TitleImproving the Utilization of Agricultural By-product as Feed forRuminant: Studies of Fermented Juice of Epiphytic Lactic AcidBacteria on Total Mixed Ration( 本文(Fulltext) )

Author(s) Yuli Yanti

Report No.(DoctoralDegree) 博士(農学) 甲第705号

Issue Date 2019-03-13

Type 博士論文

Version ETD

URL http://hdl.handle.net/20.500.12099/77941

※この資料の著作権は、各資料の著者・学協会・出版社等に帰属します。

Improving the Utilization of Agricultural By-product as

Feed for Ruminant: Studies of Fermented Juice of Epiphytic

Lactic Acid Bacteria on Total Mixed Ration

2018

The United Graduate School of Agricultural Science,

Gifu University

Science of Biological Production

(Gifu University)

Yuli Yanti

Improving the Utilization of Agricultural By-product as

Feed for Ruminant: Studies of Fermented Juice of Epiphytic

Lactic Acid Bacteria on Total Mixed Ration

Yuli Yanti

CONTENTS

CHAPTER I

General Introduction

CHAPTER II

Total Mixed Ration silage prepared from agricultural by-products and food by-

products: Improving quality of TMR silage using fermented juice of epiphytic lactic

acid bacteria

II-1. Introduction

II-2. Materials and Methods

II-3. Results

II-4. Discussion

II-5. Summary

CHAPTER III

Effect of total mixed ration ensiled with fermented juice of epiphytic lactic acid

bacteria on nutritive intake, nutrient digestibility, rumen fermentation and nitrogen

balance in ewes

III-1. Introduction

III-2. Materials and Methods

III-3. Results

III-4. Discussion

III-5. Summary

CHAPTER IV

Effect of total mixed ration ensiled with fermented juice of epiphytic lactic acid

bacteria on blood parameters, energy balance and methane emission in ewes

IV-1. Introduction

IV-2. Materials and Methods

IV-3. Results

IV-4. Discussion

IV-5. Summary

CHAPTER V

General Discussion

REFERENCES

ACKNOWLEDGEMENTS

CHAPTER I

GENERAL INFORMATION

Introduction

Crop production in the world was dominated by sugar cane, maize, paddy

(rice) and wheat (FAO, 2013). Those agriculture products paralleled with a huge by-

product whereas giving a negative impact on the environment if not managed properly.

In the tropical area, ruminant depends on cut grasses and agricultural by-products as

feed since the availability of pastures decreases in the dry season (Winugroho, 1999;

Sarnklong et al., 2010). Crop residues and agro-industrial by-products include a high

number of materials of which straw of cereals, stover from maize and sorghum, corn

cobs and bagasse. These feeds often are rich in carbohydrates in the form of cellulose

and hemicellulose (Owen and Jayasuriya, 1989; Van Kuijk et al., 2015). The use of

cereal straw for ruminant feed has been known classically for its poor nutritive value;

low energy availability and nitrogen (N) content, low intake (Madrid et al. 1997) and

low in digestibility, therefore animal production is low (Van Soest, 2006). For many

years, researchers have attempted to improve the nutritional quality of agricultural

waste as feed for ruminants. The various papers have reported treatments such as

biology, physics, chemical and enzymes as will be discussed in detail in this section.

Increased nutrient in the feed is expected to enhance animal production.

However, increasing the nutrient quality of agriculture by-product should not

only focus on the increase of animal production but also should be considered

regarding the environmental problem. As it known for many years, fermentation of

dietary carbohydrates in the rumen by methanogenic archaea produces methane gas.

Methane production tends to increase as the fiber content of feed increases (Kurihara

et al., 2007). Type of carbohydrate of agricultural by-product, especially fodder/straw,

contains the high level of fiber. It means that feed from agricultural by-product tends

to produce more methane than other feed sources (forage and legume). A ruminant is

an important source of methane emission in many countries since they are in huge

population and high methane emission rate due to their digestive system (Broucek,

2014). For many decades, reducing methane production from rumen fermentation was

considered only in the context of feed inefficiency (Abrar et al., 2016). Methane is the

second largest greenhouse gas after carbon dioxide in contribution to global warming

which is the main driving to climate change. Climate change is transforming the

planet’s ecosystems and threatening the well-being of current and future generations

(Marino et al., 2015). Energy losses as methane gas vary from approximately 2 to

nearly 12% of gross energy intake (Johnson et al., 1993). However, recently

ruminant-generated methane has become recognized as a contributor to climate

change (Abrar et al., 2016). Emission of methane from enteric fermentation were the

greatest contributor to agricultural emissions (40%) (FAO, 2014). It is influenced by

many factors such as level of feed intake, type of carbohydrate in the diet, feed

processing, addition of lipids or ionophores to the diet and alterations in the ruminal

microflora (Johnson and Johnson, 1995).

Many strategies that had been developed in mitigating enteric methane

production from ruminant to achieve both on reducing global greenhouse gas

emissions and as a means of improving feed conversion efficiency (Martin et al.,

2010). Therefore, the aim of this chapter is to provide an overview of existing

information on how to increase agricultural by-product nutrition and mitigate its

methane emission as well.

Source of agricultural by-product

The most crops production in last decade is shown in Table 1.1. The highest

production was sugarcane then followed by maize, paddy rice, and wheat. However,

the highest increased production was maize in the last ten years then followed by

sugarcane, wheat, and paddy rice. The increase in crop production has the same

meaning of increases in crop by-product. Those residues when not managed properly

would give an adverse impact on the environment. The usage of agricultural by-

product as feed for ruminant is common in the tropical country, especially by small-

scale farmers. Winugroho (1999) reported that the highest agricultural by-product

produces in India and China. Sruamsiri (2007) reported that in Chiang Mai, Thailand,

agricultural waste and agricultural by-products such as rice straw, corn stover,

soybean straw, soybean pod, soybean hull, sugarcane tops, and bagasse were utilized

as dairy feed in the dry season when no green forage is available. Agricultural by-

product has poor digestibility, and low nutritive value as ruminant’s feeds. It contains

low crude protein (2.6-4.3% dry matter or DM) and high crude fiber (70.8-97.2% DM

of neutral detergent fiber or NDF). The more detail for nutrients content of some

agricultural by-product is shown in Table 1.2.

Rice straw is the most common agricultural by-product used for ruminant feed

in the countries that are high in rice production. Rice straw production in the top 10

countries in the world is shown in Table 1.3. The farmers in these countries use rice

straw for animal feeding. However, the nutrient content from this feed is not sufficient

for ruminants to grow and to meet the maintenance needs when given as a single feed

source (Sarnklong et al., 2010). Therefore, for achieving sufficient in nutrient

requirement when rice straw is a basic diet is by improving its nutritive value or

supplementing with other high-quality feed sources (Malik et al., 2015).

Table 1.1. The major crop production in the world, 2003-2013

Crop Production (Million tones)

Relative Increase (%) 2003 2013

Sugar cane 1,379 1,898 38 Maize 645 1,017 58 Rice, paddy 587 738 26 Wheat 560 711 27

Modified from FAO (2015).

Table 1.2. Nutrients content of selected agricultural by-products

Composition Agricultural by-product

Rice strawa

Wheat strawb

Corn stoverc

Barley strawd

Bagassee

DM (%) 90.1 87.2 93.4 87.4 93.4 OM (% DM) 85.6 90.5 95.2 90.4 98.0 CP (% DM) 4.3 3.4 4.1 2.6 1.3 NDF (% DM) 70.8 80.1 71.9 77.3 97.2 ADF (% DM) 43.5 49.7 41.4 46.7 61.4 ADL (% DM) - - 6.3 9.0 13.2 EE (% DM) 1.50 0.68 1.31 1.00 0.80 Ca (% DM) - 0.14 0.40 - 0.03 P (% DM) - 0.09 0.05 - 0.10

DM: dry matter, OM: organic matter, CP: crude protein, NDF: neutral detergent fiber, ADF: acid detergent fiber, ADL: acid detergent lignin, EE: ether extracts. Source: aHayashi et al. (2007), bDe and Singh (2002), cLi et al. (2014), dMadrid et al. (1996), eRamli et al. (2005).

Table 1.3. Rice production and obtained residues of the 10 leading rice-producing countries in 2013.

Country Rice productiona (million t)

Rice huskb (million t)

Rice strawb (million t)

China 205.20 47.20 92.34 India 159.20 36.62 71.64 Indonesia 71.28 16.39 32.08 Bangladesh 51.50 11.85 23.18 Vietnam 44.04 10.13 19.82 Thailand 36.06 8.29 16.23 Myanmar 28.77 6.62 12.95 Philippines 18.43 4.24 8.30 Brazil 11.78 2.71 5.30 Japan 10.79 2.47 4.84 Total 637.04 146.52 286.67

aFAOSTAT, (2013); bCalculated from Sarnklong et al. (2010)

Improving the utilization of agricultural waste

The other possible alternative for better utilization of straw is to improve its

digestibility through treatment to break its ligno-cellulose bond or loosened to free

major portion of cellulose (Malik et al., 2015). A variety of physical, chemical,

biological, or enzyme treatments to improve straw have been studied as described

below in detail. Generally, the physical treatment applies pressure and heat in

combinations with steam or pressure to straw. The chemical treatment includes the

application of ammonia sodium hydroxide (NaOH), ammonia and urea. Biological

treatment is the method that uses acids and microorganism such as ligninolytic fungi

with extracellular ligninolytic enzymes; whereas applying enzyme includes specific

enzymes degrading cellulose and/or hemicellulose (Van Soest, 2006; Sarnklong et al.,

2010).

Physically treatments on crop residues had been investigated in some studies,

however with diverse results. Zhang et al. (2010) observed that chopped rice straw

silage had better fermentative quality than whole plant rice straw silage. Increased

particle size and physical effective fiber (peNDF) increased the time of rumination,

chewing activity, and ruminal pH, but showed no impact on feed intake (Zhao et al.,

2009). Muhammad et al. (2014) reported that rice straw treated with steam at a steam

pressure of 15.5 kgf/cm2 for 120 s increased the daily body weight gain of goats

33.02% in close house and 44.37% in open house compared with goats fed with

untreated rice straw. It is also reported that those treatment increased apparent

digestibility, feed efficiency and improved plasma volatile fatty acid profile

(Muhammad et al., 2014). However, reduced size of rice straw had no effect on

apparent digestibility, rumen fermentation and N utilization (Gunun et al., 2013). A

similar study also reported that increased particle size of rice straw or dietary peNDF

hardly affected the duodenal N flow, the values of blood chemical parameters, and the

microbial amino acid composition in the rumen of goats (Wang et al., 2011). Physical

treatments mostly are not practicable on small-scale farms since they require machine

or industrial processing. However, small grinder or chopper may be practical for these

small-scale farmers (Sarnklong et al., 2010).

Improving the value of agricultural by-product chemically have been widely

investigated whereas alkali is the most reported and easier to apply on farms

compared to acidic or oxidative agents (Sarnklong et al., 2010). Urea is also the most

used agent by small-medium holder farmers since it is not quite expensive and easy to

apply (Malik et al., 2015). The fiber content of wheat straw treated using chemical

reported by Sahoo et al. (2000, 2002) is shown in Table 1.4. The crude protein and

cellulose contents were increased as an addition of urea and storage for 21 days before

feeding, while hemicellulose and lignin contents were decreased. An in vitro

fermentation experiment of Khejornsarts and Wanapat (2010) reported that rice straw

with 3% urea or 2% urea-lime resulted in the high gas production and high

accumulation of volatile fatty acid (VFA), i.e. acetate and propionate. The dry matter

and cell wall digestibility and utilization of energy and nitrogen of wheat straw were

improved by treatment with urea and/or a mixture of urea and calcium hydroxide

followed by storage as compared to urea supplementation just prior to feeding (Sahoo

et al., 2002). Gunun et al. (2013) reported that long form straw treated with 2.5% urea

Table 1.4. Effect of chemically treatment on fiber and CP contents of wheat straw (%

DM)

Type of wheat straw CP NDF ADF ADL Hemi-cellulose

Cellulose

Wheat straw, untreated 3.5 69.1 47.8 10.1 21.3 37.7 Wheat straw, treated with 4% urea (21 days storage time)

7.8

72.8

55.3

9.5

17.6

45.7

Wheat straw, sprayed with 1.5% urea prior to feeding

7.9

68.5

46.6

10.8

21.9

35.8

Wheat straw, treated with 3% urea + 3% calcium hydroxide (21 days storage time)

7.6 70.6 54.7 9.7 15.9 45.5

CP: crude protein, NDF: neutral detergent fiber, ADF: acid detergent fiber, ADL: acid

detergent lignin.

Adapted from Sahoo et al. (2000; 2002).

resulted in improved rumen fermentation, efficiency of microbial N synthesis, feed

intake, digestibility of nutrients, milk yield of dairy cows as well as an economical

return when compared with untreated rice straw.

The combination of physical (Gamma irradiation at 200 kGy) and chemical

(5% urea concentration) treatments has the potential to increase the nutritive value of

some agricultural by-product (wheat straw and grain shell) (Al-Masri and Guenther,

1999). Likewise Banchorhorndhevakul (2001) observed rice straw and corn stalk

treated with combination of 20% urea and 10 kGy gamma gave a higher percent of

decrease in neutral detergent fiber (NDF), acid detergent fiber (ADF), acid detergent

lignin (ADL), cellulose, hemicellulose, lignin, and cutin in comparison with urea

effect only. However, it will be limiting factor in applying in farm due to the high cost

of radiation.

Wheat straw treated with calcium oxide (CaO), sodium hydroxide (NaOH)

and NaOH plus hydrogen peroxide (H2O2; AHP) improved the nutritive value of

straws compared with untreated through modification of cell wall with a subsequent

increase in digestibility by sheep (Chaudhry, 1998). On the other hand, Nasir and

Elseed (2004) found that urea-calcium hydroxide treated straw could be used for

sheep feed as an alternative to ammonia treated straw. Treatment with sodium

hydroxide at a concentration of 7% (pH~12) followed by ensiled may be regarded as

the most effective to improve the ruminal degradability of rice straw (Ghasemi et al.,

2013).

The combination of using urea-lime-treated rice straw and fed with

concentrate feed (4% urea) has ability to improve rumen ecology, rumen fermentation

efficiency, and nutrient digestibility in swamp buffaloes as reported by Nguyen et al.

(2012). Hue et al. (2008) reported that on feeding sheep with urea treated rice straw as

a based diet, the commercial concentrate could be replaced by protein source feed,

such as cassava and jackfruit foliage. Chemical treatment gives many advantages on

improving nutritive values of agricultural by-product and seems easier to apply than

physical treatment since it needs no high technologies or high-cost equipment.

However, implementation in small-scale farmer faces difficulties mainly in

developing countries since the chemical material is still relatively expensive in price

which will lead in increasing cost production.

Bacterial additive

Using microorganism as an effort to improve the nutritional value of

agricultural by-product has been studied since at the beginning of 20th century.

Biological treatment on crop residue has great potential in comparison to other

treatment; low cost and reduced pollution (Malik et al., 2015). Figure 1.1. showed the

biological treatments on agricultural by-product.

Such as by using ligninolytic fungi, including their enzymes, may be one

potential alternative to provide a more practical and environmental-friendly approach

(Sarnklong et al., 2010). Villas-Bôas et al. (2002) offered the similar solution that use

microorganisms, mainly fungi, to obtain higher protein and vitamin contents and

digestibility from agro-industrial waste. Furthermore, the growth of microbes on

lignocellulosic wastes is able to furnish all the hydrolytic enzymes often added in the

preparation of feeds, and also makes the minerals more available for absorption by the

animal (Villas-Bôas et al., 2002).

Whereas Abdel-Aziz et al. (2015) reported that mixing crop residue with

microorganisms such as lactic acid bacteria (LAB) and cellulolytic bacteria on

ensiling of crop residues is one of the methods for achieving a proper fermentation

and nutrient preservation. Combination microorganisms with actions such as

chopping, reconstitution of moisture and pressing are also potential to improve the

fermentation quality (Abdel-Aziz et al., 2015). Fermenting rice straw with

Lactobacillus fermentum resulted in better silage quality compared to bacillus and

fungi (Aspergillus niger and Saccharomyces cerevisiae) as reported by Yanti et al.

