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1

INFLUENCE OF NITRATE AND SULPHATE FEEDING ON ENTERIC

METHANE PRODUCTION IN SHEEP AND GOATS

By

MUHAMMAD ARIF

M.Sc (Hons) Animal Nutrition

A Dissertation submitted in partial fulfillment of the requirements for the

degree of

PhD

In

ANIMAL NUTRITION

2

Institute of Animal Sciences

Faculty of Animal Husbandry

University of Agriculture, Faisalabad

Pakistan

2015

To

The Controller of Examinations

University of Agriculture

Faisalabad

We, the Supervisory Committee, certify that the contents and form of

dissertation submitted by Mr. Muhammad Arif, Regd. No. 89-ag-831, have

been found satisfactory and recommend that it be processed for evaluation by

External Examiner(s) for the award of degree.

SUPERVISORY COMMITTEE

1)

Dr. Muhammad Sarwar

Chairman:

__________________

2)

Dr. Zafar Hayat

Co-Supervisor:

__________________

3

3)

Dr. Mahr un Nisa

Member:

__________________

4)

Dr. Muhammad Younas

Member:

__________________

4

Declaration

I hereby declare that the contents of thesis, title of thesis "Influence of nitrate and sulphate

feeding on enteric methane production in sheep and goats" are product of my own

research and no part has been copied from any published source (except the reference,

standard mathematical or genetic models/formulae/ protocols etc.). I further declare that this

work has not been submitted for award of any other diploma/degree. The University may take

action if information provided is found inaccurate at any stage.

______________

Muhammad Arif

89-ag-831

5

ACKNOWLEDGEMENT

I am thankful to Allah Almighty, the most Gracious and the most Beneficent, who

enabled me to accomplish this work. I am thankful to Muhammad (Peace be upon him) who

spread the message of Allah to enlighten our hearts and brains.

I would like to thank my supervisor, Professor Dr. Muhammad Sarwar (T.I,

Distinguished National Professor) for the patient guidance, encouragement and advice he has

provided throughout my time as his student.

I am also obliged to my supervisory committee for their cooperation during my

Ph.D study. I would like to thank University of Sargodha, Pakistan for awarding me

Scholarship for Ph.D. Studies under Faculty Development Program.

I pay thanks to all other staff and colleagues in the faculty and especially that of

Institute of animal nutrition and feed technology that helped me during my stay at the

campus. I am also thankful to my family that suffered much due to my studies.

6

Contents Page#

ACKNOWLEDGEMENT 6

TABLES OF CONTENTS iv

LIST OF TABLES vi

LIST OF FIGURES ix

ABSTRACT 1

Chapter 1 INTRODUCTION 2

Chapter 2 REVIEW OF LITERATURE 5

Global warming and climate change 5

Role of livestock in Global warming 11

Mitigation of enteric CH4 emission in ruminants 16

Improved feeding practices 16

Animal breeding and management changes 17

Immunisation 17

Bacteriophages and bacteriocins 18

Defaunation 18

Plant secondary compounds 19

Dietary supplements 20

Nitrate feeding 26

Nitrate and Sulphate feeding 28

Dietary Nitrate Adaptations 28

Effect of dietary nitrate on feed intake, growth

performance and rumen ecology

29

Measuring methane production 29

Chapter 3 MATERIAL AND METHODS 33

Experiment 1 33

Experiment 2 33

Experiment 3 34

Chapter 4 Effect of varying levels of potassium nitrate with or without

sulphur on enteric methane production in post weaned Lohi

sheep and Teddy goats

38

Abstract 38

Introduction 38

Materials and Methods 40

Results 42

Discussion 43

Chapter 5 Effect of different levels of calcium ammonium nitrate with or

without sulphur on enteric methane production in Lohi sheep and Teddy goats at growing age

56

Abstract 56

Introduction 57


7

Results 60

Discussion 61

Chapter 6 Effect of different levels of sodium nitrate with or without

sulphur on enteric methane production in Lohi sheep andTeddy goats at fattening age

72

Abstract 72

Introduction 73


Results 76

Discussion 77

General Conclusions 88

Chapter 7 Summary 89

Literature Cited 91

8

List of Tables Page#

Table 2.1 Important greenhouse gases: their formulae, lifetimes and

global warming potentials

7

Table 2.2 Summary of climate risks by south Asian countries 10

Table 2.3 Agriculture's contribution to global green house gas and other

emissions

13

Table 2.4 Estimated annual enteric methane emissions from the main

domesticated livestock species

15

Table 2.5 Rumen modifiers that lower enteric methane production per

unit animal product

25

Table 2.6 Methane measuring techniques 31

Table 2.7 Gasmet DX4030 (General Specifications) 36

Table 4.1 Ingredient composition of experimental diets 49

Table 4.2 Effect of different levels of potassium nitrate with or without

sulphur on Nutrient intake and their digestibility in post

weaning Lohi sheep and Teddy goat

50


sulphur on blood metabolites in post weaning Lohi sheep and

Teddy goat

51


sulphur on hematology in post weaning Lohi sheep and Teddy

goat

52


sulphur on nitrogen balance in post weaning Lohi sheep and

Teddy goat

53


sulphur on growth performance in post weaning Lohi sheep

and Teddy goat

54


sulphur on enteric methane production in post weaning Lohi

sheep and Teddy goat

55


Table 5.2 Effect of different levels of calcium ammonium nitrate with or

without sulphur on Nutrient intake and their digestibility in

growing Lohi male sheep and Teddy goat

66


without sulphur on blood metabolites in growing Lohi male


67


without sulphur on hematology in growing Lohi male sheep

and Teddy goat

68

Table 5.5 Effect of different levels of calcium ammonium nitrate with or 69

9

without sulphur on nitrogen balance in growing Lohi male



without sulphur on growth performance in growing Lohi male


70


without sulphur on enteric methane production in growing

Lohi male sheep and Teddy goat

71


Table 6.2 Effect of different levels of sodium nitrate with or without

sulphur on Nutrient intake and their digestibility in Lohisheep

and Teddy goat

82


sulphur on blood metabolites in Lohisheep and Teddy goat

83


sulphur on hematology in Lohi sheep and Teddy goat

84


sulphur on nitrogen balance in Lohi sheep and Teddy goat

85


sulphur on growth performance in Lohi sheep and Teddy goat

86


sulphur on enteric methane production in Lohi sheep and

Teddy goat

87

10

List of Figures Page#

Figure 1 Enteric fermentation emission factors in developed countries 8

Figure 2 Enteric fermentation emission factors in developing countries 8

Figure 3 Agriculture growth percentages 9

Figure 4 Livestock population 9

Figure 5 Annual livestock production growth 12

Figure 6 Enteric fermentation (Regional classification) 14

Figure 7 Enteric fermentation (Average global emissions) 14

Figure 8 Break down of global emissions by animal type, averaged over

the period 2000-2010 for enteric fermentation.

15

Figure 9 Pathways for the microbial anaerobic fermentation of glucose

in ruminants (Nolan 1999).

26

11

Abstract

The objective of this study was to examine the effect of different nitrate sources with or

without sulphur (S) in a 2×3×2 factorial arrangement under Randomized Complete Block

Design (RCBD) on growth performance and enteric methane production in Lohi sheep and

Teddy goats. This study had 3 independent experiments. In experiment 1, 48 animals (24

sheep and 24 goats), 3 months old, were randomly divided into 12 groups, 4 animals in each

group. Twelve iso-nitrogenous (crude protein 18%) and iso-caloric (2.0 ME Mcal/Kg) diets

were formulated using 0, 3 and 6% potassium nitrate (PN) with 0.4% S (anhydrous MgSO4)

or without S for both sheep and goats. The experiment lasted for 90 days. Like experiment 1,

experiment 2 and 3 were conducted using ~ 0, 1.5 and 3% calcium ammonium nitrate (CAN)

and ~ 0, 2.5 and 5% sodium nitrate (SN) with 0.4% S (anhydrous MgSO4) or without S,

respectively. The gases were measured by using infra-red biogas analyzer. In experiment 1,

both nutrient intake and digestibility in weaned animals were similar (P> 0.05) across all

diets. Similar results were observed in experiment 2 & 3 on animals at six and nine months of

age respectively. In all three experiments animals fed diet containing 6% PN, 3% CAN and

5% SN with 0.4% S showed better nitrogen balance. Enteric CH4 was 32.6% reduced

(P<0.05) in lambs and 31.9% in goats fed diet containing 6% PN and 0.4% S compared to

those fed control diet (C). Daily live weight gain of of both lambs (146 g/day) and goats (66.0

g/day) fed diet containing 6% PN and 0.4% S was the highest and best values of feed

conversion ratio (FCR) (P<0.05) were also observed in both group of animals. Enteric CH4

was 28.6% reduced (P<0.05) in sheep and 26.9% in goats fed diet containing 3% CAN and

0.4% S compared to those fed C. Each sheep fed diet containing 3% CAN and 0.4% S gained

144 g/day, whereas daily live weight gain by goats fed diet containing 3% CAN with or

without S was 58 g/day. The enteric CH4 was 19.6% reduced (P<0.05) in sheep and 18.2% in

goats fed diet containing 5% SN and 0.4% S compared to those fed C. Daily live weight gain

of both sheep and goats fed diet containing 5% SN and 0.4% S was 143 and 59 g/day,

respectively. The FCR had better values in sheep and goats fed diet containing 5% SN and

0.4% S. In all the three experiments, none of the animal showed any kind of abnormal

behaviour or signs of illness during the whole experimental period. All the nitrate sources

showed similar response with sulphates during various stages of growth in sheep and goats.

In conclusion, animals fed diets containing 6% PN with 0.4% S, 3% CAN with 0.4% S and

5% SN with 0.4% S not only gained more weight but enteric CH4 production was also

reduced in these animals compared to those fed C.

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Chapter 1

Introduction

Ruminants are considered as one of the major sources of greenhouse gases (GHG)

emission which is partially responsible for global warming (CONAM, 2001). The global

warming is considered one of the real issues of this century (FAO, 2006) because the

atmospheric tempretaure is expected to rise between 0.50C and 2.50C by 2030 (Moss et al.,

2000). The United Nations Framework Convention on Climate Change (UNFCCC, 2008)

reported expected rise between 1.80C and 4.00C by 2100. This increased temperature will

melt ice of Arctic and Antarctic poles and raise 17 to 26 cm sea level by 2030 (Moss et al.,

2000).

Ruminants emit about 80 million tonnes of methane (CH4) annually which accounts

for up to one third of the emitted CH4 worldwide (Beauchemin et al. 2008; IPCC, 2007). It is

predicted that enteric CH4 will increase over 30% from 2000 to 2020 (O’Mara, 2011). As

cattle lose 6% of their gross energy intake as CH4 (Johnson and Johnson, 1995) which lowers

animal productive efficiency. Thus, reduction of enteric CH4 would not only enhance the

animal productive efficiency but it will also mitigate environmental pollution (Beauchemin et

al., 2008). Thus, some efforts are required to develop ways and means to reduce CH4 gas

emission from ruminants.

There are many ways through which enteric CH4 can be minimized. They include

rearing of high yielding animals (Kirchgessner et al., 1995), immunisation (Hegarty, 2001),

biological control strategies (Klieve and Hegarty, 1999), various feed additives (Joblin, 1999;

Moss et al., 2000; Lopez et al., 1999; Asanuma et al., 1999; Wenk, 2003) and nutritional

manipulation (Dohme et al., 2000; Johnson and Johnson, 1995; Moss et al., 2000; Itabashi,

1994). These CH4 mitigating strategies can broadly be categorised into breeding and

management changes, usage of feed additives or specific agents and improved feeding

practices (Smith et al., 2008). Breeding and management changes are long term practices but

nutritional manipulation is short term and seems promising. Ruminants eructate 95% of CH4

through mouth and remaining 5% is removed through anus. Term enteric CH4 production

means total amount of CH4 eructated by animal in unit time. The composition of animal feed

has a potential bearing on the amount of enteric CH4 production (Ellis et al., 2008). Animals

in developed countries produce less methane per unit produced than under developed

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countries due to feeding high concentrate rations, improved feed efficiency, better breeding

strategies and good health of animals (Smith et al., 2008).

Sheep and goats are important livestock species of animal agricultural in developing

countries and they play a pivotal role in economic uplifting of any country (Devendra, 2005).

Of the world's 1,614 million sheep and 475 million goats, 65% and 95%, respectively, are

located in developing countries. Small-ruminant population is 53% in developing countries of

Asia, particularly in India and Pakistan, 33% in Africa, and 14% in Latin America (FAO,

1984). In Pakistan the livestock accounts for approximately 55.4% of the agriculture value

added and 11.9% to GDP during 2012-13. There are 28.8 million sheep and 64.9 million

goats in Pakistan which constitute almost 55% of total livestock population in the country

(Fifure 4).

Among different thin tailed sheep breeds in Pakistan Lohi sheep is considered

relatively important with approximately 15% share of total sheep population in the region

(FAO, 1997). Khan et al. (2008) reported that teddy and beetal are two most inhabited goat

breeds in Punjab, Pakistan. Enzose, derived from the enzymatic conversion of corn starch, is

a byproduct of corn milling industry. It contains 85% dextrose, with its pH ranged from 3.5-

4.5. It is a rich source of fermentable carbohydrate and is usually in-expensive (Shahzad et

al., 2009; Sarwar et al., 2004).

In developing countries like Pakistan horizontal expansion of ruminants will result in

more enteric CH4 emissions.The enteric CH4 is produced under anaerobic conditions in the

rumen, by methanogenic Archaea that gain energy by reducing CO2 with H2 to form CH4.

Nitrate could potentially replace urea in low protein diets to provide a source of rumen

ammonia and provide a hydrogen sink to reduce enteric CH4 production (Leng, 2008). On

basal diets low in crude protein, the availability of ruminal ammonia is often a primary

deficiency which limits microbial growth and thus, reduces digestibility and feed intake.

Slow and stepwise inclusion of nitrate in the diet allows the rumen micro flora to adjust and

enhance their capacity to reduce both nitrate and nitrite (Alaboudi and Jones, 1985).

The S supplementation in the diet (Leng, 2008) or cystein (Takahashi et al., 1998) inclusion

might reduce nitrite accumulation in the rumen.

Nitrate being dietary nitrogen source can provide a hydrogen sink to reduce enteric CH4

production in ruminants (Leng, 2008). However, use of nitrate as a source of rumen

14

fermentable N and as an alternative hydrogen sink to carbon dioxide had previously

been neglected due to toxic effects of nitrite which is formed as an intermediate during

the reduction of nitrate to ammonia (Leng and Preston, 2010). It has been suggested that

nitrate reduction to ammonia could be suppressed by feed-back inhibition from high ruminal

ammonia (Leng, 2008). Sulphur supplementation in the diet (Leng, 2008) or cysteine

(Takahashi et al., 1998) inclusion reduced nitrite accumulation in the rumen. Sulphate is a

reductant (ΔG 0 = -21.1 kJ/mol of hydrogen; Ungerfeld and Kohn, 2006) and will also

compete for electrons and may lower CH4 production.

In-vitro gas production technique (IVGPT) has been commonly used to measure the

enteric CH4 production in ruminants (Lovett et al., 2004; Rymer et al., 2005; Pellikaan et al.,

2011; Navarro-Villa et al., 2011; Blummel and Orskov, 1993; Bhatta et al., 2006). This

IVGPT only simulates ruminal feed fermentation and does not take into account emissions

and digestibility by the entire animal. The results obtained regarding the usage of nitrate to

mitigate CH4 from IVGPT can not be directly applied in the entire animal. Thus, the scientific

evidence regarding the use of nitrate with or without S to mitigate in-vivo CH4 production is

limited.

Therefore, the present study was planned to examine the influence of different nitrate

sources with or without sulphur (S) on growth performance and enteric CH4 production in

sheep and goats. The hypothesis was that nitrate and S will mitigate the enteric CH4

production in both sheep and goats. Both nitrate and S will give an additive effect on the CH4

reduction.

15

Chapter 2

Review of Literature

Global warming and climate change

There are many factors which affect global warming and climate change and some of

the most important of them include green house gases (GHG), aerosols (liquid or solid

particles suspended in air), land cover and solar radiation. The GHG arise both from

anthropogenic and natural sources. However, according to Intergovernmental Panel on

Climate Change (IPCC, 2001), the increase in GHG concentration due to anthropogenic

sources is the main concern. The CH4, CO2, N2O, hydro fluorocarbons (HF) perfluorocarbons

(PFC) and SF6 are six GHG (UNFCCC, 2008). Each of the above mentioned gas has a

varying global warming potential (GWP) This GWP of various GHG is shown in table 2.1.

The CH4 has 25X higher warming potential than CO2 (Foster et al., 2007; IPCC, 2007).

Higher concentrations of these GHG in troposphere trap more heat resulting in warmer

atmosphere (Solomon et al., 2007; Table 2.1).

Green House Gases

The CO2, being the potential GHG, is liberated by variety of natural and man made

sources. Its concentration in troposphere has elevated from 280 to 379 ppm in 2005 (Solomon

et al., 2007). The combustion of fossil fuels share approximately 66% and land use change

(LUC) contributes 33% CO2 in earth's environment (Solomon et al., 2007; IPCC, 2001).

Land use change is a term used to measure carbon losses from soil and plants due to soil

cultivation or deforestation. The CO2 also has large sinks. Terrestrial biosphere and oceans

remove 50 to 60% of human induced CO2 from atmosphere (Solomon et al., 2007).

The combustion of fossil fuels, ruminants, rice cultivation, landfills, coal mining

gas/oil drilling are the main anthropogenic sources responsible for CH4 emissions. In

temperate regions, plants release insignificant amount of CH4 while emissions from tropical

rain forests are high (Lower, 2006; kirchban et al. 2006).

The N2O is released both by human and natural activities. Oceans, soils of grasslands,

savannas and forests are the natural N2O emission sources. The electricity generated by fossil

16

fuel combustion, nitric acid, nylon products, livestock urine and agricultural soils are human

induced sources responsible for N2O emissions (IPCC, 2001; Forster et al., 2007). The

natural sources contribute approximately 60% N2O emission and 40% is contributed d by

human activities (Solomon et al., 2007). Forster et al. (2007) reported 18% rise of N2O

concentrations in atmosphere since 1750. Indiscriminate use of fertilizers is responsible for

higher N2O emissions (Nevison and Holland, 1997). Various plant species are responsible for

its release in earth's environment (Hakata et al., 2003). Similarly, the HFC have been utilized

as alternative to hydro CFC and CFC in refrigeration and air conditioning industry and

responsible to increase the global warming effect (Forster et al., 2007).

The CF4 is one of the naturally occurring PFC. It is normally present in very low

concentrations in earth's atmosphere (Khalil et al., 2003). Forster et al. (2007) reported

approximately 50% increase in PFC concentrations since 1960 and it is permanently

contributing towards global warming. The aluminium and electronic chip manufacturing

industry are the main anthropogenic sources of PFC (Khalil et al., 2003).

The SF6 is often used as inert tracer in oceanic or atmospheric studies and is also

utilized as an insulating gas in electric equipment (Forster et al., 2007). Lindley and

Mcculloch (2005) reported very small SF6 emission rates. Contrarily, Forster et al. (2007)

reported increasing concentrations of SF6 in atmosphere.

The global warming is one of the burning issues of the twenty first century (FAO,

2006). It is expected to rise between 0.50C and 2.50C by 2030 (Moss et al., 2000) while

United Nations Framework Convention on Climate Change (2008) reported expected rise

between 1.80C and 4.00C by 2100. Melting of ice of Arctic and Antarctic poles and thermal

expansion of ocean water will result in main sea level rise of 17 to 26 cm by 2030 (Moss et

al., 2000). Pakistan geographically is located in region where the average temperature is

higher than global averages.

In Pakistan the livestock accounts for approximately 55.4% of the agriculture value

added and 11.9% to GDP during 2012-13. There are 28.8 million sheep and 64.9 million

goats in Pakistan which constitute almost 55% of total livestock population in the country

(Figure 3 and 4). Pakistan possesses both arid and semi arid conditions and its 24% land area

receive 250-500 mm and 60% receives 250 mm of annual rain fall. Pakistan contributes a

little towards global GHG emission by sharing only 0.8%. Pakistan is ranked 135 th in global

17

GHG per capita emission. However, Pakistan is the most liable to devastated effects of

climate change and is rated 12th most vulnerable country to climate change. The average

temperature in coastal areas of Pakistan rose from 0.6 to 1.00C since 1900S, resulting in 10 to

15% decrease in precipitation. The temperatures are still elevating and it is expected that this

might reach from 1.8 to 4.00C by the end of this century. In Asia, fresh water availability is

cogitated the most serious threat arising from climate change (Table 2.2)

In 2007-08, total CH4 production from manure and enteric fermentation was 3667.4

teragrams (Tg). Buffaloes contribute 55% and both non-dairy and dairy cattle contribute 24%

of total CH4 emission (Figure 2; Table 2.4).

