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ASSESSING THE HUMANENESS AND EFFICACY OF A NEW FERAL PIG

BAIT IN DOMESTIC PIGS

Study: PC0409

Veterinary Services Division,

Institute of Medical and Veterinary Science

Study Commencement Date: 19 March, 2008

PC0409: Report 1 of 29

Table of contents

Signature Page Responsible Personnel.................................................................................................4

Abbreviations used in this Study Plan.................................................................................................5

1. Executive Summary......................................................................................................................6

2. Sponsor Representatives and Contacts........................................................................................6

3. Animal Welfare and Institutional Animal Care and Use Committee Review............................7

4. Introduction..................................................................................................................................7

5. Objective........................................................................................................................................9

6. Key Study Dates............................................................................................................................9

7. Study Design.................................................................................................................................9

8. Justifications...............................................................................................................................10

8.1 Animal Species...................................................................................................................10

8.2 Number of Animals...........................................................................................................10

8.3 Routes of Administration..................................................................................................10

9. Study Materials...........................................................................................................................10

9.1 Test Item.............................................................................................................................109.1.1 HOG-GONE® Pig Bait................................................................................................10

9.2 Control Item.......................................................................................................................109.2.1 HOG-GONE® Pig Bait................................................................................................10

10. Test System..............................................................................................................................11

10.1 Demographics....................................................................................................................11

10.2 Husbandry..........................................................................................................................1110.2.1 Housing.......................................................................................................................1110.2.2 Quarantine...................................................................................................................1110.2.3 Acclimatisation............................................................................................................1110.2.4 Environment................................................................................................................1110.2.5 Food.............................................................................................................................1110.2.6 Water...........................................................................................................................1110.2.7 Contaminants...............................................................................................................1210.2.8 Identification...............................................................................................................1210.2.9 Initial Health Status.....................................................................................................1210.2.10 Spare and Replacement Animals.................................................................................1210.2.11 Restraint.......................................................................................................................1210.2.12 Disposal.......................................................................................................................12

11. Experimental Methodology....................................................................................................13

11.1 Animal Assignment...........................................................................................................13

11.2 Fasting................................................................................................................................13

11.3 Anaesthesia........................................................................................................................13

11.4 Animal Preparation...........................................................................................................13

11.5 Surgical Interventional Procedure...................................................................................13


11.5.1 Insertion of Arterial Access Line for Blood Collection..............................................13

11.6 Postoperative Procedures and Animal Observations.....................................................1311.6.1 Recovery......................................................................................................................13

11.7 Administration of Bait......................................................................................................14

11.8 Monitoring Procedures.....................................................................................................1411.8.1 Post-Consumption of Bait...........................................................................................14

11.9 Blood Sample Collection and Processing........................................................................1411.9.1 Methaemoglobin..........................................................................................................1411.9.2 Clinical Biochemistry..................................................................................................1511.9.3 Humane Killing of Animals Not Affected by Bait......................................................15

12. Data Presentation...................................................................................................................15

13. Results.....................................................................................................................................16

13.1 Surgical Procedure............................................................................................................16

13.2 Allocation of Animals........................................................................................................16

13.3 Bait Administration and Consumption...........................................................................16

13.4 Methaemoglobin Concentrations.....................................................................................17

13.5 Clinical Pathology.............................................................................................................18

13.6 Clinical Assessment...........................................................................................................19

14. Conclusion..............................................................................................................................20

15. Acknowledgements.................................................................................................................21

Appendix 1. Clinical Pathology Results............................................................................................22

Appendix 2. Methaemoglobin Results (%)........................................................................................23

Appendix 3. Individual Clinical Records Summary.........................................................................24

Reference.............................................................................................................................................28


Signature Page Responsible Personnel

We the undersigned, hereby declare that this study was undertaken as described below, and that the

report accurately reflects the results obtained.

Study Position Name Signature Date

Study Director Ms Susan Porter _____________ 24 June 2009

Test Facility Management Dr Tim Kuchel 24 June 2009


Abbreviations used in this Study Plan.

APVMA Australian Pesticides and Veterinary Medicines Authority

CRC Co-operative Research Centre

GPARC Gilles Plains Animal Resource Centre

HR heart rate

IA intra-arterial

IM intramuscular

IMVS Institute of Medical and Veterinary Science

metHb methaemoglobin

mL millilitre

SOP standard operating procedure(s)

VSD Veterinary Services Division


1. Executive Summary

Ten pigs previously trained to eat non-toxic bait were fed either toxic bait (n=5) or non-toxic bait

(n=5). On the previous day the animals had a catheter inserted into the carotid artery to facilitate

arterial blood sampling for methaemoglobin and clinical pathology analyses.

The amount of toxic bait consumed ranged from 179 to 822 mg/Kg of body weight. All animals that

consumed the toxic bait died within two hours of consumption with the exception of the pig with the

lowest consumption (179 mg/kg), which died three hours post-consumption.

