Effect of a Simultaneous Increase in Crude Protein and Total Digestible Nutrients Contents on Late Fattening Hanwoo Steers

Recently, in Korea, efforts have been undertaken to shorten the fattening period to reduce feed costs and decrease the production of inedible fat in Hanwoo (Hong, 2016). However, shortening the fattening period may reduce the carcass weight and intramuscular fat (marbling), which may lower the farmer’s income. Therefore, there are opinions that the crude protein and total digestible nutrients (TDN) in the formula feed should be increased during the late fattening period (Jeong et al., 2010).

It was reported that an increase in the supply of crude protein through feed increased feed intake and an increase in intestinal amino acid absorption increased weight gain (Nagpal et al., 2011). In addition, Trenkle (2003) reported that increasing the crude protein content of the feed had positive effects on the feed conversion ratio (FCR), carcass weight, marbling in the finishing steers. Moreover, previous studies (Kim et al., 2013) reported that the increase in the crude protein content of the feed had a positive effect on growth performance and carcass characteristics during the late fattening period. Conversely, Chung et al., (2015) reported that the TDN level of feed was a major factor affecting the growth performance, carcass characteristics and fat deposition in beef cattle. In addition, increasing the TDN levels in the late fattening formula feed improves the dry matter (DM) digestibility, energy availability, average daily gain (ADG) and meat quality grade (Chung et al., 2015; Jeong et al., 2010).

Recent research has been centered on determining the optimal levels of crude protein and TDN for various growth stages. In addition, there are few studies on the effects of the simultaneous increase of crude protein and TDN contents in formula feed on growth performance, blood metabolites, carcass characteristics and meat composition during the late fattening period.

We hypothesized that increasing the levels of crude protein and TDN in the feed for late fattening Hanwoo steers could address the challenges in growth and meat quality, such as body weight and marbling, associated with shortened fattening periods, without adversely affecting the rumen fermentation characteristics. Therefore, this study evaluates the in vitro rumen fermentation characteristics following increases in crude protein and TDN in the formula feed and investigates the growth performance, blood metabolites, carcass characteristics and meat composition of late fattening Hanwoo steers.

Ethics statement
 
All procedures on animals were carried out in compliance with South Korea regulations (Animal and Plant Quarantine Agency Ministry of Food and Drug Safety Joint Animal Testing and/or Laboratory Animal Related Committee (IACUC, 2020) Standard Operating Guidelines).
 
Animals, treatments and management
 
A cow equipped with rumen fistula was used for the in vitro experiment. In the field trial, 30 late fattening Hanwoo steers (694.3±71.7 kg and 25 months of age) were used. The steers were randomly assigned to one of the two dietary groups: control group (16.4% crude protein and 83.6% TDN based on DM) and treatment group (17.4% crude protein and 84.6% TDN based on DM). The control and treatment groups were fed experimental feeds for approximately 4 months.

The steers were allotted into six pens (5 m × 10 m) and the floor was covered with 20 cm of sawdust Formula feed was provided twice daily (08:30 and 17:00) using an automatic feeding system (SEOCHANG 65M/M, Seochang Co, Ltd, Cheonan, Korea). The steers had free access to rice straw, water and mineral blocks. Other feeding management procedures were conducted according to the practices of the experimental farm. The ingredients and chemical composition of the experimental diets are listed in Table 1.


 
In vitro rumen fermentation
 
Ruminal fluid was collected from the ruminal fistula of a cow prior to morning feed administration. The collected rumen fluid was immediately stored at 39°C in a thermos flask, transferred to the laboratory, filtered through eight layers of cheesecloth and diluted in an in vitro buffer (Goering and Van Soest, 1970) at a ratio of 1:3. To maintain anaerobic conditions, O2-free CO2 was bubbled through the diluted rumen fluid until transferred into serum bottles. Under completely anaerobic conditions, each 60 mL of the rumen fluid was dispensed into 125 mL serum bottles containing 1 g of the experimental diet and completely sealed using a butyl rubber stopper and aluminum cap. The sealed serum bottles were incubated in an incubator at 39°C for 24 h.

The in vitro ruminal pH was measured using a pH meter (FP20, Mettler Toledo). The total gas production was measured using a pressure transducer (EA-6, SunBee Instrument, Seoul, Korea) according to the method of Theodorou et al., (1994).

The ammonia concentration was determined using the method of Chaney and Marbach (1962) and the volatile fatty acid (VFA) concentration was measured using gas chromatography (Agilent 7890A, Agilent Technology, CA, USA).

The DM degradability was calculated by washing the filter bag (F57, Ankom Technology, NY) with distilled water after incubation for 24 h drying it at 60°C for 72 h and measuring the weight.
 
