Ⅰ. INTRODUCTION

Na-bentonite (NaB) is an expanded lattice clay of the montmorillonite group of minerals (Bates and Jackson, 1980) with a high ion exchange capacity that binds a wide range of cations (Fenn and Leng, 1989). Incorporated into diets, it has improved wool growth of sheep (Fenn and Leng, 1989; Cobon et al., 1992), decreased ruminal ammonia concentrations, and improved feed and bacterial protein flow to the small intestine of ruminants (Ivan et al., 1992). Because acid buffering capacity has been correlated with total cations and total ash (Jasaitis et al., 1987), mineral buffers such as NaB might be effective in alleviating acid stress in ruminants under intensive feeding and management programs. The swelling capacity of NaB might provide a desirable habitat niche for probiotic microbes in the ruminant digestive tract. 

It is well known that probiotic microbes improve gut microbial balance, feed intake, weight gain and feed efficiency in ruminants(Yoon and Stern, 1995; Krehbiel et al., 2003). Kim et al. (2007) reported that feeding probiotic cultures improved growth, meat quantity and meat quality of Hanwoo steers. However, there is no research conducted on feeding effects of NaB and probiotics together on cattle. It is, therefore, postulated that the addition of probiotics with NaB might show a desirable effect on meat quality and quantity. 

Based on previous reports of the beneficial effects of bentonite and probiotic cultures incorporated into diets of ruminants, a study was conducted to determine the effects of these dietary additives on meat quality and quantity by Hanwoo steers. 

Ⅱ. MATERIALS AND METHODS

1. Animals and treatments

All animal care protocols were approved by the Konkuk University Institutional Animal Care and Use Committee. Twenty four-Hanwoo (Bos taurus coreanae) steers at 9 mon of age (average BW 232 kg) were allotted in groups of 4 steers to each of 6 pens. Four of the pens were located at Farm 1, and 2 at Farm 2 in Boeun County, Chungbuk Province, Korea. Steers were fed one of two rations. Each ration was fed to steers in two pens located at Farm 1 and one pen at Farm 2. The steers were fed a control diet (concentrate mix and rice straw), and a treatment diet (the control diet + 0.5-1.0% NaB + 0.5-1.0% probiotics). Periods for growing, fattening and finishing were 6, 8, and 8 mon, respectively. Diets were fed for 22 mon until the animals were slaughtered. 

Feeds and feed additives were supplied daily to the steers in a manner shown in Table 1. The concentrate mix (Table 2) was fed in a restricted manner to achieve 0.8 kg levels of average daily gain (ADG) during the growing period, and over 0.9 kg ADG during the fattening period. Animals had free access to rice straw at all times. The NaB and probiotics were top-dressed at each feeding time. Feed was supplied twice a day at 07:00 h and 18:00 h. During the finishing period, the concentrate mix was fed ad libitum and rice straw was restricted at a level of approximately 10% of concentrate mix. 

Table 1. Diets for growing, fattening and finishing periods for Hanwoo steers¹⁾

Table 2. Chemical composition of concentrate mix and rice straw^1),2),3)

Animals were observed for health status, and body weight was measured on a monthly basis throughout the study. Samples of concentrate mix and rice straw were collected every 2 wk for proximate analysis. 

The chemical composition of the commercial concentrate mix and rice straw fed to the steers is presented in Table 2. The bentonite product (Bionit, Korea Sud Chemical, Korea) used in the study was an extra-purified powder for animal use, and was composed of 75~85% Montmorillonite, 58% SiO₂, 20% Al₂O₃, 6% Fe₂O₃, 3.5% MgO, 2.5% CaO, 2% Na₂O, and 1% K₂O. Based on individual minerals, it contained 1.41% Ca, 0.04% P, 0.10% Mg, 1.78% K, 0.66% Na, 1,631 ppm Fe, 7.9 ppm Zn, 5.0 ppm Cu, and 212 ppm Mn. The pH was about 10. The swelling volume was 9 mL/g, and the cation exchange capacity was 80 meq/100 g. The specific surface area was 400-600 m²/g. The probiotics used in the study was a mixture of Bacillus subtilis and Saccharomyces cerevisiae at a viable cell concentration in excess of 10⁶ cfu/g for each culture. The cultures were grown on rice bran and the culture mixture had 85% DM, 16.0% CP, 14.0% ether extract (EE), 6.3% crude fiber (CF) and 10.3% crude ash. Based on the individual minerals, the probiotics contained 0.19% Ca, 1.41% P, 0.25% Mg, 1.79% K, 0.04% Na, 231 ppm Fe, 40 ppm Zn, 6.9 ppm Cu, and 184 ppm Mn. 

