Effects of Spent Mushroom Substrates Supplementation on Rumen Fermentation and Blood Metabolites in Hanwoo Steers

: This study was designed to investigate the effects of supplementation of spent mushroom substrates (SMS) on rumen fermentation and blood metabolites in Hanwoo steers. The experiment was conducted as a duplicated Latin square design with six Hanwoo steers (600 ± 47 kg), each permanently fitted with a ruminal cannula. There were three treatments; i) control (concentrates 4.8 kg +rice straw 1.2 kg/d), ii) Pleurotus eryngii a (PE) treatment (concentrates 4.8 kg+rice straw 0.73 kg+ Pleurotus eryngiia 1.20 kg/d) and iii) Pleurotus osteratus (PO) treatment (concentrates 4.8 kg+rice straw 0.73 kg+ Pleurotus osteratus 1.20 kg/d). There were no major effects of different dietary treatments on rumen parameters such as pH, ammonia-N, individual and total VFA production. Parameters of N utilization, including blood urea nitrogen (BUN), total protein and albumin levels, were not significantly different among the treatments, except for creatinine. Thus, the present results indicated that protein utilization was mostly unaffected by SMS treatments such as PE and PO, even though creatinine concentration was lower in PE compared with control and PO treatments (p<0.05). The present results indicate that Pleurotus eryngii and Pleurotus osteratus could be used as a forage source to replace 40% of rice straw without any negative effects on rumen fermentation and blood metabolites in Hanwoo steers. ( Key Words : Spent Mushroom, Rumen Fermentation, Blood metabolites, Hanwoo)


INTRODUCTION
Similarly to the global situation, mushroom production has been increased in Korea in response to trends in human health and well-being. The majority of the spent mushroom substrates (SMS) comprises the remains of the compost in which mushrooms are produced. The mushroom industry has been considering problems with SMS from an environmental standpoint concerning its effective disposal and recycling. Previous studies have shown the feasibility of using these kinds of waste to produce animal feed (Calzada et al., 1987;Zhang et al., 1995;Adamovic et al., 1998;Bae et al., 2006), because SMS is a nutrient-rich organic by-product of the mushroom industry. In addition, feed expenses comprise about 40% of the total product cost in fattening Hanwoo in Korea (KOSIS, 2010). Thus, dietary use of SMS in animal feed could be feasible from an economic point of view. Xu et al. (2010) recommended a SMS level of 6.5% dietary DM in a silage-based total mixed ration for wethers. Also, Kim et al. (2010) suggested that ensiled SMS could be used as an appropriate forage source in maintenance rations for ruminants, possibly due to high levels of fiber.
Therefore, this study was conducted to investigate effects of dietary SMS supplementation such as Pleurotus ostreatus or Pleurotus eryngiia on rumen fermentation and blood metabolites in Hanwoo steers. 0.73 kg+Pleurotus eryngiia 1.20 kg/d) and Pleurotus osteratus (PO) treatment (concentrates 4.8 kg+rice straw 0.73 kg+Pleurotus osteratus 1.20 kg/d). The formulation and chemical analysis of experiment feeds are shown in the Table 1. Water and trace mineral salt were available freechoice. Each experimental period lasted 14 d, with a 13 d period of adaptation to the diets, followed by a 1 d sampling period. Rice straw and/or SMS were offered at 09:00 h and concentrates were offered in two equal feeds at 09:00 and 16:00 h. Refusals were removed daily at 08:30 h and recorded.

Sample preparation and analytical methods
On the last day of each period, fermentation characteristics were determined at 0, 1, 3, 5, 7 and 9 h postmorning feeding. Rumen contents were sampled in the area of the ventral blind sac, with the composite sample being strained through four layers of cheesecloth. The pH of ruminal fluid was immediately determined using a pH meter (Coring model 530 pH meter, Artington, UK). To determinate volatile fatty acids (VFA) and ammonia-N, 1 ml rumen fluid was treated with 0.2 ml HPO 3 for 30 min and stored at -20°C. VFA concentration of rumen fluid was analyzed with gas chromatography (Varian, CP-3800, USA) according to the method of Erwin et al. (1961) after the sample was filtered through a 0.45 μm disposable micro filter. Ammonia-N concentration was analyzed by UVspectrophotometer (UVIKON 923, Double beam UV/VIS) at 630 nm according to the method of Chaney and Marbach (1962).
Blood samples were collected before morning feeding on Day 14 by jugular vein puncture into two 10-ml vacuum tubes containing K 3 -EDTA (Vacutainer, Becton Dickinson, Franklin Lakes, NJ, USA). Samples were centrifuged (5,000×g for 20 min at 4°C) and frozen at -20°C until analyzed. Blood urea nitrogen, glucose, albumin, creatinine and total protein concentrations were analyzed using an automatic blood analyzer (Express Plus, Ciba-Corning, CA, USA) according to the urease method of Rocch-Ramel (1967), the hexokinase method of Farrance (1987), the bromocresol green method of Doumas et al. (1971), the picric acid method of Husdan and Rapoport (1968), and the biuret method of Flack and Woollen (1984), respectively. Chemical composition of the diet was determined by the method of AOAC (1990). The analysis of neutral detergent fibre (NDF) and acid detergent fibre (ADF) was carried out

