TEE EFFECT OF DIETARY FLAX OIL AND ANTIOXlDANTS ON PULMONARY

HYPERTENSION AND ASCITES IN BROILER CHICKENS.

A Thesis

Presented to

The Faculty of Graduate Studies

of

The University of Guelph

by

JOHN-PETER WALTON

In partial Mfilment of requirements

for the degree of

Master of Science

December, 1998

@ John-Peter Walton, 1998

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ABSTRACT

THE EFFECT OF DIETARY FLAX OIL AND ANTIOXIDANTS ON PULMONARY HYPERTENSION AND ASCITES IN BROILER CHICKENS.

John-Peter Walton University of Guelph, 1998

Advisor: Professor E.J. Squires

This thesis is an investigation of the effect of dietary supplementation of 8ax oil and

antioxidants on pulmonary hypertension (PH), ascites, growth and haematological

parameters in broiler chickens. Under hypobaric hypoxia, the inclusion of 5% flax oi1 in

broiler diets has been previously shown to increase erythrocyte deformability, and

reduce whole blood viscosity and PH. In this thesis, when a diet containing 2.5% flax oil

was fed, it did not significantly reduce PH in broilers subjected to hypobaric hypoxia.

Using a cold temperature model, 5% Bax oil diets tended to reduce PH and ascites

compared to control birds fed a diet containing an AN blend oil, but the differences were

not significant (P > 0.05). PH was not decreased in the cold temperature model with diets

containing 2.5% flax oil, or with the supplementation of added antioxidants (vitarnins C

and E). Combining antioxidants with 5.0% flax oil increased ascites, mortality and PH

compared to feeding the 5% flax oil diet alone. When a 5% flax oil diet was fed to birds

under hypobaric conditions, treatment with cyclo-oxygenase or lipoxygenase inhibitors did

not affect PH compared to control birds. The results suggest that the decreases in PH due to

the feeding of flax oil are likely due to increases in erythrocyte defonnability, and not the

production of vasoactive arachidonic acid metabolites.

ACKNOWLEDGEMENTS

I would like to thank my advisor Dr. E.J. Squires and my advisory cornmittee, Dr.

J-L. Atkinson and Dr. R.J. Julian for their continual guidance and support during my

research at the University. Their insightful instruction and enthusiasrn was greatly

appreciated.

My thanks are extended to al1 faculty members, staff and graduate students within

the department of Animal and Poultry Science. 1 am indebted to al1 of them for their

helpful comments, advice, and fnendship. A persona1 thanks is extended to Bruce

Schumann for al1 of his time and fnendship. 1 would also like to thank my fiancee,

Joanne, for her patience, understanding and support while 1 finished my degree.

The financial support for this research, provided by the Ontario Ministry of

Agriculture, Food and Rural Affairs, and The Flax Council of Canada is greatly

acknowledged. 1 also wish to thank BASF for their contributions.

Table of Contents

C hapter 1 . 0 Literature review

...................................................................................... 1 . 1 . 0 Introduction 1

1.1.1 Pu lmonq hypertension ..................................................................... 1

1.1.2 Ascites (Pulmonary hypertension syndrome - PHs) ..................................... 3

1 .2.0 Measuring right ventncular hypertrophy ( R n ) and cardiac darnage ............... -5

1.2.1 Right ventncle to total ventncle weight V / T V ratio) .................................. 5

1 .2.2 Electrocardiography and Troponin-T ..................................................... .5

........................................ 1.2.3 Minimally invasive indices for predicting ascites 6

............... 1.3.0 Experimental models of inducing pulrnonary hypertension and ascites 6

...................................................... 1 .4.0 Hypoxia and physiological responses 8

.......................................................... 1.4.1 Incubational hypoxia and growth -8

.............................................. 1.4.2 Blood and plasma viscosity / polycythaemia 9

............................................. 1.4.3 Erythrocyte deformability (filtration index) 10

............................................................. 1.5.0 Dietary fatty acids and ascites 12

............................. 1.5.1 Omega-3 (n-3) and omega-6 (n-6) fatty acid metabolism 13

.................................................................... 1.6.0 Vasoactive compounds -1 5

........................................................ 1 .6.1 Prostaglandins and thromboxanes 1 5

.......................................... 1 .6.2 Cyclo-oxygenase and lipoxygenase inhibitors 17

................................................................................. 1 .6.3 Inflammation 18

...................................................... 1 . 7.0 Lipid peroxidation and free radicals 19

.......................................................................... 1 .7.1 Lipid peroxidation 19

............................................... 1.7.2 Free radicals and reactive oxygen species 20

1 .8.0 Antioxidant defence mechanisms ......................................................... 2 1

............................................................................. 1.8.1 Vitamin C and E 22

.................................................................. . . 1 -9 0 Factors influencing PHs -24

................................................ 1.10.0 Research hypotheses and experiments -26

1.10.1 Experiments 1 and 2 (chapter 2) ........................................................ 27

........................................................... 1.10.2 Experirnents 3 - 5 (chapter 3) 27

............................................................. Experiment 6 (chapter 4) - 2 9

Chapter 2.0 The effect of dietary flax oil and hypobaric hypoxia on pulmonary hypertension and haematological parameters in broiler chickens . (Accepted by British Poultry Science) (modified from accepted version. adapted for thesis)

2.1.0 Abstract ..................................................................................... 34

2.2.0 Introduction ................................................................................ 35

................................................................... 2.3.0 Materials and methods -36

2.4.0 Results ....................................................................................... 39

................................................................................. 2.5.0 Discussion -42

2.6.0 Conclusion .................................................................................. 47

2.7.0 Acknowledgements ....................................................................... 48

Chapter 3.0 The effect of dietary flax oil and antioxidants on ascites and pulmonary hypertension in broilers using a cold temperature mode1 . (Submitted to British Poultry Science) (modified from submitted version. adapted for thesis)

..................................................................................... . 3.1 0 Abstract -50

................................................................................ 3.2.0 Introduction -51

3 . 3.0 Matenals and methods ................................................................... .53

3.3.1 Diets ......................................................................................... 53

Chapter 5.0

........................................................................... 5.1 . 0 Final Discussion -90

........................................................................... 5.2.0 Future Research -94

..................................................................................... 6.0 References 96

..................................................................................... 7.0 Appendix -106

..................................................... 7.1 Methods and Matenals (Bond, 1996) 106

......................................................................................... 7.2 Results 108

List of Tables

1.1 Fatty acid composition of flax oil (%) ...................................................... 30

1.2 Major pulmonary oxidant scavengers ....................................................... 30

1.3 Selected arachidonic acid metabolites and their biological activities .................. .32

2.1 Composition of experimental diets (glkg) ................................................. 38

2.2 Total mortality and causes of death in broiler chickens fed diets containing flax oil or AN blend oil kept at either hypobaric or ambient atmospheric pressure ................. ..40

2.3 Effect of 2.5% dietary flax oil or A N blend oil on weekly body weight gain (g) of broilers held at hypobaric or ambient pressure ............................................... ..41

2.4 HaematoIogicaI values of broilers fed 2.5% dietary flax oil or A/V blend oil and held at hypobaric or ambient pressure ...................................... ..... ...................... 43

2.5 RV/TV ratios of broilers fed flax oil or A N blend oil diets and kept under hypobark or arnbient pressure.. .............................................................................. -44

3.1 Composition of expenmental diets ( g k g ) for the three experiments ................... 54

3.2 Incidence of ascites, and PHs and RVîTV ratios of broilers exposed to cold temperature .......................................................................................... 5 7

3.3 Experiment 1. Effects of diet on weekly and overall weight gain (ghroiler), feed intake (ghroiler), and feed conversion ratio (FCR) of broilers exposed to cold temperature ......................................................................................... -59

3.4 Experiment 1. Haematocnt, whole blood and plasma viscosity (mean k SE) of broilers fed diets containing 5% oil exposed to cold temperature ............................ 60

3.5 Experiment 2. Effects of diet on weekly and overall weight gain (ghroiier), feed intake (ghroiler), and feed conversion ratio (FCR) of broilers exposed to cold temperature. ......................................................................................... -62

3.6 Expenment 3. Effects of diet on weekly and overall weight gain (glbroiler), feed intake (ghroiler), and feed conversion ratio (FCR) of broilers exposed to cold temperature. .......................................................................................... 64

3.7 Experiment 3. Effects of diet on weekly weight gain (weeks 5 -8 and day 1 - week 8) (ghroiler), total feed intake (ghroiler), and feed conversion ratio (FCR) of broilers

..................................................................... exposed to cold temperature -65

4.1 Effect of indomethacin and meclofenemate on weekly mortality and total mortality of broilers fed 5% Bax oil subjected to hypobaric hypoxia ................................ -230

4.2 RViTV values and haematocnt of broilers fed 5% flax oil subjected to hypobaric hypoxia .............................................................................................. -8 1

4.3 Effect of indomethacin and meclofenemate on weekly weight gain and total weight gain of broilers fed 5% flax oil subjected to hypobaric hypoxia .......................... ..A2

4.4 Fatty acid composition of plasma from broilers fed 5% flax oil injected with propylene glycol (control), indomethacin, or meclofenemate subjected to hypobaric hypoxia. .............................................................................................. -83

4.5 Fatty acid composition of erythrocyte membranes of broilers fed 5% flax oil injected with propylene glycol (control), indomethacin, or meclofenemate subjected to hypobaric hypoxia .......................................................................................... - 3 4

7.1 Total rnortality and causes of death in broiler chickens fed diets containing flax oil or A N blend oil kept at either hypobaric or ambient atmospheric pressure .................. 108

7.2 Experiment 1. Effect of 5% d i e t q flax oil or AN blend oil on weekly body weight ................. gain (g) of broilers held at hypobaric or ambient pressure. (Bond. 1996) -109

7.3 Haematological values of broilers fed 5% dietary flax oil or AN blend oil and held at ................................................ hypobaric or ambient pressure. (Bond, 1996) -1 10

7.4 RVITV ratios of broilers fed flax oil or AN blend oil diets and kept under hypobaric ............................................................................. or arnbient pressure.. -1 1 1

7.5 Fatty acid composition of erythrocyte membranes from broilers fed diets containing 5% flax oil or A N blend oil and kept under hypobaric or ambient pressure. ........... -112

vii

List of Figures

1.1 Omega-3 and Omega-6 pathways of desaturation and elongation ..................... -3 I

1.2 Pathways involved in the oxygenation of arachidonic acid leading to the formation of eicosanoids ................... .. .........................-.-........................................ 32

viii

Chapter 1 .O

Literature Review

1.1.0 Introduction

Pulmonary hypertension syndrome P H s ) or ascites as it is cornmonly called, is a

costly metabolic disorder occurring in the poultry sector worldwide (Huchzermeyer et al.,

1988). Although the development of pulmonary hypertension and right ventricular

hypertrophy can be induced by high altitude and hypoxia (Sillau et a(., 1980; Julian and

Wilson, 1986; Maxwell et al., 1990; Monge and Leon-Velarde, 199 1) and produce ascites

after 2 to 3 weeks (Julian et al., 1993), the incidence o f PHs and ascites has increased at

low altitudes since the 1980's (Julian, 1993). Ascites is characterised by the

accumulation of lyrnph fluid in the abdominal cavity. The developrnent of ascites is a

complex syndrome with fast growth causing hypoxearnia (Peacock et al., 1989).

1.1.1 Pulmonary hypertension

PulmonaIy hypertension (PH) cm develop in broilers under a number of

physiological conditions. Pulmonary hypertension is caused by an increased blood flow

anaor increased resistance to blood circulation in the lung leading to excessive

pulrnonary pressure within the lung (Mirsalimi and Julian, t 991). The result of the

pulmonary arterial hypertension is right ventricular hypertrophy (RVH), Ieading to

valvular insufficiency, right vennicular failure (RVF), and finally the development of

ascites (Julian and Wilson, 1986). Myocardial hypertrophy is increased growth of heart

tissue due to an increase in ce11 size, thus cardiac performance rnay decline due to a large

ce11 volume in relation to ce11 surface (Wikman-Coffelt, 1986). Although the exact

causes of increases in vascular resistance to blood flow in hypertension remain unknown

in humans, there are several possible explanations: (1) increased levels of circulating

vasoconstnctive substances, or a deficiency of circulating vasodilator substances; (2)

increased vasoconstrictor receptor sensitivity or depressed vasodilator receptor

sensitivity; and (3) abnormal vascular structure (Julius and Egan, 1 986). Increases to

pulrnonary vascular resistance are aggravated by vasoconstriction of pulmonary blood

vessels and an elevated packed ce11 volume (PCV/haematocnt), resulting in more viscous

blood (Wideman, er al., 1994). Burton et al. (1968) reported that arterial hypertension

c m result from: 1) a major branch of the pulmonary artery being acutely obstructed, 2)

acute hypoxia, and 3) exposure to high altitude for a long period of time. There are also

additional factors which c m lead to cardiac hypertrophy, e.g. cold acclimation, renal

hypertension, and thyroxine administration (Wikman-Coffelt, 1986).

A major physiological contnbutor to the development of pulmonary hypertension

syndrome (PHs) is systemic hypoxaemia stemming from pulmonary insufficiency (Peacock

et al., 1989, 1990). Hypoxaemia stimulates erythropoiesis, polycythaemia, and increases

blood volume which contribute to PH (Burton and Smith, 1969; Burton et al., 1971;

Julian et al., 1986). Hypervolaemia and polycythaemia both increase nght ventricle

workload (Julian, 1993).

Hypertrophy of the right ventricle due to increased cardiac workload precedes

right atnoventncular valve hypertrophy, leading to valvular insufficiency and right

ventncular failure (Julian, 1993). Accompanying hypertrophy of the right ventricle of

the hem, is central venous congestion, a transudative fluid accumulation in the hepato-

pentoneal cavity (ascites), and pressure-induced cirrhosis of hepatic tissue (Wideman, et

al., 1994). In broiler chickens, this fluid escapes fiom the liver into the ventral hepato-

pentoneal cavities because of increased hydraulic pressure in the vena cava and portal

system (Julian, 1993).

Olkowski et al. (1998) found severe to moderate dilation of the both the nght and

left ventricles in ascitic broilers compared to controls. There were prominent nodules

involving valve cusps and the chordae tendineae. No unilateral left ventricular dilation

was observed, with 30% of ascitic birds having a grossly normal lefi ventricle. The

authors suggest that left-atrio-ventricular degeneration and left ventncular dilation should

be considered as possible aetiologic mechanisms involved in the development of PH.

Olkowski et al. (1998) suggest that the nse in pulmonary artery pressure observed by

Peacock et al. (1 989) rnay be in part due to a rise in pressure in the left atria.

1.1.2 Ascites (Pulrnonary hypertension syndrome - PHs)

Clinical signs of ascites include a distended abdomen, plasma-like fluid in the

abdomen (ascites), sternal recumbency, dyspnoea, poor growth rate, lethargy and possibly

cyanosis of the comb (Maxwell et al., 1986a). Birds with ascites are usually smaller than

their penrnates since growth is reduced as RVF develops (Julian, 1993). Death Erom

ascites is determined by gross postmortem changes consistent with that of the disease.

Upon necropsy, the abdomen may contain a gelatinous clot-like material, and/or clear

yellow fluid, primarily in the ventral hepato-peritoneal sac and pericardium (Julian,

1993). Excessive fluid in the abdomen, an enlarged heart, pericardial effusion with

pulmonary edema (Owen et al., 1990) are characteristic lesions of birds dying from

experimentally induced hypobaric hypoxia. Broilers with ascites secondary to RVF die

fkom respiratory failure due to the pressure of the ascitic fluid on their air sacs or from

lung oedema (Julian et al., 1989).

A marked increase in puhonary hypertension syndrome (PHs) has been noted in

broiler chickens raised at both low and high altitude; this points to the increased

metabolic oxygen requirement from hi& feed intake and rapid growth (Hernandez, 1987;

Julian et al., 1987; Odom et al., 1992). Julian et al. (1989) reported that chickens 6 to 8

weeks old are particularly susceptible to death fiom ascites. This corresponds to the period

of the most rapid growth in broilers (Peacock et al., 1990). It has been demonstrated in

very rapidly growing chickens that hypoxaemia develops, which results in PH, right

venû-icular hypertrophy (RVH), right ventncular failure (RVF), and eventually death due to

cardiac failure (Peacock et al., 1990).

Reeves et al. (1 991) demonstrated an improved artenal oxygen saturation,

decreased respiratory frequency, and an increase in tidal volume in feed restricted male

broilers. The authors also found a trend towards a reduction in r-ight ventricular

hypertrophy, and lower haematocrits in feed restricted broilers compared to non-feed

restricted broilers. Slowing growth will help reduce hypoxaemia related to pulmonary

hypertension.

Low temperature has also been implicated to increase the incidence of ascites in

broilers (Julian et al., 1989; Bendheim et al., 1 992). Vanhooser et al. (1 995) found

weight gain and feed efficiency were reduced, and the incidence of ascites was increased

in Iow atrnospheric oxygen cold stressed chicks compared to controls. Oxygen

insufficiency may develop in broilers through low atrnospheric oxygen pressure or

increased oxygen requirements for maintenance and homeostasis (Bendheim et al., 1992).

1.2.0 Measuring right ventricular hypertrophy (RVH) and cardiac damage

1.2.1 Right ventricle to total ventricle weight (RV/TV ratio)

The amount of PH can be estimated by the ratio of the weight of the right

ventricle to the total ventncular weight (RViTV ratio) (Burton and Smith, 1967; Sillau et

al., 1980; Huchzerrneyer and De Ruyck, 1986; Guthrïe et al., 2 987; Mirsalimi et al.,

1993). Birds which develop right ventricular hypertrophy will have RV/TV ratios greater

than 0.249 . The normal ratio is estimated to be less than 0.250, moderate has a range of

0.250 - 0.299, and severe pulmonary hypertension is estimated to have a ratio greater than

0.299 (Julian and Squires, 1994). Unfortunately, the birds have to be killed and the

ventricles removed and weighed to obtain such anatomical measurements.

1.2.2 Electrocardiography and troponin T

A non-invasive technique to predict the susceptibility of birds to PH and ascites is

the measure of lead II electrocardiogram (ECG) traces. Owen et al. (1990) reported that

broilers reared under hypobaric conditions (5000 m), have a statisticall y signi ficant

increase in the mean amplitude of the QRS complex of the lead II ECG, compared to

control broilers reared at 366 metres. Measurements of elevated heart-to-body-weight

and lung-to-body-weight ratios were also reported (Owen et al., 1990) in the hypobark

group of broilers which may be a result of oedema and congestion.

Electrocardiography may become a useful diagnostic technique for identifjhg

broilers susceptible to PH and ascites after an exposure to high altitude and rneasuring the

lead II ECG complexes. After a short exposure to high altitude, electrocardiography may

. be a useful screening tool to select a population of highly susceptible birds for further PH

studies or genetic selection (Owen et al., 1995).

