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Thesis (PhD(Agric))--Stellenbosch University, 2011. / ENGLISH ABSTRACT: Exogenous fibrolytic enzymes (EFE) as additives in ruminant feeds are being researched worldwide. Promising effects on dry matter intake (DMI), digestibility and production in especially dairy cows, but also feedlot steers and even sheep have been observed. However, lack of or negative effects are also reported and the need arises for clarity on the mode-of-action of EFE. Forages are characterised as being highly heterogenic and contain varying concentrations of fibre. The fibre, in turn, varies greatly in digestibility, due to the chemical as well as anatomical build-up of this complex carbohydrate. Fibre, however, presents a major source of potential energy for ruminant animals and EFE is a viable option to increase the digestibility of forages. Therefore, a study with the aim of establishing whether EFE can affect the digestibility of forages, how it affects the digestibility and the clarification of the mode-of-action was drafted. From the literature, the first objective was readily attained and clear indications exist that EFE can indeed improve animal performance when their diets are treated with such enzymes. From the current study, it was shown that EFE can alter the rate and extent of gas production of certain forages (lucerne, kikuyu and weeping love grass) and also improve the in vitro digestibility thereof (P < 0.05). This is in agreement with other research findings and it was concluded that these effects were likely exerted during the early stages of digestion. A complete feed for sheep, when treated with the EFE, showed positive effects on the in sacco digestibility, as well as on the digestion kinetics of the feed (P < 0.05). The in vitro digestibility of the complete feed was also improved due to EFE treatment (P < 0.05). The observations on in vitro digestibility were less marked when a purified xylanase, obtained from the partial purification of the EFE cocktail, was used as the sole fibrolytic enzyme treatment. It is apparent, therefore, that enzyme specificity plays a major role in obtaining positive effects on digestibility of forages and feeds. In agreement with the literature, it is proposed that the approach to improve the digestibility of forages should be to use EFE cocktails containing various enzymes, matching the complexity of the substrate. The major aim of the study was, however, an in depth investigation of the mode-of-action of EFE. This aim was approached by observing changes in plant tissue material at the histological level upon treatment with EFE and incubation in buffered rumen fluid. Results showed that EFE had subtle, yet significant effects on cell wall material for the various tissues studied (P < 0.05). The major effect observed here was that EFE had a thinning effect on the cell wall thickness (P < 0.05). It was deduced that as EFE affected the cell wall of the plant material, earlier access by microorganisms could be achieved. Also, nutrients caught in the cell wall matrix could then be released for digestion. Therefore, observations that EFE increases the rate of digestion, as well as the extent of digestion of not only fibre, but also protein, were explained by the enzyme’s action on cell wall material. It was concluded that there is definite merit in the use of EFE to improve the digestibility of ruminant feeds and that this is partly related to effects on the cell walls of the forages. The effects can be expected to occur during the early stages of digestion, thereby potentially increasing the passage rate of digesta from the rumen. Additionally, the effect of the EFE is not limited to fibre and increased digestibility of all nutrients can be expected, thereby increasing the overall digestibility of the feed. Future research should further elucidate the mode-of-action of EFE using advanced technologies routinely employed in the plant sciences. Additionally, the main potential advantage of EFE treatment lies in improving the digestibility of poor quality roughages. Unfortunately, this is an area where limited positive effects are observed and in depth investigations should be undertaken to classify the specificity and optimum conditions of EFE to better match the complexity of the substrate