The control diet (concentrate mix + rice straw) was lack in trace mineral contents as shown in Table 3. Dietary Zn levels in growing, fattening and finishing periods, respectively were lower than NRC (2000) requirement of beef cattle. Dietary Cu levels according to the periods were much lower than the requirement (NRC, 2000). When the control diet was added with NaB and probiotics, the levels of Zn and Cu (48 and 12 ppm, respectively) were higher than the NRC requirement (NRC, 2000). 

Table 3. Zn and Cu levels in the control diet according to feeding periods¹⁾

2. Sampling and chemical analysis

Feed samples taken from troughs prior to feeding were dried and ground to pass through a 1 mm screen using a Sample Mill (Cemotec, Tecator, Sweden). The content of dry matter was determined by drying samples at 105℃ for 24 h to constant weight. The contents of crude protein, EE, acid detergent fiber (ADF), and ash were determined by the AOAC methods (2000). The content of ash free neutral detergent fiber (NDF) was determined according to the method of Van Soest et al. (1991). Organic matter (%) was determined as 100 minus ash %.

Steers were withdrawn from the experimental diets 24 h before slaughter. Following a 24-h carcass chill, yield and quality grades were assigned to each carcass using Korean carcass grading standards specified in the attached list No. 4 of Korean Livestock Enforcement Regulation (KLER, 2007). The 12th to 13th rib longissimus muscle was removed, retained from each steer and frozen until later analysis. 

For mineral analysis of the rib muscle, samples were analyzed for Ca, P, Ma, K, Na, Mn, Fe, Zn and Cu by inductively coupled argon plasma emission spectroscopy (ICP-OES 5300DV, Perkin Elmer, USA) as described by Braselton et al. (1997). 

For animal health diagnosis, blood samples were taken from jugular vein of steers during the growing period and an equal portion was divided into bottles with or without anti-coagulant EDTA. Serum profiles were analyzed using an Automatic Biochemical Analyzer (Hitachi 7170A, Hitachi Ltd., Tokyo, Japan) based on photometer and ion selective electrode methods, and whole blood profiles were analyzed with an Automatic Blood Analyzer (Coulter STKS, Beckman Coulter Co., Miami, FL, USA) based on impedance and VCS (volume, conductivity, light scattering) methods.

3. Statistical analysis

Data were analyzed using farms as a block in a randomized complete block design by the General Linear Model (SAS Institute, Inc., 1990). Comparison of means between control and NaB treatment was made using studentized-t test (SAS Institute, Inc., 1990). Significant differences were detected at p <0.05. 

Ⅲ. RESULTS AND DISCUSSION

1. Meat mineral profile

The effect of the dietary treatment on the meat mineral profiles of steers is presented in Table 4. Feeding a diet (treatment) added with NaB and probiotics affected (p < 0.05) the mineral concentrations in the longissimus muscle. Compared with the control, the treatment increased concentrations of Zn, Cu, Fe, P, Mg and Na, but concentrations of Mn, Ca and K were not affected. The longissimus muscle of steers assigned to the treatment had higher levels of Zn (49.0%), Cu (100%), Fe (34.5%), P (49.3%), Mg (46.1%), and Na (43.9%) than the control group. Individual herd data showed little variation associated with the increasing rates of the specific minerals (data not presented). The mineral concentrations ranged between the values reported by Westing et al. (1985) and Williams et al. (1983). However, Cu levels in the present study were rather low compared with those reported by Salles et al. (2008). This difference was attributed to the different analytical method. In a short term study with lambs Walz et al. (1998) reported a diet with 0.75% bentonite did not affect concentrations of Zn, Cu, Mn, Fe, Ca, Mg, and K in bone, liver and kidney. 

Table 4. Mineral profiles (ppm) in the longissimus muscle of Hanwoo steers fed different diets¹⁾

The control diet in this study contained more Fe, Mn, K and Na and less Zn, Cu, and Mg (data not presented) than the dietary requirement specified in KFSEC (2007) and NRC (2000). Dietary supplementation of these deficient minerals especially for the treatment resulted in a higher Zn, Cu, and Mg retention in the longissimus muscle. Zinc and Cu are essential trace minerals and perform important biochemical functions. These minerals are deficient in typical human diet; however, Cu deficiency is more common and important in the elderly diet (Subar et al., 1998; Ma and Betts, 2000). Cao et al. (2000) reported that Zn supplementation of ruminant diets that were deficient in Zn resulted in increased Zn content of the ruminant muscle. 

