Incidence of exertional rhabdomyolysis in emus (Dromaius novaehollandiae) consequent to

transport for six hours.

D. G. Menon, D. C. Bennett, Schaefer, A.L, and K. M. Cheng*

Avian Research Centre, Faculty of Land and Food Systems, University of British Columbia,

2357 Main Mall, University of British Columbia, Vancouver, B.C., Canada V6T 1Z4.

*Corresponding author
Abstract

Many emu farms are located in areas lacking slaughter and processing facilities that can handle

these birds. Transport over distances invariably result in injuries, mortality and malaise in emus.

The attempt in this study is to examine the changes in hematology, serum biochemistry, enzymes

and corticosterone levels in emus to evaluate the systemic changes consequent to transport. Two

trials were conducted wherein emus were transported in a modified horse trailer for about 6

hours. The core body temperature of emus significantly increased (P<0.001) after transport. Both

the blood glucose, creatinine levels were increased significantly (P<0.001) at slaughter, while

albumin levels and UA: creatinine ratios were decreased irrespective of the sexes. There was

significantly higher (P<001) ALT, AST and CK levels after transport. The correlations between

baseline ALT and AST (r=0.57); after transport ALT and AST (r=0.90), ALT and CK (r=0.66),

AST and CK (r=0.57) values were highly significant. The blood calcium levels were

significantly (P=0.020) reduced so also there was increase in serum potassium and sodium

levels. The clinical picture was suggestive of the incidence of exertional rhabdomyolysis and

thus underlines the need to exercise extreme caution while handling and transporting emus.

Key words

Transport, stress, emu, biochemistry, rhabdomyolysis

Introduction

Emus are ratites native to Australia and reared for their fat and meat. Many emu farms are

located in areas lacking slaughter and processing facilities that can handle these birds. This

necessitates the transportation of the birds, often over long distances, to such facilities resulting

in many stressful and adverse conditions that not only raise welfare concerns, but can also cause

injury and mortality. Many of the critical points in the process have been examined in traditional

poultry species, but have received little attention in emus. Emus differ from other poultry in their

behavioural and physiological requirements and hence strategies to minimize welfare problems

specific for the species have to be examined.

Though all stress responses associated with transport may not be detrimental, continued

stress could produce considerable economic losses (Villa et al., 2008). It has been widely

observed that though we can assess the stress related symptoms in animals and birds, the point at

which it threatens life cannot be easily determined (Maria et al., 2004). The attempt in this study

is to examine the changes in hematology, serum biochemistry, enzymes, weight losses, injuries

and corticosterone levels in emus consequent to transport so as to evaluate the systemic changes

and then suggest methods to alleviate the same.

Materials and Methods

EXPERIMENTAL ANIMALS

The experiment was conducted in a private farm in Denholm, Saskatchewan in the month of

November. The experimental group comprised of 24 and 22 emus belonging to the age group of

4-6 years.
TRANSPORT

The total feed intake was considerably reduced during this period of the year (200g per emu

per day). Emus were randomly loaded into either of the two compartments of the horse trailer

and transported (stocking density of 0.5m2/ emu) for about 6 hours. The loading and unloading

accounted for another one hour of confinement inside the compartment. The activities of emus

during transport were continuously observed using a camera and monitor. At the end of the

journey, the emus were kept at the lairage for 15 hours before slaughter at the abattoir in the

Lacombe research Station, Alberta.

METEOROLOGICAL DATA

The mean atmospheric temperature for the week prior to transport was 2 oC, and on the day of

transport, the temperatures recorded were between 8 o and 17oC for trial 1 (warm weather) and -

55o to -1oC (cold weather). The relative humidity was comparable in the two trials, varying from

45-89%. The recording from inside the truck revealed that during transport, the temperatures

ranged from 110- 220 C.

SAMPLE COLLECTION AND PROCESSING

All the emus were tagged for identification and the core body temperature (digital

thermometer- Medline industry) was recorded in two intervals. Body weights were recorded

before, immediately after transport and after lairage for 15 hours. Blood samples were collected

24 hours prior to transport and after transport through jugular venipuncture with 21G vacutainer

needles. Blood was collected 4 ml each in lithium heparin tubes, EDTA tubes and serum

separator tubes (BD vacutainer® plus). Blood smears were made to evaluate the heterophil
lymphocyte ratio after staining with Diff quick stain. Hematocrit values or packed cell (PCV)

was determined using the standard microhematocrit method with centrifugation at 12,000 G for 5

minutes (Campbell, 1994). The tubes were stored on ice or refrigerated and the separated serum

was kept frozen till examined in the lab. Analyses of serum levels of calcium, phosphorus, total

protein, albumin, glucose, triglycerides, cholesterol, uric acid, and the enzyme activities of

alkaline phosphatase (AP), alanine aminotransferase (ALT), aspartate aminotransferase (AST),

and creatine kinase (CK) were carried out in a multichannel auto analyser (Siemens, Dimension

RXL). Plasma corticosterone (CORT) was determined by the ELISA method, using

immunoassay kit (Enzo Life Sciences ADI-900-097). The CORT reading from ELISA reader

were utilized for curve fitting with MasterPlex reader fit quantitative analysis module and

expressed in ng/mL. The body condition including injuries and feather losses were noted after

the end of the journey.

