Guidelines for the Welfare of Livestock from which Blood is Harvested for Commercial and Research Purposes

Animal Welfare Advisory Committee
c/- Ministry of Agriculture
PO Box 2526
Wellington

April 1996

Table of Content

1. Introduction

2. Legislation

3. Blood Harvesting from Livestock

4. Adverse Effects

5. Blood Harvesting Procedures Not Reviewed

6. Guidelines

6.1 Husbandry
6.2 Donor Animal Type and Temperament
6.3 Facilities and Equipment
6.4 Bleeding Technique
6.5 Volume and Frequency of Bleeding
6.6 Monitoring
6.7 Monitoring Antiserum Donors

7. Blood Harvesting from Pre-term Calves

7.1 Calves Transported to Slaughter Premises
7.2 Slaughter

References

1. Introduction

Up until now, there have been no national guidelines to safeguard the welfare of animals from which blood is harvested for commercial or research purposes. The following guidelines have been prepared for use by Animal Ethics Committees involved in consideration of these practices and for companies and research workers involved in or planning to enter this type of business.

Guidelines are given for livestock including induced calves.

2. Legislation

The 1983 amendment to the Animals Protection Act 1960 places controls on the "manipulation" of animals including blood sampling of (harvesting from) live animals. In 1987, a further amendment to the Act extended the interpretation of manipulation to include the use of animals for antiserum production.

Anyone who intends to collect blood from an animal must first comply with their institutional Code of Ethical Conduct and submit a protocol application to be approved by their institutional Animal Ethics Committee. If there is doubt whether or not a procedure requires ethical approval, advice can be obtained from the institutional Animal Ethics Committee or the National Animal Ethics Advisory Committee. Approval and on-going monitoring of each project must be in accordance with the National Animal Ethics Advisory Committee guidelines for Animal Ethics Committees.

Blood collection protocols submitted to Animal Ethics Committees should document details of animal identification, husbandry and disease control. Individual animal health records should be maintained and the training of personnel in blood removal techniques should be recorded.

The company or research institution and its veterinarian shall ensure to the Animal Ethics Committee's satisfaction that the design of the facilities, the procedure for handling of stock during bleeding and/or immunization, the equipment used, the training and competence of personnel and the procedures to monitor the animals' health and welfare comply with the guidelines below.

3. Blood Harvesting from Livestock

Blood is harvested from horses, cattle, sheep and goats in New Zealand for research purposes and for commercial purposes. Contemporary animal welfare requirements are more stringent than they have been in the past, and it is appropriate that there should be guidelines to protect the welfare of animals used in blood harvesting operations.

For the purposes of this document, blood harvesting is defined as the removal of a relatively large volume of blood over a short period of time, i.e. more than would usually be requiredfor routine diagnostic tests. (As a rule of thumb diagnostic testing would be up to 1% or more of blood volume in a period of 24 hours). If there is any doubt about whether or not a procedure constitutes harvesting, it should be referred to the institutional Animal Ethics Committee.

In recent years the number of companies collecting blood for commercial purposes has increased. Given New Zealand's favourable disease status it is expected that this trend will continue as demand from overseas markets grows, particularly from the USA and Japan. The blood harvesting industry is well suited to New Zealand and has great potential. The products are agriculturally based, value-added and export-oriented.

It is in the interests of good science as well as the welfare of the animals used that stress on the animals is kept to a minimum. If the process of harvesting is stressful for the animal because of handling, pain or discomfort, physiological changes occur which may compromise the quality of the product obtained.

In the text the word "donor" is used to indicate an animal from which blood is collected. "Must" and "shall" are used to indicate minimum standards.

3.1 Uses of Harvested Blood

Whole blood, serum, specific antiserum, "aseptic blood" and a variety of blood products are used for research purposes, in diagnostic tests and in cosmetic and pharmaceutical products including vaccine production, both in New Zealand and overseas. Some of the blood is used in techniques which replace those using laboratory animals; so the use of harvested blood can be an alternative to the use of live animals.

Specific antiserum is prepared from blood from hyperimmunised donors. The specific antibodies are used in biotechnology, medical and veterinary research.

"Aseptic blood" is collected under sterile conditions so that the blood remains free of contaminant bacteria. "Aseptic blood" is used in the preparation of blood agar plates for microbiology and in virology tests.