(2012). The acetic acid production in Lactobacillus fermentum treatment was highest

among the treatments. Whereas the lactic acid value was similar with control +

molasses and Bacillus coagulant treatment. There was also interaction effect of

microorganism type and temperature (25, 35 and 45 °C) of fermentation on lactic,

acetic and propionic acid production of fermented rice straw (Yanti et al., 2012). On

the other hand, Wang et al. (2015) found that using Lactobacillus bulgaricus

GIM1.80 as a starter cultures during wilted rice straw silage fermentation can promote

indigenous LAB activities even though it had no significant ability to change the

nutritional value of silage.

A study by Suksombat and Lounglawan (2004) indicates that some

agricultural by-product in the form of silage has a high potential to improve quality

and to utilize the various types of dairy cattle’s feeds. They also suggested that the

period of fermentation is at least 14 days and can be stored for more than six months.

Rice straw, for example, when added previous fermented juice (PFJ) as silage additive

improved fermentation quality as reported by Jin-ling et al. (2013). It was proved by

the lower pH value, lower NH3-N concentration and higher in lactic acid production

than non-additive treatment. PFJ or some papers call it FJLB (fermented juice of

epiphytic lactic acid bacteria) is additive for silage fermentation that made from single

type of grass, blended, and added by simple carbohydrates (glucose or sucrose) then

incubated for two days at 30°C. This fermented juice naturally contains a number of

species of domestic LAB which has been well known for the main role in silage

fermentation. As reported by Santoso et al. (2012), LAB that found in FJLB made

from king grass were Lactobacillus plantarum and Lactobacillus brevis. Moreover,

the application of these FJLB in rice crop residue-based silage reduced fibrous

components compared to non FJLB silage and enhanced lactic acid concentration and

in vitro organic matter digestibility (Santoso et al., 2014).

Another study on fungal treatments of fibrous agricultural by-products which

contain more than 100 g of lignin/kg organic matter (OM) showed that Ceriporiopsis

subvermispora and Lentinula edodes have a great potential to improve nutritive value

of agricultural by-products but not for poorly lignified feedstuff such as maize stover

(Tuyen et al., 2013). This result supported his previous study that both fungi and also

Phlebia eryngii have a potential to improve the nutritional value of wheat straw as a

ruminant feed, because of their capacity to degrade lignin during their vegetative

growth, without affecting cellulose to a great extent (Tuyen et al., 2012). Similarly,

Ramli et al. (2005) found that bagasse feed (combination mixture of bagasse with

wheat bran) fermented by Aspergillus sojae fungi improved the digestibility of some

fiber components (NDF, ADF or cellulose) in the feed. Therefore, the fermented

bagasse feed (FBF) could be an alternative for goat feed to save the cost and lead to

increased self-sufficiency in animal feeds (Ramli et al., 2005). Whereas Aspergillus

terreus reduced hemicellulose 32.68% and reduced cellulose 16.32% after eight days

fermentation in rice straw (Jahromi et al., 2012).

White root fungi such as Pleurotus ostreatus (P. ostreatus) had been reported

in many studies. Incubation under solid-state condition with this fungi in maize straw,

rice straw, wheat straw, and their mixture reduced the content of cell wall and

increased the content of crude protein and ruminal degradation (Fazaeli et al., 2006;

Khattab et al., 2013; Khan et al., 2015). The mutant of P. ostreatus had also resulted

in the similar performance as reported by Chalamcherla et al. (2009).

The microorganism, mainly fungi, seems to be the most potential on degrading

cell wall content of agricultural by-product. However, the application of fungi in both

big and small-scale farm still need more studies mainly related to animal health which

related to cost production, since some fungi such Aspergillus sp. has the safe dose to

be consumed by livestock. Whereas the use of FJLB seems more likely to be

implemented mainly in small-scale farms because it is easy to prepare and economical.

FJLB treatment improved the fermentation quality of agricultural by-product, but

further investigation needed when it applied in the form of total mixed ration (TMR)

that contains dry material of agricultural by-product.

Exogenous enzymes treatment

In the last two decades, concerted efforts have been devoted of using

exogenous fibrolytic enzymes (EFE) to improve forage quality and ruminant animal

performance (Adesogan et al., 2014). The various enzymes as biological treatment are

shown in Figure 1.1 These enzymes have cellulolytic and hemicelluloytic capability

to attack the lignocellulose structure of crop residue for enhancing their feeding value

(Abdel-Aziz et al., 2015). Whereas Table 1.5 shows a brief summary of enzyme

treatments in agricultural by-products. The supplementation singly or in combinations

of exogenous fibrolytic enzymes cellulase and xylanase on maize stover enhanced in

vitro DM digestibility (Bhasker et al., 2013). The addition of this exogeneous

fibrolytic enzyme also increased in vitro gas production and fermentation kinetics of

corn stover (Vallejo et al., 2016) and wheat straw (Togtokhbayar et al., 2015). The

optimum combination cellulose-xylanase-β-D-glucanase for increasing nutrient

utilization from maize stover was 25,600-25,600-0 IU/g, whereas for increasing the

total VFA and

Figure 1.1. Biological treatment for improving agricultural by-products (summarized

from the reports of Adesogan et al., 2014; Abdel-Aziz et al., 2015).

Bacterial additives - Lactobacillus - Aspergillus - Sacharomiches - Propionibacterium - Bifidobacterium - Enterococcus - Streptococcus - Bacillus - Megasphaera elsdenii - Prevotella bryantii - Trichoderma reesei F-418

Exogenous enzyme - Enzyme from Trichoderma viride - Enzyme from Pleurotus ostreatus - Amylase - Cellulase-xylanase - Cellulase-xylanase-amylase - Ferulic acid esterase - Endoglucanase-xylanase - Cellulase enzyme - ZAD® and ZADO®8

NH3-N concentration in the rumen of sheep fed 50% maize stover based TMR was

supplemented by cellulose-xylanase-β-D-glucanase 12,800-12,800-0 IU/g (Bhasker et

al., 2013). In rice straw, a combination of cellulase (7.5U/g of DM) and xylanase

(15/g of DM) was more effective in improving rumen fermentation, increasing rice

straw DM digestibility and NDF digestibility and enhancing the rumen bacterial

numbers than a single cellulase or xylanase (Mao et al., 2013). However, Eun et al.

(2006) reported that the effectiveness of exogenous enzymes was enhanced when they

were used with ammoniated rice straw rather than with untreated rice straw.

The application of EFE for improving fibrous ruminant feed often lack.

Therefore, Adesogan et al. (2014) suggested for ideal fibrolitic enzyme as follows: 1)

contain the appropriate complement of potent fibrolitic activities for improving NDF

digestibility, 2) contain appropriate amounts of cofactors, co-enzymes, and activators

(where needed) to optimize the fibrolytic activities and lack inhibitors such enzymes,

3) be resistant to degradation by ruminant proteases, 4) have a robust composition that

does not vary appreciably with the enzyme batch, 5) be sourced from a readily

culturable fungus that produces large quantities of enzymes naturally or via genetic

modification, 6) exhibit optimal and steady activity under conditions that prevail

where it will exerts its hydrolytic effect, 7) be in liquid form or dissolve rapidly and

completely in water, 8) be thermostable if it will be added during feed manufacturing,

9) maintaining its hydrolytic activity when appropriately stored for long durations,

and 10) be generally regarded as safe.

Various treatments for improving the nutritive value of agriculture by-product

including physical, chemical, biological and enzymes, has been many reported.

However, application in farm scale needs to be more investigated considering the cost

production, safety aspect, and environmental impact. Enzymes to treat crop by-

Table 1.5. Enzymes treatment in agricultural by-product Agricultural by-product

Enzymes recommended

Method Result References

Maize stover

Combination of cellulase- xylanase- β-D-glucanase 25,600-25,600-0 IU/g Cellulase-xylanase- -D- glucanase 12,800-12,800-0 IU/g

In vitro In vivo

Increase in vitro DM digestibility Increase the TVFA and NH3-N concentration in the rumen.

Bhasker et al., 2013

Corn stover 40 μg/ g DM of cellulase and xylanase

In vitro Decrease pH, increase OM digestibility, metabolizable energy, short chain fatty acid and microbial CP

Vallejo et al., 2016

Wheat straw

Xylanase 1.0 to 1.5 μL/g

In vitro and in sacco

Improve gas production, rumen NH3-N concentration and volatile fatty acids

Togtokhbayar et al., 2015

Rice straw Combination of cellulosa (7.5U/g of DM) and xylanase (15U/g of DM)

In vitro Increase DM digestibility and NDF digestibility and enhance the rumen bacterial numbers

Mao et al., 2013

Rice straw Exogeneous enzymes used with treated rice straw (ammoniated rice straw)

In vitro Enhace in vitro degradability

Eun et al., 2006

DM: dry matter, OM: organic matter, CP: crude protein, NDF: neutral detergent

fiber, TVFA: total volatile fatty acids.

product are still limited production right now, which resulted expensive in price, but it

might be different in the future trend. The combination of the treatments is also

promising to improve the quality of agricultural by-product.

Mitigating Methane emission

A huge of agricultural by-product mostly producing in tropical areas, such as

rice straw dominates in India and Indonesia. That by-product became a serious

problem because the amount increased annually due to the food demand increased as

a result of increasing in human population. Burning or stacked it in the field just bring

in another problem. Carbon monoxide and methane will release to the air and become

pollution to the environment. On the other side, the utilization of agriculture by-

product as feed for ruminant in many tropical areas seems to relieve this problem.

However, because the characteristics of crop by-product are high of fiber content,

another problem has revealed. As reported by Kurihara et al. (1997) that the methane

productions will also tend to increase when fiber content in feed increase. Methane is

produced from microbial fermentation within the rumen. The substrates for ruminal

methanogenesis are from dietary carbohydrates such as cellulose, hemi-cellulose,

pectin and starch. When these substrates are hydrolyzed, hydrogen and carbon dioxide

are produced, which are metabolized by methanogens to yield methane (Bhatta et al.,

2007).

Over the next 40 years, methane as a source of greenhouse gasses in livestock

production will increase as a consequence of increased food production unless there is

major progress in mitigation technologies (O’Mara, 2011). Strategies for reduction of

methane emissions from the rumen have been suggested by Mitsumori and Sun

(2008). Their strategies include 1) control of components in feed, 2) application of

feed additives and 3) biological control of the rumen fermentation.

The strategy of controlling the feed component only will be discussed in here

because it more related to the agricultural by-product as feed for ruminant than the

other two strategies. The type of carbohydrate fermented in the rumen influences

methane production most likely through impacts on ruminal pH and the microbial

population. Fermentation of cell wall fiber yields higher acetic:propionic acid and

higher methane losses. Methane can be reduced with diets containing higher levels of

nonstructural carbohydrate (Johnson and Johnson, 1995). That would be difficult to

apply in tropical/developing countries where agricultural by-product is still dominated

as feed for ruminant.

Many investigations concerned on mitigating methane emission using both in

vitro and in vivo technique with given treatment to straw/fodder or adding

supplementation when rice straw as a basal diet for ruminant (Table 1.6). The

reduction of methane emission was varied depends on the material used and the

treatment. Sahoo et al. (2000) observed that wheat straw treated with urea alone or

with urea plus calcium hydroxide and stored for 21 days reduced methane production

per kilogram digested organic matter per day in sheep. A study in Nellore × Guzera

beef steers fed sugarcane based diets showed that addition of 22 g nitrate/kg dry

matter in the diet reduced methane emission by 32% and increased rumen ammonia

concentration (Hulshof et al., 2012). On the other hand, fermented rice straw (FRS)

which containing lovastatin after fermentation with Aspergillus terreus, reduced total

gas, and methane production, methanogens population, but increased in vitro dry

matter digestibility compared to the non-fermented rice straw (Jahromi et al., 2012).

Liu et al. (2013) studied that sheep fed 20% concentrate and ensiled cornstalk as

roughage added to their diet had the effect of reducing the methane emission, and the

decrease in acetate:propionate ratio might cause the suppression of methanogenesis by

depriving of the hydrogen utilized by methanogens to produce methane.

A study by Chuntrakort et al., (2014) on Native Thai and Brahman crossbreed

cattle fed rice straw as a based diet resulted in a reduction in methane emission up to

50.1% by replacing concentrate feeds to oil plants such as whole cotton seed, whole

sunflower seed and coconut kernel. While a study by Ampapon et al. (2016) on

swamp buffaloes consuming rice straw, they recommended urea supplements of 60-

90 g/head/day when fed with cassava hay in order to reduce methane production by

16.8-18.8%. Supplementation using mangosteen (Garcinia mangostana) peel powder

(MSP) from 100-300 g/head/day could decrease rumen methane production about 5.5-

13.8% from control when rice straw as a based diet at lactating dairy cows (Polyorach

et al., 2016).

The combination of high fiber content roughage with non-structural

carbohydrates in the form of TMR also has the possibility to reduce methane emission.

Fermented TMR has lower ruminal methane emission in sheep than non-fermented

TMR that contains whole-crop rice and rice bran as reported by Cao et al. (2010a).

Previously in vitro study by Cao et al. (2009a) showed that supplementation with

lactic acid bacteria to TMR silage containing whole-crop rice and 30% of rice bran

has lower methane production per digestible dry matter than in control.

Mitigating methane emission in ruminants when agricultural by-product as

feed has two advantages; increasing feed (energy) efficiency and reducing pollution to

the environment as well. The application of researches above in the farmer scale

needs to be realized to support the reduction of the impact of global warming.

Table 1. 6. Methane emission from agricultural by-product as based diet Agricultural by-product

Treatment Animal Methane Emission References

Wheat straw

Urea and calcium hydroxide followed by storage

Sheep (-) 17 l/kg digested organic matter per day

Sahoo et al., 2000

Sugarcane Addition of 22 g nitrate/kg dry matter in the diet

Steers (-) 32% Hulshof et al., 2012

Rice straw Fermented rice straw with Aspergillus terreus

In vitro (-) 1.02% Jahromi et al., 2012

Cornstalk Different concentrate-to-forage ratios

Sheep (-) 5.98-7.43 L/kg DM intake

Liu et al., 2013

Rice straw Oil plant feeding Beef cattle (-) 50.1% Chuntrakort et al., 2014

Rice straw Urea supplements of 60-90 g/head/day with cassava hay as feed

Buffaloes (-) 5.8-6.5 m mol/L Ampapon et al., 2016

Rice straw Mangosteen peel powder supplement

Cows (-) 1.5 - 3.8 m mol/100ml3

Polyorach et al., 2016

DM: dry matter.

Conclusion

Agricultural by-products are still a potential source of feed for ruminant since

its nutrition could be increased by some treatments including physical, chemical and

biological methods. However, applying the treatments to small-scale farms is limited

by cost and lack of technology. Biological treatment may be the most practical

method when applied to small-holder farmers, because of its lower cost. One of

promise biological treatment is the use of FJLB. This treatment could improve the

fermentation quality of silage. The utilization of agricultural by-product as ruminant

feed also appears an environmental problem. The digestion of high content of fiber in

the rumen increased methane emission. Urea treatment or fermentation using fungi in

agricultural by-product had successful on reducing methane emission, but the using

both chemical and fungi treatment must consider to the animal health. The

combination of agricultural by-product and other nonstructural carbohydrates also has

possible to reduce methane emission. Further study is needed to be investigated on the

application of biological treatment which easier to apply in small-scale farm such as

fermented juice of epiphytic lactic acid bacteria in agricultural by-product. In this

case, TMR silage will improve its nutritive value. The effect of TMR on reducing

methane production still also need to be studied more extensively, remembering that

this gas is the major cause from livestock production sector contributing on

greenhouses effect or global warming.

Therefore, this study aimed: 1) to improve the utilization and the quality of

agriculture by-product and food by-product using FJLB as silage additive on the

fermentation quality and nutritive value of TMR silage comparing with commercial

silage additive; 2) to determine the effect of FJLB on nutrient intake, digestibility,

rumen fermentation, and nitrogen balance on ewes; 3) to determine the effect of total

mixed ration ensiled with FJLB on in vitro methane emission, energy balance and

blood parameters in ewes.