Another more recent non-invasive technique to determine the extent of cardiac

damage is to measure s e m troponin T . Studies in humans have confirmed that semm

troponin T values are a highly specific non-invasive indicator o f cardiac damage.

Troponin T is a cardiac specific protein related to the contractile tissues of strîated heart

muscle (Maxwell and Moseley, 2 995).

1.2.3 Minirnally invasive indices for predictiog ascites

A recent study (Widernan, et al., 1998) evaluated the use of rninimally invasive

indices for predicting ascites in susceptible broilers from three successive hatches

exposed to cool temperatures. The minimally invasive indices included measuring the

following parameters; percentage saturation of haemoglobin with oxygen, haematocnt,

heart rate, electrocardiogram Lead II recordings, and body weight. None of the

minimally diagnostic indices consistently differentiated ascitic from nonascitic broilers

within each of the hatches. When applied to individual birds of different sex, the

predictive value of the minirnally invasive indices may Vary due to bird to bird variation.

However, necropsy revealed elevated right:total ventncular weight ratios (RV/TV) in

ascitic cornpared to nonascitic broilers, supporting the contention that pulmonary

hypertension, and not cardiomyopathy is involved in the development of ascites induced

by Iow temperatures (Wideman, et al., 1998).

1.3.0 Experimental models of inducing pulmonary hypertension and ascites

In the past, researchers have exposed broiler chickens to high altitudes in order to

study ascites and pulrnonary hypertension. Hemandez (1987), compared the RV/TV

values of ascitic broilers raised at 2638 rn above mean sea level (m.a.s.l.), with that of

healthy broilers reared at the sarne altitude and at 220 m.a.s.1. There was a significant

increase in RV/TV in the ascitic group, compared to the healthy group of birds reared at

the sarne altitude. Also, there was a significant increase in RV/TV between the healthy

birds at 2638 m.a.s.1. compared with the healthy birds at 220 m.a.s.1. This study

reinforces Burton et al. (1968) that high altitude induces pulmonary hypertension and

right ventrîcular hypertrophy.

Ln order to induce PH, Owen er al. (1990) have demonstrated that utilizing a

hypobaric chamber to simulate altitudes of 3000, 3500, and 5000 m will produce ascites

in broilers. Bond et al. (1996 and 1997), have also demonstrated that hypobaic

chambers will produce PH and ascites in broilers when reared at 1000, 1500, and 2000

metres. The advantage of this system is that it can reproduce ascites in broilers under

controlled laboratory conditions year round.

Several other model systems exist to study pulmonary hypertension and ascites in

broilers. Cold temperature (Julian et al., 1 989), low ventilation and cool temperatures

(Enkvetchakul et al., 1993; Wideman et al., 1994), and the use of toxic levels of sodium

chloride (Julian, 1987; Mirsalimi et al., 1992) have been used to study the development

of PH and ascites. The development of PH and ascites using these different model

systems will evoke varying physiological responses depending on the model used.

Cold temperature has been shown to increase right ventricular failure and ascites,

secondary to PH in broiler chickens (Julian et al., 1989). Gleeson (1 986) demonstrated

that a drop in temperature f?om 20" C to Z 0 C, almost doubled the oxygen requirements in

adult White Leghom hens. To cope with the increased oxygen requirements, cardiac

output and blood flow increase, which may cause both a volume and pressure overload on

the nght ventricle due to increased pulrnonary arterial pressure (Julian et al., 1989). Low

ventilation is used to imitate cold weather conditions (Enkvetchakul et al., 1993), and

would produce similar effects as the cold temperature rnodels.

Ascites caused by high levels of sodium chloride is a result of decreased

erythrocyte deformability, and an increase in blood volume (hypervolaernia) (Mirsalimi

et al., 1992). It is likely sodium causes fluid retention in broilers. since body weight

showed an immediate increase in response to increasing levels of sodium chloride in the

diet (Julian, 1987). Increases in blood volume and polycythaemia, along with a decrease

in eiythrocyte deformability contribute to PH (Julian, 1993).

1 A.0 Hypoxia and physiological responses.

1.4.1 Incubational hypoxia and growth

Hypoxia or hypoxaemia can be defined as a state of reduced oxygen supply to

tissue despite adequate perfusion to that tissue (Wikman-Coffelt, 1986). It is unclear

whether incubational hypoxia initiates PH in chicks upon hatch (Odom et al., 1992), or

whether hypoxia begins as soon as the chick hatches (Owen et al., 1995). Vidyadaran et

al. (1990) compared undomesticated red jungle fowl to a modem laying strain of fowl,

and found that modem strains had a 20% reduced lung volume when standardized against

body weight, and a 25% lower anatomical diffusing capacity. Hypoxia could result fiom

the reduced capacity for oxygen uptake and lead to pulmonary hypertension and nght

ventricular hypertrophy starting on the day of hatch in susceptible broilers. As broiler

chickens grow the percentage of lung volume to body weight decreases, and rnay lead to

decreased lung capillary capacity for oxygen exchange (Julian, 1989). In addition, the

fixed lung structure of the broiler chicken, which relies on abdominal movement for air to

pass through the lungs, combined with the birds small stature, rnay contribute to the

increased incidence of pu lmonq hyperiension syndrome (Julian, 1993). Hypobaric

hypoxia c m initiate various physiological aberrations. The growth of broiler chickens in

hypobaric hypoxia is reduced compared to birds reared at arnbient pressure (Mirsalimi et

al., 1993; Bond et al., 1996 and 1997).

1 A.2 Blood and plasma viscosity / polycythaemia

Besides growth, there are nurnerous physiological changes within the vascular and

circulatory systems affected by exposure to hypobaric hypoxia. The physiological

responses to hypoxia in pouItry include hypoxaemia, increased heart rate, and increased

respiratory fkequency (Butler, 1967; Peacock er al., 1989; Reeves et al., 199 1). Often the

increase in resistance to pulmonary blood flow is due to polycythaemia that increases

blood volume and viscosity (Julian, 1993; Wideman, et al,. 1994). High altitude and

hypobaric hypoxia is known to induce polycythaemia in broiler chickens resulting in PH

and RVH (Sillau et al., 1980; Julian and Wilson, 1986; Maxwell et al.. 1990; Monge and

Leon-Velarde, 199 1). Hypoxaemia stimulates erythropoiesis and polycythaemia of

mostly Young, large, red blood cells to increase the blood volume (Julian, 1993).

Polycythaemia can also be stimuIated by high levels of dietary cobalt and result in PH;

therefore it is likely that the increase in the number of circulating erythrocytes is

responsible for the increased workload on the heart rather than vasoconstnction caused by

hypoxia (Diaz et al., 1994).

Water is termed a Newtonian fluid, whose viscosity does not change as the shear

rate is changed. Blood is classified as a non-Newtonian fluid since its viscosity increases

with shear rate decreases (Chien, 1975). The viscosity of broiler blood is less dependent

on shear rate than is blood fiom humans or horses (Fedde and Wood, 1993). Fedde and

Wideman (1996), found that the increase in apparent blood viscosity becarne greater as

the shear rate decreased at a haematocrit above 0.30, and at a haematocrit below 0.30, the

apparent viscosity of blood was nearly shear rate independent. ï h e authors found that

ascitic birds with a haernatocrit of 0.50 had more than a tsvofold increase in apparent

viscosity compared to normal birds with a haematocrit of 0.30 .

Plasma viscosity may be lower in birds with ascites compared to non ascitic birds.

The lower plasma viscosity in birds with ascites is most probably due to a loss of protein

into the ascites fluid (Maxwell et al., 1992). Fedde and Wideman (1996), suggest that the

lower plasma viscosity may be beneficial in partiaIly compensating for the increase in

viscosity due to increases in haematocrit, however, the compensation may be only minor.

Fedde and Widemsn (1996), have demonstrated that an increase in haematocrit is

accompanied by an increase in viscosity, and these two haematological variables rnay

contribute to the development of PH and ultimately ascites.

1.4.3 Erythrocyte deformability (filtration index)

Erythrocyte deformability is primarily determined by (1) cellular geometry

(surface area to volume ratio), (2) interna1 fluid viscosity (estirnated by MCHC) and (3)

membrane rigidity (viscoelastic properties) (Hakim and Macek, 1988; Mirsalimi et al.,

1992).

The mean corpuscular haernaglobin concentration (MCHC) has been found to be

an indicator of the interna1 viscosity of erythrocytes (Mohandes el al., 1980). It has been

found that as erythrocytes age, the cell volume, and length to width ratio decrease, and

subseguently the MCHC is increased (Sutera et al., 1985). These older, srnaller cells

tend to have a reduction in erythrocyte deformability (Sutera et al., 1985; Mills et al.,

1993; Bosch er al., 1994).

It has been found that decreased deformability of the erythrocytes, as measured

by the filtration index, can contribute to an increase in the arnount of PH and ascites in

broiler chickens (Mirsalimi and Julian, 199 1 ; Mirsalimi et al., 1993). Erythrocyte

deformability is based upon the measurement of the filtration time for a suspension of

10% erythrocytes to pass through a polycarbonate membrane with an average pore

diameter of 5 prn (Hakim and Macek, 1988). Blood viscosity is greater when erythrocyte

defomability is reduced (Chien et al., 1967; Doyle and Waker, 1990). It has also been

found that the flexibility of erythrocytes is reduced due to hypoxemia in several species

of mammals particularly the rat (Hakim and Macek, 1988). -41~0, because the resting

diarneter of normal erythrocytes exceeds the diameter of pulmonary capillaries, a

decrease in deformability with an increase in whole blood viscosity may also restrict the

passage of erythrocytes through the pulmonary capilIaries (Doyle and Walker, 1990).

Doyle et al. (1 989) perfused rat lungs constricted with a thromboxane A, mimetic,

and found suspensions containing stiffened erythrocytes increased Ppa (pulrnonary

artenal pressure) in the lung. Constriction at capillaries or the precapillary arterioles

would potentiate the effects of increased whole blood viscosity subsequent to the

reduction in erythrocyte deformability (Doyle et al., 1989).

The age of the erythrocytes has been shown to influence the flexibility of red

blood cells as well. The average lifespan for the avian erythrocyte is 28 to 35 days

(S turkie, 1986). It has been demonstrated in human erythrocytes that the flexibility

declines as the cells age (Sutera et al., 1985; Mills er al.. 1993; Bosch et aL, 1994).

When comparing erythrocytes of birds raised in a hypoxic environment to those raised at

arnbient pressure, a cornplicating factor might be that there is a difference in the age of

the erythrocytes behveen groups under atmosphenc pressure, as hypoxia stimulates

erythropoiesis (Sturkie, 1986), which could increase the number of younger red blood

cells in birds that are hypoxaemic.

Human studies have demonstrated erythrocyte membrane fluidity c m be

increased by dietary supplementation of omega-3 (n-3) fatty acids, narnely fish oils

(Berlin et al , 1992). This is in agreement with previous studies in which platelet

membranes in humans showed an increase in the total n-3 fatty acids with no change in

the n-6 fatty acids after supplementation with dietary flax oil (Allman et al.. 1995).

Archer er cd. (1989) found that supplementation with fish oil reduces blood viscosity and

RVH in rats. Therefore, it may be possible to improve erythrocyte deformability in

broilers by increasing the amount of unsaturated fats in the diet, thereby increasing the

omega-3 fatty acid content of the erythrocyte membrane and increasing its fluid

properties.

2.5.0 Dietary fatty acids and ascites

Previous research has indicated that the inclusion of flax oil in broiler rations was

effective in reducing mortality and the incidence of ascites at 2200m and pulmonary

hypertension at 1500 m (Bond et al., 1 W6), when exposed to hypobaric hypoxia without

reducing growth rates. Other studies (Hulan et al., 1989), found a reduction in the growth

rate of birds fed red fish meal as a source of omega-3 fatty acids. However, Phetteplace

and Watkins (1992), found no difference in the growth of fernale chicks raised on diets

containing either 50 g/kg soybean oil or menhaden fish oil as a fat source. Bond et al.

(1996) also found that there was no difference in the growth of birds fed diets containing

either flaw oil or control diets containing an anirnaihegetable (AN) blend of oil. The

incidence of ascites can be reduced by slowing the growth rate of broilers (Julian, 1993).

Flax oil may be a useful fat source which can be incorporated into broiler rations to

reduce PH and ascites without compromising growth. Other studies should be conducted

to deterrnine if other fat sources could be used to reduce PH and ascites.

1.5.1 Omega-3 (n-3) and ornega-6 (n-6) fatty acid metabolism

Linoleic acid (1 8:2(n-6)) and a-linolenic acid (1 8:3(n-3)), the precursors of the (n-

6 ) and (n-3) family of fatty acids, respectively, are essential fatty acids (EFA) and have to

be supplied by the diet (Nair ef al. 1997). On average, flax oil contains 53.3 g ram of a-

linolenic acid per 100 grarns of raw oil (Bhatty, 1995) (Table l . l). Linolenic acid is a

precursor to eicosapentaenoic acid (EPA) (20:5(n-3)), and docosahexaenoic acid @HA)

(22:6(n-3)) (Holub, 1995). These fatty acids replace arachidonic acid (AA)(20:4(n-6))

produced frorn linoleic acid, in the membranes of platelets, erythrocytes, neutrophils,

monocytes, and hepatocytes (Sirnopoulos, 199 1).

Linoleic acid and a-linolenic acid compete for desaturation, elongation and for

the A-6-desaturase activity (Figure 1.1), therefore a proper balance of fatty acids is

essential to optimize fi, EPA, and DHA in membrane phospholipids (Vence1 et al.,

1991; Nair et al., 1997) Consequently, increased arnounts of EPA would be produced

from a-linolenic acid and deposited in membrane phospholipids in place of arachidonic

acid, a fatty acid derivative of linoleic acid. As the ratio of linoIeic acid to a-Iinolenic

acid decreases (LO:LN), the arachidonic acid content of the liver and cerebellurn lipids

decreases (Applegate and Sell, 1996).

When Olomu and Baracos (199 1) fed varying dietary levels of flaxseed oil to

broiler chicks, ranging from 1.5 to 4.5%, there were increases in EPA, DHA, and

(C22:5(n-3)) in tissue lipids in proportion to the level of flaxseed oil fed. The same

authors also found that concentrations of polyunsaturated fatty acids (PUFA7s) in chick

muscle lipid were 18% when a 6% animal tallow fat source was used in the diet, and the

PUFA content increased to as much as 30% when flaxseed oil (4.5%) was fed as a lipid

source. The a-linolenic acid concentration increased fiom less than 1% of muscle lipids

to 8.9%, when 4.5% flaxseed oil was fed for 7 days (Olomu and Baracos, 199 1).

Bond et al. (1997) found that feeding varying levels of flaxseed increased stearate

(18:0), and decreased the relative percentages of oleic acid (18:l) and linoleic acid (182)

in the erythrocyte membranes of broiler chickens. This is in agreement with Olomu and

Baracos (1 991), who demonstrated that feeding flaxseed oil ranging fiom 1.5 to 4.5%

decreased the arnounts of saturated and monounsaturated fatty acids deposited in the

Sartorius muscle.

1.6.0 Vasoactive compounds

1.6.1 Prostaglandins and thromboxanes

The oxygenation products of PUFA's derived from phospholipid in the plasma

membranes produce the eicosanoid family of prostaglandins, thromboxanes, and

leukotnenes (Calder et al.. 1992). The principal substrate for eicosanoid production is

arachidonic acid, which can be metabolized by cyclo-oxygenase to yield the 2-series

prostaglandins and thromboxanes (Figure 1.2). Altematively, arachidonic acid can be

metabolized by 5-lipoxygenase to produce the Cseries leukotrienes (Calder et al.. 1992).

Table 1.3 provides a sumrnary of selected important arachdionic acid metabolites and

their biological activities.

Vasodilatory prostanoids such as prostaglandin E, (PGE,) and prostacyclin (PGI,),

which are synthesized from arachidonic acid, have been irnplicated in such disorders as

pulmonary hypertension in hurnans (Cremona and Higenbottarn, 1995). Also, Maxwell et

al. (1986 a,b) suggest an inflarnrnatory response occurs in broilers suffering from PHs.

Therefore, leukotnene B, (LTB,), a neutrophil proinflammatory substance (Wallace and

Chin, 1997), may play a role in the inflammation associated with PH and ascites in broilers.

Prostanoids are not stored by cells, but are rather synthesized in response to ceil specific

stimuli (Smith, 1989).

The fatty acids EPA and DHA inhibit the conversion of arachidonic acid to

prostaglandins in platelets (Culp et al., 1979) and the enzymatic conversion of

prostaglandin H, (PGH,) to the EPA denvative thromboxane A, (TxA,) (Needleman er

al.. 1979). Thromboxane A, is less active in humans at aggregating platelets (Lee et al.,

1985) or as a vasoconstrictor than thromboxane A? (Lewis er al.. 1986; Weber et al.,

1986). TxAL , is an important derivative of arachidonic acid and is produced mainly by

blood platelets and behaves as a vasoconstrictor and promotes platelet aggregation

Meydani, 1992). Both EPA and DHA have shown significant potential to reduce platelet

aggregation and TxA, generation (Holub, 1995). These nvo fatty acids c m be further

metabolized to the prostacyclins, PGI, and PGI, respectively, by blood vesse1 enzymes to

function as coronary relaxants (Needleman er al., 1 9 79). Birds have thrombocytes which

are fiagile nucleated cells, simila. to platelets of mamrnals (Sturkie, 1986). Bond et al.

(1996) found a decrease in PHs experienced by birds fed diets containing flax oil. If

thrombocytes behave the same as platelets with respect to EPA and DHA synthesis, then

the production of coronary relaxants could help explain the decrease in the number of cases

of PHs due to feeding flax oil.

Linoleic acid, an omega-6 fatty acid, is the precursor to arachidonate which c m

then be converted to PGI, (Needleman et a l , 1979). PGI, is synthesized kom arachidonic

acid via the cyclooxygenase pathway and is continuously released by vascular endothelium

(Cremona and Higenbottam, 1995). PGI, is a potent endogenous vasodilatory compound

and antiaggregatin; agent (Cremona and Higenbottam, 1995). It has been demonstrated

in humans suffering from primary pulmonary hypertension, that infùsing PGI, causes

vasodilation and reduces vascular resistance (Cremona and Higenbottam, 1995). The

balance between arterial wall PGI, production and platelet TxA, has been suggested as an

important factor in platelet reactivity and aggregation (Meydani, 1992). Patients with

primary pulmonary hypertension still release PGI,; however it maybe the balance

between thromboxane A, and prostacyclin (TxA2/PG12) which contribute to the disorder

(Meydani, 1992; Cremona and Higenbottarn, 1995; Nair et al., 1997). Patients suffering

fiom primary PH showed improvement with infusions of PGI, due to decreased

pulrnonary resistance and subsequent increase in cardiac output and tissue oxygen

delivery (Cremona and Higenbottarn, 1995).