All animals had methaemoglobin levels greater than 10% within 20 minutes of consuming the toxic

bait. At the time of death, the methaemoglobin levels ranged from 77.8-86.5%. The nitrite

containing toxic baits fed to the pigs in this study were efficacious. In the opinion of the authors, the

symptoms leading to death and duration of display of these symptoms would suggest that sodium

nitrite satisfies a general understanding of what a humane poison would be.

2. Sponsor Representatives and Contacts

i) SPONSOR: Invasive Animals Cooperative Research Centre (CRC), with financial

support from the Australian Federal Department of Environment,

Water, Heritage and the Arts.

Sponsor’s Representative: Dr Steven LapidgeInvasive Animals CRC 48 Oxford Terrace, Unley, South Australia 5061Tel: +61 8 8357 1222Email: [email protected]

ii) TEST FACILITY: Institute of Medical and Veterinary Science (IMVS)

Conduct of StudyAnimal Resource Centre Veterinary Services Division(GPARC) 101 Blacks Rd

Gilles Plains SA 5086AUSTRALIATel: +61 8 8261 1033

Clinical Pathology Division of Clinical Pathology Frome RoadAdelaide SA 5001


AUSTRALIATel: +61 8 8222 3414

Test Facility Management: Dr Tim KuchelHead,Veterinary Services Division, IMVSTel: +61 8 8261 1033Fax : +61 8 8261 2280Email: [email protected]

Study Director: Ms Susan PorterVeterinary Services Division, IMVS101 Blacks RdGilles Plains SA 5086AUSTRALIATel: +61 8 8161 5106Fax : +61 8 8261 2280Email: [email protected]

3. Animal Welfare and Institutional Animal Care and Use Committee Review

The study was reviewed and approved by the Animal Ethics Committee of the IMVS. The Ethics

Committee number for this study was 123/08. This study followed the requirements of the Animal

Ethics Committee of the IMVS, including the Australian Code of Practice for the Care and Use of

Animals for Scientific Purposes, 7th edition, 2004.1

4. Introduction

Feral pigs cause a large and diverse range of impacts where they occur. They cause agricultural

losses to stock and crops conservatively estimated at $106 million annually. They cause numerous

environmental impacts, including wildlife predation and habitat destruction, and have been listed as

a threatening process in NSW and nationally. They spread endemic disease and will potentially

spread exotic disease should one enter Australia.

Poisoned baiting of feral pigs is the most efficacious and cost-effective control technique. Currently

three toxins are used for feral pig control- sodium fluoroacetate (1080), warfarin and yellow

phosphorus. Each of these toxins has potential problems. 1080 is required in doses that potentially

compromises the safety of non-target species. 1080 can also lead to profuse vomiting in feral pigs

which may cause non-target risks and potential welfare compromises. 1080 in pen trials has never

killed all poisoned feral pigs, even at high doses, and the toxin has no antidote in the event of non-

target poisoning. Phosphorus causes severe clinical disease over several days and almost certainly

causes welfare compromise in poisoned feral pigs. In addition, its use pattern exposes non-target


scavengers to potential poisoning. Phosphorus use is not supported in NSW and Queensland by land

management agencies. NSW Primary Industries has assessed phosphorus as inhumane and

unacceptable for use. Warfarin is an efficacious feral pig toxin. However, it is not registered for use

by the APVMA. Agencies such as the NSW National Parks and Wildlife Service and the ACT

Parks and Conservation Service which have used the toxin under experimental permits are phasing

out its use due to animal welfare concerns. NSW Primary Industries has reviewed the toxin as

inhumane and unacceptable for use in controlling feral pigs.

The PIGOUT® project commenced in January 2004 with the aim of developing and registering a

manufactured 1080 feral pig bait that would improve the welfare and target-specificity properties of

feral pig baiting. To be successful, the bait had to be highly attractive to pigs, cheap, target-specific

and easy to use. The 1080 also had to be presented in a way that would minimise non-target

exposure and vomiting. This was achieved through the use of an internal core. Field trials conducted

in Queensland, New South Wales and South Australia have achieved 78 ± 4% (S.E.; n=9 sites)

population or activity reduction using ground baiting, with little to no non-target take. Based on

these results an Australian Pesticide and Veterinary Medicine Authority (APVMA) registration for

PIGOUT® was granted in December, 2007.

The search for improved feral pig toxins has been ongoing for many decades. A recent ‘Achilles

Heel’ review searched for physiological and biochemical weaknesses in feral pigs. One such

weakness identified was the low levels of methaemoglobin reductase in pigs, an enzyme required to

convert methaemoglobin back to its oxygen-carrying haemoglobin form. As such, pigs are

particularly sensitive to methaemoglobin forming compounds, of which sodium nitrite is perhaps the

most commonly available. Ironically, the best known use of sodium nitrite is to preserve pig meats,

such as ham, bacon and smallgoods.

