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Masters Theses Graduate School

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Dietary Supplementation of Choline and Betaine in Heat-Stressed Dietary Supplementation of Choline and Betaine in Heat-Stressed

Broilers Broilers

Kouassi Remi Kpodo University of Tennessee - Knoxville, [email protected]

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To the Graduate Council:

I am submitting herewith a thesis written by Kouassi Remi Kpodo entitled "Dietary

Supplementation of Choline and Betaine in Heat-Stressed Broilers." I have examined the final

electronic copy of this thesis for form and content and recommend that it be accepted in partial

fulfillment of the requirements for the degree of Master of Science, with a major in Animal

Science.

Michael O. Smith, Major Professor

We have read this thesis and recommend its acceptance:

Brynn Voy, Agustin Rius, Arnold Saxton

Accepted for the Council:

Carolyn R. Hodges

Vice Provost and Dean of the Graduate School

(Original signatures are on file with official student records.)

Dietary Supplementation of Choline and Betaine in Heat-Stressed Broilers

A Thesis Presented for the

Master of Science

Degree

The University of Tennessee, Knoxville

Kouassi Remi Kpodo

August 2015

ii

Copyright © 2015 by Kouassi Remi Kpodo

All rights reserved.

iii

Dedication

This thesis is dedicated to my family, my wife, brothers and sisters, and my friends whose

encouragement and moral support gave me the strength to pursue the Master’s Degree.

iv

Acknowledgements

I would like to express my sincere gratitude to Dr. Michael Smith, my graduate mentor

for giving me the opportunity to pursue my Master’s Degree under his guidance, all his teaching

and transfer of knowledge and for his investment in helping me find a job after graduation. I

would like to thank Dr. Arnold Saxton, Dr. Brynn Voy and Dr. Agustin Rius for serving on my

graduate committee. Special thanks to Dr. Henry Kattesh for all his help with corticosterone

analysis, and Dr. Virginia Artegoitia for her help with choline and choline metabolites

determination.

Thanks to my fellow graduate students Rekek Negga, Xiang Liu and Tania Torchon for

their assistance during the live bird trial, and especially to Ronique Beckford for all his help and

constructive discussions about the experiment and the analysis of statistical outputs. Thanks also

to Eddie Jarboe and Linda Miller for all their help with taking blood samples as well as to

Barbara Gillespie and Sierra Lockwood for their help with corticosterone analysis, and JARTU

staffs for helping me raise the birds.

I would not finish without expressing my appreciation to Balchem Corporation and the

University of Tennessee AgResearch which partially provided the funding and materials for this

project. Finally, thanks to my family and friends whose support and encouragement gave me the

strength to finish the program.

v

Abstract

A study was conducted to determine the effects of supplemental choline (CHO) and betaine

(BET) on broiler performance, carcass characteristics, corticosterone levels, immune organ

weights, intestinal morphology and choline metabolites under thermoneutral (TN: 23.9oC) and

heat stress (HS: 28-36oC) conditions. Eight hundred day-old chicks (400 per temperature

treatment) were assigned in groups of 10 to each of five dietary treatments: 1 (CON) the basal

diet; 2 (CHO500), basal diet plus 500 ppm methyl equivalents added CHO; 3 (CHO1000), basal

diet plus 1000 ppm methyl equivalents added CHO; 4 (BET500), basal diet plus 500 ppm methyl

equivalents added BET and 5 (BET1000), basal diet plus 1000 ppm methyl equivalents added

BET. Pen feed intake and body weights were recorded weekly. Foot pad dermatitis (FPD) was

assessed and litter samples were collected on day 42. On days 22 and 45, blood samples were

collected from eight birds per treatment to determine corticosterone and choline metabolites

concentrations. On day 52, birds were processed, spleen, bursa and thymus were weighed. Liver

and intestinal samples were collected for CHO and CHO metabolites determination and

histology (intestine). Breast color was measured and drip loss evaluated 4 and 7 days post

slaughter. Results showed that HS birds consumed 20.59% less feed, gained 23.35% less weight

and had lower feed efficiency compared to TN birds (P < 0.05) during days 22-49. Foot pad

dermatitis was decreased (P = 0.003) by CHO500 and BET1000. Drip loss 4 days post slaughter

was reduced (P = 0.04) by CHO500 (0.60%) in HS and BET1000 (0.83%) in TN birds. On day

45, HS increased (P = 0.02) corticosterone levels (3.61 ng/ml HS vs 2.16 ng/ml TN). Thymus

and bursa weights were reduced (P = 0.03) by HS. There was a diet X temperature interaction for

liver BET concentration (P = 0.03) whereas intestinal BET was increased only by diet (P = 0.02).

Liver CHO and glycerophosphocholine were increased by temperature (P = 0.01). Dietary

vi

supplementation with CHO and BET did not negate the negative impacts of HS on broiler

performance and immune system. However, they reduced FDP as well as drip loss.

vii

Table of Contents

Chapter I: Introduction .................................................................................................................... 1

References ....................................................................................................................................... 5

Chapter II: Literature Review ......................................................................................................... 7

Heat Stress in Broilers ................................................................................................................. 8

Foot Pad Dermatitis ................................................................................................................... 10

Impact of Heat Stress on Intestinal Morphology ...................................................................... 12

Heat Stress and the Immune System ......................................................................................... 13

Choline ...................................................................................................................................... 14

Betaine ....................................................................................................................................... 16

Interrelation of Choline, Betaine and Methionine Metabolism................................................. 18

References ..................................................................................................................................... 21

Chapter III: Performance of heat-stressed broilers supplemented with dietary choline and betaine

....................................................................................................................................................... 29

Abstract ..................................................................................................................................... 30

Introduction ............................................................................................................................... 31

Materials and Methods .............................................................................................................. 33

Birds and Housing ................................................................................................................. 33

Temperature Treatments ........................................................................................................ 34

Dietary Treatments ................................................................................................................ 35

Growth Performance .............................................................................................................. 35

Litter Samples and Foot Pad Dermatitis ................................................................................ 35

Processing of Birds ................................................................................................................ 40

Drip Loss ............................................................................................................................... 40

Breast Meat Color .................................................................................................................. 40

Statistical Analysis ................................................................................................................ 41

Results ....................................................................................................................................... 41

Growth Performance .............................................................................................................. 41

Litter Moisture ....................................................................................................................... 43

Foot Pad Dermatitis ............................................................................................................... 48

Breast Meat Color .................................................................................................................. 48

viii

Breast Meat Drip Loss ........................................................................................................... 48

Discussion ................................................................................................................................. 52

References ..................................................................................................................................... 55

Chapter IV: Effect of dietary choline and betaine on corticosterone levels and immune organ

weights of heat-stressed broilers ................................................................................................... 58

Abstract ..................................................................................................................................... 59

Introduction ............................................................................................................................... 60





Blood Collection .................................................................................................................... 64


Corticosterone Radioimmunoassay ....................................................................................... 68


Results ....................................................................................................................................... 69

Bursa of Fabricius, Spleen and Thymus Weights .................................................................. 69

Plasma Corticosterone ........................................................................................................... 69

Discussion ................................................................................................................................. 72

References ..................................................................................................................................... 74

Chapter V: Effect of dietary choline and betaine on choline metabolites and intestinal

morphology of heat-stressed broilers ............................................................................................ 76

Abstract ......................................................................................................................................... 77

Introduction ............................................................................................................................... 78





Blood Collection .................................................................................................................... 83


Intestinal Histology................................................................................................................ 87

ix

Choline Metabolites Analysis by HILC-MS/MS .................................................................. 87


Results ....................................................................................................................................... 88

Intestinal Histology................................................................................................................ 88

Intestinal Choline and Choline Metabolites .......................................................................... 88

Liver Choline and Choline Metabolites................................................................................. 91

Plasma Choline and Choline Metabolites .............................................................................. 93

Discussion ................................................................................................................................. 95

References ..................................................................................................................................... 98

Chapter VI: Summary and Conclusion ....................................................................................... 101

Vita .............................................................................................................................................. 103

x

List of Tables

Table 1. Nutritional composition of starter diet (%) ..................................................................... 36

Table 2. Nutritional composition of grower diet (%) ................................................................... 37

Table 3. Nutritional composition of finisher diet (%)................................................................... 38

Table 4. Effect of choline and betaine supplementation on average feed intake (kg) 1

of heat-

stressed broilers during 1-21, 22-49 and 1-49 days of age ........................................................... 42

Table 5. Effect of choline and betaine supplementation on average body weight gain (kg) 1

of

heat-stressed broilers during 1-21, 22-49 and 1-49 days of age ................................................... 44

Table 6. Effect of choline and betaine supplementation on feed efficiency (Gain:feed) 1 of heat-

stressed broilers during 1-21, 22-49 and 1-49 days of age ........................................................... 45

Table 7. Effect of choline and betaine supplementation on mortality (%) 1

of heat-stressed

broilers during 1-49 days of age ................................................................................................... 46

Table 8. Effect of choline and betaine supplementation to broilers on litter moisture content1

under thermoneutral and heat stress conditions on day 42 ........................................................... 47

Table 9. Effect of choline and betaine supplementation on foot pad score (%) 1 of heat-stressed

broilers on day 42.......................................................................................................................... 49

Table 10. Effect of choline and betaine supplementation on breast meat color (L*, a*, b*) 1

from

heat-stressed broilers ..................................................................................................................... 50

Table 11. Effect of choline and betaine supplementation on drip loss from breast meat (%) 1 of

heat-stressed broilers 4 and 7 days post slaughter ........................................................................ 51

Table 12. Composition of starter diet (%) .................................................................................... 65

Table 13. Composition of grower diet (%) ................................................................................... 66

Table 14. Composition of finisher diet (%) .................................................................................. 67

Table 15. Effect of choline and betaine supplementation on lymphoid organ weights (% body

weight) 1

of heat-stressed broilers ................................................................................................. 70

Table 16. Effect of choline and betaine supplementation on Corticosterone Concentration (ng/ml)

1 of heat-stressed broilers on days 22 and 45 ................................................................................ 71

Table 17. Temp X Day interaction on corticosterone concentration (ng/ml) 1

of heat-stressed

broilers .......................................................................................................................................... 72

Table 18. Composition of experimental diet for starter period (%) .............................................. 84

Table 19. Composition of experimental diet for grower period (%) ............................................ 85

Table 20. Composition of experimental diet for finisher period (%) ........................................... 86

Table 21. Effect of choline and betaine supplementation on intestinal villi height, crypts depth

(mm) 1

and villi height:crypt depth of heat-stressed broilers ........................................................ 89

Table 22. Effect of choline and betaine supplementation on intestinal choline and choline

metabolites (mg/100g) 1

of heat-stressed broilers ......................................................................... 90

Table 23. Effect of choline and betaine supplementation on liver choline and choline metabolites

(mg/100g) 1

of heat-stressed broilers ............................................................................................. 92

Table 24. Effect of choline and betaine supplementation on plasma choline and choline

metabolites (umol/l) 1

of heat-stressed broilers ............................................................................. 94

xi

List of Figures

Figure 1. Typical daily temperature in TN (23.9 ±1°C) and HS (28-36±1°C) rooms. ................. 34

Figure 2. Scoring system for foot pad dermatitis (Mello, et al., 2011). ........................................ 39

Figure 3. The daily temperature in TN (23.9 ±1°C) and HS (28-36±1°C) rooms. ....................... 63

Figure 4. Temperature treatment in TN (23.9 ±1°C) and HS (28-36±1°C) rooms. ...................... 82

1

Chapter I: Introduction

2

The poultry industry has evolved from a dual purpose backyard operation to a vertically

integrated industry (USDA, 2002) with two distinct production systems, broiler (meat)

production and layer (egg) production. Both systems have benefited from various advances

allowing them to produce more efficiently through improved nutrition, genetics, environment

and management practices. Despite these achievements, the industry is still facing some

problems such as the effect of heat stress. Modern poultry production is highly intensive and

occurs in large enclosed houses with high stocking density and fast growing birds. Combination

of these factors leads to an increase of heat in chicken houses. Faster growing broilers produce

more heat (Cahaner and Leenstra, 1992; Yalcin, et al., 2001) and in addition, in the United

States, which is the highest producing country, the production of broilers is mostly concentrated

in the southeastern region (USDA, 2014) where extreme summer temperature combined with

high humidity creates the ideal condition for heat related stress (Konrad, 2013).

Heat stress occurs when the body produces or absorbs more heat than it can dissipate

which can result in negative consequences. Under heat stress, chickens eliminate the extra heat

by increasing their respiratory rate. This increased respiration may result in acid/base imbalance

or alkalosis (Borges, et al., 2004; Toyomizu, et al., 2005). Prolonged heat stress reduces visceral

blood supply to the intestine and causes damages to epithelial cells in the gut there by affecting

feed digestion and nutrients absorption (Cronje, 2007). Prolonged heat stress may also disrupt

intestinal barrier increasing the likelihood of pathogenic bacteria and endotoxin entry, which can

then result in excessive inflammation, decreasing production performance, and possibly death

(Quinteiro-Filho, et al., 2012). Heat stress prior to slaughter increases hemorrhage incidence

thereby decreasing meat quality post processing (Sandercock, et al., 2001). It also disrupts

intestinal integrity (Lambert, 2009) thus rendering it less resistant to breakage during mechanical

3

processing. According to Rosenquist, et al. (2006), the highest rate of chicken carcass

contamination by campylobacter occurs during evisceration and is likely to cause food borne

illness or disease outbreak. Heat stress can be very costly to the poultry industry. In 2003, it was

estimated to cost the poultry industry approximately 128 million dollars annually (St-Pierre, et

al., 2003).

Over the years, producers have been trying to find various ways to alleviate the negative

impacts of heat stress on broilers. Management practices such as electrolyte supplementation

have been used to alleviate the negative impacts of heat stress. It has been shown that continuous

supply of electrolytes (K+ and Cl

-) improves weight gain (Smith and Teeter, 1992) and overall

performance. However, electrolytes are limited osmotic effectors and their excessive

accumulation in tissues may inhibit protein synthesis (Petronini, et al., 1992). Because of this

negative impact, an alternative to electrolytes is being sought, one such alternative being organic

osmolytes, such as choline and betaine. Choline and betaine are two important compounds that

benefit the poultry industry by improving production. Several studies have shown that not only

do they support growth but also alleviate the negative impacts of heat stress on chicken

physiology and production performances by attenuating cells dehydration (Attia, et al., 2009;

Farooqi, et al., 2005).

Choline plays various metabolic functions throughout the body including building and

maintenance of cell structure primarily as phosphatidylcholine, fat mobilization in the liver and

formation of acetylcholine (Combs Jr, 1991; McDowell, 1989). It is a component of molecules

important in the fetal development (Jones III, et al., 1999), in inflammation (Wang, et al., 2005)

and in blood clotting (Wells, 1964). Choline constitutes a source of osmolytes through its

metabolites, betaine and glycerophosphocholine, for regulating cell volume (Nakanishi and

4

Burg, 1989). Glycerophosphocholine, mainly formed from phosphatidylcholine, is the

predominant osmolyte in the kidney that protects renal medullary cells from hypertonicity of the

interstitial fluid (Nakanishi and Burg, 1989). Betaine, an important methyl group donor to the

formation of methionine, has been shown to improve lipid metabolism and production

performance of broilers (Zhan, et al., 2006). In intestinal cells subjected to osmotic disorder and

dehydration, betaine is taken up and may have a stabilizing function (Kettunen, et al., 2001). It

also stimulates intestinal epithelial cell proliferation, feed digestibility and absorption of nutrients

(Augustine and Danforth, 1999).

Choline is already being included in broiler feed at required levels. Because of their

osmotic properties, it is possible that choline and betaine could be included at higher levels.

Various researchers have investigated this possibility, however further research is warranted to

understand the integrated role of these two compounds on chicken physiology and growth

performances especially in heat stress environment. Therefore, the objectives of this experiment

were to evaluate the osmoprotectant benefits of choline and betaine on growth performance and

on the intestine of heat-stressed broilers.

5

References

Attia, Y., R. Hassan, and E. Qota. 2009. Recovery from adverse effects of heat stress on slow-

growing chicks in the tropics 1: Effect of ascorbic acid and different levels of betaine. Trop.

Anim. Health Prod. 41:807-818.

Augustine, P., and H. Danforth. 1999. Influence of betaine and salinomycin on intestinal

absorption of methionine and glucose and on the ultrastructure of intestinal cells and parasite

developmental stages in chicks infected with Eimeria acervulina. Avian Dis.:89-97.

Borges, S. A., A. V. F. da Silva, A. Majorka, D. M. Hooge, and K. R. Cummings. 2004.

Physiological responses of broiler chickens to heat stress and dietary electrolyte balance

(sodium plus potassium minus chloride, milliequivalents per kilogram). Poult. Sci. 83:1551-

1558.

Cahaner, A., and F. Leenstra. 1992. Effects of high temperature on growth and efficiency of

male and female broilers from lines selected for high weight gain, favorable feed conversion,

and high or low fat content. Poult. Sci. 71:1237-1250.

Combs Jr, G. F. 1991. The vitamins: fundamental aspects in nutrition and health. Am. J. Clin.

Nutr. 53:755-763.

Cronje, P. Year. Gut health, osmoregulation and resilience to heat stress in poultry. Proc. Proc.

19th Aust. Poult. Sci. Symp., Sydney, New South Wales, Australia.

Farooqi, H., M. Khan, M. Khan, M. Rabbani, K. Pervez, and J. Khan. 2005. Evaluation of

betaine and vitamin C in alleviation of heat stress in broilers. Int. J. Agric. Biol 5:744-746.

Jones III, J., W. H Meck, C. L. Williams, W. A. Wilson, and H. S. Swartzwelder. 1999. Choline

availability to the developing rat fetus alters adult hippocampal long-term potentiation. Dev.

Brain Res. 118:159-167.

Kettunen, H., S. Peuranen, K. Tiihonen, and M. Saarinen. 2001. Intestinal uptake of betaine in

vitro and the distribution of methyl groups from betaine, choline, and methionine in the body

of broiler chicks. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 128:269-278.

Konrad, C. E., Fuhrmann, M. 2013. Climate of the Southeast USA: past, present and future.

