In vitro Study of Urtica cannabina and Leymus chinensis on Rumen Microbial Fermentation and Gas Production

High-quality forage is essential for the growth and development of ruminants. Nowadays, the quantity of high-quality forage is insufficient in the ruminant industry in China (Xie et al., 2019). Some studies demonstrated that an unconventional forage with high nutritional values can substitute for ruminant feed sources (Iniguez, 2011; Porqueddu et al., 2016).
       
As an unconventional forage, Urtica cannabina is widely distributed in Xinjiang, Inner Mongolia and other places because of its strong vitality and adaptability (Zhang et al., 2014). Usually, the yield range of this plant is 3.6-7.8 t ha^-1 without chemical fertilizers, while under good environment and nutrition conditions, the dry matter yield of the natural U. cannabina can reach to 14 t ha^-1 (Zhang and Zhao, 2008). It’s stem and leaves are rich in protein, trace elements, cellulose and a variety of active substances (Zhang et al., 2020). Medically, it has been used in the treatment of diseases such as rheumatoid arthritis and prostatitis syndrome, etc. (Bourgeois et al., 2016; Carvalho et al., 2017). In recent years, U. cannabina has been used in livestock production. It has already been demonstrated by that U. cannabina is involved in improving morphological features of gastrointestine and nutrients digestibility in lamb (Jin et al., 2018). Humphries and Reynolds (2014) reported that Urtica dioica haylage (100 g/kg DM) increased milk production in the diet of lactating dairy cows instead of ryegrass silage. However, little is known about the effects of U. cannabina on rumen microorganisms, it may alter rumen microbial activity because it indeed contains many biologically active compounds. So we hypothesized that the addition of U. cannabina can increase rumen microbial activity in vitro. Therefore, the effects of U. cannabina to L. chinensis ratios on the rumen fermentation in vitro were investigated in the present study.

The study was carried out during the period of July 2017 to June 2018, at the Institute of Metabolic Manipulation of Herbivorous Animal Nutrition, College of Animal Science and Technology, Yangzhou University, China.

Animal management
 
The following experimental procedures, including animal ethics and usage, were approved by the Animal Welfare Committee which is managed by Yangzhou Veterinarians of the Agriculture Ministry of China.
       
Two-year-old, three healthy Xuhuai goats having permanent rumen fistulas with body weight (BW±SD) of 35±3.1 kg were chosen for the sampling purpose from the experimental farms of Yangzhou University. The diet offered to goats (dry matter amount about 3% BW) was mixed well with following contents including 70% alfalfa, 28% corn kernels and 2% soybean meal. This diet was fed to goats in equal amounts at 07:00 h and 19:00 h. They had free access to mineral licking blocks and fresh drinking water.
 
Culture substrate and experimental design
 
The samples of U. cannabina were collected early in July 2017 during the flowering stage and the samples of L. chinensis were bought from Caodu Co., Ltd. The forages were automatically collected and dried outside in an open environment in Xilinguole, Inner Mongolia region, China.
       
U. cannabina and L. chinensis were dried at 65°C to a constant mass in a force air oven and then ground finely with a Retsch ZM 100 Wiley mill (Retsch GmbH, Haan, Germany) to pass through 1 mm screen. These two kinds of refined grass were mixed in proportion as the in vitro culture substrates with the following five ratios: 0:100, 30:70, 50:50, 70:30 and 100:0 categorized into the five groups from A-E respectively. The ingredient and nutritive values of each group are shown in the Table 1.
 

 
In vitro gas production test
 
The rumen fluid samples were collected from different localities through the rumen fistula using a vacuum negative pressure device (self-constructed) before morning feeding. These samples were filtered into a thermos flask containing four layers of gauze. The flask was filled with CO₂ and heated at 39°C before collecting samples. The artificial saliva salt was configured according to Menke and Steingass (1988). The culture medium for in vitro culture of rumen microorganisms was prepared using artificial saliva salt with rumen fluid ratio 2:1. Then CO₂ was introduced for saturation purpose. The substrate of each group weighed 0.5000 g (±0.0500 g) accurately in a 150 mL fermentation bottle and 50 mL of artificial saliva and 25 mL of rumen fluid were added later on. Each treatment in sampling time was replicated three times. The culture bottle was connected to the 64-way microbial fermentation micro-product gas automatic recorder (AGRS-III, Beijing, China). The incubation temperature was set to 39°C and incubated for 24 h at constant temperature under anaerobic conditions.
 
Sample collections and chemical analysis
 
Gas production was recorded at 1, 3, 6, 12 and 24 h of incubation. The kinetics of gas production in vitro were calculated according to the exponential function model proposed by Ørskov and McDonald (1979). The culture medium at each time point was filtered through the four layers of gauze and dispensed in 5 mL centrifuge tubes. The pH value of the rumen fluid was measured immediately using an electrode pH meter (pHS-3C, Shanghai, China). The rumen fluid was immediately stored for analysis of ammonia nitrogen, bacterial and protozoal proteins at -20°C. The ammonia nitrogen concentration was measured by the phenol-sodium hypochlorite colorimetric method (Weatherbum, 1967). The bacterial and protozoal protein concentrations in rumen fluid were determined according to the method as described by Hall et al., (2001).
 
Statistical analysis
 
Statistical analysis was performed by SPSS software (version 16.0, SPSS Inc., Chicago, USA) using ANOVA procedure with posthoc Tukey’s multiple comparison test. The fixed effect was treatment and sampling time, whereas random effect in the ANOVA model was repetition. Significance was declared at p-value <0.05.