During 2006 the Invasive Animals CRC undertook pen trials to assess the lethality of sodium nitrite

to feral pigs. Gavage and bait-delivered nitrite trials were both highly successful, with a rapid and

humane death occurring in feral pigs in approximately two hours (compared to 6-8 hours with 1080)

and with generally unremarkable symptoms. Symptomatology included lethargy, un-coordination,

dyspnoea (laboured breathing), loss of consciousness and death. Half of the feral pigs tested vomited

one to three times prior to unconsciousness. These results were recently presented to the APVMA,

who were encouraged by the findings and supported the development of sodium nitrite as an

additional feral pig toxin.


The development of sodium nitrite as an additional feral pig active will increase the efficiency of

feral pig control, as it is more lethal to feral pigs than 1080 and will also be available more readily

due to its lower scheduling as a toxin; it will improve the humaneness of control, as time to death,

the duration of symptoms and the symptoms themselves are greatly reduced; and will also reduce

the non-target impact of control, as most other species are less sensitive to sodium nitrite poisoning,

and it has an effective antidote in methylene blue. As such, the principal benefit of this project will

be a greater ability of land managers to control feral pigs and in turn to reduced the impact of feral

pigs on agriculture and the environment.

5. Objective

The scientific aim of this project was to assess the humaneness of sodium nitrite toxicosis for the

lethal control of feral pigs. Pigs are particularly sensitive to methaemoglobin-forming compounds,

of which sodium nitrite is perhaps the most commonly available. Therefore, baits containing sodium

nitrite were fed to domesticated pigs, and the humaneness, progress of symptoms and physiology of

death were assessed in a controlled environment.

6. Key Study Dates

Commencement of Experimental Phase of Study: 12th March 2009

Completion of Experimental Phase of Study: 24th March 2009

Proposed Completion of Final Report: 24th April 2009

7. Study Design

Adult pigs 30-50 Kg in mass were used, which represents the average weight of a feral pig

population. Animals were fed nitrite-free baits for two to three days prior to the start of the study.

All animals were anaesthetised and an arterial catheter was inserted for blood collection. The next

day when the animals had recovered from surgery, baseline blood samples were taken (co-oximetry,

clinical pathology). One or two HOG-GONE® baits with 20 g of micro-encapsulated sodium nitrite

spread throughout the matrix were placed in the pens of the treatment animals. The controls received

the same amount of bait without the sodium nitrite. Animals were provided with water ad libitum.

All animals were watched until they had eaten the bait.

The animals were closely monitored for clinical signs of distress, and physiological changes

(respiration, haematology, biochemistry, cortisol and lactate and methaemoglobin levels) until death.


Cortisol and lactate levels were assessed to monitor the internal stress response of the animals. Any

animal still alive four hours post-consumption of bait would be humanely killed.

8. Justifications

8.1 Animal Species

This product will be used in feral pigs, therefore the pig is the only species which can be used.

8.2 Number of Animals

Five treated animals and five controls was the minimum number of pigs required to evaluate the

humaneness and efficacy of this product.

8.3 Routes of Administration

The anticipated route of administration is oral. Therefore, that route was used in this study.

9. Study Materials

9.1 Test Item

9.1.1 HOG-GONE® Pig Bait

Dose: 20 g micro-encapsulated sodium nitrite per bait

Size-range: 200 g bait

Physical Description: Brownish grey cylinders

Storage Conditions: Room Temperature

Supplier: Animal Control Technologies and the Invasive Animals CRC

Date of Manufacture: January 2009 (Pigs 1 and 5); March 2009 (Pigs 7, 8 and 9)

9.2 Control Item

9.2.1 HOG-GONE® Pig Bait

Dose: 0 g sodium nitrite per bait

Size-range: 200 g non-toxic bait matrix

Physical Description: Brownish grey cylinders

Storage Conditions: Room Temperature

Supplier: Animal Control Technologies and the Invasive Animals CRC

Date of Manufacture: March 2nd 2009


10. Test System

10.1 Demographics

Species: Pig

Strain: Domestic Large White x Landrace

Source: Pig and Poultry Production Institute, University of Adelaide

Age at Treatment: Young Adult/Adult

Weight at Initial Treatment: 30-50 kg

Number of Animals 12 females (including 2 spares)

10.2 Husbandry

10.2.1 Housing

Prior to study commencement, the animals were delivered and held at the Animal Resource Centre

(GPARC), Gilles Plains, IMVS for acclimatisation and pre-surgery assessment. The animals were

group housed, then for bait-feed training, five animals were housed individually in pens. The pens

conform to accepted Australian standards for the care of research pigs.

10.2.2 Quarantine

There were no quarantine requirements for this study.

10.2.3 Acclimatisation

The animals were acclimatised for at least three days to become familiar with their surroundings and

the bait prior to intervention.

10.2.4 Environment

The pigs were housed at ambient temperature. There was no direct control of relative

humidity. Animals were kept in natural light. Natural ventilation in the pig holding area

eliminated urea and odour.

10.2.5 Food

A commercial diet for growing pigs was fed to the animals daily after the animals had consumed

their bait.

10.2.6 Water

The pigs had access to tap water (unfiltered) ad libitum.


