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Preparation of brush border and basolateral membrane vesicles from bovine intestine for nutrient uptake studiesWilson, Jonathan Wesley January 1986 (has links)
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.
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The effect of an exogenous fibrolytic enzyme on forage digestibility parametersGoosen, Liezel 12 1900 (has links)
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.
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Assessing the feeding value of pea straw and evaluating biological methods to improve its utilisation by ruminantsMohamed, Neijat. January 1995 (has links) (PDF)
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.
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The fibrolytic potential of domestic and wild herbivores microbial ecosystems on maize stover.Fon, Fabian Nde. January 2012 (has links)
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.
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