J. Anim. Sci. 2005. 83:344-349
© 2005 American Society of Animal Science
ANIMAL GROWTH, PHYSIOLOGY, AND REPRODUCTION |
Effects of propylene glycol on carcass traits and its related gene expression in Korean native steers1
Y. K. Kim*,
H. Choi
and
K. H. Myung,2
* Animal Genetic Resources Station, National Livestock Research Institute, Namwon, Jeonbuk 590-832; and
and
Department of Animal Science, Chonnam National University, Gwangju 500-757, South Korea
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Abstract
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The effects of propylene glycol (PEG) on performance, ruminal fermentation, blood glucose and insulin, carcass traits, and abundance of IGF-1 mRNA in LM and leptin mRNA in adipose tissue were examined in 20 Korean native steers, with 10 each in control and PEG-fed groups, respectively. Propylene glycol mixed with concentrate diet was provided daily at a rate of 2.5 mL/kg BW0.75. Experimental animals were fed a concentrate diet to 1.8% of BW twice daily plus rice straw ad libitum during the 4-mo period before marketing. Daily DMI and ADG did not differ between control and PEG-fed steers. Steers receiving PEG displayed an increase (P = 0.044) in propionate concentration, whereas acetate concentration decreased (P = 0.032). Although blood glucose was not affected, serum insulin was increased (P = 0.047) by PEG feeding. Propylene glycol did not affect carcass weight, 13th-rib fat depth, marbling score, or lipid content of LM. The backfat of PEG-fed steers did not differ in leptin mRNA from control steers, whereas increased leptin mRNA was found in i.m. fat with PEG feeding. There was no treatment effect on the level of IGF-1 mRNA in the LM of the tested steers. These results indicate that the amount of PEG fed to steers was not sufficient to improve marbling score through enhanced ruminal propionate and insulin. The role of increased i.m. leptin mRNA level in PEG-fed steers remains to be further elucidated.
Key Words: Carcass Traits • Insulin-Like Growth Factor-1 • Leptin • Performance • Propylene Glycol • Steers
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Introduction
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Beef producers continuously try to decrease carcass fatness, while at the same time aim to increase i.m. adipose (marbled fat) levels to meet consumer preferences. This can be accomplished only if the factors regulating lipid deposition in i.m. adipose tissue and other fat depots differ substantially. Ruminants represent a special case with respect to de novo lipogenesis because most dietary carbohydrates are extensively fermented in the rumen, and even when grains are fed, there is little absorption of glucose from the small intestine (Rowe and Pethick, 1994
). As a consequence, acetate derived from fermentation in the rumen is thought to be the main source of carbon for fatty acid synthesis (Vernon, 1981); however, there also is evidence for some synthesis from glucose, especially via lactate (Prior, 1978; Smith, 1995). Studies by Smith and Crouse (1984) have shown that adipocytes associated with the i.m. depot have a higher reliance on glucose and/or lactate as a substrate than acetate. Administration of glucogenic precursors such as propylene glycol (PEG) is effective in decreasing plasma NEFA to prevent ketosis through early lactation in dairy cows (Christensen et al., 1997). Two reports have demonstrated at least two enzymatic pathways in animals for the conversion of PEG to glucose via lactaldehyde and lactic acid (Gupta and Robinson, 1960; Miller and Bazzano, 1965). Circulating concentrations of leptin may provide an indicator of fat content in live cattle (Geary et al., 2003) and the increased IGF-1 mRNA level in muscle has been associated with an increased rate and efficiency of muscle deposition in steers (Pampusch et al., 2003). Therefore, it might be possible to use blood concentrations of leptin and/or IGF-1 as predictors of carcass composition in beef cattle. The objective of this experiment was to evaluate the efficacy of PEG feeding once daily for 4 mo before slaughter at market live weights to increase marbling scores in Korean native steers and its effect on its related gene expression.
