Effects of a Stair-step Growth Pattern on Improvements in Meat Quality and Growth in Hanwoo Steers*

: The present study was conducted to examine the effect of a stair-stepped feed intake pattern on growth, feed efficiency, and meat quality of Hanwoo steers. Twenty-seven 11-month-old Hanwoo steers were randomly divided into three groups. The control group was fed according to the Korean steer feeding program, and the other two groups were fed according to an alternated feeding schedule of 3-2-4-2 months. During the first three months of the experiment, treatment group 1 (T1) and treatment group 2 (T2) were fed 20% and 30% less than the control group, respectively. For the following two months, the T1 group was fed 20% more than the control group while the T2 group was fed 20% less than the control group. In the third step, T1 and T2 groups were fed 20% and 10% less, respectively, than the control group for four months. In the last two months, T1 and T2 groups were fed 20% more than the control group. After the stair-step feeding trial, steers were fed concentrated feed ad libitum for five months. The altered feed intake pattern did not affect daily body weight gain. However, daily feed intake tended to decrease and growth efficiency tended to increase in the two treatment groups compared to the control group. Altered feed intake also affected blood metabolite levels. The serum glucose and BUN level of the T1 group increased in the first re-fed period compared to the T2 and control groups. The serum cholesterol level of the T2 group decreased in the first restricted-re-fed growth period compared to the T1 and control groups. The serum NEFA levels of the two treatment groups increased from the first restricted period compared to the controls. The serum insulin level of the T2 group increased in the last period compared to the T1 and control groups. Regarding meat yield index, the control group was significantly higher than the T2 group (p<0.05). Regarding meat yield grade, the carcass back fat thickness of the T2 group was significantly higher than the control group (p<0.05). In marbling score, the T1 group was the highest (4.9), followed by the control group (4.1) and the T2 group (4.0). These results indicate that using a stair-stepped growth pattern (T1) can contribute to improvements in growth efficiency and muscle marbling.


INTRODUCTION
In general, compensatory growth occurs when previously marginal-or under-fed animals are re-alimented onto a higher nutritional level. Compensatory growth following a period of malnutrition depends on the nature of the restricted diet, the severity and duration of malnutrition, the stage of development at the outset of malnutrition, the relative mature body weight (BW) of the animal, and the pattern of realimentation (Wilson and Osbourn, 1960;Abdalla et al., 1988;Jin et al., 2004).
Therefore this study was conducted to examine the effect of compensatory growth feeding on growth, feed efficiency, and meat quality of Korean native cattle.

Animals and diets
Twenty-seven, 11-month-old Hanwoo steers were divided into three groups of nine animals each based on body weight (293±22.8 kg). This trial was conducted over a period of 16 months. To avoid metabolic disease due to excessive concentrate feeding during the stair-step growth period, these steers were put through a two month adaptation period. During the experimental period, steers were fed twice daily and had free access to water and red mineral blocks. Control group steers were fed according to the Korean steer feeding program, and treatment groups were subjected to a stair-step nutrition scheme according to an alternating schedule of 3-2-4-2 months. Treatment group 1 (T1) steers were fed 20% less than the control group in the first three months of the trial, 20% more than the control group for two months, 20% less than the control group for four months, and 20% more than the control group for two months. Treatment group 2 (T2) animals were fed 30% less than the conventional diet for three months, followed by 20% less than the control group for two months, 10% less than the control group for four months, and 20% more than the control group for two months. After the stair-step feeding trial, steers were again fed concentrate ad libitum, along with 1 kg hay per day (dry matter). The roughage was mainly composed of borme hay. Experimental feeds were analyzed for DM (Dry matter), Crude fiber, CP (Crude protein), EE (Ether extract), Ca and P by the methods of AOAC (1995). The ingredients and chemical composition of the experimental diets are shown in Table 1.

Real-time ultrasound measure
Real-time ultrasound measurements were obtained from the Scanner unit (2MHz linear, Super-Eye Meat, FHK, Japan). A single image by this equipment of scanned 13 th rib rump was interpreted by video copy processor (AP-950, Mitsubishi, Japan) and computer program (Image-Pro Express, Media Cybernetics, USA) (Rhee et al., 2005).

