Thesis (M.Sc. (Agric.)) -- University of Pretoria, 2008. / Abstract in English. Includes bibliographical references.

The growing demand for meat worldwide by the increasing human population (6.8 billion) calls for an increase in livestock production as well as attention to environmental sustainability. Production increases are critical especially in Africa with the highest annual population growth rate (2.5%), where most communities rely on livestock for protein supply. Attempts by intensive livestock farming to optimize production are limited by fibrous quality feeds (roughages) and their unavailability in both developed and developing countries. The overall objective of this study was to scan both domestic and wild herbivores in search for microbial ecosystems with superior fibrolytic potential that can be used as feed additives. It was hypothesized that microbes from wild herbivore can improve fibrous feed breakdown in domesticated ruminants. Experiment 1 evaluated the use of fresh or in vitro cultured faecal inoculum (FF) from two Jersey cows as a potential substitute for rumen fluid (RF). Cultured FF was a better substitute for fresh RF as demonstrated by percentage differences in exocellulase activity (0.4%) and true degradability (TD) (7%), compared to the differences observed between fresh RF and FF for exocellulase activity (33%) and TD (14%). It was applied in subsequent experimentation because it was cost effective (no surgery and reduced sample collection time). The second experiment compared the fibrolytic competence of cultured faecal inocula from three hindgut fermenters (miniature horse (mH), horse (H) and Zebra (ZB)) in summer and winter grazing in their natural environment. Both cellulase enzyme assays (exocellulase, endocellulase and hemicellulase) and in vitro maize stover digestibility study ranked the herbivores according to their fibrolytic competence as ZB > H > mH. The effect of cultured faecal inocula from H, ZB and wildebeest (WB) and its combined systems (N1=H+WB, N2=H+ZB, N3=WB+ZB and N4=H+WB+ZB) on the fermentation of maize stover were also evaluated in vitro. Both enzyme assays and MS degradability studies showed that the combined systems were higher (P<0.01) in fibrolytic activities compared to the individual systems. The microbial ecosystems were ranked as N1 > N2 > N4 > H > ZB > WB >N3; and N3 > N1 > N4 > WB > N2 > ZB >H by their exocellulase activity and degradability parameters, repetitively. The diversity of microbial ecosystems was confirmed by numerous active carboxymethyl cellulase bands present on a carboxymethy cellulose zymograms in experiment 4. The combined microbial ecosystems contain more active and variable bands of cellulases than in the individual microbial ecosystems. Systems N3 and N1 were considered as the best inocula for rumen transinoculation studies. Experiment 5 assessed the in vivo effect of direct-fed microbials from N1 and N3 on MS degradation, ruminal fermentation characteristics and cellulase enzyme profile in sheep. Feed dry matter intake increased (P<0.03) in N1 but tended to increase when inoculated with N3. The treatments, N1 and N3 increased (P<0.05) rumen exocellulase (9.4 and 33.2%, respectively) and endocellulase (82.1 and 47.1%, respectively) specific activities but not hemicellulase activity. Maize stover degradability parameters for N3 (TD, degradability of the insoluble fraction of MS, effective degradability, total SCFA and propionate) measured after 96 h of incubation tended (P>0.05) to be numerically different (1.1, 5.4, 7.1 and 7.9%, respectively). Increase in propionate for N3 was accompanied by higher total SCFA and lower CH4. A decrease in CH4 and no difference in CO2 allow both systems to be environmentally friendly since they have been associated with global warming. These studies showed that direct-fed microbials from N1 and N3 inocula have the potential of improving the utilization of maize stover feeds in ruminants, particularly in view of its simplicity and availability which allows it to be implemented at a relatively lower cost compared to other specific strains or microbial cultures. However, more research is required to identify, purify and classify the superior fibrolytic microbes in the most active ecosystems. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2012.

The aim of this study was to determine firstly if there exist variations in fibrolysis among herbivore microbial ecosystems and secondly, the effect on fibre hydrolysis of compositing the most active systems with ruminal microbial ecosystem harvested from a Jersey cow. A literature review pointed to the complexity of carbohydrate (fibre) and how the physical and chemical nature of the forage carbohydrate can present barriers that hinder digestion in the rumen, especially its association with hemicelluloses, pectin, lignin and tannins. Fresh rumen fluid was collected from fistulated herbivores (Jersey cow and sheep) and faecal samples from non-fistulated herbivores (buffalo, horse, impala, camel, elephant, llama, sheep, wildebeest and elephant). Crude protein samples were precipitated with 60% ammonium sulfate. Sample activities were monitored and optimised by incubating with carboxymethyl cellulose (CMC) for 2 h at 39°C. The crude protein samples precipitated from the 11 herbivore microbial ecosystems were active. This was confirmed by an increase in enzyme specific activity with a decrease in total crude protein concentration. In vitro pH optimisation showed a broad range of activity for all ecosystems (4.5-8.0) but for the zebra, horse and elephant which peaked at pH 5. In experiment two (Chapter 4), seasonal variation of the enzymes (exocellulase, endocellulase, cellobiase and xylanase) were monitored through winter and summer. Enzyme specific activity of exocellulase, endocellulase, cellobiase and xylanase were determined by incubation with the specific substrates, crystalline cellulose, CMC, pNPG and xylan, respectively. The amount of reducing sugar released was used to determine the enzyme specific activity. Exocellulase analysis was suitable in winter while summer was preferred for carboxymethyl cellulase and xylanase due to their relative abundance. Cellobiase analysis did not depend on any particular season. Eleven herbivore microbial ecosystems were characterised according to their fibrolytic enzyme specific activities. Enzyme catalytic activities were calculated from kinetic parameters (Km and Vmax) obtained from Eisenthal and Cornish-Bowded plots (Chapter 5). Fibrolytic enzyme expression as well as their activities differed among the 11 ecosystems (P<O.OOOI). They were classified into three groups based on fibrolytic enzyme concentrations; group A with high enzyme concentrations (horse, impala, zebra, wildebeest and the elephant), group B with intermediate (cow, llama, camel, buffalo and giraffe) and group C with low enzyme concentrations (sheep). Exocellulase activity was reasonably correlated with endocellulase activity (r = 0.8978). Xylanase activity was also correlated with carboxymethyl cellulase actvity (r = 0.7104). Enzyme kinetic studies revealed that crude protein samples from the horse, zebra, wildebeest and elephant had the highest enzyme catalytic activities. Microbial or enzyme composite systems were created from the most active ecosystems (horse, wildebeest and zebra) in an attempt to improve the Jersey cow system. These systems were B (cow and horse), C (cow and wildebeest), D (cow and zebra) and E (cow, horse, zebra and wildebeest). The specific activities and enzyme efficiencies of these new systems were determined and compared with system A (cow). Microbial synergism of these systems was also investigated by measuring the amount of gas produced and true degradability (TD) after 72 h of incubation. The composite systems Band E were the most active fibrolytic enzyme systems while C and D were intermediate when compared to that of A. In vitro microbial synergism assays showed that systems B, D, and E had the highest potential of improving milky maize stover (MM) and nutral detergent fibre (NDF) fermentation and degradability in Jersey cows. It was concluded that: (i) fibrolytic and hemicellulolytic enzyme concentrations vary from one season to another with the changing forages; (ii) microbial fibrolytic activities vary among animals grazing on the same field or different geographical regions; and (iii) lastly microbial synergisms of active ecosystems have the potential of improving fibre hydrolysis. However, there is a need to conduct in vivo experimentation to determine the real potential of these in vitro observations. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2006.