Table 2.1 Important greenhouse gases: their formulae, lifetimes and global

warming potentials

Chemical species Formula Lifetime (yr)1 100-yr GWP2

Carbon dioxide CO2 50-2003 1

Methane CH4 12 25

Nitrous oxide N2O 114 298

Per fluoro methane CF4 50,000 7,390

HFC CHF3 270 14,8000

Sulphur hexafluoride SF6 3,200 22,800

(Solomon et al., 2007)

1 Global mean lifetime is calculated as the total atmospheric burden divided by mean global

sink of gas in steady state

2 Global warming potentials expressed on a weight basis relative to CO2 over a hundred year

time frame

3 Data from IPCC, 2001

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Figure 1 Enteric fermentation emission factors in developed countries

All estimates + 20%

Source: Default fixed emission factors adopted by IPCC (IPCC, 1996)

Figure 2 Enteric fermentation emission factors in developing countries

All estimates + 20%

Source: Default fixed emission factors adopted by IPCC (IPCC, 1996)

19

Figure 3 Agriculture growth percentages

Source: Adapted from Pakistan economic survey 2012-13

Figure 4 Livestock population

Source: Adapted from Pakistan economic survey 2012-13

20

Table 2.2 Summary of climate risks by south Asian countries

Afghanistan Bangladesh Bhutan India Nepal Pakistan Maldives Sri Lanka

Sea Level rise - High - Modest - Modest High High

Glacier retreat High High High High High High - -

Temperature rise - High High High High High Modest High

Floods more frequent - - Likely High High Likely High -

Drought more frequent Likely High some

areas

- High - Likely - -

Source: Adapted from Symposium on “Changing Environmental Pattern and its impact with Special Focus on Pakistan” by R. K. Anver,

Pakistan Engineering Congress, 2011

http://www.google.com.pk/search?tbo=p&tbm=bks&q=inauthor:%22R.+K.+Anver%22&source=gbs_metadata_r&cad=2

http://www.google.com.pk/search?tbo=p&tbm=bks&q=inauthor:%22Pakistan+Engineering+Congress%22&source=gbs_metadata_r&cad=2

21

Role of livestock in Global warming

Ruminants are considered as one of the major sources of GHG emission. They emit

about 80 million tonnes of CH4 annually which accounts for up to one third of the emitted

CH4 worldwide (Beauchemin et al. 2008; IPCC, 2007). Globally, Asia alone contributes 36%

of the enteric CH4 production (Figure 6). It is predicted that enteric CH4 will increase over

30% from 2000 to 2020 (O’Mara, 2011; Figure 7). The cattle generally lose 6% of their gross

energy intake as CH4 (Johnson and Johnson, 1995) and this indicates that CH4 production

makes livestock production inefficient. Thus, reduction in enteric CH4 would not only

mitigate environmental pollution but it will also enhance the animal productive efficiency

(Beauchemin et al., 2008). Efforts are required to develop the means to reduce CH4 emission

from ruminants which are partially responsible for global warming (CONAM, 2001). This is

a real challenge for animal nutritionists who are expected to develop feasible nutritional

strategy to reduce enteric CH4 emissions (Martin et al., 2008).

Smith et al. (2007) reported that share of agriculture in total global anthropogenic

GHG emission is about 10 to 12%. The total human induced global emissions from

agriculture in 2005 were 5.1 to 6.1 GtCO2-eq/yr and CH4‘s share was 3.3 GtCO2-eq/yr. The

concentration of global agricultural CH4 has risen by 17% for the last 15 years since 2005

(US-EPA, 2006). According to FAO (2009), global share of livestock is 36% of the gross

value of agriculture. The enteric CH4 production is considered one of the largest sources

contributing 32% of total non-CO2 emissions in 2005 (US-EPA, 2006).

World food requirement will increase 70% by 2050 and meat requirement will be

double by 2030 (FAO, 2009; Figure 5) because of ever increasing human population. It is

predicted that by year 2030, developing countries may constitute 85% of the world's

population. The population explosion in developing countries will raise the demand for meat

and milk (Figure 8) and their respective annual growth rate will be 2.5 and 2.4% from 2001

to 2030 (FAO, 2006).

22

Figure 5 Annual livestock production growth

Source: Adapted from World agriculture towards 2030/2050: the 2012 revision

(http://www.fao.org/docrep/016/ap106e/ap106e.pdf)

23

Table 2.3 Agriculture's contribution to global green house gas and other emissions

Gas Carbondioxide Methane Nitrous oxide Nitric oxide Ammonia

Main effects Climate change Climate change Climate change Acidification Acidification &

eutrophication

Agricultural source

(estimated %

contribution to total

global emissions)

Land use change

especially

deforestation

Ruminants (15)

Rice production (11)

Biomas burning (7)

Livestock (including

manure applied to

farmland) (17)

Mineral fertilizers (8)

Biomas burning (3)

Biomas burning (13)

Manure &

mineral fertilizers (2)

Livestock (including manure

applied to farmland) (44)

Mineral fertilizers (17)

Biomas burning (11)

Agricultural

emmisions as % of

total

anththropogenic

sources

15

49

66

27

93

Expected changes in

agricultural

emission to 2030

Stable or

declining

From rice:

Stable or Declining

From livestock:

rising by 60 %

35-60% increase From livestock:

rising by 60 %

Source FAO (2002)

24

Figure 6 Enteric fermentation (Regional classification)

Figure 7 Enteric fermentation (Average global emissions)

Percent average contributions to global emissions (MtCO2eq yr-1)

Source of Figure 6 & 7: The FAOSTAT database of greenhouse gas emissions from

agriculture by Francesco N Tubiello et al, 2013 Environ. Res. Lett. 8 (2013) 015009

doi:10.1088/1748-9326/8/1/015009

25

Figure 8 Break down of global emissions by animal type, averaged over the period 2000-

2010 for enteric fermentation.

Source: Adapted from Williams et al., 2008

Table 2.4 Estimated annual enteric methane emissions from the main

domesticated livestock species

Methane emissions

(kg CH4 animal-1 year-1)

Assumed average

body weight (kg)

Methane emission

g Kg BW-1year-1

Ruminants

Dairy cows 90 600 150

Beef cattle 65 400 163

Sheep 8 50 160

Goats 8 50 160

Non-ruminants

Swine 1 80 13

Poultry < 0.1 2 -

Horses 18 600 30

Source: Adapted from Sauvant, 1993

26

Mitigation of enteric CH4 emission in ruminants

There are many ways through which enteric CH4 can be minimized (Table 2.5). These

include rearing of high yielding animals (Kirchgessner et al., 1995), nutritional manipulation

(Dohme et al., 2000; Johnson and Johnson, 1995; Moss et al., 2000; Itabashi, 1994),

immunisation (Hegarty, 2001), biological control strategies (Klieve and Hegarty, 1999) and

various feed additives (Joblin, 1999; Moss et al., 2000; Lopez et al., 1999; Asanuma et al.,

1999; Wenk, 2003). These CH4 mitigating strategies can broadly be categorised into three

groups i.e. improved feeding practices, management changes and breeding, usage of feed

additives or specific agents (Smith et al., 2008; Soliva et al., 2006; Monteny et al., 2006).

Breeding and management changes are long term practices but nutritional manipulation is

short term and seems promising. The composition of animal feed has a potential bearing on

the amount of enteric CH4 production (Ellis et al., 2008). Animals in developed countries

produce less CH4 per unit produced as compared to the underdeveloped countries (Figure 1 &

2) due to feeding high concentrate rations, improved feed efficiency, better breeding

strategies and good health of animals (Smith et al., 2008).

Mitigation of enteric CH4 emission in ruminants by any dietary manipulation follows

same basic principle i.e. direct inhibition of methanogenesis, lowering of the hydrogen

production during ruminal fermentation or providing alternative H2 consuming pathways for

its use in the rumen (Martin et al., 2010). The potential of dietary strategies to reduce

CH4 emission by ruminants has been extensively reviewed (Tamminga et al., 2007;

Leng, 2008; Beauchemin et al., 2008; Martin et al., 2010; Eckard et al., 2011;

Grainger et al., 2011). Many chemicals have been used to mitigate CH4 but nitrates and

sulphates are very effective.

Improved feeding practices

The CH4 production in ruminants is also affected by feed type. In ruminants diet forage:

concentrate (F:C) has an impact on ruminal fermentation. The decreased F:C decreases the

acetate:propionate and feeding high concentrate diets might produce less CH4 production as

propionate might be the major alternative H2 sink after CH4 (Beauchemin and McGinn,

2005; Blaxter and Claperton, 1965; Fahey and Berger, 1988; Johnson and Johnson, 1995;

Lovett et al., 2003). When feeding at maintenance energy was offered, a loss of 6–7% of

gross energy intake was observed with forages and this loss was reduced to 2–3% in case of

27

high grain concentrates due to enteric CH4 emissions (Johnson and Johnson, 1995).

Improving pasture quality does not only enhance animal productivity but it also reduces

enteric CH4 in ruminants (Alcock and Hegarty, 2006; McCrabb et al., 1999; Leng, 1991).

Animal breeding and management changes

Improvement in the productive performance of animals through better management

practices and breeding, such as reduction in the number of animals, often results in the

reduction of CH4 output per unit of animal product (Boadi et al., 2004). Kebreab et al. (2007)

has proposed the direct selection of cattle for reduced CH4 production but still, it appears

impractical, because of difficult accurate measurement of CH4 emissions at a level suitable

for breeding programs. Meat producing animals with improved efficiency reach slaughter

weight at a younger age and thus have lower lifetime emissions (Lovett and O’Mara, 2003).

Adaptation of animals to reduced CH4 production can also be improved by employing

better breeding strategies. Many indigenous breeds are already acclimated to their harsh

conditions. However, in developing countries, lack of livestock production technologies

hinders the process of adaptation of animals. Adaptation strategies not only include tolerance

of animals to extreme weather conditions but also their livability, growth and reproductive

efficiency with poor nutrition, parasitic and disease resistance (Hoffmann, 2008). These

adaptation mechanisms include identification and conservation of indigenous breeds

acclimated to locally available feed resources and climatic conditions and improvement in

genetics of local breeds by cross breeding with disease resistance and heat tolerant breeds. If

climate change is faster than natural selection the risk of survival and adaptation of the new

breed becomes greater (Hoffmann, 2008).

Immunisation

Vaccination against methanogens may be another way for inhibiting methanogenesis

(Wright et al., 2004). This is based on the concept of a regular supply of antibodies to rumen

through saliva. McAllister and Newbold (2008) suggested that preparation of a recombinant

vaccine against cell surface proteins might improve the efficiency of vaccination as a CH4

inhibition method. Wright et al. (2004) observed 7.7% enteric CH4 production in sheep

vaccinated against methanogens. New vaccines against methanogenic bacteria are being

developed but are not yet in-vivo effective (Hristov et al., 2013; Williams et al., 2009; Wright

28

et al., 2004). More in-vivo work is required to develop effective vaccines which could abate

enteric CH4 production.

Bacteriophages and bacteriocins

Acetogenesis is one of the better alternatives to methanogenesis (Joblin, 1999). This

could spare from 4 to 15% energy gain to the animal. The reductive acetogenesis can be

accelerated directly by inhibiting methanogens with expected 13 to 15% energy gain (Nollet

et al., 1997). However, thermodynamically, CO2 reduction to acetate is less favorable than

enteric CH4 production (Joblin, 1999). In order to redirect H2 to acetogens instead of

methanogens in the rumen, bacteriocins may be effective. Lytic property of bacteriophages

and viruses infect both methanogenic bacteria and archaea (McAllister and Newbold, 2008)

and thus can be effective in reducing enteric CH4 production. However, Park et al. (2007)

reported that presently there are no bacteriophage which might be rumen methanogens

specific, but different bacteriophage populations are thought to be located in anaerobic

environment as in methanogenic biodigesters. Siphophages (siphoviridae phages) might

infect Methanobacterium, Methanobrevibacter and Methanococcus spp. which are

methanogens yet to be isolated from the rumen (McAllister and Newbold, 2008). Most of the

work done is still at early stages and requires extensive research for the availability of

commercial vaccines.

Defaunation

Protozoa contribute up to 50% of the fibrolytic activity in ruminants (Coleman, 1986).

Ushida et al. (1997) reported that defaunation was associated with mitigation of CH4

emission in ruminants. Hegarty (1999) associated defaunation with lower methanogenesis

due to the symbiotic interaction between protozoa and methanogens. Various effective

defaunating agents tested in-vivo, include lauric acid, coconut oil (Sutton et al., 1983;

Machmuller and Kreuzer, 1999; Hristov et al., 2004, 2009, 2011, Hollmann and Beede, 2012)

and linseed oil (Doreau and Ferlay, 1995). The defaunation in ruminants often results in

lower ruminal NH3 (Eugène et al., 2004) and higher propionate concentration (Bird et

al., 2008). The efficiency of microbial protein (MP) synthesis and its flow to the duodenum

was also improved by defaunation (Eugène et al., 2004). Therefore, the defaunation would

enhance animal performance when diets are deficient in protein. The role of protozoa in

mitigating CH4 production was reinvestigated by McAllister and Newbold (2008) who orally

administered ruminal fluid from faunated adult sheep to lambs and other group was given

29

defaunated ruminal fluid. They reported that lambs receiving defaunated ruminal fluid

emitted 26% less CH4 per kg DM intake than those receiving faunated ruminal fluid. Morgavi

et al. (2010) reported up to 10% CH4 reduction with defaunation while Hristov et al. (2013)

reported no effect of defaunation on methanogens in dairy cows treated with lauric acid. In-

vitro and in-vivo data and short and long-term defaunation experiments present contradictory

results (Ranilla et al., 2003) and the extent of defaunation in reducing methanogenesis is still

ambiguous. Variable responses of defaunation do not make this as a feasible CH4 mitigating

option.

Plant secondary compounds

Many compounds extracted from plants have been screened and utilized for

abatement of CH4 emission from the rumen by altering rumen fermentation. Most studied

compounds include saponins, tannins, essential oils and their active ingredients (Hristov et

al., 2013). Protozoa numbers were affected by use of plant extracts (Broudiscou et al., 2000).

Rhubarb (Rheum palmatum) probably restricts ruminal methanogens (García-González et al.,

2006). Ferme et al. (2007) reported that garlic oil, allyl mercaptan and diallyl disulfide

reduced CH4 production after 17 h of incubation. The in-vitro experiments indicated that

garlic oil decreased CH4 production due to its antimicrobial properties (García-González

et al., 2008, Chaves et al., 2008). Moreover in-vitro studies revealed that Diallyl disulfide

(DADS), the chief constituent of garlic oil, decreased CH4 emissions but it still requires in-

vivo authentication. The ruminal volatile fatty acid (VFA) concentration increased in lactating

cows fed diets containing 5 g garlic essential oil daily but it did not affect animal

performance (Yang et al., 2007). The allicin might have directly inhibited rumen

methanogens (McAllister and Newbold, 2008). In-vitro data indicated that tannins, essential

oils and saponins had the ability to hinder methanogenesis (Calsamiglia et al., 2007, García-

González et al., 2008). The dietary tannins reduced both voluntary feed intake & digestibility

(Grainger et al., 2009; Beauchemin et al., 2008; Min et al., 2003) and because of being toxic

to methanogens, they mitigated enteric CH4 by 13 to 16% on DMI basis (Grainger et al.,

2009; Carulla et al., 2005; Woodward et al., 2004). Lila et al. (2003) in one of the in-vitro

studies observed downward trend of CH4 production when yucca saponins were added to the

incubation medium. Cheeke (2000) reported that saopnin's strong detergent properties

reduced the number of rumen protozoa by disrupting their cell membrane (Cheeke, 2000).

Elimination of protozoa diminished CH4 production by 9 to 25% in-vitro (Newbold et

al.,1995). Significant diminution in the number of protozoa was observed in response to

http://thesaurus.zeemind.com/w/authentication

http://thesaurus.zeemind.com/w/diminution

30

yucca extract (Lovett et al., 2006). Beauchemin et al. (2008) highlighted the fact that

mitigating ability of plant saponins was source dependent. Some are more effective than

others and their use is still not cost effective. These compounds might have reduced the

growth of rumen methanogens and thus reduced CH4 emission in-vitro (Miller and Wolin,

2001). It can be concluded that plant extracts are expensive in mitigating CH4 but their effect

is not consistent and is source dependent.

Dietary supplements

Different compounds have been supplemented in the feed as additives specifically to

reduce CH4 emission from the rumen. The routinely used compounds include ionophores,

probiotics, organic acids, enzymes, halogenated compounds, hormones, fatty acids and other

chemical substances. The response of these dietary supplements is briefly described in Table

2.5.

Ionophores

In modern beef industry, monensin is the most studied and routinely used ionophore.

These have been extensively studied as a CH4 mitigating agent which can provide alternate

hydrogen sink in rumen environment. Ionophores when added to ruminant diets decreased

CH4 production and improved efficiency of feed utilization (Mathison et al. 1998). Guan et

al. (2006) reported that this reduction apparently correlated to an inhibition of ciliate

protozoa numbers in rumen fluid. Hino et al. (1993) reported that monensin depressed the

population of protozoa and cellulolytic bacteria resulting in lower CH4 production. Monensin

is a polyether ionophore which diverts the VFA-pattern in the rumen towards propionate.

This changed VFA pattern provides an alternative hydrogen sink. However, CH4 emission is

not persistent and after 2nd week no reduction in CH4 per unit of intake was observed

(Carmean et al., 1991). Similarly, Odongo et al. (2007), McCaughey et al. (1997) and Sauer

et al. (1998) reported that CH4 mitigation with repeated or prolonged use of ionophores was

not clear. The enteric CH4 can be abated up to 10% if higher doses (24-35 ppm) of

ionophores were used (Van Vugt et al., 2005; McGinn et al., 2004; Sauer et al., 1998).

Hristov et al. (2013) reported that effects of monensin could be enhanced by dietary

modifications and increasing monensin dose. It can be concluded that the overall effect of

ionophore is inconsistent and is dose and energy dependent. Although they have low CH4

mitigating potential, but are still effective.

31

Probiotics

Probiotics are frequently used as feed additive in animal production. Yeast-based

products are more common probiotics used in ruminant nutrition. The use of probiotics for

enteric CH4 abatement in ruminants have not shown significant results (McGinn et al., 2004)

but if specific strains were selected they might have given better results (Newbold et al.,

1996; Newbold and Rode, 2006). Cheng et al. (1988) observed that NH3 was generated

during nitrite reduction by Megasphaera elsdenii. Quantitative increase in microbes capable

of reducing nitrite, reduce both CH4 production and nitrite toxicity (Morgavi et al., 2010).The

Wolinella succinogenes has the ability to reduce nitrate into NH3 with little accumulation of

nitrites (Simon, 2002).The Saccharomyces cerevisiae inclusion in ruminant's diet reduced

CH4 by 10% in-vitro, but long term studies show that CH4 reduction is not consistent

(Mutsvangwa et al., 1992; Martin et al., 1989). Several other studies reported inconsistent

effects of Megasphaera elsdenii on ruminal pH and fermentation (Klieve et al., 2003;

Henning et al., 2010). The use of Candida Kefyr and Lactococcus lactis along with nitrate

supplementation was not effective to control methanogens (Takahashi, 2011). Still more

work is required to identify specific strains which may be effective in reducing enteric CH4

production. The cost effective availability of such products can be a good solution.

Organic acids

Use of organic acids as electron receptors has recently received attention. Among these,

fumarate and malate have been the most studied. In ruminants, these organic acids can

increase propionate production, consuming hydrogen in the process. Reduction of fumaric

acid may provide an alternative electron sink for hydrogen (Boadi et al., 2004). Contradictory

results have been observed in many in-vivo experiments regarding use of these compounds on

CH4 production (Bayaru et al., 2001, Wallace et al., 2006, Kolver and Aspin, 2006, Wood et

al., 2009). The role of fumarate in lowering CH4 production has been investigated both in-

vivo and in-vitro (Asanuma et al., 1999, García Martínez et al., 2005). A lot of variability has

been observed in in-vivo experiments. A few of them (Bayaru et al., 2001, Wallace et al.,

2006) showed decrease in CH4 production while others (McGinn et al., 2004, Kolver and

Aspin, 2006, Beauchemin and McGinn, 2006, McCourt et al., 2008) showed no effects.

Ungerfeld et al. (2007) did meta-analysis of in-vitro studies. They reported that fumarate is

32

often partially converted to propionate, but is also converted to acetate, liberating H2. In batch

culture, fumarate and acrylate produced the most consistent reductions in CH4 emissions. It

was also reported that fumarate was more efficient than acrylate in artificial rumens.