CHAPTER II

Total Mixed Ration silage prepared from agricultural by-products and food

by-products: Improving quality of TMR silage using fermented juice of

epiphytic lactic acid bacteria

II-1. Introduction

The agricultural by-product production in the world has been increasing as the

impact of increasing in crop production (FAO, 2015). This situation encourages us to

increase the utilization of agricultural by-product instead of discard and burning

which are resulting in environmental problem. In developing countries, mainly in

India and southeast Asia, animal production depends on agricultural by-products (Van

Soest, 2006) due to the inadequacy of forage grasses. However, agricultural by-

product is well known by its low nutrient content, and low digestibility for ruminants

feed, then finally leading to low in animal production. Agricultural by-product such as

rice straw contains high levels of structural carbohydrate (NDF>50%) and low levels

of protein (2 5%DM) (Wanapat et al., 2009) which result in low voluntary intake and

digestibility (Safari et al., 2011; Wanapat et al., 2014). Thus, improving the utilization

of agricultural by-product as feed for ruminant has been conducted for decades. The

combination of agricultural by-product and other nonstructural carbohydrates in the

form of total mixed ration (TMR) seems a promising method to improve the

utilization of this feed for ruminant. TMR feeding enhances feed intake, improves the

ecology of the rumen leading to stimulated microbial activity to digest more feed, and

then increases productivity of the animals (Wongnen et al., 2009). Ensilaging TMR is

usually carried out to avoid the loss of quality when distributing it to long distance

and storage for extended periods. Several studies suggest that making TMR silage

which contains agricultural by-products may improve the digestibility in animals (Cao

et al., 2009b, 2016; FAO, 2012); thus, TMR silage is a possible way to use

agricultural by-product for ruminant feed.

In order to achieve a well fermentation quality of silage, using additives on

preparing silage had been known for decades. The silage additive might contain lactic

acid bacteria (LAB) or simple carbohydrates. When an additive applies to silages, the

LAB increases, then they produce lactic acid that reduces the pH in the silages

rapidly. This acidic condition in silages prevents the growing of undesirable

microorganism, i.e. clostridia and mold (McDonald et al., 1991). One of silage

additives that have successfully increased the fermentation quality is FJLB or PFJ

(Ohshima et al., 1997a; Bureenok et al., 2005). The FJLB is prepared from one or

mixed grasses which macerated with water and added sugar, then incubate for two

days to multiply the amount of LAB. The use of FJLB seems more likely to be

implemented in any farms scale because it is easy to prepare and requires negligible

cost (Nishino and Uchida, 1999).

Applying the FJLB in alfalfa silage decreases pH, enhances the lactic acid

production, decreases the propionic acid and the ammonia nitrogen (Ohshima et al.,

1997ab), increases the in vitro dry matter digestibility (Wang et al., 2009), and

decreased NDF content (Denek et al., 2012). The good fermentation results also

reported in napier grass (Tamada et al., 1999), guineagrass, stylo legume, the mixture

of guinea grass and stylo legume (Shao et al., 2004; Bureenok et al., 2005, 2016), and

lucerne silage (Nishino and Uchida, 1999; Denek et al., 2011). Whereas the

application of this additive on agriculture by-product has been limited. Rice straw

sprayed with FJLB increases the crude protein content and reduce the dry matter

losses (Jin-ling et al., 2013). While the application of the mixture of FJLB and LAB

(Chikuso-1) to the TMR based on whole crop rice (WCR) resulted in well-prepared

silage (Cao et al., 2009b). The similar result has been reported when applying FJLB

solely to WCR (Takahashi et al., 2005). We hypothesized that those good effects of

FJLB in ensiling both grass and agricultural by-product may also improve the quality

of TMR silage prepared from agricultural by-product and food by-product. Therefore,

the aim of this study was to improve the utilization and the quality of agriculture by-

product and food by-product using FJLB as silage additive on the fermentation quality

and nutritive value of TMR silage comparing with commercial silage additive.

II-2. Material and methods

Ingredients

TMR silage was prepared from rice straw, corn cobs, brewer grain waste, tofu

waste, steam flaked maize and vitamin-mineral mix. Rice straw was harvested in the

last year at the Yanagido Farm, Gifu Field Science Center, Gifu University, and was

chopped into 2-3 cm length before ensiling. Brewer grain waste and tofu waste were

generated in a food manufacturer and a brewing maker, and both food by-products

and steam flaked maize were purchased from a local feed company (Minorakuren Co,

Ltd., Gifu, Japan). Cut corn cobs were obtained from another feed manufacture

(Oriental Yeast Co, Ltd., Tokyo, Japan). All of TMR ingredients were in dry type.

Vitamin-mineral mix (NAS DL05-HVE; NASU AGRI SERVICE, Inc., Tokyo,

Japan) contained 5000000 IU kg−1 of vitamin A, 1000000 IU kg−1 of vitamin D,

24000 IU kg−1 of vitamin E, 150 mg kg−1 of Co, 8000 mg kg−1 of Cu, 15000 mg kg−1

of Mn, 250 mg kg−1 of I, 20 000 mg kg−1 of Zn, 10 mg kg−1 of Se, 700 g kg−1 of Mg.

The proportion of each material for preparing total mixed ration was obtained by trial

and error methods using Excel (Table 2.1). TMR was designed for growing sheep,

and was formulated to obtain 12.4% of crude protein (CP) and 65.9% of total

digestible nutrients (TDN) to meet or exceed the nutrient requirement of growing

lamb (NRC, 2007).

Preparing silage

The TMR was packed into a polyethylene bag (420 mm × 500 mm) that

allowed air release only. Approximately 500 g of dry matter of TMR was put into the

bag. Four treatments were applied to the TMR: non-additive (CON), FJLB,

commercial additive “Si-Master AC”® (COM; Snow Brand Seed Co,Ltd., Sapporo,

Japan) and MIX (FJLB + commercial additive). The FJLB was prepared from Italian

ryegrass before harvesting as modified from Burenook et al. (2016). The Italian

ryegrass and distilled water (1:5 ratio; w:v) were blended using a food blender for two

minutes then filtered using doubled layer of cheesecloth. The 2% of glucose were

added to the filtrate and mixed thoroughly then incubated in anaerobic condition at

30°C for 48 hours. The LAB on this filtrate increased from 1.1×104 CFU ml−1 to

4.7×107 CFU −1ml after incubation (enumeration using MRS agar method). The LAB

was accounted using MRS agar method. The FJLB was added at 1% (v:w) of fresh

material as silage additives. The treatment of commercial additive followed the

company’s instruction. The commercial additive diluted with distilled water (17:1000

ratio; w:v) and sprayed 0.1% (v:w) on the TMR. Then the distilled water was added to

all bags to obtain 55% of DM content. Silages were made in five replications for each

treatment in each fermentation period. The bag silos were kept at ambient temperature

(25.3-32.6oC), and samples were taken at 2, 7, 14, 30, and 60 days after ensiling for

quality fermentation measurement. The chemical analysis of the TMR silages were

only conducted on 60 days of fermentation.

Table 2.1 Proportion of feed ingredient in total mixed ration (TMR) Ingredient Proportion (DM %) Rice straw 23 Corn cobs 23 Tofu waste 20 Brewer grain waste 14.5 Steam flaked corn 18 Vitamin and mineral premix1 1.5 Total 100 1Contained 5 000 000 IU kg−1 of vitamin A, 1 000 000 IU kg−1 of vitamin D, 24 000 IU kg−1 of vitamin E, 150mg kg−1 of Co, 8 000 mg kg−1 of Cu, 15 000 mg kg−1 of Mn, 250 mg kg−1 of I, 20 000 mg kg−1 of Zn, 10 mg kg−1 of Se, 700 g kg−1 of Mg.

Silage sampling and fermentation quality measurement

At each sampling day, the representative 20 g fresh matter of ensiled sample

was taken, macerated with 70 ml of distilled water and stored at 4oC for 12 h

(Burenook et al., 2005). Then the extract was filtered using a filter paper

(ADVANTEC No. 1, Tokyo, Japan). The filtrate was used to determine the

fermentation quality. The pH of silage was determined by using a pH meter (MP220;

METTLER TOLEDO, Tokyo, Japan) immediately after filtration. The volatile fatty

acid (VFA) content was measured by applying gas chromatography (GC–14A,

Shimadzu, Kyoto, Japan; column: ULBON HR-20M, 0.25 mmI.D. × 30 mL 0.25 μm,

flow rate 7.2 ml minute−1, injection port at 220°C, column at 200°C and detector at

220°C). Lactic acid content determined using a commercial kit (D-/L-Lactic Acid

Assay Kit; Megazyme, Wicklow, Ireland). The NH3-N was measured by indophenol

method (Weatherburn, 1967). Fleig point was measure as following equation (Denek

and Can, 2006). Fleig point from 85 to 100 shows very good quality; 60 80, good

quality; 55 60, moderate quality; 25 40, satisfying quality, and <20 is worthless

(Denek and Can, 2006).

Fleig points = 220 + (2 × DM% 15) – 40 × pH

The in vitro dry matter digestibility (IVDMD) was analyzed by batch culture

method according to Tilley and Terry (1963). Rumen fluid was collected from two

canulated Suffolk sheep before feeding. Sudan grass hay and concentrate were fed to

the sheep at the maintenance level and equal amount of the feed was offered in the

morning and evening. The rumen fluid then filtered with four layers cheesecloths and

mixed with anaerobic McDougal buffer (1:4, v:v). A 50 ml of this mixture was mixed

with one gram of sample in a 50 ml of glass veil with rubber cap and gas trap. The

glass veils incubated in shaking water bath at 39°C for 48 hours.

Chemical analysis

A representative sample of silage (120 g) was dried at 60oC to a constant

weight then grounded passed 1 mm for chemical analysis. The DM, ash, acid

detergent fiber exclusive ash (ADFom), CP, and ether extracts (EE) analysis were

performed according to the methods of AOAC (2007: protocol number, 930.15;

942.05; 973.18; 990.03 and 920.39, respectively). The neutral detergent fiber with

heat stable amylase, exclusive ash (aNDFom) was determined as described by Van

Soest et al. (1991). The non-fibrous carbohydrates (NFC) was calculated as follow:

NFC=100 CP NDF EE ash (NRC 2007).

Statistical analysis

All obtained data were analyzed using R programming 3.3.2 (R development

core team, 2016). The fermentation characteristic of silage was analyzed by two-way

factorial analysis of variance (ANOVA), in which the additive, the fermentation

period, and the additive × the fermentation period as the fixed effects. When the F

indicated the significant (P<0.05), further test was performed using the pairwise t-test

to compare the additive’s mean in each period. Further, the fermentation characteristic

and chemical compositions of silage at 60 days of fermentation were analyzed by one-

way analysis of variance (ANOVA), and the mean of additives was compared using

pairwise t test. The Bonferroni correction was used to detect the differences between

the means for each data analysis.

II-3. Results

Fermentation quality

The silage pH and lactic acid (Figure 2.1), VFA concentration (Figure 2.2),

DM losses and Fleig point (Figure 3.2) were affected by silage additives, fermentation

period and their interaction. The application of silage additive reduced pH value

throughout the fermentation period than in non-additive treatment. The lowest pH

value was in MIX then followed by FJLB, COM and CON after 60 days ensiling

(P<0.05; Table 2.2). The highest lactic acid production was in MIX treatment then

followed by FJLB, COM and CON treatment throughout the fermentation period.

After 60 days fermentation, MIX and FJLB silages had higher content of lactic acid

than in CON and COM silages (P<0.05; Table 2.2). Although the acetic acid

production fluctuates in all treatments through the fermentation period, MIX

treatment contained the higher acetic acid than other treatment after 60 days

fermentation (P<0.01). Propionic acid was detected from 14 days of fermentation in

FJLB and COM treatment, whereas it was detected at 30 days fermentation in CON

and MIX treatment. The MIX treatment showed the lowest production of propionic

acid and butyric acid among the treatments in days 60 (P<0.05). The NH3 N content

was affected by silage additive in the early two days of fermentation then no

difference afterwards. There was no interaction effect in NH3-N content until 60 days

of fermentation (P>0.05). The DM losses did not differ in all silage additive

treatments before 30 days of fermentation. However, the MIX treatment showed

lower DM losses than that in CON treatment after 60 days fermentation (P<0.01). The

TMR with FJLB treatment also showed lower DM losses at 60 days of ensiling period

compared to COM treatment (P<0.05). The Fleig point in MIX treatment was highest

until the last day of fermentation (P<0.01). The CON and COM did not differ in Fleig

point at the 2 and 7 days of ensiling period but then the COM was higher than CON

after that (P<0.01). The range score for Fleig point was 7.5 28.3, 84.3 109.5,

26.0 61.7, 99.5 134.8 for CON, FJLB, COM, and MIX, respectively. The IVDMD

was not affected by silage additives, fermentation period and their interaction (Figure

2.4).

Chemical composition

The DM, CP, aNDFom, ADFom and EE content were affected by silage

additive treatment (Table 2.3). The DM content of COM treatment was lower than

FJLB and MIX treatments (P<0.05). The application of silage additive increased the

CP content slightly in comparison to CON, but the significant difference was only

detected between MIX and CON treatment (P<0.05). The aNDFom content in MIX

treatment was the lowest among the treatments (P<0.05). The ADFom content of

MIX treatment was decreased slightly in comparison to other treatments (P<0.05).

The MIX treatment was the highest in EE content (P<0.05), followed by COM, FJLB

and CON treatment. Whereas crude ash and NFC content were not affected by the

silage additive treatments.

Figure 2.1 The pH and lactic acid concentration in TMR silages prepared from agriculture by-product and food by-product ensiled with CON ( ), FJLB (■), COM

( ), and MIX ( ) additive treatment. The data were collected at 2, 7, 14, 30, 60 days of fermentation. DM, Dry matter.

3

4

5

6

7

8

0 15 30 45 60

pH

Treatment: P<0.01 Period: P<0.01 TxP: P<0.01

0

10

20

30

0 15 30 45 60

Lac

tid a

cid

(g/k

g D

M)

Fermentation period (days)

Treatment: P<0.01 Period: P<0.01 TxP: P<0.01

Figure 2.2 The volatile fatty acid (VFA) and ammonia-nitrogen (NH3-N) concentration in TMR silages prepared from agriculture by-product and food by-product ensiled with CON ( ), FJLB (■), COM ( ) and MIX ( ) additive treatment. The data were collected at 2, 7, 14, 30, 60 days of fermentation. DM, Dry matter; TN, total nitrogen.

0

5

10

15

0 15 30 45 60

Ace

tic a

cid

(g/k

g D

M)

Treatment: P<0.01 Period: P<0.01 TxP: P<0.01

0

1

2

3

4

0 15 30 45 60

Prop

ioni

c ac

id (g

/kg

DM

)

Treatment: P<0.01 Period: P<0.01 TxP: P<0.01

0

5

10

15

20

25

0 15 30 45 60 Buty

ric a

cid

(g/k

g D

M)

Fermentation period (days)

Treatment: P<0.01 Period: P<0.01 TxP: P<0.01

0

20

40

60

80

0 15 30 45 60

NH

3-N

(g/k

g T

N)

Fermentation period (days)

Treatment: P<0.01 Period: P<0.01 TxP: P>0.01

Figure 2.3 The dry matter (DM) losses and Fleig point in TMR silages prepared from agriculture by-product and food by-product ensiled with CON ( ), FJLB (■), COM

( ) and MIX ( ) additive treatment. The data were collected at 2, 7, 14, 30, 60 days of fermentation.

0

2

4

6

8

10

0 15 30 45 60

DM

loss

es (%

DM

)

Treatment: P<0.01 Period: P<0.01 TxP: P<0.01

0

40

80

120

0 15 30 45 60

Flei

g po

int

Fermentation period (days)

Treatment: P<0.01 Period: P<0.01 TxP: P<0.01

Figure 2.4 The in vitro dry matter digestibility (IVDMD) of TMR silages prepared from agriculture by-product and food by-product ensiled with CON ( ), FJLB (■),

COM ( ) and MIX ( ) additive treatment. The data were collected at 2, 7, 14, 30, 60 days of fermentation.

