# Estimating ruminal crude protein degradation from beef cattle feedstuff

## Abstract

We estimated ruminal crude protein degradation of twelve feedstuffs commonly used in China using in vitro and in vivo methods. The in vivo net protein utilization (NPU) levels of corn, sorghum, barley, wheat, Chinese wild rye grass, corn stalk, rice straw, soybean straw, soybean meal, distillers’ dried grains with solubles (DDGS), Brewers’ spent grains, and sunflower meal were 52.57, 49.68, 65.38, 72.58, 82.41, 72.26, 68.57, 76.95, 54.75, 56.27, 29.03 and 41.88%, respectively. The linear regression between NH3-N incorporated into microbial proteins and gas production after incubation (6, 12, and 24 h) was significant (r = 0.9948 and P < 0.001, r = 0.9874 and P < 0.01, and r = 0.9912 and P < 0.01, respectively). Based on the linear regression equations, we estimated in vitro protein degradability (IVPD) and generated the regression equations between IVPD and NPU. The linear regression equations between IVPD and NPU after 6 h incubation in the energy, protein, and roughage feed groups were Y = 0.5633X + 33.20 (R2 = 0.8517, P < 0.05), Y = 0.8482X+ 34.81 (R2 = 0.8650, P < 0.05), and Y = 1.6295X − 17.70 (R2 = 0.909, P < 0.05), respectively. The in vitro gas production method is useful for the determination of protein degradation in feedstuffs.

## Introduction

Protein supply is a key factor that determines the economic success of the beef cattle industry. Due to lack of information on the protein quality of beef cattle feedstuffs in China, the beef cattle industry relies on accurate analyses. The degradation rate of feedstuff protein in rumen is an essential characteristic for the determination of the protein value of feeds1,2,3 and an important feed characteristic in nutritional models4,5,6. Particularly, cereal grains such as maize, sorghum, barely and wheat which are rich in starch have long become an important component of the diet in high-producing beef cattle in China, and they also are a valuable source of proteins, while chemical structure and protein matrix leads to large variations in the protein degradation capabilities of the cereal grains7. Similarly, chinese wild rye grass, corn stalk, rice straw and soybean straw were important forages commonly used for beef cattle production in China especially districts of with less availability of feed resources due to their advantages of large-scale production and high nutritional value8,9,10. Besides, soybean meal, DDGS, brewers’ spent grains and sunflower meal are special source of protein for beef cattle feeds in China due to economic and environmental concerns, and these by-products usually have high protein contents and can provide competitive alternatives to more traditional protein sources11.

Amongst all methods used to evaluate protein quality in ruminant feedstuffs, in vivo estimates are the most reliable. Even though the in vivo method is expensive, time consuming, and laborious, it provides accurate information on the animal response to feedstuffs. In addition, data from in vivo studies are used to validate the accuracy of in situ and in vitro methodologies12,13. The in vitro gas production technique14,15 has been use to evaluate rumen CP degradability of feeds via ammonia-N and gas production measurements recorded by incubating feedstuffs in buffered rumen fluid. By improving this technique (e.g., through method innovation, instrument upgrades, and data handling), the in vitro gas production technique has been widely used to predict digestibility of ruminant feedstuffs16,17,18,19. Even though in vivo and in vitro methods have been widely researched, there are few comparisons between in vitro and in vivo measurements of feedstuff protein degradation.

In order to make the evaluation of relationship between IVPD and NPU more accurate, we choosed three types (forages, energy feed and protein feed) with large range of the content of protein. And the objectives of this study were to (1) determine the chemical composition of twelve common beef cattle feedstuffs commonly used in China and (2) predict protein digestibility by using in vitro gas production technique and in vivo data derived from digestion trials in an attempt to assess the accuracy of the in vitro gas production technique.