Pages 8-36 in Climate of the Southeast Unites States. Ingram k., K. Dow, L. Carter, J.

Anderson, eds. Washington DC: Island Press.

Lambert, G. P. 2009. Stress-induced gastrointestinal barrier dysfunction and its inflammatory

effects. J. Anim. Sci. 87:E101-E108. doi 10.2527/jas.2008-1339

McDowell, L. R. 1989. Choline. Pages 565-596 in Vitamins in Animal and Human Nutrition.

2nd ed.

6

Nakanishi, T., and M. B. Burg. 1989. Osmoregulation of glycerophosphorylcholine content of

mammalian renal cells. Am. J. Physiol. 257:C795-801.

Petronini, P. G., E. De Angelis, P. Borghetti, A. Borghetti, and K. P. Wheeler. 1992. Modulation

by betaine of cellular responses to osmotic stress. Biochem. J 282:69-73.

Quinteiro-Filho, W., A. Gomes, M. Pinheiro, A. Ribeiro, V. Ferraz-de-Paula, C. Astolfi-Ferreira,

A. Ferreira, and J. Palermo-Neto. 2012. Heat stress impairs performance and induces

intestinal inflammation in broiler chickens infected with Salmonella Enteritidis. Avian Pathol.

41:421-427.

Rosenquist, H., H. M. Sommer, N. L. Nielsen, and B. B. Christensen. 2006. The effect of

slaughter operations on the contamination of chicken carcasses with thermotolerant< i>

Campylobacter</i>. Int. J. Food Microbiol. 108:226-232.

Sandercock, D. A., R. R. Hunter, G. R. Nute, M. A. Mitchell, and P. M. Hocking. 2001. Acute

heat stress-induced alterations in blood acid-base status and skeletal muscle membrane

integrity in broiler chickens at two ages: Implications for meat quality. Poult. Sci. 80:418-425.

Smith, M. O., and R. G. Teeter. 1992. Effects of potassium chloride supplementation on growth

of heat-distressed broilers. J. Appl. Poult. Res. 1:321-324.

St-Pierre, N., B. Cobanov, and G. Schnitkey. 2003. Economic losses from heat stress by US

livestock industries. J. Dairy Sci. 86:E52-E77.

Toyomizu, M., M. Tokuda, A. Mujahid, and Y. Akiba. 2005. Progressive alteration to core

temperature, respiration and blood acid-base balance in broiler chickens exposed to acute heat

stress. J. Poult. Sci. 42:110-118.

USDA. 2002. U.S. Broiler Industry Structure. Pages 5 National Agricultural Statistics Service,

Washigton DC.

USDA. 2014. Poultry - Production and Value 2013 Summary. Pages 14.

Wang, Y., D.-M. Su, R.-H. Wang, Y. Liu, and H. Wang. 2005. Antinociceptive effects of choline

against acute and inflammatory pain. Neuroscience 132:49-56.

Wells, I. C. 1964. A blood clotting defect in choline deficient rats. Biochim. Biophys. Acta. Gen.

Subj. 86:339-345.

Yalcin, S., S. Özkan, L. Türkmut, and P. Siegel. 2001. Responses to heat stress in commercial

and local broiler stocks. 1. Performance traits. Br. Poult. Sci. 42:149-152.

Zhan, X., J. Li, Z. Xu, and R. Zhao. 2006. Effects of methionine and betaine supplementation on

growth performance, carcase composition and metabolism of lipids in male broilers. Br. Poult.

Sci. 47:576-580.

7

Chapter II: Literature Review

8

Heat Stress in Broilers

Broiler chickens experience heat stress when their body produces or absorbs more heat

than it can dissipate. Chicken brooding temperature is approximately 35oC during the first week

of age, however, the comfort zone for chickens declines to approximately 24oC by four weeks

(Teeter and Belay, 1996). When the environmental temperature exceeds the comfort limit,

chickens suffer heat related stress resulting in physiological and metabolic changes (Borges, et

al., 2007). Chickens are homeotherms and have developed adaptation mechanisms to maintain

their core body temperature in the normal range of 40-41oC. When the environmental

temperature exceeds their comfort zone, chickens reduce their feed intake in order to decrease

the heat production associated with feed consumption and metabolism, as well as increase non-

evaporative heat loss followed by evaporative cooling (Teeter and Belay, 1996).

Non-evaporative cooling constitutes the first response of chickens to heat stress (Teeter

and Belay, 1996). Birds increase their surface area and blood supply to the periphery to dissipate

heat through radiation, convection and conduction. As the environmental temperature rises closer

to the bird’s body temperature, non-evaporative cooling is no longer efficient and chickens rely

more on evaporative cooling (Teeter and Belay, 1996) which occurs through evaporation of

water from the respiratory system. Chickens also eliminate extra heat through hyperventilation

(Borges, et al., 2007; Bottje and Harrison, 1985; Donkoh, 1989; Teeter and Belay, 1996).

However, increased respiratory rate results in exhalation of too much carbon dioxide. This

reduces carbon dioxide partial pressure in blood while bicarbonate remains relatively constant

causing an elevation of blood pH thus resulting in acid-base imbalance known as respiratory

alkalosis (Borges, et al., 2007; Smith and Teeter, 1987b). Through renal compensation, more

bicarbonate is excreted and less hydrogen ions (H+) secreted to correct the acid-base imbalance

9

(Borges, et al., 2004). Evidence suggests that in mammals (Rector Jr, et al., 1962), the decrease

in H+ secretion results in excretion of more potassium ions (K

+) which has been shown to alter

electrolytes balance. Smith and Teeter (1987b) reported 633% increase of K+ excretion in

chickens under heat stress (35oC) compared to thermoneutral (24

oC) conditions.

Several management practices have been developed and used to improve bird’s comfort

and survival thus mitigating the negative impacts of heat stress on broilers. These include house

insulation, ventilation, feed withdrawal (Mahmoud and Yaseen, 2005; Soutyrine, et al., 1998),

fasting before the onset of heat stress and acclimation of high temperature in younger birds (De

Basilio, et al., 2001; Yalcin, et al., 2001). Other techniques consist of dietary modification to

increase the energy content from fat in order to reduce heat increment under heat stress

conditions (Dale and Fuller, 1979). These approaches do not directly address the acid-base

imbalance and its subsequent electrolyte imbalance. However, several studies have shown that

electrolyte supplementation in feed or water improves performance (Ahmad and Sarwar, 2005;

Smith and Teeter, 1992), survival (Smith and Teeter, 1987a) and reduces alkalosis (Teeter and

Smith, 1986) in heat-stressed broilers. Smith and Teeter (1986) observed that, the addition of

adequate amount of ammonium chloride (NH4Cl) 0.3% or less reduced blood pH and increased

live weight gain. The supplementation of K+ as potassium chloride (KCl) increased weight gain

but had no effect on the blood pH (Teeter and Smith, 1986). The acid-base imbalance can also be

corrected by carbonating the drinking water or supplementing it with calcium chloride (CaCl2)

(Bottje and Harrison, 1985). While electrolytes have been shown to improve performance and

welfare of heat-stressed chickens, their excess may lead to adverse effects. High levels of NH4Cl

(Teeter and Smith, 1986), sodium bicarbonate (NaHCO3) and CaCl2 (Bottje and Harrison, 1985)

10

used for correcting heat stress induced alkalosis can result in acidosis and reduced body weight

gain in heat-stressed broilers.

Sodium (Na+), chloride (Cl

-) and K

+ are electrolytes that participate in the maintenance of

body ionic and water balance. In a study utilizing chick embryos, Petronini, et al. (1992) reported

that the excess accumulation of Na+ and Cl

- interfere with cells’ proliferation and protein

synthesis as opposed to organic osmolytes such as betaine. Excess electrolytes in broiler feed or

drinking water can lead to increased excreta moisture and litter wetness (Francesch and Brufau,

2004; Smith, et al., 2000) which constitute the main source of foot pad dermatitis (Greene, et al.,

1985; Mayne, et al., 2007).

Foot Pad Dermatitis

Foot pad dermatitis (FPD), also known as pododermatitis or contact dermatitis, is

characterized by inflammation and lesion on the plantar surface of the feet and toes (Ekstrand, et

al., 1997; Greene, et al., 1985; Shepherd and Fairchild, 2010). In chickens, FPD appears similar

to that in turkey with few differences. The metatarsal, digital pad and interpad spaces are

ulcerated in chickens while only the pad is affected in turkey (Martland, 1985). In the early stage

of FPD, there is discoloration of the skin (Ekstrand, et al., 1997) that progresses in severe cases

into scab covered ulcers, intraepithelial inflammation, dermal capillary thrombosis and extensive

granulation tissue (Martland, 1985). Incidence of FPD may be related to high dietary

concentration of Na+, K

+ and phosphorous (P) which increase water intake and excreta moisture

in chickens and turkeys (Abd El‐Wahab, et al., 2013; Smith, et al., 2000). According to Berg and

Algers (2004), FPD could also be a result of high protein content or be related to dietary factors

such as indigestible fats leading to sticky droppings. McIlroy, et al. (1987) stated that litter

11

quality, affected by stocking density and drinking water design could increase FPD. Poor

ventilation has been shown to increase litter moisture content (Martrenchar, et al., 2002) and

therefore FPD incidence. However, other factors could also contribute to FPD.

Foot pad dermatitis has been linked to genetics. According to Kjaer, et al. (2006) it may

be possible to decrease the incidence of FPD by genetic selection without negatively affecting

body weight. Another factor reportedly involved in FPD is biotin. However, there has been

mixed findings on its importance in the prevention of FPD. Harms and Simpson (1977) reported

that biotin supplementation decreased the severity of FPD when given to poults fed a diet

deficient in biotin and raised on wet litter as opposed to poults maintained on wet litter without

biotin supplementation. Conversely, Cengiz, et al. (2012) reported that supplemental biotin does

not minimize or prevent the development of FPD in broiler chickens.

The occurrence of FPD has significant welfare and economic implications for the broiler

industry. In severe cases, FPD lesions cause pain and walking difficulties in broilers (Berg and

Algers, 2004) with lameness (Greene, et al., 1985) thus reducing their access to feeders.

Martland (1985) suggested that turkeys experiencing severe FPD may develop pain-induced

inappetance which depresses growth rate and body weight causing economic losses for the

producer. Foot pad dermatitis causes birds to sit on their breast for long periods of time resulting

in the development of hock and breast lesions (Martland, 1985) which degrades the meat quality

(McIlroy, et al., 1987). With the recent development of an export market of chicken paw to

China and Hong Kong, in addition to welfare issue, FPD has become an important economic

component in the development of the broiler industry (USDA, 2013). Good litter management is

the key to preventing FPD (Hoffmann, et al., 2013). Litter wetness has been shown to be

increased by dietary concentration of Na+ and K

+, due to high moisture content of the excreta

12

(Smith, et al., 2000). Therefore, the use of these electrolytes to minimize the negative impact of

heat stress on broilers must be done with respect to the dietary electrolytes balance (Ahmad and

Sarwar, 2005).

Impact of Heat Stress on Intestinal Morphology

The gastrointestinal tract serves in the digestion and absorption of nutrients and as a

barrier between the luminal content and the systemic circulation. The maintenance of the

physical integrity of the intestine is primordial for its functions. The intestinal barrier is

composed of apical membrane of cells of the epithelium and the tight junction between them

(Sun, et al., 1998). Several stress factors including osmotic stress (Mongin, et al., 1976) and heat

stress (Lambert, et al., 2002) affect gut integrity. In birds, the contents of the small intestine are

always hypertonic to the plasma osmolality of about 320 mOsmol (Klasing, et al., 2002; Mongin,

et al., 1976) exerting substantial osmotic pressure on the cells lining the gut. In heat-stressed

animals such as rats (Lambert, et al., 2002) , pigs (Pearce, et al., 2012) and chickens (Quinteiro-

Filho, et al., 2010), the tight junctions between epithelial cells are disrupted increasing the

intestinal permeability to large and normally restricted molecules from the intestinal lumen to the

blood. During heat stress, blood is diverted to the periphery to dissipate the extra heat, reducing

blood flow to the viscera. Prolonged reduced blood flow causes tissue hypoxia, generation of

reactive oxygen species and damage to enterocyte membrane (Hall, et al., 1999; Lambert, 2004)

resulting in increased intestinal permeability. The intestinal lumen contains commensal bacteria

and a number of other ingested pathogens. Endotoxins from bacteria present in the intestine

usually enter the internal environment at a basal level in normal conditions. However, with

compromised intestinal integrity, the level of endotoxin entry increases resulting in greater

13

release of proinflammatory cytokines by the immune system causing further inflammation of the

intestinal epithelium (Lambert, 2009). Heat stress has also been proven to increase the absorption

of some nutrients. Pearce, et al. (2012) showed that there is an increase in glucose absorption in

heat-stressed pigs. Galactose and methionine absorption in the jejunum of chickens are increased

in response to heat stress (Mitchell and Carlisle, 1992).

Heat Stress and the Immune System

According to Selye (1976), stress is a non-specific response of the body to any stimulus

or stressor. Stress results in the activation of the hypothalamus-pituitary-adrenal axis (HPA) and

the secretion of glucocorticoid hormones: cortisol and corticosterone. Cortisol is the main

glucocorticoid in fish and most mammals, whereas corticosterone is the primary glucocorticoid

in birds, reptiles, amphibians and rodents (Cockrem, 2013). Glucocorticoids’ function in the

regulation of inflammatory response is well established. They inhibit proinflammatory cytokines

such as interleukin (IL)-12, tumor necrosis factor (TNF)-α and Interferon (IFN)-γ, and stimulate

the production of anti-inflammatory cytokines IL-10, IL-4 and transforming growth factor

(TGF)-β to control excessive immune response (Elenkov and Chrousos, 2002). An increase in

proinflammatory cytokines during excessive inflammation activates the HPA axis resulting in

increased production of glucocorticoids to reduce inflammation (Chrousos, 1995). Heat stress in

chickens has been shown to increase plasma corticosterone concentration (Beuving and Vonder,

1978; Quinteiro-Filho, et al., 2010) and a short term increase may be beneficial. In a study

conducted by Nasir, et al. (1999), adrenalectomised chickens were injected with corticosterone to

mimic an increase of corticosterone during stress. Results showed that elevated plasma

corticosterone stimulated higher feed intake, delayed food passage time and increased calcium,

14

phosphorous and glucose absorption. Garriga, et al. (2006) also reported an increase in apical

glucose transport during heat stress. Enhanced gluconeogenesis following short term

administration of corticosterone in chickens was reported by Lin, et al. (2004) and Shini, et al.

(2008a). However, a production of glucocorticoids at a suboptimal level has been shown to

impair the immune system by decreasing inflammatory response (Chrousos, 1995) or by

reducing spleen and bursa weights (Shini, et al., 2008b).

Choline

Choline, the common name for 2-hydroxy-N,N,N-trimethylethanammonium (Combs Jr,

1991), is a water soluble vitamin-like compound found in the form of phosphatidylcholine, or

sphingomyelin in most foods (Combs Jr, 1991; McDowell, 1989) with the highest concentrations

in wheat germ, dried soybean, egg yolk, pork, and glandular meat such as beef and chicken liver

(Zeisel, et al., 2003). Depending on its functions in the body, choline is metabolized to different

compounds, including phosphatidylcholine essential for cell membrane integrity (Plagemann,

1968) and transmembrane signaling (Zeisel, 1993), acetylcholine involved in neurotransmission

(Cohen and Wurtman, 1976; Wecker, 1989), and betaine which donates methyl groups for the

methylation of homocysteine to methionine or functions as organic osmolyte (Zeisel, et al.,

1989). Choline also plays an important role in fat metabolism. Its deficiency is associated with

fatty liver (Cooke, et al., 2007; Vendemiale, et al., 2001) and low body fat (Daily III, et al.,

1998) in all animal species. Phosphatidylcholine is required for the synthesis of very low density

lipoprotein (VLDL) in the liver (Yao and Vance, 1988) or further metabolized to

glycerophosphocholine predominantly in renal medullary cells where it protects against hyper

salinity of the interstitial fluid (Beck, et al., 1992).

15

Free choline is taken up via a carrier mediated pathway or by passive diffusion at low and

high concentration respectively (Herzberg and Lerner, 1973; Kamath, et al., 2003) and enter the

portal circulation. Choline is transported by three different mechanisms: high-affinity Na+-

dependent transporter that supplies choline for the synthesis of acetylcholine (Meyer Jr, et al.,

1982), low-affinity Na+-independent transporter that supplies choline for phosphatidylcholine

and other phospholipids synthesis in cell membranes, and very low-affinity Na+-independent

residual diffusion (Van Rossum and Boyd, 1998). Phosphatidylcholine, another form of choline

present in foods, is hydrolyzed in the intestine to lysophosphatidylcholine before absorption. In

the enterocytes, lysophosphatidylcholine is re-esterified to phosphatidylcholine, packed into

chylomicrons and sent to the lymphatic circulation (Le Kim and Betzing, 1976).

The signs of choline deficiency vary across animal species but the most common ones

include fatty liver, poor growth, hemorrhagic tissue and hypertension (McDowell, 1989). In

young chickens and turkeys, perosis (slipped tendon) is the primary sign of choline deficiency.

All animal species are able to synthesize sufficient amount of choline when enough methyl

groups, methionine or betaine are supplied in the diet (McDowell, 1989) except young chickens

until about 8 weeks of age (Cook, et al., 1984; Molitoris and Baker, 1976). Choline is therefore

required in poultry nutrition and has always been included in their diet to support growth and

prevent perosis. Fast growing broilers reach market weight at a minimum of 6 weeks and are

unable to synthesize adequate amount of choline required 1300 mg/kg for 0-3 weeks of age,

1000 mg/kg for 3-6 weeks of age and 750 mg/kg for 6-9 weeks of age (NRC., 1994).

Because of its multiple functions in the body and requirements for chickens, several

studies have investigated the effects of supplementation of choline in broiler diet for improving

performance. However these results have been conflicting and could be due to dietary

16

methionine contents. Rafeeq, et al. (2011) and Pillai, et al. (2006) reported that choline increases

weight gain in broiler chickens fed a methionine deficient diet. A 300 mg/kg choline

supplementation to a diet containing adequate methionine improved growth performance (Dilger,

et al., 2007; Tillman and Pesti, 1986; Tsiagbe, et al., 1987) while other studies reported no

significant effect of choline on weight gain (Baker, et al., 1983; Waldroup and Fritts, 2005).