STATISTICAL ANALYSIS

The data for body weights was analysed using the GLM procedure of Statistica, with sex and

the main effects included in the model with three time periods and all other variables with emu

as the experimental unit over the two time periods, 24 hours prior to transport and 24 hours after

transport. Body weights at the farm, immediately after transport and at slaughter were analyzed

using a 2-way, repeated measures ANOVA using Statistica 10, with the main effects of sex and

replicate, time period and their interaction effects. Least squares means and SE for all variables

are presented after accounting for the effect of injuries on emus which were scored as mentioned

in Table . All pair wise multiple comparison tests within the factors were performed at α = 0.05,

unless otherwise mentioned using multiple comparison procedures (Tukeys HSD). Relationships
between some parameters were generated using the correlation coefficient and regression

polynomials options of Statistica.

Results

The extent of injuries to skin and loss of feathers were found to differ among the emus and

hence the birds were categorised into three groups as mild, moderate and extensive. There were

two emus in each of the latter categories, while the former had 20 emus.

CORE BODY TEMPERATURE

The core body temperature of emus increased significantly (P<0.001) from 37.2 to 39.3 after

transport (Figure1), but we could not find any significant difference between the sexes.

BODY WEIGHT

The mean ± S.E of body weights of emus along with the minimum and maximum weights

recorded are presented in Figure 2. There was no effect of sex on the body weights. It could be

noted that the emus lost on an average 5.9% after transport and then gained slightly during

resting at lairage to have a net loss of 4.7% in trial 1 while the net losses were just 1.4% in trial

2. There was significant difference between all the three weights. The loss of body weight was

calculated to be 0.84% and 0.23 % per hour respectively for the two trials.

METABOLITES

The change recorded for each of the metabolite from the baseline to after transport and then

after lairage is given in Table 1 as least square mean ± S.E. None of the variables were affected
by sex. The serum total protein and albumin levels were numerically lowered while the uric acid

levels increased after transport.

SERUM ENZYMES

Though the ALT, AST and CK levels recorded in emus (Table 1) did not differ

significantly between sexes , there was a three times increase in ALT levels after transport which

further increased to more than five times the initial value at slaughter. The AST levels were

increased three to five times; and the CK levels were elevated by around 10 to and more than 15

times above the baseline levels after transport and at slaughter respectively.

CORTICOSTERONE

The baseline plasma corticosterone levels were found to be 8.7 ± 2.2 ng/mL ranging

between 0.06 - 43.3 ng/mL and at slaughter the value was found to increase significantly (P

<0.001) to 36.0 ± 3.7 ng/mL (ranging between 0.98 – 61.0 ng/mL).

HEMATOLOGY

None of the variables were affected by sex and hematocrit values (PCV) was unaffected

by transport. Transport resulted in leukocytosis, heterophilia, lymphocytopenia, eosinopenia and

the H: L ratio was increased five times (Table 1).

RELATIONSHIPS BETWEEN VARIABLES

The correlations between at slaughter ALT and AST (r = 0.76), AST and CK at slaughter (r =

0.85) were highly significant. Analyses also revealed that the baseline albumin level was
correlated to the baseline calcium (r = 0.36), while the at slaughter albumin was correlated to at

slaughter calcium levels (r = 0.56).

INJURIES AND FEATHER LOSS

There was mild to extensive feather loss on the ventral aspect of the neck, breast and

sides of legs too. On observation it was found that some emus lose balance, slip and fall down

during journeys and then they tend to sit down frequently for the rest of the journeys. While

these emus sat down, other emus would walk on them leading to the injuries on their back, rump,

and belly region.

Discussion

The overall picture of the various systemic changes consequent to handling and transport would

give us an idea of the impact of these stressors on emus. As the blood sampling itself could be

stressful to emus, every effort. Every attempt was made to minimize handling and handling time

as possible. It was not possible to differentiate the stressors from handling alone or transport

alone and hence the findings correspond to the effect of them in combination.

CORE BODY TEMPERATURE

Body temperature could be elevated after transport due to the higher metabolic rate resulting

in metabolic and energy balances being shifted to enable the bird to either avoid or confront the

stress (Elrom, 2000) and also due to the inability of the bird to thermo regulate effectively.
Increases in the rectal temperatures above normal range in transported ostriches was also

reported by Minka and Ayo (2008).

