The effect of exogenous fibrolytic enzymes on fibre digestion in ruminant animals

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digestion in ruminant animals

by

Zarinah Skippers

Thesis presented in fulfilment of the requirements for the degree of

Master of Science in Agriculture (Animal Science)

in the Faculty of AgriSciences

at

Stellenbosch University

Supervisor: Dr JHC van Zyl

Co-supervisor: Prof CW Cruywagen

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Declaration

By submitting this thesis electronically, I declare that the entirety of the work contained therein is my own, original work, that I am the sole author thereof (save to the extent explicitly otherwise stated), the reproduction and publication thereof by Stellenbosch University will not infringe any third party rights and that I have not previously in its entirety or in part submitted it for obtaining any qualification.

Date: March 2021

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Abstract

Title: The effect of exogenous fibrolytic enzymes on fibre digestion in ruminant animals.

Name: Ms Zarinah Skippers

Supervisor: Dr. JHC van Zyl

Co-supervisor: Prof. CW Cruywagen

Institution: Department of Animal Sciences, Stellenbosch University

Degree: MScAgric

Ruminants possess the ability to effectively utilise nutrients from fibrous plant matter and convert it into consumables for human use. However, digestion of plant cell walls is not complete as less than 65% of plant cell wall content is digested under optimal rumen conditions. As a result, ongoing research has been conducted on techniques for the improvement of fibre digestibility. Exogenous fibrolytic enzymes (EFE) has been proposed as a feed additive in order to improve fibre digestibility in ruminant production systems, with positive results.

The feasibility of ABO374 as an EFE, with the potential to improve fibre digestibility in ruminant animals, was evaluated by means of a comparative study with commercial product, Rumenase®. Feed substrates, lucerne hay (LH) and wheat straw (WH), were treated with a control (dH2O) and

the two EFE products. The treated feeds were left overnight before being evaluated for in vitro fibre degradation, and incubated for 0, 4, 8, 24, 48, 72, 96 and 120h. The rate and extent of dry matter (DM) and neutral detergent fibre (NDF) degradation of the treated feeds were then calculated. The results indicated improved NDF degradation of LH (P < 0.05) when compared to the control. An improvement greater than 29.9% was reported for WS fibre degradation. Despite a lack of significant difference between EFE treatments, the addition thereof improved fibre degradation, with LH most affected by ABO374 and WS most affected by Rumenase®. The supernatant ABO374 was deemed as effective as commercial product. In a second experiment, the effect of EFE pre-treatment incubation time (PIT) on fibre degradation was evaluated using three times (0.5, 3 and 12h) on three feeds (LH, WS, and total mixed ration or TMR). The enzyme-treated feed samples were evaluated by in vitro assessment inoculated with rumen fluid, as in the first experiment. Significant effects of DM degradation were reported for 0.5 and 3h PIT when compared to 12h (P = 0.03). Although the effects PIT on NDF degradation was not significant (P > 0.05), a tendency that favoured 0.5 and 3h treatment times over that of 12h was observed (P = 0.08), supporting the results of DM degradation.

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No significant differences were observed between 0.5 and 3h pre-treatment incubation times, and a 3h PIT was chosen as the preferred incubation time for practical application in ruminant production systems.

On the basis of these results, an experiment was conducted to determine the probability of replicating ABO374 using pure enzymes, and the efficacy of the replicated enzyme cocktail in ruminant fibre degradation. Enzyme activity assays were performed to determine the ratio in which amylase, endoglucanase, mannanase and xylanase occurred in ABO374. This data was used to create the replicated synthetic cocktail. The two EFE products were compared to a control treatment to determine their effects on fibre degradation. An enzyme PIT of 3h was used on LH, WS and TMR feed substrates before assessing the ruminal in vitro degradation thereof at incubation 6, 12 and 24h). Enzyme treated WS exhibited the greatest improvement in DM degradation at in vitro 6 and 12h incubation times (P = 0.02 and P = 0.04, respectively). The NDF degradation of WS showed no differences between EFE treatments and the control, although tendencies in favour of enzyme treatment (P < 0.10) were reported. The addition of ABO374 resulted in improved NDF degradability in ruminant in vitro studies, although some inconsistencies did occur. These inconsistencies could be a result of insufficient enzyme activity, enzyme stability, poor enzyme-feed interactions or exogenous enzyme competition with rumen microbes. Despite the varied results, DM and NDF degradation was improved with the addition of EFE, pre-treatment incubation times was influential in fibre degradation, and a cocktail of enzymes increased the degradability of fibre in ruminant animals. Overall, the use of EFE has the potential to be an effective tool in the aim to improve the utilisation of forage and feeds in ruminant production systems.

Key words: fibre digestibility, neutral detergent fibre (NDF), exogenous fibrolytic enzymes (EFE), pre-treatment incubation time (PIT), in vitro, ruminants.

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Opsomming

Titel: Die invloed van eksogene fibrolitiese ensieme op veselvetering in herkouende diere.

Naam: Me. Zarinah Skippers

Studieleier: Dr. JHC van Zyl

Medestudieleier: Prof. CW Cruywagen

Instansie: Department Veekundige Wetenskappe, Universiteit Stellenbosch

Graad: MSc Agric

Ruvoere vorm die hoofbron van vesel in herkouerdiëte en die opname daarvan kan gesondheid en produksie van die diere beïnvloed. Herkouers besit die vermoë om voedingstowwe uit veselagtige plantmateriaal effektief te benut en om te skakel in bruikbare produkte vir menslike gebruik. Die vertering van ruvoer is egter nie altyd volledig nie. Selfs onder normale optimale rumentoestande word minder as 65% van plantselwande verteer. As gevolg hiervan word tegnieke wat veselverteerbaarheid kan verbeter toenemend deur navorsing ondersoek. Positiewe navorsingsresultate met die gebruik van eksogene fibrolitiese ensieme (EFE) as ‘n behandelingsmetodestrategie om veselverteerbaarheid in herkouerproduksiestels te verbeter.