Measurements of feed intake and body weight
During the experiment, body weights of the steers were measured monthly, and steers were fed pre-weighed concentrate feed at 8 am and 5 pm every day. The amount of concentrate feed left uneaten by the steers was weighed one hour prior to the next day's morning feeding.

Blood sample collection and analysis
During the experiment, blood samples were collected a total of five times from the jugular vein of each steer prior to the morning feeding (0, 3, 5, 9, and 11 months after experiment start). Blood samples were centrifuged at 3,000 rpm for 15 minutes to obtain serum, which was stored at -70°C until analysis. The levels of serum glucose, BUN, and total cholesterol were measured using a TBA-40FR automated biochemical analyzer (Toshiba, Japan) according to Lee et al. (2005). Serum NEFA level was measured using a Wako Pure Chemical Industries (Osaka, Japan) commercial kit, and serum insulin level was measured using a bovine-specific insulin ELISA kit (Mercodia co, Sweden).

Analysis of real-time polymerase chain reaction (PCR)
The real time-PCR technique was used to measure mRNA expression levels for aP2, C/EBP-a, ACC, FAS, and SCD. GAPDH was used as an invariant internal control. Information about primer sequences of the specific genes for real time-PCR is given in Table 2. Total RNA was isolated from the subcutaneous fat and longissimus muscle using Trizol reagent (Invitrogen Life Technologies, USA). PCR reactions were begun at 95°C for three minutes, followed by 50 cycles of 95°C for 30 s, 60°C for 30 s, and 72°C for 30 s. The relative quantification of gene expression was analyzed using the 2 -ΔΔCT method (Livak et al., 2001).

Statistical analysis
The basic statistical model for the stair-step growth regimen has been described previously (Park et al., 1989). The experimental data were expressed as mean±SEM. Data were analyzed using SPSS software (Statistical Package for the Social Sciences; version 14.0, SSPS Inc, Chicago, USA), and treatment effects were analyzed using one-way analysis of variance (ANOVA) followed by Duncan's Multiple Range test. A p value <0.05 was considered to be statistically significant.

Growth efficiency and feed efficiency
After the two-month preliminary feeding experiment, restricted feeding on restricted -re-fed growth, early fattening for 3-2-4-2 months, and finally late fattening and feeding, the results for growing/fattening weight grades are shown in Figure 1 and Table 3. For the first restricted -refed growth period, the daily body weight gains of the control and T1 groups showed no significant difference, but that of the T2 group increased significantly (p<0.01). Regarding feed intake, the control group was significantly higher (p<0.05) than the T1 and T2 groups, but T1 was greater than T2 (p<0.05). Regarding growth efficiency, the T1 group was the highest with an efficiency of 9.73%, while the control group was 9.17% and the T2 group was 8.63%. During the second restricted -re-fed growth period, daily body weight gain of T2 increased significantly (p<0.01) compared to T1; for feed intake, T2 was the highest at 9.80 kg/d, followed by the control at 9.76 kg/d and T1 at 9.16 kg/d. Regarding feed efficiency, there was no   significant difference between T2 and T1, but both showed a higher feed efficiency than the control (p<0.01). Throughout the two restricted -re-fed growth periods, the daily body weight gain was similar between the treated groups, but feed intake, was 9.3 kg/d for the control, 8.72 kg/d for T2 and 8.67 kg/d for T1. For growth efficiency, T2 increased the most at 8.27%, T1 increased 8.21%, and the control increased 7.63%. These results were consistent with the results of stair-step growth feeding in which less feed intake results in increased feed efficiency and growth ability, possibly due to the promotion of efficient energy and protein use. Fasting animals had further enhanced weight gain upon re-fed supply compared with non-fasting animals fed the same feed, which is partially due to the reduction of maintenance requirements and increased feed use. This result is consistent with the results of stair-step growth feeding experiments with Holstein heifers during a feedrestricted feeding period, in which weight gain decreased, but during the re-fed growth period, the growth efficiency, energy, and protein use efficiency were greatly increased (Koch, 1982;Blum et al., 1985;Park et al., 1987;Park et al., 1989;Drouillard, 1990;Choi et al., 1992;Ryan et al., 1993a;Ryan et al., 1993b;Park et al., 1994;Jang et al., 1995;Ahn et al., 1996;Choi et al., 1996;Choi et al., 1997;Park et al., 1998). In addition, according to Hornick et al. (2000), growth hormone was secreted during the maintenance period and was maintained at high concentration in the re-fed growth period; also the increased secretion of insulin due to increased nutrient intake and collaboration of mass action showed re-fed growth effects.