20

30

40

50

60

70

0 15 30 45 60

IVD

MD

(%)

Fermentation period (days)

Table 2.2 Fermentative quality of total mixed ration (TMR) silage containing agriculture by-product after 60 days ensiling

Item Treatment

P-value CON FJLB COM MIX

pH 7.5a 5.4c 6.2b 4.5d <.001 Lactic acid (g kg−1 DM) VFA (g kg−1 DM)

4.9b 23.2a 7.8b 25.2a <.001

Acetic acid 6.2b 4.3b 4.2b 11.9a <.001 Propionic acid 2.0b 1.1bc 3.5a 0.5c <.001 Butyric acid 19.7a 10.0b 21.9a 1.3c <.001 NH3-N (g kg−1 TN) 58.4 61.7 73.3 58.1 0.217 DM losses (% DM) 2.7ab 1.7b 3.9a 0.9b 0.003 Fleig point 11.2d 95.7b 61.7c 134.8a <.001 IVDMD (%) 58.2 59.7 60.2 61.4 0.267 CON, control (no additive added); FJLB, fermented juice of epiphytic lactic acid bacteria additive; COM, Commercial silage additive; MIX, FJLB+COM; VFA, volatile fatty acid; NH3-N, ammonia-nitrogen; DM, dry matter; IVDMD, in vitro dry matter digestibility. The values with different superscript within the same row are significantly different at 5% level.

Table 2.3 Chemical composition of total mixed ration (TMR) silage fermented with different type of silage additives after 60 days ensiling Chemical composition

Treatment P-value

CON FJLB COM MIX DM (%) 53.5ab 54.1a 52.9b 54.5a 0.004 CP (% DM) 13.9b 14.1ab 14.1ab 15.0a 0.016 aNDFom (% DM) 53.1a 53.6a 52.5a 47.7b <.001 ADFom (% DM) 31.4a 29.5ab 30.9ab 28.9b 0.021 EE (% DM) 2.8c 3.3bc 3.8b 5.8a <.001 Crude ash (% DM) 6.2 6.1 6.3 6.3 0.565 NFC (% DM) 24.0 23.0 23.3 25.3 0.181 CON, control (non-additive added); FJLB, fermented juice of epiphytic lactic acid bacteria additive; COM, commercial silage additive; MIX, FJLB+COM; DM, dry matter; CP, crude protein; aNDFom, α-amylase neutral detergent fiber exclusive ash; ADFom, acid detergent fiber exclusive ash; EE, ether extract; NFC, non-fibrous carbohydrates (NFC= 100CP–EE–aNDFom–Crude ash). The values with different superscript within the same row are significantly different at 5% level.

II-4. Discussion

Fermentation quality

The TMR silage with FJLB additive treatment has lower pH value than non-

additive silage. This result is in line with the study of FJLB on alfalfa silage (Wang et

al., 2009) and on rice straw silage (Jin-ling et al., 2013). The low pH in silage was

occurred because of the LAB that producing lactic acid (McDonald et al., 1991). In

the present study, the FJLB treatment also has lower pH value than COM treatment,

suggesting that LAB in FJLB was more suitable for TMR silage prepared from

agricultural by-product and food by-product in comparison to LAB in COM

treatment. The pH in FJLB was associated with the result of lactic acid production in

FJLB treatment which showed higher lactic acid production than that in CON and

COM treatment.

The highest lactic acid concentration in MIX treatment might suggested that

lactic acid bacteria in the commercial product would resulted in better fermentation

quality when it combined with lactic acid bacteria in FJLB but not showed a good

result when it applied alone. Furthermore, FJLB treatment has much better lactic acid

production than COM treatment. Probably, the various species of lactic acid bacteria

were presents in FJLB (Burenook et al., 2005; Wang et al., 2009). Thus, LABs in

FJLB produced more lactic acid rapidly, mainly after seven days of fermentation, than

COM treatment, which is this commercial additive contains only two strains of lactic

acid bacteria; Lactococcus lactis and Lactobacillus paracasei. These two strains of

lactic acid bacteria in commercial additive might be not suitable in silage preparing

from agricultural by-product. The better fermentation quality from FJLB treatment

than LAB inocula additive were also reported by Tao et al. (2017) and Wang et al.

(2009) but on alfalfa silage.

Acetic acid is produced by heterofermentative bacteria such as Lactobacillus

buchneri when they convert lactic acid to acetic acid under anaerobic condition

(Bureenok et al., 2016). The acetic acid is also responsible for the acidity of feed

silage and able to improve the aerobic stability (Yitbarek and Tamir, 2014) by

decreasing the opportunistic bacteria and fungi; i.e. yeast (Wang et al., 2014). In the

present study, the acetic acid production fluctuates in all treatments through the

fermentation period. The highest acetic acid value at 60 days was in MIX treatment,

which also supported for the lowest pH value in this treatment. Extend the duration of

fermentation may increase the acetic acid level (Minh et al., 2014). However, silages

with a high concentration of acetic acid are not equal with poor fermentation and have

no adverse effects on animal intake (Kung and Shaver, 2001).

Propionic acid concentration in this study was detected after few days of

ensiling. This result in-lines with McDonald et al. (1991) who reported propionic acid

usually produced in the later stages of fermentation after the pH decreased. The

possible microorganisms that responsible for propionic acid formation are clostridia

and propionic acid bacteria (McDonald et al., 1991). The growth of these

microorganisms in FJLB and MIX treated silages seems more restricted than that in

CON and COM treatments. It showed by the lower propionic acid concentration in 60

days of fermentation.

The butyric acid concentration was detected from the 14 days of fermentation

in all treatments except in CON treatment. The COM treatment showed the highest

value of butyric acid until the 60 days of ensiling period. The high butyric acid

concentration generally indicates bad preservation of silage. The maximum level of

butyric acid in silage that accepted as good fermentation is 2 g kg−1 DM (Zhang et al.,

2013). In the present study, the MIX treatment showed good silage proven by the

butyric acid value that less than the maximum level suggested. The low pH value of

MIX treatment able to inhibit the growth of clostridia that produce butyric acid. The

concentration of butyric acid in FJLB treatment was lower than the maximum level

during 14 days of fermentation then increased rapidly afterwards. The possible

explanation for this result is that in FJLB treatment, the environment of silage was not

acid enough to support the restriction for clostridia to grow after 14 days ensiling. The

growth of clostridia will be inhibited at a pH of 4.2 (McDonald et al., 1991). Usually

the butyric acid concentration in FJLB treated silage is very low or not detected as

reported in other studies (Horiguchi et al., 2008; Bureenok et al., 2011; Denek et al.,

2011, 2012). However, Wang et al. (2009) reported that butyric acid concentration of

FJLB treatment reached more than 2 g kg−1 DM even at the first days of ensiling then

increased steadily along the fermentation period of alfalfa silage. This implied that

TMR silage prepared from agricultural by-product and food by-product would not

restrict for the growth of clostridia if the low level of pH were not achieved.

The presence of ammonia nitrogen concentration in silage reflected that

protein degradation occurs (Driehuis et al., 2001; Ying et al., 2017). Good

fermentation silage should contain a low level of ammonia nitrogen. The study using

LAB inoculant (Guo et al., 2014; Ali et al., 2017) and FJLB (Burenook et al., 2005;

Jin-ling et al., 2013) could reduce the ammonia nitrogen concentration in silages.

However, in the present study, ammonia nitrogen was detected since the early of the

ensiling process in all treatments. The MIX and FJLB treatment had higher ammonia

nitrogen content than control at the second day of fermentation, but there was no

significance different afterward among the treatments. The possibility for this

condition is might be proteolytic microbial were exist in TMR silage since the pH

(more than 4.5) were not acid enough to inhibit the growth of those undesirable

microbial (Zhang and Yu, 2017). The ammonia level in silage is a good indicator for

the presence of proteolytic clostridial activity as it is only small chance for other

microorganisms to produce it (Oladosu et al., 2016). There was also possibility of

plant enzymes (protease) contributes to the ammonia production in silage

fermentation (McDonald et al.,1991). However, in the present study the TMR was

prepared from dry agricultural by-product which has high DM content. It also

suggested that the proteolysis has occurred during wilting and its activity drops when

reach high enough DM (McDonald et al., 1991).

DM losses occurred as a result of microbial metabolism in silage. The DM

losses were not different in all silage additive treatments before 30 days of

fermentation. The reason for this condition is that before 30 days of fermentation, the

microorganism in all treatment degrading the nutrient in the TMR in the similar

amount. After that, the environment of the silage in the MIX treatment, which is more

acid than other treatment, has successful inhibited undesired microorganisms. This

result is in agreement with Zhang et al. (2015) that reduction of silage pH depressed

the loss of simple carbohydrates by undesirable bacteria. This reason also answered

why the FJLB treatment has lower DM losses than CON treatment.

The Fleig point is one of tool to evaluate the silage quality (Ziaei and Molaei,

2010). Increasing in Fleig point indicates improvement of silage quality. In the

present study, MIX treatment showed excellent quality of fermentation. TMR silage

with FJLB treatment showed very good quality. Then for COM treatment classified as

moderate quality. The TMR silage with no additive added showed worthless quality.

The IVDMD of TMR silage was not affected by the addition of the silage

additives, the fermentation period and their interaction. The study by Nishino and

Uchida (1999) which applying FJLB in lucerne silage tend to increase its in vitro

digestibility. Another study (Wang et al., 2009) reported that the addition of FJLB on

alfalfa silage had higher IVDMD than control. These difference results suggest that

applied FJLB on silage has various effects on IVDMD. Many factors affect the in

vitro digestibility, such as the concentration of fiber components. In this study, TMR

were prepared mainly from rice straw and corn cobs which has high fiber content.

Especially in rice straw, lignin and silica are the most factor that limiting its

digestibility (Van Soest, 2006). In addition, in vitro data not always can be directly

extrapolated as in vivo digestibility because of the rumen ambient peculiar (Graminha

et al., 2008).

From the fermentation quality results, adding 1% FJLB and/or mixed with

commercial LAB prior to ensiling was enough to improve TMR silage made from

agricultural by-product and industrial by-product indicated by the lower pH, high

lactic acid concentration, lower butyric acid and lower ammonia-nitrogen than control

and commercial additive treatment.

Chemical composition

After fermentation begins, anaerobic microbes degrade the nutrients in the

silage. The addition of FJLB alone or in combination with commercial LAB had a

higher DM content than that in COM additive and tend to have greater DM content

than the un-treatment silage. The higher DM content in FJLB treatment than that in

un treatment has been reported in some studies (Nishino and Uchida, 1999; Shao et

al., 2007; Denek et al., 2011). FJLB contains a huge amount of LAB that produce

lactic acid to promote on reducing pH to around 4. The low pH in silage is not

suitable for other microorganisms to grow. The present result suggested that LAB in

this commercial LAB treatment could not enough to inhibit the activity of undesirable

microorganism. These microorganisms were responsible for the nutrient losses in

COM treatment. The result was also supported by the higher pH value, butyric acid,

and ammonia nitrogen content in COM treatment.

The combination of FJLB and commercial LAB as silage additive improved

the CP content of TMR silage prepared from agricultural by-product and food

industrial by-product. However, when FJLB or COM applied alone has similar CP

content to un-treated TMR silage. This result was consistent with previous report that

FJLB additive did not improve the CP content on lucerne silage (Denek et al., 2011)

and on Napier grass (Bureenok et al., 2012). Lactic acid bacteria in MIX treatment

was more effective to restrict proteolysis during fermentation than that in FJLB and

COM treatment. The pH decline in silage is important in determining in extend of

proteolysis (McDonald et al., 1991). In MIX treatment, pH was the lowest among the

treatments through the fermentation periods. However, the low pH could not prevent

the proteolysis, which was proved by the presence of ammonia nitrogen in MIX

treatment. Moreover, lactic acid bacteria are virtually non-proteolytic bacteria, which

means LAB has low contribution on protein degradation (Kondo et al., 2016).

The aNDFom content in FJLB treatment and COM treatment was not different

from CON treatment. However, it showed the lowest content when FJLB is in

combination with commercial LAB in MIX treatment. The ADFom content was

numerically lower in FJLB and COM treatment, then significantly lower in MIX

treatment than CON treatment. It might be suggested that LAB from FJLB and COM

has a synergetic effect on reducing the fiber content of TMR silage prepared from

agricultural and industrial by-product. However, the mechanism on how lactic acid

bacteria has ability on degrading structural carbohydrates is unclear in this study

because generally lactic acid bacteria utilize nonstructural carbohydrates, such as

glucose, as fermentable substrates (McDonald et al., 1991). The studies on reducing

fiber content by lactic acid bacteria or juice that contains lactic acid bacteria also has

been reported in some studies (Yahaya et al., 2004; Denek et al., 2011, 2012; Guo et

al., 2014). The reason for this result is unclear and still need to be investigated (Guo

et al., 2014). The lower of fiber content in MIX treated silages compare to CON

treatment indicates nutritive value improvement and possible to increase the silage

digestibility in the rumen (Yahaya et al., 2004).

Clear changes were observed in EE content of TMR silage. The EE content in

Mix treatment was the highest among the treatments. The reason is unclear, though

the low pH in MIX treatment might inhibit the degradation of EE. Whereas in CON,

FJLB and COM treatment, the acidity was not enough to decrease the metabolism of

crude fat by an undesirable microorganism (yeast and mold). Further investigation is

needed to clarify this result.

There were no statistical differences among the treatments on NFC content.

The NFC fraction includes starch, mono+oligosaccharides, fructans, pectin substrates

and organic acid (Hall, 2003). Soluble sugars are converted to lactic acid and acetic

acid during fermentation by lactic acid bacteria (Kondo et al., 2016). The remaining

NFC in MIX treatment showed numerically higher than other treatments, indicating

that MIX treatment tends to inhibit the competing organism for sugar breakdown.

According to the chemical composition result, the FJLB and MIX treatment

has better ability on nutrient preserving than non-additive and commercial LAB

treatment in TMR silage prepared from agricultural by-product and food by-product.

In conclusion, low pH, high lactic acid and low butyric acid and ammonia

nitrogen contents in the silage indicates that the application of FJLB as silage additive

in TMR silage prepared from agriculture by-product and industrial by-product

improved the fermentation quality. Although the combination of FJLB and the

commercial additive treatment further improved fermentation quality and nutritive

values of the TMR silage than FJLB treatment, it would not cost-effective when

applying in any scale farm. Further studies are needed to test the effect FJLB as silage

additive of agricultural by-product on animal production.

II-5. Summary

To improve the utilization and the quality of agriculture by-product and food

by-product, the present study was conducted to determine the effect of fermented

juice of epiphytic lactic acid bacteria (FJLB) as silage additive on the fermentation

quality and nutritive value of total mixed ration (TMR) silage prepared from

agricultural and food industrial by-products comparing with a commercial silage

additive. Italian ryegrass has been used for preparing the FJLB. The applied

treatments were: 1) CON (non-additive); 2) FJLB; 3) COM (commercial silage

additive); 4) MIX (FJLB and commercial additive combination). The fermentation

quality measured at 2, 7, 14, 30 and 60 days of fermentation. The nutritive value

measured at 60 days of fermentation. The results showed that addition of FJLB to

TMR silage from agriculture by-product has better fermentation quality than COM

and CON proven by lower pH, higher lactic acid, lower dry matter (DM) losses and

higher Fleig point. There was no significant difference in in vitro dry matter

digestibility (IVDMD) among the treatments. The addition of FJLB combined with

commercial silage additive decreased the NDF and ADF content of TMR silage. In

conclusion, the application of FJLB in TMR silage prepared from agriculture by-

product and industrial by-product improved the fermentation quality. Although the

combination of FJLB and the commercial additive treatment further improved

fermentation quality and nutritive values of the TMR silage than FJLB treatment, it

would not cost-effective when applying in any scale farm.

