Abstract: This experiment was conducted to investigate the effect of fat content (high: 165 g fat kg^-1 DM and low: 25 g fat kg^-1 DM) of sodium hydroxide (40 g kg^-1 DM) or formaldehyde (3 and 6 g kg^-1 DM) treated sunflower meal on in vitro gas production parameters used mediums containing isolated rumen microorganisms including total rumen microbiota (TM), bacteria (B), protozoa (P) or fungi (F). Results showed formaldehyde (both applied concentrations) caused a significant reduction in the rate and gas production from fermentable fraction of sunflower meal samples by the isolated microbial groups. Sunflower meal with high fat concentration treated with NaOH had the highest gas production (p<0.05) when fermented by the rumen isolated micro-biota (193, 33, 89 and 175 mL 500 mg DM sample for TM, B, P and F, respectively). Gas produced from the chemically treated or untreated high fat containing sunflower meal was more than the low fat content samples. Therefore, it was concluded both fat concentration and chemical treatments used in the present study may affect the fermentation potential of sunflower meal as evaluated by the applied in vitro procedure. In addition, in vitro gas production of high and low fat content sunflower meal by isolated rumen microbiota fractions are influenced by formaldehyde and NaOH treatments.

INTRODUCTION

Rumen microorganisms based on the degradation rate of plant cell walls and degrading enzyme activities could be categorized into 3 distinct groups; bacteria, protozoa and fungi (Lee et al., 2000) have a key role in the degradation of polysaccharides of plant cell walls but cellulolytic bacteria, such as Ruminococcus albus, R. flavefaciens and Fibrobacter succinogenes are a major microorganisms responsible for ruminal digestion of plant cell walls due to their metabolic diversity and numerical predominance (Cheng et al., 1991).

Dijkstra and Tamminga (1995) reported that the protozoa digest fiber in the rumen by 5-21%. Although, the protozoa make a significant contribution to fibre digestion by rumen microorganisms, their removal allows more bacteria to colonize the plant fibers (Newbold et al., 1989).

The role of fungi to the degradation of any feed fiber in the rumen is still unclear due to the difficulty in estimation of exact biomass of anaerobic fungi in the rumen contents. However, recently a quantitative competitive PCR assays for relative quantifying rumen anaerobic fungal populations in both in vitro and in vivo systems has been proposed (Sekhavati et al., 2009). Lee et al. (2000) concluded that fungal activity was mostly responsible for cell wall degradation. Fungi accompanying methanogenic bacteria might increase the digestion rate of cellulose or hemicellulose (Bauchop and Mountfort, 1981). However, it was reported that cellulolytic activity of certain species of fungi could be inhibited by some fibrolytic Ruminococci (Bernalier et al., 1992). The production of gas during the microbial fermentation of plant material by rumen contents has been used as an indirect measure of substrate digestibility (Menke and Steingass, 1988; Chaji et al., 2009).

The objective of present experiment was to determine the effect of fat content (high: 165 g fat kg^-1 DM and low: 25 g fat kg^-1 DM) of sodium hydroxide (40 g kg^-1 DM) or formaldehyde (3 and 6 g kg^-1 DM) treated sunflower meal on in vitro gas production parameters used mediums containing isolated rumen microorganisms including total rumen microbiota (TM), Bacteria (B), Protozoa (P) or Fungi (F).

MATERIALS AND METHODS

Samples of Sunflower Meal (SM) containing different fat concentration (high: 165 g fat k g^-1 DM (HSM) and low: 25 g fat kg^-1 DM (LSM) were provided. They used as untreated or chemically treated using formaldehyde (37% solution of commercial grade formalin) at two levels of 3 and 6 g kg^-1 DM of formaldehyde or one level of sodium hydroxide (40 g kg^-1 DM).

The chemical reagents were sprayed on the samples and kept the mixture for 30 min at room temperature. Then samples were transferred into PVC bags, shacked for 5 min, sealed, evaporated and kept for 5 days at room temperature. Bags were then opened and spread the content in thin layers (4 mm) on a concrete floor to air-dried.

Experimental treatments were Untreated HSM (UHSM), NaOH treated HSM (SHSM), formaldehyde treated HSM [(F3HSM (3 g formaldehyde kg^-1 DM treated HSM) and F6HSM (6 g formaldehyde kg^-1 DM treated HSM)], Untreated LSM (ULSM), NaOH treated LSM (SLSM) and formaldehyde treated LSM [F3LSM (3 g formaldehyde kg^-1 DM treated LSM) and F6LSM (6 g formaldehyde kg^-1 DM treated LSM).

Rumen fluid was collected from four rumen fistulated sheep (body weight: 40 kg) prior to the morning feeding. Animals were daily fed 450 g concentrate, 550 g alfalfa hay and 180 g wheat straw once at day (09:00 a.m.). Rumen content was strained through four layers of cheese cloth.