REDUCTION IN BODYWEIGHT (SHRINKAGE)

This is a common feature in animal transport (Parker et al., 2003; 2007; Schaefer et al.,

2001;) which may be attributed to loss of intestinal contents, electrolyte imbalances, dehydration

and loss of muscle mass (Coffey et al., 2001; Kannan et al., 2002; Minka & Ayo, 2011;

Schwartzkopf-Genswein et al., 2007). Transport results in slight increase in the metabolism of

the animals (Earley et al., 2007; Parker et al., 2007) and as. As the journey continues, animals go

into an energy deficit and energy reserves (fat and glycogen) are broken down (European

Commission, 2002) leading to losses in body weight which is often proportional to the distance

of transport (Petracci et al., 2010). The reduction in weight may vary from 1.3 to 2.1 % during

durations for journeys from 3.5 to 5 hours (Bianchi et al., 2007) or at the rate of 0.6% per hour

for 6 hour journeys (Buhr et al., 1998). Shrink losses less than 6% could be mostly attributed to

the gut contents and dehydration (Richardson, 2005) though the effect on muscles and other

tissues cannot be overruled.

METABOLITES

Increased reliance on glucose as an energy source and increased blood glucose levels is often

observed during stress (Rosales, 1994). Hyperglycemia, as seen in our study has been reported

after transport in several studies (Bedanova et al., 2007; Parker et al., 2003; 2007; Thaxton &

Puvadolpirod, 2000; Zhang et al., 2009) mainly due to glycogenolysis by catecholamines and

glucocorticoids (Li et al., 2009). But later on these levels are reduced (Rosales, 1994; Tadich et
al., 2005; Voslarova et al., 2011) as the glucocorticoid secretion is exhausted. Higher glucose

concentrations found at slaughter in this study could also be due to the effect of catecholamines

produced during stunning (Mitchell et al., 1988).

TOTAL SERUM PROTEIN, ALBUMIN AND GLOBULIN

We could see that the values are towards the higher end and this is probably due to older age

and also that the emus had considerably lowered their water intake during the period of the year

in which, this study was undertaken. The total serum albumin levels were lower at slaughter due

to the gluconeogenesis process under the influence of stress hormones and also the fact that the

birds had not eaten feed during overnight lairage. The total protein levels were found to decrease

during stress (Vošmerová et al., 2010), especially the albumin levels (Sporer et al., 2008),

indicating nutritional deficits (Ndlovu et al., 2008) but Earley et al (2007) and Parker et al

(2007) found higher total protein levels probably due to dehydration. There have also been

studies in which these levels were unaltered (Huff et al., 2008; Wolmarans, 2011), as in ours,

indicating that there was no major breakdown of muscle proteins.

The albumin (A) to globulin (G) i.e. A) G ratio is generally slightly above one and the results

from the emus in our study revealed the same. The AG Ratio is considered to be a better

indicator of the hydration status and was found not to be affected by sex. Transportation resulted

in significant reduction of this ratio because the albumin levels were reduced while the globulin

levels were unaffected. Hyperproteinemia with a normal A:G ratio will be indicative of

dehydration. This also indicated that the dehydration in these birds was not severe as also evident

from the fact that the emus did drink water after transport.
 TGs are the major sources of energy in birds (Smith et al, 1999) Loweringand their lowering

after transport is indicative of stress from withdrawal of feed and gluconeogenesis (Huff et al.,

2008; Kannan et al., 2002; 2003; Minka & Ayo, 2009; Remage-Healey & Romero, 2001 and

Voslarova et al., 2011).

Creatinine is synthesized in the muscle from phosphocreatine, at a relatively constant rate,

depending on dietary intake and muscle mass (Olvera, 2004). The creatinine levels were found to

be significantly higher in the emus at slaughter when compared to those before transport and this

indicates increased protein turnover, though usually the creatinine production is relatively stable

and least affected by the catabolism of tissue proteins (Hochleithner, 1994). The increased

creatinine levels might be due to muscle damages (Bovera et al., 2007) during handling and

transport (European Commission, 2002).

Transportation and the associated events could lead to elevation of plasma urea levels (Earley

et al., 2007; Schaefer et al., 1997) indicating the breakdown of proteins and nucleic acids in the

muscles (Anon, 2005; Huff et al., 2008; Virden et al., 2009) on account of increased

glucocorticoid concentration or food deprivation (Minka & Ayo, 2009). There was almost

doubling of the uric acid levels after lairage and which is possibly a protective response against

oxidative changes (Hartman et al., 2006). As these values are highly variable among birds, their

significance as a sole factor is quite questionable (Huff et al., 2008). This also indicates that the

protein catabolism associated with the emus in this transport trial was probably marginal.

SERUM ENZYME LEVELS

The serum enzymes namely, ALT, AST and CK were found to increase during the damages

of tissues including muscles, brain, liver, kidney and heart. The levels of liver enzymes AST,
ALT was found to increase in the blood following damage to muscles; reduced muscular tissue

perfusion, hypoxia and fatigue, perhaps due to higher permeability of muscle membrane

following handling, loading and transportation stress (Huff et al., 2004; María et al., 2004;

Mazzone et al., 2010; Minka & Ayo, 2009; Olvera, 2004; Schaefer et al., 1997; Scope et al.,

2002; Villarroel et al., 2001). Creatine kinase (CK) is released from the muscle into the blood

stream due to damages to muscles like injuries and bruising; and their levels increase in

circulation, two hours after the physical stress (Kannan et al., 2000) and brain changes .