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Materials and Methods
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Animals and Feeding Management
All experimental procedures were approved by the Chonnam National University Animal Ethics Committee. Twenty 2-yr-old Korean native steers with an initial mean BW of 516 kg were stratified by weight, and 10 steers were assigned randomly to one of two treatments: 1) control, and 2) PEG (SKC Co., Ltd., Seoul, South Korea) fed. All steers were housed individually in separate pens and fed individually. Beginning 2 mo before the initiation of the study, steers were fed the concentrate diet (NongHyup Feed Inc., Gimjae, South Korea; Table 1) at a rate of 1.8% of BW on a DM basis twice a day, with rice straw fed separately and ad libitum. The composition of the concentrate diet met the requirements of finishing steers (NRC, 2000; KMAF, 2002). The nutritive composition of rice straw was 5.0% CP, 0.3% Ca, 0.1% P, 66.1% NDF, and 44.7% ADF on an as-fed basis. The control animals were maintained on the concentrate diet until the end of the experiment, whereas for the PEG-fed steers, 2.5 mL of PEG/(kgO.75•d) replaced 2.5 g of the concentrate feed for the 4 mo before slaughter. Animals were fasted for 24 h before slaughter, and live weight was determined. One day after slaughter, carcasses were weighed and graded to determine the 13th-rib backfat thickness, cross-sectional area of LMA, Korean Ministry of Agriculture and Forestry quality grade, and marbling score (KMAF, 2003). The LM from the 10th to 13th ribs was removed from the right side of each carcass, trimmed of external fat, ground three times, and subsampled for determination of moisture and ether-extractable lipid (AOAC, 1996).
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Table 1. Ingredient and chemical composition of concentrate diet (as-fed basis) fed to steers during the experimental period
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Rumen and Blood Sample Preparation, and Analysis
Ruminal fluid was sampled within 2 h of the morning feeding during the middle of the experimental period (wk 8). Approximately 100 mL of ruminal fluid was collected by stomach tube and strained through layers of cheesecloth. Samples of 10 mL were acidified with 0.5 mL of H2SO4 and frozen for subsequent VFA analysis. These samples were prepared as follows: 1) sample tubes were thawed and centrifuged at 20,000 xg, 4°C for 15 min; 2) 1 mL of supernatant was transferred into a microfuge tube, 0.2 mL of 25% metaphosphoric acid was added, and the mixture was vortexed before incubating at room temperature for 30 min, and; 3) supernatant fluid was transferred into a GLC sample vial for analysis by GLC (3400 CX, Varian, Walnut Creek, CA) using a 60 m x0.25 mm i.d. fused-silica capillary column (SP-2380, Supelco, Bellefonte, PA). On the same day as the ruminal fluid sampling, blood samples were obtained from the jugular vein 90 min after the feeding of PEG. The concentrations of glucose in whole blood were measured using an on-site glucose tester (Accu-Check; Roche Diagnostics GmbH, Mannheim, Germany). The analysis of insulin in blood was as described previously (Studer et al., 1993). Insulin intraassay variation was 7.8%, and interassay variation was 6.5%.
RNA Isolation and Real-Time Reverse Transcriptase-PCR (RT-PCR)
Immediately after slaughter, four steers from each group were selected on the basis of slaughter weight to sample for RNA isolation. Approximately 100 to 150 g of s.c. backfat and LM were collected from the left side of the carcass between the 10th and 13th ribs area on the day of slaughter. Samples were snap-frozen in liquid N and stored at –80°C for subsequent RNA isolation (Chomezynski and Sacchi, 1987). Thereafter, LM i.m. fat tissues were trimmed from visible fat in the muscle under liquid N vapor. Concentration of RNA was determined by absorbance at 260 nm. The integrity of RNA was determined by electrophoresis of total RNA through a 1% agarose-formaldehyde gel, followed by ethidium bromide staining to allow visualization of 28S and 18S ribosomal RNA. Real-time RT-PCR was used to measure the quantities of leptin and IGF-1 mRNA relative to the quantity of cyclophilin mRNA in total RNA isolated from backfat, i.m. fat and LM tissues from control and PEG-fed steers. Measurement of the relative quantity of the cDNA of interest was conducted using SYBER Green real-time RT-PCR Master Mix (Qiagen, Valencia, CA), appropriate forward and reverse primers (0.5 µM; Table 2), and 0.2 µg RNA as previously described (Kim et al., 2003). Assays were performed in the Rotor-Gene 2000 real-time cycler (RD-2072D, Corbatt Research, Sydney, Australia) using appropriate analysis software (Corbatt Research, Sydney, Australia) with thermal cycling parameters recommended by the manufactures (40 cycles of 15 s at 94°C and 30 s at 55°C). Titration of cyclophilin, leptin, and IGF-1 (0.5 µM) forward and reverse primers against increasing amounts of cDNA gave linear responses with slopes of –0.24.