10.2.7 Contaminants

There were no known contaminants in the food, water, or contact bedding that would be expected to

interfere with this study.

10.2.8 Identification

Each animal was uniquely numbered from 1-10. This number was sprayed onto the animal’s back,

and posted on the animal's enclosure. Any replacement animal was identified by the original

animal’s number followed by a letter, beginning with A, to indicate that it is a replacement animal.

The identification 1B would be used if animal 1A needed to be replaced. The study number and

animal number were recorded on all raw data related to each animal collected during the study.

10.2.9 Initial Health Status

All animals had a veterinary health check at the time of assignment to the study. Animals deemed

healthy by the Veterinarian were assigned to the study.

10.2.10 Spare and Replacement Animals

Spare animals were used to replace any study animal found not suitable for surgery. The spare

animals did not undergo cannulation of the carotid. No animal was replaced once five treatment

animals had been successfully dosed.

10.2.11 Restraint

Ketamine (10 mg/kg, intramuscularly [IM]) and manual restraint was used, as necessary, to

facilitate animal handling and performance of the technical procedures. The drug dosages were

documented in the raw data. Additional sedation was be achieved with isoflurane inhalant

anaesthetic delivered through a facemask. All restraint was performed according to the appropriate

IMVS SOPs. All animals were fully recovered from sedation prior to the humaneness assessment

commencing.

10.2.12 Disposal

The disposal of dead study animals was in accordance with the test facility’s standard operating

procedures. Spare animals not required for the study were humanely killed.


11. Experimental Methodology

11.1 Animal Assignment

Five animals each were randomly assigned to either the treatment or control group.

11.2 Fasting

Fasting overnight (≥8 hours) was conducted prior to anaesthesia. Food, including any dietary

supplements, was withheld. Water was not withheld. When the animals had recovered from surgery

they were fed, but were fasted again overnight prior to receiving experimental bait.

11.3 Anaesthesia

The animals were pre-medicated with Ketamine (10 mg/kg, IM). They were anaesthetised with

isoflurane, delivered through a facemask, to effect a suitable plane of anaesthesia to enable tracheal

intubation. The animals were intubated and maintained in anaesthesia with isoflurane inhalant

anaesthetic. A surgical record sheet was completed and kept on file for each animal. Drugs for

appropriate anaesthetic management were available for administration, if indicated. The drug,

dose, route, and site of administration was documented in the procedure records.

11.4 Animal Preparation

The animals’ hair was removed from the access site(s). The vascular access site was prepared for

aseptic procedures by first washing the area with povidone-iodine scrub solution to remove all

detritus, followed by wiping the area with 70% ethyl alcohol, which was allowed to dry.

Betadine™ solution was applied to the area. The area was appropriately draped to maintain a

sterile field.

11.5 Surgical Interventional Procedure

11.5.1 Insertion of Arterial Access Line for Blood Collection

When the animal was anaesthetised a cutdown procedure to isolate the carotid artery was performed.

A PVC cannula was inserted into the artery. The cannula was stitched around to the back of the

neck. The catheter was used for all blood sampling.

11.6 Postoperative Procedures and Animal Observations

11.6.1 Recovery

The animal was allowed to recover from anaesthesia and returned to its pen with access to food and

water. Postoperative bleeding was not anticipated, but if problems occur, the veterinary staff was


notified. All postoperative procedures were performed in compliance with IMVS SOPs, as required

throughout the in vivo portion of the study.

11.7 Administration of Bait

The pigs were given control baits for three days prior to their surgery to habituate them to the baits.

Following catheter placement surgery, the animals were allowed 24 hours to recover from

anaesthesia. They were then presented with either treated or control bait(s). All animals were

watched until they had eaten the bait, or showed no desire to eat any more.

11.8 Monitoring Procedures

11.8.1 Post-Consumption of Bait

The animals were closely monitored for clinical signs of distress. All observations were recorded.

11.9 Blood Sample Collection and Processing

Blood was collected via the surgically placed access line. Blood volumes represent whole blood and

are approximate amounts. Following collection and processing, samples were transferred to the

appropriate laboratory for analysis. Samples for clinical pathology analysis were collected from all

animals.

For blood sample collection via the indwelling cannula, an appropriate dead volume of blood was

withdrawn and retained. Following this, 1-10 mL of blood were withdrawn in a fresh syringe and

transferred to the appropriate tubes for analysis as per Table 1. The retained blood was replaced,

followed by a cannula flush with 5 mL of saline.

11.9.1 Methaemoglobin

Arterial blood samples (approximately 2.0 mL) were obtained (in blood gas syringes) at

specified times (Table 1 below). The samples were analysed immediately after collection using a co-

oximeter (OSM3, Radiometer, Copenhagen).


Table 1. Nominal blood collection schedule for all pigs. Sample collection increased or decreased depending on the clinical picture or methaemoglobin (MetHb) results.