being treated. / AFRIKAANSE OPSOMMING: Eksogene fibrolitiese ensieme (EFE) word tans wêreldwyd ondersoek vir die gebruik daarvan as voerbymiddels vir herkouers. Belowende effekte op DMI, verteerbaarheid en produksie van vernaam melkbeeste, maar ook voerkraalbeeste en selfs skape is al gerapporteer. Swak en selfs negatiewe effekte word egter ook waargeneem en daarom is ʼn deeglike ondersoek na die metode van werking van EFE van belang. Ruvoere word gekenmerk deurdat dit heterogeen van aard is en bevat variërende vlakke van vesel. Vesel maak op sy beurt ʼn wesenlike deel uit van die ruvoer, maar varieer baie in verteerbaarheid weens die chemiese sowel as anatomiese samestelling van hierdie komplekse koolhidraat. Ruvoer verteenwoordig egter ʼn goeie bron van potensiële energie vir herkouers en EFE word voorgestel as ʼn haalbare behandeling om die verteerbaarheid daarvan te verhoog. Dus is ʼn studie beplan met die doelwit om die effekte van EFE te definieer, hoe dit verteerbaarheid beïnvloed en die metode van werking daarvan te ondersoek. Vanuit die literatuur is dit duidelik dat daar wel baie positiewe effekte is waar ruvoere met EFE behandel is en dat diereproduksie wel bevoordeel word daardeur. Vanuit die studie is dit getoon dat die tempo en hoeveelheid gasproduksie van sekere ruvoere (lusern, kikuyu en oulandsgras) verbeter word deur EFE behandeling (P < 0.05). Hierdie bevinding was ondersteun deur verbeterde in vitro verteerbaarheid van die ruvoere (P < 0.05). Dit is in ooreenstemming met literatuur en die gevolgtrekking is gemaak dat hierdie effekte tydens die vroeëre stadia van vertering verwag kan word. ʼn Volledige skaapvoer wat met EFE behandel is, het positiewe effekte op in sacco verteerbaarheid en verterings kinetika data gehad (P < 0.05). Weereens is die in vitro verteerbaarheid van die voer verbeter (P < 0.05). Waarnemings op in vitro verteerbaarheid was veel minder opvallend wanneer ʼn gesuiwerde xylanase as enigste fibrolitiese ensiem behandeling gebruik is. Dit is dus duidelik dat ensiem spesifisiteit ʼn belangrike rol speel in die verkryging van positiewe resultate in die verteerbaarheid van ruvoere en veevoere. In ooreenstemming met literatuur word dit voorgestel dat ensiemmengsels wat verskeie ensieme bevat as EFE gebruik behoort te word ten einde aan die kompleksiteit van die substraat te voldoen. Die hoof doelwit van die studie was egter ʼn indiepte ondersoek na die metode van werking van EFE. Hierdie doelwit is benader deur die effekte van EFE op selwand strukture van plantmateriaal op ʼn histologiese vlak te ondersoek. Die ruvoere was vooraf met EFE behandel en in vitro geïnkubeer in rumen vloeistof. Die resultate het getoon dat EFE ʼn matige, dog betekenisvolle effek op die selwand materiaal van die onderskeie weefsels gehad het (P < 0.05). Die belangrikste waargeneemde effek was dat EFE ʼn verdunningseffek op die selwande gehad het. Dit is afgelei dat as EFE die selwand kan beïnvloed, mikro-organismes vroeër toegang tot die inhoud kan kry. Verder, nutriënte vasgevang in die selwand matriks raak ook beskikbaar vir vertering. Hierdie afleiding en die effek van EFE op selwande verklaar waarnemings dat EFE die tempo van vertering sowel as die vlak van vertering van nie net vesel, maar ook proteïen kan bevoordeel. Die gevolgtrekking is gemaak dat daar definitiewe meriete is in die gebruik van EFE om die verteerbaarheid van herkouervoere te verbeter en dat dit verband hou met die ensiem se werking op selwande van die ruvoere. Die effekte kan verwag word tydens die vroeë stadia van vertering om dus deurvloeitempo van digesta te verbeter. Die effek van die EFE is verder nie beperk tot vesel nie, maar positiewe effekte op ander nutriënte kan verwag word en vervolgens ʼn algehele verhoging in die verteerbaarheid van die voer. Navorsing behoort in die toekoms verder die metode van werking van EFE te ondersoek deur gebruik te maak van gevorderde tegnologie wat alledaags gebruik word in die Plantwetenskappe. Die belowendste aanwending van EFE lê in die verbetering in vertering van swak kwaliteit ruvoere. Dit is ongelukkig juis hier waar min positiewe resultate gerapporteer word en indiepte navorsing moet onderneem word om ensiem spesifisiteit en optimum kondisies te definieer sodat EFE beter opgewasse is teen die kompleksiteit van die substraat.