I.INTRODUCTION
Rumen protozoa are believed to actively participate in the fermentation processes of the diet in the rumen (Hungate, 1966), and they contribute to 20% of fiber degradation (Dijkstra and Tamminga, 1995). The ruminal protozoa are also strongly related to CH4 (methane) production during the ruminal fermentation. Removal of protozoa (defaunation) clearly reduced CH4 production when incubated with starchy feeds (Qin et al., 2012) and with forages (Qin et al., 2013). A symbiotic relationship between ruminal ciliate protozoa and methanogens was uncovered by Finlay et al. (1994), and they indicated that the symbiotic methanogens associated with the protozoa may account for 37 % of the total CH4 production.
Meanwhile, lipid supplementation to the diet has negatively affected digestibility both in the rumen and hind gut of cattle (Song et al., 1994), and even feed intake (Song et al., 1992). The ruminal fiber degradation was decreased when more than 4% of lipid was supplemented (Jenkins et al., 1989). Supplementation of crushed flaxseed (Song et al., 1994) and oil infusion to the rumen of sheep (Song et al., 1999) clearly decreased ruminal NDF (neutral detergent fiber) degradation. Ruminal degradation rate of the diet was decreased with increased degree of unsaturation (Huang et al., 1999). Unlikely to the bacteria, ruminal protozoa may have a minor contribution in bio-hydrogenation of poly unsaturated fatty acid as well as in lipid degradation (Dawson and Kemp, 1969).
This study was conducted to examine the effect of defaunation on fermentation characteristics, degradation of ryegrass (Lolium multiflorum) hay and CH4 production by rumen microbes when incubated with plant oils which are different in fatty acid composition.
II.MATERIALS AND METHODS
1.In vitro incubation
Rumen contents were obtained 2 h after the morning feeding (09:00) from three cannulated Holstein cows fed 9 kg/d total diets daily (7 kg concentrate and 2 kg ryegrass, as fed basis), twice (09:00 and 18:00 h) per day, in an equal volume. The rumen fluid was strained through 12 layers of cheesecloth to remove the feed particles. Carbon dioxide (CO2) was flushed into the strained rumen fluid for 30 seconds. Culture solution was prepared by mixing 40 ml strained rumen fluid with 40 ml McDougall’s buffer (McDougall, 1948). Sodium lauryl sulfate (0.000375 g/ml, Sigma, L5750) as a defaunation reagent was added into the mixed culture solution to remove ruminal protozoa (Dohme et al., 1999). One gram of (C18:2) linoleic acid -rich SO (sunflower oil) or (C18:3) (linolenic acid) -rich LO (linseed oil) with 0.4 g albumin emulsifier each dissolved in 1 ml buffer solution was added to 80 ml culture solution in 160 ml bottle. Culture solution without any supplements was also prepared for incubation (control). One gram of ground ryegrass (Lolium multiflorum) hay through 1 mm screen on a DM(dry matter) basis as substrate was prepared in a nylon bag (5 × 5 cm, a pore size 50 μm) and was added to the mixed culture solution of presence of protozoa (faunation) or absence of protozoa (defaunation) in the bottle. The bottles were sealed with rubber stoppers, and then were incubated anaerobically in a shaking incubator (VS-8480SR, VISON Science, Bucheon, Korea) at a speed of 135 rpm/min up to 24 h at 39°C. The in vitro incubation was made 3 times in duplicate under similar conditions. Chemical composition of ryegrass hay is shown in Table 1 and composition of major fatty acids of plant oils was shown in Table 2.