## Results

The chemical composition of twelve feedstuffs is shown in Table 1. Starch content in corn, sorghum, barley, and wheat was 66.69%, 52.40%, 55.14%, and 59.46%, respectively. Meals (soybean meal and sunflower meal) and by-products (DDGS and Brewers’ spent grain) contained more CP than cereals and roughages, less NDF and ADF than roughages, and less starch than cereals. Among the protein feed group, CP concentration was higher in soybean meal and sunflower meal than in DDGS and Brewers’ spent grain. NDF, ADF, and starch were higher in Brewers’ spent grain than in soybean meal, sunflower meal, and DDGS. Sunflower meal had the highest CF content among the protein feed group. The roughage feed group contained high levels of NDF and ADF. Soybean straw had higher CP than Chinese wild rye grass, corn stalk, and rice straw.

Protein intake, fecal protein, urinary protein, and NPU of the twelve feedstuffs are shown in Table 2. Protein intake consist of proteins provided by mixed feeds (energy feed group and protein feed group) or single feeds (roughage feed group). The type of feedstuff affected the amount of protein intake. Higher protein intake was observed in the protein feed group (1.9069 kg/d for soybean meal, 1.2455 kg/d for DDGS, 1.2198 kg/d for Brewers’ spent grains, and 0.9115 kg/d for sunflower meal), and lower protein intake was observed in the roughage feed group (0.3976 kg/d for Chinese wild rye grass, 0.6919 kg/d for soybean straw, 0.4322 kg/d for corn stalk, and 0.4779 kg/d for rice straw). Fecal protein was the highest for sorghum (0.3537 kg/d) and lowest for Chinese wild rye grass (0.1508 kg/d). Urinary protein was the highest for soybean meal (141.86 g/d) and lowest for corn stalk (18.19 g/d). NPU varied among the feedstuffs. In the protein feed group, the highest and lowest NPU values were obtained in soybean meal (82.41%) and Brewers’ spent grain (68.37%), respectively. Among the cereal grains, NPU was higher for wheat (72.58%) and barley (65.38%) than for corn (52.57%) and sorghum (49.68%). For roughages, soybean straw had the highest NPU (56.27%).

Figure 1 shows a linear regression between NH3-N incorporated into microbial proteins and gas production after 6, 12, and 24 h of incubation. The linear regression was significant, indicating that gas production from feedstuffs is an accurate estimate of incorporated ammonia N. The highest correlation coefficient was observed at 6 h, Y = 0.1331X + 0.133, R2 = 0.9896 (P < 0.05). The linear regression equations at 12 and 24 h were Y = 0.1502X – 0.7054 (R2 = 0.9750, P < 0.05) and Y = 0.0718X + 0.2872 (R2 = 0.9825, P < 0.05), respectively.

Based on the linear regression equations, estimates of IVPD were calculated (Table 3). Protein degradation during the first 6 h of in vitro incubation varied from 68.43% (wheat) to 25.65% (sorghum). After 12 h of in vitro incubation, CP degradation ranged from 80.12% (wheat) to 36.6% (corn stalk). IVPD after 24 h of in vitro incubation was the highest for wheat (87.56%) and the lowest for corn stalk (44.42%).

The relationship between IVPD and NPU after 6, 12, and 24 h of incubation are presented in Fig. 2. Regression analyses demonstrated that the linear regression equations in the three feed groups (energy feed group, protein feed group, and roughage feed group) were significant (P < 0.05) after 6 h of incubation (R2 of 0.8517, 0.8650, and 0.8909, respectively). However, increased incubation time decreased the R2 value. At 12 h, the R2 values of IVPD and NPU in the energy feed group and roughage feed group were 0.8316 and 0.8435, respectively. Additionally, there was no correlation between IVPD and NPU in the protein feed group after 12 h of incubation. Similarly, no relationship was observed between IVPD and NPU in the energy feed group, protein feed group, and roughage feed group at 24 h. When IVPD and NPU were analyzed together, the linear regression equations at 6, 12, and 24 h of incubation revealed no correlation. The equations were described by Y = 0.9848X + 16.25 with R2 = 0.5171 and P > 0.05 (6 h of incubation), Y = 0.7043X+ 20.55 with R2 = 0.3704 and P > 0.05 (12 h of incubation), and Y = 0.5958X + 19.11 with R2 = 0.2695 and P > 0.05 (24 h of incubation).