Die uitvoerbaarheid van ABO374 as ‘n EFE produk met die potensiaal om veselverteerbaarheid by herkouerdiere te verbeter was geëvalueer aan die hand van ‘n vergelykende studie met ‘n kommersiële ensiemproduk, Rumenase®. Lusernhooi (LH) en koringstrooi (KS), is behandel met 'n kontrole in die vorm van gedistilleerde water (dH2O) en die twee EFE-produkte. Die behandelde

voere was oornag gelaat voordat dit geëvalueer is vir in vitro NDF degradering, met inkubasietye van 0, 4, 8, 24, 48, 72 en 96h. Die tempo en omvang van droëmateriaal (DM) en neutraal bestande vesel (NBV) degradering op die behandelde voere is bepaal. ‘n Verhoging (P < 0.05) van NBV degradering met LH teenoor die kontrole is waargeneem. ‘n Verbetering (P < 0.05) van meer as 29,9% is vir die NBV degradering van EFE behandelde KS teenoor die kontrole bewerkstellig. Ten spyte van 'n gebrek aan ‘n betekenisvolle verskil, het die toevoeging van fibrolitiese ensieme die tempo van NBV degradering numeries verhoog. Met numeriese verbetering is lusernhooi die meeste deur ABO374 behandeling beïnvloed, terwyl WS die meeste deur Rumenase® behandeling beïnvloed is. Die ABO374 resultate was egter vergelykbaar met die van die kommersiële produk. In 'n tweede eksperiment was die effek van voorbehandelinginkubasietyd (VIT) op veselafbraak op drie tye (0.5, 3 en 12 uur) op drie substrate (LH, KS en TGR) geëvalueer. Die ensiembehandelde

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voersubstrate was geëvalueer deur in vitro-toetsing met vars rumenvloeistof. Betekenisvolle verbeteringe in DM degraderingsverhogings (P = 0.03) vir VIT 0.5 en 3 uur in vergelyking met 12 uur is waargeneem. Alhoewel die effek van VIT op NBV degradering nie betekenisvol was nie, is 'n neiging (P = 0.08) tot die verhoging van behandelingstye 0.5 en 3 uur bo dié van 12 uur waargeneem. Geen beduidende verskille is tussen 0.5 en 3 uur voorbehandelinginkubasietye waargeneem nie. Drie uur voorafbehandelingstyd is as die voorkeur VIT vir praktiese toepassing in herkouproduksiestelsels is derhalwe geslekteer.

Op grond van hierdie resultate is 'n eksperiment om die waarskynlikheid te bepaal of ABO374 met suiwer ensieme gerepliseer kon word uitgevoer. Tweedens is die vermoë van hierdie gerepliseerde ensiem-mengsel teenoor ‘n kontrole en ABO374 om versnelde veselafbraak te bewerkstellig ondersoek. Ensiemaktiwiteit is bepaal en gebruik om die verhouding waarin amilase, endoglukonase, mannanase en xilanase in ABO374 voorkom te bepaal. Die data is gebruik om ‘n vergelykbare sintetiese mengsel te skep. Die twee EFE-produkte is vervolgens met 'n kontrole (dH2O) vergelyk. 'n Ensiem voorbehandelingsinkubasietyd (VIT) van 3 uur is op LH-, KS- en

TGR-voersubstrate gebruik voordat ‘n ruminale in vitro degradering daarvan met inkubasietye van 6-, 12- en 24 uur uitgevoer is. Koringstrooi het die grootste verbetering in DM-degradering getoon wanneer dit met EFE behandel was tydens in vitro inkubasietye van 6 en 12 uur (P = 0.02 en P = 0.04, onderskeidelik). Neutraal bestande vesel degradering van behandelde KS het geen beduidende verskille tussen EFE bronne of die kontrole getoon nie. ‘n Neiging (P < 0.10) ten gunste van EFE-behandeling teenoor die kontrole is egter wel waargeneem.

Alhoewel die behandeling van voersubstrate met ABO374 teenstrydighede gelewer het, het dit wel onder sekere omstandighede tot ‘n verbetering (P = 0.05) in in vitro veselverteerbaarheid gelei. Hierdie teenstrydighede kan toegeskryf word aan onvoldoende ensiemaktiwiteit, ensiemstabiliteit, swak ensiem-voer-interaksies of kompetisie tussen eksogene ensiem en rumenmikrobes. Ten spyte van die uiteenlopende resultate, het beide DM- en NBV-degradering verbeter met die toevoeging van EFE. Die studie het ook bevestig dat VIT wel NDF degradering beinvloed terwyl 'n mengsel van ensieme die verteerbaarheid van vesel by herkouers verhoog. Oor die algemeen kan die gebruik van EFE 'n effektiewe strategie wees ten einde meer effektiewe veselbenutting vir herkouers te weeg te bring.

Sleutel woorde: veselverteerbaarheid, neutraal bestande vesel (NBV), eksogene fibrolitiese ensieme (EFE), voorbehandelinginkubasietyd (VIT), in vitro, herkouers.

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Acknowledgements

I would like to express my sincere appreciation and gratitude to all who, in some shape for form, contributed to the completion of my research. Without these people and institutions, completing this thesis would not have been possible:

• Dr. Brink van Zyl, for his support, guidance, patience, and meetings that turned into recipe swops! I have been blessed with a caring and insightful supervisor who fanned the flames of my curiosity and believed in me.

• Prof. Cruywagen, for all the knowledge, guidance, and invaluable wisdom. I count myself fortunate in having you as a co-supervisor.

• All the staff at the Department of Animal Sciences, Stellenbosch University, for the assistance in the lab, with special thanks to Tannie Beverly Ellis and Michael Mlambo for continued encouragement and kind words.

• Willem van Kerwel and the staff at Welgevallen Experimental Farm, Stellenbosch University, for assisting with the animals.

• Dr. Shaunita Rose and the staff at the Department of Microbiology, Stellenbosch University, for all the support and patience required to assist a novice in your lab.

• AFGRI Animal Feeds, for funding and support that made the research possible. • My family, for the love, support and encouragement they bestowed on me

• Fazielah Allie, who has always invested in me, be it experience, belief, or your knowledge in my quest thereof. Your love and support have been immeasurable.

• My friends, who’s continued support, encouragement, and sympathetic ears gave me strength.

• Tayla David and Sarah Davies, for all the sushi, failed runs and successful talks.

• Waldo van Rensburg, for the early mornings, late nights, shared love of food; and for being prepared to smell horrible for weeks on end.

• Craig Viljoen, for being my rock, a voice of reason, and a constant source of support and encouragement.

• My sisters, Nasreen and Hassaanah, for their love and banter kept me grounded. • My parents, Ghulaam and Rokia, for their endless love, support and sacrifice that has

carried me from the very beginning. You taught me to believe that I could do anything. I am, because of you.

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List of Abbreviations

ADF: Acid-detergent fibre ADL: Acid-detergent lignin

CF: Crude fibre CH4: Methane CO2: Carbon dioxide CP: Crude protein dH2O: Distilled water DM: Dry matter DMI: Dry matter intake DNS: Dinitrosalicylic acid

EFE: Exogenous fibrolytic enzymes EDTA: Ethylendediamine tetraacetic acid eNDF: Effective NDF

H2O: Water

LH: Lucerne hay

MPS: microbial protein synthesis NDF: Neutral-detergent fibre

NDFOM: Neutral-detergent fibre (organic matter)

NFE: Nitrogen-free extract nkat: Nanokatal

OD: Optical density OM: Organic matter

peNDF: Physically effective NDF PIT: Pre-treatment incubation time TMR: Total mixed ration

VFA: Volatile fatty acids WS: Wheat straw

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List of Figures

Figure 2.1 Schematic representation of plant cell wall development (Jung and Allen, 1995) ... 9

Figure 2.2 Comparison between the Weende sytem and Van Soest method of analysis (modified from Fisher et al., 1995) ... 19