CHAPTER III

Effect of total mixed ration ensiled with fermented juice of epiphytic lactic

acid bacteria on nutritive intake, nutrient digestibility, rumen fermentation and

nitrogen balance in ewes

III-1. Introduction

The Chapter II showed that addition of FJLB to TMR silage from agriculture

by-product has better fermentation quality than COM and CON proven by lower pH,

higher lactic acid, lower dry matter (DM) losses and higher Fleig point. Moreover, the

addition of FJLB combined with commercial silage additive decreased the NDF and

ADF content of TMR silage. However, the experiment in Chapter II was in laboratory

scale, and the application of FJLB in farm scale is needed to investigate the FJLB

effects on animal production.

An in vitro study by Yahaya et al. (2004) demonstrated that FJLB silage have

higher digestibility of DM and NDF. Similarly, in vivo study by Bureenok et al.

(2011) found that CP digestibility in ruzigrass treated with FJLB was improved and

cellulolytic population in the rumen were increased. However, there was limited

information of applying FJLB in TMR silage prepared from agricultural by-product

on animal production. I hypothesized that FJLB in TMR silage prepared from crop

waste could improve nutrient assessment in ruminant. If FJLB were applied in TMR

prepared from agricultural by-product, this additive would result in good fermentation

quality silage, as indicated by a low pH value and high lactic acid production. Good

quality silage possibly improves animal nutrient intake, as a result the nutrient

digestibility, ruminal fermentation and nitrogen retention would be improved.

Therefore, this study aimed to determine the effect of FJLB on nutrient intake,

digestibility, rumen fermentation, and nitrogen balance in ewes.

III-2. Materials and methods

All animal experimental procedures were approved by the Committee for

Animal Research and Welfare of Gifu University (#17035)

TMR were prepared from rice straw, corn stover silage, brewer grain, tofu

waste, steam flaked maize and mineral-vitamin mix. Rice straw was harvested at

Yanagido Farm, Gifu Field Science Center, Gifu University, and was chopped into 2-

3 cm length prior to ensiling. Corn stover were harvested at yellow ripe stage, then

chopped to 2-3 cm. Then, corn stover were ensilaged in polyethylene bag doubled

with a flexible container bag (100 L in volume) then stored outdoor about 2 months

until mixing to TMR. Wet brewer grain, tofu waste, steam flaked maize were

purchased from a local feed company (Minorakuren Co, Ltd., Gifu, Japan). Wet

brewer grain and tofu waste were preserved in an anaerobic condition until mixing.

Vitamin-mineral mix was the same to that in Chapter II. The nutrient content of each

material and the proportion in TMR are shown in Table 3.1 and Table 3.2,

respectively. The proportion of each material was obtained by trial and error methods

using Excel. TMR were formulated to obtain 12.5% of crude protein (CP) and 66.1%

of total digestible nutrients (TDN) to meet or exceed the maintenance requirement of

sheep according to National Research Council (2007).

Table 3.1. Ingredient composition of material for total mixed ration

Ingredient Rice straw

Corn stover silage

Brewer grain

Tofu waste

Steam flaked maize

Dry matter (%) 91.9 19.8 37.24 25.2 91.8 Crude protein (%DM) 3.6 6.9 22.3 27.3 6.9 aNDFom (%DM) 62.9 68.1 61.2 25.2 16.4 ADFom (%DM) 39.3 41.8 23.5 22.2 4.3 Extract ether (%DM) 2.0 1.8 9.8 8.4 3.7 Crude ash (%DM) 12.2 7.3 4.6 4.8 1.2 DM: dry matter, aNDFom: neutral detergent fiber with heat stable amylase and exclusive ash, ADFom: acid detergent fiber exclusive ash

Table 3.2. Proportion of material in total mixed ration

Item Proportion (% in DM) Rice straw 27.0 Corn stover silage 26.0 Brewer grain 13.5 Tofu waste 15.0 Steam flaked maize 17.0 Mineral mix 1.5 Total 100

Preparing silage

All the materials were mixed manually and added silage additive according to

the following treatments. The treatments were: CON (no silage additive added),

FJLB, COM (commercial additive: “Si-Master AC”®, Snow Brand Seed Co.,Ltd.,

Sapporo, Japan), MIX (FJLB+COM). The FJLB and commercial additive preparation

was similar to the previous section (Chapter II). The treatment of commercial additive

followed the company’s instruction. Distilled water was added in CON, FJLB and

COM treatment to adjust the moisture content similar to MIX treatment. TMR were

packed into polyethylene bag doubled with a flexible container bag (capacity 100 kg).

Silages were made in four replications for each treatment. TMR were fermented for

minimum 2 months (from 8 June – 31 August 2017) outdoors.

Feeding period

Four ewes Suffolk with canulla attached in the rumen, aged 7.1 ± 2.2 years old

and initial body weight (BW) 56.1 ± 15.2 kg, were assigned into a 4 4 Latin square

design. The ewes were housed in metabolic cage individually with range room

temperature was 13.9 – 33.4°C. The ewes in this experiment were managed according

to the guidelines of the Committee for Animal Research and Welfare of Gifu

University.

The TMR feed was offered (2% of BW on dry matter basis) twice a day (at

09:30 and 14:30) in an equal amount and had 10% of refusal feed. The amount of the

diets offered to ewes and the orts were recorded daily. The water provided ad libitum

to the ewes. Adaptation was 8 days followed by collection period for 6 days in each

period. The body weight was measured at the beginning of experimental and in the

end of each period.

Sample collection

Before adding silage additives, approximately 500 g of TMR were collected

with three replications to assess the quality of TMR pre-silage. A representative

sample of TMR (approx. 500 g) were collected just after the silo opened in the

beginning of each experimental period for fermentative quality and chemical

composition analysis. Approximately 100 g of daily feed sample was collected

throughout the sample collection period to correct the DM content for feed intake

calculation.

Rumen sample was collected at 0 hour and 4 hours after feeding at the end of

each collection period via the rumen canulla. The rumen sample filtered using four

layers of cheesecloth, then the pH of filtrate was measured immediately using pH

meter (MP220; METTLER TOLEDO, Tokyo, Japan). The filtrate was placed in a 2

ml microtube with 3 replications for each animal, and then centrifuged 3,500 × g for

10 minutes. The supernatant stored in -20°C for ammonia (NH3) and volatile fatty

acids (VFAs) analysis.

Fecal and urine were collected in the every morning and evening in each

collection period. Feces were weighed, mixed and then 10% of the feces were

sampled. Three ml of 10 N formaldehyde solution were added to the sample to stop

microbial fermentation and then stored in a freezer (-20°C). In the end of each

collection period, all feces from each animal were mixed thoroughly and 250 g of

representative sample was collected. The fecal samples were dried at 60°C for 48

hours to determine the dry matter (DM) content. Dried fecal samples were ground to

pass 1 mm-sieve and packed into sealed polyethylene bag until the chemical

composition analysis. The 100 mL of 20% sulfuric acid was added to the collected

urine everyday through collection period. The 20% of the total urine were collected

then stored in the refrigerator. In the end of each collection period, all urine from each

animal were mixed thoroughly and collected to 50 mL of plastic tube with two

replications. All urine then stored in -20°C for nitrogen content analysis.

Chemical analysis

The chemical composition of TMR silage and feces was analyzed by the same

methods. The DM, ash, acid detergent fiber exclusive ash (ADFom), CP, and ether

extracts (EE) analysis were performed according to the methods of AOAC (2007:

protocol number, 930.15; 942.05; 973.18; 990.03 and 920.39, respectively). The

organic matter (OM) was calculated as weight loss trough ashing. The neutral

detergent fiber assayed with heat stable α-amylase and exclusive ash (aNDFom) were

measured according to Van Soest et al. (1991). The nitrogen content in urine samples

were determined by Kjeldahl method (AOAC, 2007).

Fermentation quality of TMR silage was assayed same to that in Chapter II.

As a silage quality index, Fleig point was calculated as the following equation (Denek

and Can, 2006).

Fleig points = 220 + (2 × DM% 15) – 40 × pH

The supernatants from the rumen samples were thawed in room temperature

and added by 25% of meta-phosphoric acid at ratio 1:4 (meta-

phosphoric:supernatant), and then stored again in freezer (-20°C) for at least 8 hours

until analysis to remove protein. Frozen supernatants were thawed in refrigerator and

centrifuged at 10,000 × g for 5 minutes. The supernatant was placed into new tubes

and mixed with internal standard (10 mmol/L crotonic acid) in 6:1 ratio. One

microliter (μm) was injected to gas chromatography (GC–14A, Shimadzu, Kyoto,

Japan; column: ULBON HR-20M, 0.25 mmI.D. × 30 mL 0.25 μm; injection port at

250°C, column at 100°C and detector at 250°C, flow rate 15 mL/minute) for VFA

analysis.

Statistical analysis

All obtained data were analyzed using R programming 3.3.2 (R development

core team, 2016). The fermentation quality and chemical composition of TMR silage

were analyzed by one-way analysis of variance (ANOVA). The mean of additives

treatment was compared using pairwise t test. Data on intake, digestibility, rumen

parameters and nitrogen balance were subjected to ANOVA using the following

model: Yijkl = μ + Ti + Pj + Ak + eijkl, where Yijk = observation, μ = the overall

means, Ti = the fixed effect treatment feed, Pj = the fixed effect of period, Ak = the

random effect of animal and eijkl = residual error. The mean of additives was

compared using pairwise t test. The Bonferroni correction was used to detect the

differences between the means for each data analysis.

III-3. Results

Fermentation quality and chemical composition of TMR silage

The MIX treatment had a lower pH value than the CON and FJLB treatments

(P<0.01; Table 3.3). The lactic acid concentration in the MIX treatment was highest

among the treatments. However, there were no significant differences in pH among

the CON, FJLB and COM treatments. There were also no significant differences in

VFA and NH3-N concentrations among the treatments. Fleig points in the MIX

treatment were higher than those in the CON treatment (P<0.05). The nutritive value

of TMR silage showed that DM content tended to be lower in CON than in the other

treatments (P=0.079). The CP content in all TMR with silage additives tended to be

higher than in TMR without silage additive (P=0.089). The ADFom and aNDFom

contents of TMR silage were lower in the COM treatment than those in the CON

treatment; however, no significant difference was detected among the other

treatments. There were no significances differences in EE and OM contents among

the treatment groups (P>0.05).

Intake, digestibility and ruminal fermentation of TMR silages

There was no significant difference in nutrient intake and apparent

digestibility (P>0.05) in ewes fed TMR silage with different silage additive

treatments (Table 3.4).

The pH in the rumen was 7.1 before feeding and 6.8-6.9 after feeding for all

TMR silages (Table 3.5). The silage additive treatments did not affect ammonia-

nitrogen and VFA concentrations in the rumen at 0 and 4 hours after feeding. The

ewes showed similar N intake, excretion and retention, regardless of the silage

additive treatments in TMR silage (Table 3.6; P>0.05).

Table 3.3. Fermentation quality and chem

ical composition of total m

ixed ration (TMR

) silage prepared from agriculture by-product w

ith different silage additives Item

Pre-silage

Treatment

P-value C

ON

FJLB

C

OM

M

IX

Fermentation quality

pH

NA

4.6 ± 0.12 b

4.5 ± 0.20b

4.3 ± 0.09ab

4.1 ± 0.13a

0.005 Lactic acid (g/kg D

M)

NA

13.8 ± 6.5

a 17.1 ± 4.36

a 19.0 ± 8.64

a 37.0 ± 5.78

b 0.006

VFA

(g/kg DM

)

A

cetic acid N

A

23.6 ± 6.2 37.9 ± 13.9

29.2 ± 8.2 34.6 ± 14.6

0.812 Propionic acid

NA

2.9 ± 0.74

3.1 ± 0.76 3.6 ± 0.82

2.7 ± 0.31 0.799

Butyric acid

NA

2.7 ± 1.34

0.8 ± 0.20 0.3 ± 0.19

1.1 ± 0.46 0.161

NH

3 -N (g/kg TN

) N

A

54.3 ± 4.27 59.9 ± 6.73

56.6 ± 7.06 57.9 ± 4.85

0.916 Fleig point

NA

85.4 ± 6.81

a 96.5 ± 3.56

ab 108.5 ± 3.15

b 111.2 ± 2.17

b 0.004

Chem

ical composition

DM

38.1

31.8 ± 2.54 36.6 ± 0.51

37.0 ± 0.42 35.8 ± 0.86

0.079 O

M (%

DM

) 91.4

90.9 ± 0.21 91.1 ± 0.06

91.2 ± 0.05 91.3 ± 0.1

0.180 C

P (%D

M)

8.0 11.0 ± 0.23

11.4 ± 0.20 11.8 ± 0.10

11.5 ± 0.24 0.089

aND

Fom (%

DM

) 59.0

56.8 ± 0.82b

56.2 ± 1.52ab

51.6 ± 0.69a

52.7 ± 1.27ab

0.016 A

DFom

(%D

M)

35.9 34.0 ± 0.53

b 33.0 ± 0.69

ab 31.1 ± 0.22

a 31.7 ± 0.93

ab 0.030

EE (%D

M)

2.8 4.2 ± 0.41

4.2 ± 0.09 4.8 ± 0.23

4.6 ± 0.15 0.244

Values are presented as m

ean ± standard error, CO

N: control (no additive added), FJLB

: fermented juice of epiphytic lactic acid bacteria

additive, CO

M: C

omm

ercial silage additive, MIX

: FJLB+C

OM

, VFA

: volatile fatty acid, NH

3 -N: am

monia-nitrogen, C

P: crude protein,

Table 3.4. Effect of TMR

contained agricultural by-product treated with FJLB

on nutrient intake and apparent digestibility of ewes

Item

Treatment

P-value C

ON

FJLB

C

OM

M

IX

Intake (g/day/kg BW

0.75)

DM

34.2 ± 5.64

39.6 ± 2.30 38 ± 1.83

39.2 ± 4.44 0.393

OM

32.0 ± 5.69

37.3 ± 1.56 34.3 ± 2.73

35.3 ± 3.67 0.129

CP

3.8 ± 0.59 4.5 ± 0.24

4.3 ± 0.23 4.6 ± 0.66

0.347 aN

DFom

18.6 ± 2.77

21.7 ± 0.87 20.2 ± 0.85

20.8 ± 2.74 0.612

AD

Fom

11.5 ± 1.86 13.3 ± 0.44

12.3 ± 0.48 13.2 ± 1.76

0.625 EE

1.1 ± 0.22 1.4 ± 0.23

1.3 ± 0.32 1.5 ± 0.48

0.374 A

pparent digestibility (% of intake)

D

M

59.8 ± 2.73 65.6 ± 2.89

55.9 ± 6.37 62.2 ± 4.92

0.371 O

M

57.5 ± 1.92 62.9 ± 3.17

53.5 ± 6.07 59.2 ± 4.15

0.584 C

P 60.1 ± 4.6

65.8 ± 2.84 57.8 ± 8.52

63.2 ± 2.71 0.505

aND

Fom

57.6 ± 2.7 64.8 ± 4.85

49.1 ± 4.29 57.4 ± 7.13

0.101 A

DFom

56.8 ± 2.39

62.8 ± 4.30 47.3 ± 5.45

57.2 ± 6.89 0.172

EE 80.5 ± 3.01

83.7 ± 4.47 83.2 ± 5.39

85.2 ± 0.54 0.760

Values are presented as m

ean ± standard error, CO

N: control (non-additive added), FJLB

: fermented juice of epiphytic lactic acid bacteria

additive, CO

M: com

mercial silage additive, M

IX: FJLB

+CO

M, D

M: dry m

atter, CP: crude protein, O

M: organic m

atter, aND

Fom: neutral

detergent fiber with heat stable am

ylase and exclusive ash,, AD

Fom: acid detergent fiber exclusive ash, EE: ether extract

Table 3.5. Effect of TMR

contained agricultural by-product treated with FJLB

on rumen ferm

entation of ewes at 0 and 4 hours after feeding

Item

Treatment

P-value C

ON

FJLB

C

OM

M

IX

0 hour

pH

7.0 ± 0.16 7.1 ± 0.08

7.1 ± 0.06 7.1 ± 0.05

0.889 N

H3 -N

(mm

ol/L) 4.0 ± 0.41

3.7 ± 0.32 3.0 ± 0.53

3.4 ± 1.08 0.691

VFA

(mm

ol/L)