Foetal calf blood serum is used in biological tests, tissue culture and in the manufacture of vaccines such as those for canine distemper and feline enteritis. The blood from foetal and neonatal calves which have not been fed colostrum lacks the antibodies acquired from colostrum. Antibodies in blood can interfere with viral growth processes in the manufacture of vaccines and can alter the results of some diagnostic tests.

4. Adverse Effects

If too much blood is drawn too quickly or too frequently without replacement, animals may develop hyperpnoea (deep and rapid breathing) and may go into a state of hypovolaemic shock. In the longer term the removal of to much blood causes anaemia, muscle weakness, increased susceptibility to cold, reduced exercise tolerance and ill-thrift, particularly if management andnutrition are suboptimal.

If 15% to 20% of the blood volume is removed, cardiac output and blood pressure will be reduced. Removal of 30% to 40% can induce shock.

Inexpert bleeding techniques can result in bruising around the vein, haematoma formation and/or inflammation at the site.

If irritant adjuvants are used, local areas of inflammation and granulation and/or abscessation may occur. Freund's complete adjuvant consists of a suspension of killed mycobacteria in an oil base, and it is particularly irritant. In addition to local lesions at the site of inoculation, systemic granulomata can develop following inoculation of relatively large volumes of Freund's adjuvant.

Long term hyperimmunisation can cause loss of condition as a result of amyloid accumulation in the liver and kidneys. Repeat inoculation of some antigens can cause anaphylactic shock.

5. Blood Harvesting Procedures Not Reviewed

The welfare issues associated with cannulation are not discussed in this document.

If a very large proportion of circulating blood is to be harvested, collection immediately after slaughter is preferable on welfare grounds to preslaughter collection. No procedure may be carried out which involves terminal exsanguination, i.e. removal of circulating blood leading to death, unless it has been first comprehensively reviewed and approved by the institutional Animal Ethics Committee.

Some commercial operations obtain blood from animals after they have been routinely killed during processing of carcases at slaughter houses. For example blood may be collected from sheep and cattle after stunning and before bleeding-out by collection into containers. Contamination of blood by saliva and ruminal contents can be avoided by collecting blood through a needle or cannula inserted into the anterior vena cava (a major vein in the chest).

Foetal calf blood may be obtained in a similar way. When after slaughter of a cow, evisceration reveals a uterus with a near full term calf, blood may be collected from the heart of the calf by cannulation. This process occurs about 20 to 30 minutes after the cow has been killed and the foetus will have been dead for almost as long. These are post mortem procedures which do not require Animal Ethics Committee approval.

However one procedure which is considered below is the harvesting of blood from newly-killed, induced, pre-term calves which have not been fed colostrum or milk. Because the calves are not fed milk or colostrum after birth in order to provide suitable blood, the procedure is classed as a manipulation and must be approved by an Animal Ethics Committee.

It is theoretically possible to collect arterial blood, but there are considerable risks associated with such a procedure. Any such procedure must be comprehensively reviewed and approvedby the institutional Animal Ethics Committee.

6. Guidelines

6.1 Husbandry

The day-to-day management of animals used in harvesting procedures must be the responsibility of an experienced stockperson, who must ensure the provision of appropriate nutrition, shelter, parasite control, feet and teeth care.

A veterinarian must supervise the overall health of the animals. The animal health programme should be documented.

Goats, young sheep and horses are particularly susceptible to gastroenteric parasitism. Parasitism should be controlled by regular treatment with an effective anthelmintic. The requirement for treatment and its efficacy can be assessed using faecal egg counts before and after treatment.

Donor animals must be fed more than standard maintenance rations, at a level related to the intensity of the harvesting regime. Iron supplements must be given to donors supplying relatively large volumes of blood. Food supplements must be provided unless sufficient good quality pasture is available. In winter, food supplements should include concentrates as well as hay. When concentrates are fed, they should be introduced and withdrawn from the diet over a period of a few days. A record should be kept of the supplements provided.

Trace element supplements must be provided if appropriate, and veterinary advice should be obtained if there is any doubt as to what is appropriate.

Donors must have access to fresh clean water at all times.

Experienced stockpersons shall carry out regular appraisals of the animals' wellbeing and body condition. If any animal appears unwell or if its body condition is poor corrective action should be taken.

6.2 Donor Animal Type and Temperament

All animals used as repeat donors must be individually identified for monitoring purposes, and records must be kept of their health, body weight, condition, food and food supplements supplied, animal remedies given and blood volume harvested.