Supplemental choline was also shown to improve breast yield (Waldroup and Fritts, 2005) and

feed conversion ratio (Tillman and Pesti, 1986; Waldroup and Fritts, 2005).

Betaine

Betaine, a naturally occurring trimethylglycine (methylated amino acid), is found

predominantly in sugar beet, from which it was isolated for the first time, wheat bran, wheat and

spinach (Zeisel, et al., 2003). Chemically, betaine is a zwitterion quaternary ammonium

compound and a derivative of amino acid glycine (Craig, 2004). Its purified forms, anhydrous

betaine, betaine monophosphate and betaine chloride are used as feed additives (Eklund, et al.,

2005). Betaine can either be provided in the diet or synthesized by the mitochondria in the liver

and kidney from the oxidation of choline (Yamauchi, et al., 1992). As methylated amino acid,

betaine is mainly transported via betaine gamma amino butyric acid transporter (BGT-1) and

amino acid transport system A in most tissues (Petronini, et al., 2000; Yamauchi, et al., 1992). In

chickens, intestinal betaine is absorbed via Na+-dependent and Na

+-independent transport

systems. High concentration of betaine increases Na+-dependent transport resulting in higher

betaine uptake (Kettunen, et al., 2001b).

Betaine’s main functions are to serve as a methyl group donor for various methylation

reactions in the body or as osmolyte to protect cells subjected to osmotic pressure. As a methyl

17

donor, betaine does not participate in most methylations but donates methyl groups to

homocysteine to form methionine (Vincent , et al., 1946). Its osmoprotectant property is also

established in plants (Storey and Jones, 1975; Yang, et al., 2003) and in bacteria (Sutherland, et

al., 1986). In bacteria, when subjected to mild hyperosmolar environment the primary

intracellular osmoprotectant is K+. However, with increased environmental salinity, a

corresponding internal K+ concentration is deleterious to enzymes function and betaine is

accumulated in preference to K+ to restore osmotic balance (Sutherland, et al., 1986). Virtanen,

et al. (1989), studying the adaptation of young Atlantic salmon, showed that betaine in an

effective osmolyte that helps maintain ionic and osmotic balance in sea water.

Due to its methyl donor and osmoprotectant properties, physiological and nutritional

effects of betaine have been investigated in livestock on performance and carcass quality

(Sakomura, et al., 2013; Waldroup and Fritts, 2005), intestinal infection states (Kettunen, et al.,

2001a; Klasing, et al., 2002) as well as stress conditions (Attia, et al., 2009). Betaine has been

shown to promote growth in pigs by enhancing growth hormone synthesis in finishing pigs

(Huang, et al., 2007). Pigs fed conjugated linoleic acid (1%) and betaine (0.5%) increased

leanness and decreased shoulder fat deposition (Rojas-Cano, et al., 2011). In broiler chickens,

betaine supplementation at various levels indicates some improvement on performance and

carcass quality. Improved breast yield was reported by McDevitt, et al. (2000) and Waldroup, et

al. (2006) when 0.5 g/kg betaine was supplemented. Waldroup, et al. (2006) also reported that

with 500 mg/kg or 1000 mg/kg betaine supplementation there was improved feed conversion

ratio at 35 and 42 days. Betaine supplementation at 0.072% and 0.144% decreased abdominal fat

deposition (Hassan, et al., 2005). Waldroup and Fritts (2005) saw an increase in dressing

percentage at 42 days of age in broiler chickens supplemented with 1000 mg/kg of betaine.

18

Evidence strongly suggests that betaine acts as an osmolyte in intestinal cells. Betaine was

shown to improve diarrheal state and performance (Fetterer, et al., 2003) in coccidia-infected

chickens. Klasing, et al. (2002) showed that the supplementation of betaine at 0.5 g/kg and 1

g/kg improved osmolarity and restored villi length in the duodenum of infected chickens.

Supplemental betaine at 0.2% helps the duodenum maintain water balance in hypertonic

environment (Kettunen, et al., 2001a).

Interrelation of Choline, Betaine and Methionine Metabolism

Choline is considered a potential source of methyl groups along with methionine but is

not directly involved in methylation reactions unless oxidized into betaine (Borsook and

Dubnoff, 1947). Betaine donates its methyl group to homocysteine to form methionine, one of

the essential and limiting amino acids in chickens (Stucki and Harper, 1961). As reviewed by

Craig (2004), Eklund, et al. (2005) and Metzler-Zebeli, et al. (2009), methionine is first

converted to S-adenosylmethionine (SAM) by the enzyme methionine adenosyltransferase. S-

adenosylmethionine is the active methyl donor; it liberates methyl groups used in different

biosynthetic methylations, yielding S-adenosylhomocysteine. S-adenosylhomocysteine is

converted to homocysteine by the action of S-adenosylhomocysteine hydrolase. Homocysteine is

either irreversibly transformed to cysteine which can be utilized for protein synthesis or re-

methylated to methionine by other methyl group sources; betaine via betaine homocysteine-

methyltransferase (BHMT) or N5-methyl tetrahydrofolate via tetrahydrofolate-methyltransferase

(THMT).

Choline, betaine and methionine are interrelated and their ability to spare each other has

been extensively investigated in poultry nutrition with variable results. Various researchers have

19

reported significant methionine sparing effect of choline and betaine. Baker, et al. (1983),

evaluated three choline (0, 217, and 434 mg/kg) and five methionine levels (0, 0.05, 0.10, 0.15

and 0.2%) in a 3x5 factorial design and found that supplementation of choline at 217 mg/kg with

0 and 0.05% methionine increased performance in poultry. By adding 0 or 0.05% supplemental

methionine to the basal diets containing 0.35% DL-methionine, the researchers made them

slightly deficient with regard to NRC requirements (0.5%). Supplementation of betaine and

choline to a diet containing 0.37% methionine (Pesti, et al., 1979) and betaine to 76% methionine

adequate diet (Zhan, et al., 2006) improved weight gain and feed conversion ratio suggesting that

choline and betaine can spare methionine only in a small range of methionine deficiency. In a

study conducted by Dilger, et al. (2007), graded levels of choline (0, 150,300 and 1000 mg/kg),

betaine (260 and 600 mg/kg) and methionine at 1289 mg/kg was added to choline-free diet. They

found that supplementation of choline-free diet with betaine and methionine showed a minimal

choline sparing effect. However, the addition of betaine to a diet containing a minimal level of

choline resulted in a substantial increase in growth performances. They concluded that 50% of

dietary requirement of choline must be supplied as choline and the rest can be replaced by

betaine. While the 50% reduction might be questionable, it is noteworthy that because choline

has multiple functions in the body, betaine could only spare for its methyl donor property.

Studies have shown no methionine sparing effects of choline and betaine. Esteve-Garcia and

Mack (2000) conducted a study in which two levels of betaine (0 and 0.5 g/kg) and three levels

of methionine (0, 0.6 and 1.2 g/kg) were supplemented to a methionine deficient diet. They

found that unlike betaine, methionine supplementations improved weight gain and feed

efficiency confirming that betaine does not spare methionine. Similarly, Schutte, et al. (1997)

added two levels of betaine (0 and 0.04%) and three levels of methionine (0, 0.05 and 0.1%) to

20

different combinations of methionine deficient diets and reported that betaine did not improve

growth contrary to methionine supplementations.

21

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29

Chapter III: Performance of heat-stressed broilers supplemented with dietary choline and

betaine

30

Abstract


(BET) on broiler performance and carcass characteristics under different temperature conditions,

thermoneutral (TN: 23.9oC) and heat stress (HS: 28-36

oC). Eight hundred day-old mixed sex

Cobb500 (female) x Hubbard (M99 male) chicks were weighed, wing tagged and equally divided

into the two environments. Eight replicates of 10 chicks were randomly assigned to each of five

dietary treatments: treatment 1 (CON) basal diet; treatment 2 (CHO500) basal diet plus 500 ppm

methyl equivalents added CHO; treatment 3 (CHO1000) basal diet plus 1000 ppm methyl

equivalents added CHO; treatment 4 (BET500) basal diet plus 500 ppm methyl equivalents

added BET and treatment 5 (BET1000) basal diet plus 1000 ppm methyl equivalents added BET.

The corn-soy bean basal diet was formulated to meet NRC requirements for broilers. Feed intake

and body weight were recorded weekly by pen. On day 24, foot pads were assessed for health

and litter samples collected from each pen. At slaughter on day 52, breast meat was collected for

drip loss evaluation at 4 and 7 days post slaughter. Breast meat lightness (L*), redness (a*) and

yellowness (b*) were measured. Data were analyzed using mixed model analysis of variance

(SAS 9.3, Cary, NC). Results showed that HS birds consumed 20.59% less feed and gained

23.34% less weight than TN birds (P < 0.05) during 22-49 days of age. The overall feed intake

and weight gain during days 1-49 were similarly reduced in HS birds. During 1-21 days of age,

birds consuming BET500 (0.68) and BET1000 (0.67) had higher feed efficiency compared to

those consuming the CON (0.62). Heat stress decreased feed efficiency during days 22-49 (P =

0.02). HS birds (0.54) had lower feed efficiency when compared to TN birds (0.56). There was a

diet X temperature interaction (P = 0.04) on drip loss 4 days post slaughter. The lowest drip loss

occurred with CHO500 (0.60%) in HS and BET1000 (0.83%) in TN birds. Breast meat color of

31

HS birds was significantly (P = 0.02) lighter (54.21) while that of TN birds was significantly (P

= 0.004) more yellow (3.75). Temperature did not affect pododermatitis (P = 0.22) however,

there was an effect of diet (P = 0.003) with CHO500 and BET1000 showing the lowest

occurrence. In this study, breast meat drip loss was influenced by dietary CHO and BET while

meat color was affected by rearing temperature. Dietary CHO and BET supplementation did not

improve weight loss induced by HS conditions.

Introduction

Heat stress is one of the major problems facing the poultry industry (St-Pierre, et al.,

2003). During high environmental temperature and high relative humidity, chickens adapt by

reducing feed intake to decrease endogenous heat production and hyperventilating or panting to

eliminate extra heat. However, the results are detrimental for physiology with slow growth rate,

low body weight gain (Cooper and Washburn, 1998; Donkoh, 1989; Quinteiro-Filho, et al.,

2010) and respiratory alkalosis (Borges, et al., 2004; Bottje and Harrison, 1985). Several

management practices have been developed and used to improve birds’ comfort, survival and to

mitigate the negative impact of heat stress on broilers. These include housing insulation,

ventilation, evaporative cooling systems, feed withdrawal (Mahmoud and Yaseen, 2005;

Soutyrine, et al., 1998), fasting before the onset of heat stress and acclimation of high

temperature in younger age (De Basilio, et al., 2001; Yalcin, et al., 2001). These practices don’t

directly address the acid-base imbalance (respiratory alkalosis) even though they improve

broilers performances.

Respiratory alkalosis caused by excessive loss of carbon dioxide due to hyperventilation

(Borges, et al., 2007; Teeter and Smith, 1986) subsequently results in electrolyte imbalance in

32

chickens primarily due to potassium excretion. Smith et al (1987) reported a 633% increase of

K+ excretion via excreta during heat stress (35

oC) compared to thermoneutral (24

oC)

environment. Supplementing heat-stressed broilers with essential electrolytes could be beneficial

in correcting acid-base imbalance thereby improving broilers comfort and performance.

Supplementation of water with potassium chloride (KCl) was shown to increase body weight

gain (Ahmad, et al., 2008; Smith and Teeter, 1987) of heat-stressed broilers. Dietary electrolytes

balance for maximum production was estimated to be 240 mEq/kg (Borges, et al., 2003).

However, close attention must be paid to the use of dietary electrolytes to alleviate the negative

impacts of heat stress on broilers. Addition of the electrolytes Na+, Cl

-, and K

+ in drinking water

was shown to cause an increase in water consumption that resulted in litter wetness, the primary

cause of foot pad dermatitis (Berg and Algers, 2004). Additionally, excess accumulation of Na+

and Cl- may inhibit cells proliferation and protein synthesis as opposed to organic osmolytes

(Petronini, et al., 1992).

Organic osmolytes such as choline and betaine could be alternatives to electrolytes.

Currently, betaine is not required in a typical broiler diet, however choline is usually added at

different rates depending on age: 1300 mg/kg for 0-3 weeks of age, 1000 mg/kg for 3-6 weeks of

age and 750 mg/kg for 6-9 weeks of age (NRC., 1994). Betaine can be synthesized from choline

and has been shown to affect the movement of water across small intestine of broiler chicks

(Kettunen, et al., 2001). The supplementation of choline and betaine in broiler diet may be

beneficial in improving osmotic pressure and decreasing excreta moisture in heat-stressed

chickens and could improve their performance and foot health. Even though the effect of

supplemental choline and betaine on broiler performance has been the objective of several

studies, very little has been done concerning their osmoprotectant benefit if any, under heat stress

33

conditions. Therefore, this study was designed to evaluate the impact of dietary supplementation

of choline and betaine on the performance and carcass characteristics of broilers under HS

conditions.

Materials and Methods

Birds and Housing

The procedures were reviewed and approved by the University of Tennessee Institutional

Animal Care and Use Committee (IACUC), and the research was conducted at the Johnson

Animal Research and Teaching Unit (JARTU). Two adjacent rooms, thermoneutral (TN) and

heat stress (HS) were used. Each room was divided into 40 pens equipped with individual gravity

feeders, water nipple line and bedding made of shredded paper. The rooms were equipped with

HOBO®1

data loggers to record daily temperature and humidity. In addition, a humidifier was

placed in the heat stress room to ensure that humidity remained above 50%. Eight hundred day-

old mixed sex Cobb500 (female) x Hubbard (M99 male) chicks were obtained from Pilgrim’s

Pride hatchery in Cohutta, Georgia. They were vaccinated at the hatchery against Marek’s and

Gumboro diseases (VAXXITEK HVT-IBD); Escherichia coli, Salmonella typhimuriun and

Pseudomonas aeruginosa (GARASOL); Coccidiosis (COCCIVAC-B), Newcastle

(NEWHATCH C2-M) and Infectious bronchitis (MILDVAC ARK and GA 2008). Upon arrival,

the birds were weighed by groups of ten and assigned to one of each treatment.

1 Onset Computer Corporation

470 MacArthur Blvd. Bourne, MA 02532

34

Temperature Treatments

During the first 21 days, birds were brooded under similar conditions in the two rooms.

The temperature was set to 32.22oC the first week and decreased by 2.78

oC until it reached

23.9oC by the end of the third week. From day 22 to the end of the study, the temperature was

maintained at 23.9 oC in the TN room and cycled from 28

oC to 36

oC between 10:00 a.m. and

8:00 p.m. in the HS room (Figure 1). The photoperiod regimen was 23 hours of light followed by

1 hour of dark in both environments. The humidity was maintained at 50% in the HS room.

Figure 1. Typical daily temperature in TN (23.9 ±1°C) and HS (28-36±1°C) rooms.

10

15

20

25

30

35

40

Tem

p. °C

Time

HS

TN

35

Dietary Treatments

Five dietary treatments were tested. The basal formulation with choline or betaine

supplementation was used to make the following dietary treatments:

Control (CON), basal formulation

Low choline (CHO500), basal diet plus 500 ppm methyl equivalents added choline

High choline (CHO1000), basal diet plus 1000 ppm methyl equivalents added choline

Low betaine (BET500), basal diet plus 500 ppm methyl equivalents added betaine

High betaine (BET1000), basal diet plus 1000 ppm methyl equivalents added betaine

The basal diet was formulated to meet broilers energy, protein, minerals and vitamins

requirements (NRC., 1994) except for methionine which was 20% lower. The birds were fed

starter diet (Table 1) from days 1-21, grower diet (Table 2) from days 22-35 and finisher diet

(Table 3) from days 36-49.

Growth Performance

The feed was weighed and added every three days to ensure continuous access. Once per

week, the remaining feed was weighed to determine feed intake. Similarly, the birds were

weighed per pen to determine body weight gain. When a bird died, the carcass weight was

recorded and accounted for in the calculation of weekly average weight gain and feed intake.

Litter Samples and Foot Pad Dermatitis

The litter made of shredded paper was not changed during the entire experiment. On day

42, litter samples were collected at approximately the same area between the nipple lines and the

feeders and stored at -20oC for later determination of moisture content. On day 42, all the birds

were scored for foot pad dermatitis. The pad of both feet were observed, compared to necrotic

patterns and ranked using the following scores: 0-no lesions, 1-small lesion, 2-moderate lesion in

36

Table 1. Nutritional composition of starter diet (%)

Ingredients CON CHO500 CHO1000 BET500 BET1000

Corn, grain 62.86 62.86 62.86 62.86 62.86

Soybean meal 31.00 31.00 31.00 31.00 31.00

Vitamin premix1 0.0625 0.0625 0.0625 0.0625 0.0625

Filler, sand 0.20 0.105 0.01 0.143 0.0853

Choline, mg/kg2 0.00 0.095 0.19 0.00 0.00

Betaine, mg/kg2 0.00 0.00 0.00 0.057 0.1147

DL methionine 0.15 0.15 0.15 0.15 0.15

Salt 0.35 0.35 0.35 0.35 0.35

Limestone 1.10 1.10 1.10 1.10 1.10

Dical phosphate 2.10 2.10 2.10 2.10 2.10

Trace min. premix3 0.125 0.125 0.125 0.125 0.125

Fat, animal 1.90 1.90 1.90 1.90 1.90

Coban 0.0495 0.0495 0.0495 0.0495 0.0495

Lysine 0.10 0.10 0.10 0.10 0.10

Total 100.00 100.00 100.00 100.00 100.00

Nutrient Composition

ME, kcal/kg 3024.00 3024.00 3024.00 3024.00 3024.00

Crude protein, % 20.59 20.59 20.59 20.59 20.59

Calcium, % 0.98 0.98 0.98 0.98 0.98

Available Phosphorus, % 0.51 0.51 0.51 0.51 0.51

Potassium, % 0.81 0.81 0.81 0.81 0.81

Chloride, % 0.25 0.25 0.25 0.25 0.25

Sodium, % 0.16 0.16 0.16 0.16 0.16

Zinc, mg/kg 92.99 92.99 92.99 92.99 92.99

Choline, mg/kg 1236.38 2186.44 3136.44 1236.38 1236.38

Betaine, mg/kg 0.00 0.00 0.00 585.01 1147.03

Lysine, % 1.18 1.18 1.18 1.18 1.18

Methionine, % 0.47 0.47 0.47 0.47 0.47

Methionine +Cysteine, % 0.81 0.81 0.81 0.81 0.81

Threonine, % 0.76 0.76 0.76 0.76 0.76

Fat, % 4.60 4.60 4.60 4.60 4.69

Salt, % 0.53 0.53 0.53 0.53 0.53 1 Vitamin premix supplied per kg of feed: Vitamin A, 17636684 IU; Vitamin D3, 5952381 ICU; Vitamin

E, 35000 IU; Vitamin B12, 16 mg; Biotin, 176 mg; Menadione, 3858 mg; Thiamin, 3307 mg; Riboflavin,

15432 mg; Pantothenic acid, 24250 mg; Vitamin B6, 4409 mg; Niacin, 88180 mg; Folic acid, 1658 mg. 2

Choline chloride (60%), Betaine anhydrous, provided by Balchem Corporation and added at the expense

of the filler, sand.

