Abstract: This experiment was conducted to evaluate the effect of exogenous enzyme (3 g kg^-1 DM), formaldehyde (30 and 60 g kg^-1 DM) and or sodium hydroxide (40 g kg^-1 DM) on in vitro gas production parameters of sunflower meals (25 and 165 g fat kg^-1 DM) were with gas production technique and using fistulated sheep rumen fluid. Kinetics of gas production was fitted to an exponential model. Sodium hydroxide and enzyme treatments significantly increased gas production (B), Metabolizable Energy (ME), Organic Matter Digestibility (OMD), Net Energy Lactation (NEL), ammonia-N (NH₃-N) and Short Chain Fatty Acid (SCFA) but formaldehyde decreased them. The lowest of gas production was estimated for low fat sunflower meal treated with 60 g formaldehyde/kg DM (75.9 mL/500 mg DM). There was significant difference among samples for gas production rate Constant (C). Sodium hydroxide treated high fat sunflower meal had the highest of ME, OMD, NEL and SCFA (35.8 MJ kg^-1 DM, 207.5 g kg^-1 OM, 2.42 Mcal kg^-1 DM and 1.53 μmol L^-1, respectively). Ammonia-N concentration of high fat sunflower meal were the highest (40.9 mg/100 mL). The values of B, ME, OMD, NEL, NH₃-N and SCFA for low fat sunflower meal was less than high fat sunflower meal. The results showed, it may be that gas production parameters of low fat and high fat sunflower meal are influenced by exogenous enzyme, formaldehyde and NaOH.

INTRODUCTION

Gas production technique used for assessing the digestible value of the ruminant feeds. The gas production is basically by the fermentation of carbohydrates and protein can be fermented but is not the same as that of carbohydrates (Blu¨mmel and Ørskov, 1993). Sunflower meal is used as supplemental protein in dairy rations and is classified as highly degradable (Economides, 1998). Formaldehyde treatment that formed cross-links with proteins, make protein less degradable in the rumen (Ashes et al., 1984). Sodium hydroxide (NaOH) treatment, caused to reduce the degradation of protein in the rumen and to dissolve hemicellulose, to hydrolyze ester bonds between lignin (i.e., ferulic and p-coumaric acids) and hemicellulose and to swell cellulose microfibrils and improve digestibility (Canale et al., 1992). Also, Euna et al. (2006) reported using of exogenous enzymes increase digestibility.

The objective of current study was to determine in vitro gas production and estimated parameters of low fat (25 g kg^-1 DM) and high fat (165 g kg^-1 DM) sunflower meal, (LSM and HSM, respectively) as untreated and treated with exogenous enzyme, formaldehyde and or NaOH.

MATERIALS AND METHODS

Experimental samples: Two level of 30 and 60 g kg^-1 DM of formaldehyde (37% solution of commercial grade formalin) was mixed with the sunflower meals (low and high fat) for 30 min and were transferred inside sealed PVC bags and shaken for 5 min and kept for 5 days at room temperature.

After 5 days treated meals were spread in thin layers (4 mm) on the plastic and were air-dried. Treating sunflower meals with 4% solution of NaOH (40 g kg^-1 DM) was done the same but samples were kept for 48 h. Also, 3 g kg^-1 DM exogenous enzyme used for processing sunflower meals. The enzyme mixture composition was Cellulase, Xylanase, Betaglucanase, Alpha amylase, Pectinase, Phytase, Protease and Lipase as 0.03, 6.6, 10, 0.7, 0.7, 0.07, 0.5 and 3 MU kg^-1, respectively; Bioproton Pty. Ltd. Co. Therefore, experimental samples were; ULSM (Untreated LSM), SHLSM (40 g kg^-1 DM NaOH treated LSM); F30LSM (30 g formaldehyde/kg DM treated LSM); F60LSM (60 g formaldehyde/kg DM treated LSM); ELSM (3 g exogenous enzyme/kg DM treated LSM); UHSM (Untreated HSM); SHHSM (40 g kg^-1 DM NaOH treated HSM); F30HSM (30 g formaldehyde/kg DM treated HSM); F60HSM (60 g formaldehyde/kg DM treated HSM); EHSM (3 g exogenous enzyme/kg DM treated HSM).