Time Point – Post-Bait (mins) Clinical Pathology MetHb

Baseline (Pre-Bait) X X

+10, +20, +30, +40, +50 X

+60 X X

+70, +80, +90, +100, +110 X

+120 X X

+130, +140, +150, +160, +170 X

+180 X X

11.9.2 Clinical Biochemistry

Samples for clinical pathology (approximately 8 mL) were collected into a tube containing a clot

activator at the nominal times above (Table 1). Sample collection increased or decreased depending

on the clinical picture or methaemoglobin (MetHb) results. The blood samples were centrifuged and

the serum evaluated for: electrolytes, (sodium, potassium, chloride, bicarbonate), anion gap

(calculated), glucose and cortisol. Samples (approximately 2 mL) were also collected into

fluoride/EDTA tubes for plasma lactate analysis at the same times as the serum samples.

11.9.3 Humane Killing of Animals Not Affected by Bait.

It was expected that all animals in the specified weight range would not survive longer than four

hours if all of the bait was consumed. Animals still alive four hours after bait consumption, or

animals with two consecutive methaemoglobin levels lower than the previous would be

administered a suitable lethal compound (LethabarbTM), to induce death. Death was confirmed by

cessation of respiration and lack of palpable heartbeat. When all of the test-bait animals had died on a

particular day, the control animals were humanely killed.

12. Data Presentation

Data will be presented as individual values by animal. The following assessments will be reported:

a) Blood concentrations of specified parameters

b) Clinical assessment


13. Results

13.1 Surgical Procedure

All surgical procedures for the placement of the catheter into the carotid artery went well. One pig

had to have her catheter modified post-surgery. The exteriorised catheter was cut/cleaved, requiring

a joiner to be inserted. This pig was allocated to the control group to reduce the impact of any

possible blood sampling difficulties.

13.2 Allocation of Animals

The testing was conducted over two days. On the first testing day (group 1) two animals were

allocated to the toxic bait group (pigs 1 and 5), and three to the control group (pigs 2, 3 and 4). The

animals in the toxic bait group ate all of their non-toxic bait during the trial phase. On the second

testing day (group 2), there were two control animals (pigs 6A and 10), and three treatment pigs (7,

8 and 9). Pig 6A was a replacement for pig 6, who appeared to have a problem with her hind legs

when not on deep litter. All pigs ate the non-toxic bait during the trial, but pig 4 was fasted the

previous night, and then would only eat it if presented to her in small portions.

13.3 Bait Administration and Consumption

Table 2 shows the type and amount of bait administered and consumed by each animal in the study.

All animals received the same non-toxic bait. The group 1 animals (1-5) were presented with 2 baits,

and animals 1 and 5 received toxic baits manufactured in January 2009. The group 2 animals (6A-

10) were presented with one bait, and animals 7, 8 and 9 received toxic bait manufactured in March

2009.

Table 2. Bait administration to pigs.

Pig Group Weight(Kg)

Type of Bait

Bait Administered

(g)

BaitConsumed

(g)

Sodium Nitrite

consumed (g)

Dose Sodium Nitrite

(mg/Kg)1 1 44 Toxic 400 171 17.1 3892 1 38.5 Non-toxic 400 400 0 03 1 50 Non-toxic 400 230 0 04 1 44 Non-toxic 400 273 0 05 1 41 Toxic 400 337 33.7 8226 2 43.5 Non-toxic 200 173 0 07 2 35 Toxic 204.8 169.2 16.5 4728 2 39.5 Toxic 215.1 76.3 7.07 1799 2 35.5 Toxic 214.3 214.3 20 56310 2 45.5 Non-toxic 200 200 0 0


13.4 Methaemoglobin Concentrations

All animals that consumed the toxic bait had methaemoglobin concentrations greater than 10%

within 20 minutes. Figure 1. shows the individual methaemoglobin results for the animals that were

administered toxic bait, and Figure 2. shows the mean (+SEM) methaemoglobin results for the toxic

and non-toxic bait groups. Agonal breathing was associated with methaemoglobin levels above 69%

(4 of 5 pigs), while death occurred with methaemoglobin levels of 77.8-91.5%.

Figure 1. Individual methaemoglobin levels for animals fed toxic bait. Times are post-consumption of bait.

Figure 2. Mean +SEM % methaemoglobin over time for toxic and non-toxic baits. N=1-5 for toxic baits, for control bait, N=5 to 70 mins, then N=3 for all.


13.5 Clinical Pathology

There were no changes observed in any animal for renal function tests (Appendix 1). Figure 3

shows the changes in lactate over time, while Figure 4. shows the same data for cortisol. Pig 2

(control) was very sensitive to her neck being touched (necessary for blood collection), and was

agitated for the duration of the experiment.

Figure 3. Individual plasma lactate concentrations over time (post-bait consumption). Red denotes toxic bait, blue denotes non-toxic bait consumed.

Figure 4. Individual serum cortisol concentrations over time (post-bait consumption). Red denotes toxic bait, blue denotes non-toxic bait consumed.