Figure 3.1 In vitro NDF degradation (%) of lucerne hay treated with exogenous fibrolytic enzymes (Rumenase® and ABO374) compared to a control. ... 36

Figure 3.2 In vitro NDF degradation (%) of wheat straw treated with exogenous fibrolytic enzymes (Rumenase® and ABO374) compared to a control. ... 36

Figure 4.1 Effect of EFE pre-treatment incubation time on DM in vitro degradation (%). ... 48

Figure 4.2 Effect of EFE pre-treatment incubation time on NDF in vitro degradation (%). ... 48

Figure 4.3 Effect of EFE pre-treatment incubation time on NDFOM in vitro degradation (%). ... 49

Figure 5.1 Experimental design for control treatment ... 58

Figure 5.2 Enzyme activity (nkat/ml) of amylase, endoglucanase, mannanase and xylanase within ABO374 prepared on different substrates (TMR=Total mixed ration, WS= wheat straw, LH=Lucerne hay). ... 63

Figure 5.3 Extent of degradation (DM, NDF and NDFOM) on feed substrates, disregarding feed substrate and enzyme effects. ... 66

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List of Tables

Table 2.1 Detergent methods of forage fraction classification (Van Soest, 1967) ... 8

Table 2.2 Rumen microbes and their enzyme activities involved with plant cell wall degradation (Source: Derived from Dehority (1993) as cited by Wang and McAllister (2002)) ... 11

Table 2.3 Neutral detergent fibre (Van Soest, 1994). ... 18

Table 2.4 Constituents of the in vitro buffer solution (Goering and Van Soest, 1970). ... 20

Table 2.5 Composition of reduced buffer solution used in in vitro digestion (Goering and Van Soest, 1970). ... 20

Table 3.1 Chemical composition of energy-protein concentrate (supplied by Afgri Animal Feeds). ... 30

Table 3.2 Chemical composition of forages used. ... 31

Table 3.3 Treatment design. ... 32

Table 3.4 The effect of exogenous fibrolytic enzyme treatment on non-linear parameters of in vitro NDF degradation in lucerne hay and wheat straw. ... 35

Table 3.5 Statistical parameters of in vitro NDF degradation (%) per incubation time on lucerne hay treated with exogenous fibrolytic enzymes (Rumenase® and ABO374) compared to a control. .... 37

Table 3.6 Statistical parameters of in vitro NDF degradation (%) per incubation time on wheat straw treated with exogenous fibrolytic enzymes (Rumenase® and ABO374) compared to a control. .... 37

Table 4.1 Chemical composition of feed used in the evaluation of exogenous fibrolytic enzymes and the effects on in vitro fibre digestibility ... 45

Table 4.2 Rumen fluid pH readings before in vitro incubation... 46

Table 4.3 Effect of EFE pre-treatment incubation time on in vitro degradation (%)... 47

Table 4.4 In vitro ruminal degradation (%) of feed substrates (lucerne hay, wheat straw and a TMR) at different pre-treatment incubation times (0.5, 3, & 12 hours). ... 50

Table 5.1 Chemical solutions required for enzyme cultivation and enzyme activity assay (Department of Microbiology, Stellenbosch University, 2018). ... 59

Table 5.2 Substrates with complementing reducing sugars used in enzyme activity assay. ... 59

Table 5.3 Concentrated reducing sugar concentrations required to create standard curve. ... 60

Table 5.4 Rumen fluid pH readings before in vitro incubation... 62

Table 5.5 Enzyme activity (nkat/ml) of amylase, endoglucanase, mannanase and xylanase cultivated on substrates derived from animal feed. ... 64

Table 5.6 Activity (nkat/ml) of enzymes in ABO374 for each experimental run. ... 64

Table 5.7 Dry matter, neutral detergent fibre (DM and OM) in vitro degradation of feed substrates. ... 65

Table 5.8 Effect of exogenous fibrolytic enzyme treatments on DM, NDF and NDFOM degradation, disregarding the feed substrate and incubation times. ... 65

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Table 5.9 Effect of incubation time on the extent of in vitro degradation of DM, NDF and NDFOM,

disregarding feed substrate and enzyme effects. ... 66

Table 5.10 Effect of EFE treatment on feed substrate in vitro DM, NDF and NDFOM degradation,

disregarding incubation times. ... 67

Table 5.11 The effect of EFE treatment on the rate and extent of in vitro feed substrate DM

degradation (lucerne hay, wheat straw, TMR). ... 68

Table 5.12 The effect of EFE treatment on the rate and extent of in vitro feed substrate NDF

degradation (lucerne hay, wheat straw, TMR). ... 69

Table 5.13 The effect of EFE treatment on the rate and extent of in vitro feed substrate NDFOM

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CHAPTER 1 General Introduction

Ruminant animals have the innate ability to convert fibrous plant matter into milk, meat, wool and hides, which made them useful to humans for millennia. The use of ruminants in this manner can be considered as the foundation of animal agriculture (Weimer et al., 2009). Fibre in the form of forages, cultivated pastures or crop-residues comprises a major part of ruminant diets. Microbial fermentation within the rumen allows for superior utilisation of energy within normally inaccessible plant cell walls (Van Soest, 1994). In most modern commercial systems, producers substitute high concentrate diets or cereal grains for forage (Weimer et al., 2009) to improve animal production outputs (Muller, 2017). However, a deficiency of fibre in these diets increase the risk of metabolic disease such as acidosis, which affects digestive efficiency, intake and metabolism, milk fat production and long-term health (Mertens, 1997). Despite possessing the unique ability to effectively utilise fibrous plant materials, ruminants are unable to completely digest fibre (Krause et al., 2003). Less than 65% of plant cell wall content is digestible under optimal rumen conditions (Van Soest, 1994). The subtropical climate of South Africa affects forage quality and availability due to erratic rainfall and seasonal extremes (Meisner, 1997). Crop residues such as wheat straw have become essential in ruminant production systems, reducing costs and useful to maintain animals in the dry season. These roughages form the basis of ruminant diets in subtropical climates as land solely used for forage production is costly (McDonald et al., 2011). However, the poor nutritive value of crop residues, due to plant maturity and lignification, reduces the accessibility of nutrients within the cell walls and in turn negatively affects dry matter intake (DMI) and digestion rates (Meissner, 1997; Van de Vyver & Cruywagen, 2013).

As a result of poor nutritive value of fibrous feeds, decades of research led to the development of methods to improve nutrient availability of forages and roughage. Physical (milling and heat treatment) and chemical (ammonia, urea and sodium hydroxide) strategies are most commonly used. With the improvement in the fields of biotechnology, genetically manipulated crops have been developed to reduce the cell wall material fractions to improve digestibility and nutrient utilisation (Jung et al., 1995, Krause et al., 2003). Despite the success achieved by these strategies, limitations in fibre digestibility are still evident. The use of enzymes with activities that increase hydrolysis of cell wall fractions has been used to improve the digestive process and nutrient availability of feed in ruminants (Beauchemin et al., 1995; Bhat, 2000; Colombatto et al., 2003b).