3 Trace minerals premix supplied at 1.14 kg per ton of feed: Calcium, 3% min and 4% max; Manganese,

9.70%; Zinc, 7.50%; Copper, 3.53%; Iron, 4400 ppm; Iodine1586 ppm; Selenium, 240 ppm.

37

Table 2. Nutritional composition of grower diet (%)


Corn, grain 62.32 62.32 62.32 62.32 62.32

Soybean meal 31.00 31.00 31.00 31.00 31.00

Vitamin premix1 0.0625 0.0625 0.0625 0.0625 0.0625

Filler, sand 0.30 0.1884 0.0768 0.243 0.1853

Choline, mg/kg2 0.00 0.1116 0.2232 0.00 0.00

Betaine, mg/kg2 0.00 0.00 0.00 0.057 0.1147

DL methionine 0.20 0.20 0.20 0.20 0.20

Salt 0.35 0.35 0.35 0.35 0.35

Limestone 1.10 1.10 1.10 1.10 1.10

Dical phosphate 1.69 1.69 1.69 1.69 1.69

Trace min. premix3 0.125 0.125 0.125 0.125 0.125

Fat, animal 2.70 2.70 2.70 2.70 2.70

Coban 0.0495 0.0495 0.0495 0.0495 0.0495

Lysine 0.10 0.10 0.10 0.10 0.10

Total 100.00 100.00 100.00 100.00 100.00


ME, kcal/kg 3072.00 3074.00 3072.00 3072.00 3072.00

Crude protein, % 19.95 19.95 19.95 19.95 19.95

Calcium, % 0.89 0.89 0.89 0.89 0.89


Potassium, % 0.80 0.80 0.80 0.80 0.80

Chloride, % 0.25 0.25 0.25 0.25 0.25

Sodium, % 0.16 0.16 0.16 0.16 0.16

Zinc, mg/kg 92.48 92.49 92.48 92.48 92.48

Choline, mg/kg 1233.03 2349.37 3465.10 1233.02 1233.03

Betaine, mg/kg 0.00 0.00 0.00 585.01 1147.03

Lysine, % 1.18 1.18 1.18 1.18 1.18

Methionine, % 0.52 0.52 0.52 0.52 0.52


Threonine, % 0.76 0.76 0.76 0.76 0.76

Fat, % 5.38 5.38 5.38 5.38 5.38








38

Table 3. Nutritional composition of finisher diet (%)


Corn, grain 66.00 66.00 66.00 66.00 66.00

Soybean meal 27.37 27.37 27.37 27.37 27.37

Vitamin premix1 0.0625 0.0625 0.0625 0.0625 0.0625

Filler, sand 0.30 0.1884 0.0768 0.243 0.1853

Choline, mg/kg2 0.00 0.1116 0.2232 0.00 0.00

Betaine, mg/kg2 0.00 0.00 0.00 0.057 0.1147

DL methionine 0.15 0.15 0.15 0.15 0.15

Salt 0.35 0.35 0.35 0.35 0.35

Limestone 1.10 1.10 1.10 1.10 1.10

Dical phosphate 1.69 1.69 1.69 1.69 1.69

Trace min. premix3 0.125 0.125 0.125 0.125 0.125

Fat, animal 2.70 2.70 2.70 2.70 2.70

Coban 0.0495 0.0495 0.0495 0.0495 0.0495

Lysine 0.10 0.10 0.10 0.10 0.10

Total 100.00 100.00 100.00 100.00 100.00


ME, kcal/kg 3105.00 3107.00 3105.00 3108.00 3105.00

Crude protein, % 18.55 18.55 18.55 18.55 18.55

Calcium, % 0.88 0.88 0.88 0.88 0.88


Potassium, % 0.74 0.74 0.74 0.74 0.74

Chloride, % 0.25 0.25 0.25 0.25 0.25

Sodium, % 0.16 0.16 0.16 0.16 0.16

Zinc, mg/kg 91.15 91.16 91.15 91.16 91.15

Choline, mg/kg 1156.71 2273.05 3388.78 1157.25 1156.71

Betaine, mg/kg 0.00 0.00 0.00 569.98 1147.03

Lysine, % 1.08 1.08 1.08 1.08 1.08

Methionine, % 0.45 0.45 0.45 0.45 0.45


Threonine, % 0.70 0.70 0.70 0.70 0.70

Fat, % 5.48 5.48 5.48 5.49 5.48








39

one or both feet, and 3-extensive lesion in one or both feet (Figure 2). The scoring was done by

the same person for consistency. To determine litter moisture, samples were thawed at room

temperature, weighed and dried in the oven for 48 hours at 16oC. After 48 hours, the samples

were weighed and moisture content determined as difference between wet and dried litter weight

and expressed as a percent of wet litter.

Figure 2. Scoring system for foot pad dermatitis (Mello, et al., 2011).

40

Processing of Birds

Prior to harvesting on day 52, birds were deprived of feed for 12 hours to allow for the

emptying of the digestive track. Eighty birds (eight birds randomly selected from each treatment

per room), were weighed, hung on shackles, stunned with an electric knife, and then killed by

severing the jugular vein. They were allowed to bleed for 3 minutes then scalded in hot water

(59.44 oC) for 40 seconds, and their feathers removed using a de-feathering machine. Birds were

then eviscerated and carefully dissected. The carcasses were weighed and the breast meat

separated. The breasts were cut into half, weighed, individually bagged and chilled on ice for

later drip loss determination.

Drip Loss

The left half of each breast was hung in the refrigerator at 4oC for drip loss determination.

The breasts were weighed at day four and again at day seven post slaughter. Drip loss constitutes

water loss during storage and was determined by the difference in weights between two periods

of storage time. For four days post slaughter, drip loss was determined by taking the difference in

breast weights between days four and one and is expressed as a percent of day one breast weight.

Drip loss seven days post slaughter is determined using the same principle and expressed as a

percent of day one breast weight.

Breast Meat Color

Color of breast meat was determined using photo colorimeter (Konica Minolta)2 and

reported using L*a*b* color space (CIELAB) from CIE (International Commission on

Illumination). L* represents lightness and the breast meat is considered light when L* > 50.0 and

2 Konica Minolta, Inc. JP Tower,

272 Marunouchi Chiyoda-ku, Tokyo 100-7015, Japan

41

dark when L* < 45.0. Redness (a*) and yellowness (b*) represent the chromaticity coordinates.

Terms a* and b* indicate color direction: the more positive a* the higher the redness and the

more negative a* the greener the color, the more positive b* the more yellow and the more

negative b* the more blue the color (McGuire, 1992). Each color value is given by the average of

three measurements on an individual breast sample.

Statistical Analysis

The experimental design was a completely randomized design split plot with temperature

in the whole plots and diet in the subplots. Data were analyzed using mixed model analysis of

variance procedure SAS 9.3 (SAS Institute, Cary, NC, USA) and least squares means compared

using protected LSD at 5% level of significance. Data on foot pad dermatitis were analyzed

using contingency table.

Results

Growth Performance

Average feed consumption for days 1-21, 22-49 and 1-49 are presented in Table 4. There

was no diet or temperature effect nor was there an interaction on feed intake during days 1-21 (P

> 0.20). During days 22-49 there was no diet X temperature interaction on feed intake (P = 0.17).

However, temperature significantly affected feed intake (P = 0.001) with thermoneutral bird

(4.08 kg) consuming in average 20.59% more than heat stress bird (3.24 kg). The feed

consumption during days 1-49 was similarly affected only by temperature (P = 0.0001). The

thermoneutral bird average consumption (4.98 kg) was 17.67% higher than that of the heat-

stressed bird (4.10 kg).

42

Table 4. Effect of choline and betaine supplementation on average feed intake (kg) 1

of heat-

stressed broilers during 1-21, 22-49 and 1-49 days of age

Diet Temp.2

Day 1-21 Day 22-49 Day 1-49

CON TN 0.90

3.98 4.86

HS 0.82 3.33 4.15

CHO500 TN 0.90 4.13 5.04

HS 0.84 3.27 4.11

CHO1000 TN 0.92 4.03 4.96

HS 0.89 3.30 4.19

BET500 TN 0.85 3.99 4.83

HS 0.89 3.31 4.19

BET1000 TN 0.92 4.27 5.18

HS 0.86 3.00 3.86

SEM 0.047

0.16 0.18

Effect P values

Diet 0.25 0.99 0.99

Temp 0.86 0.001 0.0001

Diet x Temp 0.73 0.17 0.26

Main Effect Means

Diet

CON 0.86 3.65

4.51

CHO500 0.87 3.70 4.57

CHO1000 0.91 3.67 4.57

BET500 0.87 3.65 4.51

BET1000 0.89 3.64 4.52

SEM 0.033 0.11 0.13

Temp

TN 0.90 4.08a

4.98a

HS 0.86 3.24b

4.10b

SEM 0.023 0.098 0.11 1 Values are least squares means.

2 TN = Thermoneutral environment, HS = Heat stress environment.

a,b,c Means within same column followed by different letters differ significantly (P < 0.05).

43

Body weight gains are reported in Table 5. There was no diet or temperature effect nor

was there their interaction on body weight gain during the starter period (P > 0.50). During days

22-49, only temperature significantly affected body weight gain (P = 0.001). The average body

weight gain of heat-stressed bird (1.74 kg) was 23.35% lower compared to thermoneutral bird

(2.27 kg). During days 1-49, body weight gain was similarly affected by heat stress (P = 0.001)

with heat-stressed birds (2.86 kg) gaining 19.23% less weight that the thermoneutral bird (2.31

kg).

There was no diet X temperature interaction on feed efficiency (Table 6). During days 1-

21, BET500 (0.68) and BET1000 (0.67) increased (P = 0.04) feed efficiency compared to the

CON (0.62). During days 22-49 only temperature increased feed efficiency (P = 0.02). Heat-

stressed birds had lower feed efficiency (0.54) compared to TN birds (0.56). Overall during days

1-49, temperature tended to decrease feed efficiency (P = 0.05).

The mortality rate (Table 7) was not affected by diet or diet X temperature interaction.

However, mortality in the heat stress treatment (11.25%) was significantly higher than that of the

thermoneutral environment (3%) (P = 0.0006).

Litter Moisture

Litter moisture content expressed as a percent of wet litter weight is reported in Table 8.

There was no diet X temperature interaction on litter moisture (P = 0.17). Litter moisture was

significantly lower (P = 0.0001) in the heat stress (21.31%) room compared to thermoneutral

room (42.73%). CHO500 (29.73%) and BET1000 (29.73%) tended to lower litter moisture (P =

0.07).

44

Table 5. Effect of choline and betaine supplementation on average body weight gain (kg) 1

of

heat-stressed broilers during 1-21, 22-49 and 1-49 days of age

Diet Temp.2

Day 1-21 Day 22-49 Day 1-49

CON TN 0.57 2.19 2.75

HS 0.52 1.80 2.32

CHO500 TN 0.58 2.33 2.91

HS 0.55 1.76 2.31

CHO1000 TN 0.61 2.24 2.85

HS 0.60 1.78 2.38

BET500 TN 0.57 2.27 2.84

HS 0.61 1.77 2.38

BET1000 TN 0.64 2.30 2.94

HS 0.57 1.59 2.15

SEM 0.042 0.091 0.11

Effect P values

Diet 0.46 0.82 0.87

Temp 0.53 0.001 0.001

Diet x Temp 0.65 0.35 0.36

Main Effect Means

Diet

CON 0.54 1.99 2.54

CHO500 0.57 2.04 2.61

CHO1000 0.60 2.01 2.61

BET500 0.59 2.02 2.61

BET1000 0.60 1.94 2.54

SEM 0.029 0.065 0.077

Temp

TN 0.59 2.27a

2.86a

HS 0.57 1.74b

2.31b



a,b Means within same column followed by different letters differ significantly (P < 0.05).

45

Table 6. Effect of choline and betaine supplementation on feed efficiency (Gain:feed) 1 of heat-

stressed broilers during 1-21, 22-49 and 1-49 days of age

Diet Temp.2

Day 1-21 Day 22-49 Day 1-49

CON TN 0.63 0.55 0.56

HS 0.62 0.54 0.56

CHO500 TN 0.64 0.56 0.58

HS 0.66 0.54 0.56

CHO1000 TN 0.66 0.55 0.57

HS 0.66 0.54 0.57

BET500 TN 0.68 0.57 0.59

HS 0.68 0.53 0.57

BET1000 TN 0.69 0.54 0.57

HS 0.65 0.53 0.56

SEM 0.022 0.013 0.01

Effect P values

Diet 0.04 0.70 0.42

Temp 0.82 0.02 0.05

Diet x Temp 0.58 0.78 0.87

Main Effect Means

Diet

CON 0.62b

0.55 0.56

CHO500 0.65ab

0.55 0.57

CHO1000 0.66ab

0.55 0.57

BET500 0.68a

0.55 0.58

BET1000 0.67a

0.53 0.56

SEM 0.015 0.009 0.006

Temp

TN 0.66 0.56a 0.57

HS 0.66 0.54b 0.56




46

Table 7. Effect of choline and betaine supplementation on mortality (%) 1

of heat-stressed

broilers during 1-49 days of age

Diet Temp.2

Mortality

CON TN 7.50

HS 12.5

CHO500 TN 2.50

HS 13.75

CHO1000 TN 1.25

HS 3.75

BET500 TN 2.50

HS 11.25

BET1000 TN 1.25

HS 15.00

SEM 3.64

Effect P values

Diet 0.32

Temp 0.0006

Diet x Temp 0.54

Main Effect Means

Diet

CON 10.00

CHO500 8.13

CHO1000 2.50

BET500 6.88

BET1000 8.13

SEM 2.58

Temp

TN 3.00b

HS 11.25a

SEM 1.63 1 Values are least squares means.



47

Table 8. Effect of choline and betaine supplementation to broilers on litter moisture content1

under thermoneutral and heat stress conditions on day 42

Diet Temp.2

Day 42

CON TN 41.43

HS 26.32

CHO500 TN 41.30

HS 18.16

CHO1000 TN 45.39

HS 25.40

BET500 TN 42.67

HS 18.16

BET1000 TN 42.87

HS 15.72

SEM 2.86

Effect P values

Diet 0.07

Temp 0.0001

Diet x Temp 0.17

Main Effect Means

Diet

CON 33.88

CHO500 29.73

CHO1000 35.40

BET500 31.82

BET1000 29.30

SEM 2.02

Temp

TN 42.73a

HS 21.31b

SEM 1.86 1 Values are least squares means.



48

Foot Pad Dermatitis

Foot pad lesion score is presented in Table 9. There was no diet X temperature interaction

on foot pad lesion score (P > 0.05). Scores did not differ among temperature treatments (P =

0.22) but were affected by diets (P = 0.003). The pairwise comparisons showed that the pattern

of foot pad lesion scores for the CON did not differ from that of the rest of the diets. However,

BET1000 and CHO500 showed similar pattern with the highest score (score 0; 68.55% and

67.27 % respectively).

Breast Meat Color

The mean L*, a* and b* values for breast meat are presented Table 10. There was no diet

or diet X temperature interaction on lightness and yellowness. Lightness (54.51) was

significantly higher (P = 0.02) for HS birds compared to TN (51.73). Conversely, yellowness

was significantly higher (P= 0.004) for TN (3.75) compared to HS (2.60) birds.

Breast Meat Drip Loss

Drip loss assessed on days four and seven post slaughter is presented in Table 11. There

was diet X temperature interaction for day four (P = 0.04). The lowest drip loss occurred for

BET1000 (0.80%) and CHO500 (0.60%) in thermoneutral and heat stress environments

respectively. For seven days post slaughter, drip loss was not significantly affected by diet,

temperature or by their interaction (P > 0.30).

49

Table 9. Effect of choline and betaine supplementation on foot pad score (%) 1 of heat-stressed

broilers on day 42

Foot pad lesion score

Temp.2

0 1 2 3

HSa

58.81 15.80 16.06 9.33

TNa

58.16 20.92 13.27 7.65

Diet2

CON abc

56.29 19.87 14.57 9.27

CHO500 ab

67.27 15.76 9.70 7.27

CHO1000 c 47.30 19.59 23.65 9.46

BET500 bc

51.61 21.94 18.06 8.39

BET1000 b

68.55 15.09 8.18 8.18 1 Values are least squares means.

2 Pairwise comparisons among temperature and dietary treatments respectively,

TN = Thermoneutral

environment, HS = Heat stress environment a Temp followed by different letters differ significantly in foot pad lesion score patterns (P < 0.05).

a,b,c Diet followed by different letters differ significantly in foot pad lesion score patterns P< 0 .05).