It has been observed that these changes are associated with rhabdomyolysis in animals and

ratites. These elevations probably indicated the extent of tissue damage caused during the

handling, loading, transport and unloading of emus. Their levels in ratites are generally higher

than other birds (Doneley, 2006). Physical activities like catching are more important in

elevating plasma CK activity than transportation itself (Kannan et al., 2003). Increase in plasma

CK activity was after transport was also reported by other authors (Huff et al., 2008; Sporer et

al., 2008; Tadich et al, 2005; Van de Water et al., 2003) and these levels were lower in winter

than in summer and unaffected by lairage (Tadich et al., 2005).

CORTICOSTERONE (CORT)

Changes in corticosterone levels should be interpreted with respect to the baseline levels

(Bonier, et al., 2009). The values observed in our study indicated wide variation between the

emus and some researchers have found that CORT levels vary widely even within the individual

(Romero & Reed, 2008). Researchers have stressed the need to collect samples with minimal

handling and time of contact to get feedback free results (Romero & Reed, 2005; Romero &
Romero, 2002) as elevated CORT levels could modify the functioning of different organs and

hence alter the blood biochemistry and hematology.

HEMATOLOGY

Blood samples collected at slaughter revealed decrease in hematocrit values which were not

statistically significant similar to the findings of (Gupta et al., 2007) further confirming that there

was considerably low level of dehydration in emus at slaughter in these trials.

White blood cell (WBC) counts increased corresponding to neutrophilia in emus during the

stress (response.(Minka & Ayo, 2008; Ndlovu, et al. 2008; Wolmarans, 2011). The heterophyl:

lymphocyte (H:L) ratio is considered to be a reliable indicator of stress in many studies ( Chacon

et al., 2005; Smith et al., 2004) in assessing poultry welfare status (Singh et al., 2009) along with

other indicators (Moneva et al., 2009). Significant increases in this ratio has also been reported in

the studies conducted by Kamau, Patrick, & Mushi (2002) in ostriches and Minka & Ayo, (2008;

2011). In chicken low and high stress could be identified from the extend of change in H:L ratios

(Gross & Siegel, 2012).

Blood calcium plays an important role in blood coagulation membrane permeability, and

functioning of the nerves, muscles and heart (Harr, 2005). An appropriate ratio between calcium

and phosphorous is essential for proper bone health. Total calcium should always be interpreted

along with albumin concentrations as lower albumin levels may erroneously result in decreased

calcium levels (bound forms alone) detected during tests (Harr, 2005; Hochleithner, 1994).

Dehydration may also lead to hyperproteinaemia and associated hypercalcaemia (Hochleithner,

1994). Highly elevated calcium levels are common in egg laying females (Hoefer, 2010) and

hence the our results further confirm that the emus were approaching the breeding season.
RELATIONSHIP BETWEEN VARIABLES

The final CK levels were correlated to the final ALT (r=0.66) and AST (r=0.55) levels

indicating that the final rises in all the three are all probably attributable to the same source. The

fact that the injury scores were found to be correlated only to the final AST levels (r=0.43)

suggests that changes in AST may be contributed to the injuries inflicted on emus.

INJURIES

The extent, location of bruises and injuries on all the emus were recorded after transport. We

found that most injuries (on being walked on extensively) were sustained on the back; and the

sides of the leg of emus. There were no dislocations or fractures. The loss of feathers from the

body was classified into three categories namely, mild/none, moderate and extensive. The losses

were on either sides of the mid central line, belly and back. Six emus had mild feather losses

mostly at the neck and back. One bird had minor bruises on the leg. None of the birds had any

bruises on the head or foot pad.

EXERTIONAL RHABDOMYOLYSIS OR CAPTURE MYOPATHY

Handling, prolonged chases and transportation for hours along with the vibrations in the

truck are considered to cause lysis of the muscle cell walls and leakage of contents into the blood

stream (Paterson, 2007) leading to a clinical condition called exertional rhabdomyolysis or

capture myopathy in long legged birds ( Marco et al., 2006; N K, J, & LBusinga, Langenberg &

Carlson, 2007) including ratites (Crowther, 2002; Crowther, Davies & Glass, 2003). This

condition is characterized by the destruction or lysis of striated muscles could result in brain

damage, paralysis and death in some cases (Wotton & Hewitt, 1999). The indication of this
condition may range from an asymptomatic increase in the CK level to paralysis and death due to

electrolyte imbalances, acute renal failure and intravascular coagulation. The clinical picture

often includes increased AST and CK levels (Harr, 2005; Marco et al., 2006), hyperglycemia

and hypercalcaemia (Tully et al., 1996); hypocalcaemia and, hyperkalemia (Veenstra et al.,

1994); and heterophilic leukocytosis (Smith, Murray, & Carlos, 2005). Moreover reduced blood

uric acid: creatinine ratios (Figure 7) with elevated creatinine levels are also characteristic of

rhabdomyolysis (Linares, Golomb, & Jaojoco, 2009; Walid, 2008). Any case where there is 10

times and more increase in CK levels are indicative of exertional rhabdomyolysis (Paterson,

2007; Veenstra et al., 1994).