Exogenous fibrolytic enzymes (EFE) has proven to be effective when used as a feed additive for increased fibre digestibility. Some researchers reported effective increases in fibre degradation

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during in vitro ruminal assessments (Colombatto et al., 2003b; Eun and Beauchemin, 2007; Useni, 2011; Lunagayriya et al., 2017), while a lack of response was observed in other reports (Baloyi, 2008). Animals that were fed diets treated with EFE showed significant increases in weight gain, average daily gain and milk production (Beauchemin et al., 1999; Cruywagen and Goosen, 2004; Cruywagen and Van Zyl, 2008; Holtshausen et al., 2011), confirming beneficial effects observed with fibre degradation during in vitro studies. Despite many positive outcomes, studies contradicting the efficacy of enzyme treatments for use in ruminant diets were reported. Baloyi (2008) described a lack of effective in vitro dry matter (DM) and neutral detergent fibre (NDF) digestibility when forage was treated with two variations of exogenous enzymes. The factors that could possibly influence the efficacy of enzyme treatment on feed is however variable. Suggested factors include the type of enzyme and enzyme activity, sub-optimal pH temperatures for enzyme activity, variations in animals, pre-treatment effects, method of application, and substrate-enzyme specificity (Beauchemin et al., 2003a; Beauchemin et al., 2003b; Beauchemin et al., 2004a; Beauchemin et al., 2004b). Pre-treatment application of EFE to feed prior to ingestion by cattle was most effective as it allowed for enzyme-substrate interaction to occur (Beauchemin et al., 2003b), suggesting that cell wall degradation occurred prior to introduction to the rumen microbes. This is supported by Van de Vyver and Cruywagen (2013) who histologically evaluated the effect of EFE treated forages and observed thinning of cell walls after ruminal in vitro degradation.

An enzyme supernatant, derived from fungi discovered in South African soil, was developed by the Department of Microbiology (Stellenbosch University). The enzyme product, ABO374, underwent further experimentation by the Department of Animal Sciences (Stellenbosch University) to evaluate the potential effects the EFE could exhibit on fibre digestibility in ruminant diets. Positive results were reported on in vitro NDF degradation and gas production (Goosen, 2004; Useni, 2011), body weight and cumulative growth (Cruywagen and Goosen, 2004; Cruywagen and Van Zyl, 2008) and plant cell wall thinning (Van de Vyver and Cruywagen, 2013) when ABO374 was applied to feedstuffs. Introducing a fibrolytic enzyme additive to the diets of ruminant animals has the potential to improve the value of poor-quality feeds and creating an economical manner to increase production of meat, milk or wool for human consumption. A clear understanding of the pre-treatment incubation time (PIT) required prior to feeding is important as this needs to be practical to be used on a regular basis in practice.

To further understand enzyme use in fibre digestibility, the aim of this study was to re-evaluate the effect of an exogenous enzyme cocktail (ABO374) on DM and NDF digestibility to improve the utilisation of fibrous feed and forages in ruminants. The objectives were to:

1) evaluate the rate and extent of EFE products (ABO374 and a commercial cocktail) on the NDF digestibility of roughage by means of rumen in vitro digestibility.

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2) determine the optimal pre-treatment application time of an EFE cocktail (ABO374) on in vitro NDF digestibility.

3) determine the activity of fibrolytic enzymes within the EFE cocktail, replicate it using commercially produced pure enzymes, and compare the fibre digestibility of each by in vitro techniques.

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Useni, B.A., 2011. Effect of exogenous fibrolytic enzymes on fibre and protein digestion in ruminant animals. MSc(Agric) thesis, Department of Animal Sciences, Stellenbosch University, Stellenbosch.

Van de Vyver, W.F.J., Cruywagen, C.W.C., 2013. Exogenous fibrolytic enzymes to unlock nutrients: Histological investigation of its effects on fibre degradation in ruminants. South African J. Anim. Sci. https://doi.org/10.4314/sajas.v43i5.10.

Van Soest, P.J., 1994. Nutritional ecology of the ruminant, 2nd Edition. Cornell University Press, Ithaca, NY.

Weimer, P.J., Russell, J.B., Muck, R.E., 2009. Lessons from the cow: What the ruminant animal can teach us about consolidated bioprocessing of cellulosic biomass. Bioresour. Technol. https://doi.org/10.1016/j.biortech.2009.04.075.

Yang, W.Z., Beauchemin, K.A., Rode, L.M., 1999. Effects of an Enzyme Feed Additive on Extent of Digestion and Milk Production of Lactating Dairy Cows. J. Dairy Sci. https://doi.org/10.3168/jds.S0022-0302(99)75245-8.

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CHAPTER 2 Literature Review

2.1 Introduction

Ruminants are physiologically capable of converting low quality feed and forage into usable products for human consumption (meat, milk, leather, wool, etc.). Except in the case of high producing production animals, forages form a significant component of the ruminant diet. Forages may be consumed in the form of pasture, silage, hay, or crop residues. The ability of ruminants to utilise plant cell walls is unmatched and the economic implications cannot be overlooked. Along with its nutritional value, coarse fibre also functions in maintaining a healthy rumen by stimulating rumination (Van Soest, 1994). This improves digestion by physical break-down of feed, fermentation and digestion of feed, saliva production, and stimulating ruminal contraction. The utilisation of forages by ruminants differ by production system, ranging from pasture grazed forage to treated forages in a total mixed ration (TMR).

The efficiency of forage utilisation to be converted consumable products depend on the digestibility of the forage cell walls (Beauchemin et al., 2004a). As the digestibility of the plant cell wall content is limited to less than 65% under optimal rumen conditions (Van Soest, 1994) a large margin for improvement still exists. The use of exogenous fibrolytic enzymes (EFE) as a feed additive has improved fibre digestibility and feed utilisation in ruminant animals and has been studied since the 1960’s (Beauchemin et al., 1995). Despite positive results a lot is still unknown about exogenous enzymes and the use of it in animal diets.

2.2 Forages and fibre

Forage is the edible parts of plants, other than separated grain, that provides feed for grazing animals or that can be harvested for feeding animals (Allen et al., 2011). Fibre was defined by Mertens (1997) as the fractions of feed that are slowly digestible or indigestible, and that occupies space in the gastrointestinal tract of ruminant animals. The classification systems for forages by Van Soest (1967) has standardized fibre analysis worldwide (Table 2.1).