The biological reasons for the improved effects of dietary NaB on mineral metabolism are not apparent. The improved  bioavailability of mineral might be due to the high swelling capacity of NaB resulting in a slowing in the rate of digesta passage through the gastrointestinal tract or might be related to the high cation exchange capacity of NaB. 

The use of microorganisms in animal diets has been shown to improve mineral balance. Yoon and Stern (1995) reported that yeast culture supplementation improved retention of minerals (Cu, Fe and K) in growing ruminants and Cole et al. (1992) reported that lambs fed yeast culture tended to have a better balance of Zn and Fe. The present study showed there might be a desirable trace mineral retention when a diet was supplemented with both NaB and probiotics. This phenomenon may be possibly due to a better microbial habitat attributed to the swelled NaB in the digestive tract and also due to an improved mineral uptake by the microbes.

2. Meat characteristics

The effects of the dietary treatments on meat characteristics are presented in Table 5. The cold carcass weights were 20 kg higher for the treatment than for the control, but the difference was not significant. Also, the dietary treatment did not affect carcass yield traits including backfat thickness, longissimus muscle area, yield index and yield grade and quality traits including marbling score, meat color, fat color, texture, maturity and quality grade. For the meat quality grades the number of carcasses graded as 1⁺⁺, 1⁺, 1, 2, and 3 grades were 2, 3, 4, 2, and 1 for the control group and 1, 2, 8, 1, and 0 for the treatment group. 

Table 5. Meat characteristics of Hanwoo steers fed different diets¹⁾

Overall the dietary treatment had a beneficial effect on the carcass quality grades. In another study (Walz et al., 1998), feeding 0.75% NaB did not affect carcass weight, longissimus muscle area, backfat thickness, quality grade nor yield rade of lambs. The differences in response to NaB could be due to different feeding periods and contents. When beef steers were upplemented with Zn, carcass weights were increased (Spears and Kegley, 2002). A previous study by Kim et al. (2007) reported no effect  on meat characteristics when 1% of probiotic culture was fed to steers. 

3. Blood profiles

The blood profiles of the Hanwoo steers are presented in Table 6. For blood nutrients, the dietary treatment did not show any differences in blood concentrations of triglyceride, cholesterol, high density lipoprotein, low density lipoprotein, glucose and protein. These results indicate that fat, energy and protein metabolisms within the animal body were not affected by the treatment. 

Table. 6. Blood profiles of Hanwoo steers fed different diets¹⁾

The blood electrolytes Ca, P and K were not affected by the treatment, but blood Na and Cl levels were lower (p<0.05) compared to steers on the control diet. In a similar study (Ha et al., 1985), feeding 2% NaB to lambs did not affect concentrations of blood Ca, P, and K, but Na content decreased. Also, in another study of Pulsipher et al. (1994), feeding NaB decreased blood Na and Cl levels. The exact reason for decreased blood Na and Cl leves was not explained. But they also could be associated with non-dietary factors such as hormone levels of the animals on the different diets (Church and Pond, 1982). 

Blood enzyme analyses showed little effect of the dietary treatments on liver and kidney function because steers on the treatment had similar concentrations of blood aspartate aminotransferase and lactate dehydrogenase. Blood cell counts were not affected by the treatment. Generally, values for all blood constituents were within the normal range for healthy cattle (Church and Pond, 1982; Wallach, 1974). In a similar study, Cho et al. (2001) reported that feeding clay minerals to growing Hanwoo steers did not affect their red and white blood cell counts. Pulsipher et al. (1994) reported that feeding 42 g/d NaB to lambs did not affect their blood nutrients, electrolytes, and enzymes, but there was a decrease in serum Na and Cl concentrations. None of the steers showed abnormal health problems throughout the experimental periods. 

In summary, the addition of NaB and probiotics to the diet of Hanwoo steers increased concentrations of trace minerals (Zn, Cu, and Fe) in the longissimus muscle of steers. In conclusion, the combined use of clay mineral and probiotics in the animal diet improved mineral retention in muscle without any deleterious effects on carcass traits of Hanwoo steers. 

Ⅳ. ACKNOLEDGEMENTS

This study was conducted by means of a grant from the Agenda Research Program (PJ006852072012) of the Rural Development Administration, Korea. Special thanks go to the Boeun County Agriculture Technology in Chung-Buk province for their active support. 

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