Effects of Fattening Period on Growth Performance, Carcass Characteristics and Lipogenic Gene Expression in Hanwoo Steers

This study was conducted to investigate the effects of different fattening periods i.e. 25, 27 and 29 months of age (25 mo, 27 mo and 29 mo), on feed consumption, body weight gain, carcass parameters, and lipogenic gene expression in 45 Korean native steers (Hanwoo). Daily DM intake was higher in steers on 29 mo compared with those on 25 mo or 27 mo. Daily body weight gain was higher in steers on 25 mo compared with those on 27 mo or 29 mo during fattening and overall experimental periods. Therefore, feed conversion ratio was lower in 25 mo compared with 27 mo or 29 mo during the fattening and whole experimental periods. As expected, slaughter and carcass weights were higher in the order of 29 mo>27 mo>25 mo. Carcass yield grade was relatively lower in 29 mo reflecting higher back fat thickness compared with other treatments, while carcass quality grade was not largely influenced by the treatments. By investigation with an ultra-sound scanning technique, the marbling score was significantly and numerically higher in 25 mo compared with 27 mo or 29 mo. The mRNA levels of stearoyl-CoA desaturase (SCD) gene were gradually increased in the late fattening stages (p<0.01) and mRNA of acetyl-CoA carboxylase (ACC), ATP citrate lyase (ACL) and glucose transporter 4 (GLUT4) gene were highly expressed in 29 mo compared with 25 mo and 27 mo (p<0.05). However, gene expressions of adipocyte fatty acid binding protein 4 (FABP4) and lipoprotein lipase (LPL) were not significantly different among the treatments. Thus the present results indicated that different fattening period has no major effect on carcass characteristics, although 25 mo had a lower carcass weight compared with 27 mo or 29 mo. (


INTRODUCTION
Bovine carcass characteristics and thus beef quality are affected by various factors such as age, sex, genetics, and nutrition. In addition, meat quality is also determined by meat color, fat color, texture, and marbling (intramuscular fat) scores. Marbling plays a particularly important role in determining the juiciness and tenderness of beef, and is one of the main factors used to determine beef quality grade in Korea (Lee et al., 2001;Lee, 2004). Tatum et al. (1982) reported that marbling has been implicated as a contributing factor to beef palatability, and is used as one of the most important factor in evaluating the beef quality.
Hanwoo steers dramatically increase their marbling fat in muscle between 12 and 27 months of age (Lee et al., 2007). Generally, Hanwoo has been fattened until almost 30 months of age to improve meat quality through marbling in Korea. Chung et al. (2000) reported that lipogenesis in subcutaneous fat and intermuscular fat was higher at 30 months of age than at 24 months of age in Hanwoo bulls. Therefore, fattening period is an important factor to produce desirable beef with high proportion of marbling fat in Korea. However, it is difficult for farmers to keep their cattle until 30 months age due to increasing feed expense in recent times.
Thus, the present study was designed to investigate effects of three different fattening periods (i.e. 25, 27 and 29 months) on the growth performance, meat quality and lipogenic gene expression in relation to intramuscular fat content in Hanwoo steers.

