ligusticum chuanxiong and Pueraria lobata were obtained from Xiuzheng Pharmaceutical Co. Ltd. The residue was air-dried and then pulverized to pass through a 2-mm screen.

Animals and feeding management
A total of 24 Doper x Small Tail Han hybrid ewes with average weight of 29.64 ± 1.80 kg were randomly allocated to 3 treatment groups with 8 repetitions (8 sheep in each treatment) in a randomized complete block design based on body weight. Each group was fed a diet containing different levels of TGR; 0%, 10%, and 15% TGR. The diets were formulated according to the National Research Council (NRC, 2010) recommendations for an average daily weight gain of 200 g/day ( Table 1). The experiment lasted for 75 days with an adaptation period of 15 days, during which time; the animals were weighed, tagged and treated for ecto-and endoparasites. Diets were offered twice daily (08 and 16 h) with free access to clean drinking water. Feed and water troughs were cleaned daily and the pen kept ventilated and hygienic at all times.

Sampling and analysis
The animals were weighed before the morning feeding on the rst and last days of the feeding period, and the amount of feed supplied and rejected were recorded per animal. The average daily gain (ADG), average daily feed intake (ADFI), and feed conversion ratio (FCR) were calculated.
On the 65th day of the feeding period, the test sheep were transferred to single metabolic cages for metabolic test. Left over feed and total faecal samples were collected before the morning feeding from each animal from day 68 to 74 of the feeding period. Samples of approximately 10% of the total faeces were taken and to them were added 10% H 2 SO 4 . At the end of the metabolic test period, the samples were Ruminal uid samples were obtained from the rumen immediately after the sheep were slaughtered.
Ruminal pH was measured using a portable type pH meter (S20K,METTLER TOLEDO Switzerland) during rumen content collection. The samples were then ltered through four layers of cheesecloth and the extracts were collected into 50 mL centrifuge tubes and then centrifuged at 10,000 × g for 15 min at 4 °C. The supernatants were collected and subsequently stored at − 80 °C until analyses for ammonia nitrogen Page 7/21 (NH 3 -N) and volatile fatty acids (VFA) concentrations. The NH 3 -N concentration was determined by spectrophotometer colorimetry, and the VFA concentration was measured by gas chromatography.

Statistical analysis
Data were analysed by analysis of variance, using the General Linear Model procedure of SPSS 23.0 (SPSS Inc., Chicago, IL, USA). The means of each trait were compared by Tukey multiple comparisons and presented with the standard error of the mean. Differences were considered statistically signi cant if P ≤ 0.05.

Growth performance
The effect of dietary TGR on growth performance in Doper x Small Tail Han sheep is presented in Table 2.
The growth performance of sheep was not improved by dietary TGR supplementation. When 15% TGR was added, the average daily gain decreased signi cantly (P < 0.05), and the feed conversion ratio increased signi cantly (P < 0.05). There was no signi cant difference in the average daily feed intake between the sheep in all dietary groups (P > 0.05). Apparent nutrient digestibility As shown in Table 3, dietary TGR had no signi cant effect on the apparent digestibility of CP and ADF (P > 0.05). However, 10% TGR signi cantly increased the apparent digestibility of OM (P < 0.05) while 15% TGR signi cantly increased the apparent digestibility of EE and OM (P < 0.05), and signi cantly decreased that of NDF (P < 0.05).

Blood biochemical parameters
The effect of TGR on the blood biochemical parameters of sheep is summarized in Tables 4-7. On day 15, dietary supplementation of 10% TGR and 15% TGR signi cantly increased the concentrations of HDL-C in serum (P < 0.05), and had no signi cant effect on other biochemical parameters (P > 0.05). However, the concentrations of GLU, BUN, TG, TP, and GLB decreased, and the concentrations of ALB, IgG, and IgM increased with an increase in TGR addition (P > 0.05).    Dietary TGR supplementation did not affect the blood biochemical parameters of sheep on day 45 of the feeding period (P > 0.05). However, the concentrations of BUN and TP decreased, and the concentrations of ALB, IgA, and IgG increased with a corresponding increase in TGR addition (P > 0.05).
Compared with the control group, dietary supplementation of 10% TGR and 15% TGR signi cantly increased the concentrations of IgA in serum (P < 0.05) on day 60 of the feeding period. When 15% TGR was added, serum HDL-C and IgG concentrations increased signi cantly (P < 0.05). The concentrations of IgM in the 15% TGR group were signi cantly higher than those in the control group (P < 0.05). There was no signi cant difference in other biochemical parameters (P > 0.05).

Rumen Fermentation Parameters
As shown in Table 8, TGR at 10% had no signi cant effect on rumen pH, NH 3 -N, TVFA, acetic acid, propionic acid, and butyric acid concentrations as well as the acetic acid: propionic acid ratio in the rumen (P > 0.05). However, TGR at 15% signi cantly decreased the contents of acetic acid, propionic acid, butyric acid, and TVFA (P < 0.05), and signi cantly increased the pH, NH 3 -N concentration, and the acetic acid: propionic acid ratio (P < 0.05) in the rumen.