These findings further confirm that the changes noticed in emus after transport are mostly

due to muscle injury and hence point to the chances of rhabdomyolysis in emus and hence

warrants extreme caution while handling and transporting the same.

RECOMMENDATIONS

Administration of electrolytes and nutrients supplements to overcome the losses and

transporting emus during night or cooler weather conditions alone. Improved trailers, ramps and

chutes to facilitate easier loading and unloading are needed. Better loading and unloading

techniques to avoid injuries and minimal and careful handling to avoid instances of

rhabdomyolysis is also suggested.

Conclusion

On being transported for 7 hours in a modified horse trailer, emus lost 6 % of their body but

gained over 1 % after being rested at lairage for 15 hours. At slaughter, there was significantly
increased plasma glucose, creatinine, and heterophil: lymphocyte ratios and corticosterone levels

while the serum albumin, albumin: globulin ratio and blood calcium levels were decreased. The

serum enzymes ALT, AST and CK and the plasma corticosterone levels were significantly

elevated at slaughter. These findings suggest that transportation stress is associated with elevated

corticosterone levels and resulting in shrinkage, hypoalbuminaemia, hypocalcaemia, with

increase in serum enzymes and altered immune status in emus. Elevations in enzyme levels,

creatinine, glucose and heterophilic leukocytosis are suggestive of muscle injury and point to the

incidence of rhabdomyolysis in this species. We could not find any significant effect of on body

weight losses or any of the variables and hence this warrants the need to try a better mode of

administration and dosage for the feed supplement. This study also confirms the relevance of

heterophil: lymphocyte ratio changes as an indicator of stress in this species.

 6% 3%
47 a P< 0.001
a
46 b
Body weight (kg)

45 a

44
b Before transport
43 b
After transport
42
After lairage
41
40
39
Warm weather Cool weather
α = 0.05/3 =0.015, P = <0.001 between a and c and a and b; P = 0.005 between b and c

Figure 2. Changes in body weight

6% 3%
47 a P< 0.001
a
46 b
Body weight (kg)

45 a

44
b Before transport
43 b
After transport
42
After lairage
41
40
39
Warm weather Cool weather

Table 1 The levels of various metabolites from emus recorded at farm (baseline), after transport

and after lairage (at slaughter).

 Variable Unit Before After After lairage P value
transport transport

Mean ± SE Mean ± SE Mean ± SE

Glucose * mMol/L 8.9 ± 0.4c 12.6 ± 0.5a 10.3 ± 0.5b F2,30= 5.02, P<0.001

Total protein g/L 52.1 ± 1.0 51.8 ± 2.2 52.3 ± 1.3 NSD

Albumin * g/L 28.5 ± 0.6 28.0 ± 0.9 27.4 ± 0.5 NSD

Triglyceride mMol/L 5.1 ± 1.3a 2.0 ± 0.4a 0.9 ± 0.3b F(2,30)=4.92, P=0.01

Creatinine * µMol/L 4.9 ± 0.9b 7.6 ± 1.1b 30.7 ± 2.2a F(2,30)=35.46, P<0.001

Uric acid (UA) µMol/L 163.2 ± 11.1 186.5 ± 14.3 242.8 ± 43.5 NSD

PCV 55.6 ± 0.5 52.6 ± 0.8 53.3 ± 2.3 NSD

WBC Count x109/L 12.9 ± 1.2a 14.1 ± 0.4ab 16.9 ± 0.8b F(2,30) =6.09, P=0.006

Heterophils % 65.8 ± 2.6b 88.5 ± 1.1a 89.1 ± 1.2a F(2,30) =22.29, P<0.001

Lymphocytes % 30.1 ± 2.8a 7.3 ± 1.1b 8.0 ± 1.1b F(2,30)=19.93, P<0.001

H:L Ratio* 2.6 ± 0.4b 17.1 ± 2.9a 17.1 ± 3.4a F(2,30)=5.54, P=0.01

Basophils % 0.9 ± 0.2a 0.7 ± 0.2a 0.1 ± 0.1b F(2,30)=5.62, P=0.008

Eosinophils % 1.1 ± 0.3 0 0.8 ± 0.2

Hematocrit 53.8 ± 1.1 52.0 ± 0.8 54.3 ± 0.9 NSD

UA : creatinine* 17.0 ± 1.3a 9.1 ± 1.4b 7.5 ± 0.8b

Calcium mMol/L 4.1 ± 0.4 4.1 ± 0.4 3.6 ± 0.3 F(2,30)=7.35, P=0.003

Sodium 146.8 ± 1.3 145.9 ± 0.9 150.3 ± 0.9 NSD

Bicarbonate 23.9 ± 0.9a 21.2 ± 0.5b 26.9 ± 0.7a F(2,30)=3.75, P=0.035

Chloride 104.6 ± 0.7b 109.7 ± 0.6a 109.0 ± 0.5a F(2,30)=9.15, P<0.001

ALT IU/L 12.6 ± 1.8b 27.8 ± 4.6b 103.4 ± 26.9a F(2,30)=4.15, P=0.03

AST IU/L 197 ± 16b 508 ± 68 b 1852 ± 424a F(2,30)=4.49, P=0.02

CK IU/L 453 ± 54b 4493 ± 1067 b 15962 ± 2870a F(2,30)=6.67, p=0.001

Variables with * significantly differ at the two time periods

Figure 7 Comparison of the changes in uric acid: creatinine ratio consequent to transport
 References

Adams, D.B., & Thornber, P. M. (2008). Epidemiology, ethics and managing risks for

physiological and behavioural stability of animals during long distance transportation.