The animals used must be more than 6 months old and preferably fully grown adults. They must be in good body condition and in good health.

Horses should be at least 3 years old and at least 400 kg body weight. Horses with a body condition score of 3 or less must not be bled (see 'Code of Recommendations and Minimum Standards for the Welfare of Horses' for body condition score chart).

Sheep and goats should be at least 12 months old. Sheep should weigh at least 40 kg. Goats vary greatly in size according to breed, so a minimum weight cannot be given for the species, but they should be in good body condition.

Cattle should be at least 18 months old and should weigh at least 250 kg if 18 to 24 months of age and at least 300 kg if older.

If growing animals are used their diet must be nutritious, supplemented with iron, and fed at appropriate above-maintenance levels to allow for replacement of blood as well as steady growth.

The animals to be bled must be accustomed to handling so that they are relaxed and calm throughout the harvesting procedure. Excitement and fear can cause splenic contraction which results in altered blood parameters.

6.3 Facilities and Equipment

Facilities must be such that there is minimal risk of animals being injured by projections, wire or sharp corners.

There must be facilities and equipment on site to allow clinical examination of animals and to carry out packed cell volume and/or haemoglobin assays.

Since blood volume estimations are based on body weight measurements, accurate weighing crates or platforms should be provided. They must be provided when relatively large volumes of blood are collected, and/or when relatively large numbers of animals are used.

6.4 Bleeding Technique

The design of the bleeding facilities and methods of restraint must be such that the procedure can be carried out efficiently with the minimum of stress to the animals.

The bleeding process shall be carried out by a veterinarian or under veterinary supervision, ie by a lay person after appropriate training by a veterinarian.

Cattle and horses must be standing when bled.

It is preferable to bleed sheep and goats in the standing position, but where large numbers of sheep and goats are bled, it may be acceptable to strap them in lateral recumbency on tables. In this case the animals must have been fasted with access to drinking water for at least 6 to 24 hours before harvesting. The harvesting procedure should begin as soon as the animal is restrained, and animals should be kept under close supervision during the bleeding process to guard against inhalation of ruminal contents or the development of ruminal bloat. After release they should be allowed to return to their paddock at their own pace.

The skin over the sampling site may be clipped or shaved to facilitate placement of the needleand the site may be cleaned with disinfectant such as alcohol. It is important that time be taken to locate the vein accurately and that it be distended by gentle pressure before the needle is inserted.

A local anaesthetic should be administered carefully between the skin and vein if a large-bore needle (14 gauge or larger bore) is used.

A needle with as large a bore size as possible should be used to ensure efficient blood withdrawal without collapsing the vein, without causing haematoma formation and without causing blood pressure to drop too rapidly.

Immediately after removal of blood, all animals must have unrestricted access to water.

6.5 Volume and Frequency of Bleeding

For adult animals, not more than 15% of the estimated circulating blood volume should be removed in any 4-week period, ie 0.9% liveweight in cattle and sheep and 1.1% liveweight in goats and horses.

Circulating blood volume (litres) can be estimated from body weight (kg) using a conversion factor of 0.06 for cattle and sheep, 0.07 for goats and 0.075 for horses. As a guide, 1% of body weight is the weight of 16 to 17% of the circulating blood volume in sheep and cattle; about 13% in horses and about 14% in goats.

If more than 15% of blood volume is removed, consideration should be given to fluid replacement using lactated Ringer's solution with 5% dextrose.

In some circumstances and with the approval of the institutional Animal Ethics Committee, it may be acceptable to remove up to 25% of the blood volume in a 4-week period. The husbandry and nutrition of the animals must be appropriate for the intensity of the harvesting and full use must be made of the monitoring schemes outlined below.

Horses which are managed well seem to tolerate the removal of even larger volumes with no ill effects, and there are overseas reports of up to 8 litres of blood being collected from large horses (approximately 25% blood volume) at fortnightly intervals with no untoward effects. If harvesting on this scale is to be undertaken, the Animal Ethics Committee must then be certain that the welfare of the horses is safeguarded by appropriate husbandry, nutrition and monitoring.

For young animals, the volumes removed should be relatively less. For animals 6 months old, not more than 10% circulating blood volume should be removed, with incremental increases to the maximums above when fully grown (more than 12 months old for sheep and goats, 18 months old for cattle and 3 years old for horses).