Gas production method: Rumen fluid was supplied from two fistulated sheep in prior to the morning meal. Animals fed a 40:60 concentrate: forage (2 kg alfalfa hay and 4.5 kg corn silage) diet. About 500 10 mg sample (1.0 mm screen) incubated with 35 mL buffered rumen fluid under continuous CO₂ reflux in 100 mL calibrated glass syringes for 2, 4, 6, 8, 10, 12, 16, 24, 48, 72 and 96 h, in a water bath maintained at 39°C, according to Menke and Steingass (1988). Samples were incubated in triplicate together with 3 syringes containing only incubation medium (blank). After 96 h of incubation, the medium of each syringe used for determination ammonia-N (NH₃-N) concentration. Content of NH₃-N was determined using distillation method (Kjeltec 2300 Autoanalyzer, Foss Tecator AB, Hoganas, Sweden). Cumulative gas production data were fitted to the exponential equation:

Where:

The values of Organic Matter Digestibility (OMD) and Metabolisable Energy (ME) of samples were calculated by the equation of (Menke and Steingass, 1988), OMD (g kg^-1 OM) = 148.8 + 8.89 GP + 4.5 CP + 0.651A and ME (MJ kg^-1 DM) = 2.20 + 0.136 GP + 0.057 CP + 0.0029 CP². Short Chain Fatty Acids (SCFA) were determined by the equation reported by Getachew et al. (1999). SCFA (μmol L) = 0.0239xGP B 0.0601. The net energy of lactation (NEL, Mcal kg^-1 DM) = (1.64 + 0.0269 GP + 0.00078xGP² + 0.0051CP + 0.01325CL)/4.186 (Seker, 2002). Value of CP, A and CL were crude protein, ash and crude lipid in g/100 g DM. GP was the net gas production (mL/200 mg DM) after 24 h incubation.

Statistical analysis: Data on in vitro gas production, ME, OMD, NEL, NH₃-N and SCFA were subjected to analysis as a completely randomized design using the General Linear Model (GLM). Duncan’s multiple range test was used to compare treatment means at p<0.05.

RESULTS AND DISCUSSION

Exogenous enzyme, formaldehyde and NaOH had significant effect on in vitro gas production kinetic, ME, OMD and NEL of samples (p<0.05) (Table 1). Enzyme and NaOH treatments increased B and the highest of B (185 mL/500 mg DM) and C (0.1 mL h^-1) was for SHHSM, but formaldehyde resulted to decrease these parameters and this decrease for 60 level was >30 level. Alkali to cleave esterified bonds 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). Chen et al. (2007) reported alkali treatment increased colonization of F. succinogenes in situ experiments. The researchers reported alkali makes carbohydrates more accessible to the action of rumen microorganisms and organic matter digestibility improved (Rezaeian et al., 2005). Berger et al. (1979) concluded solubilization of hemicellulose with NaOH caused to improvement in DM digestibility. Exogenous enzymes work in synergy with rumen microbial enzymes, which containing xylanases and esterases had stimulatory effects on fibre degradation of alfalfa hay in vitro (Nsereko et al., 2000), whereas Colombatto et al. (2003) increases their hydrolytic potential within the rumen and increase digestibility of plant cell wall (Morgavi et al., 2000).

Table 1:	Gas production and estimated parameters of sunflower meal treated with exogenous enzyme, NaOH and or formaldehyde
1ULSM (Untreated LSM), SHLSM (40 g kg-1 DM NaOH treated LSM); F30LSM (30 g formaldehyde/kg DM treated LSM); F60LSM (60 g formaldehyde/kg DM treated LSM); ELSM (3 g exogenous enzyme/kg DM treated LSM); UHSM (Untreated HSM); SHHSM (40 g kg-1 DM NaOH treated HSM); F30HSM (30 g formaldehyde/kg DM treated HSM); F60HSM (60 g formaldehyde/kg DM treated HSM); EHSM (3 g exogenous enzyme/kg DM treated HSM); B: Gas production from fermentable fraction; C: Gas production rate constant; ME: Metabolizable Energy; OMD: Organic Matter Digestibility; NEL: Net Energy Lactation; SEM: Standard Error of Mean; Means within each column with different letters are significantly different (p<0.05)