2.2.1 Chemical composition of plant cells

Plant cell walls primarily comprise of structural polysaccharides, containing various sugars (e.g., xylose, mannose, glucose, galactose, etc.) that allow for varied structural compositions (Wang and McAllister, 2002). Cellulose accounts for the largest fraction of the polysaccharide structures,

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accounting for 20 - 30% of the dry weight of primary cell walls (McNeil et al., 1984). Hemicellulose, a main component in leaves and cell walls, is formed from xylans (xylose) and a β-1,4-linked backbone structure. Various side chains (ferulic acid, acetic acid, arabinose, etc.) complicates the

Table 2.1 Detergent methods of forage fraction classification (Van Soest, 1967)

Fraction Components

Cell contents (soluble in neutral detergent) Lipids

Sugars, organic acids and water-soluble matter Pectin and starch

Non-protein nitrogen Soluble protein

Cell wall constituents (insoluble in neutral detergent)

Soluble in acid detergent Hemicelluloses

Fibre-bound protein

Acid-detergent fibre Cellulose

Lignin

Lignified nitrogen Silica

hemicellulose structure with attachments to the xylose residues (McNeil et al., 1984). Xylan polymers may also be cross-linked to lignin or other hemicellulose backbones. A large network of cross-linked cellulose microfibrils derives from the linear xylan backbone that strongly binds to cellulose, interlocking with other xylan polymers (Carpita and Gibeaut, 1993). A three-dimensional matrix is formed from polysaccharides, hydroxycinnamic acids, lignins, proteins and ions that are cross-linked through ionic-, hydrogen-, and covalent bonds. These matrices trap the polysaccharide within the structure of the cell wall (Wang and McAllister, 2002). Complete degradation of cell walls therefore requires a combination of more than just hydrolytic enzymes, as enzymes with other activities have the capability to complete the cleaving of bonds retained in the matrix structure.

Plants cells consists of primary cells walls with the potential to grow thick secondary cell wall layers within the primary walls (Figure 2.1). As the secondary cell walls thicken, the deposition of certain components (ferulic acid, pectin and xylan) reduce to facilitate increased deposition of lignocellulosic components (Jung & Allen, 1995; Graminha et al., 2008). The lignification of plant tissues, a result of plant maturity, affects the structure of hemicellulose due to the high concentration found within the primary cell walls. Lignification thus changes the plant cell wall structure to form a rigid bar rier that prevents damage within the plant. Layers of epicuticular waxes, cuticle and pectin occur as outermost layers of plant cell walls and function as the first line of defense against environmental threats (dehydration, disease, etc.) (McAllister, 2002).

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2.2.2 The nutritive value of forages

The nutritive value of forages and feeds pertains to forage composition factors that affect nutritient accessibility, but are independent of voluntary intake (Fisher et al., 1995). Two factors, when biological constituents are considered, determine (1) the nutritive value of feeds and forages (the proportion of cell

wall) and (2) the degree of lignification. The nutritive value of plants declines with age as a result of lignification (Van Soest, 1994). Nutrients within the cell wall are less accessible, the quality of leaves changes and plant matter consist mostly of stems with fewer leaves. Environmental factors and forage type also influence the extent to which maturity occurs in plants (Van Soest,1994). The fibrous cell wall portion of forages consists of approximately 30 - 80% of the total dry matter. Despite fibre being a major source of nutritional energy for ruminants, less than 50% of this fraction is digestible and utilised by the animals (Hatfield et al., 1999). Agricultural crop residues, such as wheat straw, are typically high in fibre. Within these residues, cellulose and hemicellulose represent the largest fraction of the plant cell structures (Graminha et al., 2008) with lignification mainly affecting the hemicellulose fraction of the cell walls. Baumont et al. (2000) defined forage indigestibility as the maximum quantity of feed that an animal can consume when it is supplied ad libitum. Indigestibility, and effectively digestibility, depends on the age of and stage of growth of forages. These factors determine the plant cell wall structure and level lignification (Van Soest,1994), which influences intake, gut fill, and nutrient utilisation.

2.3 Ruminant digestion and fibre utilisation

The ruminant diet primarily contains forages and roughages. These feedstuffs consist mainly of polysaccharides such as cellulose and hemicellulose (McNeil et al., 1984) and typically cannot be

Figure 2.1 Schematic representation of plant cell wall development (Jung and Allen,

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broken down by conventional mammalian digestive enzymes. Ruminants have evolved to include microbial fermentation within the digestive system (McDonald et al., 2011) to facilitate fibre digestion.

2.3.1 Microbial fermentation

Ruminant feed digestion results from a symbiotic relationship between the host animal and digestive microbes that reside within the rumen. The anaerobic conditions within the rumen provides favourable conditions for microbes to digest plant cell walls through their fibrolytic enzyme secretions (Krause et al., 2003). The rumen microbiome comprises of bacteria, fungi, protozoa (Table 2.2), along with viruses, prions, bacteriophages and archea. Bacterial and fungal degradation contributes approximately 80% of the total degradation within the rumen (Dijkstra and Tamminga, 1995). Ruminal bacteria have been extensively studied with more than 2000 species identified (Firkins, 2010; McDonald et al., 2011; Puniya et al., 2015), whereas ruminal fungi are yet to be well understood. However, fungi are capable of penetrating the cuticle layer of cell walls and lignified plant tissues and produce a variety of enzymes capable of degrading a greater variety of substrates (Wang and McAllister, 2002). The various activities of fungal enzymes allow for degradation of resistant cell wall components (Useni, 2011). The activity of the enzymes confirmed to be present within the rumen are diverse and include cellulases, xylanases, -glucanases, amylases, proteases, phytases, tannases and other enzymes that degrade plant toxins. The complex structure of plant cell walls requires a diverse range of hydrolytic enzymes for complete degradation to occur.

Two models have been proposed for fibrolytic enzyme systems, after microbial synthesis. The first model suggested that enzymes act individually, yet synergistically, to digest substrate (Wood, 1992). The second model proposed that individual enzymes co-ordinate and form enzyme complexes (e.g., cellulosome). Interactions between the many rumen microbes occur as either synergy or competition for the same source of energy (Wang and McAllister, 2002). Synergy between rumen microbes can improve the efficacy of feed digestion and utilisation. Competition for the same resources does occur and can in turn reduce degradation.

The products of microbial digestion are utilised by the host animal as energy. The main products of microbial digestion include volatile fatty acids (VFA; mainly acetic acid, propionic acid and butyric acid), microbial protein, carbon dioxide (CO2) and methane (CH4). VFA absorption occurs via the

rumen wall, serving as the major energy source for mucosal tissue and the host animal. The VFA absorbed by the rumen supplies up to 80% of the animal’s energy requirements, and 67% of digested CP absorbed in the small intestine results from the rumen microbial protein (NRC, 2001). Diets high in fibre produce higher levels of acetate and butyrate, both important for milk and milk fat production. High levels of propionate results from diets high in cereals and starch (Beever and Mould, 2000).

