Systems N1 and N3 were chosen as inoculants for in sacco degradability of MS in sheep because of high TD and cellulase enzymes diversity (chapter 5 and 6). It could be questioning why N3 was chosen over WB and N2 with a higher exocellulase activity and TD values that were similar. The fermentation parameters GP, total SCFA, and propionic acid were higher in N3 by 8.4 and 24, 52.3 and 26, and 1.8 and 1.4% than in WB and N2 respectively. Higher MY and total SCFA implies more microbial protein and energy metabolites (SCFA) for the host animal (Hungate, 1984; Armentano, 1992; Wanapat, 2000; Krause et al., 2003) which are the main objectives of most research. Inoculating sheep with direct-fed microbials from N1 and N3 increased rumen exocellulase and endocellulase specific activities but not hemicellulase activity. Increased cellulase activity could be associated with three factors; i) increase in
introduction of superior fibrolytic microbes from inoculants and iii) the introduction of cellulolytic microbes growth promoting factors by inoculants (Martin and Nisbet, 1992).
Interactions among these factors are also possible e.g. the introduction of a superior cellulolytic microbial strain which has the potential of competing and colonizing an ecological niche would increase its microbial population and exert a greater influence on fibre hydrolysis.
Although the exact stimulant for the increase in cellulase activity was not conclusive, one of the objectives of this trial was achieved (increase in cellulase activity).
A lower hemicellulase activity in N1 and N3 was not expected and was very difficult to explain. However, this was probably due to partial inhibition of hemicellulolytic microbes by proteins or anti-growth factors from the game herbivores (WB and ZB). The game herbivores were previously observed with lower hemicellulase activities compared to that of the H (chapter5). Feed dry matter intake increased in N1 as observed by Krehbiel et al. (2003) but tended to increase when inoculated with N3 (Arcos-García et al., 2000; Malik and Bandla, 2010). The feed intake results are promising as most probiotics or direct-fed microbial studies tend to improve the efficiency of feed utilization than intake. In sacco degradability parameters of MS (B, PD and C) were similar between the treatments and control. These results were similar to those obtained by Aydin et al. (2009) where direct-fed microbials plus enzyme supplementation on the fattening performance of Holstein young bulls at two different initial body weights were studied. The digestibility parameters (average daily and total weight gains, final weight and DMI) measured were numerically different (1.8, 3.6, 2.9 and 0.2%
respectively) but not statistically significant. However, some of the rumen fermentation by- products measured (total SCFA, acetic acid and propionic acid), showed that N1 and N3 have the potential of increasing average daily weight gain (Aydin et al., 2009; Malik and Bandla, 2010), live weight (Adams et al., 2008) and milk production (Nocek and Kautz, 2006). This was further confirmed by a small increase in MS degradability for both N1 and N3 between the 10-25 h of incubation. The indifference in B and PD between the treatments and controls may be due to longer dosing periods. Reducing the dosing time from three days interval to daily interval may improve forage fermentation. A similar study by Paul et al. (2011) also observed a decreased in dry matter degradability and enzyme activities post dosing with direct-fed microbials. Different dosing times have been adopted by different researches; for
instance twice daily through the fistula (Nocek et al., 2002) or rationed feed (Lee et al., 2000;
Raeth-Knight et al., 2007; Arthur et al., 2010), once daily through a fistula (Raeth-Knight et al., 2007) and weekly (Paul et al., 2011). The dosing time and method of dosing depends on the research objectives (Malik and Bandla, 2010). Daily dosing was not applied in this study because microbial adaptation over time was being tested. The expectations were that, cellulolytic microbes from the inocula that survive rumen microbial hydrolysis and colonize a particular niche in the rumen would continue to exist and exert its fibrolytic activities.
One of the shortcomings of this research was that the different types of microbes in the inocula (N1 and N3), sheep rumen fluid and rumen inoculated fluid were not identified. Only the overall effect of these microbes on MS fermentation was measured. Knowledge about the different types of microbes especially the cellulolytic microbes and their population would have been very useful in explaining some of the outputs observed (higher exocellulase activity and rate of degradation of MS in N1 but lower total SCFA and PD, and an increase in exocellulase activity and total SCFA in N3 inoculated sheep). A study carried out by Paul et al. (2004) showed that increased in total tract digestibility and SCFA when supplemented with a specific fungal strain was associated with increases in cellulolytic (2.5 fold), hemicellulolytic (2.9 fold), total bacteria (2.5 fold) and fungal counts (4.4 folds). Similar results also associated with increases in microbial population have also been reported (Lee et al., 2000; Paul et al., 2011). It is also speculated that some microbes secrete proteins (peptides, oligopeptides or antibiotics) that stimulate microbial growth (Hernandez-Diaz et al., 2010).