50

Table 10. Effect of choline and betaine supplementation on breast meat color (L*, a*, b*) 1

from

heat-stressed broilers

Diet Temp L* a* b*

CON TN 53.09 2.84 4.33

HS 55.41 2.16 2.58

CHO500 TN 51.56 2.66 3.27

HS 53.19 2.73 2.01

CHO1000 TN 50.36 2.97 4.04

HS 54.57 3.06 3.43

BET500 TN 52.60 2.96 4.47

HS 54.05 2.48 3.08

BET1000 TN 51.04 2.88 2.64

HS 53.84 2.86 1.92

SEM 1.18 0.36 0.62

Effect P values

Diet 0.37 0.46 0.06

Temp 0.02 0.64 0.004

Diet x Temp 0.73 0.72 0.88

Main Effect Means

Diet

CON 54.25 2.50 3.45

CHO500 52.38 2.69 2.64

CHO1000 52.46 3.02 3.73

BET500 53.32 2.72 3.78

BET1000 52.46 2.87 2.28

SEM 0.84 0.26 0.43

Temp

TN 51.73b

2.86a

3.75a

HS 54.21a

2.66a

2.60b

SEM 0.67 0.19 0.28 1 Values are least squares means. L* = lightness; a* = redness; b* = yellowness



51

Table 11. Effect of choline and betaine supplementation on drip loss from breast meat (%) 1 of

heat-stressed broilers 4 and 7 days post slaughter

Diet Temp.2

Day 4 Post

Slaughter

Day 7 Post

Slaughter

CON TN 0.98ab

1.80

HS 0.92ab

1.80

CHO500 TN 1.00ab

1.74

HS 0.60b

1.13

CHO1000 TN 1.25ab

2.06

HS 0.90ab

1.89

BET500 TN 1.62a

2.10

HS 0.83b

1.18

BET1000 TN 0.80b

1.61

HS 1.55a

2.13

SEM 0.28

0.37

Effect P values

Diet 0.39 0.62

Temp 0.35 0.32

Diet x Temp 0.04 0.35

Main Effect Means

Diet

CON 0.94 1.80

CHO500 0.80 1.43

CHO1000 1.08 1.97

BET500 1.23 1.64

BET1000 1.18 1.87

SEM 0.17 0.26

Temp

TN 1.13 1.86

HS 0.96 1.63

SEM 0.13 0.16 1 Values are least squares means.



52

Discussion

This study was conducted to evaluate the impact of dietary supplementation with choline

and betaine on heat-stressed broilers. The performance parameters evaluated were weight gain,

feed consumption, feed efficiency and mortality. Numerous studies have reported the effect of

high environmental temperature on the performance of broilers. Heat stress has been shown to

negatively impact broiler chicken performance (Dale and Fuller, 1980; Smith and Teeter, 1987).

In the current study, heat stress decreased feed intake and weight gain during days 22-49. The

reduction in feed consumption could be due to an effort by the bird to reduce heat generated

from feed consumption, digestion and metabolism. Feed efficiency was affected by diet during

the starter phase. BET500 and BET1000 showed higher feed efficiency compared to the CON.

During 22-49 days of age, feed efficiency was decreased only by heat stress. These results are

consistent with those reported by Bartlett and Smith (2003) who found that broilers under cyclic

and chronic heat stress (23.9oC to 37

oC) had lower feed intake compared to thermoneutral birds

(23.9oC).

In this study dietary supplementation of choline and betaine to provide 500 ppm and 1000

ppm methyl equivalents to a diet low in methionine did not improve weight gain, feed intake nor

feed efficiency during the grower and finisher periods. Waldroup, et al. (2006) evaluated choline

and betaine sparing effect of methionine by adding the 500 mg or 1000 mg of choline or betaine

to the diet and reported an improvement in feed intake in birds reared in thermoneutral

environment. Esteve-Garcia and Mack (2000) fed methionine diet supplemented with 0.5 g/kg

betaine and found no positive effect on broiler performance. Similarly Rafeeq, et al. (2011)

found that addition of 700 mg/kg betaine and choline to a methionine deficient diet did not

improve performance. According to Baker, et al. (1983), the beneficial effect of choline

53

regarding its methionine sparing effect is possible only in a diet already containing a level of

choline and deficient in methionine. Although methionine level was deliberately decreased

compared to NRC requirements, choline sparing effect of methionine and its translation into

weight gain was not apparent in this study.

This study showed that mortality rate was increased by heat stress conditions. The

introduction of the temperature treatment resulted in 11.25% mortality compared to 3.00% in

thermoneutral environment. De Basilio, et al. (2001) obtained a higher mortality rate of 38%

with thermal challenged chickens. This could be due to the difference between the temperatures

used in both studies. In the current study the maximum temperature in the heat stress room was

36oC while 38

oC was used by De Basilio, et al. (2001). Supplementing diets with choline and

betaine did not reduced mortality rate. This is consistent with the results reported by Waldroup

and Fritts (2005) who found no effect of choline and betaine on broilers mortality.

Litter moisture was reduced by 50% in the heat stress room. This is not surprising due to

the fact that in the heat stress environment, the temperature cycled between 28 and 36oC. This

allowed the moisture in the litter to evaporate and the bedding to remain drier. Litter wetness has

been positively linked to foot pad dermatitis (Greene, et al., 1985; Mayne, et al., 2007). Hence, it

is very important to maintain litter dryness. Various factors can increase litter wetness and

contribute to the development of foot pad dermatitis (Francesch and Brufau, 2004). These

include infectious diseases, dietary composition and increase water consumption due electrolytes

supplementation (Lichtorowicz, et al., 2012). Water consumption was not measured in this study.

In evaluating the impact of choline and betaine supplementation on litter wetness, results showed

that CHO500 and BET1000 tended to decrease litter moisture compared to the CON. The trend

was similar in food pad scores. Even though CHO500 and BET1000 did not statistically differ

54

from the CON, they showed less incidence of foot pad dermatitis (highest score 0). Chicken feet

health is a welfare issue, and it has been shown that turkey poults with severe necrosis develop

pain induced inappetance resulting in decrease feed intake and weight gain (Martland, 1984).

Additionally, there is an increasing market of chickens’ paws in China and Japan and keeping the

highest 0 score is economically important for poultry producers.

Meat color and drip loss are important quality parameters that could influence

consumers’ selection. The results of this study showed that choline and betaine supplementation

did not affect breast meat color nor was there a diet X temperature interaction. However, heat

stress increased breast meat lightness and decreased yellowness. Similarly, in an experiment

conducted by Akşit, et al. (2006) in which broilers were exposed to cyclic temperature, breast

meat lightness was increased by heat stress. However, heat stress did not affect meat yellowness

in their study. There was a diet X temperature interaction for breast meat drip loss. This was

caused by BET1000 increasing drip loss by 0.75 (0.80 % TN to 1.55 % HS) while CHO500 and

BET500 decreased it by 0.4 (1.00% TN to 0.60 % HS) and 0.79 (1.62 % TN to 0.83 % HS)

respectively. This suggests that the supplementation of BET1000 under thermoneutral

environment and CHO500 under heat stress could reduce water loss during short term storage

(up to four days). The supplementation of BET1000 under heat stress conditions may have

caused betaine to effectively accumulate in cells and help retain water in heat-stressed birds. This

could explain the higher drip loss of breast meat from heat-stressed birds. In order to fully

appreciate the negative impact of heat stress on the meat quality, further research need to be done

including the determination of the meat pH, cook loss and shear force.

55

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Cooper, M. A., and K. W. Washburn. 1998. The relationships of body temperature to weight

gain, feed consumption, and feed utilization in broilers under heat stress. Poult. Sci. 77:237-

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Dale, N. M., and H. L. Fuller. 1980. Effect of Diet Composition on Feed Intake and Growth of

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a dual feeding program for male broilers challenged by heat stress. Poult. Sci. 80:29-36.

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Donkoh, A. 1989. Ambient temperature: a factor affecting performance and physiological

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Esteve-Garcia, E., and S. Mack. 2000. The effect of DL-methionine and betaine on growth

performance and carcass characteristics in broilers. Anim. Feed Sci. Technol. 87:85-93.

Francesch, M., and J. Brufau. 2004. Nutritional factors affecting excreta/litter moisture and

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58

Chapter IV: Effect of dietary choline and betaine on corticosterone levels and immune

organ weights of heat-stressed broilers

59

Abstract


(BET) on broiler corticosterone concentration and immune organ weights under different

temperature conditions, thermoneutral (TN: 23.9oC) and heat stress (HS: 28-36

oC). Eight

hundred day-old mixed sex Cobb500 (female) x Hubbard (M99 male) chicks were weighed,

wing tagged and equally divided into the two environments. Eight replicates of 10 chicks were

randomly assigned to each of five dietary treatments: treatment 1 (CON) the basal diet; treatment

2 (CHO500) basal diet plus 500 ppm methyl equivalents added CHO; treatment 3 (CHO1000)

basal diet plus 1000 ppm methyl equivalents added CHO; treatment 4 (BET500) basal diet plus

500 ppm methyl equivalents added BET and treatment 5 (BET1000) basal diet plus 1000 ppm

methyl equivalents added BET. The corn-soy bean basal diet was formulated to meet NRC

requirements for broilers. The birds were brooded under standard conditions for three weeks

prior to heat stress imposition. Plasma corticosterone was determined before and three weeks

after imposition of heat stress. At the termination of the study, birds were processed and immune

organs (spleen, bursa of Fabricius and thymus) collected and weighed. Data were analyzed using

mixed model analysis of variance (SAS 9.3, Cary, NC). On day 22, there was no effect of diet or

temperature and no diet X temperature interaction on corticosterone levels. Day 45

corticosterone levels (HS, 3.97 ng/ml and TN, 2.16 ng/ml) were affected by temperature (P =

0.002) while there was no diet effect or diet X temperature effect. There was day X temperature

interaction (P = 0.03) for corticosterone levels. This was caused by corticosterone levels

decreasing less by 0.6 (4.57 ng/ml to 3.97 ng/ml) in HS birds while decreasing more by 1.45

(3.61 ng/ml to 2.16 ng/ml) in TN birds from days 22 to 45. There was no diet effect or diet X

temperature interaction on immune organ weights. However, chronic heat stress significantly (P

60

= 0.03) reduced both thymus and bursa weights. Thymus (r = - 0.33, P = 0.002) and bursa (r = -

0.36, P = 0.0008) are negatively and moderately related to corticosterone levels. Betaine and

choline supplementation at 500 ppm and 1000 ppm did not protect broilers from heat stress and

did not affect immune organ weights.

Introduction

Exposure of chickens to high environmental temperature causes them to become heat

stressed. Birds respond to being stressed by decreasing feed intake which results in decreased

weight gain and feed efficiency (Cooper and Washburn, 1998; Sakomura, et al., 2013). Other

physiological changes also occur such as an increase in plasma corticosterone levels (Edens and

Siegel, 1976; Quinteiro-Filho, et al., 2012b) . Corticosterone is the main stress hormone in

chickens. The increase in corticosterone levels is the result of the activation of hypothalamus-

pituitary-adrenal (HPA) axis in avian species (Beuving and Vonder, 1978; Etches, 1976;

Quinteiro-Filho, et al., 2010) and is a response of the body to a stressor (Selye, 1976). The

stressor is detected by the cortex in the brain which activates HPA by sending a signal to the

hypothalamus. Corticotropin releasing hormone (CRH) from the hypothalamus causes the

pituitary gland to release adrenocorticotropic hormone (ACTH) which stimulates the synthesis of

glucocorticoids by the adrenal cortex (Chrousos, 1995). Glucocorticoids release is also

stimulated in response to inflammation. An increase in proinflammatory cytokines during

excessive inflammation also activates the HPA resulting in increased production of

glucocorticoids which reduces the inflammation (Chrousos, 1995).

Corticosterone serves as an indicator of stress in chickens. During acute heat stress,

plasma corticosterone increased after 70 minutes exposure but decreased rapidly to base line in

61

birds heated for 120 min (Edens and Siegel, 1975). Several studies, in which either

corticosterone was fed or ACTH injected to simulate chicken response to high temperature, have

shown that an increase in corticosterone for a short period of time may be beneficial. In a study

conducted by Nasir, et al. (1999), corticosterone was injected to adrenalectomised chickens to

mimic an increase of corticosterone during stress. They found that elevated plasma

corticosterone stimulated higher feed intake, delayed food passage time and increased calcium,

phosphorous and glucose absorption. Similarly Garriga, et al. (2006) found an increase in apical

glucose transport during heat stress. Enhanced gluconeogenesis following short term

administration of corticosterone in chickens was reported by Lin, et al. (2004b) and Shini, et al.

(2008b).

Immune organs (bursa of Fabricius, spleen and thymus) are important components of the

immune system (McArthur, et al., 1971). Thymus and bursa are central lymphoid tissues; thymus

produces small lymphocytes and is responsible for the cell mediated immunity while bursa

produces large lymphocytes and is involved in humoral immunity (Kendall, 1980). Spleen, a

peripheral lymphoid tissue, is considered a final stage of lymphocyte differentiation for the

production of memory cells involved in secondary immune response (John, 1994). According to

(Chrousos, 1995), the production of glucocorticoids at a suboptimal level has detrimental effects

on the immune system. Shini, et al. (2008a) showed that increased levels of corticosterone

decreased spleen and bursa weights. Similarly, Post, et al. (2003) reported a reduction in weight

gain and spleen weight in chickens treated with high corticosterone to simulate heat stress

conditions. Quinteiro-Filho, et al. (2012a) showed that acute heat stress increased plasma

corticosterone but found no negative impact on immune organ (spleen, thymus and bursa)

weights. Few studies have evaluated the effect of chronic heat stress on corticosterone levels in

62

broiler chickens and its effect on lymphoid organs. Quinteiro-Filho, et al. (2010) reported an

increase in corticosterone levels in chronic heat-stressed birds and a decrease in lymphoid organ

weights. Therefore the objective of the present study was to ascertain the effect of chronic heat

stress on chicken plasma corticosterone levels and immune organ weights and to determine if

dietary supplementation of choline and betaine could decrease the negative impacts of heat

stress.


Birds and Housing






HOBO®3



old mixed sex Cobb500 (female) x Hubbard (M99 male) chicks were obtained from Pilgrim’s

Pride hatchery in Cohutta, Georgia. They were vaccinated at the hatchery against Marek’s and





63






oC until it reached



oC to 36




Figure 3. The daily temperature in TN (23.9 ±1°C) and HS (28-36±1°C) rooms.

10

15

20

25

30

35

40

Tem

p. °C

Time

HS

TN

64

Dietary Treatments










starter diet (Table 12) from days 1-21, grower diet from (Table 13) days 22-35 and finisher diet

(Table 14) from days 36-49.

Blood Collection

On day 49, one bird was randomly selected from each pen in each room and blood was

collected from their brachial vein into heparinized tubes and stored on ice. Blood was

centrifuged at 2000 rpm for 10 min, and plasma stored in micro-centrifuge tubes at -80oC for

later corticosterone analysis.

Processing of Birds

Prior to harvesting on day 52, birds were deprived of feed for 12 hours to allow for the




(59.44oC) for 40 second, and their feathers removed using a de-feathering machine. Birds were

65

Table 12. Composition of starter diet (%)


Corn, grain 62.86 62.86 62.86 62.86 62.86

Soybean meal 31.00 31.00 31.00 31.00 31.00

Vitamin premix1 0.0625 0.0625 0.0625 0.0625 0.0625

Filler, sand 0.20 0.105 0.01 0.143 0.0853

Choline, mg/kg2 0.00 0.095 0.19 0.00 0.00

Betaine, mg/kg2 0.00 0.00 0.00 0.057 0.1147

DL methionine 0.15 0.15 0.15 0.15 0.15

Salt 0.35 0.35 0.35 0.35 0.35

Limestone 1.10 1.10 1.10 1.10 1.10

Dical phosphate 2.10 2.10 2.10 2.10 2.10

Trace min. premix3 0.125 0.125 0.125 0.125 0.125

Fat, animal 1.90 1.90 1.90 1.90 1.90

Coban 0.0495 0.0495 0.0495 0.0495 0.0495

Lysine 0.10 0.10 0.10 0.10 0.10

Total 100.00 100.00 100.00 100.00 100.00


ME, kcal/kg 3024.00 3024.00 3024.00 3024.00 3024.00

Crude protein, % 20.59 20.59 20.59 20.59 20.59

Calcium, % 0.98 0.98 0.98 0.98 0.98


Potassium, % 0.81 0.81 0.81 0.81 0.81

Chloride, % 0.25 0.25 0.25 0.25 0.25

Sodium, % 0.16 0.16 0.16 0.16 0.16

Zinc, mg/kg 92.99 92.99 92.99 92.99 92.99

Choline, mg/kg 1236.38 2186.44 3136.44 1236.38 1236.38

Betaine, mg/kg 0.00 0.00 0.00 585.01 1147.03

Lysine, % 1.18 1.18 1.18 1.18 1.18

Methionine, % 0.47 0.47 0.47 0.47 0.47


Threonine, % 0.76 0.76 0.76 0.76 0.76

Fat, % 4.60 4.60 4.60 4.60 4.69








66

Table 13. Composition of grower diet (%)


Corn, grain 62.32 62.32 62.32 62.32 62.32

Soybean meal 31.00 31.00 31.00 31.00 31.00

Vitamin premix1 0.0625 0.0625 0.0625 0.0625 0.0625

Filler, sand 0.30 0.1884 0.0768 0.243 0.1853

Choline, mg/kg2 0.00 0.1116 0.2232 0.00 0.00

Betaine, mg/kg2 0.00 0.00 0.00 0.057 0.1147

DL methionine 0.20 0.20 0.20 0.20 0.20

Salt 0.35 0.35 0.35 0.35 0.35

Limestone 1.10 1.10 1.10 1.10 1.10

Dical phosphate 1.69 1.69 1.69 1.69 1.69

Trace min. premix3 0.125 0.125 0.125 0.125 0.125

Fat, animal 2.70 2.70 2.70 2.70 2.70

Coban 0.0495 0.0495 0.0495 0.0495 0.0495

Lysine 0.10 0.10 0.10 0.10 0.10

Total 100.00 100.00 100.00 100.00 100.00


ME, kcal/kg 3072.00 3074.00 3072.00 3072.00 3072.00

Crude protein, % 19.95 19.95 19.95 19.95 19.95

Calcium, % 0.89 0.89 0.89 0.89 0.89


Potassium, % 0.80 0.80 0.80 0.80 0.80

Chloride, % 0.25 0.25 0.25 0.25 0.25

Sodium, % 0.16 0.16 0.16 0.16 0.16

Zinc, mg/kg 92.48 92.49 92.48 92.48 92.48

Choline, mg/kg 1233.03 2349.37 3465.10 1233.02 1233.03

Betaine, mg/kg 0.00 0.00 0.00 585.01 1147.03

Lysine, % 1.18 1.18 1.18 1.18 1.18

Methionine, % 0.52 0.52 0.52 0.52 0.52


Threonine, % 0.76 0.76 0.76 0.76 0.76

Fat, % 5.38 5.38 5.38 5.38 5.38








67

Table 14. Composition of finisher diet (%)