Acetic acid

62.4 ± 7.68 70.4 ± 18.05

56.6 ± 3.91 49 ± 8.95

0.834 Propionic acid

14.2 ± 1.27 15.1 ± 3.73

12.8 ± 1.48 12 ± 2.81

0.982 B

utyric acid 4.8 ± 0.59

5.6 ± 0.85 4.5± 0.90

3.7 ± 0.78 0.470

4 hour

pH

6.8 ± 0.15 6.8 ± 0.10

6.9 ± 0.09 6.8 ± 0.09

0.996 N

H3 -N

(mm

ol/L) 3.3 ± 0.68

3.5 ± 1.38 3.4 ± 0.65

3.3 ± 1.11 0.983

VFA

(mm

ol/L)

Acetic acid

74.7 ± 8.64 79.0 ± 4.35

73.9 ± 4.99 62.5 ± 13.42

0.176 Propionic acid

21.3 ± 4.89 18.6 ± 0.83

21.5 ± 2.29 16.1 ± 6.61

0.524 B

utyric acid 6.2 ± 1.45

5.7 ± 0.25 6.0 ± 0.86

4.8 ± 1.25 0.585

Values are presented as m

ean ± standard error, CO

N: control (no additive added), FJLB

: fermented juice of epiphytic lactic acid bacteria

additive, CO

M: C

omm

ercial silage additive, MIX

: FJLB+C

OM

, VFA

: volatile fatty acid, NH

3 -N: am

monia-nitrogen,

Table 3.6. Effect of TMR

contained agricultural by-product treated with FJLB

on nitrogen (N) balance of ew

es

Item

Treatment

P-value C

ON

FJLB

C

OM

M

IX

N balance (g/day/B

W0.75)

Intake N

0.63 ± 0.10

0.72 ± 0.03 0.68 ± 0.05

0.73 ± 0.09 0.234

Fecal N

0.24 ± 0.03 0.25 ± 0.02

0.28 ± 0.04 0.26 ± 0.04

0.803 U

rinary N

0.09 ± 0.01 0.09 ± 0.03

0.09 ± 0.02 0.08 ± 0.01

0.953 R

etention N

0.29 ± 0.07 0.39 ± 0.05

0.31 ± 0.10 0.39 ± 0.06

0.192 Proportion of N

retention to N

intake (%)

46.70 ± 4.75 53.98 ± 5.47

45.56 ± 11.89 53.42 ± 3.35

0.465

Values are presented as m

ean ± standard error, CO

N: control (no additive added), FJLB

: fermented juice of epiphytic lactic acid bacteria

additive, CO

M: C

omm

ercial silage additive, MIX

: FJLB+C

OM

.

III-4. Discussion

Fermentation quality

The MIX treatment in this study was categorized as well-preserved silage

because the pH value was around four (Cao et al., 2010). The pH in the MIX

treatment was the lowest among the treatments due to the high lactic acid

concentration in the MIX treatment. This result suggested that the combination of

lactic acid bacteria from FJLB and a commercial additive contributed to greater lactic

acid production.

With respect to VFA concentration, there was no significant difference in

VFA concentration in all the treatments. The concentration of acetic acid in all

treatments was less than 1% of DM silages, although acetic acid contributes to

improving aerobic stability by depressing the growth of yeast (Da Silva et al., 2014).

Butyric acid was detected both in the control and treated silages in the present study.

However, the butyric acid concentration in all additive-treated silage was under the

acceptable level (0.2% of DM silage; Zhang et al., 2013).

The NH3-N/TN concentrations in all the treatment silages were less than

12.5%, indicating that all treatment silages contained well-preserved fermentation

(McDonald and Wittenbury, 1973; Kung, 2010). The NH3-N in the control silage was

not different from the FJLB treatment silage in this study. This result was inconsistent

with the study by Wang et al. (2009), who found that FJLB additive in alfalfa silage

had a lower NH3-N content than that of the control. A possible reason for these

different results is that CON in this study had a lower pH (4.6) than that in the other

study. Since we used fermented corn stover and tofu waste as ingredients of TMR

silage, the original ingredients may contain enough LAB. Thus, CON showed a

relatively low pH value and inhibited the further breakdown of protein.

The Fleig point is a method for assessing fermentation quality based on the

DM content and pH value of silage. The Fleig points in the MIX and COM treatments

were higher than those in the CON treatment. However, according to Ziaei and

Molaei (2010), all silage in this study was categorized as very good quality since the

value was greater than 85.

DM and CP contents in the silage additive treatment including FJLB were

slightly greater than those in the CON treatment (P=0.079), suggesting that FJLB has

the ability to inhibit DM losses and CP degradation. This result is in line with that of

previous studies (Denek et al., 2011 and 2012) that found DM content in FJLB-

treated silage was higher than that in un-treated silage. The higher CP content in the

FJLB treatment than in the control was also reported in elephant grass (Yahaya et al.,

2004) and rice straw silage (Jin-ling et al., 2013).

The FJLB treatment in this study showed no clear distinction with CON

treatment (P>0.05) in NDFom and ADFom content. This result is in agreement with

some previous studies that showed applying FJLB as silage additive did not affect the

NDF and ADF content of alfalfa silage (Wang et al., 2009) and ruzigrass silage

(Bureenok et al., 2011). Structural carbohydrates in NDFom and ADFom are not

fermentable substrates for lactic acid bacteria in silage (McDonald et al., 1991).

However, studies by Denek et al. (2011 and 2012) found that FJLB treatment had

lower NDF and ADF contents than those of un-treated alfalfa silage. Therefore, it is

suggested that the lower cell wall content in the FJLB treatment compared to the

control in Denek’s study was a result of the hydrolysis brought about by the action of

some bacteria that are able to breakdown cellulose and hemicelluloses (Wanapat et

al., 2013) or by enzymes that are present in FJLB. However, the details of this

mechanism need to be further investigated.

Nutrient intake and digestibility

The DM, OM, CP, aNDFom, ADFom and EE intakes were not affected by

silage additive treatment in the TMR silage prepared from agricultural by-product,

suggesting that all TMR silage in this study has similar palatability. This finding

might be explained by the fact that all TMR silages in this study were categorized as

well-preserved silage based on the Fleig point. This result was in agreement with that

of a previous study (Bureenok et al., 2012), which found that FJLB treatment in

ruzigrass silage did not affected total intake in cows.

The apparent digestibility of DM, OM, CP, aNDFom, ADFom and EE was not

affected by silage additive treatments. Yahaya et al. (2004) found that FJLB treatment

in elephant grass silage increased the DM and NDF digestibility in situ compared to

control and acetic acid treatments. Bureenok et al. (2011) found that the addition of

FJLB in ruzigrass silage increased the digestibility of CP compared to that of the

control. Another study (Takahashi et al., 2005) found that whole crop rice straw

treated with FJLB and crushing improved the digestibility of fibrous components

compared to the control. This inconsistency with the present study might be explained

by the fact that, that in the present study, all treated silages were well-preserved,

including that of the CON treatment. Wet tofu waste, brewer grain and corn stover

silage as the ingredients of TMR in the present study might contain a sufficient level

of LAB, although I did not analyze the LAB in the material. However, the presence of

LAB in tofu waste and brewer grain was documented by Tanaka et al. (2001), and

LAB in corn stover was also documented by Wang et al. (2017). Thus, we supposed

that the doses of FJLB in this present study were not sufficient to have a positive

effect on the TMR silage over the effect of the original materials.

Rumen fermentation

The fermentation characteristics in the rumen were not affected by treatment.

The pH in all silages was 7.1 before feeding and 6.8-6.9 after feeding and was within

the normal range (6.5-7.0) for optimal microbial digestion of fiber and protein (Huyen

et al., 2012).

The silage additive treatments did not affect the ammonia-nitrogen

concentrations in the rumen. According to Satter and Slyter (1974), the minimum

ammonia nitrogen concentration in the rumen is 2.94 mmol/l to supply the rumen

microbes with sufficient N for protein synthesis. All the ruminal ammonia-nitrogen

values in the present study were more than the minimum level, indicating that they

were sufficient for microbial protein synthesis. This result is in line with that of

Burenook et al. (2016), who found that ammonia-nitrogen concentration was more

than the minimum concentration in goats after applying FJLB in stylo legume, guinea

grass and their mixture. The present experiment was also in line with previous studies

that applied FJLB in ruzigrass (Bureenok et al., 2011) or napier grass silage

(Bureenok et al., 2012), resulting in similar rumen NH3-N concentrations in cows. In

the present study, as the TMR silage has similar fermentation quality, ewes showed

similar nutrient intake, which in turn led to a similar result of the rumen NH3-N

concentration.

The silage additive treatments also did not affect VFA concentrations in the

rumen, and the total VFA concentrations in the present study were in the normal

range (70-130 mmol; Huyen et al., 2012). This result might relate to similar nutrient

intakes among the four treatments. Takahasi et al. (2005) reported a similar result; the

ruminal VFA concentration in sheep fed with FJLB-treated whole rice straw silage

did not differ from the control sheep. In addition, the level of DM intake in the present

experiment was too low to influence the process of rumen fermentation (Bureenok et

al., 2012). Compared to some studies, DM intake in the present study was lower and

thus might not influence the VFA concentration. The study by Yani et al. (2015)

reported the DM intake in sheep was 43.8-45.4 g/day/kg BW0.75, whereas Ishida et al.

(2012) reported that DM intake in sheep was 49.27-50.58 g/day/kg BW0.75. In their

studies, the effect of treatment on ruminal fermentation was detected as a higher DM

intake in sheep.

Finally, sheep showed similar N intake, excretion and retention, regardless of

the treatment. The similar N balance in the present study is due to the same quality of

silages, which did not influence rumen microbial protein synthesis or the utilization of

amino acids. This result was consistent with the study by Takahashi et al. (2005), in

which FJLB was added to whole rice straw, and Horiguchi and Takahashi (2007),

who found that FJLB treatment in green soybean stover did not affect nitrogen intake,

fecal or urine nitrogen or nitrogen retention in sheep. However, Cao et al. (2002)

showed improvements in nitrogen retention in dry cows fed a total mixed ration

composed of alfalfa silage treated with FJLB, but FJLB did not affect nitrogen intake

or the fecal and urinary excretion of nitrogen. These differences might be caused by

the different material used as feed in their studies and the present study.

The FJLB as a silage additive in TMR made from agricultural by-product had

similar fermentation quality to that of non-additive TMR silage. If the FJLB

combined with a commercial additive, the fermentation quality was improved. The

application of FJLB additive in agricultural by-product silage has no negative effects

on nutrient intake, nutrient digestibility and nitrogen balance in ruminants. The

present study suggests that doses of FJLB should be further investigated, although

original materials containing a certain amount of lactic acid bacteria contribute to the

improvement of the quality of TMR silage prepared from agricultural by-products.

III-5. Summary

The goal of this study was to determine the effects of fermented juice of

epiphytic lactic acid bacteria (FJLB) on the quality of total mixed ration (TMR) silage

that contained agricultural by-products, and nutrient digestibility, rumen fermentation

and nitrogen balance in ewes. TMR was prepared from rice straw, corn stover silage,

brewer grain, tofu waste, steam flaked corn, and a mineral mixture. The treatments

consisted of silage additives added to TMR: CON (no silage additive), FJLB

(fermented juice of epiphytic lactic acid bacteria), COM (commercial additive), and

MIX (FJLB + COM). Four cannulated ewes were assigned into 4 × 4 Latin square

design. The MIX treatment produced a lower (P<0.01) pH than CON and FJLB

treatment and higher (P<0.01) lactic acid concentration than did the other treatments.

Fiber content in the COM treatment was lower (P<0.05) than that in the other

treatments. Although, silage quality index (Fleig point) was higher in MIX and COM

than in CON, all silages had a good quality. The chemical compositions in FJLB

treatment were similar (P>0.05) to other treatments. Thus, any silage additives did not

affect intake, digestibility, rumen fermentation and nitrogen balance. In conclusion,

the FJLB additive in TMR silage prepared from agricultural by-products slightly

improves its quality as well as other additives, and did not give negative effect on

nutrient digestibility, rumen fermentation and nitrogen balance in ewes.

CHAPTER IV

Effect of total mixed ration ensiled with fermented juice of epiphytic lactic

acid bacteria on in vitro methane emission, blood parameters and energy balance

in ewes

IV-1. Introduction

As shown in Chapter III, the fermentation quality of TMR silage treated with

FJLB had similar quality to the non-additive silage. The FJLB treatment did not

reduce the fiber content in TMR silage. This result indicates that carbohydrates

degradation did not occurred enough in FJLB treatment. This result was in line with

some studies. Applying FJLB in alfalfa silage (Wang et al., 2009) and in Napier grass

(Burenook et al., 2012) did not significantly decrease their fiber content. However,

other studies (Denek et al., 2011, 2012) reported that FJLB treatment reduced the

structural carbohydrates content in alfalfa silage. These different results might be

caused by the different materials used. In my experiment, the food by-product as

TMR material has original LAB that might cause the effect of FJLB on fiber

degradation was not clear. The different result of FJLB application indicates that

FJLB might has possibility on improving the fiber content of silage. The improved

fiber content will improve the energy utilization and reduce the methane emission as

well.

As suggested in Chapter I, the critical issue when using agricultural by-

products as a feed for ruminants is methane emission. In the process of fermentation

in the rumen, 2-12% of ingested gross energy is converted to methane, which leads to

lowers the efficiency of feedstuff utilization (Anantasook and Wanapat, 2012). Feed

that contains many structural carbohydrates, such as rice straw, has lower digestibility

(Yuangklang et al., 2017), and produces more methane emission in the rumen than

feed contain low non-structural carbohydrates (Archimède et al., 2011). The result of

Chapter III suggested that the fibrous content in FJLB treatment was similar to CON

treatment. Whereas, other studies showed that FJLB treatment improve the fiber

content of silage, as pointed previously. The FJLB silage was also increased the

digestibility of DM and NDF as reported by Yahaya et al. (2004). Feed that contains

many structural carbohydrates produces more methane emission in the rumen than

feed contained low non-structural carbohydrates (Archimède et al., 2011). Methane

has been known as the one of global warming cause. In the other side, methane

emission also considered as energy loss in ruminant diet. FJLB combined with lactic

acid bacteria (Chikuso-1) in in vitro study by Cao et al. (2009) decreased the methane

production per digestibility DM. However there was limited information of the FJLB

effect alone on methane production. Therefore, it may suggest that TMR silages with

FJLB mitigates the methane emission and improve energy utilization of agricultural

by-products. To the author knowledge there a scare study of the effect of FJLB

treatment as silage additive in TMR on the blood metabolites and not yet reported on

energy balance. The objective of this study was to determine the effect of TMR

ensiled with FJLB on in vitro methane emission, energy balance and blood parameters

in ewes.

IV-2. Materials and Methods

All animal experimental procedures were approved by the Committee for

Animal Research and Welfare of Gifu University (#17035)

TMR was prepared from rice straw, corn stover, brewer grain, tofu waste, steam

flaked maize and mineral-vitamin mix. Rice straw was purchased from a local

supplier (Ishiji Co, Ltd., Takayama, Japan). Corn stover were prepared from yellow

ripe stage then chopped to 2-3 cm before ensiling. Steam flaked maize, wet brewer

grain and tofu waste were purchased from a local feed company (Minorakuren Co,

Ltd., Gifu, Japan). The wet brewer grain and tofu waste were preserved in anaerobic

conditions until mixing. Vitamin-mineral mix was same as that in Chapter II. The

nutrient content of each material and the proportion in TMR is shown in Table 4.1 and

Table 4.2, respectively. The proportion of each material was obtained by trial and

error methods using Excel. TMR was formulated to obtain 12.5% of crude protein

(CP) and 66.1% of total digestible nutrients (TDN) to meet or exceed the nutrient

requirement of growing lamb according to National Research Council (2007).

