Assessment of in vitro Gas and Methane Production of Diet Fortified with Yeast and Lactobacilli spp.

The effect of dietary fortification of two levels of bakers yeast and yeast plus Lactobacilli against negative control and positive control (antibiotic) was assessed extent of gas production (a + b) differed significantly (p < 0.05). For methane gas, D3 recorded the highest (5.00 ml / 200 mg DM) and D2 had the lowest (1.67 ml / 200 mg DM). All other fortifications were higher in methane gas than control. The present study demonstrated the potential of probiotics especially when mixed at 5 g level in improving degradation.


INTRODUCTION
Livestock in the tropics subsist mainly on low quality roughages and this leads to low quality of output not forgetting the increase in population growth, dwindling grazing lands converted to cultivation of foods to meet the urgent human needs resulting in decreased land for fodder cultivation and forcing livestock to depend on alternate feed resources [1].
Methane production during anaerobic fermentation of nutrients in the rumen is an essential metabolic but nutritionally wasteful process which represents 2 to 15% of gross energy loss [2]. Reducing methane production is an important goal of ruminant nutritionists not only for reducing greenhouse gases and global warming but also for improving the efficiency of animal production [3].
Hence, several new technologies are being tried to improve their digestibility and utilization. One of such effort in recent years is supplementation of Probiotics to rations of livestock since it presents an attractive alternative to the use of chemical and hormonal promoters. They are known to improve the utilization of cellulosic materials, health, productivity and reproduction [1].
Studies had shown that the in vitro gas production was improved when supplemented with probiotics [4,1,5]. According to [6] the total gas production was higher in yeast supplemented groups under in vitro system. Methane production had been reported to increase by addition of yeast [7].
Mixed probiotics (yeast and lactobacilli) have been reported to record a higher in vitro gas production when compared with control and yeast supplemented diet. For methane gas, mixed probiotics supplemented diet was lower than control and sole Lactobacilli supplemented diet [4].
The objective of the present study was to further validate the effect of yeast and Lactobacilli in combination on in vitro gas production and methane.

Experimental Site
Experiments were conducted at the Departments of Animal Science, University of Benin, Edo state and University of Ibadan, Oyo state.

Collection and Preparation of Experimental Diets
Concentrate diet was formulated as presented in Table 1 and mixed with Panicum maximum in a ratio of 60:40 (i.e. Roughage to concentrate). The Panicum maximum served as a basal diet while concentrate as a supplement. Bakers' yeast by Angel TM was used as yeast source for D3 and D4 while yeast plus Lactobocilli was manufactured by Zoom TM and used as fortification for D5 and D6 diets.

In vitro Gas Production Procedure
In vitro gas production technique [8] was used to describe the extent of gas production from treatment diets. Rumen liquor was obtained with the help of a stomach tube, transferred into pre heated thermos flask, strained through a sieve cloth and flushed with CO 2 . The buffer containing NaHCO 3 + Na 2 HPO 4 + KCl + NaCl + MgSO 4 .7H 2 O + CaCl 2 .2H 2 O was used and kept in the incubator for warming prior to being mixed with rumen fluid (1:4) as inoculums, all under continuous flushing with streams of CO 2 . About 200 mg of the substrate was measured and introduced into the syringe after removing the plunger. Incubation was carried out at 39±1ºC and volume of gas production was measured at 3 hourly intervals for 24 h. At post incubation period, 4 ml of NaOH (10M) was introduced to estimate methane production as reported by [9]. The organic matter digestibility (%), metabolizable energy (ME, MJ kg/DM) and short chain fatty acid (SCFA, mmol/L) were calculated also degradability, partitioning factor and microbial biomass were estimated from the substrate truly degraded in vitro (mg).

Chemical and Statistical Analysis
Dried and ground samples of the feed were used for chemical analysis. Crude protein, crude fibre, ether extract and ash were determined according to methods of [10]. Neutral detergent fibre (NDF), Acid detergent fibre (ADF) and Acid detergent lignin (ADL) were determined using the methods of [11]. Data collected were statistically analyzed using [12] and the design was completely randomized.