According to their results both fumarate and malate have lower mitigating effect than

nitrates. Kolver et al. (2004) reported that in continuous fermenters with forage as substrate,

fumarate mitigated CH4 production by 38%. Contrarily feeding fumarate (1% of DM intake)

did not reduce CH4 production in growing beef cattle (McGinn et al., 2004; Beauchemin and

McGinn, 2006). O’Mara (2004) showed non-significant reduction in CH4 when 3% malate

was added to the diet of dairy cattle. It appears that it would be uneconomical even if organic

acids are supplemented in diet (Newbold et al., 2005; McAllister and Newbold, 2008). In

contrary to fumarate, malate, acrylate and dicarboxylic acids are not cost effective as a

mitigation agents and show response only at high doses (McAllister and Newbold, 2008). It

can be concluded that long term effect of these compounds is yet to be established. Moreover,

these compounds are expensive to be used as CH4 mitigant.

Enzymes

Enzymes, like cellulases and hemicellulases can be considered as an abatement

strategy. They enhance fiber digestion in ruminants and reduce enteric CH4 production by

28% in-vitro and 9% in-vivo (Beauchemin et al., 2003; Beauchemin et al., 2008). Recently,

Hristov et al. (2013) reported that enzymes may improve digestibility and animal production,

resulting in decreased fermentable organic matter, thus lowering enteric CH4 production in

ruminants. Contrarily, Chung et al. (2012) reported increased CH4 production per unit of

DMI, in lactating cows using exogenous enzymes endoglucanase and xylanase. The use of

enzymes in paper, textile and food processing in large quantities ensures its bulk availability

at cost effective (Beauchemin et al., 2008). It can be concluded that use of enzymes as CH4

mitigating option is not feasible because of their lower affectivity.

Halogenated compounds

These are some chemical compounds with specific inhibitory effect on rumen

microbes. These are halogenated compounds which include bromochloromethane,

chloroform, 2-bromoethane sulfonate and cyclodextrin. These compounds inhibit

methanogenesis by reacting with coenzyme B (cobamine), which functions at the last step of

the methanogenic pathway (Shima et al., 2002; McCrabb et al., 1997; Chalupa, 1977).

33

Bromochloromethane and chloroform are potent halogenated compounds used as enteric CH4

inhibitors. Higher doses can reduce 91% CH4 on DMI basis (Tomkins and Hunter, 2004).

Other researchers (Immig et al. 1996; Lila et al., 2004; Mitsumori et al., 2011; Knight et al.,

2011) reported up to 50% CH4 reductions with these compounds in ruminants. Immig et al.

(1996) suggested that animals should be pre-adapted to these compounds for long term

efficacy. Reduced intake, side effects & transitory effects limit their use as better CH4

mitigant (Van Nevel and Demeyer, 1995; Wolin et al., 1960). There was sudden drop in CH4

production in cows administered chloroform but enteric CH4 production gradually increased

at later stages (Knight et al., 2011). A 60% CH4 reduction was observed in cows (Haisan et

al., 2013) supplemented with 3-nitro oxypropanol whereas 24% was observed in sheep fed

diets with similar compounds (Martinez-Fernandez et al., 2013). Bromochloromethane is a

banned compound therefore cannot be suggested as CH4 mitigant. Moreover, compounds like

chloroform are known carcinogens therefore cannot be adopted. These are effective CH4

mitigants but because of hazards regarding their use, they cannot be recommended.

Bovine somatotropin (bST)

Bovine somatotropin (bST) and hormonal growth implants are genetically engineered

metabolic modifiers approved for use in some countries. They do not specifically suppress

CH4 formation, but by improving animal performance (Bauman, 1992; Schmidely, 1993),

they can reduce emissions per-kg of animal product (Johnson et al., 1992; McCrabb, 1997).

Their use is controversial, consumer acceptability is a major issue.

Fatty acids

Dietary fats include tallow, prilled fat, various calcium salts of fatty acid and oil

seeds. Supplementation of these compounds may reduce DMI which in combination with

increased productive performance results in better feed efficiency and, consequently, lower

CH4 emissions. Many studies have shown that the inclusion of dietary fat can mitigate CH4

production in ruminants (Machmüller, 2006, Jordan et al., 2006b, Martin et al., 2008).

Eugene et al. (2008) reported that reduced ruminal CH4 production in cows fed diets

containing fat was because of reduced DMI by cows. Quantitatively less amount of hydrogen

is produced per unit of feed when higher levels of fat are added in ruminant's diet which

consequently decreased CH4 emissions. When sunflower oil was fed approximately 5% of

DMI, it reduced enteric CH4 production by 22% (McGinn et al., 2004). Feeding canola oil

lowers feed intake and fibre digestibility consequently reducing animal productivity

34

(Beauchemin and McGinn, 2006). Martin et al. (2008) found that dietary inclusion of linseed

in ruminant's diet lowered enteric CH4 production and animal productivity. In order to prove

the sustainability of these products more research is required. Odongo et al. (2007b) found

that when myristic acid was supplemented at 5% of diet DM, lactating dairy cows decreased

36% of daily CH4 emissions. Eugene et al. (2008) and Martin et al. (2010) proposed

increasing fat in ruminant’s diet as a promising strategy to mitigate enteric CH4 production.

Types of fatty acids affects methanogenesis in ruminants (Czerkawski et al., 1966b, Prins et

al., 1972). Czerkawski et al. (1966a) reported that the addition of C18 fatty acids reduced

enteric CH4 emissions to the degree of unsaturation. In-vitro studies revealed that specific

medium chain fatty acids lowered methanogenesis (Dohme et al., 2001). During in-vitro

studies when cyclodextrin complexes of capric (C10:0) or caprylic (C8:0) acid were

incubated with rumen fluid, significant CH4 reductions were observed (Ajisaka et al.,

2002). Inclusion of 40 mg of capric acid with the β-cyclodextrin carrier to 60 mL

medium resulted in 60% while the inclusion of 20 mg of capric acid resulted in 40%

reduction in CH4 production. Similar observations were provided by Goel et al. (2009) who

observed 45 and 88% reduction in CH4 production when 20 and 30 mg of capric acid were

added to 50 mL incubation medium, respectively. According to Beauchemin et al. (2008), 5

to 6% enteric CH4 was reduced in animals fed diets containing 1% fat on DM basis. Martin et

al. (2010) reported that 37 to 52% enteric CH4 could be abated if animals were fed fat

supplemented diets. Thus, it can be concluded that lipids cannot be considered as better CH4

mitigating option as they may cause significant drop in feed intake in ruminants.

35

Table 2.5 Rumen modifiers that lower enteric methane production per unit

animal product

Strategy Mode of action comments

Defaunation Decreases: Protozoa; H2;

archaea

Microbial adaptation

Saponins e.g yucca schidigera Decreases: Protozoa; H2;

archaea


Tannins e.g sainfoin Decreases:Protozoa;

archaea


More concentrate & starch ( or

Algae) in diet

Increases propionate;

H2 sink

Competes with monogastrics

PUFA, e.g Linseed C 18:3 fish

oil, EPA, DHA

Decreases celluololysis;

small H2 sink

Dose dependent; DMI may

drop

Saturated fatty acids e.g C12:0;

C14:0

Archaea inhibition Decreases DMI

Organic acids e.g Fumaric,

malic

H2 sink Small effect; expensive

Reduction of nitrate and

sulphate

H2 sink Persistent;

toxic intermediates

Ionophores e.g Monensin Increases propionate;

H2 sink


Enzymes, yeast and probiotics Increases propionate;

H2 sink,pH

Varying results

Other plant extracts e.g garlic

,eucalyptus

Archaea inhibition Microbial adaptation

Immunization against archaea Archaea inhibition More research required

Bacteriocins & archael viruses Archaea inhibition More research required

Short chain nitrocompounds Archaea inhibition; H2

sink

More research required

Source: Adapted from Boadi et al., 2004, Beauchemin et al., 2008 and Martin et al., 2010

36

Nitrate feeding

Nitrate can replace CO2 as an electron acceptor. This reduces nitrate to nitrite and then

to NH3. Nitrate is reduced to nitrite due to NADH reduction and assimilatory nitrite

ammonification results in reduction of nitrite to NH3 coupled with ATP formation (Simon,

2002; Figure 9). One mol nitrate reduces CH4 in equivalent amount which in turn produces 1

mol of NH3 (Leng, 2008).

NO3 +4H2 +2H+ → NH4+ +3H2O

4H2+CO2 →CH4+2H2O

Figure 9: Pathways for the microbial anaerobic fermentation of glucose in ruminants

(Nolan 1999).

Dissimilatory nitrate reduction to NH3 (DNRA) and assimilatory nitrate reduction to

NH3 (ANRA) are two distinct pathways responsible for nitrate reduction in anaerobic systems

(Leng, 2008).

4H+

Glucose

Glucose

Glucose-6-P

2 Pyruvate

2 Acetyl CoA

2 Acetyl-P

2 AcetateAcetate

Butryl-CoA

Butryl-P

ButyrateButyrate

2 Lactate

2 Lactyl-CoA

2 Acrylyl-CoA

2 Propionyl-CoA

2 PropionatePropionate

2 Oxaloacetate

2 Malate

2 Fumarate

2 Succinate

2 Succinyl-CoA

2 Methylmalonyl-CoA

2 Propionyl-CoA

2 PropionatePropionateNAD +

NAD+

H2 MethaneMethane

ATP

2 ATP

ATP

NADH

NADH

NADH NADH

H+

2 NADH

H+

NADH

ATP

NADH

CO2 CO2

37

NO3-+2H+ → H2O+NO2

-

NO2-+6H+ → H2O+ NH3

Organisms capable of DNRA and ANRA use formate and hydrogen as the common

electron donors

3HCO-2+NO2

-+5H+=3 CO2+NH+4+2H2O

3H2+NO2-+2H+ = NH4

+ +2H2O

In most of the experiments, the animals were not adapted to nitrate (Sar et al., 2004; Sar

et al., 2006), instead the nitrate was injected intraruminally. The nitrate and nitrite reduction

in rumen fluid increased by 3-10 X as sheep become accustomed to increasing dietary nitrate

intake (Allison and Reddy, 1984) and this resulted in increase number of nitrate reducing

bacteria (NRB). Carver and Pfander (1974) reported that period of 21 days was required by

the sheep to adapt to diet with 3.2% KNO3. The nitrate addition to ruminant diet inhibits CH4

production and the effect is more pronounced in animals adapted to nitrate (Allison et al.,

1981; Allison and Reddy, 1984). Considerable amount of work has been done under in-vitro

conditions (Leng, 2008; Leng and Preston, 2010). If scientists working with in-vitro

techniques had worked in close association with those working with in-vivo techniques

(Flachowsky and Lebzien, 2009), this could have enhanced the practicability of this

technique. Therefore some in-vivo work is required to determine the efficacy of nitrates and

sulphates in the mitigation of enteric CH4 production in ruminants. The goal is not to stop

CH4 emissions, but rather, redirecting hydrogen into more beneficial pathways (Hegarty et

al., 2007).

Morgavi et al. (2010) reported that the thermodynamic conversion of nitrate into NH3

was more favourable than the formation of CH4 if nitrate was present in ruminant diet.

Methanogenesis can strongly be inhibited by use of nitrates in all systems of fermentation in

rumen or bio digestors (Hungate 1965; Allison et al., 1981; Akunna et al., 1994). However,

prior to its practical application, methods must be explored to reduce toxicity risks induced

by intermediates of sulphate and nitrate metabolism (Perdok and Newbold, 2010).

Denitribacterium detoxificans and Wolinella succinogenes, (sulphate-reducing bacteria) make

use of sulphate, as electron acceptor (Weimer, 1998; Anderson et al., 2000; Simon, 2002)

other than CO2 to oxidise hydrogen (Morgavi et al., 2010).

38

Nitrate and Sulphate feeding

In anaerobic environments nitrate-sulphate interrelationship is complex. Sulphate is

reduced to hydrogen sulphide (H2S) which does not accumulate appreciably in rumen fluid

and is converted to CH4. High nitrate load reduces sulphate to H2S but may cause nitrate

toxicity in ruminants (Leng, 2008).

Approximately 8% of nitrate load, suddenly introduced directly into the rumen was

recovered in urine (Leng, 2008). Thermodynamically, sulphate reduction is more favourable

than methanogenesis (Ungerfeld and Kohn, 2006). Sulphate concentration in the medium and

the residence time within the digestor are the factors which determine the extent of decrease

in CH4 production from sulphate addition (Isa et al.,1986).

Dietary Nitrate Adaptations

The methemoglobinemia can be caused by high doses of nitrate in ruminant diets

resulting in the decrease of the blood capacity to transport oxygen to animal tissues (Bradley

et al., 1939, Lewis, 1951 as cited in Leng, 2008). In the past, many scientists (O’Hara and

Fraser, 1975; Crawford et al., 1966; Deeb and Sloan, 1975; Ruhr and Osweiler, 1986;

Setchell and Williams 1962 as cited in Leng, 2008) highlighted the toxic role of nitrate in

ruminants and they mainly focused on the variability of lethal doses. Methemoglobin

(MetHb) levels of 30 to 40% of haemoglobin (Hb) and higher results in clinical toxicity signs

(Bruning-Fann and Kaneene, 1993). Intraruminal administration of 0.9 g of nitrate/Kg of

body weight (0.75 BW) per day to a sheep in a single load caused MetHb levels of 18.4% of

Hb (Sar et al., 2004). The MetHb levels of over 30% of Hb was also observed when sodium

nitrate (SN) was pulse-dosed into the rumen of sheep at a rate of 1.1 g nitrate/Kg 0.75 BW

per day (Takahashi et al., 1998). Feeding nitrate (0.47 g of nitrate/kg of BW per day) to

sheep increased nitrite reduction rates from 25 to 62 nmol/min per mL of rumen fluid,

whereas nitrate reduction rates increased 26-times (from 4.5 to 117 nmol/min per mL)

compared with sheep fed no additional nitrate in their diets (Allison and Reddy ,1984).

Slow introduction of high nitrate diets in sheep (1.5 g of nitrate/Kg of BW per day)

also showed similar results (Alaboudi and Jones,1985). For unadapted ruminants, nitrate

concentrations more than 0.5% of DM in forages can be detrimental (Bruning-Fann and

Kaneene, 1993), but it appears that adaptation increases rumen microbial population or

improves their nitrite-reducing capacity. Fukui et al. (1980) observed that after 24 h storage

in a refrigerator 36.1% of the original amount of MetHb recovered while in another study

39

Sleight and Sinha (1968) showed that 24-h refrigeration of guinea pig blood resulted in

reductions of over 50% in MetHb. The MetHb values might not, therefore, represent the

actual values at the time of sampling. However, clinical signs of methemoglobinemia

must be observed during the experiment. There are also some reports indicating no clinical

signs of methemoglobinemia even when sheep were fed high doses of nitrate in their diets

(Carver and Pfander, 1974; Alaboudi and Jones, 1985 cited in Leng, 2008).

Effect of dietary nitrate on feed intake, growth performance and rumen ecology

Reduced dry matter intake in animals fed diets containing nitrates was reported by

Leng (2008). Negative effects on feed intake by sheep were observed when dietary

nitrate exceeded 3% of feed DM (Bruning-Fann and Kaneene,1993) . In-vitro studies by

Marais et al. (1988) demonstrated that reduction in feed intake might be co-related to a

nitrite-induced depression of forage cell wall digestion. The accumulation of nitrite in rumen

fluid reduces cell wall fermentation and therefore potentially subsides energy digestibility. As

cited in Leng (2008), Sokolowski et al. (1969) found that addition of 3.2% KNO3 with or

without added S to a concentrate based diet of lambs reduced their growth rates.

Dairy cows fed high nitrate diets shifted the VFA profile from propionate to acetate

with significant reduction in butyrate concentration (Farra and Satter, 1971). Allison and

Reddy (1984) also reported similar results when sheep were fed nitrates. Alaboudi and Jones

(1985) reported shift of VFA profile from butyrate to acetate when nitrates were added in

sheep diet. Sheep fed nitrate revealed quantitative decrease of methanogens in rumen fluid

(Allison et al., 1981).

Measuring methane production

It is very complex to measure CH4 from individual animals, because of its gaseous

properties. Different techniques developed to measure CH4 emissions (Table 2.6) from

ruminants include respiration chambers (Frankenfield, 2010), CH4 estimations from the

VFA production (Hegarty and Nolan, 2007), ventilated hood techniques (Odongo et al.,

2007), isotopic (Hegarty et al., 2007), non-isotopic tracer techniques (Johnson et al.,

1994), tunnel technique (Murray et al., 2007), SF6 technique (Grainger et al., 2007) and

IVGPT (Navarro-Villa et al., 2011). Respiration chambers have been used for the last 100

years to study the energy metabolism of animals (Johnson et al., 2003).

40

The SF6 tracer gas technique and respiration chambers are the two most prevalent or

established methodologies to estimate CH4 production ruminants. Although use of respiration

chambers can be considered as standard method but it is very expensive and cannot be

applied on large number of animals. The SF6 method is easy and is mostly used in range

cattle. However, it gives more variable results of CH4 emission than chamber measurements.

Johnson et al. (1994) observed 7% lower CH4 emission with the SF6 technique than with

chambers with cattle. However, others have shown slightly higher values with the SF6

technique than chambers (Grainger et al., 2007; Boadi et al., 2002) and yet other studies have

found much higher values with the SF6 technique than chambers (Pinares-Patino et al., 2011;

Ulyatt et al., 1999; Pinares- Patino et al., 2008; Fredeen et al., 2004). Moreover, SF6 is a very

potent GHG (23,800 X the Global Warming Potential of CO2; Table 2.1). Its use is also

banned in many countries. Among newly developed CH4 measuring techniques,

commercially developed Open respiratory device is based on the use of tracer gas with lower

GWP as compared to SF6 (Zimmerman et al., 2011).

Micrometeorlogical method is another technique of CH4 estimation which is based on

measuring CH4 from animals in isolated pens where there is no restriction on animal activity.

Pens are equipped with open-path lasers to estimate CH4 concentrations (Harper et al., 1999).

The IVGPT simulates fermentation of feed in rumen, not emissions and digestibility by the

animal. In ruminants, knowledge regarding VFA concentrations is not considered sufficient

to predict the amount of CH4 generated or energy availability to animal as it can be

influenced by anabolic usage of VFA for microbial growth.

Recently, a new method for estimating enteric CH4 production from ruminants has

been developed. It is based on the use of naturally emitted CO2 as a tracer gas instead of SF6

(Storm et al., 2012; Madsen et al., 2010). This technique is easy, fast, cost effective and less

labour intensive. By this technique, data from more number of animals can be collected in

limited period of time. Also the results from animals are in their natural environment.

However, this technique is yet to be tested against standard respiration calorimetry chamber

technique. In conclusion, the respiration chamber technique is the best however, technique

based on the use of naturally emitted CO2 as a tracer gas is more feasible in developing

countries.

41

Table 2.6: Methane measuring techniques

Breed Data Source Methane collection technique

Beef Birkelo et al. (1986) Whole animal calorimetry

Beef Okine et al. (1989) Hood calorimetry

Beef Varga et al. (1990) Whole animal calorimetry

Beef Reynolds et al. (1991) Whole animal calorimetry

Beef Hironaka et al. (1996) Whole animal calorimetry

Beef McCaughey et al. (1997) SF6

Beef McCaughey et al. (1999) SF6

Beef Reynolds and Tyrrell (2000) Whole animal calorimetry

Beef Westberg et al. (2001) SF6

Beef Boadi and Wittenberg (2002) SF6

Beef Boadi et al. (2002) SF6

Beef Boadi et al. (2004) SF6

Beef McGinn et al. (2004) Whole animal calorimetry

Beef Beauchemin and McGinn (2005) Whole animal calorimetry

Beef Sangkhom et al. (2012) CO2 as tracer gas (Gasmet)

Beef Sophal et al. (2013) CO2 as tracer gas (Gasmet)

Beef Hulshof et al. (2010) SF6

Beef Phuong et al. (2012) CO2 as tracer gas (Gasmet)

Dairy Coppock et al. (1964) Whole animal calorimetry

Dairy Tyrrell and Moe (1971) Whole animal calorimetry

Dairy Moe et al. (1973a) Whole animal calorimetry

Dairy Moe et al. (1973b) Whole animal calorimetry

Dairy Moe and Tyrrell (1977) Whole animal calorimetry

Dairy Moe and Tyrrell (1979a) Whole animal calorimetry

Dairy Belyea et al. (1985) Mask calorimetry

Dairy Holter et al. (1986) Whole animal calorimetry



Dairy Tyrrell et al. (1992) Whole animal calorimetry

Dairy Sauer et al. (1998) Micrometeorological mass balance technique

Dairy Waldo et al. (1997) Whole animal calorimetry

Dairy Westberg et al. (2001) SF6

Dairy Boadi and Wittenberg (2002) SF6

42

Dairy Zijderveld et al. (2011) Respiration indirect Calorimetric Chambers

Goats Sophea and Preston (2011) CO2 as tracer gas (Gasmet)

Goats Silivong et al. (2011) CO2 as tracer gas (Gasmet)

Goats Trinh phuc Hao et al. (2009) CO2 as tracer gas (Gasmet)

Sheep Zijderveld et al. (2010) Indirect Calorimetry respiration chamber

Sheep Thanh et al. (2012) CO2 as tracer gas (Gasmet)

Sheep Nolan et al. (2010) Open-Circuit respiration chambers

43

Chapter 3

Materials and Methods

The study was conducted at University College of Agriculture, University of

Sargodha, Pakistan. Sargodha is located at latitude 32.0836° North, longitude 72.6711° east

with altitude 194 meters. This study had 3 independent experiments of 90 days each.