Growth performance and blood metabolites
 
The body weight (BW) was measured at 2-month intervals using a cattle scale before administration of the morning feed. The ADG was calculated based on the BW difference and the number of feeding days. Feed intake was calculated by measuring the quantity of residual feed before administration of the morning feed. The FCR was calculated based on the dry matter intake (DMI) and ADG values.

Every two months, 3 mL of blood was drawn from the jugular vein of the steers using an 18-gauge needle attached to a heparin-coated Vacutainer (Becton-Dickinson, Franklin Lakes, NJ, USA). Immediately after collection, samples were chilled and transported to the lab within 6 hours. Following centrifugation at 1,250 × g for 10 minutes to extract plasma, samples were processed with an automatic blood analyzer (Hitachi 7020, Hitachi Ltd., Tokyo, Japan) for assessing levels of metabolites.
 
Carcass characteristics
 
All steers were slaughtered to assess the meat yield traits (carcass weight, back-fat thickness and rib-eye area) and quality traits (marbling score, meat color, fat color, texture and maturity) of the carcasses at the local slaughterhouse. Carcass characteristics were determined from the sirloins of each carcass. Meat graders evaluated the carcass traits according to the Korean carcass grading system (MAFRA, 2018).
 
Beef quality measurements
 
Muscle pH was assessed after carcass quality grading using a portable pH meter (Testo 206-pH2, Testo AG, Lenzkirch, Germany). After 30 min of blooming at 4°C, the meat surface color was measured using a chromameter (CR400, Minolta Camera, Osaka, Japan) and values were expressed as lightness (L*), redness (a*) and yellowness (b*) (CIE, 1978).

The drip and cooking losses were measured according to the procedure described by Honikel (1998). Following the measurement of cooking loss, the same samples were subsequently utilized for the Warner-Bratzler shear (WBS) analysis, employing a method adapted from AMSA (2015).
 
Sensory quality evaluation
 
Beef steaks were heated until the core temperature reached 71°C using an oven (180°C) and then stored in a water bath (54°C) until the evaluation. Ten trained panelists were used for sensory quality analysis in this study. Sensory panel training was performed for at least six months (1-2 times a week, 1 hour per session) according to the AMSA guidelines (2015) and all items were evaluated using a 9-point scale.
 
Fatty acid composition
 
The fatty acid composition of the longissimus muscle was determined using a method adapted from Folch et al., (1957), analyzed with a chromatography-mass selective detector (GC, Agilent 7890N, USA; MSD, Agilent 5975A, USA). The GC setup included a DB-WAXetr column (300 m × 0.25 mm ID, 0.25 µm film), helium as the carrier gas at a flow rate of 1 ml/min and temperatures set at 210°C for the injection port, 260°C for the detector and 200°C for the oven, with a split ratio of 10:1.
 
Statistical analysis
 
Statistical analysis to obtain averages and standard differences were performed using IBM SPSS (Statistical Package for the Social Sciences, SPSS Inc. Chicago, IL, USA). Statistically significant differences between the treatments were determined using the t-test, with P<0.05 indicating significance.

In vitro rumen fermentation
 
Table 2 the effect of a simultaneous increase in crude protein and TDN contents of the formula feed on the rumen parameters. The pH and ammonia concentrations were similar between the two groups. Gas production was slightly, but insignificantly, higher in the treatment group than in the control group. There was no effect of crude protein and TDN contents in the formula feed on the VFA concentrations and DM degradability.



Based on the findings, it can be concluded that as the availability of nutrients in the feed for the rumen increases, there is an elevation in the production of volatile fatty acids, ammonia, gases and the DM degradability (Dey et al., 2022). However, an excessive increase in soluble carbohydrates leads to a decrease in rumen acidity and negatively affects rumen metabolism (Keles and Demirci, 2011). Nevertheless, this study found no differences in such rumen fermentation characteristics, suggesting that the cause might be the minimal difference in non-fibrous carbohydrate content (Table 1) and a similar level of nutrients available in the rumen between the two groups.
 
Growth performance and blood metabolites
 
Table 3 shows the effect of a simultaneous increase in crude protein and TDN contents of the formula feed on the growth performance and blood metabolites concentrations of the late fattening Hanwoo steers. No differences were observed in ADG, formula feed intake, rice straw intake and FCR between the control and treatment groups. The concentrations of the blood metabolites were similar between the two groups.



