Brush border and basolateral membrane vesicles were isolated by subjecting homogenized mucosal cells from bovine small intestine to a divalent cation aggregation followed by a series of differential and density gradient centrifugations. Membrane marker enzyme assays were used to monitor the effectiveness of the fractionation procedure. Enrichments were determined by comparing the enzyme specific activities of the membrane fractions to the homogenate. Sodium-potassium adenosine triphosphatase and alkaline phosphatase served as the enzyme markers for the basolateral and brush border membranes, respectively. Basolateral membrane vesicles enriched 11.1 fold were isolated from the interface of the 31 and 34% sucrose bands of a discontinuous sucrose gradient. Brush border membranes enriched 10.1 fold were isolated from the surface of the 28% sucrose band of a discontinuous sucrose gradient. The use of frozen rather than fresh mucosal tissue in the isolation procedures was found to enhance the purification of basolateral and brush border membrane fractions. The transport capabilities of vesicles were demonstrated by incubating vesicles with radiolabeled substrate, then separating the vesicles and transported substrate from the incubation buffer by filtration. Substrate uptakes were quantified by liquid scintillation counting. Basolateral membrane vesicles were observed to accumulate substrate into an osmotically active space and to have Na⁺-dependent alanine transport capabilities. The use of basolateral and brush border membrane vesicles as tools to investigate nutrient uptake allows the investigator to manipulate both the extravesicular and intravesicular environments, thus making possible the evaluation of the complex interactions which are involved in nutrient transport mechanisms. / M.S.

Thesis (MScAgric) -- Stellenbosch University, 2004. / ENGLISH ABSTRACT: The ruminant has the ability to utilize forages more efficiently than any other production animal. The utilization of forage fibre is an important aspect of ruminant production systems, as this is the main source of energy available to the animal. The availability of high-fibre forage nutrients is, however, restricted by cell wall degradability, and since low quality forages contribute a great deal to ruminant production systems worldwide, the improvement of this degradation process is of major economic importance. The use of exogenous fibre degrading enzymes has been proposed as a means of enhancing this process, with positive results being obtained from in vitro studies incorporating exogenous enzyme preparations. Positive in vivo results with regard to forage digestibility and other animal production parameters have consequently also been obtained following the addition of exogenous fibre-degrading enzyme preparations to the ruminant diet. Two initial screening experiments were undertaken in order to identify fungal enzyme preparations that may have a positive effect on in vitro fibre degradability. The initial screening employed an in vitro organic matter digestibility technique, and was successful in identifying at least six enzyme preparations displaying enhanced digestibility results that were statistically significant. A second in vitro gas production procedure was used to confirm results obtained from organic matter digestibility assays, as well as to increase screening capacity in order to evaluate new enzyme preparations more time-efficiently. Statistical analysis of results obtained from the secondary screening identified various enzyme candidates producing promising results. Only one of these, Abo 374, proved to be statistically superior to the control and other enzyme preparations. A growth trial was subsequently conducted to assess the performance of this enzyme in vivo. The trial involved individual feeding of 32 Dohne Merino ram lambs grouped according to weights into four groups consisting of 8 lambs each. Each group represented a specific application level of enzyme to the wheat straw component of a high fibre diet, amounting to 10, 5, or 1 ml enzyme supematant/kg straw. The enzyme was diluted with water at appropriate rates to obtain an application rate of 300ml/kg straw. The fourth (control) group was treated with water at the same application rate. The trial was conducted over a period of six weeks, during which feed intakes, weekly weight gains, as well as feed conversion efficiencies were recorded. Results suggested significant weight gains in the high (10ml/kg) and medium (5ml/kg) treatment groups, indicated by a P-value of 0.04. Similarly, feed conversion efficiencies were improved for above-mentioned groups (P=0.05), while feed intakes did not differ significantly between the four experimental groups. / AFRIKAANSE OPSOMMING: Die herkouer besit die vermoee om ruvoere beter as enige ander produksiedier te kan benut. Die gebruik van ruvoervesel is 'n belangrike aspek van herkouer produksiesisteme, aangesien ruvoere die hoof bron van energie aan die herkouer verskaf. Die beskikbaarheid van hoe-vesel ruvoer nutriente word egter beperk deur die degradeerbaarheid van die selwand, en aangesien lae kwaliteit ruvoere 'n groot bydrae tot wereldwye herkouer-produksiesisteme maak, is die moontlike verbetering van hierdie degraderingsproses van groot ekonomiese belang. In 'n poging om hierdie verteringsproses te help bevoordeel, is die gebruik van eksogene veselverterende ensieme ondersoek, en positiewe resultate is verkry wanneer hierdie ensieme in in vitro studies gebruik is. Goeie verbeterings ten opsigte van ruvoer verteerbaarheid en ander diereproduksie parameters is ook verkry deur middel van in vivo studies waar eksogene ensieme by die ruvoer van herkouers gevoeg IS. Twee eksperimente is ondemeem in 'n poging om ensiempreparate wat 'n moontlike positiewe effek op in vitro veselvertering mag he, te identifiseer. Die eerste, 'n in vitro organiese materiaal verteerbaarheid tegniek, was suksesvol in die identifisering van minstens ses ensiem preparate wat statisties betekenisvolle verbeterings ten opsigte van verteringsresultate geproduseer het. 'n Tweede in vitro gasproduksie prosedure is vervolgens gebruik om resultate verkry vanaf die eerste tegniek, te bevestig, asook om evalueringskapasiteit te vergroot en sodoende, nuwe ensiempreparate meer tydseffektief te evalueer. Statistiese evaluering van resultate verkry uit die tweede in vitro tegniek het 'n reeks ensieme met positiewe resultate opgelewer. Een van hierdie, Ab0374, het statisties betekenisvolle resultate ten opsigte van die kontrole, sowel as ander ensieme getoon. In 'n volgende eksperiment is 'n groeiproef gedoen om die effektiwiteit van hierdie ensiem in vivo te toets. In die proef is 32 Dohne Merino ramlammers op grond van hul gewig in vier groepe van agt skape elk verdeel, en individueel gevoer. Die groepe het verskillende toedieningsvlakke van die toetsensiem, toegedien tot die koringstrooi komponent van 'n hoe-vesel dieet, ontvang. Toedieningsvlakke was 10, 5, of lml ensiemkonsentraat/kg strooi. Elke groep se ensiemkonsentraat is verdun met die toepaslike hoeveelheid water om 'n toedieningsvlak van 300ml ensiemoplossing/kg koringstrooi te verkry. 'n Vierde groep is behandel slegs met water teen dieselfde toedieningsvlak, en het gedien as 'n kontrole. Die eksperiment is oor 'n periode van 6 weke uitgevoer. Tydens die proeftydperk is voerinnames, weeklikse gewigstoenames, sowel as voeromsetverhoudings, gedokumenteer. Resultate het betekenisvolle gewigstoenames in die hoe (lOml/kg) en medium (5ml/kg) groepe opgelewer, aangedui deur 'n P-waarde van 0.04. Voeromsetverhoudinge het ook verbeteringe getoon vir bogenoemde twee groepe (P=0.05), terwyl voerinnames nie merkbaar tussen die vier groepe verskil het nie.

Copies of the author's previously published articles inserted. Bibliography: leaves 142-159. Provides information on the significance of pea straw as a ruminant feed and the potential of two biological techniques for improving its feeding value.

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.