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Table 2.2 Rumen microbes and their enzyme activities involved with plant cell wall degradation (Source:

Derived from Dehority (1993) as cited by Wang and McAllister (2002))

Organism Degradative activity

Cellulolytic Hemicellulolytic Pectinolytic

Bacteria Fibrobacter succinogenes + + + Ruminococcus albus + + + Ruminococcus flavefaciens + + + Butyrivibrio fibrisolvens + + + Eubacterium cellulosolvens + Clostridium longisporum + + Clostridium locheadii + + Prevotella ruminantium + Eubacterium xylanophilum + Ruminobacter amylophilus + Succinimonas amylolytica + Succinivibrio dextrinosolvens + Selenomonas ruminantium + Selenomonas lactilytica + Lachnospira multiparus + Streptococcus bovis + + Megasphaera elsdenii + + Protozoa Eudiplodinium maggii + + + Ostracodinium dilobum + + + Epidinium caudatum + + Metadinium affine + + + Eudiplodinium bovis + + + Orphryoscolex caudatus + + + Polyplastron multivesiculatum + + + Diplodinium pentacanthum + Endoploplastron triloricatum + Orphyroscolex tricoronatus + Ostracodinium gracile + Entodinium caudatum + + Isotricha intestinalis + + + Isotricha prostoma + Fungi Neocallimastix frontalis + + + Neocallimastix patriciarum + + + Neocallimastix joyonii + + Caecomyces communis + + + Piromyces communis + + + Orpinomyces bovis + + Ruminomyces elegans + +

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The microbial digestion of feed has been described as the Inside-Out concept (Cheng et al.,1991). As the most readily digestible tissues are located within the plant structure, intact plants are digested slowly. The mechanical break-down of fibre, due to mastication or feed processing, allows rumen microbes to access the nutrient-rich cell contents via the stomata, or via the spaces created by mastication.

2.3.2 Factors affecting fibre digestion

Despite advanced capabilities, digestion and utilisation of fibre in ruminants is not optimal. Fibre obtained from faeces was found to be fermentable by Krause et al. (2003), a clear indication that improvements in fibre digestibility can be made. The reasons for incomplete fibre digestion can broadly be described as the influence of physical and biochemical barriers of ingested feedstuffs and ruminal retention time (Wang and McAllister, 2002). The efficiency in plant cell wall degradation greatly depends on the interaction between rumen microbes and the anaerobic fermentation conditions within the rumen (Cruywagen and Goosen, 2004).

The rumen environment is a large contributing factor in fibre digestion. The growth and maintenance of fibrolytic microbes however require optimal rumen conditions. Under normal conditions the rumen ranges close to neutral, between pH 6 and pH 9, with fibre digestibility being negatively affected at a pH below 5.5 (Weimer, 1996). Interactions of competition and synergy between rumen microbes create a secondary environmental factor within the rumen that influences digestion (Weimer, 1996). The health and physiological status of the animal also influences digestibility as the nutritional requirements differ with these factors. Rumination and mastication stimulate saliva production which contain bicarbonates and phosphates that work as a buffer to maintain optimal ruminal pH (Castillo-González et al., 2014). A sufficient fibre content of the feed is required to ensure chewing activity for optimal pH and the maintenance of a healthy rumen environment (NRC, 2001). The rumen temperature averages at 39 ºC (Van Soest, 1994).

The form of fibre plays a significant role in ruminant nutrition as coarse fibre is required to sustain healthy rumen function (Cruywagen and Goosen, 2004). Fibre coarseness influences the rate of digestion, and retention time of feedstuffs within the rumen, and in turn influences the available energy to the animal per time unit (Van Soest, 1994). Longer ruminal retention times allow for extended periods for fibre digestion to occur. Particle size is the main determining factor in ruminal retention, as larger feed particles remain in the rumen for longer periods (Wang and McAllister, 2002). The physical characteristics of fibre are crucial for optimal rumen function, with amount and effectiveness important for rumen fermentation (Mertens, 1997). NDF can be divided into physically effective NDF (peNDF) and effective NDF (eNDF). Effective NDF describes the physical characteristics (particle size) that influence rumen content and chewing activity. Effective NDF

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further relates to the ability of the feed to provide nutritive value. Chewing activity also promotes saliva production and improves the buffering capacity of the animal (NRC, 2001).

2.4 Technological advancements for improved fibre digestibility

Advancements in technology has continued to drive innovation to optimize fibre digestibility and utilisation in ruminants. Several strategies, such as physical (milling and heat treatment), chemical (ammonia, urea and sodium hydroxide) and biological treatments have been used for decades to improve the nutritive value of feedstuffs. The use of traditional chemical treatments became less common with the development of new treatment methods and research that proved certain treatments to be harmful. Sodium hydroxide (caustic soda), for example, is corrosive (Kellaway et

al., 1978) and is not used that often anymore, although some feed companies still treat low quality

roughages, such as wheat straw, with sodium hydroxide. New treatment methods to improve DMI and bioavailability of nutrients remain an important focus of forage quality research. Genetic manipulation of crops to reduce cell wall material concentrations, and thus fibre concentration, has been investigated (Jung & Allen, 1995; Krause et al., 2003). Microwave-assisted alkali (MAA) pre-treatment combined the traditional alkali pre-pre-treatment with microwave irradiation for improved efficacy of cell wall hydrolysis in wheat straw (Zhu et al., 2006). Enzymes in addition to MAA pre-treatments further improved the hydrolysis potential of wheat straw (Patel et al., 2017).

2.4.1 The use of enzymes as a feed additive

The use of exogenous enzymes to improve feed utilisation in ruminants has been used with proven potential. Research shows that the supplementation of fibrolytic enzymes in dairy cow, feedlot cattle and sheep diets have significant potential to improve digestion and production (Beauchemin et al., 2003; Cruywagen and Goosen, 2004; Cruywagen and Van Zyl, 2008; Bala et al., 2009). A review paper focused on cellulases and related enzymes in biotechnology was done by Bhat in 2000. In the review the use of enzymes, cellulases and hemicellulases, indicated great potential applications for both monogastric and ruminant animal production systems. The forage used for ruminant feed, containing cellulose, hemicellulose and lignin, is more complex than cereal-based diets used for monogastric livestock. Enzyme preparations that contain high levels of cellulase and hemicellulose activity have indicated improvements in feed utilisation, milk yields and body weight gain (Bhat., 2000).

2.4.2 Commercial enzymes

Commercial enzyme products typically contain a combination of enzymes with varied activities (Colombatto et al., 2003b). Cellulases and hemicellulases are commonly used as research has been

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focused on the major structural polysaccharides, namely cellulose and hemicellulose (Van Soest, 1994). Variation between the types of cellulases and hemicellulases allow for differences between commercial enzyme products and the manner in which they are produced. Production of these cocktails depend on individual enzyme activity and the potential effects it may have on fibre degradation (Beauchemin et al., 2004b). Other fibrolytic enzymes that provide secondary activities (amylases and proteases) can also be found within commercial enzyme products.

2.5 Exogenous fibrolytic enzymes in ruminant nutrition

The success of a ruminant farming system greatly depends on the effective utilisation of feed, as feed contributes the greatest fraction of production costs. Forage forms a significant part of ruminant feed requirements and the effective utilisation thereof can play an important role to improve production. Despite ruminants possessing the ability to utilise nutrients from fibrous plant matter (Silva et al., 2016), it is not optimal. Most forages have a cell wall content of 40 – 70% of the total DM with less than 65% cell wall digestibility in the total digestive tract despite optimal conditions (Van Soest, 1994).