Veterinaria Italiana, 44(1), 165-176.

Bedanova, I., Voslarova, E., Chloupek, P., Pistekova, V., Suchy, P., & Al, E. (2007). Stress in

broilers resulting from shackling. Poultry Science, 86(6), 1065-1069.

Ben-Shahar, A. R. (n.d.). Introduction to the Physiology of Stress The “Fight or Flight” response,

the hormones and the part of adrenaline in the stress response. Retrieved January 12, 2010,

from http://www.imt.co.il

Bianchi, M., Petracci, M., Sirri, F., Folegatti, E., Franchini, A., & Meluzzi, A. (2007). The

influence of the season and market class of broiler chickens on breast meat quality traits.

Poultry Science, 86(5), 959-963.

Bonier, F., Martin, P. R., Moore, I. T., & Wingfield, J. C. (2009). Do baseline glucocorticoids

predict fitness? Trends in ecology & evolution (Personal edition), 24(11), 634-642.

Bovera, F., Moniello, G., De Riu, N., Di Meo, C., Pinna, W., & Nizza, A. (2007). Effect of diet

on the metabolic profile of ostriches (Struthio camelus var. domesticus). Tropical Animal

Health and Production, 39(4), 265-270.

Buhr, R. J., Northcutt, J. K., Lyon, C. E., & Rowland, G. N. (1998). Influence of time off feed on

broiler viscera weight, diameter, and shear. Poultry Science, 77(5), 758-764.

Businga N K, Langenberg J, & Carlson L. (2007). Successful treatment of capture myopathy in

three wild greater sandhill cranes (Grus canadensis tabida). Journal of Avian Medicine

Surgery, 21(4), 294-8.

Chacon, Garcia-Belenguer, &V., & Maria. (2005). Effect of transport stress on physiological

responses of male bovines. DTW. Deutsche tierärztliche Wochenschrift, 112(12), 465-469.

Schaper.

Coffey, K., Coblentz, W., Humphry, J., & Brazle, F. (2001). Review: Basic Principles and

Economics of Transportation Shrink in Beef Cattle. The Professional Animal Scientist,

17(4), 247-255. American Registry of Professional Animal Scientists.

Crowther, C. (2002). Transport of ratites – implications for welfare. Surveillance, 29(4), 18-20.

Crowther, C., Davies, R. & Glass, W. (2003). The effect of night transportation on the heart rate

and skin temperature of ostriches during real transportation. Meat Science, 64(4), 365-370.

Doneley, B. (2006). Management of Captive Ratites. In G. Harrison & T. Lightfoot (Eds.),

Clinical Avian Medicine Volume II (pp. 124-156).

Earley, B., Murray, M., & Prendiville, D. . (2007). Animal Transport Developing optimum

animal handling procedures and effective transport startegies in the food production chain

to improve animal welfare and food quality. Animals (pp. 1-69). Dunsany, Co.Meath.

Elrom, K. (2000). Handling and transportation of broilers: welfare, stress, fear and meat quality -

II Stress. Israel Journal of Veterinary Medicine, 55(2), 1-11.

European Commission. (2002). The welfare of animals during transport ( details for horses ,

pigs , sheep and cattle ) . Management (pp. 1-130). Report of the Scientific Committee on

Animal Health and Animal Welfare.

Gross, W. B., & Siegel, H. S. (2012). Evaluation of the heterophil/lymphocyte ratio as a measure

of stress in chickens. Avian diseases, 27(4), 972-9.

Gupta, S., Earley, B., & Crowe, M. A. (2007). Effect of 12-hour road transportation on

physiological, immunological and haematological parameters in bulls housed at different

space allowances. Veterinary Journal, 173(3), 605-616.

Harr, K. E. (2005). Diagnostic value of Biochemistry. In G. J. Harrison & B. F. Gwen (Eds.),

Clinical Avian Medicine Volume1 (1st ed., Vol. 365, pp. 647-665). Spix Publishing.

Hartman, S., Taleb, S. A., Geng, T., Gyenai, K., Guan, X., & Smith, E. (2006). Comparison of

Plasma Uric Acid Levels in Five Varieties of the Domestic Turkey, Meleagris gallopavo.

Poultry Science, 85(10), 1791-1794.