Animals and diets
Forty five Hanwoo calves, 3 months of age and weighing an average of 94.2±6.4 kg, were distributed into 3 groups of 15 calves, each with an individual feeding system (Calan system, Seil Tech, Korea). The calves were assigned to 3 different fattening periods, which lasted for 25 months (25 mo), 27 months (27 mo) and 29 months (29 mo), respectively. In the treatments, 3 pens (5.3×10.6 m) which had concrete floors with sawdust bedding were arranged with 15 calves per pen. The growth trial was conducted with the animals at 4 months of age after a one-month adaptation period. The present study was categorized into the following periods: early growing (4-6 months age), late growing (7-12 months age), early fattening (13-17, 18 and 20 months age in 25 mo, 27 mo and 29 mo, respectively), and late fattening (18-25 months age, 19-27 months age and 21-29 months age in 25 mo, 27 mo and 29 mo, respectively). During the growing and early fattening periods, animals were offered a commercial concentrate at 1.6%-1.8% of body weight, and they were offered concentrate ad libitum during the late fattening period until slaughtered. During the early and late growing periods, animals were offered commercial concentrate at 1.5-1.6% of body weight. In the early fattening period, animals were offered commercial concentrate at 1.7-1.8% of body weight. The ingredients and chemical composition of the experimental concentrate offered at different growth stages are presented in Table 1. Klein grass hay (Fanicium Contoratom L.) was offered from 2 kg/animal/d (1.8% of BW) to 4 kg/animal/d (1.3% of BW) between 4 and 12 months of age. From 13 to 15 months of age, the hay was changed to rice straw, and animals were offered hay at 2.0, 1.5 and 1.0 kg/animal/d, and rice straw at 2.0, 2.1, and 2.2 kg/animal/d at 13, 14 and 15 months of age, respectively. From 16 months of age only rice straw was offered at 2.2 kg/animal/d. The amount of rice straw offered was decreased with increasing age, and it was restricted to 0.6 kg/d during the late fattening period. The contents of DM, crude protein, ether extract, NDF and ADF of hay were 91.5%, 10.9%, 1.7%, 71.6% and 39.5%, respectively. The contents of DM, crude protein, ether Feed conversion ratio was expressed as average feed intake per daily body weight gain.

Slaughter and carcass assessment
Marbling score was predicted between the 13 th thoracic and 1 st lumbar vertebrae of steers using ultra sound scanning equipment (Falco 100, 3.5 MHz, 18 cm linear probe, Pie Medical, Netherlands) at 18, 20, 22, 24, 25, 27 and 29 months of age before they were slaughtered, The steers on 25 mo, 27 mo, and 29 mo treatments were slaughtered at 25, 27 and 29 months of age, respectively, according to the procedure of APGS (2007). Their carcass characteristics such as yield grade and quality grade were assessed at 24 h post-mortem by a carcass grader of the Animal Products Grading Service (APGS, 2007), Korea.
Quality grades (marbling, meat color, fat color, texture, and overall mature score) and yield grades (cold carcass, fat thickness, and Longissimus muscle area) were recorded. Live weights were determined immediately before slaughter. After a 24-h chill, cold carcass weights were measured and then the left side of each carcass was cut between the last rib and the first lumbar vertebrae to determine quality grade. The quality grade was determined by assessing the degree of marbling and firmness in the cut surface of the rib eye, in relation to the maturity and fat color of the carcass. The rib eye area was measured from Longissimus muscle taken at the 13 th rib and back fat thickness was also measured at the 13 th rib. Yield index was calculated as follows: Yield index: 68.184-(0.625×back fat thickness (mm))+(0.130× Longissimus muscle area (cm 2 ))-(0.024×dressed weight (kg)). The degree of marbling was evaluated with the Korean Beef Marbling Standard, and the scores of meat color and fat color were made using the color standard (APGS, 2007). The scores for texture and maturity were made using the APGS reference index (APGS, 2007). The grading ranges were 1 to 9 for marbling score with higher numbers for better quality (1 = devoid, 9 = abundant); meat color (1 = brightly cherry red, 7 = extremely dark red); fat color (1 = white, 7 = dark yellow); texture (1 = soft, 3 = firm); Maturity (1 = youthful, 9 = mature). The Longissimus muscles were taken and immediately frozen in liquid nitrogen, and stored at -80°C until analysis of lipogenic gene expression.