2.5.1 Enzymes and ruminant diets

The use of exogenous fibrolytic enzymes has been extensively studied for the purpose of improving fibre digestibility, feed intake and animal production parameters (Yang et al., 1999; Lewis et al., 1999; Beauchemin et al., 2002; Krause et al., 2003; Cruywagen and Goosen, 2004). Enzymes have been defined as “proteins that catalyze the degradative reactions in biological systems” in a review paper by Hristov et al. (1998). When referring to ruminant feeds, exogenous enzymes catalyse the degradative reactions of feed to ultimately release nutrients to be used by the host animal or any other microorganisms within the rumen. The use of enzymes depends on:

• the stability of the enzymes in the feed, either during or after processing occurs; • the ability of the enzymes to hydrolyze plant cell wall polysaccharides; and • the ability of the animals to efficiently utilise the products (Bhat., 2000).

Synergy between the endogenous enzymes produced by rumen microbes and the exogenous enzymes introduced to the animal was documented by Beauchemin et al. (2004a). A net effect was observed when exogenous fibrolytic enzymes (EFE) were added, creating an increase in enzyme activity that exceeded the additive effects of the individual constituents involved.

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2.5.2 Factors affecting exogenous enzyme function and activity

Enzyme activity is influenced by temperature, pH, ionic strength, substrate type and substrate concentration (Beauchemin et al., 2003). Temperatures above 60 ºC will decrease the efficiency enzyme activity, with total enzyme destruction occurring above 100 ºC (McDonald et al., 2011). All enzymes have temperatures at which activity is optimum. The majority of enzymes function effectively within a pH range of 6 and 7, although many are capable of functioning below pH 6. Enzyme activities for commercial products are typically measured at the manufacturer’s discretion. An approximate temperature of 60 C and a pH ranging between 4 and 5 are optimal conditions for the majority of commercial cellulases (Coughlan, 1985). However, these conditions are not representative of rumen conditions, which average at 39 C with a pH range between 6.0 and 6.7 (Van Soest, 1994). Some commonly used exogenous enzymes function optimally at lower pH levels than that of ruminal enzymes, suggesting that the addition of such enzymes may prove beneficial when the rumen pH is sub-optimal for optimal function of rumen microbes (Beauchemin et al., 2004a). According to Beauchemin et al. (2003) the most effective method of application for EFE treatment is the pre-treatment of feed before it is made available to the animal. This method allows for the enzymes to effectively attach to feed particles and initiate the release of reducing sugars (Hristov et al., 1996).

2.5.3 Enzyme specificity

Enzyme-substrate specificity is widely recognized as enzymes function optimally under different conditions (Beauchemin et al., 2004b). Colombatto et al. (2003b) examined 22 commercial enzyme products to evaluate biochemical characteristics and in vitro degradation of lucerne hay and corn silage. The results indicated that enzyme efficacy differed with each substrate (Colombatto et al., 2003b). Enzymes applied to lucerne hay seemingly exerted activity during a period of pre-treatment, whereas enzymes applied to corn silage were exclusively active after exposure to ruminal fluid. This may provide an explanation on how enzyme specificity functions as different modes of action occurs on different substrates under different pH and temperature conditions.

2.6 ABO374

An exogenous fibrolytic enzyme cocktail, derived from a strain of Aspergillus fungi in South Africa, was developed by the Department of Microbiology (Stellenbosch University) in collaboration with the Department of Animal Sciences (Stellenbosch University). The product, known as ABO374, was characterised by Cruywagen and Van Zyl (2008) as a fibrolytic enzyme cocktail, grown on wheat straw, and mainly containing xylanase (296  0.07 U/mg protein), endoglucanase (1.44  0.39 U/mg

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protein) and mannanase (1.10  0.37 U/mg protein) enzyme activity. Enzyme activity was determined using the dinitrosalicylic acid method (Miller et al., 1960; Bailey et al., 1992).

Between 2001 and 2006, a series of trials were done by in an Innovation Fund project by a consortium consisting of the Departments of Microibiology and Animal Sciences at Stellenbosch University, the Department of Microbiology at the University of the North (UNIN), the CSIR, Voermol and later Meadow Feeds. The project was funded by the then Department of Arts, Culture, Science and Technology (DACST) and the purpose was to find a strain of fungus that would produce a viable mix of fibrolytic enzymes that could be used in the animal feed industry in South Africa. Around 450 lignocellulolytic fungi and yeast strains indigenous to South Africa were collected. From a pre-selection of 150 strains, 35 strains showed a positive response in in vitro digestibility assays on wheat straw and Eragrostis curvula hay substrates. Of the 35 strains, seven performed better than the industrial standard Trichoderma reesei RUT C30. Eventually, a specific strain of Aspergillus, which was termed ABO374, performed consistently better than the other six strains (information obtained from a 2005 Consortium report, provided by Prof CW Cruywagen, Department of Animal Siences, Stelenbosch University).

An approximate of 300 fungal strains were initially cultivated to obtain enzyme-containing supernatants by the Department of Microbiology, Stellenbosch University. A total of 150 strains were selected for further screening by the Department of Animal Sciences, Stellenbosch University. Cruywagen and Goosen (2004) conducted the screening of the selected supernatants to identify promising fungal strains for possible use in the animal feed industry. Two phases of screening were used: (1) in vitro digestibility and (2) in vitro gas production (GP). Results indicated that ABO374 was superior, increasing the cumulative GP by >10% at 18 hours (Cruywagen and Goosen, 2004). Van de Vyver and Cruywagen (2013) investigated the mode of action and histological effects of ABO374 on plant tissue degradation over a 24 h incubation period. Four plant materials were used for the histological evaluation, namely: weeping love grass, kikuyu leaf, lucerne hay and wheat straw. Plant tissue samples were cut into 20 µm cross sections and fixed to microscope slides. Slides were treated with ABO374 and incubated anaerobically for 0, 6, 12 and 24h in buffered rumen fluid. After incubation, the samples were observed and quantified using image analysis software. Thinning of cell walls of kikuyu and weeping love grass metaxylem, phloem and adaxial epidermis tissue was observed on EFE treated samples. The EFE treatment also resulted in a thinner epidermis of lucerne hay.

Further research on ABO374 was conducted to evaluate its fibrolytic effects on animal production, with the majority of findings yielding positive results (Cruywagen and Goosen, 2004; Cruywagen and Van Zyl, 2008; Useni, 2011; Van de Vyver, 2011). Siginificant weight gains and feed conversion efficiencies were recorded with Döhne Merino lambs in treatment groups that received medium (5

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ml/kg) and high (10 ml/kg) enzyme inclusion levels (Cruywagen and Goosen, 2004). Cruywagen and Van Zyl, 2008) evaluated the effect of ABO374 on South African Mutton Merino lambs and observed an increase in animal performance (FCR, growth and cumulative weight gain).