## Discussion

The approach we used in this study aimed to analyze the correlation between gas production and NH3-N incorporation into microbial proteins as an indicator of protein degradation. Gas production can be used to evaluate metabolisable energy content of feedstuffs15, which in turn determines the synthesis of rumen microbial protein. Dietary protein is eventually degraded into ammonia N in the rumen via enzymatic activity of ruminal microorganisms. However, ammonia from protein degradation and microbial catabolism, which occur simultaneously in the rumen, make it difficult to identify IVPD of feedstuffs from ammonia release. To eliminate the effect of ruminal microbial protein synthesis during fermentation, we used a method to eliminate the effect of ruminal microbial protein synthesis during fermentation. Raab et al. (1983) reported that the amount of NH3-N released when no fermentable carbohydrates are available and consequently no bacterial protein synthesis can take place can be represented by the intercept of the linear regression between NH3-N concentration and gas production. The results can be corrected by the difference between this intercept and NH3-N content in the blank (rumen fluid without substrate14. In our earlier studies, we attempted to determine protein degradability using techniques described by Broderick (1987) and Raab et al.14,38. However, the methods were complicated and cumbersome and had limitations on the batch culture used. In this study, we classified ruminal ammonia N into (1) incorporated ammonia N, which is used for microbial degradation of protein, and (2) free ammonia N. There was a high correlation between ammonia N incorporated into microbial proteins and gas production, suggesting that gas production of feedstuffs is an accurate estimate of incorporated ammonia N. Therefore, we hypothesize that free ammonia N and incorporated ammonia N represent the N obtained from feeds.

The in vivo method is the gold standard for determining ruminal CP degradability. The weak correlation between the in vivo and in vitro methods is due to variations in the latter. The quality of rumen fluid has a great impact on in vitro gas production technology. Our previous studies found that different sources of rumen fluid and sampling time had no effect on IVPD. However, in this study, feedstuffs were not incubated in defaunated rumen fluid. Ignoring the presence of protozoa in rumen fluid may influence in vitro measurements of feedstuff. Lorenz et al. (2011) reported that when soybean meal was incubated as a protein source, the removal of protozoa in vitro by centrifugation minimized the recirculation of bacterial protein by protozoa40. However, Rymer et al. (2005) observed that rumen fluid continuously flushed with CO2 during incubation may adversely affect gas production by excluding adherent cellulolytic bacteria from the rumen inoculum45. Furthermore, protein degradability has been generally estimated at the end-point of incubation, which provides inadequate information of the degradation pattern over time. To compare the ruminal degradability of feedstuffs, we need to obtain kinetic parameters, which could be offered by modified gas production instruments. Pellikaan et al. (2011) and Elberg et al. (2018) modified the in-vitro gas production instrument and reported total gas production and a precise description of in-vitro gas kinetics44,46. There is a need to generate a standardized approach of the in vitro gas production technique.

In summary, there were significant correlation between IVPD and NPU in the same feed type, and in vitro gas production method could be used for rapid determination of protein digestibility. More samples are needed for building more accurate relationship between IVPD and NPU in different feed type prediction for further study.

## Methods

### Feed samples

Twelve feedstuffs were obtained from Beijing (Hebei Province, P. R. China), air-dried, and ground in a hammer mill to pass through a 2-mm sieve for chemical analysis. In the in vivo method, the feedstuffs were divided into three groups: (1) energy feed group (cereals): corn, sorghum, barley, and wheat; (2) roughage feed group: Chinese wild rye grass, corn stalk, rice straw, and soybean straw; (3) protein feed group (meal and by-products): soybean meal, distillers’ dried grains with soluble (DDGS), Brewers’ spent grains, and sunflower meal.