The strained and free feed residual of rumen fluid was used as total rumen microbiota. Rumen isolated protozoa was obtained using centrifuge procedure (1000 RPM, 5 min). The isolation of ruminal fungi was carried out from non protozoa strained rumen fluid using antibacterial agent (streptomycin sulfate, penicillin G, potassium and chloramphenicol (0.1 mg m^-1 each). Ruminal bacteria was isolated from non protozoa strained rumen fluid using antifungal agent (benomyle (500 ppm L^-1) and metalaxyle (10 mg L^-1). Antibiotics and the other chemical reagents applied to isolate rumen bacteria and fungi were added as 0.1 mL per 1 mL of in vitro gas production medium culture. In vitro method of Menke and Steingass (1988) was used to determine gas produced from each ruminal microbial fractions. Approximately, 500 mg of each sample (1.0 mm screen) were incubated with 35 mL buffered rumen fluid containing TM, B, P or F under continuous CO₂ reflux in a 100 mL calibrated glass syringes for 2, 4, 6, 8, 10, 12, 16, 24, 48, 72 and 96 h in a water bath msaintained at 39°C. Each sample in 4 replicates was incubated in 3 run together with three syringes containing only incubation medium (blank). After 96 h of incubation, the culture fluid of each sample was carefully removed into centrifuge tubes and pH values were immediately measured. Cumulative gas production data were fitted to the exponential equation:

Where:

Data of gas production parameters and pH were subjected to analysis as a completely randomized block design using the general linear model procedure by SAS (1996). Duncan’s multiple range test was used to compare the means at p<0.05.

RESULTS

The gas production parameters (b and c) of chemically untreated and treated of low and high fat sunflower meal samples by total rumen microorganism, protozoa, bacteria and fungi over 96 h incubation are shown in Table 1. Formaldehyde at both concentration applied (3 and 6 g kg^-1 DM) caused a significant decrease (p<0.05) in gas production from fermentable fraction. However, NaoH caused to increase the gas production parameters when samples were incubated with isolated ruminal mirobiota.

The highest and the lowest gas production by the rumen microbial fractions were obsereved when sunflower meal containing both levels of fat was treated with NaoH and 6 g formaldehyde, respectively (p<0.05). The highest gas production for all the samples evaluated was observed in medium containing TM which was followed by B whereas, a less gas volume was produced by F and P. The patterns of gas production parameters influenced by the fat content were similar between samples fermented by various rumen isolated microbial groups. The effect of formaldehyde, sodium hydroxide and fat content of the sunflower meal samples on in vitro pH of the medium pH containing rumen isolated microbial groups shown in Table 2. The lowest and the highest pH were detected in medium used sunflower meal treated with sodium hydroxide and 6 g formaldehyde, respectively (p<0.05).

Medium pH for low fat sunflower meal samples was higher than those of high fat sunflower meal (p<0.05). There was a significant difference in medium pH values when various rumen isolated microbial groups were considered. The lowest pH values were observed in medium of TM groups and the highest was for P.

Table 1:	Gas production parameters of untreated or chemically treated (sodium hydroxide or formaldehyde) of low and high fat sunflower meal using isolated rumen microbial fractions
*Experimental treatments were untreated HSM (UHSM), NaOH treated HSM (SHSM), formaldehyde treated HSM [F3HSM (3 g formaldehyde kg-1 DM treated HSM) and F6HSM (6 g formaldehyde kg-1 DM treated HSM)], untreated LSM (ULSM), NaOH treated LSM (SLSM) and formaldehyde treated LSM [F3LSM (3 g formaldehyde kg-1 DM treated LSM) and F6LSM (6 g formaldehyde kg-1 DM treated LSM)]. **When mean±SEM of a treatment in each row and each column was greater than mean±SEM of the others, it is considered as significant (p<0.05)

Table 2:	Main effect of fat content and chemically processing applied on in vitro gas production medium pH (96 h incubation) of sunflower meal using isolated rumen microbial fractions
*Experimental treatments were Untreated HSM (UHSM), NaOH treated HSM (SHSM), formaldehyde treated HSM [F3HSM (3 g formaldehyde kg-1 DM treated HSM) and F6HSM (6 g formaldehyde kg-1 DM treated HSM)], untreated LSM (ULSM), NaOH treated LSM (SLSM) and formaldehyde treated LSM [F3LSM (3 g formaldehyde kg-1 DM treated LSM) and F6LSM (6 g formaldehyde kg-1 DM treated LSM)]. **When mean±SEM of a treatment in each row and each column was greater than mean±SEM of the others, it is considered as significant (p<0.05)

DISCUSSION

The main objective of the present experiment is to evaluate the effect of fat concentration of untreated or chemically treated sunflower meal samples on in vitro gas production parameters when rumen isolated microbial groups used as inoculants. Previously, it was reported that in situ ruminal degradation of sunflower meal was influenced by fat content and chemically processing applied (Mohammadabadi et al., 2008). The amount of gas produced from rumen microbial fermentation of a feedstuff under in vitro condition is closely related to the digestibility and therefore to the energetic value of any feed for ruminants (Menke et al., 1979). Under the condition of the present experiment, NaOH caused an increase in the rate and gas production from fermentable fraction of the experimental samples when isolated microbial groups applied. Maximum gas volume was observed for rice straw treated with NaOH (Chen et al., 2007). Corn stover treated with 3% NaOH had higher (p<0.05) fiber and dry matter digestibility than did the untreated samples (Anderson et al., 1981). It was indicated that hemicellulose present in the untreated wheat straw was solubilized (32.5%) upon NaOH treatment (Lesoing et al., 1981). Bas et al. (1989) reported DM and NDF digestion of the straw treated with an alkali was greater than untreated straw (82 and 85%, respectively).