In bleeding programmes scheduled to last over a year and in which relatively large volumes of blood are taken, consideration should be given to resting the donor animals for 4 weeksonce or twice a year, preferably in winter.

Any departure from these recommendations must first be approved by the Animal Ethics Committee.

6.6 Monitoring

The well-being of donor animals must be monitored by visual appraisal/clinical examinations and body weight measurements should also be used. For animals from which relatively large volumes of blood are taken (25% or more of blood volume in a 4-week period), body weights must be monitored and haematological parameters such as packed cell volume (PCV) must be shown to be in the normal range immediately before blood collection.

6.6.1 Visual Appraisal/Clinical Examination

Immediately before bleeding, every animal must be subject to close visual appraisal or examination by an experienced stockperson. No animal which appears light in condition or which shows malaise or any other sign of ill-health shall be bled. The animals should be monitored after release for evidence of ill-effects. Animals which show signs of ill-health at any stage should be examined by an experienced stockperson or veterinarian who will determine if treatment and modification of, or withdrawal from, the programme is necessary.

6.6.2 Body Weights

All animals must be in good body condition throughout the programme. The body weight of each animal should be recorded before the programme begins, and weights should be recorded before each sampling. If the weight of any animal falls significantly below its pre-programme level that animal shall be withdrawn from the programme.

Body weights must be recorded as above to monitor the well-being of animals from which relatively large volumes of blood are taken, i.e. >25% in a 4-week period.

6.6.3 Packed Cell Volume Assay

PCVs as well as body weights must be used to monitor the well-being of animals used to provide relatively large volumes of blood, i.e. >25% of the blood volume in a 4-week period.

Individual baseline PCV concentrations must be established for these animals before the start of the programme. If the baseline value of any animal does not lie within the normal range shown in the table below, the animal must be withdrawn from the programme.

Just before each bulk sampling, the PCV of each animal must be measured again. If a low PCV is encountered in any animal, blood must not be harvested from that animal. It should be not be bled for a further 6 weeks.

Each time blood samples are taken for PCV assay, the animals must be relaxed and calm, because livestock, especially horses, undergo splenic contraction when excited causing an increase in the PCV concentration. Some customers require blood with a PCV above a given level. In these cases it is imperative that the PCV is not artificially elevated by excitement or dehydration.

Any departure from these recommendations must first be presented to and approved by the Animal Ethics Committee.

Normal PCVs (haematocrit levels) (%) are as follows: [Duncan and Prasse (1986) Veterinary Laboratory Medicine]

Horses	32-48
Cattle	24-46
Sheep	27-45
Goats	22-38

6.6.4 Disposal

Any animal culled from a bleeding programme for whatever reason must be disposed of in a humane manner.

6.7 Monitoring Antiserum Donors

Visual appraisal, clinical examinations, body weights and haematological parameters such as PCV must be used as outlined above to monitor the well-being of antiserum donors. In addition, there are concerns relating to the hyperimmunisation procedure which must be addressed.

The inocula used to produce high antibody concentrations in donor animals can cause adverse reactions for a variety of reasons. The adjuvant used maybe irritant. Freund's complete adjuvant (FCA), which is an emulsion of mycobacterial cell walls in oil, is particularly irritant and can cause local granulomata, local ulceration and/or systemic granulomata. The antigen used can provoke a local reaction. Other factors which will affect the host's reaction include the volume given relative to the size of the animal, the site (e.g. brisket or rump), the route (e.g. subcutaneous or intraperitoneal), the interval between inoculations and the number of inoculations given.

At least twice a week for 4 weeks, the injection sites must be observed by an experienced stockperson and/or veterinarian for evidence of an adverse reaction.

If any injection site appears painful or ulcerated lesions develop veterinary advice must beobtained.

Animals must be withdrawn from the programme if they develop lameness or any other abnormal signs related to the inoculations, such as anorexia, lethargy, malaise, or loss of weight. Reinstatement shall be at the discretion of the veterinarian/Animal Ethics Committee.

Alternatives to the use of FCA and other irritant substances must be used wherever possible. For example, STM (Span Tween Marcol)* is an adjuvant with immunogenic properties equivalent to FCA but it is far less irritant. Freund's Incomplete Adjuvant (FIA) is less irritant than FCA. The use of FCA followed by FIA use in booster injections may be acceptable.

There must be sound scientific justification for the use of FCA.