2.Measurement and analysis
Incubation was stopped by removing the bottles from the shaking incubator at 3, 6, 12 and 24 h, and pH of culture solution was immediately measured. At the same time an aliquot of culture solution (0.8 ml) was collected from each bottle for ammonia and VFA (volatile fatty acid) analyses. Ammonia concentration was determined by the method of Fawcett and Scott (1960) using a spectrophotometer (Optizen 3220UV, Mecasys Co. Ltd., Daejeon, Korea). The 0.8 ml culture solution was mixed with 0.2 ml 25% phosphoric acid and 0.2 ml pivalic acid solution as the internal standard for the VFA analysis. The VFA concentration was determined by a gas chromatograph (GC, HP5890 series II, Hewlett Packard Co. USA) equipped with a FID (flame ionization detector). The oven temperature for VFA analysis was 120°C, and the temperatures of the injector and detector were maintained at 170°C and 200°C, respectively. A 30-m fused silica capillary column (HP-FFAP, 19091F-112, 0.32 mm i.d. USA) was used. Ultra high purity He was used at a flow rate of 50 ml/min. Identification and quantification were carried out by the external standard method following injection of a VFA standard (Supelco 47058, WSFA-4, USA) of known concentration. Total gas production was measured at each incubation time through the 3-way stopcock connected to culture bottles. A gas sample was transferred to a 5 ml vacuum tube and analyzed for CH4 and CO2 by gas chromatography (YL 6100 GC, Young Lin Instrument Co., Korea) equipped with FID and TCD (thermal conductivity detector). A 30 m silica capillary column (Agilent HP-PLQT Q, 19095P-Q04, 0.54 mm i.d., USA) was used to identify CH4 and CO2 peak analysis. The oven and injector temperatures for gas analysis were 100°C and 150°C, respectively, and temperatures for FID and TCD detector were kept at 230°C and 150°C, respectively. The N2 (nitrogen) gas was used as carrier gas at a flow rate of 30 ml/min. The nylon bag containing feed residue after incubation was washed with tap water and dried at 60°C for 48 h in the drying oven to measure DM degradation. The CP (crude protein), EE (ether extract), and ash of feeds (ryegrass hay) prior to incubation and feed residue after incubation were analyzed according to AOAC methods (1995). The NDF (neutral detergent fiber) was analyzed by the methods of Van Soest et al. (1991).
3.Estimation of effective degradability in vitro
Percent disappearance of DM and NDF at each incubation time was calculated from the remained portion after incubation in the rumen. Disappearance rate was fitted to the equation of Ørskov and McDonald (1979): Y(t) = a + b(1-e－ct)
Where Y(t) is the proportion of the incubated material degraded at time t; ‘a’ is the water soluble and instantly degradable fraction; ‘b’ is the potentially degradable fraction; ‘c’ is the fractional rate of degradation of fraction b. Non-linear parameters a, b and c were estimated by an iterative least square procedure to calculate effective degradability of DM(EDDM) and NDF (EDNDF) according to the following equation (Ørskov and McDonald, 1979): Effective degradability = a + (b×c)/(c+r)
Where ‘r’ is the fractional outflow rate and a hypothetical fractional outflow rate of 0.05 h was used for estimation of effective degradability.
4.Statistical analyses
The present study was shown as 2×3 factorial design that represent two fractions (faunation and defaunation) and three treatments (control, SO or LO supplementation).
Data were analyzed using the GLM(general linear models) procedure of SAS (V 9.1, 2002). Six treatments were replicated twice per time and repeated 3 times. For each variable measured at each time, replicates were averaged, and the total number of observations was 6 (treatments) × 3 (times) = 18 observations. The 18 observations obtained were subjected to least squares analysis of variance according to the following models:
Yijk = μ + τi + Sj + Ok + (τi × Sj) + εijk
Where Yijk is observation, μ is the overall mean, τi is the effect of oil treatment (i=1-3), Sj is microbial effect (j=1-2), Ok is the jth incubation time, (τi × Sj) = interaction effect between oil and microbe and εijk is the error term. Data between treatments were compared by S-N-K (Steel and Torrie, 1980) and significant differences were declared at p<0.05.