1.6.2 Cyclo-oxygenase and lipoxygenase in hibitors

In order to study the physiological effects of eicosanoids (prostaglandins,

thromboxanes, and leukotrines), specific inhibitors can be used to block cyclo-oxygenase

or lipoxygenase activity. Selectively blocking a specific metabolic pathway may help

elicit which eicosanoid compounds contribute to the development of pulmonary

hypertension.

Non-steroidal anti-inflarnmatory drugs (NSAID's) such as ibuprofen,

indomethacin, aspirin, and meclofenemate prevent the production of eicosanoids by

inhibiting cyclo-oxygenase or lipoxygenase activity (Smith, 1989; Meade el al., 1993;

Laneuville et al., 1994). Indomethacin and meclofenemate cause irreversible inactivation

of cyclo-oxygenase, and thus, new enzyme synthesis is required before more prostanoids

c m be produced (Smith, 1989).

The conversion of arachidonic acid to prostaglandin endoperoxide H2 (PGK) is

mediated by cyclo-oxygenase (PGG/H synthase) , a membrane protein of prostanoid-

forming cells (Smith, 1989). There are two isoenzymes of prostaglandin endoperoxide

(PGH or PGG/H) synthase (Meade et al., 1993). PGHS -1 is probably involved with

cellular "housekeeping" and vascular homeostasis, while PGHS -2 may produce

prostanoids involved in inflammation (Meade et al., 1993 ; Laneuville et al., 1994). The

level of PGGN synthase protein has been shown to regulate prostanoid formation (Smith,

1989). Leukotriene A, can be converted to LTB, by LTA, hydrolase (Smith, 1989).

In an in vitro expression experiment, LaneuviIIe et al. (1994), measured the

products formed by [L- ''Cl arachidonate. Meclofenemate inhibited human PGHS- 1 and

hunan PGHS-2 activity, but not as effectively as indomethacin. PGHS -1, also called

COX- 1 (cyclo-oxygenase- 1) is constituitively expressed and involved in modulating

blood flow and mucosal defence factors. PGHS-2, also called COX-2 (cycio-oxygenase-

2) is inducible and largely responsible for the spthesis of inflamrnatory prostanoids

(Wallace and Chin, 1997). Peri et al. (1995) decreased prostaglandin synthase-2

synthesis (COX-2) in newbom pigs using NS-398. It may be possible to selectively

inhibit COX-2 prostanoids without comprirnising beneficial COX- 1 prostanoids.

1.6.3 Inflammation

There is considerable evidence that various eicosanoid compounds denved fkom

arachidonic acid participate in inflarnrnatory reactions (Calder ei al., 1992; Wallace and

Chin, 1997). Maxwell el al. (1986a) found that heterophils and monocytes were

increased at the expense of lymphocytes in ascitic broilers compared to controls.

Heterophils are a type of leukocyte in broiler chickens, or classified as neutrophils in

marnmals (Sturkie, 1986). Maxwell et al, (1986b) also found increases in immature and

mature heterophils in the hearts and livers of ascitic broilers. Many of these changes are

the result of PH and not the cause of PH (Julian, 1993). The inflammatory response c m

be amplified by neutrophils, since these cells have the capacity to release chemotaxins

such as leukotriene B, (LTB,) (Wallace and Chin, 1997). LTB, can also stimulate

neutrophils in return to release reactive oxygen metabolites, which could contribute

additionally to tissue injury (Wallace and Chin, 1997). Therefore, by shifting fatty acid

metabolism fiom arachidonic acid synthesis to the omega-3 senes is beneficial, and may

help reduce vasoconstriction and inflammation associated with arachidonic acid derived

eicosanoids.

1.7.0 Lipid peroxidation and free radicals

1.7.1 Lipid peroxidation

Bottje et al. (1995) found a significant correlation (r = .18, Pc.05) between RVlTV

values and plasma lipid peroxide values. Lipid peroxidation is initiated with the generation

of reactive compounds such as reactive oxygen species @OS) including hydrogen peroxide,

hydroxyl radical, superoxide, hydroperoxides, or other &ee radicals. When the ROS exceed

the antioxidant capability of cells or tissues, lipid peroxidation ensues (Horton and

Fairhurst, 1987).

Lipid peroxidation caused by the excess production of free radicals c m be rneasured

quantitatively by the arnount of malondialdehyde in a tissue or organ. Malondialdehyde

content is measured by the concentration of thiobarbituric acid reactive substance (TBARS)

(nrnol of TBARS 1 mg protein) (Gutteridge and Halliwell, 1990). Diaz-Cruz et al. (1996)

found an increase in the concentration of TBARS in the liver and cardiac tissue of broiIers

exhibiting ascites. This is indicative of lipid peroxidation in the h e m and liver, initiated by

eIevated levels of reactive oxygen species. Maxwell et al. (1996), compared the

ultracytochemical characteristics of ascitic broiler hearts to those of flock-mate controls.

The authors observed heart tissue from ascitic broiiers had myofibillar degeneration.

mitochondrial hyperplasia and increased nurnbers of hydrogen peroxide deposits located

within the mitochondrial matrices.

1.7.2 Free radicals and reactive oxygen species

Lipid peroxidation fonns lipid-fkee radicals capable of advancing the process of

lipid peroxidation unless they are rapidly reduced (Heffher and Repine, 1989). Vitamin E is

a lipid soluble vitarnin that represents the principle defence against oxidant-induced

membrane damage (Heffner and Repine, 1989; Bottje and Wideman, 1995). Vitamin E (a-

tocophero! radical (a-T-)) prevents the reaction of unsaturated lipid-f?ee radicals with other

unsaturated fatty acid compounds which form damaging hydroperoxides, and more lipid-

£kee radicals (Boxer, 1990). The a - T breaks the damaging lipid-free radical chain reaction

and c m be recycled back to its reduced state by the reduction with ascorbate (vitamin C)

(Hefher and Repine, 1989). The dietary suppIementation of vitarnin E may help reduce

f7ee radical membrane damage, which may be related to the darnage caused by PHs.

Other factors can exacerbate conditions leading to the development of PHs.

Maxwell et al. (1986a and 1990) dernonstrated that tissues of birds with PHs show

inflamrnatory ce11 infiltration. The inflarnrnatory response c m be arnplified by neutrophils,

since these cells have the capacity to release chemotaxins such as leukotriene B, (LTB,)

(Wallace and Chin, 1997). LTB, can aiso stimulate neutrophils in return to release reactive

oxygen metabolites (ROSS) creating an endless darnaging cycle.

Bottje and Wideman (1995) suggest birds with PHs expenence physio10,aical

conditions sirnilar to ischemia-reperfusion injury sustained dunng hypoxia. During the

metabolism of hypoxanthine, xanthine oxidase reduces oxygen to superoxide (McCord,

1985). The superoxide radical is a fairly good reductant capable of initiating chain reactions

that can chemically modiQ cellular proteins, amino acids, and lipids (McCord, 1985;

Babbs, 1988). Therefore, hypoxia can lead to the development of ROSS responsible for

massive tissue damage similar to rapid reoxygenation during the reperfüsion phase (Brown

and Hall, 1992). Several intracellular antioxidants exist to cope with the ROS insult,

namely, catalase, superoxide dimutase (SOD), and an array of enzymes involved with the

glurathione (GSH) redox cycle (Heffher and Repine, 1989). The interaction between

vitamin E, ascorbate and GSH, is crucial to the fûnctioning of the antioxidant defence

network (Table 1.2). The vitarnin E f7ee radical requires ascorbate to convert it back to its

reduced form, and GSSG may be reduced back to GSH by vitarnin C (Bottje and Wideman,

1995).

1.8.0 Antioxidant defence mechanisms

Histopathology studies by Maxwell et al. (1986a, 1990) indicated that tissues of

ascitic birds and hearts of experimentally-induced hypoxic birds, had elevated nurnbers of

inflammatory cells as a result of PH. Activated white blood cells can produce a variety of

reactive oxidants into surrounding tissues, which may have the ability to alter the

antioxidant status of the tissue (Enkvetchakul et al., 1993). Enkvetchakul et al. (1993) also

found that concentrations of both vitamin E and C, plus glutathione (GSH) were

comprornised in broilers with PHs. Low levels of these antioxidants are associated with

darnaging oxidative tissue reactions. The depression of these antioxidants may play an

important role in the development of PHs or potentiate the situation M e r (Enkvetchakul

et al., 1993). It may be necessary to supplement vitarnin E early to broilers due to

inefficient absorption of lipid soluble nutrients dunng posthatch. This may also be

responsible for the declining vitamin E concentrations in the liver and plasma as broilers

age (AppIegate and Sell, 1996). Vitamin C is not usually found in poultry rations.

However, under environmentally, pathologically, or nutritionally stressful conditions,

vitarnin C may become an essential nutrient for chickens (Pardue and Thaxton, 1986).

Ln addition to the development of ascites, hypoxia will also depress feed intake in

broiler chickens (Enkvetchakul et al., 1993). The depressed feed intake will alter the

antioxidant statu of the broiler further, which is under oxidant stress due to cardiac

insuficiency. The depressed feed intake may exacerbate any potential antioxidant

deficiency and increase the reactive oxygen insult.

1.8.1 Vitamin C and E

Bottje et al. (1995) found supplementing broilers with 15 mg of a-tocopherol in

the form of an implant fiom O to 3 weeks of age reduced the five-week cumulative PHs

mortality. The five-week cumuIative PHs mortality decreased by maintaining tissue

antioxidant concentrations and alleviating processes leading to lipid peroxidation.

However, when Bottje et al. (1997) performed a similar expenment feeding supplemental

dietary dl-a-tocopheryl acetate ranging f?om O to 87 mgkg it did not reduce PHs mortality.

The researchers suggest the lack of effect on PHs rnay be due to timing, chemical form,

and arnount of tocopherol supplied to the birds.

Ladmakhi et al. (1997) reared broilers at a relatively low ambient temperature with

the addition of vitarnin C (500 mgkg) in the diet had no effect on growth, feed intake or

feed conversion. When Bottje et al. (1997) fed supplemental dietary dl-a-tocopheryl

acetate ranging from O to 87 mg&g of feed, it aiso had no effect on body weight, feed

intake, or feed efficiency. These results indicate that vitamin C and E can be added to

broiler rations at these incorporation rates without negatively affiecting feed intake or feed

conversion.

Hollander (198 1) found that the rate of absorption of fat soluble vitarnins declined

when linoleic acid (Cl 8:2) and linolenic acid (C18:3) were added in the perfusate of rat

small intestine. Polyunsaturated fatty acids increase the physical size of the micelles and

decrease the access to the absorptive ce11 membrane of the enterocyte (Hollander, 1981),

compared to smaller micelles containing less PUFA. Also, a decline in the activity of fatty

acid binding protein in the enterocyte of broilers fkom hatch to 2 weeks of age (Sel1 et al.,

1986), may be paflially responsible for the inefficient absorption of lipid soluble

compounds. If the broiler small intestine behaves similar to rat, antioxidant absorption may

be impaired depending on the fat source in the ration. Therefore, the levels of

polyunsaturates in poultry rations should be monitored closely as not to impair lipid soluble

vitamin absorption. This may be of inrerest especially when animalhegetable blends of oil

are utilized and the composition not always known.

Hollander (1981) also demonstrated that a-tocopherol absorption eom the

gastrointestinal tract is only 60 to 80% efficient. In an experiment using swine, Chung ef al.

(1992) found that dietary D-a-tocopherol was more effectively absorbed and retained than

the DL-a-tocopheryl acetate form. Also, the biological activity of D-a-tocopherol was

2.44 IU/mg, versus DL-a-tocopheryl acetate of 1 .O Wmg. Therefore, the biochemical

composition of the compound will impact on its absorption and biological activity in the

animal. A major limitation to prophylactic vitamin E therapy is the complex

pharmacokinetics involved in the delivery of lipophilic antioxidants since it takes several

weeks to achieve small increases in vitamin E tissue levels (Heffner and Repine, 1989).

Due to the water solubility of ascorbate, it is found widely in both the extracellular

and intracellular spaces (Heffher and Repine, 1989). AI-Taweil and Kassab (1 990) found

that the haernatocrit increased when birds were fed higher levels of ascorbate (450 mgkg).

It is postulated that ascorbate might also affect the protein content of semm (Al-Taweil and

Kassab, 1990). If vitamin C is incorporated into broiler rations at this level (45ûmgkg) or

higher, it may increase whole blood and possible plasma viscosity. It is known that blood

viscosity is mostly influenced by the percent of circulating red blood cells (Sturkie, 1986).

The elevated haematocrit would be responsible for the increased whole blood viscosity

caused by the ascorbate. Polycythaernia is known to increase the workload on the heart

(Diaz et al., 1994).

1.9.0 Factors influencing PHs

Enkvetchakul ef al. (1993) found concentrations of both vitarnin C and E, plus

glutathione were compromised in broilers with PHs. However, when Bottje et al. (1997)

performed an experiment feeding supplemental dietary dl-a-tocopheryl acetate rangïng

from O to 87 mgkg, it did not reduce PHs rnortality. Therefore, before antioxidants are

incorporated into broiler rations to reduce PH and ascites, the chernical form, and timing of

antioxidant supplementation requires further investigation.

The type of fat in the diet cari affect the incidence of PHs. Squires and Summers

(1993), suggest lipid peroxidation might play a role in the development of PHs.

Unsaturated fatty acids tend to decrease, and saturated fats tend to improve the

gastrointestinal absorption of lipid soluble vitarnins (HoIlander, 198 1). The addition of

antioxidants to fat sources may help stabilize the oil and reduce oxidation. Wang et al.

(1 997) found broilers supplemented with SantoquinB (ethoxyquin at 125 ppm) exhibited

higher duodenal GSH at 3 and 7 wks, and higher ileal GSH at 3 wks than birds not

receiving ethoxyquin. Lowering the oxidative stress in the gastrointestinal tract may

spare endogenous tissue antioxidants, and in turn help reduce PHs.

The excessive use of sodium chloride should be avoided in broiler rations and

water supply systems. Sodium chloride has been shown to reduce erythrocyte

deformability and increase blood volume (Julian 1987; Mirsalimi et al., 1992).

Decreasing erythrocyte deformability has been associated with an increase in PH, and

increases in the incidence of ascites in broiler chickens (Mirsalimi and Julian, 199 1).

Excessive sodium chloride in the diet rnay be arneIiorated with the addition of vitarnin C

(ascorbic acid). Ascorbic acid enhances the removal of N a - via the unne, thereby

reducing the level of sodium ions in the serum (Lewin, 1976). This process is effective in

removing excess sodium ions in humans and rnight be applicable to broiler chickens.

Cold ternperature (Julian et al., 1989; Enkvetchakul et al., 1993; Wideman er al.,

1991; Walton er al., 1998a), and low ventilation combined with cool temperatures

(Enkvetchakul et al., 1993; Wideman et al., 1994) have been used to study the

developrnent of PH and ascites. As a means of reducing and preventing PH and ascites,

controlling both ventilation and temperature within the birds comfort zone should be of

importance to the broiler producer.

Maxwell et al. (1986a, 1990) found that heterophils and monocytes were

increased at the expense of lymphocytes in ascitic broilers compared to controls, which is

indicative of in.flamrnation. Broiler chickens should be protected from diseases which

may have an inflamrnatory component or affect the pulmonary system. Routine

vaccination may help decrease the incidence of disease (e.g. infectious bronchitis virus)

and possibly PHs.

Rapid growth has ofien been associated with increases in birds with ascites

(Julian, 1993). Peacock et al. (1990) demonstrated in very rapidly growing broiler

chickens that hypoxaemia develops, which is associated with PH and in tum leads to right

ventncular failure (RVF), and eventually death due to cardiac failure. Therefore, if

growth c m be slowed, perhaps by feeding less energy rations, then the incidence of

ascites and PHs may be reduced (Julian, 1993). The potential problem with this solution

is that it will take longer for the bird to reach market weight.

1.10.0 Research hypotheses and experiments

Several studies were conducted either using hypobaric chambers to produce

hypoxaemia, or exposing the birds to cold ternperature, to induce pulmonary hypertension

and ascites. This thesis will attempt to answer the following questions:

Does the inclusion of 2.5% flax oil in a broiler ration reduce ascites due to

pulmonary hypertension in birds subjected to hrpobanc hypoxia?

Secondly. c m the dietary supplementation of antioxidants (vitamin C and E),

especially in diets containing flax oil, reduce pulmonary hypertension and ascites in

broiler chickens exposed to cold ternperature?

Lastly, is the decrease in pu lmonq hypertension and ascites from feeding flax

oil due to the production of arachidonic acid metabolites via cyclo-oxygenase and

lipoxygenase?

1.10.1 Experirnents 1 and 2 (chapter 2)

Flax oil has been show^ to alter the composition of erythrocyte membranes,

increasing fluidity, and improving their deformability (Bond, 1996). The objective of

these studies was to determine if the incorporation of 2.5% flax oil into a broiler mash

starter ration would reduce the incidence of PH and ascites by increasing erythrocyte

deformability without compromising growth. Birds were fed a 2.5% animal vegetable

(AN) blend of oil, which served as a control diet, compared to a ration containing 2.5%

flax oil. Several pens were kept at ambient atmospheric pressure to serve as control pens.

PH was induced by subjecting the broiler chickens to hypobaric hypoxia for four weeks.

Measurement of several haematological parameten and weekly growth rates was

conducted to determine if blood related parameters or growth rates were responsible for

any reduction in PH. Haernatological measurements included haemoglobin content of

erythrocytes, mean corpuscular haemoglobin concentration (MCHC), haernatocrit, whok

blood and plasma viscosity, and erythrocye deformability. The degree of PH was

assessed by measuring the right ventricle to total ventricular weight (RWTV).

1.10.2 Experiments 3 - 5 (chapter 3)

Vitamin C and E have been found to be lower in birds with PHs (Enkvetchakul et

al. 1993). The purpose of these expenments was to determine the effect of feeding 5% f lac

oil or 2.5% flax oil, along with supplemental antioxidants dunng the starter and grower

periods on the incidence of pulrnonary hypertension (PH) and ascites in meat type chickens

exposed to cold temperatures. This paper is composed of three separate trials al1 utibzing a

control diet of an animalhegetable ( A N ) blend of oil, 5.0% or 2.5% flax oil, supplemental

vitarnin C and E, and a combination of Bax oil plus antioxidants (flax/anti). The two

antioxidants used were vitamin E supplemented at 50 mgkg diet (dl-a- tocopheryl acetate),

and vitarnin C at 2000 mgkg diet (L-ascorbic acid). The two antioxidants tvere added to

the diets as welI as the usual mineral and vitarnin premix. Vitamin E was supplied at 11 .O

mg/kg in the premix and the premix contained no vitamin C. Each of these diets was then

supplemented with vitamin C and E (antioxidants). A total of four diets, replicated three

times were tested per trial, each having an AN diet with or without antioxidants, and a

flax oil diet with or without supplemental antioxidants.