While serum cortisol concentrations were increased in all treated animals, four of the five toxin-

baited animals showed signs of distress (gasping, paddling) for only 5-10 minutes prior to evidence

of reduced consciousness (dilated pupils, reduction or loss of reflexes). The pig which consumed the

lowest dose of nitrite showed these signs for approximately 20 minutes before losing consciousness.

It is not possible to ascribe a welfare index to changes in cortisol concentrations when a metabolic

insult such as methaemoglobinaemia is induced. Methaemoglobinaemia causes substantial

stimulation of the hypothalamo-pituitary-adrenal axis by way of a developing cerebral hypoxia and

changes in alertness. In this context cortisol is a non-specific indicator of overall stimulation and

cannot be(easily) directly related to welfare outcomes.

13.6 Clinical Assessment

The animals fed the non-toxic bait remained normal throughout with the exception of Pig #2, who

was agitated, particularly when her neck was touched. It was thought that the suture attaching the

catheter-holding bag to her neck may have been too tight.

All animals fed the toxic bait died. Four of the five died within 64±13 min (SEM) of bait

consumption, the remaining pig (pig number 8 which consumed the lowest dose) died within three

hours of bait consumption. Table 3 shows the dose of sodium nitrite and time to death for the five

pigs fed toxic bait, and the time for major clinical signs to develop. These data are summarised in

Figure 5. A more detailed summary of clinical records is shown in Appendix 3.

Table 3. Dose of sodium nitrite and time taken (min) post-consumption of bait to develop major clinical signs and death in the five pigs fed toxic bait.

Time to occur (min)Pig Dose Sodium

Nitrite (mg/Kg)Loss of righting reflex

Vomiting Paddling/Thrashing

Agonal breathing

Death

1 389 38 - 81 96 1005 822 36 36/41 44 48 547 472 20 - 26 28 398 179 134 137/139 156 172 1779 563 48 43 53 - 64


0 10 20 30 40 50 60 70 80

Time since bait consumed (mean ± SE)

Death

Terminal symptoms / Loss of consciousness

Labored breathing

Emesis (50% of pigs)

Unsteadiness

Fig. 5 Symptoms of sodium nitrite toxicosis in four pigs involved in the independent welfare assessment. A fifth pig received a very marginal dose and took 3 hours to die, but showed no symptoms for the first 2 hours.

14. Conclusion

The nitrite containing toxic baits fed to the pigs in this study were efficacious and resulted in an

apparently humane death. Biochemical changes other than the rise in lactate and cortisol, were not

different between test and control animals. The rise in blood lactate concentration was consistent

with the level of methaemoglobinaemia. Skeletal muscle metabolism becomes anaerobic as oxygen

debt develops because of the reduced oxygen carrying capacity of red blood cells containing

methaemoglobin. Further studies would be required to determine which organs contribute most to

the lactate load. Lactate at 25 mmol/L is not in itself associated with pain or discomfort, however.

The rise in serum cortisol was significant compared to baseline values in the test animals, giving an

8-10-fold increase after consumption of bait. It is of interest that one of the controls had significantly

elevated cortisol levels, and this was attributed to stress associated with soreness of the neck,

exacerbated by blood collection.

It is the opinion of the authors that the development of methaemoglobinaemia as a result of sodium

nitrite ingestion leads to a state of unconsciousness without a prolonged preliminary excitatory state.


The behavioural evidence suggests that death from nitrite intoxication is an acceptable method of

humane killing for large scale feral animal culling.

15. Acknowledgements

This study was funded by a grant from the Australian Department of the Environment, Water,

Heritage and the Arts. Day 2 of the trial was observed by Dr Bidda Jones, Chief Scientist, Royal

Society for the Protection of Cruelty to Animals, Australia, who also had input into the trial design.

Day 6 of the trial was observed by Mr Alan Learmonth from the IMVS Animal Ethics Committee.

We thank Frank Gigliotti (General Dogs Body P/L) for taking the methaemoglobin measurements,

and Dr Simon Humphrys (IACRC) for assisting in the Day 6 trials.


Appendix 1. Clinical Pathology ResultsPig 1 Pig 5 Pig 7 Pig 8 Pig 9

Toxic bait B/L +1hr +1.7

hr*

B/L +0.67

hr

+0.85

hr *

B/L +0.48

hr

+0.63

hr

B/L +1hr +2hr +2.7hr +2.9 hr +3hr B/L +1hr +1.2 hr

Sodium mmol/L 140 139 147 140 141 146 139 141 142 140 142 142 140 141 148 143 142 145

Potassium mmol/L 4.5 4.7 7.6 4.5 7.2 9.3 4.7 7.9 7.5 4.2 4.4 4.5 4.3 5.1 6.2 3.1 6.1 6.6

Chloride mmol/L 103 104 106 102 104 106 104 107 108 103 105 105 103 103 106 116 108 106

Bicarb mmol/L 29 28 7 28 30 10 27 16 6 24 30 28 13 6 2 16 15 6

Anion gap mmol/L 12 12 42 14 14 39 13 26 36 17 11 14 28 37 46 14 25 40

Glucose mmol/L 4.1 4.4 12.4 4.4 5.0 14.6 4.4 10.6 12.0 5.1 4.8 5.5 15.8 21.4 21 3.2 8.7 13