Changes in blood metabolites and insulin
During two restricted -re-fed growth experiment periods, the blood metabolite changes shown in Figure 2 were observed. The level of blood glucose (Figure 2A) in the first re-fed growth period of the T1 group increased significantly compared to the control and T2 groups. In addition, during the entire experiment the T1 group showed higher blood glucose levels than T2; after the re-fed growth period, the blood glucose level of T2 was higher than the control group. This result is similar to that of Park et al. (1987) for the stair-step growth of Holstein fattening. Furthermore, the blood concentration of glucose has a tendency to decrease with increased age. According to Stufflebeam et al. (1969), in an experiment using 58-weekold heifers which were divided into three groups of 16 depending on the level of energy supply and fed an experimental diet for 224 days, blood glucose concentrations decreased gradually with growth but did not have any relationship with energy intake.
For BUN ( Figure 2B), the T1 group maintained a low level during the feeding period, and showed a rapid increase during the re-fed growth period (p<0.05). However, during the two maintained feeding periods, a rapid decline in BUN concentration was noted, which increased significantly in the re-fed growth period compared to the control group (p<0.05). The results of some studies (Park et al., 1987;Choi et al., 1996) are similar to the low level observed herein during the feeding period, but increased sharply in the re-fed growth period. Huntington et al. (1996) showed that as the combination of feed supply increased, BUN concentration also increased. Moreover, Philippe et al. (2005) showed that the growth of fatty tissue increased significantly outside of the muscle tissue during the re-fed growth period. Based on these results, the storage capacity of fat tissue is considered higher than the protein synthesis capacity while using the energy provided in the re-fed growth period. Therefore, animals experiencing re-fed growth take in an excessive amount of feed yielding a fat synthesis rate higher than that of protein synthesis, upsetting the body's energy and protein balances. Increased ammonia produced during metabolism is converted to urea in the liver causing an increased BUN concentration.
The total blood cholesterol density ( Figure 2C) of the control group increased significantly compared to T1 and T2 groups during the restricted feeding period (p<0.05, p<0.01, respectively). However, T1 and control groups showed no significant difference during the re-fed growth period, which is consistent with the results of Park et al. (1988). Furthermore, Arave et al. (1975) reported that as growth and feed energy intake increased, blood cholesterol levels also increased. In addition, total blood cholesterol increased with animal age, but decreased with high content of protein in feed (Park et al., 1980(Park et al., , 1983. In this experiment, during the two maintenance re-fed periods there was no significant difference between the treated groups. The T1 and T2 groups had higher cholesterol concentrations in their blood due to excessive feed intake compared to the control group. From the changes in serum NEFA concentration ( Figure  2D) during the first re-fed period, T1 and T2 groups showed and maintained higher values than the control group (p<0.05), which was consistent with the results of Blum et al. (1985). In addition, Yambayamba et al. (1996) showed that in the maintenance period serum NEFA concentration of heifers increased significantly, but with increased feed intake the NEFA concentration decreased. Hornick et al. (2000) reported that during the restricted feeding period increased secretion of growth hormone promoted breakdown of fat to maintain body energy balance, which then increased the serum NEFA concentration. In the current experiment, however, the rapidly increasing energy intake during the re-fed growth period also increased blood NEFA concentrations that were maintained at high level regardless of the reduction in energy intake. Emery (1979) showed that in the ruminant body fat accumulation was mainly due to accumulation of triglycerides and NEFA in blood in dynamic equilibrium. Pothoven et al. (1975) reported that fatty acid-mobilizing lipase activity in fat tissue increased as fattening progressed. Therefore, in our experiment, during the second maintenance re-fed period the serum NEFA concentration increased.