Corn, grain 66.00 66.00 66.00 66.00 66.00

Soybean meal 27.37 27.37 27.37 27.37 27.37

Vitamin premix1 0.0625 0.0625 0.0625 0.0625 0.0625

Filler, sand 0.30 0.1884 0.0768 0.243 0.1853

Choline, mg/kg2 0.00 0.1116 0.2232 0.00 0.00

Betaine, mg/kg2 0.00 0.00 0.00 0.057 0.1147

DL methionine 0.15 0.15 0.15 0.15 0.15

Salt 0.35 0.35 0.35 0.35 0.35

Limestone 1.10 1.10 1.10 1.10 1.10

Dical phosphate 1.69 1.69 1.69 1.69 1.69

Trace min. premix3 0.125 0.125 0.125 0.125 0.125

Fat, animal 2.70 2.70 2.70 2.70 2.70

Coban 0.0495 0.0495 0.0495 0.0495 0.0495

Lysine 0.10 0.10 0.10 0.10 0.10

Total 100.00 100.00 100.00 100.00 100.00


ME, kcal/kg 3105.00 3107.00 3105.00 3108.00 3105.00

Crude protein, % 18.55 18.55 18.55 18.55 18.55

Calcium, % 0.88 0.88 0.88 0.88 0.88


Potassium, % 0.74 0.74 0.74 0.74 0.74

Chloride, % 0.25 0.25 0.25 0.25 0.25

Sodium, % 0.16 0.16 0.16 0.16 0.16

Zinc, mg/kg 91.15 91.16 91.15 91.16 91.15

Choline, mg/kg 1156.71 2273.05 3388.78 1157.25 1156.71

Betaine, mg/kg 0.00 0.00 0.00 569.98 1147.03

Lysine, % 1.08 1.08 1.08 1.08 1.08

Methionine, % 0.45 0.45 0.45 0.45 0.45


Threonine, % 0.70 0.70 0.70 0.70 0.70

Fat, % 5.48 5.48 5.48 5.49 5.48








68

then eviscerated and the carcasses weighed. Immune organs (bursa of Fabricius, spleen and

thymus) were carefully dissected and weighed. The weight of each organ was calculated as a

percent of total body weight.

Corticosterone Radioimmunoassay

Radioimmunoassay was performed to determine corticosterone concentration in plasma.

Total plasma corticosterone was analyzed in duplicate using ImmuChemTM

Double Antibody

Corticosterone 125

RIA Kit4. The procedures described in the radioimmunoassay kit were

followed. Frozen plasma samples were thawed at room temperature, centrifuged to precipitate

the fat content and then vortexed for 5 seconds to remix the supernatant. Plasma was diluted 1:4

in order to line up with the standard curve for more accurate count. Precipitates were counted

using the Perkin Elmer 1470 Automated Gamma Counter5. The intra and inter coefficient of

variation (CVs) were 15.45 and 18.64% respectively for low (1.93 ng/ml) and 16.85 and 19.62%

respectively for high (10.61 ng/ml) corticosterone standards.


The experimental design was a completely randomized design (CRD) split plot with

temperature in the whole plots (rooms) and diet in the subplots (pens). Data were analyzed using

mixed model analysis of variance procedure SAS 9.3 (SAS Institute, Cary, NC, USA) and least

squares means compared using protected LSD at 5% level of significance. The analysis of the

change in corticosterone levels over time was performed using repeated measures. Correlation

was used to study the relation between corticosterone and lymphoid organ weights.

4 MP Biomedicals, LLC

Diagnostics Division, 13 Mountain View Avenue Orangeburg, NY 10962 5 Perkin Elmer, Wallac Oy, P.O. Box 10

Turku, Finland

69

Results

Bursa of Fabricius, Spleen and Thymus Weights

Bursa of Fabricius, spleen and thymus weights expressed as a percent of body weights

are presented in Table 15. There was no diet X temperature interaction (P > 0.10) for either of

the lymphoid organ weights. Spleen weight was not affected by treatments (P > 0.30). Thymus

weight was not affected by diet (P = 0.34), however it was decreased (P = 0.03) by heat stress

(0.22% HS vs 0.27% TN). Bursa of Fabricius weight was decreased (P = 0.03) by heat stress

(0.16% HS vs 0.13% TN). Diet tended (P = 0.06) to increase bursa weight with birds consuming

BET500 having higher weights (0.18%) compared to the rest of the diets.

Plasma Corticosterone

Data on plasma corticosterone concentrations have been log transformed, back

transformed means and standard errors are reported (Table 16 and Table 17). On day 22, prior to

the imposition of elevated temperature, corticosterone concentrations determined as base line

were statistically similar for both HS and TN birds. On day 45, there was no diet (P = 0.97) or

diet X temperature interaction (P = 0.16) on plasma corticosterone concentration. Corticosterone

level in HS (3.97 ng/ml) birds was significantly (P = 0.002) higher compared to TN (2.26 ng/ml)

birds (Table 16). There was temp X day interaction (P = 0.03) for corticosterone (Table 17). A

Pearson correlation among thymus, bursa, spleen weights and corticosterone day 45, showed a

weak and negative correlation between corticosterone and spleen (r = - 0.03, P = 0.78) whereas

thymus (r = - 0.33, P = 0.002) and bursa (r = -0.36, P = 0.0008) were negatively and moderately

related to corticosterone.

70

Table 15. Effect of choline and betaine supplementation on lymphoid organ weights (% body

weight) 1

of heat-stressed broilers

Diet Temp.2

Bursa Thymus Spleen

Cntrl TN 0.14 0.32 0.07

HS 0.15 0.21 0.09

Cho500 TN 0.16 0.27 0.08

HS 0.12 0.27 0.08

Cho1000 TN 0.14 0.21 0.07

HS 0.12 0.24 0.07

Bet500 TN 0.19 0.29 0.08

HS 0.16 0.21 0.08

Bet1000 TN 0.16 0.26 0.07

HS 0.11 0.16 0.06

SEM 0.02 0.03 0.01

Effect P values

Diet 0.06 0.34 0.31

Temp 0.03 0.03 0.99

Diet x Temp 0.39 0.10 0.39

Main Effect Means

Diet

Cntrl 0.14 0.26 0.08

Cho500 0.14 0.27 0.08

Cho1000 0.13 0.22 0.07

Bet500 0.18 0.25 0.08

Bet1000 0.14 0.21 0.07

SEM 0.01 0.02 0.01

Temp

TN 0.16a

0.27a

0.08

HS 0.13b

0.22b

0.08

SEM 0.01 0.02 0.004 1 Data are least squares means.



71

Table 16. Effect of choline and betaine supplementation on Corticosterone Concentration

(ng/ml) 1

of heat-stressed broilers on days 22 and 45

Diet Temp.2

Day 22 Day 45

Cntrl TN 3.63 2.97

HS 4.34 3.39

Cho500 TN 3.88 2.05

HS 3.10 4.99

Cho1000 TN 2.68 2.02

HS 6.68 4.00

Bet500 TN 4.41 2.19

HS 5.48 4.06

Bet1000 TN 3.69 1.92

HS 4.06 3.91

SEM 0.84 0.62

Effect P values

Diet 0.49 0.97

Temp 0.12 0.002

Diet x Temp 0.07 0.16

Main Effect Means

Diet

Cntrl 3.97 3.04

Cho500 3.47 3.05

Cho1000 4.23 2.85

Bet500 4.91 2.98

Bet1000 3.87 2.74

SEM

Temp

0.58 0.41

TN 3.61 2.16b

HS 4.57 3.97a

SEM 0.41 0.34 1 Data are back transformed least squares means.



72

Table 17. Temp X Day interaction on corticosterone concentration (ng/ml) 1

of heat-stressed

broilers

Temp.2

Days Corticosterone

HS 22 4.57 a

HS 45 3.97 a

TN 22 3.61 a

TN

Pooled SEM

Effect

Temp x Day

45 2.16 b

0.35

P value

0.03 1 Data are least squares means.



Discussion

The results of his study suggest that during chronic HS corticosterone levels increased

and remained elevated compared to thermoneutral environment. This is consistent with the

results of a study by Quinteiro-Filho, et al. (2010). These researchers reported that broilers

subjected to 31 and 36oC for seven days had higher corticosterone levels compared to control

birds raised at 21oC. Similarly, Geraert, et al. (1996) reported that plasma corticosterone

increased and remained elevated in chickens exposed to 32oC for two weeks. Studies have also

shown positive effects related to increased corticosterone levels. An increase in plasma

corticosterone during acute HS enhances gluconeogenesis (Lin, et al., 2004a). Increased plasma

corticosterone following corticosterone feeding to mimic stress conditions also elicited an

increase in glucose absorption from the intestine (Nasir, et al., 1999). Generally, and as seen in

this study, prolonged high temperature or chronic HS causes plasma corticosterone to remain

elevated longer than normal and this could be detrimental to the immune system. In the absence

73

of infection and external stressors other than high temperature, it appears that corticosterone

levels naturally decreases with age, in birds reared under TN environment. In the present study,

birds received a prophylactic dose of an anti-coccidia agent (Coban) in their diet, and no

infection was seen during the entire experiment. Hence, the sustained elevation of corticosterone

was due to HS.

Based on the result of the present study, there is a moderate and negative correlation

between corticosterone and lymphoid organ (thymus and bursa) weights. Heat stress decreased

thymus and bursa of Fabricius weights while spleen weight was unchanged compared to the TN

environment. This could be due to the increase in corticosterone level. These results are

consistent with those reported by Bartlett and Smith (2003) and Quinteiro-Filho, et al. (2010)

who found a decrease in thymus and bursa weights. However, in their studies spleen weight was

decreased by high temperature as well. The bursa of Fabricius and thymus are two gut associated

lymphoid tissues (John, 1994) involved in the adaptive immunity of chickens. Although this

study did not specifically determine and evaluate adverse effects of decreased lymphoid organ

weights on the immune functions, a reduction in their capacity to produce enough immune cells

for the adaptive immunity can be predicted.

The results of the study suggest that chronic HS causes an increase in corticosterone and

reduces lymphoid organ weights, the combination of which may impair the ability of heat-

stressed chickens to mount an appropriate immune response. Dietary supplementation of CHO

and BET did not prevent the increase of plasma corticosterone and the reduction of immune

organ weights in heat-stressed broilers.

74

References

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immunocompetence of broilers under heat stress. Poult. Sci. 82:1580-1588.

Beuving, G., and G. Vonder. 1978. Effect of stressing factors on corticosterone levels in the

plasma of laying hens. Gen. Comp. Endocrinol. 35:153-159.

Chrousos, G. P. 1995. The Hypothalamic–Pituitary–Adrenal Axis and Immune-Mediated

Inflammation. N. Engl. J. Med. 332:1351-1363. doi doi:10.1056/NEJM199505183322008

Cooper, M. A., and K. W. Washburn. 1998. The relationships of body temperature to weight

gain, feed consumption, and feed utilization in broilers under heat stress. Poult. Sci. 77:237-

242. doi 10.1093/ps/77.2.237

Edens, F. W., and H. S. Siegel. 1976. Modification of Corticosterone and Glucose Responses by

Sympatholytic Agents in Young Chickens During Acute Heat Exposure. Poult. Sci. 55:1704-

1712. doi 10.3382/ps.0551704

Edens, F. W., and H. t. Siegel. 1975. Adrenal responses in high and low ACTH response lines of

chickens during acute heat stress. Gen. Comp. Endocrinol. 25:64-73.

Etches, R. 1976. A radioimmunoassay for corticosterone and its application to the measurement

of stress in poultry. Steroids 28:763-773.

Garriga, C., R. R. Hunter, C. Amat, J. M. Planas, M. A. Mitchell, and M. Moretó. 2006. Heat

stress increases apical glucose transport in the chicken jejunum. Am. J. Physiol. Regul. Intgr.

Comp. Physiol. 290:R195-R201.

Geraert, P. A., J. C. F. Padilha, and S. Guillaumin. 1996. Metabolic and endocrine changes

induced by chronic heat exposure in broiler chickens: Biological and endocrinological

variables. Br. J. Nutr. 75:205-216. doi 10.1079/bjn19960125

John, J. L. 1994. The avian spleen: a neglected organ. Q. Rev. Biol.:327-351.

Kendall, M. D. 1980. Avian thymus glands: a review. Dev. Comp. Immunol. 4:191-209.

Lin, H., E. Decuypere, and J. Buyse. 2004a. Oxidative stress induced by corticosterone

administration in broiler chickens (Gallus gallus domesticus): 1. Chronic exposure. Comp.


Lin, H., E. Decuypere, and J. Buyse. 2004b. Oxidative stress induced by corticosterone

administration in broiler chickens (Gallus gallus domesticus): 2. Short-term effect. Comp.


McArthur, W., J. Chapman, and G. Thorbecke. 1971. Immunocompetent cells of the chicken I.

Specific surface antigenic markers on bursa and thymus cells. J. Exp. Med. 134:1036-1045.

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Nasir, A., R. Moudgal, and N. Singh. 1999. Involvement of corticosterone in food intake, food

passage time and in vivo uptake of nutrients in the chicken (Gallus domesticus). Br. Poult.

Sci. 40:517-522.


Post, J., J. Rebel, and A. Ter Huurne. 2003. Physiological effects of elevated plasma

corticosterone concentrations in broiler chickens. An alternative means by which to assess the

physiological effects of stress. Poult. Sci. 82:1313-1318.




Quinteiro-Filho, W., M. Rodrigues, A. Ribeiro, V. Ferraz-de-Paula, M. Pinheiro, L. Sa, A.

Ferreira, and J. Palermo-Neto. 2012a. Acute heat stress impairs performance parameters and

induces mild intestinal enteritis in broiler chickens: Role of acute hypothalamic-pituitary-

adrenal axis activation. J. Anim. Sci. 90:1986-1994.

Quinteiro-Filho, W. M., M. V. Rodrigues, A. Ribeiro, V. Ferraz-de-Paula, M. L. Pinheiro, L. R.

Sa, A. J. Ferreira, and J. Palermo-Neto. 2012b. Acute heat stress impairs performance

parameters and induces mild intestinal enteritis in broiler chickens: role of acute

hypothalamic-pituitary-adrenal axis activation. J. Anim. Sci. 90:1986-1994. doi

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Sakomura, N. K., N. A. A. Barbosa, F. A. Longo, E. P. da Silva, M. A. Bonato, and J. B. K.

Fernandes. 2013. Effect of dietary betaine supplementation on the performance, carcass yield,

and intestinal morphometrics of broilers submitted to heat stress. Braz. J. Poultry Sci. 15:105-

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Selye, H. 1976. Forty years of stress research: principal remaining problems and misconceptions.

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Shini, S., P. Kaiser, A. Shini, and W. L. Bryden. 2008a. Biological response of chickens (Gallus

gallus domesticus) induced by corticosterone and a bacterial endotoxin. Comp. Biochem.

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Shini, S., P. Kaiser, A. Shini, and W. L. Bryden. 2008b. Differential alterations in ultrastructural

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76

Chapter V: Effect of dietary choline and betaine on choline metabolites and intestinal

morphology of heat-stressed broilers

77

Abstract

The objective of this study was to evaluate the effects of supplemental choline (CHO) and

betaine (BET) on production of choline metabolites and on the intestinal morphology of broilers

under different temperature conditions, thermoneutral (TN: 23.9oC) and heat stress (HS: 28-

36oC). Eight hundred day-old mixed sex Cobb500 (female) x Hubbard (M99 male) chicks were

weighed, wing tagged and equally divided and placed into each of the two environments. Eight

replicates of ten chicks were randomly assigned to each of five dietary treatments in each

environment: treatment 1 (CON) the basal diet; treatment 2 (CHO500) basal diet plus 500 ppm

methyl equivalents added CHO; treatment 3 (CHO1000) basal diet plus 1000 ppm methyl

equivalents added CHO; treatment 4 (BET500) basal diet plus 500 ppm methyl equivalents

added BET and treatment 5 (BET1000) basal diet plus 1000 ppm methyl equivalents added BET.

The corn-soy bean basal diet was formulated to meet NRC requirements for broilers. On day 45,

blood was collected from eight birds per treatment while liver and intestinal samples were

collected on day 52. Samples were analyzed for CHO and CHO metabolites. Intestinal histology

was evaluated. Data were analyzed using mixed model analysis of variance (SAS 9.3, Cary, NC).

Results showed that neither diet nor temperature had any effect on villi height, crypt depth or

villi height:crypt depth ratio nor was there any diet X temperature interaction. Intestinal betaine

was affected by diet (P = 0.01) with CHO1000 (6.36 mg/100g) and BET1000 (6.40 mg/100g)

higher than the other treatments. The diet X temperature interaction for intestinal choline tended

(P = 0.06) to be significant while glycerophosphocholine (GPC), total lysophosphatidylcholine

(TLPC), total phosphatidylcholine (TPtCho), phosphocholine (PCho) and sphingomyelin (Sm)

were unaffected. Plasma BET was highest (P = 0.0001) for CHO1000 (233.59 umol/l) and

BET1000 (219.75 umol/l) while plasma CHO and PCho were unaffected.