Hochleithner, M. (1994). Biochemistries. In B. . Ritchie, G. . Harrison, & L. . Harrison (Eds.),

Avian Medicine (pp. 223-245). Wingers Publishing, Inc., Lake Worth, Florida.

Hoefer, H. . L. (2010). Basic Avian Clinical Pathology Testing. West Hills Animal Hospital,

Huntington, New York. ievcli.com, 1-4.

Huff, G. R., Huff, W. E., Rath, N .C., Anthony, N. B., & Nestor, K. E. (2008). Effects of

Escherichia coli Challenge and Transport Stress on Hematology and Serum Chemistry

Values of Three Genetic Lines of Turkeys. Poultry Science, 87, 2234-2241.

Kamau, J. M., Patrick, B. T., & Mushi, E. Z. (2002). The effect of mixing and translocating

juvenile ostriches (Struthio camelus) in Botswana on the heterophil to lymphocyte ratio.

Trop Anim Health Prod, 34(3), 249-56 ST - The effect of mixing and translocatin.

Kannan, G., Terrill, T. H., Kouakou, B., Gelaye, S., & Amoah, E. A. (2002). Simulated

preslaughter holding and isolation effects on stress responses and live weight shrinkage in

meat goats. Journal of Animal Science, 80(7), 1771-1780.

Li, Y., Cai, H. Y., Liu, G. H., Dong, X. L., Chang, W. H., Zhang, S., Zheng, a J., et al. (2009).

Effects of stress simulated by dexamethasone on jejunal glucose transport in broilers.

Poultry Science, 88(2), 330-337.

Linares, L. A., Golomb, B. A., & Jaojoco, J. A. (2009). The modern spectrum of

rhabdomyolysis: drug toxicity revealed by creatine kinase screening. Current Drug Safety,

4, 181-7.

Lumeij, J. T., & Overduin, L. M. (1990). Plasma chemistry references values in psittaciformes.

Avian pathology journal of the WVPA, 19(2), 235-244.

Marco, I., Mentaberre, G., Ponjoan, A., Bota, G., Mañosa, S., & Lavín, S. (2006). Capture

myopathy in little bustards after trapping and marking. Journal of wildlife diseases, 42(4),

889-91.

Maria, G., Escoc, J & Alados, C. . (2004). Complexity of behavioural sequences and their

relation to stress conditions in chickens (Gallus gallus domesticus): a non-invasive

technique to evaluate animal welfare. Applied Animal Behaviour Science, 86(1-2), 93-104.

Mazzone, G., Vignola, G., Giammarco, M., Manetta, a C., & Lambertini, L. (2010). Effects of

loading methods on rabbit welfare and meat quality. Meat science, 85(1), 33-39.

Minka, N S, & Ayo, J. O. (2008). Assessment of the stresses imposed on adult ostriches (Struthio

camelus) during handling, loading, transportation and unloading. Veterinary Record,

162(26), 846-851.

Minka, N. S., & Ayo, J. O. (2011). Modulating Role of Vitamins C and E against Transport-

Induced Stress in Pullets during the Hot-Dry Conditions. ISRN Veterinary Science, 2011, 1-

7. doi:10.5402/2011/497138
Mitchell, G., Hattingh, J., & Ganhao, M. (1988). Stress in cattle assessed after handling, after

transport and after slaughter. Veterinary Record, 123(8), 201.

Ndlovu, T., Chimonyo, M., Okoh, A. I. and, & Muchenje, V. (2008). A comparison of stress

hormone concentrations at slaughter in Nguni , Bonsmara and Angus steers. Journal of

Agricultural Research, 3(2), 96-100.

Olvera, J. R. . (2004). Capture, restraint and transport stress in Southern chamois Modulation

with acepromazine and evaluation using physiological parameters. Bellaterra, 1-154.

Parker A .J, Dobson, G. P., & Fitzpatrick, L. A. (2007). Physiological and metabolic effects of

prophylactic treatment with the osmolytes glycerol and betaine on Bos indicus steers during

long duration transportation. Journal of Animal Science, 85(11), 2916-2923.

doi:10.2527/jas.2006-193

Parker, A. J., Hamlin, G. P., Coleman, C. J., & Fitzpatrick, L. A. (2003). Quantitative analysis of

acid-base balance in Bos indicus steers subjected to transportation of long duration. Journal

of Animal Science, 81(6), 1434-1439.

Paterson, J. (2007). Capture Myopathy. In G. West, D. Heard, & & N. Caulkett (Eds.), Zoo

Animal & Wildlife Immobilization and Anesthesia (pp. 115-122). Blackwell Publishing.

Petracci, M., Bianchi, M. A., & Cavani, C. (2010). Pre-slaughter handling and slaughtering

factors influencing poultry product quality. World’s Poultry Science Journal, 66(01), 17–

26.

Remage-Healey, L., & Romero, L. M. (2001). Corticosterone and insulin interact to regulate

glucose and triglyceride levels during stress in a bird. American Journal of Physiology.,

281(3), 994-1003.

Richardson, C. (2005). Reducing cattle shrink. OMAFRA, (05), 63-64.