Changes in blood concentration of insulin are shown in Figure 2E. During the first maintenance growth feed period, blood insulin concentrations of T1 and T2 groups decreased compared to the control group (p<0.05), and in the re-fed supply period insulin concentration tended to increase in T1 and T2. During the second re-fed growth period, the insulin level of the T2 group was significantly increased compared to the control and T1 groups (p<0.05). The insulin level of the T1 group showed a tendency to increase compared to the control. Insulin concentration is known to have a correlation with feed or energy intakes (Bassett, 1972). Hornick et al. (2000) reported that increased feed intake during the re-fed growth period resulted in a sudden increase of insulin concentration in the blood. Jang et al. (1995) reported that during stair-step growth feeding of Holstein cattle, plasma insulin concentrations were lower than in the control group, but higher concentrations were noted during re-fed growth; Blum et al. (1985) reported a similar result. The results of the present experiment agree with previous reports. This indicates that during the maintenance period insulin secretion decreases, but as nutrient intake and feed intake are increased insulin secretion also increases and promotes the synthesis of protein and fat tissues. Rhee et al. (2005) reported that, by using an early prediction system with ultrasound, flesh and meat yields could be evaluated. Using ultrasound in this experiment, we performed early prediction on 24-month-old Korean native steers. The evaluation results are shown in Table 4; marbling score was highest in the T1 group, followed by the control and T2 groups. For back fat thickness, the T1 group was significantly lower than T2. After slaughter at 28 months, the marbling score of the T1 treated group was higher than those of the control and T2 groups. For back fat thickness, T1 showed no significant difference with either the control or T2 groups, while T2 was significantly higher than the control group. According to these results, animals that experienced multiple re-fed growth periods had an increased ability to synthesize fat than did the control and T2 groups in the early fattening phase; however these same animals had a decreased ability for synthesis of subcutaneous fat. Fattening, according to the marbling score, increased in the treated groups, but the back fat thickness of T1 and T2 increased more than that of the control group (p<0.05). These results are consistent with the results of  Means with different superscripts in the same column differ significantly (p<0.05). 1 Means±SEM of 9 steers. 2 Grading ranges are 1 to 7 for marbling score with higher numbers for better quality, and 1 to 7 for meat and fat colors, 1 to 3 for firmness, maturity and grade with lower numbers of better quality. T1: stair-step growth pattern, T2: restricted -re-fed. Dulloo et al. (2002) who found that in the re-fed growth period the body weight gain from fat tissue increased more than the body weight gain from muscle tissue. This result is due to the body's reaction to favour nutrient storage to help assure survival in periods of malnutrition.
Carcass characteristics of experimental cattle of each treated group are shown in Table 5. In T2 and T1 groups these were higher than for the control group. For meat yield grade and carcass back fat thickness, the T2 group scored significantly higher than the control (p<0.05), but the T1 stair-step treated group did not show a significant difference. For meat yield index, the control group was significantly higher than the T2 group (p<0.05), while the T1 group did not show a significant difference. For Grade A scoring, the control group was the highest at 66.7%, followed by T1 at 44.4%, and T2 at 11.1%. For marbling score, which is the most important determinant of flesh grade, the T1 group had the highest (4.9), followed by the control (4.1), and T2 (4.0) groups. According to the results, during late fattening the T2 group increased in daily body weight gain and feed efficiency compared to T1 and control groups; fat synthesized by the re-fed growth effect was mainly stored as subcutaneous fat resulting in a lower meat yield grade.

