Influence of dietary crude protein content on fattening performance and nitrogen excretion of Holstein steers

Abstract The objective was to investigate the influence of crude protein (CP) content in a fattening diet on feed intake, body weight gain, nitrogen excretion, and carcass traits in Holstein steers. Steers (initial body weight 241 ± 26 kg) consumed feed with the following CP content: (a) 17.7% during the early period (from 7 to 10 months of age) and 13.9% during the late period (from 11 to 18 months of age) (HIGH, n = 3), and (b) 16.2% during the early period and 12.2% during the late period (LOW, n = 4). The CP intake was lower in the LOW than the HIGH group. Urinary and total nitrogen excretion in the late period tended to be lower (p < .10) in the LOW than the HIGH group. However, growth performance and carcass traits were not affected by dietary CP content. Free histidine and total amino acid contents in the longissimus thoracis muscle tended to be higher (p < .10) in the HIGH than the LOW group, however, the CP contents were not affected by dietary CP content. The results of this experiment suggest that decreasing dietary CP to 16% (early period) or 12% (late period) of dry matter would reduce nitrogen excretion from Holstein fattening farms without affecting productivity.


| INTRODUC TI ON
Reducing the environmental impact of the livestock industry has become an important issue. In particular, the reduction in greenhouse gas (GHG) emissions is required worldwide. Nitrous oxide is a GHG that is generated from livestock manure (Petersen, 2018;Powers & Capelari, 2016). The emission of nitrous oxide is reduced by decreasing the amount of urinary nitrogen (N) excretion associated with dietary crude protein (CP) level (Bao, Zhou, & Zhao, 2018). Therefore, decreasing the dietary CP content to the appropriate level is important for minimizing environmental impacts such as GHG and ammonia emissions without reducing productivity.
In contrast, Holstein steers, the major fattening cattle in Japan, have a higher body growth rate and higher CP requirements than other breeds (National Agriculture and Food Research Organization, 2009). In Japan, the CP content of the diet for fattening cattle is generally higher than the required amount, and Holstein steers are fed a fattening diet with relatively high CP content (National Agriculture and Food Research Organization, 2009). From the relationships of N intake and urinary N excretion of fattening Holstein steers based on reports (Choumei, Terada, & Hirooka, 2006;Terada, Abe, Nishida, & Shibata, 1998), it is possible that decreasing the dietary CP content can reduce N excretion, especially urinary N excretion on Japanese cattle fattening farms. However, no studies have examined the effects of decreasing dietary CP content on fattening performance and N excretion of Holstein steers in recent fattening programs in Japan.
The object of this study was to investigate the influence of CP content in a fattening diet on the feed intake, body weight gain, carcass traits, and N excretion of Holstein steers.

| Cattle and feeding management
Seven Holstein steers (average age: 7 months) were assigned to a high-CP (HIGH, average body weight (BW) 230 kg, n = 3) or a low-CP (LOW, average BW 249 kg, n = 4) concentrate feeding group. Steers were kept on average from 7 to 18 months of age, including an early period (from 7 to 10 months of age) and a late period (from 11 to 18 months of age). Steers were typically group fed in pens at 7 and 8 months of age and fed individually from 9 to 18 months of age. BW was measured weekly before morning feeding. At an average of 18 months of age, steers were slaughtered in a commercial slaughter facility. Carcass traits were evaluated according to standard procedures of the Japan Meat Grading Association. All animal treatments were approved by the Animal Care and Use Committee of the Institute of Livestock and Grassland Science, NARO.
The chemical composition and ingredients of the diet are presented in Table 1. Steers were fed the concentrate mix and hay according to the feeding program shown in

| Blood and muscle samples
Blood samples for measuring urea N were taken from the jugular vein into a heparinized tube every month before morning feeding from 6 months of age before the experiment to 18 months of age. Plasma was separated by centrifugation at 1,760 g for 20 min at 4°C and stored at −30°C until analyzed. The plasma urea N concentration was determined by the urease-GLDH method (Sanritsu Co., Ltd, Chiba, Japan).
The longissimus thoracis (LT) muscle around the 7th rib was taken from the carcass and reserved in a refrigerator at 4°C until 14 days after slaughter. The muscle samples were analyzed for moisture, crude fat, and CP contents. Free amino acids (AA) in muscles were determined using an automatic AA analyzer (JLC-500/V; JEOL Ltd., Tokyo, Japan) after deproteinization by the addition of 5% perchloric acid.

| Nitrogen balance trials
After acclimatizing the steers to the facilities and feeds in advance, steers moved to individual pens or stanchion stall, and then the N balance trials were conducted. To measure the N excretion and retention at 9 months of age in the early period, steers were kept for 7 days in individual pens, including the last 3 days sampling period.
Steers were given up to 8.5 kg of a concentrate mix and up to 2 kg of timothy hay daily. Water was available ad libitum. At 13 months of age in the late period, steers were kept for 11 days in a stanchion stall, including the last 3 days sampling period. Steers were given up Therefore, urinary N excretion was estimated as follows.
where BW is the body weight, kg; Cr is the urinary creatinine concentration, mg/L; and UN is the urinary N concentration, g/L.
The feed and fecal acid-insoluble ash (AIA) concentration was analyzed by the 4 N HCl method (TeradaIwasaki, Tano, & Haryu, 1979). The fecal weight was estimated using the AIA concentration as an internal marker. Therefore, the fecal N excretion was estimated as follows.
where AI is the AIA intake, g; FA is the fecal AIA concentration, %; and FN is the fecal N concentration, %.