Preparing silage

All the materials were mixed manually and added silage additive according to

the following treatments. The treatments were: CON (no silage additive added),

FJLB, COM (commercial additive: “Si-Master AC”®, Snow Brand Seed Co.,Ltd.,

Sapporo, Japan), MIX (FJLB+COM). The FJLB were prepared from Italian ryegrass

modified from Burenook et al. (2016) and was same method with that in Chapter II.

The commercial additive diluted with distilled water (17:1000 ratio; w:v) and sprayed

0.1% (v:w) on the TMR. Distilled water was added in CON, FJLB and COM

treatment to adjust the moisture content similar to MIX treatment. TMR were packed

into polyethylene bag doubled with flexible container bag (capacity 100 kg). Silages

were made in four replications for each treatment. TMR were fermented for minimum

2 months (from 27 October – 18 December 2017) outdoors.

Table 4.1. Ingredient composition of material for TMR Ingredient Rice

straw Corn stover

Brewer grain

Tofu waste

Steam flaked maize

Dry matter (%) 90.9 30.6 38.3 25.3 90.5 Crude protein (%DM) 4.1 4.1 20.5 24.9 7.1 aNDFom (%DM) 69.8 67.0 59.7 24.8 14.7 ADFom (%DM) 48.5 43.4 28.9 22.8 4.6 Extract ether (%DM) 2.0 1.8 9.8 8.4 3.7 Crude ash (%DM) 10.9 9.4 3.8 4.5 1.4 Energy (kJ/g DM) 17.6 17.8 22.0 21.4 18.6 DM: dry matter, aNDFom: neutral detergent fiber with heat stable amylase and exclusive ash, ADFom: acid detergent fiber exclusive ash

Table 4.2. Proportion of material in TMR Item Proportion (% in DM) Rice straw 25 Corn stover 23 Brewer grain 19.5 Tofu waste 19 Steam flaked maize 12 Mineral mix1 1.5 1 Vitamin-mineral mix (NAS DL05-HVE; NASU AGRI SERVICE, Inc., Tokyo, Japan) contained 5,000,000 IU kg−1 of vitamin A, 1,000,000 IU kg−1 of vitamin D, 24,000 IU kg−1 of vitamin E, 150 mg kg−1 of Co, 8,000 mg kg−1 of Cu, 15,000 mg kg−1 of Mn, 250 mg kg−1 of I, 20,000 mg kg−1 of Zn, 10 mg kg−1 of Se, 700 g kg−1 of Mg.

Feeding period

Four Suffolk ewes with canulla attached to the rumen in this experiment was

the same to ewes in Chapter III. These ewes aged 7.5 ± 2.2 years old and initial body

weight (BW) 51.0 ± 19.5 kg, and were assigned into 4 4 Latin square design. The

ewes were housed in metabolic cage individually with ambient temperature was 0.7 –

13.5°C.

The TMR feed was offered (2% of BW on dry matter basis) twice a day (at

09:30 and 14:30) in an equal amount and had 10% of refusal feed. The amount of the

diets offered to ewes and the orts were recorded daily. Water was provided ad libitum

to the ewes. Adaptation was 8 days followed by collection period for 6 days in each

period. The body weight was measured at the beginning of experiment and in the end

of each period.

Sample collection

Before adding silage additives, approximately 500 g of TMR were collected

with three replications to assess the quality of TMR pre-silage. A representative

sample of TMR (approx. 500 g) were collected just after the silo opened in the

beginning of each experimental period for chemical composition analysis.

Approximately 100 g of daily feed sample was collected throughout the sample

collection period to correct the DM content for feed intake calculation.

The blood sample was collected at the last day of each collection period.

Blood samples were collected in 9 ml vacuum tubes (TERUMO Venoject II,

TERUMO corporation) 4 hours after feeding by jugular venipuncture. The samples

then centrifuged at 800 × g for 20 minutes at room temperature. Serum was separated

and stored in triple microtubes at -20°C until analysis.

Fecal and urine were collected and handled same to that in Chapter III.

Chemical analysis

The chemical composition of TMR silage and feces was analyzed by the same

methods to the previous chapters. The DM, ash, acid detergent fiber exclusive ash

(ADFom), CP, and ether extracts (EE) analysis were performed according to the

methods of AOAC (2007: protocol number, 930.15; 942.05; 973.18; 990.03 and

920.39, respectively). The organic matter (OM) was calculated as weight loss trough

ashing. The neutral detergent fiber assayed with heat stable α-amylase and exclusive

ash (aNDFom) were measured according to Van Soest et al. (1991). The crude protein

in urine samples were determined by Kjeldahl method (AOAC, 2007).

The fermentation quality of TMR silage was assayed in the same methods to

chapter II. Lactic acid content determined using a commercial kit (D-/L-Lactic Acid

Assay Kit; Megazyme, Wicklow, Ireland). The NH3-N of silage and rumen fluids was

measured by indophenol method (Weatherburn 1967). As a silage quality index, Fleig

point was calculated as the following equation (Denek and Can, 2006).

Fleig points = 220 + (2 × DM% 15) – 40 × pH

An automated clinical chemistry analyzer (DRI-CHEM 4000V, FUJIFILM

Corporation, Tokyo) was used to determined serum glucose, urea, total protein,

triglyceride and cholesterol.

In vitro Methane production

The methane emission was analyzed by in vitro method according to Matsui et

al. (2013). Rumen fluid was collected from three canullated Suffolk sheep before

feeding to minimalize individual effects. Sudan grass hay and concentrate were fed to

the sheep at the maintenance level and equal amount of the feed was offered in the

morning and evening. The rumen fluid were collected before morning feeding then

filtered with four layers cheesecloths and mixed with anaerobic pre-warmed

McDougal buffer (2:1, v/v). A 50 ml of this mixture was mixed with one gram of

sample in a 50 ml of anaerobic glass veil which tightly sealed using a butyl rubber

stopper and an aluminum cap. Each treatment has three replications. The glass veils

incubated in shaking (150 rpm) water bath at 39°C for 24 hours. Bottle with no

substrate were also incubated for calculating methane production.

The cumulative gas volume produced during fermentation was measured at 24

h after incubation, using a glass syringe fitted with a 23-G needle via a Luer-lock 3-

way stopcock. The headspace gas composition was determined using a gas

chromatograph (GC–14A, Shimadzu, Kyoto, Japan; column: ULBON HR-20M, 0.25

mmI.D. × 30 mL 0.25 μm). Helium gas was used as the carrier gas at a 50 mL min -1

flow rate. The temperature of the injection port was 200°C. The detector temperature

was 200°C, whereas the column temperature was 170°C. A 0.5-mL aliquot of

headspace gas was injected by a gas-tight syringe. Methane gas volume in milliliters

was calculated as described by Lopez and Newbold (2007).

Each incubated sample was transferred to a glass filter to obtain residue. The

residue was dried in a drying oven at 80°C for 48 h and then weighed calculating in

vitro dry matter digestibility. Methane per digested dry matter (DDM) was calculated

by dividing methane production with DDM mass (Abrar et al., 2016).

Energy utilization calculation

The gross energy (GE) concentration of the samples was determined with a

bomb calorimeter (CA-4PJ, Shimadzu Corp., Kyoto, Japan). Digestible energy (DE)

was computed from difference gross energy intake and fecal energy. Metabolizable

energy (ME) was computed from DE minus the urinary energy (UE) and energy

losses from methane. Methane production was estimated by the following equation

according to Blaxter and Clapperton (1965).

CH4 = 3.67 + 0.062D

Where D is the apparent digestibility of the energy of the feed.

Statistical analysis

All obtained data were analyzed using R programming 3.3.2 (R development

core team, 2016). The fermentation quality, chemical composition and in vitro

methane production of TMR silage were analyzed by one-way analysis of variance

(ANOVA). Data on feed intake, digestibility, blood parameters, and energy balance

were analyzed using the following mathematical model. Yijkl = μ + Ti + Pj + Ak +

eijkl, where Yijk = observation, μ = the overall means, Ti = the fixed effect silage

additive treatment, Pj = the fixed effect of period, Ak = the random effect of animal

and eijkl = residual error. The mean of silage additive treatment was compared using

pairwise t test. The Bonferroni correction was used to detect the differences between

the means for each data analysis.

IV-3. Results

The Fermentation quality and chemical composition of TMR ensilaged with

FJLB are shown in Table 4.3. All the parameters observed in fermentation quality

were not affected by silage additives (P>0.05), except for acetic acid concentration.

The COM and MIX treatment has higher acetic acid concentration than CON

treatment (P<0.01). The chemical composition of TMR silage were not affected by

the silage additives (P>0.05). However, the DM content in FJLB treatment tended to

be higher than that in COM treatment (P=0.06).

In Table 4.4, effect of different silage additives in TMR silage prepared from

agriculture by-product on in vivo nutrient digestibility and in vitro methane

production are presented. The DM, CP, aNDFom and ADF digestibility were not

affected by the silage additives (P>0.05). The in vitro methane production was also

unaffected (P>0.05) by the treatments. Blood parameters of ewes fed TMR silage are

presented in Table 4.5. There were no significant differences among the treatments on

total protein, triglyceride, cholesterol, glucose and blood urea nitrogen in ewes. It

suggested that there was no impact of silage additive on blood parameters. Table 4.6

shows the energy utilization of ewes fed TMR silage with different silage additive.

The silage additives did not show clear effect (P>0.05) on the GE intake, FE, DE,

energy digestibility, UE, CH4 energy, ME and metabolizability in ewes.

IV-4. Discussion

Silage fermentation quality

The Fleig point in all silages was over 97.99, indicating that all TMR silages

in the present experiment have very good quality fermentation (Denek and Can,

2006). This might be caused by the material that was used in this experiment. Wet

type of food by-products including tofu waste and brewer grain contained LAB that

contributed to produce organic acid and then depressing the pH value. Actually,

Tanaka et al. (2001) reported that before fermentation tofu cake and brewer grain

contained LAB 9 × 107 and 6.4 × 107 cfu/g, respectively. With these numbers, it

would be sufficient to produce lactic acid and lowering pH to achieve good

fermentation quality because well preserved silage will be obtained when LAB level

reaches 105 cfu/g (Cao et al., 2016).

Acetic acid in the COM and MIX treatment showed higher than that in CON

treatment. LAB in COM additives were Lactococcus lactis that generally considered

as homofermentative LAB (McDonald et al., 1991; Gallo et al., 2018) and

Lactobacillus paracasei as well (Nkosi et al., 2010). However according to Gollomb

and Marco (2015), L. lactis possesses an enzyme required for heterofermentative

metabolism. It suggested that this microorganism contributes to the high acetic acid

production in COM treatment. Acetic acid has been known increases the aerobic

stability in silage (Danner et al., 2003) because it could inhibit the growth of acid-

tolerant yeast (Gallo et al., 2018). Li et al. (2016) found that increasing in acetic acid

can suppress fungal growth in silage inoculated with heterofermentative LAB. Other

parameters in silage fermentation quality did not differ among the treatment. Again,

this was because non-additive treatment also contained sufficient LAB for getting

favorable fermentation.

Table 4.3. Fermentation quality and chem

ical composition of TM

R ensilaged w

ith fermented juice of epiphytic lactic acid bacteria

Item

Treatment

P-value C

ON

FJLB

C

OM

M

IX

Fermentation quality

pH

4.58 ± 0.13

4.41 ± 0.13 4.25 ± 0.10

4.33 ± 0.10 0.303

Lactic acid (g/kg DM

) 31.51 ± 3.40

37.43 ± 3.78 37.80 ± 4.36

40.15 ± 2 0.379

VFA

(g/kg DM

)

Acetic acid

13.22 ± 2.88b

16.72 ± 0.78ab

21.46 ± 1.59a

24.22 ± 1.21a

0.005 Propionic acid

0.89 ± 0.34 0.92 ± 0.27

0.96 ± 0.31

1.08 ± 0.36 0.976

Butyric acid

0.46 ± 0.09 0.27 ± 0.10

0.42 ± 0.15 0.1 ± 0.06

0.125 N

H3 -N

(g/kg DM

) 0.18 ± 0.01

0.15 ± 0.01 0.19 ± 0.02

0.18 ± 0.03 0.217

Fleig point 97.99 ± 5.55

106.23 ± 4.86 109.35 ± 3.03

108.52 ± 3.94 0.301

Chem

ical composition

D

M

38.0 ± 0.33 38.8 ± 0.44

37.1 ± 0.45 38.4 ± 0.35

0.055 C

P (%D

M)

12.5 ± 0.16 12.9 ± 0.28

13.2 ± 0.20 13.1 ± 0.31

0.227 aN

DFom

(%D

M)

48.9 ± 1.08 47.6 ± 1.41

47.7 ± 0.74 46.9 ± 0.34

0.581 A

DFom

(%D

M)

30.5 ± 0.80 30.4 ± 0.74

30.3 ± 0.57 30.0 ± 0.36

0.946 EE (%

DM

) 4.55 ± 0.16

4.73 ± 0.20 4.84 ± 0.08

4.82 ± 0.12 0.412

Crude A

sh (%D

M)

8.2 ± 0.27 8.3 ± 0.18

8.2 ± 0.21 8.3 ± 0.07

0.938 C

ON

: control (no additive added), FJLB: ferm

ented juice of epiphytic lactic acid bacteria additive, CO

M: C

omm

ercial silage additive, MIX

: FJLB

+CO

M, V

FA: volatile fatty acid, N

H3 -N

: amm

onia-nitrogen, CP: crude protein, aN

DFom

: α-amylase neutral detergent fiber exclusive ash,

AD

Fom: acid detergent fiber exclusive ash, EE: ether extract, D

M: dry m

atter. The values with different superscript w

ithin the same row

are significantly different at 5%

level. Values are m

ean ± standard error.