In order to induce pulrnonary hypertension (PH) and asci tes, a low temperature

protocol was modified fkom Julian et al. (1989). The temperature in the room was lowered

starting at Day 35 from 2 1 O C to 1 SOC , and at Day 42 £tom 15°C to 10°C , until the end of

the experiment.

PH was assessed by measurement of the RV/TV ratios. Mortality, ascites, g r o ~ h ,

feed intake, feed conversion, whole blood viscosity, plasma viscosity, and the erythrocyte

packed ce11 volume (haematocrit) were also measured. At the end of the trials the birds

were killed humanely and the hearts removed to calculate the RV/TV ratios. Birds which

died dunng the trial were necropsied for ascites and the RVlTV calculated.

1.10.3 Experirnent 6 (chapter 4)

Various arachidonic acid metabolites such as prostaglandin E, (PGE,) and

prostacyclin (PGIJ have been implicated in PH in humans (Cremona and Higenbottarn,

1995). Thromboxane Al, the predominant arachidonic acid metabolite fiom platelets is

fomed frorn PGHZ, and is a potent platelet aggregator (Sigal, 199 1). By inhibiting cyclo-

oxygenase and lipoxygenase, we may be able to elicit whether eicosanoids might be

involved in PH and ascites in broiIer chickens. The objective of this experirnent was to

investigate the effect of inhibiting cyclo-oxygenase and lipoxygenase, which catalyse to

form prostaglandins and leukotrienes, on ascites mortality, pulmonary hypertension,

growth performance and haematological parameters in broi ler chic kens. The birds were fed

a diet containing 5% flax oil and were exposed to hypobaric hypoxia for 4 weeks. In

order to study the physiological effect of eicosaniod substances, 2 specific metabolic

inhibitors were used. The birds were divided into 3 treatment groups per hypobanc

charnber and received subcutaneous injections of either propylene glycol a control

vehicIe, indomethacin, or meclofenemate.

Meclofenemate is a dual inhibitor and blocks 5-lipoxygenase, and cyclooxygenase

1 and II. Indomethacin inhibits cyclooxygenase selectively over lipoxygenases (Smith,

1989; Meade er al., 1 993; Laneuville et al., 1994). At the end of the trial, RV/TV ratios

were evaluated along with haematocrit and fatty acid composition of plasma and

erythrocyte membranes.

Table 1.1 Fatty acid composition of flax oil (%).

Fatty acid Designation Fractions 1 1 %

Paimitic 1610 6 Stearic 18:O 6 Oleic 18:l (n-9) 20 Linoleic 1 8 :2 (n-6) 13 Alpha-lino Ienic 18:3 (n-3) 53

(Data refer to fractions 21 %) (Hornstra, 1983)

Table 1.2 Major pzclmonary oxidant scavengers. (Heffner and Repine. 1989)

Category S tmcture Tissue site Actions

GSH

Fat soluble vitamin Lipid membranes, extracellular fluids

Water soluble vitamin Found in intraceliular and extracellular fluids

Trip ep tide Intracellular

Converts h ydrox y 1 radical, superoxide anion, and lipid peroxyl radicals to less reactive forms. Breaks lipid peroxidation chain reactions.

Scavenges hydroxyl radical and superoxide anion. Neutralizes neutrophil oxidants and contributes to the regeneration of vitamin E.

Reacts with hydroxyl radical and superoxide anion. Substrate in GSH redox cycle.

Figure 1.1 Omega-3 and omegc-6pathways ofdesaturation and elongation. (Kinsella eî al., 1990)

n-6 Polyunsaturated fatty acids n-3 polyunsaturated fatty acids (omega-6) (omega-3)

(eayzme) ' Linoleic ( 1 8 :2) a-Linolenic (1 8:3)

1 elongase -------------------

Dihomo-~-Lino lenic 1

Eicosapentaenoic (20:3) (EPA)(20:5)

1 Enzymes involved in the desaturation and elongation of fatty acids.

Figure 1.2 Pathways involved in the oxygenation of arachidonic acid Zeading ro the formation of eicosanoids. (Smith, 1989)

Arachidonic acid

Prostaglandin Endoperoxide H,

W H 2

HPETEs

I Leukotnene A, + LTB,

f

Prostanoids Prostaglandins and thromboxanes Leukotrienes (PDG?, PGE:, PGF2, PGr2, TxAI) (LTC,, LTD,, LTE,)

Table 1.3 Selecfed aruchidonic acid merubolires und rheir biologicnl activities. (Sigal. 1991)

Metabolite Enzymes involved in synthesis Major biological action

Thromboxane AI

Prostacyclin PGIz

Leukotnene B,

Cyclo-oxygenase, PGD synthase

CycIo-oxygenase, PGE synthase

Cyclo-oxygenase, thromboxane AZ synthase

Cyclo-oxygenase, PGI synthase

5- lipoxygenase, LTA, hydrolase

Bronchoconstriction

Bronchodilation and vasodilation

Bronchoconstnction, vasoconstrition and platelet aggregation

Vasodilation, increase in vascu1a.r permeability and inhibition of pIatelet aggregation

Leukocyte migration, adhesion and activation.

Chapter 2.0

The effect of dietary flax oil and hypobaric hypoxia on pulmonary hypertension and h aematological parameters in broiler chickens.

Accepted by British Poultry Science (Modified fiom accepted version, adapted for thesis) (For cornparison purposes, data was included fiom Bond (1996) see appendix)

1 Department of Animal and Poultry Science and ' ~ e ~ t . of Pathobiology, University of Guelph,Guelph, Ontario, Canada N1 G 2W 1

2.1.0 Abstract 1. Two expenments were conducted with broiler chickens using

hypobaric charnbers and control pens feeding diets containing 2.5% flax oil, or control

diets with equivalent amounts of animahegetable (AJV) blend oil for 4 weeks. The

effect of these diets on haematological parameters and the extent of right ventricular

hypertrophy (RVH) leading to ascites were determined.

2. Overall growth rate was not consistently affected by dietary treatrnent, although

feeding the 2.5% flax oil diet reduced weight gain in week 4 of one experiment. Feeding

the 2.5% flax oil diet did not significantly reduce RVH in birds exposed to hypobaric

conditions compared to feeding control diets.

3. Feeding the 2.5% flax oil diet under hypobanc conditions did not significantly reduce

haematocrit, haemoglobin content, MCHC, or erythrocyte deformability compared to

feeding an A N blend of oil. Under hypobaric conditions feeding the flax oil diet

increased whole blood viscosity compared to feeding control diets.

2.2.0 INTRODUCTION

P u l m o n q hypertension (PH) in broiler chickens can occur as a result of increased blood

flow (increased cardiac output) or from an increase in the resistance to blood flow

through the lungs (Mirsalimi and Julian, 199 1). The result of pulmonary hypertension is

nght ventricular hypemophy (RVH) leading to nght ventricular failure, and finally the

development of ascites (Julian and Wilson, 1986). Death from ascites is determined by

gross postmortem changes inchding excessive fluid in the abdomen, an enlarged heart

and pericardial effusion with pulmonary edema (Owen et al., 1990). The arnount of PH

c m be estimated by the ratio of the weight of the right ventncle to the total ventricular

weight of the heart (RVITV ratio) (Sillau et al., 1980; Huchzermeyer and De Ruyck,

1986; Guthrie et ai.. 1987; Mirsalimi et al., 1993).

Often the increase in resistance to pulmonary blood flow is due to polycythaemia,

which increases blood volume and viscosity (Julian, 1993; Wideman et al.. 1994). High

altitude and hypobanc hypoxia are known to induce polycythaemia in broiler chickens

resulting in PH and RVH (Sillau er al., 1980; Julian and Wilson, 1986; Maxwell er a l ,

1990; Monge and Leon-Velarde, 199 1). Hypoxaernia stimulates erythropoiesis, resulting

in an accumulation of mostly Young, large, red blood cells to increase the blood volume

(Julian, 1993). Polycythaemia can also be stimulated by high levels of dietary cobalt and

results in PH; therefore, it is likely that the increase in the number of circulating

erythrocytes is responsible for the increased workload on the heart rather than

vasoconstriction caused by hypoxia @iaz et al., 1994).

It has also been found that decreased deformability of the erythrocytes, as

measured by the filtration index, c m increase the amount of PH and ascites in broiler

chickens (Mirsalirni and Julian, 199 1 ; Mirsalimi et al., 1993). The defomability of

erythrocytes is reduced by hypoxaemia (Hakim and Macek, 1988). Studies with humans

have demonstrated that erythrocyte Buidity c m be increased by dietary supplementation

of omega-3 (n-3) fatty acids £kom fish oils (Berlin et al., 1992). Archer et al. (1989) have

found that supplementation with fish oil reduces blood viscosity and RVH in rats.

However, other studies (Hulan et al., 1989) reported a reduction in the growth rate of

birds fed red fish meal as a source of omega-3 fatty acids. This is important, since the

incidence of ascites can be reduced by slowing the growth rate of broilers (Julian, 1993).

However, previous research frorn this laboratory has indicated that the inclusion of 10%

Bax oil in broiler rations was effective in reducing mortality and the incidence of ascites

at 2200 m and pulmonary hypertension at 1500 rn without affecting growth performance

(Bond et al., 1996).

The objective of the present study was to deterrnine whether the inclusion of 2.5%

flax oil in a broiler starter ration would also reduce the incidence of PH. The effect of

dietary fl av oil on growth rate, haematological parameters, including haemoglobin

content, mean corpuscular haemoglobin content, haernatocnt, whole blood viscosity, and

erythrocyte deformability was also determined. For cornparison purposes, data was

included from Bond (1996) who conducted a 5% flax oil experiment (see appendix).

2.3.0 MATERIALS AND METHODS

Two separate expenments were conducted. In each experirnent, day-oId male

broiler chicks purchased fiom a commercial hatchery were weighed, wingbanded and

divided randomly into four treatrnent groups. Two groups of birds were held in

hypobaric chambers and two groups were held in control pens at normal atmospheric

pressure, each pen was divided in half to replicate each of the dietary treatments within

each atmospheric condition twice. In each type of housing, one group of birds was fed a

flax oil diet and one group of birds was fed a control diet with the same inclusion level of

animavvegetable oil (Table 2.1). In experiments 1 and 2, oil was included in the diets at

25 g/kg and simulated altitudes of 2400 m and 1600 m respectively were used in the

hypobaric chambers. The equiprnent used has been descnbed previously (Bond et al.,

1996). Water and feed were available at al1 times under continuous lighting.

Temperature was maintained at a level cornfortable for the birds throughout the

experiments.

The birds were weighed initially on day I and then weekly for 4 weeks. A total of

100 birds were used in 4 treatment groups containing 2 replicates each in each

experirnent. The diets used contained 2.5% oil (Table 2.1, diets 1 and 2). In expenment

L, six birds randomly selected fiom each treatment group were bled on day 27 and used

ro detemine HCT, haemoglobin content and whole blood viscosity. In experiment 2, six

birds randornly selected f?om each treatrnent group were bled on day 28-30 for analysis

of filtration index. The methods used to determine filtration index (a measure of

erythrocyte defomability), whole blood viscosity, HCT, and haernoglobin were

previously descnbed in Bond et al. (1997). The mean corpuscular haemoglobin content

(MCHC) was calculated by dividing the haemoglobin value by the haernatocrit

measurement. The filtration index is based upon the time for a 10% suspension of

washed erythrocytes to pass through a polycarbonate membrane filter with an average

pore diarneter of 5 Fm. An increase in the filtration index indicates a decrease in

Table 2.1 Composition of erperimental diets (gk&

ExperÏments 1 and 2 Ingredients Diet 1 Diet 2

2.5% A N 2.5% flax oil

Soybean rneal (480 g CPkg) Corn Fat AN' Flax oi17 Limestone Dicalcium Phosphate. DL-Methionine Salt Vitarnin mix ' Mineral mix ' 2.5 Calculated nutrient content CP 2 14.3 ME (MJkg) 12.72 Cnide fat 49.3 Calcium 9.6 Available P 4.2 Methionine 5.1 Lysine 12.0

N V animallvegetable blend oil (stabilised with ethoxyquin 125~J1000L) Stabilised with ethoxyquin, 1 25g/1 000L; kindly provided by Grand Valley Fortifiers,

Cambridge, Ontario 'supplied per kilogram of diet: vitamin A, 8000 IU; cholecalciferol, 1600 IU; vitarnin E, 1 I .Omg; vitarnin K, 1.5 mg; v i t amid i z , 13 ug; thiamine, 4.0 mg; riboflavin, 9.0 mg; niacin, 26 mg; pantothenic acid, 1 1 .O mg; fo tic acid, 1.5 mg; biotin, 0.25 mg; choline equivaIents, 900 mg; selenium, 0.1 mg; Mn (MnSOa HzO), 55 mg; 1 [CaI(03)? HzO], 30 mg; Cu (CuSO4 7H20), 5 mg; Zn (ZnS04 7H20), 50 mg; ethoxyquin, 125mg.

deforrnability of the erythrocytes.

The birds were euthanatized on day 30 by cervical dislocation. The nght ventricle

was then carehlly separated fkorn the lef€ ventricle and septum, and weighed to calculate

the right ventricle to total ventricular ratici (RV/TV). Sick birds were removed and

eutl~anatized, and broilers that died during the experiment were necropsied.

Each expenment was designed as a split-plot with diets nested within each of the

atmospheric conditions. Statistical analysis was accomplished using the General Linear

Models procedure of the SAS hstitute (1985) and the least significant difference (LSD)

used to compare means with significance set at P < 0.05. Initial body weight was

introduced as a covariate to analyze body weight. The error term was diet interacted with

pen nested within each atrnosphere (arnbient or hypobaric).

2.4.0 RESULTS

The rnortality for the expenments is summarïzed in Table 2.2. In expenment 1,

there was one bird with PHs in the AN blend oil hypobaric group and none in the flax

oil hypobaric group. However, in experiment 2 there were three mortalities due to PHs in

the flax oil hypobaric group.

Overall weekly weight gain is surnmarized in Table 2.3. In week 4 of expenment

1, the flax oil groups gained less weight (Table 2.3) (P < 0.05), but this was not observed

in Expenment 2. Hypobaric hypoxia also did not significantly alter weight gain dunng

the sarne period.

Table 2.2 Total rnortaliy and causes of death in broiler chickens fed diets contnining Jar oïl or A/V blend oil and kept at either hypobaric or arnbient atrnospheric pressure.

Expenments 1 and 2 ' Number of Birds in Mortality / Cause group

Experiment

Flax oil Hypobaric

PJV blend oil Hypobaric

Fiax oil Arnbient

AN blend oïl Arnbient

O 3PHS 1 culled 1 PHs 1 culled 1 culled

1 SDS 1 SDS 1 starveout 1 SDS O

Hypobaric conditions simulating an altitude of XOOm (experiment 1) and 1600111 (expenment 2) .LI Pulmonary hypertension syndrome (RV/TV ratio > 0.299 or ascites fluid present)

Sudden death syndrome or dead in good condition Birds were culled due to leg problems

Table 2.3 Effect of 2.5% dietaryflar or A N btend oit on weekly body weight gain (g) of broilers held at hypobaric or ambient pressure (Means f SEM)

Expenment 1 ' Treatrnent Week 1 Week 2 Week 3 Week 4

AN blend oil Hypo baric

Flax oil Hypobaric

NV btend oil Ambient

Flax oil Arnbient

Experirnent 2

Treatment Week 1 Week 2 Week 3 Week 4

A N blend oil Hypobaic

Flax oil Hypobaric

AN blend oil Ambient

Flax oil Hypobaric

1 Hypobaric conditions simulating an altitude of 2400 rn ' Hypobaric conditions simulating an altitude of 1600 m a-b Means within a column for the same experiment with different superscripts differ significantly at P < 0.05

Haemoglobin content, MCHC, and HCT were not significantly different in

Expenment 1 (Table 2.4) among the hypobaric groups. Haemoglobin and HCT were

higher (P < 0.05) in both hypobaric dietary treatments compared to ambient control

groups. MCHC did not differ (P > 0.05) arnong broilers in the two atmospheric

conditions. Whole blood viscosity was higher (P < 0.05) in the hypobaric flax group

compared to hypobaric control, but did not differ among groups at arnbient pressure

(Table 1.4). The filtration time was increased in birds under hypobaric hypoxia (P < 0.05)

compared to those in pens at arnbient atrnospheric pressure.

The degree of pulmonary hypertension, as rneasured by the RVITV ratio, is given

in Table 2.5. Hypobaric hypoxia increased the RV/TV ratio (P -= 0.05) compared to

arnbient pressure groups in experiment 1 , but not at the lower simulated altitude of 1600

m used in experiment 2. Feeding the 2.5% flax oil diet did not significantly reduce the

RVITV ratios under hypobaric conditions compared to feeding the AN blend diet in

experiment 1 or 2.

2.5.0 DISCUSSION

Bond et al. (1 996) found no difference in the growth of birds fed diets containing

high levels of flax oïl and control diets containing an equivalent arnount of an AN blend

of fat, which is largely consistent with the results reported here. Kowever, feeding diets

containing flaxseed, rather than flax oil, reduced the growth rate of broilers (Bond et al.,

1997). Decreasing the growth rate decreases the oxygen demand of the tissues and thus

reduces PH and ascites in broiler chickens (lulian, 1993). It is therefore apparent that the

reduction in the arnount of PH under hypobaric conditions when the 5% flax oil diet

Table 2.4 Haematological values of broilers fed 2.5% dielaryjTnr or A N blend oil and held ar hypobaric or arnbient pressure (Means S E M )

Parame t er A N blend oil FIax oil A N blend oil Flax oil Hypobaric Hypobaric Ambient Arnbient

MCHC ' (g/l) 335.4 121.3

HCT ' (%) 39.6" k0.2

Whole blood 1 .8gb viscosity ' (cps) k0.03

Filtration 18.7" indexL (seconds)

1 Expenment 1 - hypobaric conditions simulating an altitude of 2400 m ' ~ x ~ e r i m e n t 2 - hypobaric conditions simulating an altitude of 1600 m a-b Means within a row with no cornmon superscript differ significantly at P < 0.05

Table 2.5 R V/TV ratios of broilers fedflar or A/V b l e d oil diets and kept under hypobaric or arnbient pressure (Means I SEM).

Arnbient pressure Hypobaric pressure

A N blend diet Flax oil diet A N blend diet Flax oil diet - - -- - -

Experiment 1 ' 0.22 1 a + -008 0.225" -t .O08 0.309~ + .O09 0.283~ i .O08

Experiment 2 0.1 9zab r .O 1 O 0.184~ + -01 1 0 . 7 3 5 ~ ~ ç -012 0.249" + .O 13

' Hypobaric conditions simulating an altitude of 2400 rn Hypobaric conditions simulating an altitude of 1600 rn

a< Means within a row for each experiment with no cornmon superscript differ sigificantly at P < 0.05

rather than the AN blend diet was fed was not due to a reduction in growth rate of the

birds (see appendix).