Quantitative real-time RT-PCR
Total RNA was prepared from each Longissimus muscle (100 mg) using 1 ml of TRIzol reagent (Invitrogene Life Technologies, USA) according to the manufacturer's instructions. For real-time RT-PCR analysis, 500 ng of RNA was reverse transcribed in a 20-μl reaction volume using a RT-PCR high kit (Toyobo, Japan) as described in the RT-PCR high kit protocol. We analyzed the expression levels of lipogenic-related genes such as adipocyte fatty acid binding protein 4 (FABP4), glucose transporter 4 (GLUT4), lipoprotein lipase (LPL), acetyl-CoA carboxylase (ACC), stearoyl-CoA desaturase (SCD) and ATP citrate lyase (ACL) in Hanwoo Longissmus muscle. The primer sets were designed with the Primer3 out program, and the sequences of the sets are shown in Table 2. The amplification was performed in a total volume of 50 μl included 5 μl cDNA (125 ng), each 1 μl of 10 pmol/μl forward and reverse primer, 25 μl SYBR Green Master Mix (Toyobo, Japan), and 18 μl distilled water using a 7500 Real time PCR system (Applied Biosystems, USA) as follows: 50°C for 2 min, 95°C for 1 min, and 40 cycles of 95°C for 15 s, 58°C for 15 s, and 72°C for 32 s. Following amplification, a melting curve analysis was performed to verify the specificity of the reactions. The end point used in the real-time RT-PCR quantification, C T , was defined as the PCR threshold cycle number. All samples were examined for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal control, and quantities of each gene were presented as the 2 -ΔCt which was calculated using the ΔCt value (Ct value of sample-Ct value of GAPDH).

Statistical analysis
Statistical analysis for all dependent variables by treatments as environmental effects was performed using the GLM procedure (Version 8.1; SAS Inst. Inc., Cary, NC). Significant differences among treatments were determined by Duncan's multiple range test (Duncan, 1955) at a level of p<0.05.

Growth performance
Final body weight of steers on 25 mo was lower (p<0.05) compared with 27 mo or 29 mo treatments ( Table   Table 3. Effect of fattening period on intake, body weight (BW) gain and feed conversion ratio of Hanwoo steers 3). During the growing and early fattening periods, daily body weight gain was not significantly different among the treatments. Daily body weight gain during the late fattening period and for the overall experimental period was in the order 25 mo>27 mo>29 mo (p<0.05). Total DM intake by steers was higher (p<0.05) on 25 mo compared with 27 mo or 29 mo during the early growing period. However, during the late growing period, total DM intake was higher (p<0.05) in steers on 29 mo compared with 25 mo or 27 mo. During the early fattening period, concentrate intake was higher (p<0.05) and hay intake was lower (p<0.05) in steers on 29 mo compared with 25 mo or 27 mo. Total DM intake was greater in steers on 29 mo followed by 27 mo and 25 mo during the early fattening period (p<0.05). Although concentrate intake was not significantly different among the treatments, rice straw and total feed intakes were higher (p<0.05) in 29 mo compared with other treatments during the late fattening period. During the overall period, concentrate and total DM intakes were higher in 29 mo (p<0.05), while rice straw/hay intake was higher in 25 mo compared with other treatments (p<0.05) during the overall period. Chu et al. (2003) reported similar results that concentrate and rice straw intakes during growing, early and late fattening, and the whole period were 2.60, 5.14, 8.07 and 5.02 kg/d, and 2.87, 2.91, 2.41 and 2.58 kg/d, respectively. In the present study, during the growing period average daily body weight gain and daily feed intake was 0.8 kg/d and 5.4 kg/d/animal, respectively. Similarly, Cho et al. (2001) reported that daily body weight and feed intake of Hanwoo steers during the growing period was 0.7 kg/d and 5.5 kg/d/animal, respectively. Feed conversion ratio in steers fattened for different periods was similar during the early growing period, however, it was lower for the late growing period (p<0.05) in the order 27 mo>25 mo>29 mo. During the whole fattening period, feed conversion was lower in 25 mo compared with 27 mo or 29 mo (p<0.05). In addition, feed conversion ratio was lower (p<0.05) in 25 mo or 27 mo compared with 29 mo during the whole period. Thus, 25 mo and 27 mo had a beneficially lower feed conversion ratio compared with 29 mo.