The mechanisms of such proteins are very complex but can be explain in very simple terms.
Some of the mechanisms include the suppression of lactic acid producing microbes activity, stimulating lactic utilising microbes to use up lactic acid and inhibiting microbes competing for the same ecological niche (Baah et al., 2009). All these factors would contribute to a net increase in the dominant cellulolytic microbes hence an increase in fibre breakdown.
Rumen fermentation products measured after incubation with direct-fed microbials vary from one study to another. In this study, total SCFA increased in N3 (Dey et al., 2004) but decreased in N1 which is similar to those observed by Paul et al. (2010). Short chain fatty acids proportion also varied among the treatment and the control. The variation observed in
molar proportion of SCFA in N3 and N1 were similar to those observed by Paul et al. (2010).
This implies that different inocula may affect the rumen differently depending on the type of microbes it harbours. These microbes may affect rumen fermentation by either increasing or decreasing one of the following parameters: total SCFA, acetic acid, propionate acid, butyric acid, CH4 and CO2 (Sehgal et al., 2008; Shelke et al., 2009; Mamen et al., 2010; Paul et al., 2010). Inoculating sheep with N3 decreased CH4 production which is similar to the results obtained by Paul et al. (2010). Improving forage utilization without increasing CH4 production will be a major achievement as CH4 production by ruminants has been linked with global warming. Although the total methane contribution of all the factors that leads to global warming is less than 2%, it is very essential as it is 21 times more effective than CO2 (Song et al., 2011). Therefore, identifying the superior fibrolytic strains and those that secrete factors which promote the proliferation of fibrolytic microbes would improve forage fermentation and utilization in herbivores.
8.6 Conclusion
Laboratory cultured faecal inoculum is a better substitute for rumen fluid than fresh faecal inoculum. In vitro fermentation of MS and cellulases enzyme assays by laboratory cultured faecal inocula from H, mH and ZB showed that hindgut microbial species from both wild and domestic herbivores may be different in their fibrolytic potential. These inocula vary in their ability to degrade fibre in vitro with the ZB being the most active, followed by H and lastly mH. Further investigation on the fibrolytic competence of microbes in chapter 5 where H, WB, ZB and its combined systems were investigated in vitro, showed that the combined systems N1 (H+WB), N2 (H+ZB), N3 (WB+ZB) and N4 (H+WB+ZB) fibrolytic competence were higher than those of the individual systems based on their exocellulase activities and true degradability of maize stover. The digestibility parameters TD, GP, PF, total VFA and B, ranked the microbial ecosystems according to their fibrolytic potential as N3 > N1 > N4 > WB
> N2 > ZB >H.
The fibrolytic competences of the different inocula (H, WB, ZB, N1, N2, N3 and N4) were
cellulose zymograms. Systems N1 had the highest number of proteins with carboxylmethy cellulase activities. Although cultured CW, mH, N2, N4, WB, ZB and H had almost the same number of carboxylmethy cellulase bands, fewer bands were observed with the same molecular weights showing the diversity of these systems. The activities of sheep cellulases increased while hemicellulase did not when inoculated with N1 and N3 systems. Although PD and B were similar between N1 and N3, its C and Total SCFA values shows that, these ecosystems possess the potential of improving fibre utilization in ruminants.
Achievements in this study include; i) the preservation of faecal inoculum in the laboratory using very simple laboratory technique (reduces the cost on daily collection of faecal inocula for fermentation studies), ii) establishing microbial competence variation on MS fermentation between hindgut fermenters grazing in the same field, iii) identification and creation of synergistic (combined) systems with variable potential of digesting forages and iv) the identification of system N1 and N3 with the highest potential of improving fibre breakdown in ruminants. The major advantage of this study is its simplicity and availability to all livestock farmers for the improvement of rumen fermentation especially for straws in the developing countries where quality feeds and resources are limited.