Romero, L. M, & Reed, J. . . (2005). Collecting baseline corticosterone samples in the field: is

under 3 min good enough? Comparative Biochemistry and Physiology Part A:, 140(1), 73-

79.

Romero, L. Michael, & Romero, R. C. (2002). Corticosterone Responses in Wild Birds: the

Importance of Rapid Initial Sampling. The Condor, 104(1), 129. doi:10.1650/0010-

5422(2002)104[0129:CRIWBT]2.0.CO;2

Romero, L., & Reed, J. M. (2008). Repeatability of baseline corticosterone concentrations.

General and Comparative Endocrinology, 156(1), 27-33.

Rosales, A. G. (1994). Managing stress in broiler breeders: a review. The Journal of Applied

Poultry Research, 3(2), 199-207.

Schaefer, A. ., Dubeski, P., Aalhus, J., & Tong, A. (2001). Role of nutrition in reducing

antemortem stress and meat quality aberrations. Journal of Animal Science, 79(E-Suppl),

E91.

Schwartzkopf-Genswein, K. ., Booth-McLean, M. E., Shah, M. A., Entz, T., Bach, S. J., Mears,

G. J., Schaefer, A. L., et al. (2007). Effects of pre-haul management and transport duration

on beef calf performance and welfare. Applied Animal Behaviour Science, 108(1-2), 12-30.

doi:10.1016/j.applanim.2006.11.012

Scope, A., Filip, T., Gabler, C., & Resch, F. (2002). The influence of stress from transport and

handling on hematologic and clinical chemistry blood parameters of racing pigeons

(Columba livia domestica). Journal Information, 46(1).

Smith, F. ., West, N. ., & Jones, D. R. (1999). The Cardiovascular System. In P. . Sturkie (Ed.),

Sturkie’s Avian Physiology (5th ed., p. 140-).

Smith, K. M. ., Murray, S., & Carlos S. (2005). Successful Treatment of Suspected Exertional

Myopathy in a Rhea ( Rhea americana ). Journal of Zoo and Wildlife Medicine, 36(2), 316-

320.

Sporer, B.K. R., Weber, P. S .D., Burton, J. L., Earley, B., & Crowe, M. A. (2008).

Transportation of young beef bulls alters circulating physiological parameters that may be

effective biomarkers of stress. Journal of Animal Science, 86(6), 1325-1334.

Tadich, N., Gallo, C., Bustamante, H., Schwerter, M., & Vanschaik, G. (2005). Effects of

transport and lairage time on some blood constituents of Friesian-cross steers in Chile.

Livestock Production Science, 93(3), 223-233.

Thaxton, J .P., Puvadolpirod, S. (2000). Model of physiological stress in chickens. Response

parameters. Poultry Science, 79(3), 363-369.

Tully, T. N., Hodgin, C., Morris, J. M., Williams, J., & Zebreznik, B. (1996). Exertional

Myopathy in an Emu ( Dromaius novaehollandiae ). Journal of Avian Medicine and

Surgery, 10(2), 96-100.

Veenstra, J., Smit, W. M., Krediet, R. T., & Arisz, L. (1994). Relationship between elevated

creatine phosphokinase and the clinical spectrum of rhabdomyolysis. Nephrology Dialysis

Transplantation, 9(6), 637-641.

Villa, P. ., Iannetti, L., Di Francesco, C., Di Pasquale, A., Fiore, G., & Caporale, V. (2008).

Quality management for the road transportation of livestock. Veterinaria italiana, 44(1),

187-200.

Voslarova, E., Chloupek, P., Vosmerova, P., Chloupek, J., Bedanova, I., & Vecerek, V. (2011).

Time course changes in selected biochemical indices of broilers in response to pretransport

handling. Poultry science, 90(10), 2144-52. doi:10.3382/ps.2011-01473

Vošmerová, P., Bedáňová, I., Chloupek, P., Chloupek, J., Suchý, P., Večerek, V., & Vosmerova,

P. (2010). Transport-induced Changes in Selected Biochemical Indices in Broilers as

Affected by Ambient Temperatures. Acta Veterinaria Brno, 79(9), S41-S46.

doi:10.2754/avb201079S9S041

Walid, M. S. (2008). Blood urea nitrogen/creatinine ratio in rhabdomyolysis. Indian Journal of

Nephrology, 18(4), 173-4. Medknow Publications. doi:10.4103/0971-4065.45295

Wolmarans, W. . (2011). The Effect of Transport on Live Weight Loss , Meat Quality.

differences. Stellenbosch University.

Wotton, S. B., & Hewitt, L. (1999). Transportation of ostriches-a review. The Veterinary Record,

145(25), 725-731.

Zhang, L., Yue, H. Y., Zhang, H. J., Xu, L., Wu, S. G., Yan, H. J., Gong, Y. S., et al. (2009).

Transport stress in broilers: I. Blood metabolism, glycolytic potential, and meat quality.

Poultry Science, 88(10), 2033-2041. doi:10.3382/ps.2009-00128

Annexure

Feed supplement composition