Experiment No. 1

In this experiment, 48 male animals at post weaned age (24 Lohi lambs and 24 Teddy

goats), were randomly divided into 12 groups having in each 4 animals. Each group was

maintained in a separate pen measuring 0.30m x 0.30m. Twelve iso-nitrogenous (crude

protein 18%) and iso-caloric (2.0 ME Mcal/Kg) diets were formulated using 0, 3 and 6% PN

with or without 0.4% S (anhydrous MgSO4). Nonprotein nitrogen was same across all diets

(Table 4.1). The total mixed rations were offered twice a day and fresh clean water was made

available round the clock during experimental period. This experiment lasted for three

months including 15 days of adaptation. Animals were gradually acclimated to the diets with

nitrate and S. Animals were fed separately. The animals were weighed fortnightly.

Experiment No. 2

In this experiment, 48 male animals at growing age (24 Lohi sheep and 24 Teddy

goats of approximately six months of age), were randomly divided into 12 groups having in

each 4 animals. Each group was maintained in a separate pen measuring 0.30m x 0.30m.

Animals were fed separately. Twelve iso-nitrogenous (crude protein 18%) and iso-caloric

(2.0 ME Mcal/Kg) diets were formulated using ~0, 1.5 and 3% CAN with or without 0.4% S

(anhydrous MgSO4). Nonprotein nitrogen was same across all diets. The control diet CAN0-

S0 contained neither CAN nor S. Whereas CAN0-S4, CAN1.5-S0, CAN1.5-S4, CAN3-S0

and CAN3-S4 diets had 0% CAN and 0.4% S, 1.5% CAN and 0% S, 1.5% CAN and 0.4% S,

3% CAN and 0% S and 3% CAN and 0.4% S, respectively (Table 5.1). The total mixed

rations were offered twice a day and fresh clean water was made available round the clock

during experimental period. This experiment lasted for three months including 15 days of

adaptation. Animals were gradually acclimated to the diets with nitrate and S. Animals were

fed separately. The animals were weighed fortnightly.

44

Experiment No. 3

In this experiment, 48 male animals (24 Lohi sheep and 24 Teddy goats of

approximately nine months of age), were randomly divided into 12 groups, 4 animals in each

group. Each group was maintained in a separate pen measuring 0.30m x 0.30m. Animals

were fed separately. Twelve iso-nitrogenous (crude protein 18%) and iso-caloric (2.0 ME

Mcal/Kg) diets were formulated using ~0, 2.5 and 5% SN with or without 0.4% S (anhydrous

MgSO4). Nonprotein nitrogen was same across all diets. The control diet SN0-S0 contained

neither SN nor S. Whereas SN0-S4, SN2.5-S0, SN2.5-S4, SN5-S0 and SN5-S4 diets had 0%

SN and 0.4% S, 2.5% SN and 0% S, 2.5% SN and 0.4% S, 5% SN and 0% S and 5% SN and

0.4% S, respectively (Table 6.1). The total mixed rations were offered twice a day and fresh

clean water was made available round the clock during experimental period. This experiment

lasted for three months including 15 days of adaptation. Animals were gradually acclimated

to the diets with nitrate and S. Animals were fed separately. The animals were weighed

fortnightly.

Weight gain of each animal was determined by the difference between weights at 90

days minus weight at 0 day. In order to increase the versatility and accuracy of experiment

different nitrate sources were supplemented in the animal diet with or without S. It was also

observed whether chemical nature of nitrates affects animal health when supplemented with

or without S or not.

Data Collection

The daily feed intake was recorded and representative samples were taken and

analyzed for DM and CP using the procedures described by AOAC (1990). The Neutral

detergent fiber (NDF) and acid detergent fiber (ADF) were determined by the methods

described by Van Soest et al. (1991).

Digestibility and nitrogen balance trials were conducted during the last week of

experiment. For digestibility and nitrogen balance trials, all animals were shifted to

metabolic crates for 7 days to ensure complete collection of feces and urine. Locally made

metabolic collection crates, each measuring 1.5m x 1.2m x 1.5m, were used to collect the

urine and feces of individual animal and then urine and feces were mixed by animal.

Metabolic collection crates consisted of a collection tray and two plastic urine collection

bowls. Removable trays were fitted on floor of metabolic collection crates and animals stood

45

on these trays. Removable urine collection bowls were set beneath the floor of metabolic

collection crates. During total collection method, urine was collected in urine collection

bowls through the small hole at the bottom of the collection tray. These bowls had measured

amount of solution acidified with 50% H2SO4 to avoid N losses during collection (Nisa et al.,

2004). Feces and urine were collected, weighed and representative samples were stored at -

20oC for further analysis. At the end of collection period, urine and feces samples from

individual pens were thawed, composited and homogenized. Composite samples were dried

at 55oC and ground through 1-mm screen. Feed and fecal samples were analyzed for NDF

and ADF by the methods described by Van Soest et al. (1991) and DM and CP were

determined by methods described by AOAC (1990). At the end of experiment, one hour after

morning feed, blood samples (10 mL/animal) were collected by jugular vein punctured into

vacutainer tube containing 81 µL of 15% EDTA solution and analyzed in local pathological

laboratory for blood urea nitrogen, glucose, creatinine using cobas c 111 analyzer (Roche),

hematology was determined using Sysmex pocH-100i and methemoglobin (MetHb; Evelyn

and Malloy, 1938).

The enteric CH4 was analyzed at the end of the experiment using method described

by Madsen et al. (2010) in which naturally emitted CO2 was used as tracer gas (Storm et al.,

2012). Data of enteric methane production was recorded one hour after each feeding.

Methane production gradually increases and reaches peak values during first hour of feeding

and then decreases gradually (Sar et al., 2004). Each animal was kept in an especially

designed closed enclosure (each measuring 1.5 m x 1.2 m x 1.5 m) for 15 minutes and

enteric methane production was measured during the last 5 minutes with the help of infra red

CH4 analyzer (Gasmet Dx-4030, Finland Table 2.7). The enclosure was made airtight.

Arrangements were made for gases measurement with the help of probe. The probe, for

measurement of gases was inserted from the side of closed chamber. For Zero point

calibration nitrogen gas was used. Efforts were made to maintain no change in temperature

or air pressure during data collection. Moreover, In order to minimize stress factor all

animals were made accustomed to enclosure 15 days prior to data collection.

The CO2 and CH4 in background air were measured at the same time. Measuring the

CH4 to CO2 ratio combined with the measuring of total CO2 produced, the amount of CH4

was calculated as under:

CH4:CO2 = (a-b)/(c-d)

46

Where a is CH4 concentration in mixed eructed gas plus air, c is CO2 concentration in

mixed eructed gas plus air, b is CH4 in background air and d the CO2 in background air.

As feed intake did not differ among the treatments it was assumed that CO2

production was also similar and could be used as internal marker as described by Madsen et

al. (2010).

Methane reduction was derived from the equation proposed by Leng and Preston

(2010) in which it was assumed that:

If the CH4 production rate is A, the CO2 entry rate B is same on all diets, and the ratio

of CH4 to CO2 is R, then the following equations apply:

For urea-fed animal…………………..…. AU (Urea) = B * R1

For the nitrate-fed animal……………..….AN (nitrate) = B * R2

The % CH4 reduction rate is then…………B(R1-R2) / BR1*100

Table 2.7: Gasmet DX4030 (General Specifications)

Measurement Principle: FTIR (Fourier Transform InfraRed) spectroscopy

Measuring Parameters:

Zero point calibration 24 hours, calibration with nitrogen gas

Zero point drift 2% of smallest measuring range per zero-point calibration interval

Sensitivity drift None

Linear derivation 2% of smallest measuring range

Temperature drift 2% of smallest measuring range per 100 C change, temperature

measured and compensated

Pressure influence 1% change of measuring value for 1% sample pressure change,

ambient pressure changes measured and compensated

Response time Typically < 120s, depending on the gas flow and measurement time

Gas Inlet and Outlet

conditions:

Gas temperature Ambient temperature (0-500C) non-condensing

Flow rate 120-360 l per hour

Gas filteration Filteration of particulates included in the sample probe

Sample gas pressure Ambient

Sample pump Flow 2 l/min for ambient air only

47

Statistical analysis

The data collected were analyzed using 2×3×2 factorial arrangement under

Randomized Complete Block Design (RCBD). The data were analyzed by methods described

by Steel et al. (1996). Treatments were compared by Tukey’s test using statistical software

Statistix 8.1.

48

Chapter 4

Effect of varying levels of potassium nitrate with or without sulphur on

enteric methane production in post weaned Lohi sheep and Teddy goats

Abstract

Ruminants are considered as one of the major sources of greenhouse gases emission.

The study was conducted to evaluate the influence of varying levels of potassium nitrate (PN)

with or without S on the growth performance and enteric CH4 production in weaned Lohi

lambs and Teddy goats in a 2×3×2 factorial arrangement under Randomized Complete Block

Design. In the experiment, 48 male animals at post weaned age (24 Lohi lambs and 24 Teddy

goats), were randomly divided into 12 groups, 4 animals in each group. Twelve iso-

nitogenous and iso-caloric diets were formulated. Nonprotein nitrogen was same across all

diets. The control diet PN0-S0 contained neither PN nor S whereas PN0-S4, PN3-S0, PN3-

S4, PN6-S0 and PN6-S4 diets had 0% PN and 0.4% S, 3% PN and 0% S, 3% PN and 0.4% S,

6% PN and 0% S and 6% PN and 0.4% S, respectively. The Gases were measured by using

infra-red biogas analyzer. The nutrient intake and digestibility in both weaned lambs and

goats were similar (P> 0.05) across all diets. The animals fed diet containing 6% PN with

0.4% S showed better nitrogen balance. Daily live weight gain of of both lambs (146 g/day)

and goats (66.0 g/day) fed diet containing 6% PN and 0.4% S was the highest and best values

of FCR (P<0.05) were also observed in both group of animals. Both in lambs and goats non-

significant differences (P> 0.05) were observed in blood metabolites including blood urea

nitrogen, glucose and Creatinine. Similar trend (P> 0.05) was observed in hematology. None

of the animals suffered from methemoglobinemia or polioencephalomalacia. Enteric CH4 was

32.6% reduced (P<0.05) in lambs and 31.9% in goats fed diet containing 6% PN and 0.4% S

compared to those fed C.

Introduction

Ruminants are considered as one of the major sources of GHG emission. They emit

about 80 million tonnes of methane (CH4) annually which is one third of the emitted CH4

worldwide (Beauchemin et al., 2008; IPCC, 2007). The CH4 does not only cause

environmental problems, but it also makes animal production inefficient. Beauchemin et al.

49

(2008) reported enhanced animal productive efficiency when enteric CH4 was reduced. Thus,

enteric CH4 production in ruminants was reduced through nutritional manipulation (Dohme et

al., 2000; Johnson and Johnson, 1995; Moss et al., 2000; Itabashi, 1994) or use of various

feed additives (Joblin, 1999; Moss et al., 2000; Lopez et al., 1999; Asanuma et al., 1999;

Wenk, 2003). Nitrate being dietary N source can provide a hydrogen sink to reduce enteric

CH4 production in ruminants (Leng, 2008). However, use of nitrate as a source of rumen

fermentable N and as an alternative hydrogen sink to CO2 had previously been neglected due

to its toxicity. Nitrate is converted into nitrite as an intermediate during its reduction in the

rumen (Leng and Preston, 2010). It has been suggested that nitrate reduction to NH3 could be

suppressed by feed-back inhibition from high NH3 levels in rumen fluid (Leng, 2008).

Sulphur supplementation in the diet (Leng, 2008) or cysteine (Takahashi et al., 1998)

inclusion reduced nitrite accumulation in the rumen. Sulphate is a reductant (ΔG 0 = -21.1

kJ/mol of hydrogen; Ungerfeld and Kohn, 2006) and will also compete for electrons and

may lower CH4 production.

The SF6 and in-vitro gas production techniques are the most commonly used techniques

to measure enteric CH4 production in ruminants. The SF6 method is the most commonly used

technique in most of experiments but its results of measuring CH4 emission are more variable

than that of chamber measurement (Pinares-Patino et al., 2011; Johnson et al., 1994;

Grainger et al., 2007; Boadi et al., 2002). That is why the most of the studies used to reduce

enteric CH4 were conducted in-vitro (Lovett et al., 2004; Rymer et al., 2005; Pellikaan et al.,

2011; Navarro-Villa et al., 2011; Blummel and Orskov, 1993; Bhatta et al., 2006). However,

the in-vitro technique simulates ruminal fermentation of feed only and it does not take into

consideration other factors including CH4 formation, its emission and digestibility by the

entire animal.

Thus, there is a dire need to measure the CH4 reduction directly from ruminants and a

method, which is based on the use of naturally emitted CO2 as a tracer gas (Storm et al.,

2012; Madsen et al., 2010) to estimate CH4 emission directly from ruminants seems

promising. However, the scientific evidence regarding the use of nitrate to mitigate CH4

emission directly from ruminants is limited. Therefore, the present study was planed to

determine the effect of varying levels of PN with or without S on growth performance and

enteric CH4 production in weaned Lohi sheep and Teddy goats fed roughage based diet. The

50

hypothesis was that nitrate and S will mitigate the enteric CH4 production in both sheep and

goats. Both nitrate and S will give an additive effect on the CH4 reduction.


Experimental site


Sargodha, Pakistan.

Experimental animals and feeding

In this experiment, 48 male animals at post weaned age (24 Lohi lambs and 24 Teddy

goats), were randomly divided into 12 groups having in each 4 animals. Each group was

maintained in a separate pen measuring 0.30m x 0.30m. Animals were fed separately. Twelve

iso-nitrogenous (crude protein 18%) and iso-caloric (2.0 ME Mcal/Kg) diets were formulated

using 0, 3 and 6% PN with or without 0.4% S (anhydrous MgSO4). Nonprotein nitrogen was

same across all diets (Table 4.1). The total mixed rations were offered twice a day and fresh

clean water was made available round the clock during experimental period. The animals

were weighed fortnightly. This experiment lasted for three months including 15 days of

adaptation. Animals were gradually acclimated to the diets with nitrate and S. The daily feed

intake was recorded and representative samples were taken and analyzed for DM and CP

using the procedures described by AOAC (1990). The NDF and ADF were determined by the

methods described by Van Soest et al. (1991).


experiment by using total collection method. For this purpose, all animals were shifted to


metabolic collection crates, each measuring 1.5 m x 1.2 m x 1.5 m, were used to collect the

urine and feces of individual animal. Metabolic collection crates consisted of a collection

tray and two plastic urine collection bowls. Removable trays were fitted on floor of

metabolic collection crates and animals stood on these trays. Removable urine collection

bowls were set beneath the floor of metabolic collection crates. Urine was collected in urine

collection bowls through the small hole at the bottom of the collection tray. These bowls had

51

measured amount of solution acidified with 50% H2SO4 to avoid N losses during collection

(Nisa et al., 2004). Feces and urine were collected, weighed and representative samples were

stored at -20oC for further analysis. At the end of collection period, urine and feces samples

from individual pens were thawed, composited and homogenized. Composite samples were

dried at 55oC and ground through 1-mm screen. Feed and fecal samples were analyzed for

NDF and ADF by the methods as described by Van Soest et al. (1991) and DM and CP were

determined by the methods as described by AOAC (1990). At the end of experiment blood

samples (10 mL/animal) were collected by jugular vein punctured into vacutainer tube

containing 81 µL of 15% EDTA solution and analyzed in local pathological laboratory for

blood urea nitrogen, glucose, creatinine using cobas c 111 analyzer (Roche) and

methemoglobin (MetHb; Evelyn and Malloy, 1938).

The enteric CH4 was analyzed at the end of the experiment by using method as

described by Madsen et al. (2010) in which naturally emitted CO2 was used as tracer gas

(Storm et al., 2012). Data of enteric methane production was recorded one hour after each

feeding. Methane production gradually increases and reaches peak values during first hour of

feeding and then decreases gradually (Sar et al., 2004). Each animal was kept in an

especially designed closed enclosure (each measuring 1.5 m x 1.2 m x 1.5 m) for 15 minutes

and enteric methane production was measured during the last 5 minutes with the help of infra

red CH4 analyzer (Gasmet Dx-4030, Finland Table 2.7). For Zero point calibration nitrogen

gas was used. In order to minimize stress factor all animals were made accustomed to

enclosure 15 days prior to data collection. The CO2 and CH4 in background air were

measured at the same time. Measuring the CH4 to CO2 ratio combined with the measuring of

total CO2 produced, the amount of CH4 was calculated as under:

CH4:CO2 = (a-b)/(c-d)

Where a is CH4 concentration in mixed eructed gas plus air, c is CO2 concentration in

mixed eructed gas plus air, b is CH4 in background air and d the CO2 in background air.


production was also similar and could be used as internal marker as described by Madsen et

al. (2010).



52

If the CH4 production rate is A, the CO2 entry rate B is same on all diets, and the ratio

of CH4 to CO2 is R, then the following equations apply:




The hypothesis was that nitrate and S will have mitigating effect on the enteric CH4

production. Both nitrate and S will give an additive effect on the CH4 reduction.


The data collected were analyzed using 2×3×2 factorial arrangement under RCBD.

The data were analyzed by methods described by Steel et al. (1996). Treatments were

compared by Tukey’s test using statistical software Statistix 8.1.

Results

The nutrient intake and digestibility in both weaned lambs and goats were similar (P>

0.05) across all diets (Table 4.2). The animals fed diet containing 6% PN with 0.4% S

showed better nitrogen balance (Table 4.5). Daily live weight gain was 138, 138, 142, 142,

142 and 146 g/day in Lohi lambs and was 62, 62, 61, 62, 65 and 66 g/d in Teddy goats fed

PN0-S0, PN0-S4, PN3-S0, PN3-S4, PN6-S0 and PN6-S4 diets, respectively (Table 4.6).

The FCR in lambs fed PN0-S0, PN0-S4, PN3-S0, PN3-S4, PN6-S0 and PN6-S4 diets were

5.3, 5.3, 5.2, 5.2, 5.2 and 5.0, respectively. Moreover, the FCR were 7.4, 7.4, 7.4, 7.3, 7.0 and

6.9 in goats fed PN0-S0, PN0-S4, PN3-S0, PN3-S4, PN6-S0 and PN6-S4 diets, respectively

(Table 4.6). The best feed conversion values (P<0.05) were observed in animals fed PN6-S4

diet. Both in lambs and goats non-significant differences (P> 0.05) were observed in blood

metabolites including blood urea nitrogen, glucose and Creatinine (Table 4.3). Similar trend

(P> 0.05) was observed in hematology (Table 4.4). None of the animals suffered from

methemoglobinemia or polioencephalomalacia. The difference in enteric CH4 reduction was

0, 1.99, 18.98, 20.41, 27.21 and 32.65% in Lohi lambs and 0, 3.95, 15.77, 24.18, 25.81 and

31.91% in Teddy goats fed PN0-S0, PN0-S4, PN3-S0, PN3-S4, PN6-S0 and PN6-S4 diets,

respectively (Table 4.7). Both lambs and goats fed diet containing 6% PN and 0.4% S

showed better enteric CH4 reduction as compared to other diets (Table 4.7).

53

Discussion

In this study all the diets were supplemented with either nitrate or urea at iso-

nitrogenous concentrations as the main fermentable nitrogen source. Feed intake did not

differ among the treatments but better weight gains with higher levels of nitrate indicated that

PN was slightly better fermentable N source than urea in the diet. Nitrogen retention was

slightly better both in sheep and goats when nitrate was non-protein N source rather than

urea. Trinh phuc Hao et al. (2009) used highest level of PN upto 5.33% in ruminant diet.

Other researchers (Thanh et al., 2012) in a sheep experiment used maximum 4% PN against

1.8% urea (% DM basis) in ruminant diets. The present study was conducted to evaluate

whether PN at 6% with 0.4%S could safely be supplemented in ruminant diet without any

toxicity.

Nutrient Intake and Nutrient digestibility

Our findings were consistent with Zijderveld et al. (2010) and Sophea and Preston

(2011) who reported that dietary nitrate did not affect feed intake by sheep and goats.

Similarly, Sangkhom et al. (2012) and Phuong et al. (2012) reported that animals fed diets

containing nitrate (or) urea ate the same amount of DM. The lack of difference in DMI in this

study could be ascribed to slow conversion of nitrates into nitrites which did not accumulated

in the rumen, consequently there was no nitrite-induced depression of forage cell wall

digestion. As mentioned earlier, in ruminants, nitrate is reduced to NH3 by certain rumen

microbes, with nitrite as an intermediate product but under some circumstances the rate of

nitrite formation may exceed the rate of nitrite reduction to NH3, resulting in elevated nitrite

levels thus causing methemoglobinemia. Slow adaptation of dietary nitrates might have

resulted in the development of rumen microflora that possibly had higher capacity to reduce

nitrite. Non-significant changes in hematology and blood metabolites indicated same health

status of all animals. In this experiment, none of the animal showed any kind of abnormal

behaviour or signs of illness during the whole experimental period. This indicated that none

of the animals suffered from nitrate or S toxicity.