78

Glycerophosphocholine was highest for CON (18.60 umol/l). However, it was not statistically

different from CHO500 (11.53 umol/l). Plasma TPtCho was increased (P = 0.02) by temperature

in HS birds (11744.00 umol/l) compared to TN birds (5908.08 umol/l). There was a diet X

temperature interaction for plasma TLPC (P = 0.04) resulting from the CON TLPC decreasing

by 49.06 (82.83 TN to 33.77 umol/l HS) while BET1000 TLPC only decreased by 4.55 (47.9 TN

to 43.35 umol/l HS). Liver acetylcholine (Acho), TLPC, TPtCho and Sm contents were not

affected by diet, temperature or their interaction (P > 0.05). There was a diet X temperature

interaction (P = 0.03) for liver betaine content. Under HS, BET1000 (17.90 mg/100g) was

significantly higher than the other diets while in the TN environment, CHO1000 was the highest

(14.49 mg/100g). Liver CHO and GPC contents were affected by temperature which increased

(P = 0.01) CHO by 27.95% (7.14 mg/100g TN vs 9.91 mg/100 HS) and GPC (P = 0.02) levels

by 23.02% (3.88 mg/100g TN vs 5.04 mg/100g HS). In this study, CHO metabolites were

affected by temperature, while intestinal morphology was not.

Introduction

Choline is found in the form of phosphatidylcholine or sphingomyelin in most foods

(Combs Jr, 1991; McDowell, 1989) with the highest concentrations in wheat germ, dried

soybean, egg yolk, pork, and glandular meat such as beef and chicken liver (Zeisel, et al., 2003).

Choline is metabolized into different compounds (betaine, glycerophosphocholine, acetylcholine,

phosphatidylcholine among others) throughout the body. Betaine donates a methyl group to

homocysteine to form methionine (Borsook and Dubnoff, 1947) or serves as an osmolyte;

glycerophosphocholine also serves as an osmolyte (Nakanishi and Burg, 1989); acetylcholine is

involved in neurotransmission (Combs Jr, 1991; McDowell, 1989) whereas phosphatidylcholine

79

is essential for cell membrane integrity (Plagemann, 1968) and transmembrane signaling (Zeisel,

1993). Choline plays important metabolic, physiological and structural functions in the body

therefore meeting its requirement at every step in the development is essential. Additionally, up

to eight weeks of age (broilers are processed at 6-7 weeks) chickens are unable to synthesize

enough endogenous choline to meet their requirements (Cook, et al., 1984; Molitoris and Baker,

1976) making dietary choline very crucial. It is important to identify and reduce factors that may

affect birds’ health and their ability to efficiently absorb and utilize choline.

Several stressors including osmotic stress and high temperature affect gut integrity. These

conditions may decrease feed intake and feed digestibility thus reducing choline absorption and

its distribution into different pathways. In birds, the contents of the small intestine are always

hypertonic to plasma osmolality (320 mOsmol) (Klasing, et al., 2002; Mongin, et al., 1976).

During heat stress, water excretion increases (Belay and Teeter, 1993) causing water imbalance.

Combined, these conditions exert substantial osmotic pressure on intestinal cells resulting in

decreased cell volume and perturbation of cells’ activity including enzymatic processes and

protein synthesis (Petronini, et al., 1992). To restore the osmotic balance, inorganic and organic

osmolytes are used by cells. Inorganic osmolytes are used in initial response to osmotic

imbalance but their excessive accumulation in cell can be detrimental (Petronini, et al., 1992). In

bacteria, at low osmotic pressure, potassium (K+) is preferably used to restore the balance.

However, when the external osmolarity is increased, proportional accumulation of K+ is

deleterious for cell activities and betaine is used in preference to K+

to restore cell turgor

(Sutherland, et al., 1986). Similarly, Petronini, et al. (1992) demonstrated that under

hyperosmotic conditions, unlike organic osmolytes, excessive accumulation of inorganic

osmolytes (Na+, Cl

- and K

+) disrupt enzymatic activities and protein synthesis.

80

Among organic electrolytes such as polyols, sugar, amino acids and methylamines,

betaine has been shown to be the most effective in restoring cell volume, enzyme activity and

protein synthesis (Petronini, et al., 1992). Kettunen, et al. (2001a) suggested that betaine

accumulates in the intestinal cells, and plays an important role in osmoregulation in the small

intestine by controlling water movement across the intestinal epithelium. Several studies have

also proven the importance of betaine on the intestine of infected chickens. Betaine has been

shown to improve water balance and decrease the osmolarity in the duodenum of coccidia

infected chickens (Klasing, et al., 2002), stabilize intestinal mucosal structure and protect against

coccidia infections (Kettunen, et al., 2001b). The osmolytic properties of betaine could be

expected to improve osmotic pressure and protect the intestinal structure in broiler chickens

subjected to heat stress.

Similarly in mammals, glycerophosphocholine, predominantly formed from

phosphatidylcholine in the kidney, protects renal medullary cells from hyper tonicity of the

interstitial fluid (Nakanishi and Burg, 1989). Both, betaine and glycerophosphocholine are

choline metabolites and their relative importance in providing osmoprotectant benefits in the

intestine of chickens in unknown. The impact of heat stress on choline and choline metabolites in

the body have not been investigated. In addition, given the different functions of choline in the

body, evaluating the impact of heat stress on its different metabolites may be fundamental in

understanding the importance of supplemental choline and betaine to improve performance of

heat-stressed broilers. Therefore the objectives of this study were to evaluate the effects of

supplemental choline and betaine on choline metabolites, intestinal morphology and the relative

importance of betaine and glycerophosphocholine osmoprotectant properties on the intestine of

broilers under different temperature conditions, thermoneutral and heat stress.

81


Birds and Housing






HOBO®6



old mixed sex Cobb500 (female) x Hubbard (M99 male) were obtained from Pilgrim’s Pride

hatchery in Cohutta, Georgia. They were vaccinated at the hatchery against Marek’s and








oC until it reached



oC to 36




82



Figure 4. Temperature treatment in TN (23.9 ±1°C) and HS (28-36±1°C) rooms.

10

15

20

25

30

35

40

Tem

p. °C

Time

HS

TN

83

Dietary Treatments










starter diet (Table 18) from days 1-21, grower diet (Table 19) days 22-35 and finisher diet (Table

20) from days 36-49.

Blood Collection

On day 49, one bird was randomly selected from each pen in each room and blood was

collected from their brachial vein into heparinized tubes and stored on ice. Blood was

centrifuged at 2000 rpm for 10 min, and plasma stored in micro-centrifuge tubes at -80oC for

later choline metabolites analysis.

Processing of Birds

Prior to harvesting on day 52, birds were deprived of feed for 12 hours to allow the




(59.44oC) for 40 seconds, and their feathers removed using a de-feathering machine.

84

Table 18. Composition of experimental diet for starter period (%)


Corn, grain 62.86 62.86 62.86 62.86 62.86

Soybean meal 31.00 31.00 31.00 31.00 31.00

Vitamin premix1 0.0625 0.0625 0.0625 0.0625 0.0625

Filler, sand 0.20 0.105 0.01 0.143 0.0853

Choline, mg/kg2 0.00 0.095 0.19 0.00 0.00

Betaine, mg/kg2 0.00 0.00 0.00 0.057 0.1147

DL methionine 0.15 0.15 0.15 0.15 0.15

Salt 0.35 0.35 0.35 0.35 0.35

Limestone 1.10 1.10 1.10 1.10 1.10

Dical phosphate 2.10 2.10 2.10 2.10 2.10

Trace min. premix3 0.125 0.125 0.125 0.125 0.125

Fat, animal 1.90 1.90 1.90 1.90 1.90

Coban 0.0495 0.0495 0.0495 0.0495 0.0495

Lysine 0.10 0.10 0.10 0.10 0.10

Total 100.00 100.00 100.00 100.00 100.00


ME, kcal/kg 3024.00 3024.00 3024.00 3024.00 3024.00

Crude protein, % 20.59 20.59 20.59 20.59 20.59

Calcium, % 0.98 0.98 0.98 0.98 0.98


Potassium, % 0.81 0.81 0.81 0.81 0.81

Chloride, % 0.25 0.25 0.25 0.25 0.25

Sodium, % 0.16 0.16 0.16 0.16 0.16

Zinc, mg/kg 92.99 92.99 92.99 92.99 92.99

Choline, mg/kg 1236.38 2186.44 3136.44 1236.38 1236.38

Betaine, mg/kg 0.00 0.00 0.00 585.01 1147.03

Lysine, % 1.18 1.18 1.18 1.18 1.18

Methionine, % 0.47 0.47 0.47 0.47 0.47


Threonine, % 0.76 0.76 0.76 0.76 0.76

Fat, % 4.60 4.60 4.60 4.60 4.69








85

Table 19. Composition of experimental diet for grower period (%)


Corn, grain 62.32 62.32 62.32 62.32 62.32

Soybean meal 31.00 31.00 31.00 31.00 31.00

Vitamin premix1 0.0625 0.0625 0.0625 0.0625 0.0625

Filler, sand 0.30 0.1884 0.0768 0.243 0.1853

Choline, mg/kg2 0.00 0.1116 0.2232 0.00 0.00

Betaine, mg/kg2 0.00 0.00 0.00 0.057 0.1147

DL methionine 0.20 0.20 0.20 0.20 0.20

Salt 0.35 0.35 0.35 0.35 0.35

Limestone 1.10 1.10 1.10 1.10 1.10

Dical phosphate 1.69 1.69 1.69 1.69 1.69

Trace min. premix3 0.125 0.125 0.125 0.125 0.125

Fat, animal 2.70 2.70 2.70 2.70 2.70

Coban 0.0495 0.0495 0.0495 0.0495 0.0495

Lysine 0.10 0.10 0.10 0.10 0.10

Total 100.00 100.00 100.00 100.00 100.00


ME, kcal/kg 3072.00 3074.00 3072.00 3072.00 3072.00

Crude protein, % 19.95 19.95 19.95 19.95 19.95

Calcium, % 0.89 0.89 0.89 0.89 0.89


Potassium, % 0.80 0.80 0.80 0.80 0.80

Chloride, % 0.25 0.25 0.25 0.25 0.25

Sodium, % 0.16 0.16 0.16 0.16 0.16

Zinc, mg/kg 92.48 92.49 92.48 92.48 92.48

Choline, mg/kg 1233.03 2349.37 3465.10 1233.02 1233.03

Betaine, mg/kg 0.00 0.00 0.00 585.01 1147.03

Lysine, % 1.18 1.18 1.18 1.18 1.18

Methionine, % 0.52 0.52 0.52 0.52 0.52


Threonine, % 0.76 0.76 0.76 0.76 0.76

Fat, % 5.38 5.38 5.38 5.38 5.38








86

Table 20. Composition of experimental diet for finisher period (%)


Corn, grain 66.00 66.00 66.00 66.00 66.00

Soybean meal 27.37 27.37 27.37 27.37 27.37

Vitamin premix1 0.0625 0.0625 0.0625 0.0625 0.0625

Filler, sand 0.30 0.1884 0.0768 0.243 0.1853

Choline, mg/kg2 0.00 0.1116 0.2232 0.00 0.00

Betaine, mg/kg2 0.00 0.00 0.00 0.057 0.1147

DL methionine 0.15 0.15 0.15 0.15 0.15

Salt 0.35 0.35 0.35 0.35 0.35

Limestone 1.10 1.10 1.10 1.10 1.10

Dical phosphate 1.69 1.69 1.69 1.69 1.69

Trace min. premix3 0.125 0.125 0.125 0.125 0.125

Fat, animal 2.70 2.70 2.70 2.70 2.70

Coban 0.0495 0.0495 0.0495 0.0495 0.0495

Lysine 0.10 0.10 0.10 0.10 0.10

Total 100.00 100.00 100.00 100.00 100.00


ME, kcal/kg 3105.00 3107.00 3105.00 3108.00 3105.00

Crude protein, % 18.55 18.55 18.55 18.55 18.55

Calcium, % 0.88 0.88 0.88 0.88 0.88


Potassium, % 0.74 0.74 0.74 0.74 0.74

Chloride, % 0.25 0.25 0.25 0.25 0.25

Sodium, % 0.16 0.16 0.16 0.16 0.16

Zinc, mg/kg 91.15 91.16 91.15 91.16 91.15

Choline, mg/kg 1156.71 2273.05 3388.78 1157.25 1156.71

Betaine, mg/kg 0.00 0.00 0.00 569.98 1147.03

Lysine, % 1.08 1.08 1.08 1.08 1.08

Methionine, % 0.45 0.45 0.45 0.45 0.45


Threonine, % 0.70 0.70 0.70 0.70 0.70

Fat, % 5.48 5.48 5.48 5.49 5.48








87

Birds were then eviscerated and the carcasses weighed. Liver samples from both lobes were

collected, snap frozen with liquid nitrogen in cryogenic vials and stored at -80oC. The small

intestine was carefully separated, and two samples were collected: a 5 cm section was cut above

the Meckel’s diverticulum, cleaned in 10% saline buffer solution and snap frozen with liquid

nitrogen in cryogenic vials and stored at -80oC for later choline metabolites analysis, and another

5 cm section from the ileum was rinsed with 10% saline buffer and stored in formaldehyde at

room temperature for later histology.

Intestinal Histology

Histological sections from the intestinal samples were embedded in paraffin and stained

with hematoxylin and eosin. The tissue slides were observed under microscope EVOS® imaging

systems and photographs of three villi per slide and per bird showing complete villi and crypts

were taken (10x0.25 magnification). Villus length from the tip to the base of the villus and crypt

depth from the base of villus to the thin muscularis mucosae were measured in millimeter using

Imagej software.

Choline Metabolites Analysis by HILC-MS/MS

Plasma, liver and intestinal samples were extracted for choline and choline metabolites

analysis. The samples were kept on ice during the extraction process. First, 1 ml of extraction

solvent made of chloroform, methanol and water in a 1:2:0.8 ratio, was added to100 mg of

ground intestinal and liver tissues, 200 ul of plasma and 40 ul of standard solution. Samples were

centrifuged (16000rpm, 28620xg) at 4oC for 5min. The resulting supernatant was transferred into

separate glass vials. The extraction process was repeated twice for each sample and the

supernatant transferred into the same vial. The supernatant for each sample was dried under

constant nitrogen steam and the solid extract re-dissolved in 5 ml of methanol. The final

88

solutions were analyzed for choline metabolites using the HILIC LC-MS/MS method adapted by

(Artegoitia, et al., 2014).


The experimental design was a completely randomized design split plot with temperature

in the whole plots (rooms) and diet in the subplots (pens). Data were analyzed using mixed

model analysis of variance procedure SAS 9.3 (Cary, NC, USA) and least squares means

compared using protected LSD at 5% level of significance.

Results

Intestinal Histology

Intestinal villi height, crypts depth and villi height:crypt depth ratio are presented in

Table 21. There was no diet X temperature interaction for villi height, crypt depth and villi

height:crypt depth ratio (P > 0.2). Neither diet nor temperature affected crypt and villi:crypt

ratio, however, HS tended (P = 0.07) to decrease villi length (1.39 mm TN vs 1.26 mm HS).

Intestinal Choline and Choline Metabolites

Choline and CHO metabolites concentrations in the intestine are reported in Table 22.

Intestinal GPC, TLPC, TPtCho, PCho and Sm were not affected (P > 0.15) by temperature, diet

and diet X temperature interaction. There was no diet or diet X temperature interaction effect (P

> 0.1) on Acho however, temperature tended (P = 0.07) to increase it (0.83 in HS vs 0.62

mg/100g in TN).

There was no diet X temperature interaction for intestinal BET (P = 0.57), nor was there a

temperature effect (P = 0.34). However, intestinal BET content was affected by diet (P = 0.02).

89

Table 21. Effect of choline and betaine supplementation on intestinal villi height, crypts depth

(mm) 1

and villi height:crypt depth of heat-stressed broilers

Diet Temp.2 Villi Crypt Villi:Crypt

CON TN 1.55 0.16 10.63

HS 1.28 0.13 9.97

CHO500 TN 1.34 0.14 10.34

HS 1.21 0.14 9.22

CHO1000 TN 1.34 0.14 9.74

HS 1.39 0.15 10.07

BET500 TN 1.35 0.16 8.88

HS 1.23 0.13 10.64

BET1000 TN 1.35 0.16 9.17

HS 1.21 0.14 9.35

Pooled SEM 0.30 0.02 0.81

Effect P values

Diet 0.25 0.96 0.85

Temp 0.07 0.10 0.87

Diet x Temp 0.22 0.58 0.56

Main Effect Means

Diet

CON 1.41 0.15 10.30

CHO500 1.28 0.14 9.78

CHO1000 1.36 0.15 9.91

BET500 1.29 0.14 9.76

BET1000 1.28 0.15 9.26

Pooled SEM 0.06 0.01 0.63

Temp

TN 1.39 0.15 0.38

HS 1.26 0.14 0.42

Pooled SEM 0.04 0.01 0.40 1

Data are least squares means. 2TN = Thermoneutral environment, HS = Heat stress environment.

90

Table 22. Effect of choline and betaine supplementation on intestinal choline and choline

metabolites (mg/100g) 1


Metabolites2

Diet Temp3

BET CHO GPC Acho4

TLPC4

TPtCho4

PCho Sm

CON TN 3.89 11.23 3.42 0.76 12.40 18473.75 24.94 144.47

HS 4.96 11.47 4.83 1.06 15.49 21863.73 25.00 161.79

CHO500 TN 5.24 9.51 4.27 0.52 13.52 12177.63 25.45 177.74

HS 4.27 9.94 4.06 0.85 14.37 14887.65 27.73 169.13

CHO1000 TN 7.02 9.16 3.97 0.49 9.56 12829.29 24.71 168.43

HS 5.71 10.38 4.03 0.69 9.72 12900.75 25.29 157.29

BET500 TN 5.02 12.19 4.23 0.51 10.44 13301.08 26.86 163.73

HS 4.59 9.30 4.27 0.79 8.43 10447.52 26.32 177.41

BET1000 TN 6.73 14.43 3.85 0.90 13.50 15600.19 27.38 179.34

HS 6.08 13.25 4.09 0.80 15.79 23656.70 28.42 176.55

Pooled SEM 0.75 1.09 0.55 0.15 3.24 4547.97 1.38 16.01

Effect P values

Diet 0.02 0.10 0.98 0.14 0.24 0.18 0.17 0.53

Temp 0.34 0.71 0.38 0.07 0.78 0.63 0.48 0.88

Diet x Temp 0.57 0.06 0.61 0.58 0.93 0.79 0.87 0.84

Main Effect Means

Diet

CON 4.43b

11.35 4.12 0.90 13.86 20097.39 24.97 153.13

CHO500 4.75b

9.72 4.17 0.66 13.94 13464.63 26.60 173.43

CHO1000 6.36a

9.77 4.00 0.58 9.64 12864.97 25.00 162.86

BET500 4.81b

10.74 4.25 0.64 9.38 11788.27 26.59 170.57

BET1000 6.40a

11.84 3.97 0.85 14.60 19210.65 27.90 177.95

Pooled SEM 0.53 0.77 0.39 0.11 2.28 3186.17 0.98 11.32

Temp

TN 5.58 10.50 3.95 0.62 11.77 14304.33 25.87 166.74

HS 5.12 10.87 4.26 0.83 12.36 15967.12 26.55 168.43

Pooled SEM 0.34 0.69 0.25 0.08 1.48 2422.05 0.66 7.98 1 Data are least squares means.