This study showed that the protein content in the formula feed had little effect on the ADG of late fattening Hanwoo steers, similar to the report by MAFRA (2007). Furthermore, Jeong et al., (2010) and Chung et al., (2015) also reported no significant difference in ADG and FCR with an increase in crude protein or TDN (approximately 2%) in the formula feed. Similarly, this study also found that increasing crude protein and TDN levels in the compound feed did not affect growth performance, which is consistent with previous research findings. Similar to this study, Paek et al., (2005) reported that an increase in the TDN content (increased from 72% to 74%) in the formula feed during the late fattening period did not affect the ADG or FCR and suggested further studies (more than 3% TDN difference). Conversely, some studies (Kim, 2015; Jin et al., 2012) reported that the increase in TDN level in the formula feed increased the DMI and ADG. This difference might be due to the differences in TDN content in the formula feed among studies.

It was reported that there was no difference in the concentrations of blood metabolites after an increase in TDN in the formula feed during the late fattening period (Cho et al., 2019; Chung et al., 2015). Similarly, in this study, the simultaneous increase in TDN and crude protein contents of the formula feed had no effect on the concentrations of blood metabolites during the late fattening period.
 
Carcass characteristics
 
Table 4 shows the effect of a simultaneous increase in crude protein and TDN contents of the formula feed on the carcass characteristics of the late fattening Hanwoo steers. The carcass weight and yield index were higher in the treatment group than in the control group, but there was no significant difference between the groups. The back-fat thickness was slightly, but not significantly, lower in the treatment group than in the control group. There was no effect of dietary treatment on marbling score, meat color, fat color, texture, or maturity.



Kim et al. (2013) reported that an increase in the crude protein content in the formula feed during the late fattening period positively affected the back-fat thickness and marbling score. Chung et al. (2015) reported that the back-fat thickness of Hanwoo steers slaughtered at 26 and 30 months of age was higher in the high energy treatment than in the control. However, this study showed no correlation between TDN in the formula feed and back-fat thickness. This might be because there was no difference in DMI due to limited feeding the formula feed in all groups (Table 2). Similar to this study, previous studies (Cho et al., 2019; Chung et al., 2015) showed that the difference in TDN levels in formula feed during the late fattening period affected the carcass weight.
 
Beef quality measurements, sensory evaluation and fatty acid composition
 
Table 5 shows the effect of a simultaneous increase in crude protein and TDN contents of the formula feed on the meat quality characteristics of the Hanwoo steers. Between the control and treatment groups, no differences were observed in pH, surface color, drip loss and cooking loss in the longissimus muscle. The shear force was slightly, but not significantly, lower in the treatment group than in the control group.



Kim and Jung (2007) reported that the better the marbling score, the lower the shear force and cooking loss, but the higher the water holding capacity. Also, it is reported that the cooking loss of beef decreases as the meat quality level and intramuscular fat level increases (Ozutsumi, 1994). Kim et al.  (2013) reported that the cooking loss of beef was lowered due to an increase in marbling after an increase in the crude protein content in the formula feed. However, this study showed no difference in drip loss, cooking loss, or shear force because there was no change in marbling from the change in crude protein and TDN contents of the formula feed.

Table 6 shows the effect of a simultaneous increase in crude protein and TDN contents of the formula feed on the sensory quality characteristics in the beef of the Hanwoo steers.



No differences were observed in tenderness or sensory characteristics of the longissimus muscle between the control and treatment groups

The simultaneous increase in crude protein and TDN in the formula feed during the late fattening period did not affect the sensory quality characteristics of cooked beef in this study, presumably due to the similar marbling scores between the two groups (Table 4). This is supported by previous studies (Bown et al., 2016; Schaefer et al., 1986) showing that there were generally no differences in juiciness, flavor and off-flavor in beef at the same marbling degree.

Table 7 shows the effect of a simultaneous increase in crude protein and TDN contents of the formula feed on the fatty acid composition in the beef of the Hanwoo steers. The overall fatty acid compositions were similar between the two groups.



Jeong et al. (2010) reported that the contents of stearic, myristoleic and oleic acids was increased by a simultaneous increase of crude protein and TDN content in the formula feed during the late fattening period. However, this study found no difference in the fatty acid composition of the longissimus muscle after the change in crude protein and TDN content of the formula feed, presumably because there was no difference in the marbling level and meat quality grade. Previous studies (Kim, 2006) support the results of this study by indicating that the fatty acid composition of beef affects the marbling degree and meat quality grade.

In this study, increasing the crude protein and TDN in the formula feed to compensate for the shortened fattening period did not negatively affect the rumen fermentation characteristics. However, it also did not exhibit a positive impact on growth performance, carcass characteristics and meat quality, which are crucial for productivity and profitability. To address these issues, further research is suggested, potentially extending the experimental period (starting in the growing stage or early fattening stage) or increasing the crude protein and TDN levels beyond the current study’s parameters.

All authors declare that they have conflict of interest.