Urea mmol/L 3.6 3.7 4.6 2.8 3.5 3.8 2.8 3.6 3.8 2.8 2.8 2.9 3.4 3.5 3.7 2.7 3.8 4.1

Creatinine mol/L 83 89 116 82 98 128 87 119 127 72 74 76 90 98 112 40 87 98

Lactate mmol/L 0.6 2.2 22.6 0.7 5.8 21.1 0.70 12.6 21.2 0.8 0.7 2.3 14 19.4 24.5 0.2 12.3 20

Cortisol nmol/L 47 358 435 68 327 451 94 282 385 63 213 278 261 328 350 41 262 357

Pig 2 Pig 3 Pig 4 Pig 6A Pig 10Non-toxic bait B/L +1hr +2hr B/L +1hr B/L +1hr B/L +1hr +2hr +3 hr B/L +1hr +2hr

Sodium mmol/L 140 142 141 141 142 142 143 141 140 141 143 140 141 142

Potassium mmol/L 4.7 4.5 4.5 4.4 3.9 4.3 4.0 4.3 3.7 3.9 4.2 4.3 4.0 4.4

Chloride mmol/L 102 101 100 101 100 102 102 102 99 99 101 104 103 103

Bicarb mmol/L 28 30 33 29 36 30 36 30 31 31 33 26 28 29

Anion gap mmol/L 15 16 12 15 10 14 9 13 14 15 13 14 15 14

Glucose mmol/L 4.8 5.0 4.7 4.5 5.0 5.1 5.6 4.0 4.3 4.8 5.1 4.3 5.0 4.8

Urea mmol/L 3.0 2.6 2.8 3.7 3.3 4.4 3.8 2.2 2.3 2.5 2.8 3.1 2.9 2.9

Creatinine mmol/L 71 71 69 81 74 106 92 80 80 78 79 91 96 88

Lactate mmol/L 1.7 1.3 1.8 1.3 2.8 0.7 1.7 0.9 1.3 1.2 1.8 0.7 1.7 1.1

Cortisol nmol/L 283 417 201 53 47 104 85 78 206 145 126 52 49 40

* denotes peri/post-mortem sample


Appendix 2. Methaemoglobin Results (%)

Time (mins)

Pig # B/L 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180

Toxic bait

1 0.5 11.7 21.3 32.8 43.4 51.5 60.2 68.7 86.5

5 0.4 16.4 31.0 46.0 60.4 77.8

7 0.5 35.7 56.7 73.3 82.4

8 0.5 6.0 11.4 16.1 21.4 28.0 35.7 42.5 48.6 54.0 58.5 62.7 66.0 69.1 70.8 74.8 76.4 78.8 83.5

9 0.7 5.8 18.9 33.2 47.3 60.4 70.4 79.9

Non-toxic bait

2 0.4 0.5 0.3 0.3 0.7 0.4 0.5 0.4 0.4 0.3 0.3 0.3 0.3

3 0.6 0.4 0.4 0.5 0.4 0.3 0.2

4 0.5 0.4 0.5 0.5 0.4 0.2 0.2

6A 0.4 0.7 0.7 0.5 0.6 0.6 0.6 0.6 0.5 0.4 0.5 0.6 0.5 0.5

10 0.4 0.5 0.5 0.5 0.4 0.3 0.4 0.4 0.4 0.3 0.4 0.4 0.3


Appendix 3. Individual Clinical Records Summary

Pig

IDDay

Time

(mins)Observation

1 2

0 44 kg HR 120 bpmGiven 2x200g toxic baits. Ate 171 grams, didn’t consume all bait.

25 Blood starting to turn dark38 Fell over, tried eating more food. HR 150 bpm47 Started to make snorting sounds53 Very lethargic, HR 160 bpm73 Heavy breathing81 Frothing at mouth, heavy panting, thrashing around, limb stiffness, rolled86 Dilated pupils, very heavy breathing90 Very weak thready pulse, agonal breathing, HR 96 bpm, evidence of heart block91 Cardiac output (on auscultation – 1 cardiac sound) tongue blue, agonal breathing93 Pupils dilated96 agonal breathing, pupils dilated,

100 No heartbeat, DEAD 1 hr 40 mins

5 2

0 41 kg HR 108 bpmGiven 2x200g toxic baits, ate 337grams

21 Pale HR 192 bpm, fully alert31 Little wobbly on feet, pale, HR 150 bpm36 Lying down, chomping, vomited, started paddling, grey with bluish nose.41 Reduced consciousness, vomited

44 Thrashing, unresponsive, vocalisation, dribbling, still reacts to corneal touch but no concept of it happening, pupils glazed and dilated, snoring sounds.