The nutrient digestibility in the present study is consistent with the findings of Thanh

et al. (2012) who reported that nitrate (or) urea as NPN source did not affect nutrient

digestibility in sheep. Nolan et al. (2010) also reported non-significant results of dry matter

digestibility (DMD) when nitrates were used as feed additive. There are many factors which

54

affect feed digestibility but feed intake is one of the most important. The digestibility in the

present study did not change simply because feed consumption remained unaltered across all

diets.

Nitrates are also known to affect the digestibility of forages in-vitro (Marais, 1980).

Nitrite is a well recognized antimicrobial agent but the mechanism of bacterial inhibition has

not been satisfactorily elucidated (Yarbrough et al. 1980). In this study, similar digestibility

across all the treatments in both trials might be attributed to the absence of nitrite

accumulation in the rumen. Nitrite accumulation inhibits the growth of some bacterial species

which decreases feed digestibility. This reduced digestibility reduces VFA production and

this in turn reduces microbial biomass which then reduces intake by the animal. During

digestion process, the concentrations of branched chain acids decreases which is essential for

the growth of cellulolytic rumen bacteria. According to the findings of Marais et al. (1988),

nitrite is the prime factor responsible for reducing digestibility in ruminants, fed high nitrate

diets. This reduces the solubility of the structural components, such as hemicellulose and

cellulose. The nitrite negatively affects the xylanolytic and cellulolytic microbial population

with a concomitant reduction in xylanase and cellulase activity. Values for xylanase and

cellulase activity could each be due to the action of more than one enzyme. The nitrite affects

cellulolytic microbes possibly by altering the energy metabolism. It inhibits microbes which

produce ATP via electron transport systems but has no effect on bacteria which lack

cytochromes and rely on glycolysis for ATP generation (Marais et al., 1988)

However, Hulshof et al. (2010) reported reduced DMI by animals fed diets containing

nitrates. This reduced DMI by animals might be due to ruminal nitrite accumulation in beef

cattle fed sugarcane-based diets. Similar results were reported by other researchers (Bruning-

Fann and Kaneene, 1993; Weichenthal et al., 1963).

Statistically non-significant results were observed in DMI with 0.4% S

supplementation in the diets of sheep and goats. Possibly the animals on sulphate treatments

were fed a considerable amount of S in the diet (4 g of S/kg of DM). Silivong et al. (2011)

reported reduced intake by goats fed diets containing 0.8% S. In another study, Phuong et al.

(2012) reported DMI decreased when 0.8% S was supplemented in cattle diets. Higher

concentrations of S supplementation than NRC recommendations i.e 4 g of S/kg of DM

might have increased its bitterness in taste , which reduced intake. The toxic production of

H2S also affects the feed intake. High levels of H2S in the rumen and the subsequent

inhalation of hydrogen sulfide increases the risk of polioencephalomalacia (Gould, 1998).

55

In this study we did not observe even a single case of polioencephalomalacia. Thus 4 g of

S/kg of DM indicates optimal amount of S required for microbial growth.

Abatement of enteric methane

Diets containing PN in combination with S reduced ~32% enteric CH4 production

from both Lohi sheep and Teddy goats. The results of this study supported the findings of

Binh Phuong et al. (2011), Thanh et al. (2011), Sangkhom et al. (2011), Jeong et al. (2005)

Bozic et al. (2009), Kluber Conrad (1998), Guo et al. (2009), Guangming et al. (2010) and

Mohanakrishnan et al. (2008) who reported reduced CH4 production in animals fed diets

containing nitrates. Our results are also in agreement with Leng and Preston (2010) who

observed that nitrate supplementation in ruminant diet reduced enteric methane production.

Similar results were reported by Leng (2008) who reported10% enteric CH4 reduction in

animals fed diets containing 1% PN.

Dissimilatory nitrate reduction to NH3 (DNRA) and assimilatory nitrate reduction to

NH3 (ANRA) are two distinct pathways responsible for nitrate reduction in anaerobic system.

Nitrate and nitrite reduction in rumen fluid increases number of nitrate reducing bacteria.

Nitrate-reducing bacteria uptake hydrogen, resulting in a lower electron pool which is

necessary for population density of methanogens. This reduced methanogenic activity might

be an alternative explanation for the reduced methane production. The thermodynamic

conversion of nitrate into NH3 is more favourable than the formation of CH4 if nitrate is

included in ruminant diet (Morgavi et al., 2010). One mol nitrate reduces CH4 in equivalent

amount which in turn produces 1 mol NH3 (Leng, 2008). Reduction of nitrate also draws

hydrogen away from other processes such as propionogenesis. Theoretically, 1 mole of

nitrate reduces 1mole of CH4 as 4 moles of hydrogen are redirected towards nitrate reduction,

which is equivalent to a reduction of methane emission by 25.8 g for each 100 g of nitrate

fed.

In this study sulphate inclusion rate was according to the maximum

recommendations as indicated by NRC (4 g of S/kg of DM; NRC, 2001). Results of

the study show that sulphate is effective in decreasing CH4 production. None of the animals

showed clinical signs of polioencephalomalacia during the trial.Thus it can be assumed that

there was no H2S toxicity. Sulphate reduction to H2S consumes 8 electrons and thus

offers the same potential per mole to reduce CH4 emissions as nitrate. Stoichiometrically, the

full reduction of 100 g sulphate to H2S would reduce CH4 production by 16.7 g. Methane

56

generated from sulphate supplementation is dependent on two important factors i.e sulphate

concentration in the medium and the residence time within the digester (Isa et al., 1986). At

low sulphate concentration relatively more electrons were directed toward sulphate reduction

compared with a high sulphate concentration. Increased residence time increases the flow of

electrons from methanogenesis toward sulphate reduction. High rumen liquid passage rate in

both sheep and goat might explain the lower than expected use of hydrogen for sulphate

reduction. The sulphate reduction is thermodynamically more favourable than

methanogenesis (Ungerfeld and Kohn, 2006) and the additive effect of both nitrate and

sulphate in the present study reduced CH4 production in both sheep and goats fed PN6-S4

diet. It appeared that adaptation improved the nitrite-reducing capacity of rumen microbes.

Non-significant changes in hematology and blood metabolites indicated same health status of

all animals. In this experiment, none of the animal showed any kind of abnormal behaviour or

signs of illness during the whole experimental period. This indicated that none of the animals

suffered from nitrate or S toxicity.

Sangkhom et al. (2012) reported that dietary nitrates reduced 27% enteric CH4

emission. Similarly, Zijderveld et al. (2010) reported 32% CH4 reduction and Nolan et al.

(2010) showed 23% CH4 reduction in sheep fed diets containing nitrate when compared to

those fed diets containing urea. Hulshof et al. (2010) also observed 32% CH4 reduction in

cattle fed diets containing nitrate when compared to those fed diets containing urea. Sophal et

al. (2013) reported 43% enteric CH4 reduction in cattle while Sophea and Preston (2011)

observed 60% reductions in goats. However, Zijderveld et al. (2011) reported 16% CH4

reduction in dairy cows fed diets containing nitrates and they also noticed some ill effect on

the health of those cows.

Inconsistent results in CH4 mitigation might be due to various CH4 measuring techniques.

Various researchers (Hulshof et al., 2010) used SF6 technique and Zijderveld et al. (2011)

used calorimetric chambers. Nolan et al. (2010) measured CH4 via VFAs. Sophal et al.

(2013), Sophea and Preston (2011) and Sangkhom et al. (2012) determined CH4 reduction by

the use of naturally emitted CO2 as a tracer gas. Moreover, Hulshof et al. (2010) used

sugarcane-based diets which might have initiated nitrite production which affected CH4

reduction. The SF6 method is the most commonly used technique in most of the experiments

but it gives more variable results of CH4 emission than chamber measurements (Pinares-

Patino et al., 2011; Johnson et al., 1994; Grainger et al., 2007; Boadi et al., 2002).

57

Growth performance

Daily live weight gain and better feed efficiency of both sheep or goats in this study are in

accordance with Trinh phuc Hao et al. (2009) who reported higher weight gain in goats when

nitrates replaced urea as the source of fermentable N. Dietary nitrate supplementation

resulted in better reduction of enteric CH4 as compared to urea, which might have increased

weight gains. Sangkhom et al. (2012) also reported higher growth and better feed efficiency

by cattle fed diets containing nitrate than those fed diets containing urea. In both trials

animals were fed similar amounts of feed. Nitrate fed sheep and goats might have resulted in

reduced heat production possibly due to reduced metabolizability of the ingested gross

energy, from a reduced conversion of metabolizable energy into heat, potentially resulting in

an increase in energy retention, or from a combining effect of the two. As in this study we did

not measure metabolizable energy intake the distinction cannot be made. However,

arguments for both options can be made.

Blaxter and clapperton (1965) mentioned that enteric CH4 mitigation improved the feed

nutritive value resulting into better animal performance. Beauchemin et al. (2008) also

reported that ruminant productive efficiency could be improved by reducing enteric CH4

production. If nitrite does not accumulate in the rumen, it will produce ruminal NH3

production at a rate equal to assimilatory growth rate of microbial mass.The electron transfer

of NH3 during microbial cell synthesis conserves more energy in the rumen end products than

when electrons are donated to CH4 which is excreted out. This additional energy stored

during microbial synthesis might have increased microbial growth efficiency which resulted

into higher feed efficiency of diets containing nitrate than those containing urea (Leng, 2008).

However, in contrast to the present study, some workers (Zijderveld et al., 2010; Thanh et

al., 2012) reported reduced enteric CH4 emission in sheep fed diets containing nitrates but

this CH4 abatement did not affect growth performance of sheep. Similar results were reported

by Zijderveld et al. (2011) in dairy cows. Sophea and Preston (2011); Nguyen Ngoc Anh et

al. (2010) and Trinh phuc Hao et al. (2009) also repoterd reduction in CH4 production but

they did not report any increase in growth rates in goats. Sophal et al. (2013), Phuong et al.

(2012), Ngoc Huyen Le Thi, (2010) and Do Thi Than Van et al. (2010) also showed lack of

growth in cattle fed diets containing nitrates. Tillman et al. (1965) reported that nitrate

reduced growth rates without toxicity symptoms.

58

Blood Chemistry

None of the sheep or goat showed any kind of abnormal behaviour or signs of illness

during the whole experiment. In present study, blood metabolites and haematology showed

non-significant difference among treatments. The animals did not show any symptom of

nitrate or nitrite toxicity because either they were slowly acclimated or adjusted to nitrate

feeding or the nitrate to S ratios in the feed were possibly balanced in such a manner that they

maintained the activity of S reducing bacteria (SRB) which in turn reduced nitrite to NH3

(Leng, 2008). Carver and Pfander (1974) and Alaboudi and Jones (1985) reported that

supplementation of elevated doses of nitrate did not affect the health of the animal and had no

symptoms of nitrate toxicity. No ill effects were observed in most of the feeding trials with

young animals when nitrates were supplemented in their diets (Trinh phuc Hao et al., 2009;

Nguyen Ngoc Anh et al., 2010; Ngoc Huyen Le Thi, 2010). Zijderveld et al. (2010) reported

minor signs of methemoglobinemia when only nitrate was supplemented in the sheep diet

while no signs were observed when nitrate and sulphate were used together. Nolan et al.

(2010) also reported similar trend. Nitrate feeding to non-acclimated animals may lead to

nitrite accumulation in the rumen. Nitrite when diffused into the blood oxidizes ferrous ions

in haemoglobin (Hb) to ferric ions to form MetHb (Zijderveld et al., 2011) which decreases

its oxygen carrying capacity and in acute cases may be fatal (Zijderveld et al., 2010).

Elevated nitrate doses in the diet of ruminants caused the signs of methemoglobinemia

(Bradley et al., 1939; Lewis, 1951) but clinical toxicity symptoms appear only when MetHb

level is 30 to 40% of Hb (Bruning-Fann and Kaneene, 1993). Blood urea nitrogen

concentrations are very high for goats possibly due to breed difference. Moreover, its

numbers can vary greatly between different locations and different laboratories.

Conclusion

Diets containing PN in combination with S did not only reduce ~32% enteric CH4

production from both Lohi sheep and Teddy goats but it also improved their growth

performance

59

Table 4.1 Ingredient composition of experimental diets

1PN0, PN3 and PN6 stand for potassium nitrate at 0, 3 and 6%, respectively. S0 and S4 stand for 0

and 0.4% sulphur, respectively.

Ingredients

Diets1

PN0 PN3 PN6

S0 S4 S0 S4 S0 S4 Wheat Straw 35.00 35.00 35.00 35.00 35.00 35.00

Cotton seed meal 10.00 10.00 10.00 10.00 10.00 10.00

Rice bran 10.00 10.00 10.00 10.00 10.00 10.00

Maize gluten Meal 30% 5.00 5.00 5.00 5.00 5.00 5.00

Hay (Lucerne) 21.00 21.00 21.00 21.00 21.00 21.00

Enzose 14.00 14.00 14.00 14.00 14.00 14.00

Anhydrous Mag. Sulphate 0.00 1.50 0.00 1.50 0.00 1.50

Sodium Chloride 1.00 1.00 1.00 1.00 1.00 1.00

Di-Calcium Phosphate 2.00 2.00 2.00 2.00 2.00 2.00

Potassium nitrate 0.00 0.00 3.00 3.00 6.00 6.00

Urea 1.70 1.70 0.85 0.85 0.00 0.00

Chemical composition (%)

Dry Matter 88.08 89.58 90.23 91.73 92.38 92.38

Crude Protein 18.00 18.00 17.99 17.99 17.98 17.98

Neutral Detergent Fiber 44.36 44.36 44.36 44.36 44.36 44.36

Acid Detergent Fiber 31.38 31.38 31.38 31.38 31.38 31.38

Metabolizable Energy (Mcal/kg) 2.06 2.06 2.06 2.06 2.06 2.06

60

Table 4.2 Effect of different levels of potassium nitrate with or without sulphur on Nutrient intake and their

digestibility in post weaning Lohi sheep and Teddy goat

Items

Lohi lambs Teddy Goat

SEM2

Significance3 Diets1

PN0 PN3 PN6 PN0 PN3 PN6

S0 S4 S0 S4 S0 S4 S0 S4 S0 S4 S0 S4 A B AB ABC

Nutrient Intake, g/d

DM 741a 742a 742a 742a 741a 735a 455b 457b 456b 458b 456b 456b 2.13 NS NS NS *

CP 134a 133a 133a 134a 133a 132a 82b 82b 82b 82b 82b 82b 0.44 NS NS NS *

NDF 329a 329a 329a 329a 329a 326a 202b 203b 202b 203b 203b 202b 0.99 NS NS NS *

ADF 233a 233a 233a 233a 232a 231a 143b 143b 143b 144b 143b 143b 0.64 NS NS NS *

Digestibility,%

DM 55.0de 56.0bcde 54.7e 55.5cde 55.2cde 56.7abcde 59.5abc 60.0ab 58.0abcde 59.0abcde 59.2abcd 60.5a 0.89 NS NS NS *

CP 65.5c 65.8c 64.8c 64.8c 64.5c 63.0d 72.5a 72.8a 71.8a 71.8a 71.5a 70.0b 0.27 * * * *

NDF 53.0ab 52.0ab 52.5ab 53.3a 51.0ab 52.8ab 50.5ab 49.8ab 50.0ab 52.0ab 49.8b 51.3ab 0.85 NS NS NS *

ADF 41.0 40.0 40.5 41.3 39.5 40.8 41.5 41.8 42.0 44.0 41.0 43.3 0.92 NS NS NS NS

1PN0, PN3 and PN6 stand for potassium nitrate at 0, 3 and 6%, respectively. S0 and S4 stand for 0 and 0.4% sulphur, respectively. 2SEM stand for Standard error mean. 3A, B, C and ABC stand for Nitrate levels, Sulfur levels, Species and their Interaction, respectively while AB stand for Interaction of nitrate levels

with sulphur levels. NS stand for Non significant (P>0.05) and * stand for Significant (P<0.05). a,b,c Means in a row with different superscripts differ significantly (p<0.05). DM, CP, NDF and ADF stand for Dry matter, Crude Protein, Neutral detergent fibre and Acid detergent fibre respectively.

61

Table 4.3 Effect of different levels of potassium nitrate with or without sulphur on blood metabolites in post weaning

Lohi sheep and Teddy goat

Items

(mg/dL)


SEM2

Significance3

Diets1



BUN 19.3a 18.5a 18.3a 19.0a 18.5a 19.0a 26.5b 26.5b 26.5b 26.5b 26.0b 26.5 b 0.57 NS NS NS *

Blood Glucose 73.0a 73.5a 74.5a 73.8a 73.3a 72.3a 94.0b 96.3b 95.5b 96.5b 96.5b 96.0b 0.86 NS NS NS *

Creatinine 2.4a 2.5a 2.4a 2.4a 2.5a 2.4a 1.6b 1.7b 1.5b 1.6b 1.5b 1.6b 0.09 NS NS NS *


with sulphur levels. NS stand for Non significant (P>0.05) and * stand for Significant (P<0.05). a,b,c Means in a row with different superscripts differ significantly (p<0.05). BUN stand for Blood urea nitrogen.

62

Table 4.4 Effect of different levels of potassium nitrate with or without sulphur on hematology in post weaning Lohi


Items


SEM2




Haemoglobin (g/dL) 14.0a 13.5abc 14.0a 14.3a 13.5abc 13.8ab 11.3cd 11.3cd 11.0d 11.5bcd 11.0d 11.0d 0.48 NS NS NS *

Methemoglobin (%) 1.7 1.8 2.0 2.0 2.0 2.2 2.2 2.0 2.0 2.0 1.8 1.7 0.14 NS NS NS NS

Neutrophils (%) 45.3a 43.8a 45.0a 44.3a 44.5a 44.0a 38.5b 39.0b 37.5b 37.5b 39.5b 39.3b 0.96 NS NS NS *

Lymphocytes (%) 43.3a 47.5a 46.8a 46.3a 46.5a 44.5a 54.0b 53.5b 56.3b 56.3b 53.0b 54.0b 0.92 * NS * *

Monocytes (%) 3.8 3.3 2.8 3.5 2.8 3.8 2.8 2.5 2.3 2.0 3.0 2.5 0.50 NS NS NS NS

Eosinophils (%) 6.5 4.3 4.8 4.5 4.8 6.3 4.3 4.5 3.8 4.0 4.0 3.8 0.82 NS NS NS NS

Basophils (%) 1.3 1.3 0.8 1.5 1.5 1.5 0.5 0.5 0.3 0.3 0.5 0.5 0.38 NS NS NS NS

Platelets (k/µL) 576.3 558.8 561.3 567.5 577.5 563.8 547.5 558.8 551.3 548.8 561.3 541.3 10.58 NS NS NS NS


with sulphur levels. NS stand for Non significant (P>0.05) and * stand for Significant (P<0.05). a,b,c Means in a row with different superscripts differ significantly (p<0.05).

63

Table 4.5 Effect of different levels of potassium nitrate with or without sulphur on nitrogen balance in post weaning

Lohi sheep and Teddy goat

Items

(g/day)


SEM2

Significance3

Diets1



Nitrogen Intake 21.3a 21.4a 21.4a 21.4a 21.3a 21.2a 13.1b 13.2b 13.1b 13.2b 13.1b 13.1b 0.06 NS NS NS *

Faecal Nitrogen 7.4c 7.4c 7.6b 7.6b 7.6b 7.8a 3.7 e 3.6e 3.7e 3.8e 3.8e 3.9d 0.03 * * * *

Urinary Nitrogen 6.6ab 6.7a 6.2bc 6.3abc 6.2bc 5.6d 6.2 bc 6.3abc 6.1c 6.1c 5.9cd 5.7d 0.08 * * * *

Nitrogen Balance 7.3b 7.3b 7.6ab 7.5ab 7.5ab 7.8b 3.2 cd 3.3cd 3.3cd 3.3cd 3.4cd 3.5c 0.05 * * * *


with sulphur levels. NS stand for Non significant (P>0.05) and * stand for Significant (P<0.05). a,b,c Means in a row with different superscripts differ significantly (p<0.05).