III.RESULTS
Prior to each incubation, it was found that live protozoa were virtually absent when the culture solution was examined under the microscope, thus it was confirmed that sodium lauryl sulfate could be an effective agent against protozoa. The effect of oils or defaunation on pH, NH3-N and VFA are presented in Table 3.
pH from defaunation was increased for all treatments from 6 h incubation times (p<0.01-0.001) compared with those from fauantion. Concentration of ammonia-N from defaunation is higher than that from faunation at 3 h (p<0.001), 12 h (p<0.05) and 24 h (p<0.001) incubation times. Defaunation decreased (p<0.01-0.001) total VFA concentration at all incubation times. Molar proportions of C2 (acetate, p<0.05-0.001) and butyrate (p<0.01-0.001) were also decreased by defaunation at all incubation times. Molar proportion of C3 (propionate), however, was increased by defaunation at all incubation times (p<0.001). Thus the rate of C2 to C3 was decreased by defaunation at all incubation times (p<0.001). Frequency of oil effect in difference of VFA composition was not consistent by incubation times while interaction between oil supplementation and defaunation was seldom appeared in most observation items at most incubation times.
Defaunation decreased values of degradation parameters a and b (p<0.001), and in vitro EDDM (p<0.001) of ryegrass hay compared with relevant values of faunation (Table 4). Similar trends to EDDM were observed in ED of NDF degradation. Oil supplementation only increased degradation parameter c of DM and degradation parameter a of NDF. Interaction between oil supplementation and defaunation was found from degradation parameter b (p<0.001), and EDs of DM and NDF (p<0.001).
Defaunation decreased total gas, CH4 production, CH4 % in total gas and CH4/CO2 at all incubation times (p<0.001), and CO2 production from 12 h incubation times (p<0.001) while clearly increased CO2 % in total gas at all incubation times (p<0.001, Table 5). Oil supplementation decreased total gas (p<0.05-0.001), CH4 production (p<0.001) and CH4 % in total gas (p<0.001) at all incubation times, and CH4/CO2 (p<0.001) from 6 h incubation times while increased CO2 production (p<0.05-0.001) and CO2 % in total gas (p<0.001) at all incubation times compared with corresponding values of control. Interactions between oil supplementation and defaunation were observed mainly from production of total gas (p<0.001), CO2 (p<0.001) and CH4 (p<0.01-0.001) at 12 h and 24 h incubation times.
IV.DISCUSSION
Kiran and Mutsvangwa (2010) showed that elimination of protozoa from the rumen lowered dietary CP degradation and resulted in a decreased concentration of NH3-N. This indicates the active proteolytic activity of protozoa in the rumen. But in the present study, increased NH3-N concentration by defaunation may be due to the fact that defaunating agent (sodium lauryl sulfate) killed protozoa cells, and the dead protozoa might be lysed by bacteria, thus led to a higher NH3-N concentration rather than bacterial CP analyses of ryegrass hay. The present result in concentration of NH3-N is in agreement with Qin et al. (2012).
The concentration of total VFA is, generally, positively correlated with feed digestibility. In the present study, decreased EDDM of ryegrass hay by defaunation could be closely related with the reduced concentration of total VFA, confirming that rumen protozoa actively participate in the fermentation processes of the diet in the rumen (Hungate, 1966). Oil supplementation in association with faunation in the current study further decreased total VFA concentration. The decreased total VFA concentration as one possible reason may be attributed to the toxicity of plant oils to the protozoa (Ivan et al., 2001), resulting in reduced EDDM and EDNDF. Qin et al. (2012) observed that oil supplementation or defaunation influenced the VFA profiles by shifting in a decreased molar proportion of C2 and C4 but an increased molar proportion of C3, thus resulted in a decreased ratio of C2 to C3. It may indicate that these treatments led to alteration of microbial communities and further medication of fermentation pattern. It could also be explained that defaunation and oil supplementation cause a higher partial pressure of hydrogen due to inhibition of CH4 production and thus stimulated another hydrogen consuming pathway (such as C3 production) to maintain optimal hydrogen levels.