48 Agonal gasps, chomping, thrashing. Unconscious.50 HR 160 bpm, evidence of heart block54 DEAD 54 minutes


Pig ID Day Time

(mins) Observation

76

0 35 kg HR 104 bpmGiven 1 bait (204.8g), Ate 169.2 grams

3 Pallor evident, lying down, HR 100 bpm thready pulse6 Stood for 30 seconds with encouragement, lying down again, eyes closed sleepy looking9 Up on haunches, wobbly, unsteady10 HR 116 bpm, lying down distinct pallor20 Unable to rise, looking grey, HR 132, grunting23 Trying to get up but can’t, leapt into air after twitching26 Grunting, slight paddling28 Deep breath, agonal breathing29 Paddling, corneal reflex present30 Puffing, eyes shut32 Pupillary reflex, eyelid reflex still present33 Pupillary reflex absent, eyelid reflex still present36 Unresponsive to noise39 Inaudible heart beat, DEAD 39 mins

Pig ID Day Time

(mins) Observation

8 6

0 39.5 kg HR 92 bpmFed one toxic bait (215.1g). Ate 76.3 grams

3 Looks normal HR 104 bpm13 Normal looking45 Lying down and grinding teeth, given non-toxic bait ate all47 HR 13255 Kneeling56 Lying down, looks tired, eyes closed67 Reluctant to stand but rising unassisted77 HR 152 bpm, very quiet, remained like this for some time.

122 Chomping and salivating127 Slightly pale and bluish snout134 Attempting to stand but unable, tremors in hind legs,137 Smacking lips and frothing at mouth, vomited, stood up then lay down, grunting139 Stood unassisted, vomited and laid down again, (propped on front legs then flopped down)


6

140 HR 176 bpm, corneal reflexes normal

8

144 Tremors and shivering151 Laid on side (first time), attempted to rise152 HR 154 bpm, chomping and laboured breathing155 Urinating, lip smacking, neck spasms156 Paddling with head pulled back, rigid legs162 Still rigid, semi – paddling, tremors, lip smacking, grunting with eyelid and pupillary reflexes normal167 HR 148 bpm, regular heart beat, eyelid and pupillary reflexes present and normal, panting increased, increased paddling of hind legs172 HR 148 bpm, Increased paddling, very vocal, thrashing, gasping, agonal breathing174 Heart stopped then started again, reflexes gone, not conscious176 HR inaudible177 DEAD 2 hours 57 minutes

Pig ID Day Time

(mins) Observation

9 6

0 35.5 kg HR 110 bpmAte 214.3 grams toxic bait (ate all)

12 Ate some food looks OK14 HR 120 bpm, greyish pallor, quiet16 Lying down, able to rise unassisted21 Lays down, able to stand but wobbly22 HR 124 bpm24 Pale and grey, lying down, very quiet26 HR 12427 Got up snuffled around then lay down in 20-30 seconds28 Smacking lips30 Unsteady on feet32 Lying down36 Up on feet and walking then lay down39 Sleepy and shutting eyes41 Stood unassisted, smacking lips42 HR 164, slight frothing at mouth


Pig ID

Day Time(mins)

Observation

9 6

43 Vomited, bait material present but not a lot, walking but unsteady46 urinated48 Fell over49 Tried to rise but fell over and leant against cage, lip smacking, grunting50 Tried to rise but fell over, dilated pupils, all reflexes present51 Slow pupillary reflex but present52 Muscle rigidity, tremors in neck53 Vigorous paddling54 HR 140 bpm, prostrate and splaying of limbs, pupillary reflex present55 Panting and paddling, chronic contractions56 Vigorous paddling, sluggish pupillary reflex, eyelid reflex present57 Vigorous paddling, vocalising, very sluggish eyelid reflex, pupillary reflex absent59 Eyes closed, no response to noise, very slight eyelid reflex62 Ventricular fibrillation63 HR 160 bpm, Very weak heart beat64 No heart beat, no reflexes, DEAD 1 hour 4 minutes

Pig ID

Day Time(mins)

Observation

2 2 0-2.25 hr 38.5 Kg, ate 2x200g non-toxic baits. Very grumpy when neck touched. +1 hr HR 140 bpm (active), +1.5 hr 96 bpm (resting). Humanely killed 2 hr 15 mins. Normal throughout (apart from agitation when neck touched).

3 2 0-1.25 hr 50 Kg, ate 230/400g non-toxic baits. HR 100 bpm. Normal throughout. Humanely killed 1 hr 15 mins.4 2 0-1.1 hr 44 Kg, ate 273/400g non-toxic baits. HR 100 bpm. Normal throughout. Humanely killed 1 hr 5 mins.

6A 6 0-3.5 hr 43.5 Kg, ate 173/200g non toxic bait. HR 92 bpm. +45 min HR 100 bpm. Normal throughout (slept most of time). Humanely killed 3.5 hr.

10 6 0-2.13 hr 45.5 Kg. Ate 200g non-toxic bait. HR 101. Normal throughout. Humanely killed 2 hr 8 minutes.


Reference

Australian Code of Practice for the Care and Use of Animals for Scientific Purposes, 7th edition,

National Health and Medical Research Council, Australian Government, 2004


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