Bond (1996) found that the haematocrit, haemoglobin concentration, and the

RV/TV ratios of broilers were increased in response to hypobaric conditions simulating

an altitude of 2000 m, indicating that the birds were polycythaernic. Polycythaemia is

known to increase the workload on the heart (Diaz et al.. 1994). Findings £?om

experiments 1 and 2 suggest that the increase in PH in response to hypobaric pressure, as

indicated by the increased RV/TV ratios, was due to hypoxaemia induced polycythaemia.

Decreased deformability of erythrocytes (higher filtration index) has been fomd

to contribute to increased PH and incidence of ascites in broiler chickens (Mirsalimi and

Julian, 199 1). Bond (1996) found that, under hypobaric conditions, the haernoglobin

content, haematocrit, whole blood viscosity and the erythrocyte filtration index were

reduced with the inclusion of 5% flax oil compared to the AN blend oi1 in the diet. These

effects of the 5% flax oil diet occurred without a reduction in MCHC (see appendix).

However, the same results were not found when 2.5% flax oil was fed. The MCHC has

been related to the intemal viscosity of elythrocytes (Mohandes et a l , 1980), so the

changes in the filtration index for the erythrocytes cannot be attributed to a change in the

intemal viscosity of the cells.

Hypoxaernia reduces erythrocyte deformability in several species of rnammals,

particularly the rat (Hakim and Macek, 1988), and this is consistent with the results firom

Bond (1996). Feeding the 5% AN blend oil diet under hypobaric conditions resulted in

decreased erythrocyte deformability and an increase in the content of saturated fatty acids

in erythrocyte membranes cornpared to feeding the same diet at ambient pressure (see

appendix). However, feeding the 5% flax oil diet under hypobaric conditions rather than

the AN blend diet reduced the ratio of saturated fatty acids to unsaturated fatty acids in

erythrocyte membranes (see appendix). This increased content of unsaturated fatty acids

likely increases the Buidity of the membranes and alters membrane function to increase

the deformability of the erythrocytes. This could explain the reduction in the whole

blood viscosity that was found under hypobaric conditions when the 5% flax oil diet was

fed. These factors together would decrease the resistance to blood flow and improve the

movement of the erythrocytes through the capillaries, thus improving oxygen transport

and decreasing PH. Feeding a 2.5% flax oïl diet was not effective in reducing PH and did

not affect the MCHC, haernoglobin content, haematocnt, filtration index in birds under

hypobaric conditions.

The ratio of n-3 to n-6 fatty acids was increased in the erythrocyte membranes

with the inclusion of 5% flax oil in the diet (see appendix). Flax oil contains high

arnounts (57% of total fatty acids) of a-linolenic acid (ALA, 18:3 n-3) and lower

arnounts (16% of total fatty acids) of linoleic acid (1 8:2 n-6) (Bhatty, 1995). ALA is an

essential n-3 fatty acid that is a precursor for eicosapentaenoic acid (EPA, 20:5 n-3), and

docosahexaenoic acid (DHA, 22:6 n-3), while linoleic acid is an n-6 fatty acid and a

precursor of arachidonic acid (AA, 20:4 n-6) (Holub, 1995). Linoleic acid and ALA

compete for desaturation and elongation by the sarne enzyme systems, so that a proper

balance of these fatty acids in the diet is essential to optirnize the contents of AA and

EPAIDHA in the membranes. AA is the precursor of prostaglandins, thromboxane A?,

and leukotrienes, many of which promote platelet aggregation and vasoconstnction v a i r

el al., 1997). EPA and DHA can be M e r metabolized to the prostacyclins, PG4 and

PG12 respectively, by blood vesse1 enzymes to hnction as coronary relaxants

(Needleman, 1979). Thus, a higher ratio of n-3/n-6 fatty acids in the diet reduces the

formation of endogenous compounds that increase the resistance to blood flow and

increase the production of compounds that reduce the resistance to blood flow. However,

the importance of these compounds in the altenng the resistance to blood flow in broiler

chickens has not been determined.

Maxwell et al.. (1986a,b) reported an increase in immature and mature heterophils

in the hearts and livers of ascitic broilers compared to controls, suggesting that an

inflarnrnatory response occurs in birds suffering from PHs. Many of these changes are

the result of PH, rather than the cause of PH (Julian, 1993). There is considerable

evidence that various eicosanoid compounds derived from arachidonic acid participate in

inflammatory reactions (Calder et al., 1992; Wallace and Chin, 1997). The inflammatory

response c m be arnplified by neutrophils, since these cells have the capacity to release

chemotaxins such as leukotriene BJ (LTBJ) and reactive oxygen metabolites, which could

contribute additionally to tissue injury (Wallace and Chin, 1997). Therefore, shifting fatty

acid meiabolism from arachidonic acid synthesis by a higher n-3/n-6 fatty acid ratio in

the diet may reduce inflammatory responses associated with ascites.

2.6.0 CONCLUSION

It is apparent that the aetiology of ascites and PH in broilers is multi-factorial. The

exact influence that an increase in the deformability of erythrocytes has on decreasing the

resistance to pulmonary blood flow is unknown. Others (Wideman et al., 1994) have

suggested that vasoactive compounds also play an important role in reducing the cardiac

workload. More research is required to determine the mechanism by which flax oil

reduces PH in broiler chickens under hypobaric hypoxia. In particular, the potential role

of eicosanoid compounds derived from fatty acids in flax oïl in reducing pulmonary

hypertension and ascites in broiler chickens needs to be determined.

2.7.0 ACKNOWLEDGMENTS

Funding for this research was provided by the Ontario Ministry of AMculture,

Food and Rural Affairs (OMAFRA), the Ontario Chicken Marketing Board, the Canadian

Chicken Marketing Agency and the Flax Council of Canada.

Chapter 3.0

The effect of dietary flau oil and antioxidants on ascites and pulmonary hypertension in broilers using a cold temperature model.

Submitted to British Poultry Science (modified from submitted version, adapted for thesis)

' ~ e p r t m e n t of Animal and Poultry Science and ' ~ e ~ a r t m e n t of Pathobiology, University of Guelph, Guelph, Ontario, Canada NI G 2 W 1 .

3.1.0 Abstract 1. Three experiments were conducted using a cold temperature model to

induce pulmonary hypertension (PH) and ascites in broiler chickens. The effect of feeding

diets containing 2.5% or 5% flax oil cornpared to control diets with an equivalent amount of

animalhegetable (AN) blend oil, with and without antioxidants (vitamin C and vitamin E)

supplementation was studied. The amount of PH was assessed by the ratio of the right

ventricle weight to total venhicle weight (RVITV ratio).

2. Growth rate, feed intake, feed conversion, haematocrit, whole blood viscosity and

plasma viscosity were measured. In al1 three expenments, there was no significant effect of

dietary fat source or suppIementa1 antioxidants on total feed intake or feed conversion.

There was no significant effect of dietary fat source on the incidence of ascites or PH,

although a trend for a decrease was seen when the 5% flax oil diet was fed compared to the

control diet.

3. experiment 1, the test diets contained 5% oil and were fed dunng the grower period

only. Birds fed the flax oil diet supplemented with antioxidants had increased incidence of

ascites, PH, haematocrit, whole blood viscosity, plasma viscosity, and decreased weight

gain compared to those fed the flax oil diet without antioxidants. These effects were not

seen in experiment 2, when the test diets containing 2.5% oil were fed during the grower

period. However, in experirnent 3, when the test diets containing 2.5% oil were fed Eom

day 1 to week 8, birds fed the control diet supplemented with antioxidants had the highest

incidence of PH compared to those fed the control diet alone.

4. To conclude, feeding diets containing 5% flax oil tends to reduce the incidence of PH

and ascites in a coid temperature model. Supplementing diets with a combination of vitarnin

E and vitamin C does not reduce the incidence of ascites and PH and, in fact, reduced the

effectiveness of the 5% Bar oil diet in alleviating ascites and PH.

3.2.0 INTRODUCTION

Ascites (waterbelly) is characterised by the accumulation of lymph fluid in the

abdominal cavity. In broiler chickens, this fluid leaks £?om the liver into the ventral hepato-

peritoneal cavities because of increased hydraulic pressure in the vena cava and portal

system (Julian, 1993). A major physiological contribution to the development of

pulmonary hypertension syndrome (PHs) is systemic hypoxia stemming fkom pulmonary

insufficiency (Peacock er al., 1990). The progressive hypoxia develops from increased

oxygen demand by the tissues due to increased metabolic rate or rapid gro~vth

(Huchzermeyer and DeRuyck, 1986; Julian, 1 98 7).

Several mode1 systems have been used to study ascites and PH. Hypobaric

chambers (Owen et al., 1990; Julian and Squires, 1994; Bond et al., 1996; Walton et al.,

1998b), cold temperature (Julian et al., 1989), low ventilation and cool temperatures

(Enkvetchakul et al., 1993; Wideman et al., 1994), and the use of toxic levels of sodium

chloride (Mirsalimi et al., 1992), have been used to induce ascites and pulmonary

hypertension. Bond er al. (1996) demonstrated that incorporating flax oil as a fat source in

broiler diets reduced pulmonary hypertension and the number of ascitic birds exposed to

hypobaric hypoxia. Other workers have reported that dietary polyunsaturated fats from fish

oil results in reduced mortality, blood viscosity and nght ventricular hypertrophy in rats

(Archer et al., 1989). Erythrocyte membrane fluidity can be increased by dietary

supplementation of omega-3 (n-3) fatty acids, (Berlin et al.. 1992) which can improve the

flexibility of the erythrocyte and potentially help reduce PH. Al-Taweil and Kassab

(1990) found the number of ascites cases due to high levels of sodium chloride decreased

when vitamin C was incorporated into broiler rations.

Histopathology studies by Maxwell et al. (1986% 1990) indicated that tissues of

ascitic birds and hearts of experimentally induced hypoxic birds had elevated numbers of

inflarnmatory cells. Activated white blood cells can produce a variety of reactive oxidants

into swounding tissues, which rnay have the ability to alter the antioxidant status of the

tissue (Enkvetchakul et al., 2993). Enkvetchakul et al. (1993) also found that

concentrations of both vitamin E and C, plus glutathione were compromised in broilers with

PHs. Low levels of these antioxidants are associated with darnaging oxidative tissue

reactions and may play an important role in the development of PHs or potentiate the

situation further (Enkvetchakul et al., 1993). When Hollander (1981), added two

polyunsaturated fatty acids to the perfusate of rat small intestine, fat soluble vitamin

uptake declined. This may fûrther increase the need for supplemental antioxidants when

polyunsaturated fatty acids are used as a fat source in broiler rations.

Increased lipid peroxide concentrations could contribute to the development of PHs

by altering arachidonic acid metabolism (Bottje et al., 1995). Vasodilatory prostanoids such

as prostaglandin E, (PGE,) and prostacyclin (PGI,), which are synthesized from arachidonic

acid, have been implicated in such disorders as pulmonary hypertension (Cremona and

Higenbottarn, 1995).

The purpose of these expenments was to determine the effect of feeding diets

containing 5% or 2.5% Bax oil, with or without supplemental antioxidants, vitarnin E and

vitarnin C, during the starter and grower periods on the incidence of pulmonary

hypertension (PH) and ascites in meat type chickens using a cold temperature model. This

was assessed by rneasurement of the R V f N ratios as well as mortality, growth, blood and

plasma viscosity, and the erythrocyte packed ce11 volume (haematocnt).

3.3.0 MGTERIALS AND METHODS

3.3.1 Diets

This report is composed of three separate experiments, with four dietary treatments

in each experiment. The diets contained either 5% or 2.5% flax oil, with a control diet

containing an equivalent arnount of an animdvegetable (AN) blend of oil. (Table 3.1).

Both of these diets were either fed as is or supplemented with vitamin E (dl-a-tocopherol

acetate) at 50 mgkg of diet, and vitamin C at 2000 mgkg of diet (BASF Corp., Animal

Nutrition Division, Georgetown, Ontario, Canada.). AI1 of the diets contained the usual

mineral and vitarnin premix, which supplied Vitarnin E at 1 1 .O mgkg of diet. The diets

used in experiment 1 contained 5% oil while the diets used in expenments 2 and 3

contained either 2.5% flax oil plus 0.5% A N blend oil or 3% A N blend oil. In experiments

1 and 2, the test diets were fed only during the grower phase and a commercial broiler

starter diet (22% protein, and 12.85 MJkg of ME) was fed for the first 4 weeks. In

experiment 3, test diets were fed during both the starter and grower phases.

3.3.2 Animals

In each of the experiments, 360 one-day old male broilers obtained from a local

hatchery were weighed, wingbanded and randomly placed into the four dietary treatments

containing three replicates of 30 birds on each diet. The birds were provided with water and

feed ad libitum, and maintained under continuous lighting. Sick and dead birds were

Table 3.1 Composition of experimental diets for the three experiments

Experiment I Experiment 2 and 3 Ingredients

(d'kg) NV diet ' FIax diet AfV diet ' Flax diet

Soybean meal (480 g CPkg) Corn Fat A/V1 FIax oil' Limestone Dicalcium Phosphate D L-Methionine Salt Vitamin mix Minera1 mix

Calculated nutrient content (gkg) CP 205.8 ME (M.J/kg) 13.22 Cnide fat 73 -8 Calcium 9.7 Available P 4.3 Methionine 4.3 Lysine 12.2

Grower 307.0

62 1.2 30.0

- 15.0 15.0

0.8 3.0 5.0 2.5

201.1 12.9 1 55.1 9.5 4.2 4.0 11.1

Starter 356.4

576.6 5.0

25 .O 15.0 15.0

0.8 3 .O 5 .O 2.5

221.1 f 2.66 48.7 9.6 4.2 4.4 12.6

Grower 307.0

62 1.2 5.0 35 .O 15.0 15.0

O. 8 3 .O 5.0 2.5

20 1.1 12.91 55.1 9.5 4.2 4.0 11.1

' AN represents anirnalhegetable blend of oil (stabilised with ethoxyquin, 125g/1000L) ' S tabilised with ethoxyquin, 1 X g / I 000L; kindly provided by Grand Valley Fortifiers, Cambridge, Ontario '~upplied per kilogram of diet: vitarnin A, 8000 IU; cholecalciferol, 1600 IU; vitamin E, 1 1 .Omg; vitamin K, 1.5 mg; vitamid3 ,z, 13 ug; thiamine, 4.0 mg; riboflavin, 9.0 mg; niacin, 26 mg; pantothenic acid, 1 1 .O mg; folk acid, 1.5 mg; biotin, 0.25 mg; choline equivalents, 900 mg; seleniurn, 0.1 mg; Mn (MnSO, H20), 55 mg; 1 [CaI(O,), HzO], 30 mg; Cu (CuSO, 7H20), 5 mg; Zn (ZnSO, 7H,O), 50 mg; ethoxyquin, 125mg. ' Represents animalhegetable blend oil ( N V blend) diets. The A N b1encUantioxidant diet contained supplemental dl-a-tocopherol acetate at 50 mgkg and vitarnin C at 2000 m g k g diet. ' Represents flax oil diets. The flax oiVantioxidant diets contained supplemental dl-a- tocopherol acetate at 50 mgkg and vitamin C at 2000 mgkg diet.

removed fiom each pen and necropsied for evidence of PHs and ascites. Weekly body

weight and feed intake was recorded fiom week 4 to week 8 in experiments 1 and 2 and

fiom day 1 to week 8 in experiment 3.

A low temperature protocol, modified From Iulian et al. (1989), was utilized to

induce pulmonary hypertension (PH) and ascites. Typically the temperature in the roorn

was lowered starting at day 35 korn 2 1 OC to 1 S O C , and at day 42 from 15OC to 1 O°C ,until

the end of the experiment at day 56. The birds were then killed hurnanely after elecûicai

stunning and the hearts removed, tnmrned of fat, and the right venh-icle was carefully cut

away from the left ventricle and septum. These were weighed to determine the right

ventricle to total ventricle ratio (RVRV). At the time of slaughter birds with ascites were

noted and recorded.

3.3.3 Sampling

Blood was collected on days 54 and 55 of expenment 1 from the main brachial wing

vein and placed into heparinized tubes. A srnall sample of whole blood was centrifuged for

5 minutes using an International micro-capillq centrifuge (International Equipment

Company, Needham Kts., Mass.), and the haematocrit measured. The viscosity of whole

heparinized blood and plasma was measured on a I ml sample of each using a Wells

Brookfield Synchro-Lectric cone/plate viscometer (Brookfield Engineering Labs, Inc.,

Stoughton, Mass.) at 40°C. Plasma viscosity was measured following centrifugation of the

whole heparinized blood at 1750 x g for 10 minutes at room temperature.

3.3.4 S tatistical Analysis

A one-way anova was performed to determine sources of variation due to diet and

pen, with pen nested within diet. Initial weight of the birds was used as a covariate in

analysing the growth data. A log transformation was used to normalize the R V m

distribution. Cornparisons of diet, log RV/TV ratios, haematocrit, whole blood and plasma

viscosity, weekly weight gain, feed intake and feed conversion were made by Least square

means analysis (LSD). The chi-square test for homogeneity of rnortality by diet and ascites

was conducted and, when significant, was also used in painvise cornparisons of treatments.

The statistical analysis was accomplished using General Linear Models of SAS (SAS

Institute, 1985). Data are expressed as the rnean f standard error. A probability level of

less than 0.05 was considered significant.

3.4.0 RESULTS

3.4.1 Experiment 1

In this experiment, the birds were raised on a commercial starter for 28 days then

switched to the 4 test grower diets containing 5% oil for weeks 4 through 8 (Table 3.1).

The average room temperature from day 28 to day 42 was 19.0 O C , while the average

temperature fiom day 42 to day 56 was 14.0 OC

A total of 33 birds had ascites, with the flax oiVantioxidant group having

significantly more ascitic birds than the flax oil group (Table 3.2) (chi-square < 0.027). Of

the 17 birds that died kom ascites, the majority of them died in the last two weeks of the

experiment (data not show). The incidence of PHs birds (ascites fluid present or a RWTV

ratio greater than 0.299) was also significantly higher in the flax oiVantioxidant treatrnent

Table 3.2 Incidence of ascites and PHS and R V/TV ratios of broilers aposed to cold temperature.

Experiment A N blend Flax oil PtrV blend oiV FIax oiV TotaI oil Antioxidants Antioxidants

Ascites ' 1 (nurnber of 2 birds) 3

PHs ' 1 (number of 2 birds) 3

RV/TV ' ratio 1 (Mean k SE)

PHs = pulmonary hypertension syndrome (ascites fluid present or RV/TV > 0.299) la" Values with no comrnon superscript differ within a row per expenment (chi-square 0.05 ) ' Values with no common superscript differ within a row per experiment ( P < 0.05 )

(chi-square < 0.05), cornpared to the other three dietary beaûnents. The RV/TV for the

flax oïl group was significantly lower (P < 0.05) than the flax oiuantioxidant fed birds.

Total weight gain, feed intake, and feed conversion are surnrnarized in Table 3.3 .