### Chemical analysis

Feeds and animal feces were analyzed for dry matter (DM), ash, and ether extract (EE) according to AOAC (2012) methods. Crude protein (CP), acid detergent insoluble N (ADIN), and neutral detergent insoluble N (NDIN) were determined by the Dumas method using an N Analyzer (Rapid N III, Elementar, Germany) and a nitrogen-to-protein conversion factor of 6.25. Neutral detergent fiber (NDF), acid detergent fiber (ADF), and acid detergent lignin (ADL) were analyzed using an A220 Fiber Analyzer (ANKOM Technology Corp., Macedon, NY, USA). NDF was assayed using alpha-amylase. Both NDF and ADF contained no residual ash. Urinary N was analyzed according to AOAC (2012) methods.

### In-vitro Gas production method

Rumen fluid was collected from three Angus steers (approximately 320 kg body weight) fitted with permanent rumen cannulas. The animals were fed a total mixed ration which consisted of 40% steam-flaked corn, 40% corn stalk silage, 4.5% cottonseed meal, and 13.5% brewer’s grains (DM basis) and 0.5% salt and 0.5% premix (2.4 g/kg Mg, 7.6 g/kg K, 12.0 g/kg Fe, 1.0 g/kg Cu, 200 mg/kg Mn, 650 mg/kg Zn, 2 mg/kg Se, 22 mg/kg I, 9 mg/kg Co, 121,000 IU/kg vitamin A, 37,400 IU/kg vitamin D, and 55 IU/kg vitamin E) twice a day with ad libitum access to water. Animal care and use were approved and conducted according to standards established by the College of Animal Science and Technology, CAU, Beijing, P. R. China (permit number DK1402006). In vitro incubation was carried out according to the procedure reported by Menke et al. (1979). Samples of rumen fluid were collected prior to the morning feeding, passed through four layers of cheesecloth into a pre-warmed vacuum bottle, and transported immediately to the laboratory of Beef Cattle Research Center of China Agricultural University. The rumen fluid was mixed with buffer solution in a 1:1 (v/v) ratio (Menke et al., 1979) under a continuous flux of CO2 to maintain anaerobic conditions and maintained at 39 °C.

Five levels of substrate were prepared based on the amount of soluble starch: 0 mg DM, 40 mg DM, 80 mg DM, 120 mg DM, and 160 mg DM. An equal amount of (NH4)2SO4 (10 mg N) was added to a glass syringe. Artificial rumen culture solution (30 mL, containing 25 mg of soluble starch and 25 mg of maltose) was added and incubated at 39 °C. Gas production was measured at 6, 12, and 24 h incubation. The syringe was transferred to an ice bath to prevent further microbial activity. The fermentation mixture was centrifuged at 1,000 g for 10 min. The resulting supernatant was used for the determination of incorporated NH3-N.

The feed samples in each syringe contained 200 mg DM and <10 mg N. If the N content of the sample was >5%, the additional supplementation of corn starch would result in a DM content of 200 mg; conversely, if the N content of the feed sample was <5%, only 200 mg of this sample needed to be weighed separately. Incubations were performed in triplicate. Blank samples consisted of three syringes with artificial rumen culture solution.

### In vivo Method

Twelve Angus steers (320 ± 20 kg body weight) fitted with permanent rumen cannulas (CAU Beef Cattle Research Center, Beijing, P. R. China) were kept individually in cages. The animals had ad libitum access to water and were assigned to one of three groups (each group consisted of four steers) based on their body weight in a 4 × 4 Latin square design. An adaptation period (15 d) was followed by a sample collection period (5 d). During the collection period, fecal and urine samples were collected after the morning feeding. Animal care and use were approved and conducted according to standards established by the College of Animal Science and Technology, CAU, Beijing, P. R. China (permit number DK1402006).

The steers were fed the experimental diets at 1.9% BW on a DM basis in two equal portions at 08.00 and 16.00. To prevent the occurrence of digestive abnormalities such as rumen acidosis by feeding a single feed, the first group (energy feed group) and the third group (protein feed group) were fed a mixed diet of 4.2 kg of feed and 1.8 kg of Chinese wild rye grass; the second group (roughage feed group) was fed the single feed. To meet nutrient balance, 2% rumen buffer (NaHCO3:MgO = 2:1), 0.5% stone powder, and 0.5% salt of feed intake on a DM basis and 30 mg/kg Rumensin was added to ensure normal ruminal fermentation.