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Table 2. Forward and reverse primers for real-time polymerase chain reaction for leptin, insulin-like growth factor-1, and cyclophilin mRNA
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Statistical Analyses
Data were analyzed as a completely randomized design, with individual steers serving as the experimental unit. Each diet was compared by t-test (SAS Inst., Inc., Cary, NC). The following statistical model was used in the analysis:
where Yij = dependent variable (general observation), µ= the overall mean, Ti = effect of ith treatment, and eij = error term.
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Results and Discussion
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Performance
Body weights for steers fed control and PEG-enriched diets are shown in Table 3. No significant treatment response was found to dietary PEG supplementation for the duration of the trial. The DMI also did not differ between control and PEG steers. The initial and finishing BW of the PEG-fed animals were 572.3 and 677.5 kg, respectively, and therefore, the mean dose of PEG was 296 and 333 mL/d, respectively. All animals consumed PEG-containing concentrate within 90 min of offering. This result concurs with observations in dairy cows fed 341 mL/d of PEG; these cows consumed almost the same amount of concentrate as the control animals (Christensen et al., 1997). Thus, the quantity of PEG provided in the current study did not seem to decrease concentrate intake markedly in the finishing phase of the growth cycle of beef steers. Dietary PEG decreased total feed consumption by 7.3%, resulting in G:F ratios of 0.09 and 0.10 in the control- and PEG-fed steers, respectively. The results suggest that PEG had almost the same nutritive values as the concentrate diet in this study, as there was no difference in final BW between the two groups (Table 3).
Rumen VFA, Serum Glucose, and Insulin
The VFA concentrations in the rumen during the middle period of PEG feeding are shown in Table 4. The propionate concentration increased (P = 0.044), whereas acetate concentration deceased (P = 0.032) as a result of PEG feeding, which confirms the results of Emery et al. (1964), who reported an increase of more than 10% in ruminal propionate with PEG feeding to lactating dairy cows. The ratio of acetate:propionate also was increased (P = 0.042) by PEG feeding. Blood glucose tended to be increased (P = 0.161) by PEG, which was similar to the results of Grummer et al. (1994), in which plasma glucose was increased in a dose-dependent manner by its inclusion in the diet. However, the smaller effect in the present study suggests that PEG is more effective in increasing blood glucose when cattle are experiencing nutrient deprivation than when cattle are well fed as in the present trial (Table 5). This small increase in blood glucose seems have been caused mainly by PEG being metabolized directly in tissues, as Emery et al. (1964) indicated that more than 99% of PEG administrated was metabolized in this way. Although blood glucose concentration was not significantly altered, insulin was increased (P = 0.047) in steers receiving PEG, presumably due to the nonsignificant increase in blood glucose and the significant increase of ruminal propionate. Dietary PEG has increased serum insulin in previous studies with cows (Grummer et al., 1994; Studer et al., 1993).
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Table 4. Effect of propylene glycol (PEG) on ruminal VFA and the acetate-to-propionate ratio of Korean native steers (mol/100 mol)
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Carcass Characteristics
As expected, cold carcass weights of PEG steers did not differ from those of control animals (Table 6). Feeding PEG to the steers tended to increase LM area by 11% (84.5 vs. 89.9 cm2). In terms of carcass quality, 30% of the PEG-treated steer carcasses graded 1+, and 40% graded as 1 on the Korean Animal Products Grading System (KMAF, 2003). In contrast, 10% of the carcasses from control steers graded 1+, and 40% graded 1 (Table 6). The mean marbling scores were 5.8 and 6.2 for control and PEG-fed steers, respectively. Although there was an increase from 14.56 to 15.23% in ether-extractable fat in LM with PEG feeding, it did not differ between treatments. Adipose tissue depots in ruminants are thought to develop in the order of abdominal, intermuscular, s.c., and finally the i.m. depot (Vernon, 1981). The studies of Smith and Crouse (1984) showed that adipocytes associated with marbling have a higher reliance on glucose and/or lactate as a substrate than acetate. Thus, although neither marbling score nor lipid content of the LM was significantly increased, the trend toward an increase of muscle fat content is consistent with the increase in greater numbers of carcasses with a higher quality grade in the PEG-fed steers (Table 6), especially during the late fattening period.