Feed intake and feed conversion did not differ among treatments, but the total weight gain

of the flax oiWantioxidant group was significantly lower (P < 0.05) than the other three

dietary treatments. In week 8 of the experiment, the flax oiVantioxidant goup gained less

weight (P < 0.05), consumed less feed, and had poorer feed conversion efficiency (Table

3 -3).

Data for haematocrit, whole blood and plasma viscosity can be found in Table 3.4 .

Supplementing the flax oil diet with antioxidants increased the haematocnt, whole blood

viscosity and plasma viscosity of the birds. On the other hand, supplementing the AN

blend diet with antioxidants did not affect haematocrit and whole blood viscosity and

lowered the plasma viscosity. Haematocnt was correlated with whole blood viscosity (r =

0.72, P < 0.0001) but not plasma viscosity. Haematocrit was also found to be correlated

with the RV/TV ratio (r = 0.23, P c 0.05).

3.4.2 Experiment 2

In this expenment, birds were fed a commercial starter diet for the first 4 weeks,

then the 4 test grower diets containing 3% oil were fed fiom week 4 to week 8 (Table 3.1).

According to the temperature protocol, on day 35 the temperature in the room was lowered

to average 15.9 OC from day 35 to day 41 and 12.2 OC 60m day 42 to day 56.

A total of 39 birds had ascites in the expenment (Table 3.2) with no significant

dietary effect on the incidence of ascites. Of the 26 birds that died from ascites, the rnajority

Table 3.3 Experirnent 1. Eflecrs of diet on weekly and overall weight gain (g/broiZer), feed Nltake (g/broiler). and feed conversion ration (FCR) of broilers aposed to cold temperature.(Zeust square means followed by (SE))

Week

5 6 7 8

Weekly gain (gramsibroiler) A N blend oil 5% Flax oil AN blend/Antioxidants Flax oiYAntioxidants

Feed intake (grarnshroiler) AN blend oil 5% Flax oil AN blend/Antioxidants Flax oiVAntioxidants

FCR (kg feed/kg gain) A N blend oil 5% Flax oil NV blend/Antioxidants Flax oil/Antioxidants

Total gain

2434" 2395" 2406" 2 1 83b

Total intake 5238 5219 5268 5066

Average 2.20 2.2 f. 2.22 2.5 1

. . -. - . - - - - - - - - - . . - - - . -

Values with no cornmon superscript differ within a column ( P < 0.05 )

Table 3.4 Experiment 1. Haematocrit , whole blood and plasma viscosity (mean - + SE) of broilers fed diets containing 5% oil and exposed IO cold temperature.

Diet A N Blend Flax oil A/V blend / Flax oil

Antioxidants /Antioxidants

Haematocrit (%)

Whole blood viscosity 3-58" (CPS) +o. 1

Plasma viscosity (cps) 1-01" +o.o 1

"' Values with no common superscnpt differ within a row ( P < 0.05 )

of them died in weeks 7 and 8 of the experiment (data not shown). There was also no

significant dietary effect on the incidence of PHs or the RV/TV ratios, although there was a

tendency for reduced PH in the flax oil group compared to the control group fed the A N

blend oil (P < 0.068) (Table 3.2).

The effects of dietary fat source and supplemental antioxidants on total weight gain,

feed intake, and feed conversion are shown in Table 3.5 . In week 7, the antioxidant group

had an improved feed conversion compared to flax and flax oiYantioxidants (P < 0.05)

(Table 3.5). However, by the end of the expenment there was no significant difference

among the dietary treatments relating to feed and growth parameters.

3.4.3 Experiment 3

In this expenment, test diets containing 3% oil were fed during both the starter and

grower phases (Table 3.1). From day 35 to day 42 the average room temperature was

lowered to 14.9 OC, and until the end of the experiment the room temperature averaged

1 O.7"C.

A total of 27 cases of ascites were found with 13 of them detected at slaughter. The

incidence of ascites was significantly greater in the group fed the flax oil diet supplemented

with antioxidants compared to the control group fed the diet with A N blend oil (chi-square

< 0.05) (Table 3.2). The controYantioxidants group had the highest number of birds with

PHs, and was significantly different fYom the control group (chi-square < 0.05). The

majonty of deaths attributed to PHs occurred in the last 2 weeks of the expenment, with no

significant difference observed arnong the dietary treatments (data not shown).

Supplementing the AN blend oil diet with antioxidants also significantly increased the

Table 3.5 E~eriment 2. Effects of diet on weekly and overall weight gain (ghroiler), feed intake (g/broiler), and feed conversion ratio (FCR) of broilers erposed to cold temperature. (least square means followed by (SE))

Weekly gain (ghroiler) A N blend oiI 644 (18) 623 (42) 568 (65) 577 (74) FIax oil 607 (14) 660 (22) 582 (7) 453 (1 1) AN blendlAntioxidants 626 (29) 653 (23) 672 (18) 427 (57) Flax oiVAntioxidants 605 (6) 641 (29) 565 (16) 508 (26)

Feed intake (ghoiler) NV blend oil 1229 (17) 1400 (54) 1 194 (76) 1355 (53) Flax oil 1 186 (33) 1438 (55) 1326 (87) 1346 (36) AN blend/Antioxidants 1212 (37) 1394 (41) 1262 (60) 1397 (35) Flax/Antioxidant 1201 (21) 1394 (27) 1266 (30) 1282 (49)

FCR (kg feedkg gain) AN blend oil 1.91 (.03) 2.26 (.07) 2.13 "b(.14) 2.42 (-31) Flax oiI 1.95 (.03) 2.18 (.01) 2.28 "(.12) 2.97 (-01) A N blend/Antioxidants 1.94 (-03) 2.14 (.03) 1.88 (. 13) 3.4 1 ( 3 ) Flax oiVAntioxidants 1.99 (-02) 2.18 (.09) 2.25 "(.09) 2.53 (.03)

Total gain 241 1 2301 2379 3320

Total intake 5178 5296 3265 5 143

Average 2.20 2.37 2.50 3.23

- - - - -

Values with no cornmon superscript differ within a column ( P < 0.05)

R V f N ratio cornpared to the control diet alone (P < 0.05) (Table 3.2).

No significant dietary effect was f o n d with respect to total weight gain, total feed

intake or feed conversion (Tables 3.6, and 3.7) for weeks 1 through 8.

3.5.0 DISCUSSION

The results indicate that feeding diets containing 5% flax oil alone tended to reduce

PH and ascites due to cold exposure compared to feeding diets containing an equivalent

arnount of AN blend oil. Feeding diets containing 2.5% flax oil did not affect the incidence

of ascites or PH compared to feeding the control diets, even when the test diets were fed for

the entire 8 week growth period. Surprisingly, supplementing the 5% flax oil diet with

vitarnin E and vitamin C removed this effect and increased the incidence of ascites and PH.

Our results support those of Bond et al. (1996) who found that the inclusion of 10% flax oil

decreased the incidence of ascites and reduced PH in broilers subjected to hypobaric

hypoxia. Other work from our lab has demonstrated that 2.5% flax oil diets were not

effective in reducing ascites or PH in broilers under hypobaric hypoxia (Walton et al.,

1998b).

The possible mechanisms by which flax oil reduces PH and ascites in broilers

include increasing the defonnability of the erythrocytes or the production of vasoactive

compounds to reduce blood pressure. It has also been shown that decreased deformability

of erythrocytes, as measured by the filtration index, can contribute to an increase in the

arnount of PH and ascites in broiler chickens (Mirsalimi and Julian, 1991; Mirsalimi et

al, 1993). Feeding 5% flax oil increases the content of unsaturated fatty acids in

Table 3.6 Experirnent 3. Effects of diet on weekly and overall weight gain (g/broiler), feed intake (ghroiler), and feed conversion ratio (FCR) of broilers exposed to cold temperature. (leusr square means followed by (SE))

Diet Week

Weekly gain (ghroiler) A N blend oil 116 (3) Flax oil 1 17 ( -2 ) AN blend.Antioxidants 1 1 8 (-6) Flax oiilhtioxidants 1 17 (3)

Feed intake (ghroiler) A N blend oil 206 (1 5) Flax oil 184 (17) AN blend/Antioxidants 183 (1) Flax oiVAntioxidants 162 (1 7)

FCR (kg feeàkg gain) A N blend oil 1.79 (-1 8) Flax oil 1.57 (.15) AfV blend/Antioxidants 1.56 (.004) FIax oil/Antioxidants 1.40 (. 16)

Total gain 1339 1214 1408 1257

intake 3234 2263

Average 1.70 1.77 1-61 1-70

pp - --

Values with no common superscript differ within a column ( P < 0.05 )

Table 3.7 Experiment 3. Effects of diet on weekly weight gain (weeks 5 - 8 and day I - week 8) (g/broiZer), total feed intake (ghrder) , and feed conversion ratio (FCR) of broilers erposed to coid temperature. (least square rneans followed by (SE))

Week

5 6 7 8

Weekly gain (ghroiler) AN blend oïl 606 (10) 635 (10) 589 (17) 529 (26) Flax oil 565(47) 621(15) 599(2) 481(34) A N blend/Antioxidants 582 (14) 622 (5) 618 (57) 544 (47) Flax oivhtioxidants 564 (8) 6 15 (4) 547 (33) 495 (45)

Feed intake (ghroiler) AN blend oil 1 176 (30) 1351 (23) 1458 (33) 1525 (50) Flax oil 1128(26) 1297(28) 1417(15) 1470(32) NV blend/Antioxidants 1 169 (23) 1303 (34) 1440 (27) 1544 (1 7) Flax oil/Antioxidants 1 15 1 (1 7) 1305 (7) 1395 (25) 1489 (65)

FCR (kg feed/kg gain) AN blend oil 1.94 (.02) 2.13 (-01) 2.48 (.03) 2.89 (-08) Flax oil 2.01 (-12) 2.09 (-005) 2.37 (.02) 3.07 (. 15) AN blendhtioxidants 2.0 1 (. 12) 2.09 (.06) 2.36 (. 17) 2.89 ( 2 7 ) Flax oil/Antioxidants 2.04 (.03) 2.12 (-02) 2.56 (. 1 1 ) 3 .O4 (. 17)

Total gain

2359 2266 2366 232 1

To ta1 intake 5510 5312 5456 5340

Average 2.36 2.39 2.34 2.44

- --

Values with no cornmon superscnpt differ within a column ( P c 0.05 )

e ~ h r o c y t e membranes, thereby increasing the fluidity of the membranes and increasing

the deforrnability of the erythrocytes (Bond, 1996; Walton et al., l998b).

The metabolism of n-3 fatty acids to vasoactive compounds might also be important

in reducing ascites and PH. FIax oil contains a-linolenic acid, a precunor to

eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Both EPA and DHA have

shown significant potential to dampen platelet aggregation and formation of thromboxane

A,, a pro-aggregatory vasoconstrictor Wolub, 1 995). Birds have thrombocytes which are

fragile nucleated ceils, similar to platelets of mammais (Sturkie, 1986). If thrombocytes

behave the same as platelets with respect to EPA and DHA synthesis, then the production of

coronary relaxants couid help explain the decrease in the number of birds with PHs afier

feeding diets containing 5% flax oil.

We found no significant effect on weekly weight gain, feed intake or feed

conversion fkom including 2.5% flax oil or antioxidants into the rations. in Expenment 1,

the group fed the 5% flax oil diet suppiemented with antioxidants had more cases of ascites

in the last few weeks of the experiment; subsequently feed intake and weekly weight gain

was reduced. Previous work from our laboratory (Bond et al., 1996) aiso showed no

difference in growth rate between birds fed diets containing flax oil or a commercial A N

blend of oil. Phetteplace and Watkins (1992) found no difference in the growth of female

chicks raised on diets containing either 50 g/kg soybean oil or menhaden fish oil as a fat

source. This is important, since the incidence of ascites and PH can be reduced by

decreasing growth rate (Julian, 1993).

In Expenment 1, haematocrit, whole blood and plasma viscosity, and RVEV values

were significantly higher in the goup fed the flax oil diet supplemented with antioxidants

compared to birds fed the diet containing flax oïl alone. This result was somewhat

surpnsing, given that the increased levels of polyunsaturated fatty acids in the flax oil diets

would normally increase the requirement for dietary antioxidants to prevent lipid

peroxidation. Squires and Summen (1993) suggested that lipid peroxidation might play a

role in the development of PHs. Lipid peroxidation is initiated with the generation of

reactive oxygen species including hydrogen peroxide, hydroxyl radical, superoxide,

hydroperoxides, and other free radicals. In tum, lipid fiee radicals are generated f?om

unsaturated fatty acids, which are capable of advancing the process of lipid peroxidation

unless they are rapidly reduced (Heffner and Repine, 1989). Vitamin E is a lipid soluble

vitarnin that represents the principle defence against oxidant-induced membrane darnage

(He&er and Repine, 1989; Bottje and Wideman, 1995). The vitamin E (a-tocopherol)

radical breaks the darnaging lipid fiee radical chain reaction and can be recycled back to its

reduced state by the reduction with ascorbate (vitarnin C) (Hefher and Repine, 1989;

Boxer, 1990). When the lipid peroxidation exceeds the antioxidant capability of the

tissues, tissue degeneration results (Horton and Fairhurst, 1987). Maxwell et al. (1996)

found that lipid peroxides may be involved in the degeneration of cardiac tissue.

The increase in haematocnt and viscosity of whole blood and plasma that is seen

with antioxidant supplementation of the flax oil diet may have been partially due to the high

level of ascorbate (2000 mgkg) used in our diets. Al-Taweil and Kassab (1990) reported

an increase in haematocrit and senun protein content when birds were fed ascorbate at 450

mgkg feed. It is known that blood viscosity is influenced by the number of circulating red

blood cells (Sturkie, 1986) and polycythaemia is known to increase the workload on the

heart (Diaz ef aL, 1984). The increase in blood viscosity may explain why the group fed the

flax oil diet supplemented with antioxidants had a higher incidence of ascites and PH than

the birds fed the flax oil diet without supplemental antioxidants. However, this does not

explain why these effects were not seen with antioxidant supplementation of the AfV blend

diet. Ladmakhi et al. (1997) found that dietary vitamin C at 500 mgkg did not increase

haematocrit and reduced the incidence of ascites caused by low temperature and thyroxine

(T,) supplementation. These authors suggest that vitamin C can affect thyroid function and

metabolic rate-

In spite of an increase in haematocrit, Al-Taweil and Kassab (1990) found a

decrease in ascites caused by high levels of sodium chloride when broilers were fed diets

supplemented with vitamin C ranging from 150 to 450 m g k g diet. These different effects

of vitarnin C on ascites are probably due to the different methods used to induce ascites.

Ascites caused by high levels of sodium chloride is a result of decreased ejthrocyte

deformability and an increase in blood volume (Mirsalimi et al., 1992). Vitamin C has been

suggested to help in the excretion of cations, particularly sodium, (Le~vin, 1976). If this

occurs in broilers, ascorbate would be expected to counteract the ascites due to high levels

of dietary sodium. Ascites due to cold temperature or hypobaric hypoxia is caused by

polycythaemia due to insufficient oxygen in the tissues (Julian, 1993). In these cases, any

increase in haematocrit caused by dietary ascorbate would be expected to increase PH and

ascites.

We found no differences in ascites and PH frorn feeding the test diets for 8 weeks

(Experiment 3) compared to feeding the test diets only in the grower penod (Expenment 2).

The combination of vitarnin E and vitamin C was not effective for alleviating ascites or

reducing PHs, but rather tended to increase the severity using this cold temperature model.

Bottje et al. (1995) demonstrated that providing a 21-day time released a-tocopherol

implant reduced the 5-wk cumulative PHs mortality in broilers by rnaintaining tissue

antioxidant concentrations and alleviating lipid peroxidation. However, when Bottje et al.

(1997) performed a similar expenment feeding O to 87 mg/kg supplemental dietary dl-a-

tocopherol acetate, it did not reduce PHs mortality. These researchen suggest the lack of

effect on PHs may be due to timing, chernical fom, and arnount of tocopherol supplied to

the birds. A major limitation to prophylactic vitamin E therapy is the complex

pharmacokinetics involved in the delivery of lipophilic antioxidants. It takes several weeks

to achieve small increases in vitarnin E tissue levels (Heffher and Repine, 1989). In view of

our findings experiments are needed to detemine the effectiveness of using only vitamin E

as the antioxidant supplement in diets containing 5% flax oil.

3.6.0 CONCLUSION

To conclude, we have found a tendency for decreased ascites and PH due to cold

exposure in broiler chickens by including 5% flax oil in the diet. However, supplementing

the flax oïl diet with a combination of the vitamin C (2000 mglkg diet) and vitarnin E (50

rngkg diet) removes the beneficial effects of the flax oil. In light of these results, more

research is required to determine the effects of individual antioxidants in broiler rations and

their efficacy in reducing PH and ascites.

3.7.0 ACKNOWEDGEMENTS

Funding for this research was provided by the Ontario Minisûy of Agriculture, Food

and Rural Affairs (OMAFRA) and the Flax Council of Canada. Their financial support was

greatly appreciated. The vitamin C and E were generously supplied by BASF Canada,

Georgetown, Ontario.

Chapter 4.0

The effect of cyclo-oxygenase and iipoxygenase inhibitors on pulrnonary hypertension in broilers fed 5% dietary flax oil su bjected to hypobaric hypoxia.

4.1.0 Abstract 1. An experirnent was conducted to determine the effects of uihibiting the

synthesis of vasoactive arachidonic acid metabolites on pulmonary hypertension (PH) in

broiler chickens. Birds were kept in hypobaric chambers and fed a 5% flax oil diet for 4

weeks. The birds were injected every other day for four weeks subcutaneously with either

indomethacin, meclofenemate, or propylene glycol vehic le. The effect of these inhibitors on

growth, haematological parameters and the degree of pulmonary hypertension was

evaluated.

2. Weekly body weight gain and total weight gain was not significantiy affected by

injections of indomethacin or meclofenemate compared to control. The degree of pulmonary

hypertension as measured by the RVrW ratio, or haematocrit was not significantly different

behveen the treatment groups

3. There was no significant effect of indomethacin or meclofenemate treatment on the fatry

acid composition of plasma, but there were significant effects on the fatty acid composition

of erythrocyte membranes. Eicosapentaenoic acid (EPA), clupanodonic acid, and

docosahexaenoic acid (DHA), were al1 Iower in the inhibitor treated groups compared to

the control group. The stearate and linolenate levels also increased (P< 0.05) in the

inhibitor treated broilers. These results demonstrate that the metabolism of fatty acids was

altered by injecting indomethacin and meciofenemate, and this did not reduce the dietary

effectiveness of flax oil in reducing PH in broiler chickens.

4.2.0 INTRODUCTION

Pulmonary hypertension can develop in broiler chickens due to a number of

physiological conditions. A major factor is the inability of the pulrnonary vasculature of

broilers to cope with relatively small increases in cardiac output (Wideman et al., 1996).

hcreased blood volume through the pulmonary vasculature, with decreases in transit tirne,

lead to less time for gas exchange in the tissues (Julian, 1993; Bottje and Wideman, 1995).