64

Table 4.6 Effect of different levels of potassium nitrate with or without sulphur on growth performance in post

weaning Lohi sheep and Teddy goat

Items


SEM2

Significance3

Diets1



Weight gain (g/d) 138b 138b 142b 142b 142b 146a 62d 62d 61d 62d 65de 66d 0.77 * * * *

Feed consumed (g/d) 741a 742a 742a 742a 741a 735a 455b 457b 456b 458b 456b 456b 2.13 NS NS NS *

Feed conversion ratio 5.3c 5.3c 5.2cd 5.2cd 5.2cd 5.0d 7.4a 7.4a 7.4a 7.3a 7.0b 6.9b 0.07 * NS * *

1PN0, PN3 and PN6 stand for potassium nitrate at 0, 3 and 6%, respectively. S0 and S4 stand for 0 and 0.4% sulphur, respectively. 2SEM stand for Standard error mean. 3A, B, C and ABC stand for Nitrate levels, Sulfur levels, Species and their Interaction, respectively while AB stand for Interaction of nitrate levels with sulphur levels. NS stand for Non significant (P>0.05) and * stand for Significant (P<0.05). a,b,c Means in a row with different superscripts differ significantly (p<0.05).

65

Table 4.7 Effect of different levels of potassium nitrate with or without sulphur on enteric methane production in

post weaning Lohi sheep and Teddy goat

Items


SEM2




Animal

(CH4 in ppm) 3.3a 3.3a 3.1abc 3.1abc 3.0bc 2.9c 3.3a 3.3a 3.1abc 3.0 bc 3.0 bc 2.9c 0.07 * NS * *

Animal (CO2 in ppm)

543.8a 543.8a 545.0a 545.0a 543.8a 547.5a 503.8b 503.8b 505.0b 506.3b 503.8b 507.5b 2.96 NS NS NS *

CH4 (Animal

- Air) : CO2

(Animal -

Air) 0.010cde 0.010cde 0.008def 0.008def 0.007ef 0.006f 0.014a 0.013ab 0.012abc 0.011bcd 0.010cd 0.009cdef 0.001 * NS NS NS

CH4

Reduction

(%) 0 1.99 18.98 20.41 27.21 32.65 0 3.95 15.77 24.18 25.81 31.91

Air (Methane gas in ppm) = 1.95; Air (carbon dioxide gas in ppm) = 405 1PN0, PN3 and PN6 stand for potassium nitrate at 0, 3 and 6%, respectively. S0 and S4 stand for 0 and 0.4% sulphur, respectively. 2SEM stand for Standard error mean. 3A, B, C and ABC stand for Nitrate levels, Sulfur levels, Species and their Interaction, respectively while AB stand for Interaction of nitrate levels with sulphur levels.NS stand for Non significant (P>0.05) and * stand for Significant (P<0.05). a,b,c Means in a row with different superscripts differ significantly (p<0.05).

66

Chapter 5

Effect of different levels of calcium ammonium nitrate with or without

sulphur on enteric methane production in Lohi sheep and Teddy goats at

growing age

Abstract

Mitigating enteric methane production from ruminants is an important issue of this era.

Feeding diets containing nitrates with or without sulphates could be used to lower enteric

CH4 production. This study was conducted to evaluate the influence of varying levels of

calcium ammonium nitrate (CAN) with or without S on the growth performance and

enteric CH4 production in growing Lohi sheep and Teddy goats in a 2×3×2 factorial

arrangement under Randomized Complete Block Design. In this experiment, 48 male

animals (24 Lohi sheep and 24 Teddy goats of approximately six months of age), were

randomly divided into 12 groups, 4 animals in each group. Twelve iso-nitogenous and iso-

caloric diets were formulated. The control diet CAN0-S0 contained neither CAN nor S.

Whereas CAN0-S4, CAN1.5-S0, CAN1.5-S4, CAN3-S0 and CAN3-S4 diets had 0%

CAN and 0.4% S, 1.5% CAN and 0% S, 1.5% CAN and 0.4% S, 3% CAN and 0% S and

3% CAN and 0.4% S, respectively. The Gases were measured by using infra-red biogas

analyzer. The nutrient intake and digestibility in both sheep and goats were similar (P>

0.05) across all diets. The animals fed diet containing 3% CAN with 0.4% S showed better

nitrogen balance. Enteric CH4 was 28.6% reduced (P<0.05) in sheep and 26.9% in goats

fed diet containing 3% CAN and 0.4% S compared to those fed C. Each sheep fed diet

containing 3% CAN and 0.4% S gained 144 g/day, whereas daily live weight gain by goats

fed diet containing 3% CAN with or without S was 58 g/day. Both in sheep and goats non-

significant differences (P> 0.05) were observed in blood metabolites including blood urea

nitrogen, glucose and Creatinine. Similar trend (P> 0.05) was observed in hematology.

None of the animals suffered from methemoglobinemia or polioencephalomalacia.

67

Introduction

In ruminants, the excess hydrogen is generated as a result of rumen fermentation

which is normally required to be removed for efficient and continuing microbial growth

(Immig, 1996). Methanogenic Archaea is generally active to eradicate hydrogen from

rumen by reducing CO2 to CH4 and H2O. Beauchemin et al. (2008) reported that for most

of the feeds utilized by ruminants, methanogenesis is ultimate route of the disposal of

hydrogen during anaerobic fermentation in the rumen. Enteric CH4 production inflicts a

dietary energy loss to the animal (Johnson and Johnson, 1995). Methane is also a potent

greenhouse gas responsible for global warming (Steinfeld et al., 2006). These realities

have compelled animal nutritionists to explore new aspects for strategic supplementation

in animal feed to abate CH4 production from ruminants.

One of the better options is to redirect rumen hydrogen into such a process that

produces useful products for the ruminants. Nitrate can be used as feed additive to lower

enteric CH4 production. They are converted into NH3 after using ruminal hydrogen, thus

reduce CH4 production. Various in-vitro (Guo et al., 2009, Allison and Reddy, 1984) and

in-vivo (Takahashi et al., 1998, Sar et al., 2004) studies have shown reduced CH4 yields.

Still it appears that more in-vivo work is required to evaluate the effect of nitrate as a

source to mitigate CH4 (Leng, 2008). However, use of nitrates as possible hydrogen sink to

CO2 has always been discouraged due to expected toxic effects of nitrite, which is

generated as an intermediate during the nitrate reduction to NH3 (Lewis, 1951). Leng

(2008) also suggested that S supplementation in ruminants diet might reduce the

accumulation of nitrate in the rumen. Sulphate as a reductant may also depress enteric CH4

production (Ungerfeld and Kohn, 2006).So the application of nitrates with Sulphate may

reduce the enteric CH4 production in ruminant animals.

Various commonly used in-vivo techniques to determine CH4 production include

measurements in confined respiration chambers (Frankenfield, 2010), CH4 estimations

from the VFA production (Hegarty and Nolan, 2007), ventilated hood techniques

(Odongo et al., 2007), isotopic (Hegarty et al., 2007) and naturally emitted CO2 as a

tracer gas in animals instead of SF6 (Madsen et al., 2010). According to Storm et al.

(2012), use of naturally emitted CO2 as a tracer gas to determine CH4 production is most

promising and economical method in developing countries.

68

Therefore, the present study was planed to determine the effect of varying levels of

CAN with or without S on growth performance and enteric CH4 production in growing

Lohi sheep and Teddy goats fed roughage based diet. The hypothesis was that nitrate and S

will mitigate the enteric CH4 production in both sheep and goats. Both nitrate and S will

give an additive effect on the CH4 reduction.


Experimental site


Sargodha, Pakistan.


In this experiment, 48 male animals at growing age (24 Lohi sheep and 24 Teddy goats of

approximately six months of age), were randomly divided into 12 groups having in each 4

animals. Each group was maintained in a separate pen measuring 0.30m x 0.30m. Animals

were fed separately. Twelve iso-nitrogenous (crude protein 18%) and iso-caloric (2.0 ME

Mcal/Kg) diets were formulated using 0, 1.5 and 3% CAN with or without 0.4% S

(anhydrous MgSO4). Calcium ammonium nitrate is made by adding calcium carbonate to a

slurry of ammonium nitrate resulting in a mixture of calcium nitrate and residual

ammonium nitrate. It crystallizes as a hydrated double salt: 5Ca (NO3)2•NH4NO3•10H2O.

In the experiment fertilizer grade CAN was used, it contained 8% calcium and 25%

nitrogen. Nonprotein nitrogen was same across all diets. The control diet CAN0-S0

contained neither CAN nor S. Whereas CAN0-S4, CAN1.5-S0, CAN1.5-S4, CAN3-S0

and CAN3-S4 diets had 0% CAN and 0.4% S, 1.5% CAN and 0% S, 1.5% CAN and 0.4%

S, 3% CAN and 0% S and 3% CAN and 0.4% S, respectively (Table 5.1). The total mixed

rations were offered twice a day and fresh clean water was made available round the clock

during experimental period. The animals were weighed fortnightly. This experiment lasted

for three months including 15 days of adaptation. Animals were gradually acclimated to

the diets with nitrate and S.


analyzed for DM and CP by using the procedures described by AOAC (1990). The NDF

and ADF were determined by the methods described by Van Soest et al. (1991).

69


experiment by using total collection method. For this purpose, all animals were shifted to


metabolic collection crates, each measuring 1.5 m x 1.2 m x 1.5 m, were used to collect

the urine and feces of individual animal. Metabolic collection crates consisted of a

collection tray and two plastic urine collection bowls. Removable trays were fitted on

floor of metabolic collection crates and animals stood on these trays. Removable urine

collection bowls were set beneath the floor of metabolic collection crates. Urine was

collected in urine collection bowls through the small hole at the bottom of the collection

tray. These bowls had measured amount of solution acidified with 50% H2SO4 to avoid N

losses during collection (Nisa et al., 2004). Feces and urine were collected, weighed and

representative samples were stored at -20 oC for further analysis. At the end of collection

period, urine and feces samples were thawed, composited and homogenized. Composite

samples were dried at 55 oC and ground through 1-mm screen. Feed and fecal samples

were analyzed for NDF and ADF by the methods as described by Van Soest et al. (1991)

and DM and CP were determined by the methods as described by AOAC (1990). At the

end of experiment blood samples (10 mL/animal) were collected by jugular vein

punctured into vacutainer tube containing 81 µL of 15% EDTA solution and analyzed in

local pathological laboratory for blood urea nitrogen, glucose, creatinine using cobas c

111 analyzer (Roche) and methemoglobin (MetHb; Evelyn and Malloy, 1938).

The enteric CH4 was analyzed at the end of the experiment by using method as

described by Madsen et al. (2010) in which naturally emitted CO2 was used as tracer gas

(Storm et al., 2012). Data of enteric methane production was recorded one hour after each

feeding. Methane production gradually increases and reaches peak values during first hour

of feeding and then decreases gradually (Sar et al., 2004). Each animal was kept in an

especially designed closed enclosure (each measuring 1.5 m x 1.2 m x 1.5 m) for 15

minutes and enteric methane production was measured during the last 5 minutes with the

help of infra red CH4 analyzer (Gasmet Dx-4030, Finland Table 2.7). For Zero point

calibration nitrogen gas was used. In order to minimize stress factor all animals were

made accustomed to enclosure 15 days prior to data collection.

The CO2 and CH4 in background air were measured at the same time. Measuring

the CH4 to CO2 ratio combined with the measuring of total CO2 produced, the amount of

CH4 was calculated as under:

70

CH4:CO2 = (a-b)/(c-d)

Where a is CH4 concentration in mixed eructed gas plus air, c is CO2 concentration

in mixed eructed gas plus air, b is CH4 in background air and d the CO2 in background air.


production was also similar and could be used as internal marker as described by Madsen

et al. (2010).



If the CH4 production rate is A, the CO2 entry rate B is same on all diets, and the

ratio of CH4 to CO2 is R, then the following equations apply:



The % CH4 reduction rate is then…………B (R1-R2) / BR1*100







Results

The nutrient intake and digestibility in both sheep and goats were similar (P> 0.05)

across all diets (Table 5.2). The animals fed diet containing 3% CAN with 0.4% S showed

better nitrogen balance (Table 5.5). Daily live weight gain was 138, 139, 139, 141, 141 and

144 g/day in Lohi sheep and was 54, 55, 57, 56, 58 and 58 g/d in Teddy goats fed CAN0-

S0, CAN0-S4, CAN1.5-S0, CAN1.5-S4, CAN3-S0 and CAN3-S4 diets, respectively

(Table 5.6). The FCR in sheep fed CAN0-S0, CAN0-S4, CAN1.5-S0, CAN1.5-S4, CAN3-

S0 and CAN3-S4 diets were 9.0, 8.9, 9.0, 8.8, 8.9 and 8.7, respectively. Moreover, the

FCR were 11.4, 11.3, 10.8, 10.9, 10.6 and 10.7 in goats fed CAN0-S0, CAN0-S4,

71

CAN1.5-S0, CAN1.5-S4, CAN3-S0 and CAN3-S4 diets, respectively (Table 5.6). The best

feed conversion values (P<0.05) were observed in sheep fed CAN3-S4 diet. However, best

FCR was observed in goats fed CAN3-S0 diet. Both in sheep and goats non-significant

differences (P> 0.05) were observed in blood metabolites including blood urea nitrogen,

glucose and Creatinine (Table 5.3). Similar trend (P> 0.05) was observed in hematology

(Table 5.4). None of the animals suffered from methemoglobinemia or

polioencephalomalacia. The difference in enteric CH4 reduction was 0, 2.31, 2.61, 11.80,

11.22 and 28.68% in Lohi sheep and 0, 1.88, 2.65, 10.89, 16.33 and 26.95% in Teddy goats

fed CAN0-S0, CAN0-S4, CAN1.5-S0, CAN1.5-S4, CAN3-S0 and CAN3-S4 diets,

respectively (Table 5.7).

Discussion

In this study CAN was fed in diet upto 3.14% of DM which is high as compared to

Zijderveld et al. (2010) who in a sheep experiment fed both CAN and sulphate @ 2.6% of

DM.


The results of Sophea and Preston (2011) and Zijderveld et al. (2010) are in

accordance with the results of this study. They observed that nitrate supplemented diets did

not affect the feed intake in goats and sheep. Similar trend was also reported by Sangkhom

et al. (2012) who observed no difference in DMI in cattle fed diets supplemented with

nitrate or urea as NPN source.In a study with dietary nitrates Phuong et al. (2012)

observed reduced intake with sulphate supplemented diets when included at the rate of

0.8% S. Similar results were also reported by Silivong et al. (2011) who in their

experiment on goats observed lower intake by animals fed diets with 0.8% S. Similar DMI

in this study might be due to lower accumulation of nitrite in the rumen, which is produced

as an intermediate product from dietary nitrates. Nitrite accumulation in the rumen is

generally considered responsible for lower DMI.

However, higher DMI by animals fed urea rather than nitrates was also observed

by same workers. They suggested that lower intake of feed might be due to the palatability.

Our results were not in agreement with those of Hulshof et al. (2010) who reported that

nitrate supplemented diets reduced the feed intake. In their experiment, beef cattle were

72

fed sugarcane-based diets which might favoured Ruminal nitrite accumulation, resulting in

lower feed intake. Bruning-Fann and Kaneene (1993) also reported similar results.

Ruminal nitrite accumulation lowers feed digestibility as it inhibits some of the bacterial

colonies. This reduced digestibility lowers VFA production which in turn negatively

affects microbial biomass resulting in reduced intake by the animal.

Our nutrient digestibility results are in concordance with those of Thanh et al. (2012) who

in a sheep trial observed similar digestibility with either urea or nitrate as NPN source. In

another experiment, Nolan et al. (2010) also observed no differences in DMD when

nitrates were supplemented in diets. Feed intake is one of the important factors which

affect nutrient digestibility. In this study we observed similar intake across all the diets.

This might resulted in unaltered digestibility among all the diets.


The results of present study agreed with the findings of Phuong et al. (2011),

Sangkhom et al. (2011), Guo et al. (2009) and Guangming et al. (2010) who observed CH4

reduction in animals fed nitrate supplemented diets. Leng and Preston (2010) who reported

that nitrates rather than urea resulted in better enteric CH4 reductions also supported our

findings. Our results were in consistent with the findings of Leng (2008) who observed

that dietary inclusion of 1% PN in ruminant diet could result in 10% enteric CH4

reduction. If animals are slowly acclimated to nitrate supplemented diets, it favours

quantitative increase of nitrate reducing bacteria in the rumen. According to Morgavi et al.

(2010) dietary nitrate in ruminant's diet favours NH3 production rather than CH4 formation.

One mol nitrate could reduce similar amount of CH4, thus producing one mol of NH3

(Leng, 2008). Non-significant changes in hematology and blood metabolites indicated

same health status of all animals. In this experiment, none of the animal showed any kind

of abnormal behaviour or signs of illness during the whole experimental period. This

indicated that none of the animals suffered from nitrate or S toxicity. This indicated that

animals became adapted to nitrates thus resulting in improved nitrate reducing capacity of

ruminal microbes.

Variable results in enteric CH4 reduction were reported by various researchers.

Zijderveld et al. (2010) and Nolan et al. (2010) reported 32 and 23% CH4 reductions

respectively in sheep fed nitrate supplemented diets. Sangkhom et al. (2012) reported 27%

enteric CH4 reductions. Sixty percent reductions were observed by Sophea and Preston

73

(2011) in goats. Zijderveld et al. (2011) observed only 16% CH4 reductions in dairy.

However, Hulshof et al. (2010) and Sophal et al. (2013) noticed 32 and 43% enteric CH4

abatement respectively in cattle fed nitrate supplemented diets.

The variability among the results of enteric CH4 production might be attributed to

various CH4 estimating techniques. Hulshof et al. (2010) and Zijderveld et al. (2010)

measured CH4 production by SF6 technique. However Zijderveld et al. (2011) used

calorimetric chambers to estimate CH4 emissions. Nolan et al. (2010) observed CH4 by

measuring VFAs. Sangkhom et al. (2012) determined enteric CH4 production using CO2 as

a tracer gas. Among various techniques SF6 is mostly used in-vivo technique but it gives

variable results than calorimetric chambers (Pinares-Patino et al., 2011; Grainger et al.,

2007; Johnson et al., 1994).

Growth performance

In this study we observed better daily live weight gains and feed conversion ratios in

animals fed diets containing higher levels of nitrate as compared to those fed urea

supplemented diets. Similar results were reported by Sangkhom et al. (2012) and Trinh

phuc Hao et al. (2009). Nitrates as compared to urea resulted in better enteric CH4

reduction, which might improved the average daily weight gain of animals. Contrary

results were reported by Zijderveldet al. (2010) and Thanh et al. (2012) who observed no

differences in growth performance in sheep fed nitrate supplemented diets. Zijderveld et

al. (2011) in their study on dairy cows also reported similar results. In a goat trial Sophea

and Preston (2011) also observed no increase in growth rates. Sophal et al. (2013) and

Phuong et al. (2012) reported enteric CH4 reductions with nitrate supplemented diets but

did not observe any improvement in growth rates. Tillman et al. (1965) in their study

found that nitrates negatively affect the growth rates without any toxicity signs.

Enteric CH4 reduction improves the nutritive value of the feed resulting into better

animal growth performance (Blaxter and clapperton, 1965).Similar findings were also

observed by Beauchemin et al. (2008) in their study. If there is no nitrite accumulation in

the rumen it favours the emission of ruminal NH3, at the rate which is equivalent to

assimilatory growth rate of ruminal microbes. As compared to CH4 formation, electron

transfer of NH3 conserves more energy in ruminal end products, during microbial cell

74

synthesis. This stored additional energy might favoured microbial growth resulting into

better feed efficiency with nitrate supplemented diets (Leng, 2008).

Blood Chemistry

In this study we did not observe any abnormal animal behaviour or symptoms of

illness. We found no difference in blood metabolites or haematology among nitrate or urea

supplemented diets. No nitrite toxicity observed during the experiment was possibly either

because animals were slowly adapted to nitrate feeding or the nitrate:S ratio in the diet

might be balanced in a way that the activity of S reducing bacteria was maintained it

promoted reduction of nitrite to NH3 (Leng, 2008). Our observations were in concordance

with findings of other researchers (Carver and Pfander, 1974; Alaboudi and Jones, 1985).

Minor signs of methemoglobinemia were reported by Zijderveld et al. (2010) when sheep

were fed nitrate supplemented diets. Similar findings were also reported by Nolan et al.

(2010). Feeding dietary nitrates abruptly without acclimation may lead to nitrite

accumulation in the rumen which may result in nitrate toxicity. Nitrite in blood oxidizes

ferrous ions in haemoglobin (Hb) to ferric ions to form MetHb (Zijderveld et al., 2011).

This condition in acute cases might be lethal (Zijderveld et al., 2010). Higher inclusion

levels of nitrate can develop symptoms of methemoglobinemia (Bradley et al., 1939).

Blood urea nitrogen concentrations are very high for goats possibly due to breed

difference. Moreover, its numbers can vary greatly between different locations and

different laboratories.

Conclusion

Diets containing CAN in combination with S did not only reduce enteric CH4

production from Lohi sheep but it also improved their growth performance. As compared

to Teddy goats, Lohi Sheep showed better enteric CH4 reductions by using the diets

containing CAN in combination with S.

75


1CAN0, CAN1.5 and CAN3 stand for calcium ammonium nitrate at ~ 0, 1.5 and 3%, respectively.