Leptin and IGF-1 Expressions in Tissues
The backfat of steers offered the PEG supplement over the 160 d did not differ statistically in leptin mRNA levels from control steers (Figure 1); however, LM i.m. fat leptin mRNA levels in PEG-fed steers were increased 75% (P < 0.01) relative to those of controls (Figure 2). Geary et al. (2003) also reported positive correlations between serum leptin and marbling score, and the increased leptin may reflect increased i.m. fat that would be consistent with the higher quality grade (1+) and a tendency for increased marbling scores (Table 6
) in PEG-fed steers. Nonetheless, this work has to be considered preliminary due to a small sample size (n = 4) for leptin mRNA. On the other hand, the inconsistent result of backfat leptin mRNA levels and backfat thickness (Table 6) cannot be completely reconciled. This is most likely due to increased i.m. fat deposition over the latter stages of the growth cycle (Vernon, 1981), and its dependence on glucose derived from PEG for lipogenesis (Smith and Crouse, 1984; Table 5). Longissimus muscle IGF-1 mRNA levels were not affected by PEG feeding (Figure 3). In other studies, the key anabolic growth factor, IGF-1, increased sharply in muscle in which ADG was increased by 34% in feedlot steers (Pampusch et al., 2003) in response to steroid implantation. In the current study, as steers of both treatments were provided almost the same amount of energy, resulting in equivalent ADG, it was not surprising that muscle IGF-1 mRNA levels did not differ between the two treatment groups.
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Figure 1. Subcutaneous backfat tissue leptin mRNA level in Korean native steers that were untreated (control) or fed 2.5 mL/(kg0.75•d) of propylene glycol (PEG) over 120 d at the end of fattening period. The data are expressed as a percentage of the value observed in the control. The value of mRNA was 100 and 57% for control and PEG groups, respectively. Each bar was normalized by the quantity of cyclophilin from the same tissue. The bar graph represents the mean ±SEM of four steers.
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Figure 2. Longissimus i.m. fat leptin mRNA level in Korean native steers that were untreated (control) or fed 2.5 mL/(kg0.75•d) of propylene glycol (PEG) over 120 d at the end of fattening period. The data are expressed as a percentage of the value observed in the control. The value of mRNA was 100 and 175% for control and PEG groups, respectively. Each bar was normalized by the quantity of cyclophilin from the same tissue. The bar graph represents the mean ±SEM four steers. The asterisks indicate that the mean for PEG-fed steers differs (P <0.01) from control steers.
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Figure 3. Longissimus muscle IGF-1 mRNA level in Korean native steers that were untreated (control) or fed 2.5 mL/(kg0.75•d) of propylene glycol (PEG) over 120 d at the end of fattening period. Data are expressed as a percentage of the value observed in the control. The value of mRNA was 100 and 125% for control and PEG groups, respectively. Each bar was normalized by the quantity of cyclophilin from the same tissue. The bar graph represents the mean ±SEM of four steers.
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Implications
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Under the conditions of this study, it seems that feeding propylene glycol to growing beef cattle to increase glucose availability is not a viable method for increasing the proportion of intramuscular fat in the carcass. The increased intramuscular leptin mRNA levels in propylene glycol-fed steers do indicate, however, that this important adipocyte hormone may play a role in this process. Further investigation of this relationship is therefore warranted.
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Footnotes
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1 This research was funded by the Technology Development Program for Agriculture and Forestry, Ministry of Agriculture and Forestry, Republic of Korea (Grant No. 102035-2). 
2 Correspondence: 4-431 Animal Science (phone: +82-62-530-2122; fax: +82-62-530-0431; e-mail: khmyung{at}chonnam.ac.kr).
Received for publication March 8, 2004.
Accepted for publication November 4, 2004.
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Abstract
Introduction