Chemical Composition of Feedstuff
The chemical composition of feed stuff/diet is presented in Table 2. The crude protein content ranged from 10.15% -11.55%. The crude protein (CP) values obtained in this study was higher than the critical value of 7.70% or 70 g/Kg recommended for small ruminants [13] and also within range for the minimum protein requirement of 10 -12% recommended by [14] for ruminants. Table 3 shows the in vitro gas production of diets fortified with probiotics. There was no significant (p > 0.05) difference amongst the treatments in terms of gas production at the third (3 rd ) h. However, from the 6 th to the 24 th h D6 recorded the highest gas volumes over the other treatments but similar to other treatments except for D2 from 6 th to 24 th h. The diet with antibiotics (D2) recorded the lowest gas production at all the hours. Gas volume increased with increasing hours for all the treatments signifying a high microbe action on the substrates; moreover a higher mixed probiotics (D6) recorded a higher gas volume when compared with control and D4. There was no significant (p>0.05) difference in gas production for control against test diets (D3 to D6) except in D2.

g/d; Gas production from soluble fraction (a), insoluble fraction (b); rate of constant of gas production (c) and time (t)
However, as also observed in Table 4 showing in vitro fermentation characteristics of probiotics fortified diets, D6 recorded the highest gas volume (14.67 ml) numerically when compared with control D1 (12.00 ml) but significantly (P<0.05) higher than that of D2 (3.67 ml). The result obtained have is in agreement with [15], who reported that total gas increased with increase yeast supplementation. However, only at 5 g level of D4 and D6 that the total gas surpassed control (D1), therefore, indicating a better nutrient availability for rumen microorganisms [16]. Yeast plus Lactobacilli supplemented in roughage to concentrate diet (60:40) elicited a higher total gas volume when compared with yeast alone and Lactobacilli supplementations as reported by [4]. The high gas production seen in D4 and D6 might be due to the stimulatory effect of a higher dosage of the probiotics on microorganisms that improved degradability and in turn the gas profile. Fig. 1 shows the graphical illustration of in vitro gas production of diets fortified with probiotics at 24 h incubation.
The same pattern of total gas production is observed for estimated parameters (i.e. ME, OMD and SCFA) in Table 5. Highest inclusion yeast alone and yeast plus Lactobacilli elicited higher values over control (D1) and D2 recorded the least in all the parameters. The reason for these can not be farfetched and can be attributed to the effect of antibiotics in microorganisms (i.e. reducing the amount of methanogens) [17] thereby affecting degradation rate and in turn gas volumes and estimated parameters.

Effect of Fortification on Methane Gas
Results of the present study indicated (Fig. 2) that methane production was decreased drastically (1.67 ml) in D2 while D3 recorded the highest volume (5.00 ml). Methane gas reduced by 45.50% in D2 while probiotic fortified (D3-D6) increased by 36.24%, 27.25%, 8.99% and 18.26% respectively when compared with control value. The reduction seen in D2 was due to reduction in methanogenesis as a result of antibiotic effect on protozoan population which may have led to a reduced availability of hydrogen ions for methane production by methanogens [18]. Yeast plus Lactobacilli at 2.5 g was effective in reducing methane and this is in contrast with the findings of [4] who reported that probiotics (yeast vs. yeast Lactobacilli) did not reduce methane production.
The result from the present study agreed with that of [7] who reported that methane production was increased by the addition of yeast. This was observed in D3 and D4 which were higher than those of D5 and D6 (5 and 4.67 ml vs. 4 and 4.34 ml respectively).  Fig. 3 shows the percentage (%) of degraded and undegraded substrate after 24 h incubation. As observed from the figure, D6 was degraded the most (172.00 mg) while D2 recorded the least (124.67 mg) significantly (p< 0.05) when compared with D6. In terms of undegraded substrate D2 had the highest (75.33 mg) while D6 recorded the least (28.00 mg). Since probiotics have been shown to improve gas production and also acts as an activator of ruminal fermentative processes [4,19] it therefore invariably would mean that degradation will also increase and indicate a better nutrient availability for rumen microorganisms [16].

CONCLUSION
The results of the in vitro gas production study indicated that the yeast plus Lactobacilli fortification at 5g/d had a significant impact on gas production and estimated parameters when compared with control and other probiotic fortified diets. Also it further validates the remarkable potential of probiotics in mixed culture (i.e. yeast and Lactobacilli) over single yeast in small ruminant diets/rations through its stimulatory and buffering effect in the rumen that brings about higher gas production and degradation.