S0 and S4 stand for 0 and 0.4% sulphur, respectively.

Ingredients

Diets1

CAN0 CAN1.5 CAN3

S0 S4 S0 S4 S0 S4 Wheat Straw 35.00 35.00 35.00 35.00 35.00 35.00

Cotton seed meal 10.00 10.00 10.00 10.00 10.00 10.00

Rice bran 10.00 10.00 10.00 10.00 10.00 10.00


Hay (Lucerne) 21.00 21.00 21.00 21.00 21.00 21.00

Enzose 14.00 14.00 14.00 14.00 14.00 14.00


Sodium Chloride 1.00 1.00 1.00 1.00 1.00 1.00


Calcium ammonium nitrate 0.00 0.00 1.57 1.57 3.14 3.14

Urea 1.70 1.70 0.85 0.85 0.00 0.00


Dry Matter 88.08 89.58 88.80 90.30 89.52 91.08

Crude Protein 18.00 18.00 17.99 17.99 17.98 17.98




76

Table 5.2 Effect of different levels of calcium ammonium nitrate with or without sulphur on Nutrient intake and

their digestibility in growing Lohi male sheep and Teddy goat

Items

Lohi Sheep Teddy Goat

SEM2

Significance3

Diets1

CAN0 CAN1.5 CAN3 CAN0 CAN1.5 CAN3


Nutrient Intake,g/d

DM 1242a 1243a 1246a 1241a 1243a 1243a 618 b 616 b 618 b 617 b 618 b 616 b 1.73 NS NS NS *

CP 224 a 224 a 224 a 223 a 224 a 224 a 111 b 110 b 111 b 110 b 111 b 110 b 0.33 NS NS NS *

NDF 551 a 551 a 553 a 550 a 552 a 552 a 274 b 273 b 274 b 274 b 274 b 273 b 0.78 NS NS NS *

ADF 390 a 390 a 391 a 389 a 390 a 390 a 194 b 193 b 194 b 194 b 194 b 193 b 0.60 NS NS NS *

Digestibility,%

DM 53.0de 54.0bcde 52.8e 53.5cde 53.3cde 54.8abcde 57.5abc 58.0ab 56.0abcde 57.0abcde 57.3abcd 58.5 a 0.89 NS NS NS *

CP 65.5c 64.5cd 64.5cd 64.5cd 64.5cd 63.0d 72.5a 72.5a 72.0a 72.3a 71.3ab 70.0b 0.31 * * * *


ADF 43.0 42.0 42.5 43.3 41.5 42.8 43.5 43.8 44.0 46.0 43.0 45.3 0.92 NS NS NS NS

1CAN0, CAN1.5 and CAN3 stand for calcium ammonium nitrate at ~ 0, 1.5 and 3% respectively. S0 and S4 stand for 0 and 0.4% sulphur, respectively. 2SEM stand for Standard error mean. 3A, B, C and ABC stand for Nitrate levels, Sulfur levels, Species and their Interaction, respectively while AB stand for Interaction of nitrate levels with sulphur levels. NS stand for Non significant (P>0.05) and * stand for Significant (P<0.05).a,b,c,d,e,f Means in a row with different superscripts differ significantly (p<0.05). DM, CP, NDF and ADF stand for Dry matter, Crude Protein, Neutral detergent fibre and Acid detergent fibre respectively.

77

Table 5.3 Effect of different levels of calcium ammonium nitrate with or without sulphur on blood metabolites in


Items

(mg/dL)


SEM2

Significance3

Diets1



BUN 19.0b 18.5b 19.0b 18.3b 18.5b 19.3b 26.5a 26.0a 26.5a 26.5a 26.5a 26.5a 0.57 NS NS NS *

Blood Glucose 72.3b 73.3b 73.8b 74.5b 73.5b 73.0b 96.0a 96.5a 96.5a 95.5a 96.3a 94.0a 0.86 NS NS NS *

Creatinine 2.4b 2.5b 2.4b 2.4b 2.5b 2.4b 1.6a 1.5a 1.6a 1.5a 1.7a 1.6a 0.09 NS NS NS *

1CAN0, CAN1.5 and CAN3 stand for calcium ammonium nitrate at ~ 0, 1.5 and 3% respectively. S0 and S4 stand for 0 and 0.4% sulphur, respectively. 2SEM

stand for Standard error mean. 3A, B, C and ABC stand for Nitrate levels, Sulfur levels, Species and their Interaction, respectively while AB stand for

Interaction of nitrate levels with sulphur levels. NS stand for Non significant (P>0.05) and * stand for Significant (P<0.05).a,b,c,d,e,f Means in a row with

different superscripts differ significantly (p<0.05). BUN stand for blood urea nitrogen.

78

Table 5.4 Effect of different levels of calcium ammonium nitrate with or without sulphur on hematology in growing

Lohi male sheep and Teddy goat

Items


SEM2

Significance3

Diets1



Haemoglobin (g/dL) 13.7ab 13.5abc 14.2a 14.0a 13.5abc 14.0a 11.0d 11.0d 11.5bcd 11.0d 11.3cd 11.3cd 0.47 NS NS NS *


Neutrophils (%) 44.0abc 44.5a 44.3ab 45.0a 43.8abcd 45.3a 39.3cde 39.5bcde 37.5e 37.5e 39.0de 38.5e 0.96 NS NS NS *

Lymphocytes (%) 44.5a 46.5a 46.3a 46.8a 47.5a 43.3a 54.0b 53.0b 56.3b 56.3b 53.5b 54.0b 0.92 * NS * *






stand for Standard error mean. 3A, B, C and ABC stand for Nitrate levels, Sulfur levels, Species and their Interaction, respectively while AB stand for Interaction of nitrate levels with sulphur levels. NS stand for Non significant (P>0.05) and * stand for Significant (P<0.05).a,b,c,d,e,f Means in a row with different superscripts differ significantly (p<0.05).

79

Table 5.5 Effect of different levels of calcium ammonium nitrate with or without sulphur on nitrogen balance in


Items

(g/day)


SEM2

Significance3

Diets1




Faecal Nitrogen 12.4b 12.7b 12.7b 12.7b 12.7b 13.3a 4.9d 4.9d 5.0cd 5.0cd 5.1cd 5.4c 0.09 * * * *

Urinary Nitrogen 16.1a 15.7a 15.7a 15.5a 15.6a 14.9b 10.0c 9.9cd 9.7cd 9.8cd 9.6cd 9.9d 0.11 * * * *

Nitrogen Balance 7.4b 7.4b 7.4b 7.5ab 7.5ab 7.7a 2.9c 2.9c 3.1c 3.0c 3.1c 3.1c 0.05 * * * *


stand for Standard error mean. 3A, B, C and ABC stand for Nitrate levels, Sulfur levels, Species and their Interaction, respectively while AB stand for

Interaction of nitrate levels with sulphur levels. NS stand for Non significant (P>0.05) and * stand for Significant (P<0.05).a,b,c,d,e,f Means in a row with

different superscripts differ significantly (p<0.05).

80

Table 5.6 Effect of different levels of calcium ammonium nitrate with or without sulphur on growth performance in


Items


SEM2

Significance3

Diets1



Weight gain (g/d) 138b 139b 139b 141ab 141ab 144a 54c 55c 57c 56c 58c 58c 0.95 * NS * *

Feed consumed (g/d) 1242a 1243a 1246a 1241a 1243a 1243a 618 b 616 b 618 b 617 b 618 b 616 b 1.73 NS NS NS *

Feed conversion ratio 9.0d 8.9d 9.0d 8.8d 8.9d 8.7d 11.4 a 11.3ab 10.8bc 10.9abc 10.6c 10.7c 0.10 * NS * *

1CAN0, CAN1.5 and CAN3 stand for calcium ammonium nitrate at ~ 0, 1.5 and 3% respectively. S0 and S4 stand for 0 and 0.4% sulphur, respectively. 2SEM stand for Standard error mean. 3A, B, C and ABC stand for Nitrate levels, Sulfur levels, Species and their Interaction, respectively while AB stand for Interaction of nitrate levels with sulphur levels. NS stand for Non significant (P>0.05) and * stand for Significant (P<0.05).a,b,c,d,e,f Means in a row with different superscripts differ significantly (p<0.05).

81

Table 5.7 Effect of different levels of calcium ammonium nitrate with or without sulphur on enteric methane

production in growing Lohi male sheep and Teddy goat

Items


SEM2




Animal

(CH4 in ppm) 45.2b 44.2bc 44.2bc 40.2de 37.3f 33.1g 50.2a 49.3a 49.1a 45.2b 42.3cd 37.6ef 0.57 * * * *

Animal (CO2 in ppm) 865.8a 865.8a 867.0a 867.0a 865.8a 869.5a 865.8a 825.8b 827.0b 828.3b 825.8b 829.8b 2.93 NS NS NS *

CH4 (Animal

- Air) : CO2

(Animal -

Air) 0.094c 0.092c 0.092c 0.083de 0.077e 0.067f 0.115a 0.113a 0.112a 0.103b 0.097bc 0.085d 0.0014 * * * *

CH4

Reduction

(%) 0 2.31 2.61 11.80 18.22 28.68 0 1.88 2.65 10.89 16.33 26.95

Air (Methane gas in ppm) = 1.97; Air (carbon dioxide gas in ppm) = 408,

1CAN0, CAN1.5 and CAN3 stand for calcium ammonium nitrate at ~ 0, 1.5 and 3% respectively. S0 and S4 stand for 0 and 0.4% sulphur, respectively. 2SEM stand for Standard error mean. 3A, B, C and ABC stand for Nitrate levels, Sulfur levels, Species and their Interaction, respectively while AB stand for Interaction of nitrate levels with sulphur levels. NS stand for Non significant (P>0.05) and * stand for Significant (P<0.05).a,b,c,d,e,f Means in a row with different superscripts differ significantly (p<0.05).

82

Chapter 6

Effect of different levels of sodium nitrate with or without sulphur on

enteric methane production in Lohi sheep and Teddy goats at fattening

age

Abstract

Ruminants are major contributorsof methane (CH4) production. Serious global efforts are

required to reduce CH4 produced through their digestive tract, which is generally termed as

enteric methane production. Feeding diets containing nitrates and/or sulphates could be

used to reduce enteric methane production. The study was conducted to evaluate the

influence of varying levels of sodium nitrate (SN) with or without S on the growth

performance and enteric methane production in Lohi sheep and Teddy goats in a 2×3×2

factorial arrangement under Randomized Complete Block Design. In the experiment, 48

male animals (24 Lohi sheep and 24 Teddy goats of approximately nine months of age),

were randomly divided into 12 groups, 4 animals in each group. Twelve iso-nitogenous

and iso-caloric diets were formulated. The control diet SN0-S0 contained neither SN nor S.

Whereas SN0-S4, SN2.5-S0, SN2.5-S4, SN5-S0 and SN5-S4 diets had 0% SN and 0.4%

S, 2.5% SN and 0% S, 2.5% SN and 0.4% S, 5% SN and 0% S and 5% SN and 0.4% S,

respectively. Nonprotein nitrogen was same across all diets. The Gases were measured by

using infra-red biogas analyzer. The nutrient intake and digestibility in both sheep and

goats were similar (P> 0.05) across all diets. The animals fed diet containing 5% SN with

0.4% S showed better nitrogen balance. The enteric CH4 was 19.6% reduced (P<0.05) in

sheep and 18.2% in goats fed diet containing 5% SN and 0.4% S compared to those fed C.

Daily live weight gain of both sheep and goats fed diet containing 5% SN and 0.4% S was

143 and 59 g/day, respectively. The best feed conversion values (P<0.05) were observed in

sheep fed both SN2.5-S4 and SN5-S4 diets. However, best FCR was observed in goats fed

SN5-S0 and SN5-S4 diets. Both in sheep and goats non-significant differences (P> 0.05)

were observed in blood metabolites including blood urea nitrogen, glucose and Creatinine.

Similar trend (P> 0.05) was observed in hematology. None of the animals suffered from

methemoglobinemia or polioencephalomalacia.

83

Introduction

Among various challenges of XXI century, continuous enteric CH4 emission from

ruminants is a serious threat to climate change. Forster et al. (2007) pointed out that CH4

has 25 times more global warming potential than CO2. It is predicted that growing demand

of livestock will boost up enteric methane production resulting in its 30% growth by year

2020 (O’Mara, 2010). O’Mara (2010) highlighted the importance to develop new means to

abate enteric CH4 in ruminants. In ruminants, the excess hydrogen which is generated as a

result of rumen fermentation is normally required to be removed for efficient and

continuous microbial growth (Beaucheminet al., 2008). This hydrogen is removed by

reducing CO2 into CH4 which is eventually emitted by eructation. Methane emission is also

an energy loss (Johnson and Johnson, 1995). This CH4 emission can be reduced by

substituting the CO2 with another electron acceptor. Nitrates can replace CO2 as an

electron acceptor. They are reduced to nitrite and then to NH3. One mol nitrate reduces

CH4 in equivalent amount which in turn produces one mol of NH3 (Leng, 2008).

So, the supplementation of nitrates in diet may reduce the CH4 emission by

ruminants. However, in past their use was limited because of possible risk of nitrate/nitrite

toxicity. While, Leng (2008) proposed the addition of nitrates to roughage based diet may

significantly reduce enteric methane production without causing nitrate toxicity, in slowly

acclimatized animals. Furthermore, he also stated that supplementation of S in nitrate

containing diets may also be effective in reducing nitrite accumulation in rumen. He urged

the need of more in-vivo work to evaluate the efficiency of nitrates and sulphates to abate

CH4 production in various ruminant animals.

It is very complex to measure CH4 production from individual animals because of its

gaseous properties. One of the important considerations while attempting these

experiments is the precision and accuracy of CH4 measuring methods or techniques.

Various methods used to determine CH4 production from animals include respiration

chambers (Frankenfield, 2010), CH4 estimations from the VFA production (Hegarty

and Nolan, 2007), ventilated hood techniques (Odongoet al., 2007), isotopic (Hegarty

et al., 2007), non-isotopic tracer techniques (Johnson et al., 1994) and tunnel

technique (Murray et al., 2007). Recently a new method for estimating CH4 emissions

from ruminants was found to be more effective. It is based on the use of naturally emitted

CO2 as a tracer gas (Storm et al., 2012; Madsen et al., 2010).

84

Therefore, the present study was planed to determine the effect of varying levels of

SN with or without S on growth performance and enteric methane production in fattening

Lohi sheep and Teddy goats fed roughage based diet. The hypothesis was that nitrate and S

will mitigate the enteric CH4 production in both sheep and goats. Both nitrate and S will

give an additive effect on the CH4 reduction.


Experimental site


Sargodha, Pakistan.


In this experiment, 48 male animals (24 Lohi sheep and 24 Teddy goats of

approximately nine months of age), were randomly divided into 12 groups, 4 animals in

each group. Each group was maintained in a separate pen measuring 0.30m x 0.30m.

Animals were fed separately. Twelve iso-nitrogenous (crude protein 18%) and iso-caloric

(2.0 ME Mcal/Kg) diets were formulated using 0, 2.5 and 5% SN with or without 0.4% S

(anhydrous MgSO4). Nonprotein nitrogen was same across all diets. The control diet SN0-

S0 contained neither SN nor S. Whereas SN0-S4, SN2.5-S0, SN2.5-S4, SN5-S0 and SN5-

S4 diets had 0% SN and 0.4% S, 2.5% SN and 0% S, 2.5% SN and 0.4% S, 5% SN and

0% S and 5% SN and 0.4% S, respectively (Table 6.1). The total mixed rations were

offered twice a day and fresh clean water was made available round the clock during

experimental period. The animals were weighed fortnightly. The total experimental period

was 90 days including 15 days of adaptation. Animals were gradually acclimated to the

diets with nitrate and S.


analyzed for DM and CP using the procedures described by AOAC (1990). The NDF and

ADF were determined by the methods described by Van Soest et al. (1991).


experiment. For digestibility and nitrogen balance trials, all animals were shifted to

85


metabolic collection crates, each measuring 1.5 m x 1.2 m x 1.5 m, were used to collect

the urine and feces of individual animal and then urine and feces were mixed by animal.

Metabolic collection crates consisted of a collection tray and two plastic urine collection

bowls. Removable trays were fitted on floor of metabolic collection crates and animals

stood on these trays. Removable urine collection bowls were set beneath the floor of

metabolic collection crates. During total collection method, urine was collected in urine

collection bowls through the small hole at the bottom of the collection tray. These bowls

had measured amount of solution acidified with 50% H2SO4 to avoid N losses during

collection (Nisa et al., 2004). Feces and urine were collected, weighed and representative

samples were stored at -20oC for further analysis. At the end of collection period, urine

and feces samples from individual pens were thawed, composited and homogenized.

Composite samples were dried at 55oC and ground through 1-mm screen. Feed and fecal

samples were analyzed for NDF and ADF by the methods described by Van Soest et al.

(1991) and DM and CP were determined by methods described by AOAC (1990). At the

end of experiment blood samples (10 mL/animal) were collected by jugular vein

punctured into vacutainer tube containing 81 µL of 15% EDTA solution and analyzed in

local pathological laboratory for blood urea nitrogen, glucose, creatinine using cobas c

111 analyzer (Roche) and methemoglobin (MetHb; Evelyn and Malloy, 1938).

The enteric CH4 was analyzed at the end of the experiment using method described

by Madsen et al. (2010) in which naturally emitted CO2 was used as tracer gas (Storm et

al., 2012). Data of enteric methane production was recorded one hour after each feeding.

Methane production gradually increased and reached peak values during first hour of

feeding and then decreased gradually (Sar et al., 2004). Each animal was kept in an

especially designed closed enclosure (each measuring 1.5 m x 1.2 m x 1.5 m) for 15

minutes and enteric methane production was measured during the last 5 minutes with the

help of infra red CH4 analyzer (Gasmet Dx-4030, Finland Table 2.7). For Zero point

calibration nitrogen gas was used. In order to minimize stress factor all animals were

made accustomed to enclosure 15 days prior to data collection.

The CO2 and CH4 in background air were measured at the same time. Measuring

the CH4 to CO2 ratio combined with the measuring of total CO2 produced, the amount of

CH4 was calculated as under:

86

CH4:CO2 = (a-b)/(c-d)

Where a is CH4 concentration in mixed eructed gas plus air, c is CO2 concentration

in mixed eructed gas plus air, b is CH4 in background air and d the CO2 in background air.


production was also similar and could be used as internal marker as described by Madsen

et al. (2010).



If the CH4 production rate is A, the CO2 entry rate B is same on all diets, and the

ratio of CH4 to CO2 is R, then the following equations apply:










Results

The nutrient intake and digestibility in both sheep and goats were similar (P> 0.05)

across all diets (Table 6.2). The animals fed diet containing 5% SN with 0.4% S showed

better nitrogen balance (Table 6.5). Daily live weight gain was 138, 139, 142, 143, 141 and

143 g/day in Lohi sheep and was 55, 54, 57, 57, 59 and 59 g/d in Teddy goats fed SN0-S0,

SN0-S4, SN2.5-S0, SN2.5-S4, SN5-S0 and SN5-S4 diets, respectively (Table 6.6). The

FCR in sheep fed SN0-S0, SN0-S4, SN2.5-S0, SN2.5-S4, SN5-S0 and SN5-S4 diets were

12.7, 12.5, 12.3, 12.2, 12.3 and 12.2, respectively. Moreover, the FCR were 13.9, 14.1,

87

13.5, 13.5, 13.0 and 13.0 in goats fed SN0-S0, SN0-S4, SN2.5-S0, SN2.5-S4, SN5-S0 and

SN5-S4 diets, respectively (Table 6.6). The best feed conversion values (P<0.05) were

observed in sheep fed both SN2.5-S4 and SN5-S4 diets. However, best FCR was observed

in goats fed SN5-S0 and SN5-S4 diets. Both in sheep and goats non-significant differences

(P> 0.05) were observed in blood metabolites including blood urea nitrogen, glucose and

Creatinine (Table 6.3). Similar trend (P> 0.05) was observed in hematology (Table 6.4).

None of the animals suffered from methemoglobinemia or polioencephalomalacia. The

difference in enteric CH4 reduction was 0, 2.20, 3.09, 8.06, 12.46 and 19.62% in Lohi

sheep and 0, 3.12, 3.94, 7.66, 11.55 and 18.27% in Teddy goats fed SN0-S0, SN0-S4,

SN2.5-S0, SN2.5-S4, SN5-S0 and SN5-S4 diets, respectively (Table 6.7).

Discussion

Ngoc Huyen Le Thi, (2010) compared SN, ammonium nitrate and urea as sources

of fermentable nitrogen in a basal diet of NaOH treated rice straw and cotton seed meal.

Amount of SN, ammonium nitrate and urea used were 6.6%, 3% and 2.2% of DM

respectively. In this study lower levels of SN (~ 5% SN) were evaluated with 0.4S.

Previously Phuong et al. (2012) and Silivong et al. (2011) in their studies used higher

levels nitrate and sulphate which affected feed intake.