Fecal samples were collected on sampling days, and 20% of the total fecal weight was collected. Fecal samples were collected into bags containing 10% tartaric acid to prevent the loss of ammonia nitrogen (N) and stored at −20 °C. At the end of each collection period, fecal samples were pooled and weighed. The pooled fecal samples were naturally air-dried, ground to a 1-mm screen size, and stored at 0–4 °C. Urine samples were collected in urinary bags. The bag, which was designed by a research team at the China Agricultural University (CAU; Beijing; P. R. China), consists of a funnel, a catheter, and latex tubes. Urine volume was measured daily, and the urine samples were passed through a gauze. The urine samples were collected in 600 mL of 6 N HCl to prevent the loss of ammonia nitrogen (N) and stored at 4 °C.

### Statistical analysis

IVPD was estimated for each feed after each incubation period in the GP experiment via linear regression of incorporated ammonia N (y, mg) vs. gas production (x, mL) (Meng et al.). The relationship between ammonia N incorporated into microbial proteins and gas production was quantitatively reflected by the regression coefficient b. IVPD was calculated at 6, 12, and 24 h using the following equation,

$${\rm{IVPD}}=\frac{({\rm{GP}}-\mathrm{GP}\,\,\mathrm{of}\,\,{\rm{blank}})\ast b+{\rm{free}}\,{\rm{ammonia}}-{\rm{N}}}{\mathrm{total}\,\,{\rm{N}}}\ast 100 \%$$

Data obtained from the in vitro and in vivo methods were statistically analyzed using EXCEL while taking into account the net protein utilization (NPU) of the four roughage samples (Chinese wild rye grass, soybean straw, corn stalk, and rice straw) according to the following statistical model,

$${\rm{NPU}}=\frac{(\mathrm{Intake}\,\,\mathrm{CP}\,-\mathrm{Fecal}\,\,\mathrm{CP}\,-{\rm{Urine}}\,{\rm{CP}})}{(\mathrm{Intake}\,\,\mathrm{CP}\,-{\rm{Fecal}}\,{\rm{CP}})}\times \frac{(\mathrm{Intake}\,\,{\rm{CP}}-{\rm{Fecal}}\,{\rm{CP}})}{{\rm{Intake}}\,{\rm{CP}}}$$

Protein degradation data obtained from corn, sorghum, barley, wheat, soybean meal, DDGS, Brewers’ spent grain, and sunflower meal were analyzed according to the concentration of CP in Chinese wild rye grass using the follow equation,

$$\begin{array}{c}{\rm{NPU}}=\frac{({\rm{Intake}}\,{\rm{CP}}-{\rm{Fecal}}\,{\rm{CP}}-{\rm{Urine}}\,{\rm{CP}}+\mathrm{Chinese}\,\,{\rm{wildrye}}\,{\rm{grass}}\,{\rm{CP}}\times (1-{\rm{Chinese}}\,{\rm{wildrye}}\,{\rm{grass}}\,\mathrm{CP} \% ))}{({\rm{Intake}}\,{\rm{CP}}-\mathrm{Fecal}\,\,{\rm{CP}})}\\ \,\times \frac{({\rm{Intake}}\,{\rm{CP}}-{\rm{Fecal}}\,{\rm{CP}})}{\mathrm{Intake}\,\,{\rm{CP}}}\end{array}$$The regression analysis between the two methods was determined by the CORR and REG procedures of SAS(version 9.4). Duncan’s significant difference test procedure was used to determine differences among means. Significance was set at P ≤ 0.05.

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## Author information

The study was designed by Zhenming Zhou, Qingxiang Meng and Hao Wu. Sample processing was carried out by Chang Liu, Wanbao Chen and Yan Li. Data analysis was done by Chang Liu and Deyong Li. The manuscript was written by Chang Liu, assisted by all co-authors.

Correspondence to Zhenming Zhou.

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