2 Metabolites: betaine (BET), free choline (CHO), glycerophosphocholine (GPC), acetylcholine (Acho),

total lysophosphatidylcholine (TLPC), total phosphatidylcholine (TPtCho), phosphocholine (PCho),

sphingomyelin (Sm). 3 TN = Thermoneutral environment, HS = Heat stress environment.


4 Data were log transformed and back transformed means reported.

91

Both BET1000 and CHO1000 are statistically similar (6.36 and 6.40 mg/100g) but higher than

the CON (4.43 mg/100g), CHO500 (4.75 mg/100g) and BET500 (4.81 mg/100g).

Neither diet nor temperature affected intestinal CHO content (P > 0.10). However, their

interaction tended to have a significant (P = 0.06) impact. This was caused by BET1000

decreasing intestinal CHO content by 1.18 (14.43 mg/100g TN to 13.25 mg/100g HS) whereas

CHO500 increased it by 0.43 mg/100g (9.51 mg/100g TN to 9.94 mg/100g HS).

Liver Choline and Choline Metabolites

Liver choline and choline metabolites concentration are reported in Table 23. There was

no temperature, diet or diet X temperature interaction on liver Acho, TLPC, TPtCho and Sm

content (P > 0.05). However, there was diet X temperature interaction for betaine (P = 0.03).

This was caused by BET1000 increasing liver betaine content by 6.17 (11.74 mg/100g TN to

17.91 mg/100g HS) whereas both CHO1000 and CON decreased it by 1.27 (14.49 mg/100g to

13.22 mg/100g) and 0.68 (5.98 mg/100g TN to 5.30 mg/100g HS) respectively. Similarly, PCho

was affected by the interaction between diet and temperature (P = 0.04). This was due to CON

and BET1000 increasing liver PCho content by 8.39 (17.57 mg/100g TN to 25.96 mg/100g HS)

and 7.47 (15.69 mg/100g TN to 23.16 mg/100g HS) respectively whereas CHO500 and

CHO1000 decreased liver PCho by 0.58 (20.08 mg/100g TN to 19.50 mg/100g HS) and 1.32

(21.41 mg/100g to 20.09 mg/100g HS) respectively.

Liver CHO and GPC were not affected by diet and there was no diet X temperature

interaction (P > 0.05). However, the CHO content was significantly higher (P = 0.01) in HS birds

(9.91 mg/100) compared to TN birds (7.14 mg/100g). Similarly, GPC content for HS birds (5.04

mg/100g) was significantly higher (P = 0.02) than that of TN birds (3.88 mg/100g).

92

Table 23. Effect of choline and betaine supplementation on liver choline and choline metabolites

(mg/100g) 1


Metabolites2

Diet Temp3

BET CHO GPC Acho TLPC TPtCho PCho Sm4

CON TN 5.98de

7.99 4.78 0.70 8.17 18475.00 17.57bcd

140.88

HS 5.30e

8.81 5.94 0.90 8.96 26361.00 25.96a

143.46

CHO500 TN 6.72de

6.31 3.00 0.58 7.20 13523.00 20.08bcd

126.69

HS 8.07de

9.49 4.89 0.92 8.51 21738.00 19.50bcd

140.34

CHO1000 TN 14.49ab

6.58 3.72 0.60 9.00 17322.00 21.41abc

130.61

HS 13.22b

9.80 4.59 0.78 7.94 17865.00 20.09bcd

145.95

BET500 TN 8.95cd

7.78 3.12 0.59 8.21 12877.00 16.37cd

140.71

HS 8.20cd

8.95 5.42 0.77 8.36 18387.00 19.37bcd

142.78

BET1000 TN 11.74bc

7.06 5.17 0.61 7.34 15684.00 15.69d

137.30

HS 17.91a

2.49 4.37 0.97 8.85 18713.00 23.16ab

441.19

Pooled SEM 1.22 1.17 0.75 0.15 0.58 4679.46 2.03 93.52

Effect P values

Diet 0.001 0.46 0.28 0.76 0.74 0.66 0.35 0.86

Temp 0.16 0.01 0.02 0.12 0.14 0.09 0.04 0.34

Diet x Temp 0.03 0.21 0.30 0.89 0.18 0.92 0.04 0.93

Main Effect Means

Diet

CON 5.64 8.40 5.36 0.80 8.57 22418.00 21.76 142.17

CHO500 7.39 7.90 3.95 0.75 7.85 17631.00 19.79 133.51

CHO1000 13.85 8.19 3.95 0.69 8.47 17593.00 20.75 138.28

BET500 9.07 8.37 4.27 0.68 8.29 15632.00 17.88 141.74

BET1000 14.82 9.77 4.67 0.79 8.09 17199.00 19.43 289.24

Pooled SEM 0.86 0.82 0.53 0.11 0.41 5515.43 1.43 66.13

Temp

TN 9.58 7.14b

3.88b

0.61 7.98 15576.00 18.23 135.24

HS 10.74 9.91a

5.04a

0.87 8.52 20613.00 21.62 202.74

Pooled SEM 0.56 0.67 0.34 0.11 0.26 2093.11 1.06 42.28 1 Data are least squares means.

2 Metabolites: betaine (BET), free choline (CHO), glycerophosphocholine (GPC), acetylcholine (Acho),

total lysophosphatidylcholine (TLPC), total phosphatidylcholine (TPtCho), phosphocholine (PCho),


a,b,c,d Means within same column followed by different letters differ significantly (P < 0.05).

4 Data were rank transformed and untransformed means reported.

93

Plasma Choline and Choline Metabolites

Plasma choline and choline metabolites concentration are presented in Table 24. There

was no diet or temperature effect or their interaction on PCho and Cho (P > 0.05). There was no

diet X temperature interaction (P = 0.10) on plasma GPC. However, both diet and temperature

significantly (P < 0.05) affected plasma GPC. Chickens consuming the CON diet (18.60 umol/l)

and BET500 (11.53 umol/l) had significantly higher plasma GPC concentration compared to the

rest of the diets (7.89 to 10.20 umol/l). Heat-stressed birds had significantly lower plasma GPC

(8.58 umol/l) compared to TN birds (13.87 umol/l).

Plasma TPtCho was not affected by diet, nor was there a diet X temperature interaction

(P > 0.05) whereas temperature significantly (P = 0.02) decreased TPtCho by 5835.82 umol/l

(11744.00 TN vs 5908.08 umol/l HS). There was no diet or diet X temperature interaction for

plasma Sm (P > 0.05) but temperature tended (P = 0.05) to increase it (339.94 TN vs 369.29

umol/l HS).

Betaine was not affected by temperature, nor was there a diet X temperature interaction

(P > 0.05). However, betaine level was significantly (P = 0.0001) decreased by diet. Birds

consuming BET1000 (219.75 umol/l) and CHO1000 (233.59 umol/l) had significantly higher

betaine concentration compared to the rest of the diets (99.29 to 166.67 umol/l). There was a diet

X temperature interaction for plasma TLPC (P = 0.04). This was caused by the CON decreasing

plasma TLPC by 49.06 (82.83 TN to 33.77 umol/l HS) while BET1000 decreased only by 4.55

(47.9 TN to 43.35 umol/l HS).

94

Table 24. Effect of choline and betaine supplementation on plasma choline and choline

metabolites (umol/l) 1


Metabolites2

Diet Temp3

BET CHO GPC TLPC TPtCho PCho Sm

CON TN 98.31 15.48 21.23 82.83a

15873.00 2.99 315.98

HS 100.27 14.16 15.97 33.77c

7404.34 2.90 375.07

CHO500 TN 161.98 15.10 10.19 66.09ab

9552.04 2.64 304.64

HS 160.05 15.49 5.59 36.09c

6404.87 2.37 354.08

CHO1000 TN 245.71 17.99 12.16 47.15bc

12329.00 3.20 337.32

HS 221.47 14.94 8.25 39.20c

5420.88 2.31 384.88

BET500 TN 187.28 13.94 14.59 47.75bc

8727.52 2.93 361.63

HS 146.06 16.30 8.48 32.43c

5761.40 3.57 380.85

BET1000 TN 213.08 16.18 11.16 47.90bc

12239.00 2.24 380.14

HS 226.43 13.50 4.62 43.35bc

6548.90 3.02 351.11

Pooled SEM 14.75 1.65 3.89 8.39 2318.04 0.51 23.49

Effect P values

Diet 0.0001 0.79 0.045 0.174 0.07 0.59 0.39

Temp 0.35 0.56 0.04 0.005 0.02 0.93 0.05

Diet x Temp 0.32 0.32 0.10 0.04 0.67 0.40 0.34

Main Effect Means

Diet

CON 99.29c

14.82 18.60a

58.30 11638.00 2.94 345.53

CHO500 161.02b

15.29 7.89b

51.09 7978.45 2.51 329.36

CHO1000 233.59a

16.46 10.20b

43.18 8874.92 2.76 361.10

BET500 166.67b

15.12 11.53ab

40.09 6244.46 3.25 371.24

BET1000 219.75a

14.84 7.89b

45.62 9393.94 2.63 365.63

Pooled SEM 10.43 1.17 2.74 5.94 1319.79 0.37 16.61

Temp

TN 181.27 15.74 13.87a

58.34 11744.00a

2.80 339.94

HS 170.86 14.88 8.58b

36.97 5908.08b

2.83 369.20

SEM 7.67 1.01 1.74 4.53 1585.91 0.28 10.51 1 Data are least squares means.

2 Metabolites: Betaine (BET), free choline (CHO), glycerophosphocholine (GPC), total

lysophosphatidylcholine (TLPC), total phosphatidylcholine (TPtCho),phosphocholine (PCho),


a,b,c Means within same column followed by different letters differ significantly (P < 0.05).

95

Discussion

The villi height, crypt depth and villi:crypt ratio have been used in numerous studies to

assess the overall digestive and absorptive capacity of the small intestine. An increase in

villi:crypt ratio was shown to be positively correlated to the intestinal digestive and absorptive

capacity in pigs (McDonald, et al., 2001), in dogs (Kuzmuk, et al., 2005) and in chickens

(Burkholder, et al., 2008). The results of the present study showed that villus height, crypt depth

and villi height:crypt depth were unchanged in response to diet. However, there was a trend for

high temperature to decrease villi height in the ileum. This could decrease the digestive and

absorptive capacity of heat-stressed broilers. These results are consistent with the findings of

Quinteiro-Filho, et al. (2012) who reported no changes in villus height, crypt depth and villus

height:crypt depth ratio. However the results are not consistent with those reported by Mitchell

and Carlisle (1992) who found that villi height decreased by 19% in birds subjected to 35oC for

14 days. Acute heat stress has also been shown to decrease crypt depth as reported by

Burkholder, et al. (2008), in birds subjected to 30oC for 24h. These differences are probably due

to the fact that in the current experiment, birds were exposed to cycling temperature for longer

period of time (28 vs 14 days and 24h respectively) and might have adapted to the stress

condition.

In the present study, heat stress had no impact on most choline metabolites in the liver

except that it increased choline and GPC. Heat stress has been shown to increase de novo

lipogenesis which occurs primarily in the liver of chickens and to enhance fat deposition

(Geraert, et al., 1996; He, et al., 2015). Choline is involved in the synthesis of TPtCho needed for

the formation of very low density lipoprotein (VLDL), which is necessary for triglycerides

export from the liver (Kouba, et al., 1995). With an increase in lipogenesis during heat stress, the

96

increase in choline level seen in the present study may be facilitating fat removal from the liver.

Similarly, an increase in GPC in the liver could be explained by its osmolytic property.

According to Kwon, et al. (1995), GPC is an osmolyte mainly known to protect renal cells

against hypertonicity. Therefore its increase in the liver cells could be seen as a mechanism to

restore or maintain osmotic balance. Unlike GPC, liver betaine was not affected by temperature

which suggests that GPC was preferably used in heat stress conditions to maintain osmotic

balance in the liver. Conversely, in previous studies, betaine has been shown to protect

hepatocytes kupffer cells from hyper osmosis in rat (Zhang, et al., 1996). However, there was a

diet X temperature interaction for liver betaine content with BET1000 increasing it by 34.45 %

under heat stress environment compared to thermoneutral. This is consistent with the results

reported by Saarinen, et al. (2001) who found that dietary betaine was efficient in increasing

hepatic betaine concentration. However, in their study birds were not exposed to stress

conditions. Similarly, Kettunen, et al. (2001b) reported that betaine accumulates in liver when

the diet is supplemented with higher levels of betaine. Therefore dietary supplementation of

betaine at 1000 ppm can be used to increase liver betaine content in broilers under heat stress

conditions.

Choline metabolites GPC, TPtCho, PCho, TLPC and Sm in the intestinal tissue were not

affected by diet, temperature or by their interaction in the current study. Similarly, there was no

temperature or diet X temperature interaction on intestinal betaine. However, it was affected by

diet. Intestinal betaine was increased by dietary supplementation of betaine and choline at 1000

ppm. This is consistent with the results reported by Kettunen, et al. (2001a) who found that

betaine accumulates in the small intestine and helps retain water in hyperosmotic saline solution.

In the current study, betaine did not accumulate in the intestinal tissue of heat-stressed broilers.

97

This could be explained by the fact that heat stress condition did not severely increase osmotic

pressure on intestinal cells. Previous studies on heat stress impact on broilers did not evaluate

intestinal betaine content. However, when broilers were challenged with coccidia, (Kettunen, et

al., 2001b) reported an accumulation of betaine in the intestinal tissues. This difference may be

due to the fact that unlike heat stress, coccidiosis increases water loss through diarrhea resulting

in a more severe increase in osmotic pressure. Because heat stress did not affect betaine and GPC

in the present study it was not possible to determine their relative importance in providing

osmotic benefits on the intestine of heat-stressed broilers. Even though there was not a

significant diet X temperature interaction on intestinal choline content, the interaction showed an

increasing trend. All the diets except BET500 and BET1000 increased intestinal choline content

in heat-stressed chickens.

Plasma choline and PCho were not affected by diet and temperature or by their

interaction while plasma betaine content was increased only by diet. During heat stress there was

a large decrease in plasma GPC content compared to thermoneutral environment. This could be

explained by the fact that GPC was retained in the liver to serve as an osmolyte as shown by the

higher amount of GPC in the liver. Plasma TLPC was decreased in broilers reared under HS

conditions compared to those reared under TN conditions. The largest difference was seen

between TN and HS for the CON compared to the difference between TN and HS for CHO1000

or BET1000. This could suggest that during heat stress, supplementation with CHO1000,

BET1000 could limit the decrease in plasma TLPC. This is important for the body to maintain its

ability to prevent hemorrhaging since TLPC is one to the major components in blood clotting

(Xu, et al., 2013) and could thus have an impact on carcass quality. Plasma TPtCho was not

affected by diet and there was no diet X temperature interaction but was decreased by heat stress.

98

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101

Chapter VI: Summary and Conclusion

102

The results of this study indicate that heat stress reduced feed intake and body weight

gain while increasing mortality, corticosterone levels and reducing thymus and bursa of

Fabricius weights. The supplementation of choline and betaine to heat-stressed broilers did not

improve growth performance or suppress the negative impact of high environmental temperature

on immune organ weights and corticosterone levels. Heat stress increased lightness and reduced

yellowness of breast meat color. Intestinal morphology showed no impact of heat stress on crypt

depth and villi height:crypt depth ratio. However heat stress tended to increase villi height.

Dietary supplementation of choline and betaine to heat-stressed broilers did not affect intestinal

villi height and crypt depth. Most choline metabolites were unaffected by diet or temperature or

by their interaction in the intestine, liver and plasma. However, supplemental choline and betaine

increased intestinal betaine contents. There was no effect of high temperature or diet by

temperature interaction for intestinal betaine and glycerophosphocholine contents however liver

choline and glycerophosphocholine were increased by heat stress. Adding choline and betaine to

provide 500 and 1000 ppm methyl equivalents respectively could increase increased liver betaine

contents in heat-stressed broilers. Plasma glycerophosphocholine was decreased by temperature

and supplementing with choline or betaine did not increase its content compared to the CON.

Meat color and drip loss are important qualities that might influence consumers’ choices.

However, further studies are needed including determination of the meat pH, cook loss and shear

force to fully appreciate the effect of heat stress and the supplementation of choline and betaine

on the meat quality. In this study, supplementation of choline and betaine did not improve

growth performance of heat-stressed broilers, however they improved foot heath and decreased

meat drip loss four days post slaughter.

103

Vita

Kouassi R. Kpodo was born in Kissidougou (Guinea). He obtained a degree in Agronomy/option

animal production from the College of Agronomy in Togo. After graduation, he worked for non-

governmental agencies where he managed and implemented developmental programs that

focused on the integration of animal husbandry and leguminous plants to improve soil fertility in

rural areas. Kouassi emigrated in the USA in 2008 and decided to further his education in animal

science. In 2012, he received a bachelor degree in animal science from the University of

Tennessee, and was offered the opportunity to pursue a Master’s degree with Dr. Michael Smith

as mentor.

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