In present study we observed that nitrate fed animals ate the same amount of DM

as those fed urea in their diets. These observations are similar to those reported by

Sangkhom et al. (2012) who observed similar DMI in cattle fed nitrates or urea as NPN

source in their diets. Consistent findings of Zijderveld et al. (2010) in sheep trial and

Sophea and Preston (2011) in goat trial also supported the findings of present study. The

probable reason to this might be the slow conversion of nitrates to nitrite which as a result

did not accumulate in the rumen. Generally nitrite accumulation in the rumen is considered

responsible for reduced DMI. Phuong et al. (2012) observed non-significant difference in

DMI by animals fed nitrate or urea in their diet but reported decreased intake with sulphate

supplemented diet. Similar finding was also reported by Silivong et al. (2011) who

observed reduced molasses intake when supplemented by sulphates at the rate of 0.8% in

the diet. In our present study sulphate was included at a rate of 0.4% S which was lower as

compared to that of diets used by Phuong et al. (2012) or Silivong et al. (2011) in their

88

studies on cattle or goats respectively. Sulphur addition at higher doses might have caused

bitterness in taste of the diets, which could have resulted in reduced intakes. Other findings

(Hulshof et al., 2010; Bruning-Fann and Kaneene, 1993; Weichenthal et al., 1963) did not

agree with our present results who reported that nitrate supplemented diets negatively

affect DMI. Nitrite accumulation depresses the colonies of some bacterial species growth,

which could lower the feed digestibility. It negatively affects VFA production, reduced

microbial biomass thus lowering intake by the animal. Results by Thanh et al. (2012)

supported our findings on nutrient digestibility. They reported similar digestibility with

nitrate or urea supplemented diets. Our results were also supported by the findings of

Nolan et al. (2010) who used nitrates as feed additive in diets. Feed intake can be

categorized as important factors among many others which influence digestibility. In this

study feed consumption remained unchanged which might have resulted in similar

digestibility.


In this study we observed significant enteric CH4 reductions with nitrate supplemented

diets. Various studies (Phuong et al., 2011: Sangkhom et al., 2011; Guo et al., 2009)

supported our findings. Our observations are also consistent with reports of Leng (2008)

who inferred that 1% inclusion of PN could reduce enteric methane production up to 10%

in ruminants. Findings of Leng and Preston (2010) also supported our observations who

reported nitrates as better option to abate enteric methane production rather than urea.

Generally dissimilatory nitrate reduction to NH3 and assimilatory nitrate reduction to NH3

are two pathways considered responsible for nitrate reduction in anaerobic system. Nitrate

reduction in ruminal fluid encourages the growth of nitrate reducing bacteria.

Thermodynamically nitrate conversion to NH3 is more favourable than CH4 formation, if

ruminants were fed nitrate containing diets (Morgavi et al., 2010). According to Leng

(2008) one mol nitrate can produce one mol of NH3 by reducing CH4 in equivalent

amounts. As reported by Ungerfeld and Kohn (2006) thermodynamic conversion sulphate

to hydrogen sulphide gas is more favourable than methanogenesis. We therefore concluded

in this study that both nitrate and sulphate supplementation in diets gave an additive effect

resulting in better enteric methane production. Nitrate adaptation in this study improved

nitrite-reducing capacity of ruminal microbes. This is evident from our present study

findings that we did not observe even a single incidence of nitrite toxicity, even at higher

89

levels of nitrate inclusion. Inconsistent results in CH4 abatement with dietary nitrates were

reported by many researchers. In sheep trial Zijderveld et al. (2010) observed 32% enteric

CH4 reduction. Contrarily Nolan et al. (2010) observed 23% CH4 reduction in the same

specie fed nitrate containing diets as compared to those fed urea as NPN. In cattle fed diets

with nitrate as feed additive Hulshof et al. (2010) observed 32% CH4 reduction. Maximum

60% CH4 reduction were observed in goats by Sophea and Preston (2011) while minimum

16% CH4 reduction were observed in dairy cows fed nitrate supplemented diets. The

possible reason for variable results might be attributed to CH4 measuring technique in

animals. Calorimetric chambers (Zijderveld et al., 2011) and SF6 technique (Hulshof et al.,

2010) is mostly applied techniques for CH4 estimation from animals. Enteric CH4

production can also be estimated via VFAs (Nolan et al., 2010). Sophal et al. (2013) and

Sangkhom et al. (2012) estimated enteric methane production using technique proposed by

Madsen et al. (2010) in which CO2 is used as tracer gas. Diet composition might also

affect rumen nitrite accumulation. This is evident from the work of some researchers

(Hulshof et al., 2010; leng, 2008). Hulshof et al. (2010) observed that sugarcane based

favoured nitrite accumulation in the ruminal fluid. Other researchers (Johnson et al., 1994;

Grainger et al., 2007) found variable results of CH4 estimation from SF6 method as

compared to chamber measurements.

Growth performance

In this study we observed better daily live weight gains and feed conversion ratios

with diets supplemented with 5% SN with 0.4% S. These results are similar as mentioned

by Sangkhom et al. (2012) who reported better weight gains in cattle fed nitrate

supplemented diets as compared to urea as NPN source. In a goat trial Trinh phuc Hao et

al. (2009) also observed similar trend in weight gains when nitrate rather than urea was a

part of the diet. Our results did not agree with the reports of Thanh et al. (2012) who

observed enteric CH4 reductions in sheep fed nitrate supplemented diets but did not find

any weight gains. Trinh phuc Hao et al. (2009) also reported similar weight gains in goats

fed nitrate containing diets. Results were further supported by Zijderveld et al. (2011)

working on dairy cows. According to Tillman et al. (1965) nitrate might be responsible for

reduced growth rates. Methane mitigation improves nutritive value of feed, which in turns

resulted into better animal performance (Blaxter and clapperton, 1965). Similar findings

were also reported by Beauchemin et al. (2008).

90

These findings suggest that, if nitrite accumulation did not occur in the rumen, it might

have resulted in NH3 production at a rate equal to assimilatory microbial growth rate.

During this process the electron transfer of NH3 preserves more energy in ruminal end

products as compared to CH4 emission. This conserved energy might have resulted in

increased growth efficiency of ruminal microbes. Thus improving the feed efficiency of

the nitrate supplemented diets (Leng, 2008).

Blood Chemistry

The non-significant results of blood metabolites and haematology during the study

interpreted that none of the animals had nitrate or nitrite toxicity throughout the study.

During the whole study animal remained healthy without any abnormal behaviour. Present

study data made it possible to predict that either slow nitrate acclimation of animal or

balance between nitrate to S ratios tended to maintain the activity of both nitrate reducing

bacteria and S reducing bacteria (Leng, 2008). The present results close by related to the

observations of Alaboudi and Jones (1985) and Ngoc Huyen Le Thi (2010) who reported

no evidence of nitrate or nitrite toxicity even at higher levels of inclusion in the diet. Minor

incidence of nitrate toxicity was reported by Zijderveld et al. (2010). Methemoglobinemia

condition develops only when MetHb level is 30 to 40% of Hb (Bruning-Fann and

Kaneene, 1993). Blood urea nitrogen concentrations are very high for goats possibly due to

breed difference. Moreover, its numbers can vary greatly between different locations and

different laboratories.

Conclusion

Diets containing SN in combination with S did not only reduce enteric CH4

production from Lohi sheep but it also improved their growth performance. As compared

to Teddy goats, Lohi Sheep showed better enteric CH4 reductions using the diets

containing SN in combination with S.

91


1SN0, SN2.5 and SN5 stand for sodium nitrate at ~ 0, 2.5 and 5% respectively. S0 and S4 stand for

0 and 0.4% sulphur, respectively.

Ingredients

Diets1

SN0 SN2.5 SN5 S0 S4 S0 S4 S0 S4

Wheat Straw 35.0 35.0 35.0 35.0 35.0 35.0

Cotton seed meal 10.00 10.00 10.00 10.00 10.00 10.00

Rice bran 10.00 10.00 10.00 10.00 10.00 10.00


Hay (Lucerne) 21.00 21.00 21.00 21.00 21.00 21.00

Enzose 14.00 14.00 14.00 14.00 14.00 14.00


Sodium Chloride 1.00 1.00 1.00 1.00 1.00 1.00


Sodium Nitrate 0.00 0.00 2.45 2.45 4.90 4.90

Urea 1.70 1.70 0.85 0.85 0.00 0.00


Dry Matter 88.08 89.58 89.68 91.18 91.28 92.78

Crude Protein 18.0 18.0 18.0 18.0 18.0 18.0




92

Table 6.2 Effect of different levels of sodium nitrate with or without sulphur on Nutrient intake and their

digestibility in Lohi sheep and Teddy goat

Items


SEM2

Significance3

Diets1

SN0 SN2.5 SN5 SN0 SN2.5 SN5


Nutrient Intake,g/d

DM 1740a 1738a 1736a 1736a 1735a 1738a 766b 768b 767b 766b 765b 769b 2.85 NS NS NS *

CP 313a 313a 313a 312a 312a 313a 138b 138b 138b 138b 138b 138b 0.57 NS NS NS *

NDF 772a 771a 771a 770a 770a 771a 340b 341b 340b 340b 339b 341b 1.32 NS NS NS *

ADF 546a 545a 546a 545a 545a 546a 241b 241b 241b 240b 240b 241b 0.89 NS NS NS *

Digestibility,%

DM 58.5abc 59.0ab 57.0abcde 58.0abcd 58.3abcd 59.5a 54.0de 55.0bcde 53.8e 54.5cde 54.3cde 55.8abcd 0.63 NS NS NS *

CP 63.5c 62.5cd 62.5cd 62.5cd 62.5cd 61.0d 70.5a 70.5a 70.0a 70.3a 69.3ab 68.0b 0.31 * * * *


ADF 43.5 41.8 42.8 44.0 44.8 43.8 41.8 42.0 41.0 43.8 41.5 42.3 0.92 NS NS NS NS

1 SN0, SN2.5 and SN5 stand for sodium nitrate at ~ 0, 2.5 and 5% respectively. S0 and S4 stand for 0 and 0.4% sulphur, respectively. 2SEM stand for Standard error mean. 3A, B, C and ABC stand for Nitrate levels, Sulfur levels, Species and their Interaction, respectively while AB stand for Interaction of nitrate levels with sulphur levels. NS stand for Non significant (P>0.05) and * stand for Significant (P<0.05). a,b,c Means in a row with different superscripts differ significantly (p<0.05). DM, CP, NDF and ADF stand for Dry matter, Crude Protein, Neutral detergent fibre and Acid detergent fibre respectively.

93

Table 6.3 Effect of different levels of sodium nitrate with or without sulphur on blood metabolites in Lohi sheep and

Teddy goat

Items

(mg/dL)


SEM2

Significance3

Diets1



BUN 18.3a 18.5a 19.3a 19.0 a 18.5 a 19.0 a 26.5b 26.5b 26.5b 26.0b 26.5b 26.5b 0.57 NS NS NS *

Blood Glucose 74.5a 73.3a 73.0a 72.3 a 73.5a 73.8a 96.0b 95.5b 94.0b 96.5b 96.5b 96.3b 0.86 NS NS NS *

Creatinine 2.4a 2.5a 2.4a 2.4a 2.5a 2.4a 1.6b 1.5b 1.6b 1.5b 1.6b 1.7b 0.09 NS NS NS *

1 SN0, SN2.5 and SN5 stand for sodium nitrate at ~ 0, 2.5 and 5% respectively. S0 and S4 stand for 0 and 0.4% sulphur, respectively. 2SEM stand for Standard error mean. 3A, B, C and ABC stand for Nitrate levels, Sulfur levels, Species and their Interaction, respectively while AB stand for Interaction of nitrate levels with sulphur levels. NS stand for Non significant (P>0.05) and * stand for Significant (P<0.05).a,b,c Means in a row with different superscripts differ significantly (p<0.05). BUN stand for blood urea nitrogen.

94

Table 6.4 Effect of different levels of sodium nitrate with or without sulphur on hematology in Lohi sheep and Teddy

goat

Items


SEM2

Significance3

Diets1



Haemoglobin (g/dL) 14.0a 13.5abc 14.0 a 13.8ab 13.5abc 14.3a 11.0d 11.0d 11.3cd 11.0d 11.5bcd 11.3cd 0.48 NS NS NS *


Neutrophils (%) 45.0a 44.5a 45.3a 44.0abc 43.8abcd 44.3ab 39.3cde 37.5e 38.5e 39.5bcde 37.5e 39.0de 0.96 NS NS NS *

Lymphocytes (%) 46.8a 46.5a 43.3a 44.5 a 47.5 a 46.3a 54.0b 56.3b 54.0b 53.0b 56.3b 53.5b 0.92 * NS * *





1 SN0, SN2.5 and SN5 stand for sodium nitrate at ~ 0, 2.5 and 5% respectively. S0 and S4 stand for 0 and 0.4% sulphur, respectively. 2SEM stand for Standard error mean. 3A, B, C and ABC stand for Nitrate levels, Sulfur levels, Species and their Interaction, respectively while AB stand for Interaction of nitrate levels with sulphur levels. NS stand for Non significant (P>0.05) and * stand for Significant (P<0.05). a,b,c Means in a row with different superscripts differ significantly (p<0.05).

95

Table 6.5 Effect of different levels of sodium nitrate with or without sulphur on nitrogen balance in Lohi sheep and

Teddy goat

Items

(g/day)


SEM2

Significance3

Diets1




Faecal Nitrogen 18.1b 18.6b 18.6b 18.6b 18.6b 19.3a 6.6c 6.6c 6.7c 6.6c 6.9c 7.1c 0.12 * * * *

Urinary Nitrogen 24.7a 24.1ab 23.9b 23.8b 23.9b 23.1c 12.5de 12.6d 12.3de 12.4de 12.1de 11.9e 0.14 * * * *

Nitrogen Balance 7.3 c 7.4ac 7.5ab 7.6a 7.4ac 7.7a 2.9e 2.9e 3.0de 3.0de 3.0de 3.1d 0.04 * * * *

1 SN0, SN2.5 and SN5 stand for sodium nitrate at ~ 0, 2.5 and 5% respectively. S0 and S4 stand for 0 and 0.4% sulphur, respectively. 2SEM stand for

Standard error mean. 3A, B, C and ABC stand for Nitrate levels, Sulfur levels, Species and their Interaction, respectively while AB stand for Interaction of

nitrate levels with sulphur levels. NS stand for Non significant (P>0.05) and * stand for Significant (P<0.05).a,b,c Means in a row with different superscripts

differ significantly (p<0.05).

96

Table 6.6 Effect of different levels of sodium nitrate with or without sulphur on growth performance in Lohi sheep

and Teddy goat

Items


SEM2

Significance3

Diets1



Weight gain (g/d) 138c 139bc 142a 143a 141ab 143a 55e 54e 57de 57de 59d 59d 0.62 * NS * *

Feed consumed (g/d) 1740a 1738a 1736a 1736a 1735a 1738a 766b 768b 767b 766b 765b 769b 2.85 NS NS NS *

Feed conversion ratio 12.7de 12.5de 12.3e 12.2e 12.3e 12.2e 13.9ab 14.1a 13.5bc 13.5bc 13.0cd 13.0cd 0.11 * NS * *

1 SN0, SN2.5 and SN5 stand for sodium nitrate at ~ 0, 2.5 and 5% respectively. S0 and S4 stand for 0 and 0.4% sulphur, respectively. 2SEM stand for Standard error mean. 3A, B, C and ABC stand for Nitrate levels, Sulfur levels, Species and their Interaction, respectively while AB stand for Interaction of nitrate levels with sulphur levels. NS stand for Non significant (P>0.05) and * stand for Significant (P<0.05). a,b,c Means in a row with different superscripts differ significantly (p<0.05)

97

Table 6.7 Effect of different levels of sodium nitrate with or without sulphur on enteric methane production in Lohi


Items


SEM2

Significance3

Diets1



Animal

(CH4 in ppm) 65.2bc 63.8c 63.4cd 60.2de 57.3e 53.1f 70.2a 68.1ab 67.6ab 65.2bc 62.3cd 58.0e 0.64 * * * *

Animal (CO2 in ppm)

1134.8a 1134.8a 1136.0a 1136.0a 1134.8a 1138.5a 1094.8b 1094.8b 1096.0b 1097.3b 1094.8b 1098.8b 2.93 NS NS NS *

CH4 (Animal - Air) : CO2

(Animal - Air)

0.087d 0.085de 0.0845de 0.080ef 0.076f 0.070g 0.0998a 0.0965ab 0.0955ab 0.0920bc 0.0880cd 0.0813e 0.0010 * * * *

CH4

Reduction (%)

0 2.22 3.09 8.06 12.46 19.62 0 3.12 3.94 7.66 11.55 18.27

Air (Methane gas in ppm) = 1.98; Air (carbon dioxide gas in ppm) = 410

1 SN0, SN2.5 and SN5 stand for sodium nitrate at ~ 0, 2.5 and 5% respectively. S0 and S4 stand for 0 and 0.4% sulphur, respectively. 2SEM stand for Standard error mean. 3A, B, C and ABC stand for Nitrate levels, Sulfur levels, Species and their Interaction, respectively while AB stand for Interaction of

nitrate levels with sulphur levels. NS stand for Non significant (P>0.05) and * stand for Significant (P<0.05).a,b,c Means in a row with different superscripts differ significantly (p<0.05).

98

General Conclusion

Comapared with urea, nitrate reduced enteric methane production in both species.

There were no differences among the three nitrate salts (Potassium nitrate, Calcium

ammonium nitrate and Sodium nitrate). Diets containing nitrate in combination with sulphur

did not only reduce enteric CH4 production from both Lohi sheep and Teddy goats at different

growing stages but it also improved their growth performance. Enteric CH4 reductions were

better in Lohi sheep as compared to Teddy goats fed diet containing nitrate in combination

with sulphur. It appeared that adaptation improved the nitrite-reducing capacity of rumen

microbes. Non-significant changes in hematology and blood metabolites indicated that same

health status of all animals. In all the three experiments, none of the animal showed any kind

of abnormal behaviour or signs of illness during the whole experimental period. This

indicated that none of the animals suffered from nitrate or S toxicity.

99

Chapter 7

Summary

The present study, comprising of three independent experiments, was designed to

evaluate the the effect of different nitrate sources with or without sulphate on enteric CH4

production and growth performance of sheep and goats at different growing stages. In first

weaning experiment impact of varying levels of PN with or without S on nutrient intake,

digestibility, growth performance, blood metabolites, hematology, nitrogen balance and

enteric CH4 reduction were examined in forty eight, male, weaned animals (Twenty four

each, Lohi Lambs and Teddy goats). In the second trial same experiment was repeated by

using varying levels of CAN at growing age. Animals in this experiment were of

approximately six months of age. Similarly, in the third trial animals of approximately nine

months of age were selected and the study was conducted by using different levels of SN

with and without sulphate.

In experiment 1, the nutrient intake and digestibility in both weaned lambs and goats

were similar (P> 0.05) across all diets. The animals fed diet containing 6% PN with 0.4% S

showed better nitrogen balance. Daily live weight gain of of both lambs (146 g/day) and

goats (66.0 g/day) fed diet containing 6% PN and 0.4% S was the highest and best values of

FCR (P<0.05) were also observed in both group of animals. Enteric CH4 was 32.6% reduced

(P<0.05) in lambs and 31.9% in goats fed diet containing 6% PN and 0.4% S compared to

those fed C.

In experiment 2, The nutrient intake and digestibility in both sheep and goats were

similar (P> 0.05) across all diets. The animals fed diet containing 3% CAN with 0.4% S

showed better nitrogen balance. Enteric CH4 was 28.6% reduced (P<0.05) in sheep and

26.9% in goats fed diet containing 3% CAN and 0.4% S compared to those fed C. Each sheep

fed diet containing 3% CAN and 0.4% S gained 144 g/day, whereas daily live weight gain by

goats fed diet containing 3% CAN with or without S was 58 g/day.

In experiment 3, The nutrient intake and digestibility in both sheep and goats were

similar across all diets. The animals fed diet containing 5% SN with 0.4% S showed better

nitrogen balance. The enteric CH4 was 19.6% reduced in sheep and 18.2% in goats fed diet

containing 5% SN and 0.4% S compared to those fed C. Daily live weight gain of both sheep

and goats fed diet containing 5% SN and 0.4% S was 143 and 59 g/day, respectively. The

100

best feed conversion values (P<0.05) were observed in sheep fed both SN2.5-S4 and SN5-S4

diets. However, best FCR was observed in goats fed SN5-S0 and SN5-S4 diets.

Non-significant changes in hematology and blood metabolites indicated same health

status of all animals. In all the three experiments, none of the animal showed any kind of

abnormal behaviour or signs of illness during the whole experimental period. This indicated

that none of the animals suffered from nitrate or S toxicity. All the fed nitrate sources showed

similar response with sulphates during various stages of growth in sheep and goats. In

conclusion the roughage based diets with nitrates and sulphate reduced enteric CH4

production, both in sheep and goats.

101

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