• FCA must not be given intravenously or intraperitoneally;
• it must not be used more than once in any animal;
• it must not be used in horses;
• not more than 5 ml of antigen/emulsion should be used in total;
• the dose should be subdivided so that not more than 2 ml is given at each site.

Any departure from the above must first be presented in full to, and approved by, the Animal Ethics Committee. In particular the use of repeated doses of FCA must be justified by pilot trials that indicate that this regime gives a significantly better immunological response than use of FCA followed by FIA.

6.7.1 Disposal

Unless it has been determined by an expert in veterinary public health that animals which have been used as antiserum donors may re-enter standard farming systems, all animals at the end of each programme must be killed humanely and their carcases disposed of so that no part is used for human consumption.

7. Blood Harvesting from Pre-term Calves

Induced calves are those born up to 6 weeks prematurely to cows which have been induced to calve by administration of drugs. With the approval of the institutional Animal Ethics Committee, induced calves can be killed for commercial blood harvesting either on the farm or at a slaughter house. In both procedures, blood is collected from the heart after the calf has been rendered unconscious.

Unless calves are killed within 12 hours of birth, electrolytes and glucose (300-400 ml/feed) should be fed at 8 hour intervals from birth during daylight hours and on the morning of slaughter. The time from birth to slaughter must be less than 24 hours.

On the farm they should be kept in warm dry pens (not roadside bobby calf pens) with straw or sawdust on the floor until collected for transport or slaughter.

At all times, calves should be moved gently and carried or taken by trolley when possible.

7.1 Calves Transported to Slaughter Premises

The time from birth to slaughter should be a maximum of 36 hours, which means that only calves born in the 24 hours before collection are suitable.

The drivers and other persons handling the calves from the time they leave the farm should be trained to minimise stress on the calves. The driver should drive carefully to help prevent bruising during the journey.

The calves should travel either in individual plastic or fibreglass bins in compartments big enough to allow them to stand and lie comfortably, and designed to help prevent bruising or chilling. Non-slip flooring should be provided. Very small calves should be transported individually, while bigger near-term calves can be transported in compartments with no more than 3 calves per compartment.

Compartments, bins and holding pens at the slaughter house should be cleaned at least once a day.

The duration of the journey must not exceed 4 hours.

Calves must be killed on the day they leave the farm.

After arrival at the slaughter house, the calves should be killed as soon as possible.

Calves should not be washed unless very dirty. If cleaning is considered necessary, they should be gently washed in warm water, dried and replaced in their crates or a warm dry draught-proof waiting area for inspection.

Calves which do not pass ante-mortem inspection must be killed as soon as possible and they should be placed in warm, dry, draught-free pens until killed.

7.2 Slaughter

Calves must be killed by a captive bolt pistol and care should be taken to position the gun correctly (see 'Code of Recommendations and Minimum Standards for the Welfare of Animals at the Time of Slaughter at Licensed and Approved Premises'). A seizure characterised by tonic spasms and clonic convulsions is indicative of an effective stun. If there is any doubt about the efficacy of the shot, the calf must be restunned immediately. The process should besupervised and monitored by a veterinarian or MAF Inspector.

Good maintenance of the captive bolt pistol is essential.

Effective electrical stunning causes cardiac arrest and so electrical stunning is not suitable for the harvesting for blood from the heart.

References

Archer, RK (1965), Haematological Techniques for Use on Animals, Blackwell Scientific Oxford.

Duncan, JR and Prasse, KW (1986), Veterinary Laboratory Medicine, 2nd edition, Iowa State University Press.

Rose, M (1990), "Venepuncture: Problems and Pitfalls", ACCART News, vol 3 (4), pp 3-4

Higgins, A (1990), Animal Health Trust, Newmarket, England — Correspondence.

Canadian Council on Animal Care, Guidelines on Acceptable Immunological Practices (1991).

British Home Office, undated, Whole Blood Volume in Laboratory Animals

* Span Arlacel (Sigma) Tween (Serva) Marcol (Esso): Bakhout, A, van Gaalen, C, van der Heyen, PK (1981) A selected water-in-oil emulsion: Composition and usefulness as immunological adjuvant. Veterinary Immunology and Immunopathology 2: 491-500.

"Code of Recommendations and Minimum Standards for the Welfare of Animals Transported Within New Zealand", No. 15, November 1994, Animal Welfare Advisory Committee.

"Code of Recommendations and Minimum Standards for the Welfare of Horses", No. 7, February 1993, Animal Welfare Advisory Committee.