Carcass characteristics
Slaughter and carcass weights of steers were increased as fattening period was increased (p<0.05) ( Table 4). In carcass yield traits, rib-eye area was similar in steers on different treatments, whereas back fat thickness was significantly higher and yield index was lower in 29 mo compared with 25 mo or 27 mo (p<0.05). In the yield grade of 25 mo, appearances of A, B, and C grades were 60%, 40%, and 0%, respectively. The A, B and C grades in 27 mo were 53%, 47%, and 0%, respectively, whereas the A, B and C grades in 29 mo were 33%, 47%, and 20%, respectively. Similar to the present results, Kim et al. (2005) reported that quantity grade A was decreased with increasing age of Hanwoo steers. In quality traits, marbling score, fat color, texture, and maturity were similar in steers on different treatments. Meat color was beneficially lower in the order 27 mo<25 mo<29 mo (p<0.05). The numerically higher marbling score with increasing fattening period could be related to the higher back fat thickness as  Nade et al. (2003) reported that the deposition of body fat such as back fat might be influenced by the feed intake. Yang and Ahn (2001) reported that back fat thickness and marbling score were increased as carcass weight was increased in Hanwoo steers. Thus, marbling score and back fat thickness could be activated as increasing fattening period with feed intake. The appearance of 1 ++ , 1 + and 1 quality grade of beef was 87%, 87%, and 93% in 25 mo, 27 mo and 29 mo, respectively. The best 1 ++ grade appearance was 7%, 13%, and 7% in 25 mo, 27 mo and 29 mo, respectively. The present results for meat quality were supported by the marbling score by ultra sound scanning examination which was significantly and numerically higher in 25 mo compared with 27 mo or 29 mo (Table 5). Therefore, intramuscular fat may have been deposited from 25 months of age and consistently lasted until steers were 29 months of age.

Expression levels of lipogenic-related genes
Lipogenesis encompasses the processes of fatty acid synthesis and subsequent triglyceride synthesis and takes place in both liver and adipose tissue, and various enzymes take part in this metabolism (Kersten, 2001). According to Lee et al. (2006) lipogenic-related genes were significantly increased in 27 months-old compared to 12 months-old Hanwoo steers. These reports suggested that expression of lipogenic-related genes may be related with overall fat deposition in cattle. Among these genes, SCD is the major enzyme responsible for conversion of saturated fatty acids into monounsaturated fatty acids in mammalian tissues. Previous reports showed that SCD was a good indicator for intramuscular fat content and fatty acid composition (Taniguchi et al., 2004;Wang et al., 2008). In the present study, the expression level of the SCD gene was significantly increased during the late fattening stages (p<0.01, Figure 1). In addition, mRNA of ACC, ACL and GLUT4 gene were highly expressed in 29 mo compared with 25 mo and 27 mo (p<0.05). However, FABP4 and LPL genes were not significantly different among the treatments.
Although expression of lipogenic-related genes may differ from various body fat pads or organs, unlike the present results lipogenesis in subcutaneous fat and intramuscular fat was higher in 30 months-old Hanwoo bulls compared with those at 24 months of age (Chung et al., 2000). Smith and Crouse (1984) reported that expression level of the ACL gene increased with increasing age of Angus×Hereford crossbred steers. However, the age of these animals, which ranged between 9 and 18 months, was different from that of the present experimental animals. Grading ranges are 1 to 9 for marbling score with higher numbers for better quality.

Figure 1.
Expression levels (2 -ΔCt ) of lipogenic-related genes in Hanwoo Longissimus muscle, determined using real-time RT-PCR. a, b, c As above in Table 3; Abbreviations are defined in Table 2.
Cattle undergo a dramatic increase in intramuscular fat during a late-finishing stage that occurs between 18 and 26 months of age. In Wagyu (Japanese Black) cattle, approximately 20% of the total intramuscular fat is added between 12 and 26 months (Nishimura et al., 1999) rather than after 27 months of age. In the present study, expression levels of lipogenic-related genes were little different in essence, although the patterns of gene expression were different among the muscle samples. Thus, these findings indicate that rate of lipogenesis made slow progress after the age of 27 months in Hanwoo cattle.

CONCLUSION
The present findings indicated that different fattening periods resulted in similar carcass characteristics and marbling score in Hanwoo steers. In addition, expression of several lipogenic genes in the Longissmus muscle varied with different fattening periods without consistent effects. Therefore, the present results indicated that different fattening period has no major effect on carcass characteristics, although 25 mo had lower carcass weight compared with 27 mo or 29 mo.