Changes in fat metabolism-related gene expression
For sirloin and back fat tissue of Korean native steers, the results of fat cell differentiation and fat synthesis gene expression are shown in Table 6. Back fat tissue and the sirloin quality of beef are important factors which determine meat quality. Marbling score is one very important factor for determining the grade of beef in the United States (USDA, 1989), Japan (JMGA, 1988), and Korea. The growth of fat tissue is determined by the differentiation and size of fat cells. CCAAT/enhancer binding protein (C/EBPs) belongs to the basic-leucine zipper transcription factor family and with PPARrγ, is known to perform important functions in the expression of genes such as ap2 and the terminal differentiation of fat precursor cells (Grimaldi, 2001). In this experiment, C/EBP-a mRNA expression level in muscle tissue of the T1 group was higher than that of the T2 or control groups. In back fat tissue, the T1 group showed lower levels than the control group and significantly lower levels than the T2 group (p<0.05). These results showed that differentiation of fat cells in muscle tissue of the T1 group was more active than in the control and T2 groups; on the contrary, that of the back fat cell in the T1 group was less active than the control and T2 groups. Adipocyte protein 2 (ap2) is called the adipocyte fatty acid-binding protein and is an important transcription factor in the terminal differentiation stage of fat precursor cells, which is controlled by PPARγ and C/EBPa (Macdougald and Lane, 1995). Separated fat synthesized by the liver and absorbed from feed is moved within muscle and fat cells (Sul and Dong, 1998). Because in these experiments we analyzed the gene expression level of ap2, we also analyzed the use of late fattening energy sources. The result was that aP2 mRNA expression in muscle tissue of the T1 group was higher than that of the control and T2 groups, while in fat tissue, T2 and T1 groups showed greater aP2 expression than the control group. These results showed that the use of separated fatty acid by muscle and fat cells was increased in the T1 group, and separated fatty acid within back fat tissue was increased in the T2 group. Acetyl-CoA formed through β-oxidation in mitochondria acts as a precursor of fat synthesis, which is converted into malonyl-CoA by acety-CoA carboxylasea (ACC). Finally, palmitate is synthesized as a long chain fatty acid form by fatty acid synthase (FAS). Synthesized long chain fatty acid is converted into monounsaturated fatty acid by stearoyl-CoA desaturase and accumulates in muscle and fat tissue as triglycerides (Sul and Dong, 1998).
In muscle and fat tissues, acetyl-CoA carboxylase (ACC) is known as a key enzyme to control fat synthesis rate. De novo lipogenesis begins with the carboxylation of acetyl-CoA and its conversion into malonyl-CoA (Murray et al., 2000). The ACC gene is known to react very sensitively to nutrition level. In this experiment, the ACC level of the T1 group was higher than that of the control and T2 groups, not only in muscle tissue but also in back fat tissue, although there was no significant difference. The late fattening period of stair-step feeding causes growth of muscle and fat tissue to increase with the induction of ACC gene expression resulting in the accumulation of fat in muscle and subcutaneous fat tissues. FAS (fatty acid synthase) mRNA expression in muscle as well as back fat tissue was higher in the T1 group than in the control and T2 groups. FAS is an important enzyme controlling the synthesis of fat and is very sensitive to nutritional status. Clarke et al. (1990) showed that when rats were fasting for 1-2 days, fatty acid synthase (FAS) mRNA in rat liver decreased four-fold, while the amount of FAS mRNA increased several times after 48 hours of fasting and re-feeding with a high carbohydrate diet. According to Mariashiara et al. (1995), the FAS mRNA level of rats increased rapidly after 48 hours fasting and re-feeding. Therefore, considering that the T1 group experienced several restricted/re-fed cycles, an increase of FAS mRNA expression upon intake of nutrients at the end of the fattening cycle was observed and facilitated fat synthesis both in muscle and fat tissues.
Stearoyl-CoA desaturase (SCD) is the major enzyme controlling the synthesis of unsaturated fatty acid in higher animals. SCD uses palmitic acid (16:0) and stearic acid (18:0) as substrates to synthesize palmitoleic acid (16:1) and oleic acid (18:1), respectively. Palmitoleic acid (16:1) and oleic acid (18:1) are the major components of neutral fat (triglycerides). In muscle tissue of this experiment, there were no significant differences among the three groups, but the T1 group showed the highest muscle content, while the T2 group showed the most back fat tissue. The re-fed growth effect on the late fattening feed intake of the T2 group yielded significant results. In meat grades, the T2 group showed a significantly higher score than did the T1 group. Upon late fattening, the T2 group developed fat tissue more actively than muscle tissue. Considering these results, in the muscle tissue of the T1 group, the differentiation of fat cells and use of free fatty acid was increased. However, in back fat tissue, differentiation of fat cells decreased, but use of separated fatty acid increased. In the T2 group, the differentiation of fat cells and use of separated fatty acid increased more in fat tissue than in muscle tissue. Even though there was no significant difference in gene expression level of fatty acid synthetic enzymes among the groups, there was an increase in the back fat tissue of the T2 group. This result may be due to the long-term free feed intake of the T1 and control groups, but for the T2 group, high feed intake gave greater results due to the initial feed restrictions.