| Statistical analysis
Differences in all data were examined by ANOVA with the dietary treatment as a factor, supported by SAS Add-In 7.1 for Microsoft Office (SAS Institute Japan Ltd., Tokyo, Japan). Values of p < .05 and p < .10 were considered to indicate significance and tendency, respectively.

| RE SULTS
There were significant differences (p < .01) in CP intake among treatments, and the dietary CP content of consumed feed was 17.7% for the HIGH group or 16.2% for the LOW group in the early period, and 13.9% for the HIGH group or 12.2% for the LOW group in the late period (Table 3). In the early period, the dry matter intake (DMI) and TDN were significantly higher (p < .01) by 0.1 kg/day in the LOW group, but the dietary CP content did not affect the DMI and TDN in the late and total period (Table 3). There were no feed differences in BW, average daily gain, and gain-to-feed ratio ( Table 3).
The plasma urea N concentration tended to be low or significantly lower in the LOW group from 8 to 18 months of age ( Figure 1).
Regarding the N balance trial, there was a difference only in the N intake in the early period (Table 4), but in the late period, decreasing the dietary CP content significantly reduced the N intake (p < .01) and tended to reduce (p < .10) urinary N excretion and total N excretion (Table 5).
Data related to productivity, such as carcass weight, rib-eye area, rib thickness, subcutaneous fat thickness, and yield score, were not affected by dietary CP content (Table 6). The dietary CP content did not change the meat quality, such as the beef marbling standard, beef color standard, beef fat color standard, beef meat brightness, beef meat firmness (SHIMARI), beef meat texture (KIME), beef fat brightness, and quality (Table 6).
The moisture, crude fat, and CP contents of the LT muscle around the 7th cross section were not affected by dietary CP content (Table 6). Although there were no statistical differences in many free AA contents of the LT muscle, the free histidine, total AA, and total non-essential AA contents tended to be higher (p < .10) in the HIGH group than in the LOW group (Table 6).   N concentration is highly related to N excretion (Kohn, Dinneen, & Russek-Cohen, 2005), and it was estimated that N excretion during the early period would also decrease in the LOW group. Therefore, it is expected that N excretion can be reduced by decreasing the dietary CP content to an appropriate value throughout the fattening period.

| D ISCUSS I ON
Considering the results of the N balance trial and the CP intake during the feeding period, the total N excretion during the fattening period is about 10% lower in the LOW group than in the HIGH group.
In the GHG inventory of Japan, nitrous oxide emissions are calculated by multiplying the amount of N contained in manure by the emission factor for each type of manure treatment method (Ministry of the Environment, Japan, and Greenhouse Gas Inventory Office of Japan (GIO), Center for Global Environmental Research (CGER), National Institute for Environmental Studies (NIES), 2020). If the manure treatment method is the same, it is expected that nitrous oxide emissions will be reduced by about 10% in the LOW group as compared to the HIGH group, as well as total N excretion.
The free AA content in meat is focused on as taste components of meat and influenced by storage conditions (Watanabe, Ueda, & Higuchi, 2004). Moreover, Koutsidis et al. (2008) reported that the difference of free AA content in meat between a beef breed and a dairy breed was small, but the effect of feed was significant. Iwamoto, Iwaki, and Oka (2010) reported that the dietary CP content in the early fattening period did not affect the free AA content in the LT muscle. On the other hand, the effect of differences in dietary CP content throughout the fattening period on beef free AA content has not been reported in the past. In the present study, most free AA contents of the LT muscle were not affected by the dietary CP content, but free histidine, total AA, and total non-essential AA tended to be lower in the LOW group than in the HIGH group. Ueda et al. (2007) reported that most free AA concentrations in meat were negatively correlated with fat content. In this study, the average crude fat content in meat was 13% in the HIGH group and 14.9% in the LOW group. The protein content in meat was 20.1% in the HIGH group and 19.9% in the LOW group. Ueda et al. (2007) reported a negative correlation between fat content and free AA content even in low-fat meat (<23%) with a relatively constant protein content. Therefore, in the present study, dietary CP content affected the free total AA content in meat, but fat content in meat may also have affected. The free AA are related to the taste of meat (Watanabe et al., 2004), and histidine has a bitter taste (Kawai, Sekine-Hayakawa, Okiyama, & Ninomiya, 2012). However, most AA contents, including histidine, are not expected to exceed the taste threshold (Schiffman, Sennewald, & Gagnon, 1981). Therefore, it is not clear whether the difference in AA of this experiment had a significant effect on the actual taste. In terms of productivity, carcass traits, such as carcass weight and meat quality, were similar with both treatments. Therefore, it was considered that the dietary CP content for fattening Holstein steers does not need to be higher than 16% in the early period and 12% in the late period.
The GHG reduction is important for the sustainability of the beef industry (Gleason & White, 2019), and GHG is reduced by decreasing excessive N excretion (Eckard, Grainger, & Klein, 2010). Generally, the dietary CP content is set higher than the CP requirement for fattening cattle in Japan. In this experiment, dietary CP contents of 16% for the early period and 12% for the late period were sufficient to reduce N excretion without affecting productivity. These results suggest that the dietary CP content of fattening Holstein steers should be reduced to at least this experimental level. The results of this experiment also suggest that N excretion can be reduced from cattle fattening farms in Japan, which contributes to GHG reduction.