Evaluation of enzymatic activities in rumen fluid and respective in vitro digestibilities

Thesis (MSc)--Stellenbosch University, 2018.

ENGLISH ABSTRACT: Rumen enzymatic activity studies and in vitro digestibility studies are potentially correlated and affected by similar factors. However, performing enzymatic activity studies parallel to in vitro digestibility studies is hindered by the lack of a standard method of analysis and by the lack of knowledge surrounding the variables that could potentially influence the enzymatic activity. It is known that the composition of the ruminal microbial population is responsive to changes in various natural and feed related factors, but the dynamic between the microbial population and enzymatic activity within the rumen, and factors affecting enzymatic activity are not well defined. The objective of the first experiment was to determine the effect of blending and sonication on enzymatic (amylolytic, lichenase, endoglucanase, xylanase, protease) activity and the effect of blending on in vitro digestibility (starch, neutral detergent fibre, nitrogen) when compared to simple filtration (control). Both blending and sonication significantly and consistently increased amylase, lichenase, endoglucanase and xylanase activity with no significant difference between the two enzyme extraction methods. For protease activity, only sonication was able to significantly increase the activity compared to the control. For the in vitro analysis, blending was unsuccessful in liberating amylolytic microorganism associated with the particulate fraction and might have negatively affected the microbial population responsible for neutral detergent fibre (NDF) and protein digestion. The objective of the second experiment was to determine if diurnal patterns in starch, NDF and protein digestibility and amylolytic, fibrolytic and proteolytic activity, respectively, exist. Two of the cows were restricted from feed for 8-hours overnight, and the other two continued to receive their feed ad libitum, to isolate but also quantify the effects of a possible different feeding behaviour at night. Rumen fluid was sampled before the morning feeding, and subsequent collections were every 4-hours for a 24-hour period. When fitted to a cosine function, all the parameters tested followed a daily pattern that was sensitive to the availability of feed overnight, although the parameters responded differently to the feeding treatment. The highest activities for amylase, lichenase, endoglucanase and xylanase were observed at the time points subsequent to milking and feed delivery, suggesting that the cows adapted their feeding behaviour to the time of feed delivery and milking. Protease activity was unaffected by either feeding treatment or possible feeding behaviour, although when fitted to a cosine function it did display a daily pattern that was sensitive to the availability of feed overnight. The patterns displayed by in vitro digestibility of starch, NDF and nitrogen, across the various fluid collection time points, were much more variable than expected and could be attributed to the fact that a higher number of variables affect the final results. The acrophases observed in the enzymatic analysis did not correspond to those observed in the in vitro analysis. Therefore, different interpretations should be given to enzymatic activities and in vitro digestibility values and the time of rumen fluid collection relative to feeding time should always be included when rumen fluid is utilised for research or commercial purposes.

AFRKAANSE OPSOMMING: Rumen ensiemaktiwiteite en in vitro verteringstudies is potensiëel gekorreleer en beïnvloed deur soortgelyke faktore, alhoewel die uitvoering van ensiem aktiwiteitstudies in parallel met in vitro verteringstudies belemmer word deur die gebrek aan ‘n standaard ontledingsmetode en die nodige kennis aangaande die veranderlikes wat potensiëel die ensiemaktiwiteite kan beïnvloed. Dit is bekend dat die samestelling van die rumen mikrobepopulasie reageer op verskeie natuurlike en voerverwante faktore, maar die dinamika tussen die rumen mikrobepopulasie, ensiemaktiwiteite, en die faktore wat ensiemaktiwiteite beïnvloed word nie goed verstaan nie. Die doel van die eerste eksperiment was om die effek van vermenging en sonikasie op ensiem- (amilase, ligenase, endoglukanase, xilanase, protease) aktiwiteit en die invloed van vermenging op in vitro verteerbaarheid (stysel, neutraal-onoplosbare vesel, stikstof) te bepaal wanneer dit vergelyk word met eenvoudige filtrasie (kontrole). Beide metodes het amilase-, ligenase-, endoglukanase- en xilanase-aktiwiteit betekenisvol en deurlopend verhoog, met geen betekenisvolle verskil tussen die twee ensiemontrekkingsmetodes nie. Slegs sonikasie was in staat om protease-aktiwiteit betekenisvol te verhoog in vergelyking met die kontrole. Vir die in vitro analise was vermenging nie in staat om amilolitiese mikroörganismes wat met die partikelfraksie geassosieer is te skei nie en het moontlik die mikrobepopulasie wat verantwoordelik is vir neutraal-onoplosbare vesel (NDF)- en proteïenvertering negatief beïnvloed. Die doel van die tweede eksperiment was om vas te stel of dag-nagritme patrone onderskeidelik in stysel-, NDF- en proteïenverteerbaarheid en amilolitiese-, fibrolitiese- en proteolitiese-aktiwiteit bestaan. Twee koeie is vir 8-uur oornag gevas en die ander twee is ad libitum gevoer. Die doel was om die invloed van moontlike verskille in voedingsgedrag in die nag te ondersoek en indien dit bestaan, te kwantifiseer. Rumenvloeistof is versamel voor die oggend voerperiode en die daaropvolgende versamelings is elke 4-uur vir ‘n 24-uur periode herhaal. Toe die data met ‘n kosinusfunksie gepas is het al die parameters wat getoets is’n dag-nagritme patroon gevolg wat beïnvloed is deur die beskikbaarheid van voer snags. Die hoogste amilase-, ligenase-, endoglukanase-vi en xilanase-aktiwiteit is waargeneem by tydpunte wat gevolg het na melking en voerlewering, wat waarskynlik beteken dat die koeie hulle voedingsgedrag aangepas het tot die tyd van melking en voerlewering. Protease-aktiwiteit is nie beïnvloed deur voer-behandeling of deur voedingsgedrag nie, alhoewel dit ‘n dag-nagritme patroon gevolg het wat beïnvloed is deur die beskikbaarheid van voer snags soos aangedui deur die kosinusfunksie. Die patrone wat waargeneem is by die in vitro verteerbaarheid van stysel, NDF en stikstof oor die verskeie tydpunte was meer variërend as wat verwag is en dit kan toegeskryf word aan die groter hoeveelheid veranderlikes wat die finale resultate beïnvloed het. Die akrofases wat waargeneem is in die ensiemanalises het nie ooreengestem met dié wat waargeneem is in die in vitro analises nie. Daarom moet verskillende interpretasies gegee word aan ensiemaktiwiteit en in vitro verteerbaarheidswaardes en die tyd van rumenvloeistof versameling moet altyd aangedui word wanneer rumenvloeistof gebruik word in navorsing of vir kommersiële doeleindes.

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