Statistical analysis
Statistical analyses were performed using the GLM procedure of SAS (2002) in a 3×3 Latin square design with two replicates. Means among treatments were compared by least significant difference (LSD) test.
The model used to analyze blood metabolites was Y ijk = μ+α i +β j +γ k +e ijk where Y ijk = observation, μ = mean, α i = treatment effect, β j = animal effect, γ k = period effect, and e ijk = residual error.
The effects included replicate, treatment, animal and period. Significance was declared at p<0.05.

Rumen parameters
SMS from PE and PO had no major influence on rumen fermentative parameters such as pH, ammonia-N, total and individual fatty acid concentrations (Figures 1 and 2). On every sampling time post-feeding, rumen pH values were not significantly different among the treatments (Figure 1a) except at 3 h post-feeding when the rumen pH was higher in the order of 6.64 Control>6.47 PE>6.39 PO (p<0.05). The rumen pH tended to decrease from 0 to 3 h post feeding, and tended to increase from 3 h up to 9 h post-feeding. Overall, rumen pH values among the treatments ranged between 6.4 and 7.0, which were above the low pH limiting value for microbial fermentation (Figure 1a) (Cardozo et al., 2000(Cardozo et al., , 2002, and indicated that SMS from PE or PO supplementation was an alternative to forage in maintaining a stable rumen pH. Ammonia-N concentration was not significantly different among the treatments at 0, 1, 3, 5 and 7 h, but the concentration was lower (p<0.05) in the control compared with PE and PO at 9 h post-feeding (Figure 1b). In addition, the ammonia-N concentrations of all treatments increased within 1 h of feeding and then declined gradually. The present results showed that there was no major effect of dietary SMS from PE or PO on ammonia-N concentration. The concentrations of total and individual volatile fatty acids were similar across the treatments, but were decreased in the control compared to both PE and PO treatments at 5 h (p<0.05) post-feeding. According to Fazaeli and Talebian Masoodi (2006), inclusion of spent compost straw up to 20% of the diet did not affect the digestibility of nutrients which may reflect the absence of a negative effect of dietary inclusion SMS on rumen fermentation. Although the present study did not investigate digestibility of nutrients, we could conclude that 40% SMS from PE or PO of rice straw does not decrease rumen microbial fermentative activity.
Therefore, based on the results of rumen fermentation parameters, the present study suggests that dietary supplementation with SMS, such as PE and PO, as a forage source could replace 40% of rice straw. Further research will be needed to determine growth performance and digestibility of nutrients with SMS supplements from PE or PO.

Blood metabolites
Effects of dietary SMS from PE or PO on blood metabolites are shown in Table 2. Blood glucose concentration was not significantly different across the treatments. Blood glucose is one of the most common metabolites used to assess the energy status of cattle (Ndlovu et al., 2007). Propionate derived from rumen fermentation is considered to be the major gluconegenic precursor in fed ruminants. Therefore, blood glucose concentration shows a similar trend to ruminal propionate concentration. Usually, albumin and total protein have low variability in blood (Ndlovu et al., 2007). As reported in other studies (Chumpawadee et al., 2006;Javaid et al., 2008), BUN concentration shows a similar trend to ruminal ammonia-N. Thus, the present results indicate that protein utilization is mostly unaffected by SMS from PE or PO. Creatinine concentration was lower in PE compared with control and PO treatments (p<0.05). Previous studies reported that blood creatinine concentrations vary with diet, breed, muscle mass, sex and season (Otto et al., 2000;Miller et al., 2004;Grunwaldt et al., 2005;Hammond, 2006). The reduced concentration of creatinine in the present study may be explained by prolonged active tissue protein catabolism.

CONCLUSION
The present study showed that there were no major differences in rumen fermentative parameters such as pH, ammonia-N concentration and volatile fatty acids, and blood metabolites such as glucose, urea-N, total protein, albumin and creatinine among the treatments. Thus, it should be possible to replace 40% of rice straw as a forage source with SMS from PE or PO for ruminant feed without any severe negative metabolic effects, which could thereby economically reduce feed expense and product cost in the Hanwoo industry.

ACKNOWLEDGMENTS
This work was carried out with the support of "Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ007474)" Rural Development Administration, Republic of Korea, and partially supported by a grant from the ARPC (Agricultural R&D Promotion Center, Korea). Also, this work was supported by research funds of Chonbuk National University in 2010.