Effects on the kinetics of gas production in vitro
 
In our study, gas production of different groups increased with fermentation time (Fig 1). Whereas gas production was increased in the groups A and C at 24 h (P<0.05). The data in Table 2 show that the gas produced from soluble fraction (a) was higher (P<0.05) in groups D and E (1.83 and 2.10 mL/g DM), whereas the gas produced from insoluble but fermentable fraction (b) produced was lower (P<0.05) in group E (41.01 mL/g DM). The highest value was registered for group A (62.76 mL/g DM). The rate of gas production (c) was higher (P<0.05) in group A (0.201 mL/g DM), whereas the lowest value for this fraction was observed in group E (0.029 mL/g DM). The reason might be that L. chinensis was a high-quality forage with a higher NDF content (Table 1) and the rapid degradation of soluble carbohydrates increased the gas production rate in the early stages (Cone and van Gelder, 1999). The rate of gas production (c) in group C was higher than groups B, D and E, This could be due to the reason that the proper addition of U. cannabina can promote the balance of energy and protein in the substrate, which further promote the reproduction of rumen microorganisms (Tang et al., 2005) and thus increase the gas production rate.




       
Similarly, the potential gas production (a + b) was higher for group A than group E (P<0.05). Khazaal et al., (1993) pointed out that feed intake is mainly explained by the rate of gas production (c), which affects the pass rate of feed through the rumen and the potential gas production (a + b) is related to the degradability of the feed. Therefore the higher values of potential gas production in groups A and C might indicate a better nutrient availability for rumen microorganisms (Nsahlai et al., 1994).
 
Effects on the fermentation parameters in vitro
 
In Fig 2(a), the pH value in all groups was within the normal range during the 24 h fermentation. However, the pH values at 1 h, 3 h and 6 h were increased in groups A and C and at 24 h were observed highest in group C (P<0.05). The reason might be that the crude protein of U. cannabina was higher and the digestible protein was up to 70%. A certain addition of U. cannabina can increase the digestible protein in the fermentation substrate and a large amount of protein degradation promotes the increase of ammonia nitrogen concentration and finally lead to the increased the pH value. It was found that addition of U. cannabina (100 mg/g) can prevent the effects of rumen acidosis effectively, by increasing the pH value of rumen fluid to 30% in vitro (Kleim et al., 2017). Similarly, Arroquy et al., (2004) reported that as the ratio of rumen degradable protein (RDP) increased, the pH value was also increased linearly within the normal range.
 

       
In Fig 2(b), the ammonia nitrogen concentration in all groups was all within the normal range during the 24 h fermentation. However, the ammonia nitrogen in groups D and E was higher at 3 h, 6 h, 12 h and 24 h. The concentration of ammonia nitrogen is determined by the rumen microbial decomposition of proteins and the rate at which ammonia is used to synthesize microbial proteins. Groups D and E contained higher CP and NFCs contents (Table 1), which lead to higher ammonia nitrogen synthesis. At 24 h, the ammonia nitrogen concentration in group C decreased significantly (P<0.05), due to the increased ratio of NDF and NFCs and decreased lignin concentration (Table 1) leading to suitable conditions for microbial growth and protein synthesis (Shen et al., 2017).
 
Effects on the contents of bacterial and protozoal proteins in vitro
 
As it can be seen in Fig 2 (c,d), the concentration of bacteria and protozoa protein was higher in groups A and C at 1 h, 3 h and 6 h. The reason might be that L. chinensis has high-quality forage contents which are more conducive for microbial growth. Stern et al., (1994) reported that energy and nitrogen balance and simultaneous release of them in the diet determine the growth of rumen microbes, which could change the synthesis efficiency of MCP (bacteria and protozoa protein). In the beginning, the supply of energy and nutrients in the substrate was sufficient to meet the needs of the microorganisms. Fermentation produced a large amount of energy and precursors required for the synthesis of self-proteins by microorganisms such as ammonia, volatile fatty acids and ATP (Van Soest, 1994). As time went on, the gradual consumption of nutrients and energy and the proliferation of bacteria had reached a stationary stage. In addition, the phagocytosis of protozoa was enhanced, the production of bacterial proteins in the rumen began to decrease and it might also be caused by bacteriolysis (Sherwood et al., 2012).
       
The comparison among the groups showed that with the increased ratio of U. cannabina and the decreased ratio of L. chinensis, the bacterial and protozoal protein concentrations were decreased. However, this concentration was observed highest in group C at 24 h. This could be due to the certain complementary interaction effect of U. cannabina and L. chinensis nutrients (Haddad, 2000), which promotes the balance of energy and protein in the substrate by influencing the overall increased microbial protein synthesis. It can also be inferred that with the passage of culture time the protozoal protein concentration fluctuated greatly in the groups D and E. It is speculated that some pharmacological effects of U. cannabina changes the rumen microbiota (Yang et al., 2013).

Based on the results of current experiment, the 50:50 ratio of Urtica cannabina and Leymus chinensis increased the gas production in goat rumen microorganisms in vitro. Besides, this group increased the pH value and microbial protein contents; and decreased the ammonia nitrogen concentration in culture solution. Therefore, the 50:50 ratio of Urtica cannabina and Leymus chinensis could potentially be favorable for fermentation and gas production of rumen microorganisms as shown in the in vitro studies. 

We are thankful to the National 13^th Five-Year Plan Key Research and Development Program (2018YFD0502100), National Natural Science Foundation (31402119) and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), P.R. China for supporting the current study.