Table 4.4. Effect of different silage additives in TMR

silage prepared from agriculture by-product on in vivo nutrient digestibility and in vitro

methane production

Parameters

CO

N

FJLB

CO

M

MIX

P-value

In vivo nutrient digestibility (%)

D

MD

69.5 ± 1.68

67.4 ± 2.24 68.3 ± 3.13

67.4 ± 3.39 0.763

CPD

69.3 ±1.72

67.9 ± 2.09 71.8 ± 3.07

67.2 ± 3.52 0.925

aND

Fom

66.1 ± 2.28 62.4 ± 2.92

61.3 ± 5.49 62.2 ± 4.09

0.568 A

DF

64.5 ± 1.34 60.6 ± 2.67

61.1 ± 4.79 61.6 ± 3.03

0.473 In vitro

C

H4 production (m

L/g DM

) 22.1 ± 0.42

21.0 ± 0.58 20.6 ± 0.42

20.7 ± 0.46 0.151

DM

D (%

) 30.9 ± 1.87

33.2 ± 3.03 32.6 ± 3.62

33.0 ± 3.25 0.944

OM

D (%

) 24.8 ± 1.98

27.1 ± 3.37 26.6 ± 4.01

27.0 ± 3.5 0.956

CH

4/DM

D

72.0 ± 4.98 64.4 ± 4.49

65.4 ± 7.56 64.3 ± 6.56

0.768 C

H4/O

MD

98.6 ± 9.20

87.5 ± 8.97 91.6 ± 16.87

88.0 ± 11.97 0.910

DM

: dry matter; D

MD

: dry matter digestibility; O

MD

: organic matter digestibility;

Values are m

ean ± standard error

Table 4.5. Serum param

eters of ewes fed TM

R silage w

ith different silage additive

Values are m

ean ± standard error

Parameters

Treatment

P-value C

ON

FJLB

C

OM

M

IX

Glucose (m

g/dL) 60.6 ± 2.40

57.4 ± 2.90 53.3 ± 3.19

55.0 ± 6.12 0.686

Cholesterol (m

g/dL) 110.0 ± 11.24

102.4 ± 6.44 92.3 ± 26.38

90.4 ± 9.65 0.584

Triglyceride (mg/dL)

35.9 ± 8.53 28.9 ± 7.87

26.8 ± 9.34 25.5 ± 7.01

0.374 Total protein (g/dL)

6.0 ± 0.51 5.8 ± 0.35

5.3 ± 0.35 5.5 ± 0.59

0.564 U

rea nitrogen (mg/dL)

20.2 ± 2.19 21.3 ± 3.28

17.9 ± 2.88 20.0 ± 2.64

0.459

Table 4.6. Effect of different silage additives in TMR

silage prepared from agriculture by-product on energy utilization in ew

es Param

eters C

ON

FJLB

C

OM

M

IX

P-value

DM

I (g/kg BW

0.75/day) 48.3 ± 3.25

45.9 ± 3.37 39.5 ± 5.19

44.2 ± 1.9 0.404

GE intake (M

J/kg BW

0.75/day) 0.9 ± 0.06

0.9 ± 0.06 0.8 ± 0.07

0.8 ± 0.04 0.415

FE (MJ/kg B

W0.75/day)

0.3 ± 0.03 0.3 ± 0.03

0.2 ± 0.04 0.2 ± 0.02

0.638 D

E (MJ/ kg B

W0.75/day)

0.6 ± 0.03 0.6 ± 0.03

0.5 ± 0.04 0.6 ± 0.05

0.663 Energy digestibility (%

) 71.6 ± 1.55

69.8 ± 1.85 70.5 ± 2.85

69.9 ± 3.21 0.897

UE (M

J/kg BW

0.75/day) 0.03 ± 0.003

0.03 ± 0.003 0.04 ± 0.014

0.03 ± 0.05 0.822

CH

4 energy (MJ/100 M

J GEI)

8.1 ± 0.09 8.0 ± 0.11

8.0 ± 0.17 8.0 ± 0.19

0.912 C

H4 energy (M

J/kg BW

0.75/day) 0.07 ± 0.004

0.07 ± 0.004 0.06 ± 0.005

0.07 ± 0.004 0.786

ME (M

J/BW

0.75/day) 0.5 ± 0.03

0.5 ± 0.02 0.4 ± 0.03

0.5 ± 0.05 0.490

Energy metabolizability (%

) 59.4 ± 1.48

57.5 ± 1.68 57.0 ± 3.93

58.4 ± 3.45 0.880

DM

I: dry matter intake; G

E: gross energy; FE: fecal energy; DE: digestible energy; U

E: urine energy; M

E: metabolizable energy.

Digestibility and methane production

Some studies reported the effect of FJLB on nutrient digestibility in ruminant.

The application of FJLB improved CP digestibility of ruzigrass silage in cows

(Bureenok et al., 2011). Yahaya et al. (2004) also reported in vitro DM and NDF

digestibility were improved in tropical elephant grass. Similarly, fibrous component

clearly improved in whole crop rice ensilaged with FJLB (Takahashi et al., 2005). In

contrast, there were no improvement in nutrients digestibility in the present study. The

similar result among the treatment on nutrient digestibility might be related to the

similar quality of TMR silage as shown in Table 4.3. This result is in line with the

previous reports that added FJLB as silage additive in Napier grass silage did not

improve the nutrient digestibility (Bureenok et al., 2012) and in vitro DM digestibility

of rice straw were not improved by the addition of LAB inoculant from incubated

king grass extract (Santoso et al., 2014). Those different result suggested that

different of material for preparing FJLB, the dose of FJLB and the materials of silage

will affect the digestibility in both in vitro and in vivo assessment.

In vitro methane production did not differ among the treatment. There was

limited information on the effect of FJLB application on ruminal methane production.

Cao et al. (2009a) reported that supplementation with lactic acid bacteria reduced the

methane production per in vitro digestible DM on TMR silage prepared from whole

crop rice, commercial concentrate, tofu cake, rice bran, and green tea. Similarly, in

vitro ruminal methane production was decreased 8.6% by adding LAB (Lactobacillus

plantarum Chikuso-1) in TMR with whole crop rice (Cao et al. 2010a). They suggest

that the higher lactic acid in the fermented TMR is the key factor on producing

ruminal propionic acid, then accordingly, lowered methane production (Cao et al.,

2012). In the present study, all silage has similar lactic acid concentration; thus, the

methane production was not different among the treatments.

Blood parameters

The FJLB, COM and MIX treatment in the present study did not affect serum

glucose, cholesterol, triglyceride, total protein and urea nitrogen. The absence of any

effect of silage additive on blood parameters in the present study could be due to

similar qualities of TMR silages that lead to similarities in nutrient digestibility and

uptake.

The normal range of blood glucose value in sheep is considered 25-50 mg/dL

at any time of day (Reid, 1950). However, Marcias-Cruz et al. (2014) reported that

blood glucose level in ewe lamb were ranged 79-84 mg/dL. In the present study, the

blood glucose level ranged 53.3-60.6 mg/dL. Although, low and high levels of blood

glucose suggest energy deficiency of animals, the present result indicated that the

energy supply was sufficient to meet the ewe’s requirements.

The total cholesterol was not affected by the silage additives. In general,

normal value of blood cholesterol is 52-76 mg/dL (Kaneko et al., 1983). The values

of blood cholesterol in the present study (90.4-110.0 mg/dL) were higher than normal

range. This high level of cholesterol in the present study might be due to the high

content of EE (4.55-4.82% DM) in the TMR silage. An increase in the plasma

cholesterol level is necessary to support the transport of large circulating quantities of

PUFA and total lipids (Rufino et al., 2018). Thus, this relatively high EE in the

present study contributes to the metabolism of EE metabolites (Cônsolo et al., 2017).

Serum total protein were also not affected by the treatments. The normal value

of serum total protein value in ruminant is 6.0-7.9 g/dL (Kaneko et al., 1983). The

serum total protein in the silage additive treatments were slightly lower than the

normal value. The lower in serum total protein reflects a fast rate of ruminal

degradation of crude protein, and decreased quantities of protein available to absorb

in the small intestine lead to low protein absorption into blood (Przemyslaw et al.,

2015). The higher rumen degradable protein may attribute to higher N losses in the

urine then lead to decrease in N retention (Singh et al., 2015). It suggested that TMR

silage in this study has more degradable protein in the rumen and less protein

absorption the digestion tract afterwards. However, this relatively low total protein is

not negative for animal health.

Serum urea nitrogen in present study were ranged 17.9-20.2 mg/dL. There

were no silage additive effects on serum urea nitrogen. Blood urea nitrogen

concentration were closely related to dietary CP concentration (Koike et al., 2010;

Aliyu et al., 2012). Specifically, degradable protein produces excess levels of urea in

the rumen. In the present study, tofu waste in the TMR silage includes relatively high

level of degradable protein. This might lead to the higher serum urea nitrogen.

Although appropriate serum urea nitrogen level is not known in ewes, Ferguson et al.

(1993) reported that under 20 mg/dl in serum urea nitrogen did not have negative

effect on reproduction of dairy cows.

Regarding the blood parameters result, the energy supply was sufficient to

meet the ewe’s requirements. However, there might be no positive effect on nitrogen

retention.

Energy balance

GE intake was similar in ewes fed TMR silage with different silage additive.

This result might be caused by the similar quality of TMR silage in all treatment and

suggests that all silage has similar palatability. The FE loss was not different in all

silage additive treatment. These similar values of FE loss were likely due to similar

DMI because FE loss is associated with DMI (Hales et al., 2015). Accordingly, the

DE and the proportion of DE to GE were not different among the treatments. This is

because similar chemical composition in all TMR silages led to have a similar rumen

degradation and nutrient absorption in the intestinal tract.

Methane emission in ruminants is greatly affected by crude fiber content in

DM intake. Due to no statistical difference in aNDFom and NDFom intake, the

methane emissions did not different among the treatments. This result was in

accordance with the result of in vitro methane production in the previous section.

Predicted energy losses as methane in this study was 0.08 of GE intake, and this value

agreed with the value of predicted methane in Adesogan et al. (1998) that often

quoted for feeds at maintenance. An in vivo study by Chuntrakort et al. (2014) in

cattle fed rice straw based diet contained cassava chip and soybean meal found the

methane emissions was 0.102 of GE intake. Whereas in finishing beef cattle offered

maize silage was 0.073-0.084 of GE intake (Mc Geough et al., 2010). This result

implied that TMR silage prepared from agriculture by-products does not have any

adverse effect on methane emission.

In conclusion, TMR silage prepared from agricultural by-product and wet food

by-product is an available way to make good quality of silage. FJLB did not affect

effectively on TMR that contained original lactic acid bacteria. From the results of

blood parameters and energy utilization in the present study, it can be concluded that

TMR silage prepared from agriculture by-products with FJLB has no negative effect

on animal health, methane emission and energy utilization. The FJLB did not improve

methane emission and energy utilization in sheep.

IV-5. Summary

This aim of this study was to determine the effect of total mixed ration (TMR)

ensiled with fermented juice of epiphytic lactic acid bacteria (FJLB) on fermentation

quality, in vitro methane production, blood parameters and energy utilization in ewes.

TMR was prepared from rice straw, corn stover, brewer grain, tofu waste, steam

flaked corn, and a mineral-vitamin mixture. The treatments consisted of silage

additives added to TMR: CON (no silage additive), FJLB (fermented juice of

epiphytic lactic acid bacteria), COM (commercial additive), and MIX (FJLB + COM).

Four cannulated ewes were assigned into 4 × 4 Latin square design. The chemical

composition of TMR silage were not affected by the silage additives (P>0.05).

However, the DM content in FJLB treatment tended to be higher than that in COM

treatment (P=0.06). The COM and MIX treatment has higher acetic acid

concentration than CON (P<0.01). The dry matter digestibility (DMD), crude protein

digestibility (CPD), aNDFom and ADF digestibility were not affected by the silage

additives (P>0.05). The in vitro methane production was also unaffected (P>0.05) by

the treatments. There were no significance differences among the treatments on blood

total protein, triglyceride, cholesterol, glucose and urea nitrogen in ewes (P>0.05).

The silage additives did not have clear effect (P>0.05) on the gross energy intake

(GEI), fecal energy (FE), digestible energy (DE), urine energy (UE), CH4 energy,

metabolizable energy (ME) and energy metabolizability in ewes. It can be concluded

that FJLB used in this experiment has no negative effect on animal health, methane

emission and energy utilization in ewes. Thus, adding FJLB to the TMR silage did

not show further improvement of the silage, methane emission and energy utilization

in ewes.

CHAPTER V

General Discussion

The use of FJLB as a silage additive has been successfully on increasing the

fermentation quality in some grasses. However, there were limited information about

the application of this silage additive on TMR that prepared from agriculture by-

product and food by-product. Generally, agricultural by-products contain less

nutrition than common forages. Using FJLB made of local LAB may contribute to

improve the fermentation quality of the TMR silage comparing with common forages.

We hypothesized that FJLB will improve the fermentation quality of TMR silage, and

increase the animal production. Therefore, this study has been conducted to determine

the effect of FJLB on improving the utilization of agriculture by-product in the form

of TMR through assessment on fermentation quality, nutrient digestibility, rumen

fermentation status, nitrogen balance, methane emission and energy utilization.

Fermentation quality of TMR silage

In Chapter II, showed that addition of FJLB to TMR silage from agriculture

by-product has better fermentation quality than COM and CON proven by lower pH,

higher lactic acid, lower dry matter (DM) losses and higher Fleig point. Whereas in

Chapter III and IV, the FJLB treatment has similar fermentation quality to CON

treatment. Moreover, according to the score of Fleig point, all silage categorized as

very good quality silage. The different result in Chapter II and in Chapter III and IV

supposed caused by the different DM content of material used as ingredient of TMR

silage. In Chapter II, all material used was in dry type that might less contained

original LAB than in Chapter III and IV. Considering the results of this study, food

by-products that contained a decent amount of original LAB contribute improvement

of silage quality. Although, FJLB and other silage additives may have positive effect

on silage quality, combining agricultural and food by-product with the form of TMR

silage may useful for improving utilization of agricultural by-products as a feed for

ruminants.

Nutrient intake, digestibility and rumen fermentation

The DM, OM, CP, aNDFom, ADFom and EE intake in Chapter III were not

affected by the silage additive treatment in TMR silage prepared from agricultural by-

product, suggesting that all TMR silage in this study has similar palatability. The

apparent digestibility of DM, OM, CP, aNDFom, ADFom and EE were also not

affected by the silage additive treatments in Chapter III and IV as well. It might be

explained that in the present study all treated silages were well preserved, including

CON treatment.

The silage additive treatments did not affect ammonia-nitrogen concentrations

in the rumen, but the values were more than minimum level, indicating it sufficient

for microbial protein synthesis. Thus, combining agricultural and food by-products

with the form of TMR silage is effective to supply enough nitrogen to the rumen. The

silage additive treatments did not affect VFA concentrations in the rumen, although

the total VFA concentrations were in normal range. However, the level of intake was

nearly maintenance level in the present study. Several other studies suggested that

higher intake in the silage with FJLB increased total VFA concentration. Thus, further

investigation is needed whether the TMR with FJLB treatment improve ruminal

fermentation.

Nitrogen balance, methane emission and Energy utilization

Similar N intake, excretion and retention regardless the treatment has been

shown in Chapter III. The similar N balance due to the same quality of silages did not

influence on the rumen microbial protein synthesis and on the digestion and

utilization of protein. The no difference in serum total protein corroborated the result

of N balance.

The in vitro methane production was also unaffected by the treatments. Again,

all silages in the present study produced relatively high lactic acid and was good

fermentation quality. The higher lactic acid in the fermented TMR is the key factor on

producing ruminal propionic acid, and then, lowered methane production. Thus, it can

be explained that as all silage has similar lactic acid concentration, the methane

production was not different among the treatments.

There were no any effects detected on energy utilization in ewes fed TMR

silage with different silage additive. It might be caused by the similar quality of TMR

silage in all treatment. The blood glucose value also supply the energy in a sufficient

level to meet the ewe’s requirement. Predicted energy losses as methane in this study

was 0.08 of GE intake, and this value agreed with the value of normal methane

emission at maintenance. TMR silage prepared from agriculture by-products does not

have any adverse effect on methane emission and energy utilization. The FJLB did

not improve methane emission and energy utilization in sheep which means contrary

to the hypothesis.

Overall, the application of 1% of FM FJLB in TMR silage prepared from

agricultural by-product improved the fermentation quality when the material is dry

type, which might be less contain original LAB. However, the FJLB effect was not

clear when applying in wet type of material, such as corn stover silage, wet brewer

grain and wet tofu waste. Although the similar qualities of TMR silage will not

sufficient enough to improve the animal production, the results imply that

combination of agricultural and food by-products which contains original LAB

improve fermentation quality of TMR silage. Further investigation is needed in

increasing the doses of FJLB used and the also by using the difference combination

material for TMR silage other than wet type feed.

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ACKNOWLEDGEMENTS

First of all, I grateful to The Almighty Allah for establishing me to complete

this study.

I would like to thank Directorate General of Resources for Research,

Technology and Higher Education, Republic of Indonesia for granting doctoral

scholarship trough Beasiswa Pendidikan Pascasarjana Luar Negeri (BPLN

101.21/E4.4/2015).

I would like to express my sincere gratitude to my supervisor, Professor

Masato YAYOTA for giving me an opportunity to study in his laboratory, and for his

valuable guidance, constructive advice and excellent supervision from the beginning

to completion of my study.

I wish to express my deepest appreciation to Professor Akemi YAMAMOTO,

for her helpful suggestion, comments and correction throughout my study.

I wish to express my sincere gratitude to Professor Tomohiro SASANAMI,

Laboratory of Cell Biology, Shizuoka University for his helpful suggestion and kind

advice during my study.

I wish to thanks to all the student member of Animal Nutrition Laboratory,

whose were kindly helping and supporting my experiment. Their encouragement,

assistance and hospitality during my stay in Japan are unforgettable.

I wish to express my thanks to my mother for continuing mental support

during my study. Especially I wish to thanks to my husband and my son for their

understanding, encouragement and loves during the completion of my study. I also

wish to thanks to my brothers, my sisters and my friends for continuing support

throughout my study.