Discussion
Chinese herbal medicines are rich in nutrients and biologically active substances (Hossain et al., 2012). In addition to disease prevention, treatment, and enhancing the body's immunity, they can also improve feed conversion e ciency and promote animal growth and development (Qiao et al., 2016). Zhong et al. (2012) found that the addition of Astragalus polysaccharides to the diet increased the daily feed intake of lambs. Also, Qiao et al. (2016) found that the addition of Chinese herbal medicine increased the average daily gain, nal body weight and feed conversion e ciency of Holstein dairy cows. The results of an experiment on the various levels of a Chinese herbal medicine complex on the growth performance of simulated Taiwan country chickens showed that the complex at 0.3% increased both average daily weight gain and feed conversion ratio . In contrast to the above experiments, the results of the present experiment indicated that dietary supplementation of 10% TGR did not affect the growth performance of sheep. However, 15% TGR supplementation decreased the average daily gain and increased the feed conversion ratio of sheep. This may have been related to the effect of puerarin, an active ingredient in Tongmai Granule, on weight loss and regulation of fat metabolism . However, its related mechanism needs further study.
Digestibility is a major indicator of the absorption and utilization of feed nutrients in animals, and is in uenced by factors such as feed quality and animal feeding (Lin et al., 2006;Eiras et al., 2014). At present, there is little research on the effect of TGR on the apparent digestibility in ruminants. However, studies have shown that Chinese herbal medicine has the effect of enhancing animal body metabolism and improving nutrient digestibility and utilization. For example, the apparent digestibility of organic matter, crude protein and acid detergent ber increased when heat stressed beef cattle diet was supplemented with a traditional Chinese medicine prescription (Song et al., 2014). In the present study, the digestibility of dietary CP, ADF and NDF decreased with an increase in TGR addition. Conversely, the digestibility of NDF in the 15% TGR group was signi cantly lower than that in the control group. This result is inconsistent with that of Kim et al. (2002) who reported that the addition of dried Wormwood (Artemisia sp.; a traditional Chinese medicine) increased the feed intake and apparent digestibility of dry matter, total digestible nutrients, crude protein and crude bre in sheep. The decreased digestibility in the present study may be due to the fact that Tongmai granules contain more brous substances. In this experiment, the addition of TGR increased the apparent digestibility of organic matter and crude fat. This agrees with ndings by Qiao et al. (2012) who found that the addition of Fructus Ligustri Lucidi extract to sheep diet increased the digestibility of dry matter and organic matter and had no effect on CP, ADF, and NDF digestibility in sheep. This suggests that TGR can stimulate rumen degradation of non-structural carbohydrates in sheep.
Blood biochemical parameters usually re ect the health of an animal. These parameters are vital indicators of the physiological and nutritional status of animals (Alagawany and EI-Hack, 2015). Blood glucose level is a direct response of animals' body to sugar absorption, transport, and metabolic homeostasis. In the current study, dietary inclusion of TGR had no signi cant effect on serum TC, TG and GLU concentrations. The results indicate that TGR supplementation has no adverse effects on the heart and liver, as well as the occurrence of hyperlipidemia in sheep, because TC and TG concentrations in the blood are strongly associated with cardiovascular diseases (Chanjula and Cherdthong, 2018). However, the serum GLU concentrations had a downward trend in the present study, which may be related to the activity of Pueraria, the main medicinal ingredient in TGR, in lowering blood glucose (Xie and Du, 2011).
The levels of serum LDL-C and HDL-C are important indicators that re ect the lipometabolic status in animals. HDL-C has a protective effect on blood vessels, which is generally considered to be good cholesterol and resistant to atherosclerosis, whereas LDL-C is prone to arteriosclerosis when it appears at high levels (Chen et al., 2016). The results of this study indicate that there was no signi cant difference in serum LDL-C content from day 15 to day 60 of the experimental period. On day 15, the 10% and 15% TGR groups signi cantly increased the HDL-C content, and on day 60, the 15% TGR group signi cantly increased the HDL-C content, suggesting that dietary TGR addition could improve the cardiovascular system of sheep. This may be due to TGR's ability to activate blood circulation and treat diseases such as coronary heart disease and atherosclerosis.
BUN Ruminal pH is an important indicator that re ects the level of rumen fermentation, and mainly affected by factors such as the type of diet and the amount of saliva secreted (Anantasook et al., 2013). Low ruminal pH has been shown to have a negative effect on bre digestion and attachment of bacteria to plant cell walls (Cheng et al., 1984). In the present study, ruminal pH was signi cantly increased with the addition of 15% TGR and ranged from 5.83 to 6.31. These values were optimal for rumen fermentation (Dalley et al., 1997). The increase of pH may be due to the decrease in rumen TVFA. This indicates that dietary addition of TGR has the potential to regulate the e ciency of nutrient digestion and the rumen fermentation pattern.  (Owens and Bergen, 1983). In the current study, the rumen NH 3 -N concentration in each group was within the normal range, which can meet the needs of rumen microbial growth. However, dietary inclusion of 15% TGR signi cantly increased the concentration of ruminal NH 3 -N. These ndings indicate that supplementation of high levels of TGR may result in an increased NH 3 -N concentration, which in turn may affect microbial fermentation of feed in the rumen.
Volatile fatty acids are the main products of carbohydrate fermentation by various microorganisms in the rumen. They play the role of maintaining the rumen environment and providing energy for the animals' body. In the present study, dietary inclusion of 10% TGR did not affect the concentration of TVFA but they had an increasing tendency. It shows that adding a suitable amount of TGR can help rumen microorganisms to produce more VFA, which can provide more energy Declarations