Vasodilatory prostanoids such as prostaglandin Ei (PGE,) and prostacyclin (PGI?)

have been irnplicated in puhonary hypertension in humans, although the significance of

these findings in the pathogenesis of PHs remains speculative (Cremona and Higenbottam,

1995). Prostacyclin is a potent endogenous vasodilator and decreases platelet aggregation.

PGIz is synthesized from arachidonic acid via the cyclo-oxygenase pathway, and is

continuously reieased by vascular endothehm (Cremona and Higenbottarn, 1995).

Another important derivative of arachidonic acid is the eicosanoid thromboxane

(Tx)A2. TxA2 is produced mainly by blood platelets and behaves as a vasoconstrictor and

promotes platelet aggregation. The balance between arterial wall PGIz production and

platelet TxA? has been suggested as an important factor in platelet reactivity and

aggregation (Meydani, 1992). Patients with prirnary pulmonary hypertension still release

PG12, however it may be the balance between TxAz and prostacyclin (TxA2/PGI~) which

contribute to the disorder (Meydani, 1992; Crernona and Higenbottam, 1995).

Previous research has demonstrated that the inclusion of flax oil in broiler rations

was effective in reducing mortality and the incidence of ascites at 2200m and pulmonary

hypertension at 1500m without affecting growth (Bond er al., 1996). Flax oil contains a-

linolenic acid a precursor to eicosapentaenoic acid (EPA) plus docosahexaenoic acid

@HA). Both EPA and DHA have s h o w significant potential to darnpen platelet

aggregation and TxAz formation in humans (Holub, 1995). Avian blood contains

thrombocytes which are fragile, nucleated cells similar to platelets of mammals (Sturkie,

1986). If thrombocytes behave similarly as platelets of mammals with respect to EPA and

DHA synthesis, then the production of coronary relaxants with a subsequent decrease in

vasconstrictive agents, may help reduce PH in broiler chickens.

Mirasalimi and f ulian (1 99 l), found that decreased deformability of erythrocytes cari

contribute to increases in PH and ascites in broiler chickens. Hypoxaemia has also been

found to reduce erythrocyte flexibility (Hakim and Macek, 1988). The source of dietary fat

can affect the phospholipid fatty acid composition of erythrocyte membranes (Mills et al.,

1993; Sarkkinen et al., 1994; A g e n et al., 1995). It is possible to alter the fluidity of the

erythrocyte membrane by changing dietary fat sources, making the membranes more

flexible (Bond, 1996).

in order to study the potential effect of eicosanoid substances on PHs in broiler

chickens, inhibitors c m be used to block the activity of cyclo-oxygenase or 5-lipoxygenase.

The objective of this preliminary study was to investigate the effect of inhibiting the

synthesis of arachidonic acid metabolites on ascites mortality, puimonary hypertension as

rneasured by the right ventricle to total ventricle weight (RVTTV), growth performance and

haematological parameters in broiler chickens.

4.3.0 MATEMALS AND METHODS

4.3.1 Anirnals / housing / diet

Forty-two day-old male broiler chicks purchased fiom a commerciaI hatchery were

individually weighed, wingbanded and divided randomly into three treatment groups.

Seven birds were allotted to each treatment group. Al1 aeatment groups were replicated

once in each of the 2 hypobaric chambers, Al1 the birds were fed a 5% flax oil diet (Table

3.1, experirnent 1 - flau diet). An altitude of 1740m was simulated in the hypobaric

chambers. The equipment used has been descnbed previously p o n d et al., 1996). The

birds were raised under continuous lighting, wïth ad libitum feed and water. The

temperature was rnaintained at a cornfortable level for the birds throughout the 4 week

experiment. A continuous hypoxic environment was maintained throughout the experiment

except for 30 minutes per day while the birds were fed. Sick birds were rernoved and

euthanatized, and broi lers that died during the expenment were necropsied.

4.3.2 Inhibitors

The treatment groups consisted of (1) a control group injected with propylene

glycol (Sigma Chernical Co. St. Louis, MO.), (2) a group injected with indomethacin and

(3 ), a group inj ected with meclofenemate. The indomethacin and mec10 fenemate (Biornol

Research Laboratories Inc., Plymouth Meeting, PA.) were dissoived in propylene glycol to a

concentration of 5 mg /ml. Based on work by Johnson et al. (1993) who injected adult

chickens (500 - 650 g r m s ) intrapentoneally with 5 mg of indomethacin, an initial dose of

10mg / kg was injected subcutaneously in the breast region every other day for four weeks.

AAer three injection penods the dose was decreased to 5 mg / kg for the remainder of the

experiment due to several deaths attributed to the 10 mg / kg initial dose. As the birds grew,

the concentration of the inhibitors was adjusted from jmg/rnl to 20 mgml to minimize the

volume injected (0.1 ml to 0.3ml). The control goup was injected with the same volme as

the two inhibitor groups.

4.3.3 Sarnple collection and analysis

On day 28, 3-5 ml of blood was collected into heparinized tubes From the main

brachial wing vein of each bird. The birds were bled after an overnight fast. A small

sample of whole blood was centrifuged for 5 minutes using an International micro-

capillary centrifuge (International Equipment Company, Needham Hts., Mass.), and the

haematocrit measured. The heparinized tubes were then centrifuged at 1000 x g, and the

plasma was separated from the white and red blood ce11 Iayers. The plasma and

erythrocytes were stored at -20" C for further analysis.

The fatty acid composition of erythrocyte membranes \vas determined according

to Burton et al. (198 1). A 1 ml volume of red bIood cells was resuspended in 5ml of

NaH2P04 buffer (pH = 8.0) and centrifuged at 290 x g at 4 O C for 5 minutes. This

procedure was repeated using 5ml of 5mM NaH2POJ. 2.5 mM NaH2P04, and 1 2 5 mM

NaH2P04 buffers. The final erythrocyte pellet was separated by centrifugation at 10 000

x g at 4 O C for 25 minutes. The erythrocyte membranes were extracted using IO ml

chloroform:methanol(2: 1, vol/vol) (Folch, et al., 1957) and the fatty acid composition

determined by gas-liquid chromatography afler transmethylation of the fatty acyl chains

present into their corresponding methyl esters using 0.1 ml of Meth Prep II (Alltech

Associates Inc., Deerfield, IL.). Heptadecaenoic acid (Cl 7:O) was used as an interna1

standard. A Varian 3400 gas chromatograph , with a 654 data system was used to

analyze the samples. Helium was used as a carrier gas. The column was a DB 225 (J &

W Scientific, Brockville, Ont.), with a length of 3Om, an intemal diameter of 0.Xrn1-n and

a 0.15 pn phase thickness. The injector and detector temperatures were 250°C with a

column temperature of 150°C, increasing until a final temperature of 220" C was reached

(mn time 57 minutes). Identification of individual fat@ acids was made by cornparkg

samples to known standards.

Plasma fatty acids were extracted from a 1 ml sample of plasma using 10 ml

chloroform:methanol (2: 1, voVvo1) (Folch er al., 1957) and the fatty acid composition

determined by gas-liquid chromatography. The same procedure for gas-liquid

chromatography of erythrocyte membranes, as outlined above tvas used to determine the

plasma fatty acid composition.

The birds were euthanatized on day 28 by cervical dislocation following blood

collection. Each bird was examined for the presence of ascites and then the heart was

carefully rernoved fiom each bird. The right ventncle was then carefully separated from

the lefi ventricle and septum, and weighed to determine the right ventncle to total

ventricular weight (RVTTV).

4.3.4 Statistical analysis

The experiment was a completely randomized design with birds randomly

assigned to each treatment and chamber, with charnber nested within treatment.

Statistical analysis was accomplished using the General Linear Models procedure of the

SAS Institute ( 2 985), and the least significant difference (LSD) used to compare means

with significance set at P c 0.05 . Adjusted means were used due to unequal replication

arnong the parameters. Specific contrats were also used to analyze the data. Initial body

weight was introduced as a covariate to analyze weekly body weight gain and total

weight gain.

4.4.0 RESULTS

The mortality for the experiment is summarizec i in Table 4.1. Birds were

routinely checked throughout the day with deaths in the inhibitor groups occumng 10 to

12 hours post injection during the first week (dose 10 mg I kg). No broilers died in the

control group. Necropsy revealed no ascites in any of the dead birds or in the groups

euthanatized at the end of the experiment.

Pulmonary hypertension, as measured by the RV/TV ratio is given in Table 4.2 .

The use of indomethacin or meclofenemate did not significantly alter the RV/TV ratio

from control. Haematocrit, was not significantly different between the treatment groups

(Table 4.2).

Weekly weight gain and total weight gain is summarized in Table 4.3 . Al1 birds

were fed the same 5% flax oil diet for 4 weeks. Injections of indomethacin or

meclofenemate did not significantly affect weekly weight gain or total weight gain

compared to the control treatment receiving injections of propylene glycol.

The fatty acid composition of plasma (Table 4.4) showed no significant

differences between indomethacin or meclofenemate, compared to control. The plasma

levels of palmitic acid (C16:0), linoleic acid (Cl 8:2(n-6)), homo-y-linolenic acid

(C20:3(n-6)), arachidonic acid (C20:4(n-6)), EPA (C20:5(n-3)), (C22:5(n-3)), and DHA

(C22:6(n-3)) were not significantl y different arnong the three treatment groups of

broilers. The relative percentage of oleic acid (1 8: 1 n-9)) was higher in the plasma of the

control birds compared to meclofenemate and indomethacin treated birds (P< 0.05). The

relative percentages of total n-3, total n-6, n-3/n-6, saturates, unsaturates, and the ratio of

saturates to unsaturated fatty acids in the plasma was not significantly different between

the three treaiment groups.

The fatty acid composition of erythrocyte membranes is summanzed in Table 4.5.

Indomethacin and meclofenemate showed increases (Pc 0.05) in stearic acid (C 18:0), and

u- linolenic acid (C18:3(n-3)). compared to control. Oleic acid (Cl 8: l(n-9)), was higher

(Pc 0.05) in the control than the two inhibitor groups. EPA and DHA was significantly

lower in the inhibitor treated birds compared to control (Pc 0.05). There was no

significant difference between the three treatments for palmitic acid, linoleic acid,

arachidonic acid, clupanodonic acid (C22:5(n-3)) or total n-3 fatty acids. The relative

percentage of total n-6, the amounts of saturated and unsaturated fatty acids, and the

ratio of saturates to unsaturates, in the meclofenemate and indomethacin groups were

significantly lower (P < 0.05) than the control groups. Meclofenemate injections resulted

in a higher n-3 to n-6 ratio (P~0.05) than the indomethacin treatment, but was not

different than control.

Table 4.1 Eflect of indornethacin and meclofenerna~e on weekly rnortality and total rnortaliv of broilers fed 5%@ oil subjected to hypobaric hypoxia!

Treatment Week 1 Week 2 Week 3 Week 4 To ta1 Mortaiity

Meclofenamate 6 O O O 6

- . - -

1 Hypobaric conditions sirndating an altitude of 1740 rn

Table 4.2 R V / W values and haernatocrit of broilers fed 5%flax oil subjected to hypobaric hypoxia. (rnean k SE)'

Parameter Control Indomethacin Mec lofenemate

Hematocrit (%)

' Hypobaric conditions simulating an altitude of 1740 m

Table 4.3 Effect of Nldornelhacin und rneclofenernafe on weekly weighr gain und rotal wei ht gain of broilers fed 5%f7ax oil subjected to hypobaric hypoxia. (gmms) (rnean f 7 SE)

Treatment Week 1 Week 2 Week 3 Week 4 Total gain

Control

Indomethacin

Meclo fenemate

' Hypobaric conditions simulating an altitude of 1740 m

Table 4.4 Faty acid composition ofplasma fiom broilers fed j%flax oil injected with propylene glycol, indornethacin, or meclofenemare subjected to hypobanc hypoxia!

Treatrnent Control Indomethacin Meclo fenemate

(N= 1 4) W=9) (N=8)

Fatty acid

Fatty acid percent

C14:O

C16:O

C18:O

C18:l (n-9)

C l 8 2 (n-6)

Cl 8:3 (n-3)

C20:3 (n-6)

C20:4 (n-6)

C20:5 (n-3)(EPA)

C225 (n-3)

C22:6 (n-3)@KA)

Total n-3

Total n-6

n-3 / n-6

Saturates

Unsaturates

Saturates / Unsaturates

' Hypobaric conditions sirnulating an altitude of 1740 rn a-b Means within a row with no comrnon superscript differ significantly at P < 0.05

Table 4.5 Fatty acid composition of erythrocyte membranes of broilers fed S % f l a r oil injected with propylene glycol. indornethacin, or meclofenemale subjected to hypobaric hypoxia. '

Treatment Control Indomethacin Meclofenemate

(N=14) (N=9) m=8)

Fatty acid

C16:O

C18:O

C18:l (n-9)

C 18:2 (n-6)

C18:3 (n-3)

C20:4 (n-6)

C205 (n-3)(EPA)

C22:S (n-3)

C22:6 (n-3)@HA)

Total n-3

Total n-6

n-3 / n-6

Saturates

Unsaturates

Saturates / Unsaturates

Fatty acid percent

1 Hypobaric conditions simulating an altitude of 1740 m a-b Means within a row with no common superscript differ significantly at P < 0.05

4.5.0 DISCUSSION

Currently, no studies have been completed using cyclo-oxygenase or 5-

lipoxygenase inhibitors to investigate the pathogenesis of pulmonary hypertension in

broiler chickens. Research into the pathogenesis of pulmonary hypertension in people

has identified alterations in levels of prostacyclin and thromboxane A2 (Cremona and

Higenbottarn, 1995). Although the exact role of prostanoids in the developrnent of

pulmonary hypertension remains speculative, this preliminary study was conducted in

order to detemine if similar vasoactive agents, are involved in PH of broiler chickens.

Arachidonic acid is the principal substrate for the production of various

eicosanoids. Arachidonic acid can be metabolized by cyclo-oxygenase to yield the 2-

senes prostaglandins and thromboxanes, and by 5-lipoxygenase to produce the 4-series

leukotrienes (Calder et al., 1992). Indomethacin inhibits cyclo-oxygenase selectively

over lipoxygenases, and preferentially inhibits prostaglandin synthase- 1 (PGHS- 1) over

prostaglandin-î synthesis (PGHS-2) (Meade et al., 1993). Meclofenemate is a dual

inhibitor, capable of inhibiting 5-lipoxygenase and PGHS- 1 and PGHS-2 (Meade et al.,

1993). B y inhibiting the synthesis of vasoactive arachidonic acid metabolites, one can

determine if eicosanoid production is involved in the reduction of ascites by flax oil.

Prostaglandin El (PGE,) and prostacyclin (PG12) are synthesized from

arachidonic acid and cause vasodilation of blood vessels. These prostanoids have been

implicated in pulrnonary hypertension in people (Cremona and Higenbottarn, 1995).

Thromboxane A2 (TxA2), another arachidonic acid metabolite promotes vasoconstriction

and platelet aggregation (Meydani, 1992). It rnay be the balance between thromboxane

A2 and prostacyclin which contribute to the pathogenesis of PH (Meydani, 1992;

Crernona and Higenbottam 1995; Nair et al., 1997). Another eicosanoid produced from

the 5-lipoxygenase pathway, specifically leukatriene B4 (LTB4) (Wallace and Chin,

2 997), a proinflammatory substance, may also be involved in the pathophysiology of PH.

It appears rneclofenernate and indornethacin do not have a significant effect on

plasma fatty acid composition compared to control. There were some individual fatty

acid differences behveen the indomethacin and meclofenemate treated broilers, which

may be a reflection of the variation within each bird. The meclofenemate and

indornethacin injections did have a profound effect on the fatty acid composition of

erythrocyte membranes. From the erythrocyte membrane fatty acid composition it is

difficult to show that meclofenemate and indornethacin inhibited the conversion of

arachidonic acid to its metabolites. The relative percent of arachidonic acid remained

unchanged in the three treatment groups. It was expected that arachidonic acid levels

would increase in the inhibitor treated groups if production of its metabolites were

decreased; however, this was not the case. Eicosapentaenoic acid (EPA), clupanodonic

acid. and docosahexaenoic acid (DHA), were al1 lower in the indomethacin treated group

compared to the control group. a- Linolenic acid, the precursor to EPA and DHA, was

significantly higher in the inhib itor treated birds compared to control broilers. The higher

levels of a- linolenic acid than EPA and DHA, suggest that indomethacin and

meclofenemate interfere with the metabolism of a- linolenic acid to EPA and DHA. The

inhibitors might interfere with the desaturase enzyme, however, little is known about the

activities and properties of desaturases in human tissues (Kinselia et al., 1990). The

stearate Ievels also increased in the inhibitor treated broilers. The increase in the relative

percentage of stearate is due to a decrease in the relative percentage of oleic acid. The

reason for the changes in these fatty acids might be reIated to an effect o f the inhibitors

on the desaturase enzyme responsible for desaturation of stearate to oleic acid.

The higher percentage of unsaturates in the control group compared to

indomethacin and meclofenemate, is due to the decrea ses in EPA, clupanodonic acid,

and DHA in the inhibitor treated birds. The higher percent of saturates in the inhibitor

groups compared to the control is Iikely due to the increase in stearate in the erythrocyte

membranes.

The degree of pulmonary hypertension c m be estimated by the ratio of the weight

of the right ventricle to the total ventricular weight of the heart (RV/TV ratio) (Sillau et

al., 1980; Huchzemeyer and De Ruyck, 1986; Guthrie et al., 1 98 7; Mirsalimi et al.,

1993). Injections of indomethacin or meclofenemate did not significantIy change the

RV/TV ratio or haematocnt from the control injected group of broilers. Although

specific prostanoid substances involved in the pathogenesis of PH were not measured, it

be can assurned that their production was decreased by the inhibitors. Pulmonary

hypertension was not increased by treatrnent with these inhibitors under hypobaric

conditions. Bond (1996) has shown flax oil reduces PH compared to feeding a diet

containing an animalhegetable blend of oii. Thus, it appears that the production of

vasoactive compounds are not involved in the reduction of PH and ascites in broiler

chickens fed flax oil diets.

Erythrocyte deformability is another possible rnechanisrn involved in the

aetiolo,ay of pulmonary hypertension and ascites in broilers. It has been demonstrated that

decreased deformability of erythrocytes, as measured by the filtration index (time to pass

throuzh a nucieopore membrane), can contribute to an increase in the amount of PH and

ascites found in broiler chickens (Mirsalimi and Julian 199 1 ; Mirsalimi et al., 1993).

Erythrocyte deformability is one of the major contributing factors related to blood

viscosity (Chien et al.. 19671, and whole blood viscosity is greater when the

deformability of erythrocytes is reduced (Chien et al., 1967; Doyle and Waiker, 1990).