Defaunation is known to decrease fiber digestion (Ushida and Jouany, 1990) and protozoa were generally responsible for 20% of fiber digestion (Dijkstra and Tamminga, 1995). The results of the present study are consistent with the finding of above reports. This could be simply attributed to the contribution of fibrolytic enzymes by the rumen protozoa (Takenaka et al., 2004). Meanwhile, EDDM and EDNDF of ryegrass hay in the present study were not affected by oil supplementation in the faunated culture solution. Result of another research (Pavan et al., 2007), however, has shown that oil supplementation reduced NDF digestion as PUFA (polyunsaturated fatty acid) have the direct toxicity to fibrolytic bacteria (Maczulak et al., 1981) through coating feed particle and inhibition of attachment of fibrolytic bacteria on feed particle (Dong et al., 1997). Supplementation of oil in the present study seemed to be less adverse effect on those bacteria. This could be due to the fact that plant oil was added into the culture solution in form of emulsion, which failed to result in prevention of bacterial colonization on the surface of fiber. In addition, oil in combination with defaunation resulted in an increased EDNDF compared with control. This could be closely related to the fact that elimination of protozoa and their associated methanogens led to an inefficient inter-species hydrogen transfer between fibrolytic bacteria and methanogens. Cheng et al. (2009) has proved that the presence of methanogens could maintain a lower hydrogen level and be beneficial for the fiber degradation. Thus, addition of oils rich in PUFA to defaunation culture solution may supply an alternative pathway for hydrogen sink via microbial bio-hydrogenation which indirectly promoted fiber digestion. Ruminal degradation rate of the diet was decreased with increased degree of unsaturation (Huang et al., 1999) but in the current study, no difference was found in degradation of DM and NDF of ryegrass hay between C18:2 rich sunflower oil and C18:3 rich linseed oil.
Measurement of gas production is considered as indirect indicator for fermentation kinetics. Jin et al. (2012) demonstrated that gas production was closely correlated with the disappearance rate of dietary DM in the rumen. In the present study, defaunation significantly reduced total gas production. This could be attributed to the relative lower EDDM caused by defaunation. It has been recognized for many years that elimination of rumen protozoa can effectually reduce CH4 production by interruption of interspecies hydrogen transfer between protozoa and their ecto- and endo-symbiotically associated with methanogens (Finlay et al. 1994; Hegarty, 1999; Qin et al., 2012). Meanwhile, oil supplementation was considered to be a promising dietary strategy for reduction in CH4 production and has been widely reported by in vitro (Li et al., 2011) and in vivo (Jordan et al., 2006; Li et al., 2009) studies. In the present study, SO or LO supplementation to faunatedor defaunated culture solution reduced CH4 production. Previous studies have also observed the suppression of CH4 production by SO (McGinn et al., 2004) and LO (Martin et al., 2008) added to the diet. The toxicity of oil to protozoa could partly be responsible for the reduction of CH4 production as Nagaraja et al. (1997) observed decrease in protozoal numbers with addition of oil lipid. However, decreased CH4 production was also found from oil supplementation in combination with defaunation. It may be due to a direct toxic effect of PUFA rich in plant oil on methanogens via consumption of metabolic hydrogen for bio-hydrogenation (Machmüller et al., 2003). Thus, it implied that shift in metabolic hydrogen toward bio-hydrogenation of PUFA may inhibit the normal metabolic activities of all the species of methanogens since the protozoa associated and free-living methanogens showed the different physiological characteristics (Tokura et al., 1997). It also indicated that the effects of oil supplementation are mediated by the simultaneous elimination of protozoa. One of possible reasons might be that defaunation increased the number of B. fibrisolvens and thus enhanced the utilization of metabolic hydrogen for bio-hydrogenation, especially on forage-based diet which resulted in fibrolytic bacteria as the predominant rumen microbes.
The result of this study showed that defaunation combined with oil supplementation might cause an alteration of microbial communities and further medicate the fermentation pattern, resulting in both reduction of degradation of ryegrass hay and CH4 production. No difference, however, was observed in all the examinations between SO and LO.