Fig. 1:

The effect of processing of sunflower meal with exogenous enzyme, NaOH and or formaldehyde on in vitro NH3-N content. ULSM (Untreated LSM), SHLSM (40 g kg-1 DM NaOH treated LSM); F30LSM (30 g formaldehyde/kg DM treated LSM); F60LSM (60 g formaldehyde/kg DM treated LSM); ELSM (3 g exogenous enzyme/kg DM treated LSM); UHSM (Untreated HSM); SHHSM (40 g kg-1 DM NaOH treated HSM); F30HSM (30 g formaldehyde/kg DM treated HSM); F60HSM (60 g formaldehyde/kg DM treated HSM); EHSM (3 g exogenous enzyme/kg DM treated HSM)

The researchers reported that enzyme products indicated that some xylanases and proteases improved in vitro degradation of alfalfa hay. The effect of enzyme, NaOH and formaldehyde on C fraction was significant (p<0.05) that El-Waziry et al. (2005) concluded protected soybean meal decreased C. Protect of samples by formaldehyde decreased B that El-Waziry et al. (2005) reported decrease B for protected soybean meal. Fraction of C of F30HSM and F60HSM was significantly higher than F30LSM and F60LSM. Value of B of HSM was higher than LSM that prove the findings of Getachew et al. (2001) reported addition yellow grease and corn oil increased in vitro gas production (MachMueller et al., 1998) explained, the reduction of protozoa (rumen protozoa is importance in methane formation) by sunflower seed might be partly decrease gas production.

Thomas et al. (1979) reported a reduction in DM degradability of meals and seed by formaldehyde (10-20 g kg^-1 CP). Reduce of ME and NE for treated soybean meal reported by El-Waziry et al. (2005). Despite of high fat of HSM, OMD value of HSM was more than LSM but MachMueller et al. (1998) concluded the inclusion of fatty feeds (4-5%), caused to decrease 4% fermentation and reduced the quantity OMD.

Fig. 2:

The effect of processing of sunflower meal with exogenous enzyme, formaldehyde and or NaOH on in vitro Short Chain Fatty Acids (SCFA). ULSM (Untreated LSM), SHLSM (40 g kg-1 DM NaOH treated LSM); F30LSM (30 g formaldehyde kg-1 DM treated LSM); F60LSM (60 g formaldehyde/kg DM treated LSM); ELSM (3 g exogenous enzyme/kg DM treated LSM); UHSM (Untreated HSM); SHHSM (40 g kg-1 DM NaOH treated HSM); F30HSM (30 g formaldehyde/kg DM treated HSM); F60HSM (60 g formaldehyde/kg DM treated HSM); EHSM (3 g exogenous enzyme/kg DM treated HSM)

Proccessing of sunflower meal with enzyme and NaOH increased NH₃-N content, but formaldehyde decreased it compared with untreated sunflower meal and 60 level of formaldehyde had the lowest of NH₃-N content (Fig. 1). This result suggested by Nishino et al. (1995) for aldehydes. McNiven (1995) observed, little change was shown in ruminal ammonia by 25 and 50 g NaOH kg DM. Fat content had no effect on NH₃-N production but Getachew et al. (2001) showed depression in NH₃-N by fat, due to reduce proteolysis and deamination of amino acids in the rumen. Low NH₃-N concentration with coconut oil probably was partly a result of the absence of the protozoa and a suppression of the fiber-degrading methanogenic bacteria (MachMueller et al., 1998). Treatment of enzyme and NaOH increased SCFA value and formaldehyde decreased it (Fig. 2). There is positive correlation between SCFA and gas production, about 54% of the total gas volume, CO₂ was produced from buffering the SCFA (Menke and Steingass, 1988).

CONCLUSION

The result showed exogenous enzyme and NaOH improved B, OMD, ME, NEL, NH₃-N and SCFA and formaldehyde decreased these parameters in compared with untreated samples. Therefore, based on the present data, formaldehyde is not recommended to treat SFM, when much fermentation is a goal of the feeding strategy. Also, this experiment showed ether extract content of sunflower meal had not negative effect on gas production and the other estimated parameters.