Dietary fat source can affect the phospholipid fatty acid composition of erythrocyte

membranes (Mills et al., 1993; Sarkkinen et al., 1994; Agen et al., 1995). Berlin et al.

(1992) dernonstrated that erythrocyte membrane fluidity can be increased by

supplementation of a diet with n-3 fatty acids, specifically fish oils. Sïmilar results were

found by Bond (1996), who showed that feeding 5% flax oil to broiler chickens subjected to

hypobaric hypoxia increased erythrocyte deformability. hcreasing the proportion of

unsaturated fatty acids in the erythrocyte membrane likely increases the fluidity of the

membrane, and makes the erythrocytes more flexible. The percentage of unsaturates was

significantly higher in the control compared to treatment with the two inhibitors; however, it

had no significant effect on RV/TV. The relative percent change in the unsaturates in this

experirnent may not have been high enough to alter the properties of the erythrocyte

membrane and affect deformability.

Injecting meclofenemate or indornethacin did not have a negative impact on

growth. No signifiant difference was found with respect to weekly weight gain, or total

weight gained throughout the experiment. The overall growth of the birds in this

experiment is similar to previous studies using 5% flax oil and hypobaric conditions

(Bond, 1996).

When the initial dose of the inhibitors was 10 mgkg, a higher mortality rate was

noted in the indomethacin and meclofenemate treated birds compared to the controls.

Since al1 the deaths occurred within 12 hours post injection, the deaths were attributed to

the injection of the two inhibitors and the dose was decreased to 5 mgkg. Pen et al.

(1995) treated pigs with NS-398, a novel PGHS-2 inhibitor, a decreased rate of

prostaglandin synthesis by purified cerebral microvesseIs of the newborn (1-2-days-old)

by approximately 65% and ofjuvenile pigs (4-7-weeks-old) by 30% was observed.

Higher levek of cerebral prostanoids in the newborn affect cerebral blood flow (Peri er

al., 1995). By injecting the birds at an early age we may have altered cerebral prostanoid

metabolism, and changed blood flow in the brain. Also, other factors may have

contnbuted to chicks dying from the injections of indomethacin and meclofenemate, such

as a change in body temperature or a decrease in the ability to thermoregulate. Johnson

er al. (1993) studied the effects of central and penpheral prostaglandins involved in the

physiological effects of lipopo lysaccaride (LPS) in chickens injected with indomethacin.

The hyperthermia caused by LPS activation involves the prostaglandin system in the

brain of the chicken (Johnson et al., 1 993). Since prostaglandins are involved in

temperature regulation, then the injections of indornethacin and meclofenemate may have

down reguiated prostaglandins responsible for temperature homeostasis.

4.6.0 CONCLUSION

Results from this preliminary study suggest that vasoactive arachidonic acid

metabolites are not involved in PH in broilers. Further research is necessary to investigate

the role of eicosanoids and their possible involvement in the aetiology of PH and ascites.

Other expenments should be conducted using other dietary fat sources, recognizing the

importance of erythroc yte deformability and related haematological parameters involved

in the aetiology of pulrnonary hypertension and ascites.

Chapter 5.0

5.1 .O Final Discussion

This thesis is composed of several experiments. First, the effect of dietary

supplementation of 2.5% Bax oil on pulmonary hypertension, ascites, weight gain, feed

intake, and haematological pararneters in broiler chickens exposed to hypobaric hypoxia

were investigated. Then, studies using 2.5% and 5% flax oil with and without

supplemental antioxidants (vitamin C and E), during the starter and grower penods in

broiler chickens were conducted using a cold temperature model. The same pararneters

were measured as in the first trials using hypobaric hypoxia. Lastly, an experiment using

indomethacin and meclofenemate was completed to determine the effects of inhibiting

the production of arachidonic acid metabolites on PH in broilers fed 5% flax oil

subjected to hypobaric hypoxia. Haematocrit, weight gain, RV/TV, plasma and

erythrocyte membrane fatty acid composition were measured.

One of the hypotheses was fatty acids fiom flax oil can be incorporated into

erythrocyte membranes, thereby increasing the fluidity of the membrane and improving

deformability. Previous research indicates that under hypobaric hypoxia the inclusion of

5% flax oil into a broiler ration reduces nght ventricular hypertrophy and pulmonary

hypertension (Bond, 1996). However, when 2.5% flax oil was fed to broiler chickens

subjected to hypobaric hypoxia, a reduction of PH did not occur.

Bond (1996) found an increase in erythrocyte deformability, as well as a decrease

in whole blood viscosity and haematocrit when 5% flax oil was fed to broiler chickens.

Incorporation of 2.5% flax oil did not significantly change the defomability of the

erythrocytes of broilers subjected to hypobaric hypoxia. These are important findings

since defotmability and haematocrit affect whole blood viscosity and have a large impact

on the development of pulmonary hypertension and ascites.

Diet can affect the fatty acid composition of erythrocyte membranes. As

indicated in appendix 1, feeding 5% flax oil increased the proportion of unsaturated fatty

acids in the erythrocyte membranes compared to feeding an equivalent diet containing an

animaVvegerable blend of oil. The ratio of n-3 to n-6 fatty acids in erythrocyte

membranes also increased in the 5% flax oil treatment groups compared to controls

(appendix 1). The increase content of unsaturated fatty acids likely increases the fluidity

of the erythrocyte membranes and increases the deformability of the erythrocyte. More

deformable erythrocytes decrease the resistance to blood flow through the capillaries,

thus improving oxygen transport to the tissues and decreasing pulmonary hypertension.

The use of 5% flax oil in broiler diets did not compromise growth in broilers

subjected to hypobaric hypoxia (Bond, 1996). Feeding 2.5% flax oil decreased weight

gain, and increased the incidence of ascites in broilers under hypobanc conditions. The

reduction in PH experienced by broiler chickens fed 5% flax oil was not a result of

slowing down growth, since slowing growth rates will cause a decrease in the incidence

of ascites. The improvement in erythrocyte deforrnability is a more likely the reason for

the decrease in ascites.

Fatty acid analysis of erythrocyte membranes from broilers fed a 5% fiax oil diet

and subjected to hypoxic conditions showed significant increases in the levels of a-

Iinolenic acid and eicosapentaenoic acid (EPA) (Bond, 1996; Walton et al., 1998b). a-

Linolenic acid is the precursor to eicosapenatenoic acid (EPA), and docosahexaenoic acid

(DHA ). Both EPA and DHA have been shown to reduce platelet aggregation and the

production of TxAZ. a vasoconstrictive prostanoid (Holub, 1995). The fact that

eicosanoids are involved in PH in humans, and the fact a reduction in PH using flax oil

was found, lead to the hypothesis that besides deformability decreasing PH, other

mechanisms might be involved.

Therefore, an expenment was designed to test the effects of inhibiting cyclo-

oxygenase and 5-lipoxygenase in broiler chickens fed a 5% flax oil ration exposed to

hypobaric conditions. lndomethacin and meclofenemate inhibit the conversion of

arachidonic, DHA and EPA to eicosanoid denvatives. Plasma fatty acid composition,

and weight gain did not significantly differ among the treatment groups. Erythrocyte

fatty acid membrane composition showed a significant difference between the hvo

inhibitors and the control. The inhibitors might interfere with the desaturase enzymes,

thereby altering the metabolism of fatty acids.

No significant difference was found in RV/TV or haematocrit behveen the

treatment groups. The results from this preliminary study are inconclusive as to whether

eicosanoid agents have a significant role in the development of PH in broiler chickens.

Other mechanisms such as erythrocyte deformability may be involved in the aetiology of

PHs.

Enkvetchakul et al. (1993) found that tissue concentration of both vitarnin E and

C, plus glutathione were compromised in broilers with pulmonary hypertension

syndrome. Therefore, the last hypothesis tested was if supplemental antioxidants with

flax oil, or an A N blend oil reduce PH and ascites in broilers subjected to cold

temperature. In al1 three experiments there was no significant effect of dietary fat source

or supplemental antioxidants on total feed intake or feed conversion. Similar to the

results of the hypobaric experiments, feeding 2.5% flax oil during the grower period, or

nom day 1 to week 8 (starter and grower period) did not reduce PH. There was a trend

for a decrease in PH when 5% flax oil was fed compared to the control diet containing an

equivalent arnount of A N blend oil. Supplementing 5% flax oil with vitarnin E and

vitarnin C removed this beneficial effect and increased the incidence of ascites and PH.

The reason for the increase in ascites and PH when 5% flax oil was combined

with the antioxidants remains unclear. These results are surpnsing, given that the

increased levels of polyunsaturated htty acids in flax oil would normally increase the

requirements for dietary antioxidants to prevent lipid peroxidation. There is some

evidence that the addition of vitarnin C can cause elevations in haematocrit (Al-Taweil

and Kassab, 1990). This might help explain why combining 5% flax oil and supplemental

antioxidants increased the incidence of ascites and PH, or negated the beneficial effects

of flax oil. However, the same results were not found when the antioxidants were

combined with an AN blend of oil.

To conclude, using two different models to induce PH and ascites, 2.5% flax oil

does not reduce PH. The incorporation of 5% flax oil into a broiler ration will reduce the

incidence of ascites and PH in broiler chickens exposed to hypobaric hypoxia (Bond,

1996), and tends to reduce PH in broilers subjected to cold temperature. Supplementing

diets with the combination of vitamin E and vitamin C does not reduce the incidence of

ascites and PH and, in fact, reduces the dietary effectiveness of the 5% flax oil in

reducing ascites and pulmonary hypertension. The addition of dietary supplernental

antioxidants removes the beneficial effect of 5% flax oil in a broiler ration. Improved

erythrocyte defomability is Iikely the major contributing factor for the reduction in

ascites and pulmonary hypertension found in these experiments. The results from the

preliminary inhibitor experiment suggest that arachidonic acid metabolites are not

involved in the aetiology of PH and ascites in broiler chickens.

5.2.0 Future Research

The inclusion of5% fla~ oi1 into a broiler ration will reduce the incidence of

ascites due to pulmonary hypertension in broiler chickens subjected to hypobaric hypoxia

(Bond, 1996), whereas, 5% flax oil only tends to reduce PH in broilers subjected to cold

temperatures. Since oil quality is a factor, and may have contributed to the deaths in the

2.5% flax oil hypobaric experiment, a number of hypobaric experirnents should be

conducted to test the various effects of oxidised flax oil on pulmonary hypertension and

the incidence of ascites in broiler chickens. Fatty acid analysis of the oils used in these

experiments will be conducted to determine their composition.

Since erythrocyte membrane composition can reflect dietary fatty acid changes,

O ther po lyunsaturated fat sources should be evaluated in broi lers and the erythrocyte

deformability measured. These experiments will help elicit which oils may be suitable to

feed to broiler chickens to increase erythrocyte defomability, thereby reducing PH and

ascites. Malondialdehyde levels could be measured to deterrnine the amount of lipid

peroxidation occumng in the tissues and related back to oil quality and lipid peroxide

values.

Regarding the use of supplemental antioxidants in broiler chicken rations, a

number of issues need to be resolved since we have shown that combining vitamin C and

vitarnin E, together with flax oil, increases the incidence of ascites and pulmonary

hypertension. Experiments using only one antioxidant and flax oil should be conducted

to determine if the negative effects of using the antioxidants were related to vitarnin C,

vitarnin E, or if it was due to an additive effect of combining the antioxidants with the

flax oil. Experiments using antioxidants and other polyunsaturated oils should be

considered to determine if there was a flax oil - antioxidant interaction accounting for the

increase in ascites and PH.

More studies using cyclo-oxygenase and iipoxygenase inhibitors should be done

using other dietary fat sources with varying degrees o f unsaturation and facty acid

composition to determine which fat sources are effective in reducing PH and ascites.

Along with measuring erythrocyte membrane fatty acid composition, erythrocyte

defomability should be measured. Studies such as the one descnbed above will help

determine the significance of fat source on erythrocyte defomability and its involvement

in the development of ascites and pulmonary hypertension.

Lastly, since one of the goals of poultry production is to produce a quality product

for the consumer, taste test panels need to be completed to determine the acceptability of

flax oil supplernentation in broiler chickens. Besides the reduction in ascites and PH in

broiler chickens, the consumer can benefit as well from the added anti-thrombogenic and

positive cardiovascular properties associated with n-3 fatty acid supplementation.

6.0 References

Agen, J.J., Torrnala, M-L., Nenonen, M.T. & Hannïnen, 0.0. (1995) Fatty acid composition of erythrocyte, platelet, and S e m lipids in strict vegans. Lipids, 30: 354-369.

Allrnan, M.A., Pena, M.M. & Pang, D. (1995) Supplementation with flaxseed oil versus sunflowerseed oil in healthy young men consuming a low fat diet: effects on platelet composition and function. European Journal of Clinical Nutrition, 49: 169-1 78.

Al-Taweil, R.N. & Kassab, A. (1990) Effect of dietary vitamin C on ascites in broiler chicks. International Journal of Vîtamin Research. 60: 366-3 7 1.

Applegate, T.J. & Sell, J.L. (1996) Effect of dietary linoleic to linolenic acid ratio and vitamin E supplementation on vitamin E status of poults. Portltry Science, 75: 88 1-890.

Archer, S.L., Johnson, G.J., Gebhard, R.L., Castlemen, W.L., Levine, AS., Westcott, J-Y., Voelkel, N.F., Nelson, D.P. & Weir, E.K. (1989) Effect of dietary fish oïl on lung lipid profile and hypoxic pulmonary hypertension. Journal of Applied Physiologv, 66(4): 1662- 1673.

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7.0 Appendix

For cornparison purposes this appendix contains data from Bond (1 996) pertaining to chapter 2 of this thesis.

7.1 Methods and Materials (Bond, 1996)

This experiment utilized four treatment groups of 25 birds each with diets

containing 5% added fat. Birds in the hypobaric chambers were exposed to a simulated

altitude of 1950 m. The birds were weighed initially on day Z and then weekly for 4

weeks. Six blood samples were collected fkom each group on day 26 fiom the main

brachial vein into heparinized blood tubes and used to determine erythrocyte

deformability. Six more blood sarnples were collected on day 27 and used to determine

whole blood viscosity. Six additional blood sarnples were collected on day 28 and used

to determine the haematocrit (HCT), haemoglobin content, and fatty acid analysis of the

erythrocyte membranes. The methods used to determine filtration index (a measure of

erythrocyte deformability), whole blood viscosity, HCT, haemoglobin content and fatty

acid analysis were previously descnbed in Bond et a l , (1 997). The mean corpuscular

haemoglobin content (MCHC) was calculated by dividing the haemoglobin value by the

haematocrit measurement. The filtration index is based upon the time for a 10%

suspension of washed erythrocytes to pass through a polycarbonate membrane filter with

an average pore diameter of 5 Fm. An increase in the filtration index indicates a decrease

in deformability of the erythrocytes.

The birds were euthanatized on day 28 by cervical dislocation. The right ventricle

was then carefully separated f?om the left venh-icle and septum, and weighed to calculate

the right ventncle to total ventricular ratio (RV/TV). Sick birds were rernoved and

euthanatized, and broilers that died during the experiment were necropsied.

The expenment was analyzed as a randomized block design with groups of birds

blocked into atrnospheric pressures. Statistical analysis was accomplished by using the

General Linear ModeIs procedure of the SAS Institute (1985) and the method of least

significant difference (LSD) with significance set at P c 0.05. Adjusted means for body

weights were used with initial weights introduced as a covariate. The unadjusted means

for body weight are reported in the results. The HCT was used as covariate to analyze

whole blood viscosity. Fisher's exact test was used to analyze the distribution of R V n V

ratios between the dietary treatment groups.

7.2 RESULTS

Table 7.1 Total mortaliw and causes of death in broiler chickens fed diets containing fTar oil or A/V blend oiZ and kept at either hypobaric or ambient atmospheric pressztre. (Bond, 1996)

-

5% Flax oil ' Number of Birds in group Mortality / Cause

Flax oil H ypo baric

A/V blend oil Hypobaric

Flax oil Ambient

AN blend oil Ambient

3 culled I SDS*

2 PHS* 1 starveout 1 culled

3 culled

2 culled I necrotic enteritis

' Hypobaric conditions simulating an altitude of 1960 m A Sudden death syndrome or dead in good condition

Pulmonary hypertension syndrome (RV/TV ratio > 0.299 or ascites fluid present) Birds were culled due to leg problerns

Table 7.2 Effect of 5% dietary flar or A N blend oil on weekly body weighr gain (g) of broilers held at hypobnric or nmbient pressure (Means S E M ) . (Bond. 1996)

Treatment Week 1 Week 2 Week 3 Week 4

AN blend oil 77Sa 168.8 285.gb 367.8 ~ypobaric ' t 3 .O 47.2 t 14.9 k 14.3

Flax oil 74.gab 165.9 292.7b 349.1 Eiypo baric ' 52.8 5 9 t 10.3 I l 7.9

A N blend oil 83 .Oa 177.4 333.6" 400.0 ~rnbient' +3 -2 k9.4 k 14.4 524.4

Flax oil 68.6b 166.8 294.2b 369.1 ~ r n b ien t' k3.1 t8 -2 k12.0 f 18.6

1 Hypobarïc conditions simulating an altitude of 1960 rn ' Arnbient atmospheric pressure (altitude 295 m) a-b Means within a column with different superscripts differ significantly by (Pc0.05)

Table 7.3 Huematological values of broilers fed 5% dietavflar or AN b l e d oil and held at hypobaric or arnbient pressure (Means -+SEM). (Bond, 1996) ' Parameter NV blend oil FIax oil NV blend oil Flax oil

H ypo b aric Hypobaric Ambient Ambient

Haemoglobin (dl>

MCHC (g/l)

HCT (%)

Whole bIood viscosity (cps)

Filtration index (seconds)

1 Hypobanc conditions sirnulating an altitude of 1 960 m 1-c Means within a row with no common superscript differ significantly at P < 0.05

Table 7.4 R V/TV ratios of bruiters fedflax or A/V b l e d oil diets and kepf under hypobaric or ambient pressure (Meons -+SEM). (Bond, 1996)

Arnbient pressure Hypobaric pressure

A N blend diet Flax oil diet AN blend diet Flax oil diet

5% Flax oil '

' Hypobaric conditions simulating an altitude of 1960 m 3-C Means within a row for each expenment with no common superscript differ significantly at P < 0.05

Table 7.5 Fatty acid composition of erythrocyte membranes /rom broilers fed diets containing S%f lar or A/V blend oil and kept under hypobaric or ambient pressure. (Means f SEM). (Bond, 1996)

Fatty acid content (percent) n-3 n-6 n-3/n-6 Saturated Unsat. Samnsat

A N blend oil ~ ~ ~ o b a r i c l

Flax oi1 ~ ~ ~ o b a r ï c '

AN blend oil Ambient

Flax oil Arnbient

1 Hypobaric conditions simulating an altitude of 1960 rn 3% Means within a column with no common