The complete composition of the ABO374 enzyme cocktail is currently unknown and it is believed to contain a great number of enzyme varieties. Cruywagen and Van Zyl (2008) determined the main enzymes within this cocktail, but a more complete profile of the enzyme content is required to better understand this EFE. The potential uses of ABO374 are seemingly not limited for use in ruminant feeds. Bhat (2000) described the uses of EFE in both ruminant and monogastric animal feeds. Hydrolases are mainly used for the elimination of anti-nutritional factors (ANF) in grains, to improve the nutritional value of feed or to supplement the digestive enzymes of the animals (Galante et al., 1998). According to Galante et al. (1998) improvements in FCR, growth and bodyweight, intake and production can be expected when EFE’s are used.

2.7 Methods of analysis

The analysis of feed and the potential digestibility of feed has developed over many years. Many effective methods exist and are used globally.

2.7.1 Proximate analysis

The Weende classification system, commonly known as proximate analysis, has been used for over approximately 150 years. The components of the proximate analysis are divided into six fractions (Fisher et al., 1995):

• Moisture • Ash

• Crude protein (CP) • Ether extract (EE) • Crude fibre (CF)

• Nitrogen-free extracts (NFE)

The model is simple and repeatable though it has been criticized for being outdated and imprecise. Concern pertains to the carbohydrate fraction, which is divided into crude fibre and nitrogen-free extracts (McDonald et al., 2011). The proximate analysis interprets CF to be formed by all dietary cellulose, hemicellulose and lignin (Cherney, 2000) and Van Soest (1994) showed that crude fibre has soluble and insoluble fractions, namely neutral detergent solution (NDF) and acid detergent solution (ADL), that do not separate during the proximate analysis. More accurate methods have since been developed to characterize feed expressed by nutrient requirements (McDonald et al.,

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2011). An alternative detergent method, specifically intended for high fibre feedstuffs, was developed by Van Soest (1967). The Van Soest method of feed fractionation divides feed dry matter into three fractions, namely, (1) completely available, (2) partly available due to lignification and (3) unavailable fractions. The identification of the insolubility of feed to neutral detergents (NDF) and acid detergents (ADF) in turn created a possible method for predicting feed intake and the nutritive value thereof (Mould, 2003) (Figure 2.2).

2.7.2 NDF

The insoluble fibre fraction in feed can be measured as NDF and includes cellulose, hemicellulose and lignin (Van Soest et al., 1991). NDF is used to measure the cell wall content of feed, enabling available energy from feed to be expressed as inversely correlated to the NDF fraction with the feed (Buxton and Mertens, 1995). NDF contributes significantly to rumination, rumen fill, passage rate, feed intake and it forms the rumen mat that stimulates rumen function (Van Soest et al., 1991). It is therefore the most crucial fibre fraction in ruminant diet analysis and vital in feed formulation.

Table 2.3 Neutral detergent fibre (Van Soest, 1994).

Reagent 1 L Volume

Part 1

Sodium lauryl sulphate 30 g

2-Ethoxyethanol 10 ml

dH2O 500 ml

Part 2

EDTA 18.61 g

Sodium borate decahydrate 6.81 g

dH2O 200 ml

Part 3

Disodium hydrogen phosphate 4.56 g

dH2O 100 ml

Mix Parts 1, 2 and 3 separately. Combine all parts and make up to 1 L using distilled H2O.

The process of determining the NDF fraction of feed was initially developed for forages. Fibrous feed samples were treated with a boiling neutral detergent solution (Table 2.3) to remove cell contents and the remaining residue represented the structural cell wall components (cellulose, hemicellulose and lignin), as well as proteins, fibre-bound nitrogen, minerals and plant cuticle. The detergent removes pectin (Van Soest, 1994). The inclusion of heat-stable -amylase to assist with starch removal was later introduced (McDonald et al., 2011) and allowed for NDF determination in starch-containing feeds. It corrected for starch contamination which could result in overestimation of the NDF value (Beever and Mould, 2000). The ANKOM fibre analyzer and analysis method, developed

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by ANKOM Technology Corporation (Fairport, NY, USA), is currently used as an alternative method to determine fibre fractions in feeds.

2.7.3 Techniques for in vitro analysis

Many in vitro techniques have been developed for laboratory analysis by using rumen fluid, buffers (Table 2.4), commercial enzymes or chemical solvents. ANKOM Technology Corporation (Fairport, NY, USA) developed the ANKOM DAISYll_{as a method for in vitro digestibility. Feed samples (sealed}

in filter bags) are placed in jars containing a buffered medium and rumen fluid as inoculant for varied time periods, continuously rotating within the insulated incubator. The use of filter bags allow ed for large sample sizes and different feed types (forages, grains, etc.) to be incubated in the same digestion jar, as well as preventing the filtration of residues when estimating the in vitro digestibility of the feed samples. The DAISYll_{is a convenient and efficient in vitro method for evaluating ruminant}

feed digestibility.

The Van Soest buffer solution was prepared as described by Goering and Van Soest (1970), comprising of a medium solution and a reducing solution (Table 2.5). The medium and reducing solutions were prepared 18 hours before in vitro incubation occurred and stored separately in sealed flasks. Both solutions were gassed using CO2 to maintain an anaerobic environment before storage

at 39 C to match normal rumen temperatures (Van Soest, 1994). The two solutions were combined one hour prior to the onset of in vitro incubation. summarises the constituents of the in vitro buffer solution.

Figure 2.2 Comparison between the Weende sytem and Van Soest method of analysis (modified from

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Table 2.4 Constituents of the in vitro buffer solution (Goering and Van Soest, 1970).

Macro mineral solution Reagents 1 L Volume

Distilled water (ml) 1000

Na2HPO4 anhydrous (g) 5.7

KH2PO4 anhydrous (g) 6.2

MgSO4 • 7H2O (g) 0.59

NaCl (g) 2.22

Micro mineral solution Reagents 100 ml Volume

CaCl2 • 2H2O (g) 13.2

MnCl2 • 4H2O (g) 10

CoCl2 • 6H2O (g) 1

FeCl3 • 6H2O (g) 8

Buffer solution Reagents 1 L Volume

NH4HCO3 (g) 4

NaHCO3 (g) 35

Table 2.5 Composition of reduced buffer solution used in in vitro digestion (Goering and Van Soest, 1970).

Medium solution 2 L Volume

Tryptose (g) 5

Buffer solution (ml) 2.5

Macro mineral solution (ml) 500

Micro mineral solution (ml) 0.250

Resazurin 0.1% w/v (ml) 500

Reducing solution 100 ml Volume

Cysteine hydrochloric acid (g) 0.625

KOH pellets (g) 10

Sodium sulphide anhydrous (g) 0.625

2.7.4 Enzyme assays

Enzyme activity assays are typically used to characterize enzymes and confirm the presence of an enzyme within a sample. Enzyme activity is measured by the disappearance (hydrolysis) of substrate, or the formation of product over time. The reducing sugar assay is a method commonly used to determine the amount of reducing sugars in a solution, which can be used to calculate