The alkali solution caused to hydrolyse the ester linkages between lignin and the cell wall polysaccharides mainly hemicellulose (Chesson, 1981). This makes carbohydrates and hemicellulose more accessible to the action of rumen microorganisms followed by significant improvements in organic matter digestibility and consequently increases the dry matter intake by animals (Nakashima and Orskov, 1989). Alkali solution caused to cleave esterified bounds within cell wall structure, reduce the physical enmeshment of cellulose and solubilize the inhibitory phenolic compounds; thereby enhancing enzymatic saccharification and facilitates microbial colonization of plant cell walls and improve the ruminal degradation (Euna et al., 2006).

In addition any alkali solution provide a condition to increase the colonization of bacteria in cell wall, increase fungi and bacteria populations in rumen fluid or solid fractions, increase accessible locations for microbial attach and enzymes of carboxymethyle cellulolase, aviclase and xlylanase (Chen et al., 2007). Gould (1984) proposed that a dilute solution of alkali react with lignocellulosics to yield partially delignified products that are highly susceptible to enzymatic and microbial attach. The enhanced degradability has been ascribed to a solubilisation of total phenols (Chesson, 1981), arabinoxylans and cellulose (Lindberg et al., 1984) and arising from the cleavage of alkali-labile lignin-carbohydrate linkages (Alexander et al., 1987).

Under the present experiment condition, the protection of the experimental samples by formaldehyde decreased both b and c parameters. El-Waziry et al. (2005) reported a reduction in both b and c fractions for 3% tannic acid-protected soybean meal. Under present study condition, formaldehyde decreased the gas production parameters (b and c) when used at 6 g kg^-1 compared with the less concentration. Present data show that the bacterial group generates a larger volume of gas production than fungal or protozoal group after 96 h fermentation. Rumen cellulolytic bacteria are believed to be responsible for most of the feed digestion in the rumen because of their numerical predominance and metabolic diversity (Cheng et al., 1991; Lee et al., 2000).

Concluded that fungal activity was responsible for most of cell wall degradation and degradation by anaerobic bacteria was significantly less than that of fungal. Anaerobic ruminal fungi interact with hydrogen-utilizing bacteria such as methanogens (Bauchop and Mountfort, 1981). The quantitative estimation revealed that a quarter to one-third of fiber breakdown in the rumen is of protozoal origin (Joblin, 1990).

Data of present experiment showed the highest gas production was observed in medium containing TM. Hidayat et al. (1993) reported that the highest rate of gas production was observed in fungi+protozoa. These controversial results about the relative contributions of rumen microbial groups to the degradation of plant cell walls may be associated with ways to isolate different microbial groups. Onodera et al. (1988) reported that the addition of protozoa to bacteria increased ruminal cellulose digestion but Newbold et al. (1989) demonstrated that ruminal cellulolytic activity of particle-associated populations was not affected by ruminal protozoa. Hidayat et al. (1993) reported that when the extent of hay and barley straw digestion by bacteria was maximal, the addition of either protozoa or bacteria did not further stimulate digestion.

Joblin (1990) concluded that in vitro examination of short-term protozoan-fungus incubations revealed that protozoa ingested fungal rhizoids and immature sporangia, therefore, in vitro studies that protozoa in the rumen probably interact negatively with anaerobic fungi (Joblin, 1990).

The results by Lee et al. (2000) showed that when the fungal fraction was incubated with the protozoal fraction, a steady decline (15.58%) in the degradation rate was observed at the end of the incubation period. Another possible explanation is that fungal sporangium can be degraded by protozoal chitinolytic enzymes (Morgavi et al., 1994).

Results of the present experiment, showed that sunflower meal treated with sodium hydroxide caused to decrease the medium pH; the highest pH was for sunflower meal treated with 6 g formaldehyde. Medium pH for low fat sunflower meal was more than those of the high fat sunflower meal samples incubated by all microbial fractions which confined the findings by Getachew et al. (2001) who reported that the addition of yellow grease and corn oil increased in vitro degradation and gas production but decreased pH. Machmueller et al. (1998) reported the reduction of protozoa by sunflower seed might decrease gas production. Results of the present study, indicated that the values of pH among various microbial groups were different.

CONCLUSION

It was observed that both in vitro gas produced from the fermentable fraction and constant rate of gas production were influenced by the fat content and the chemically processing applied to the samples evaluated.