An Australian Perspective of TCO2 in Harness Racing

Dr Diane Ryan
Deputy Chairperson
Harness Racing New South Wales

Plasma Total Carbon Dioxide (TCO2)

The Australian Rules of Harness Racing states that a horse shall be presented for a race free of prohibited substances. Many registered, unregistered and illegal medications, substances and their metabolites are listed as prohibited substances under these Rules. A smaller number of naturally occurring substances is listed as being prohibited if a threshold level of the substance in a horse’s system is exceeded. Plasma Total Carbon dioxide (TCO2) concentration is one such substance.

The TCO2 concentration is the total amount of carbon dioxide that can be liberated from blood plasma. Carbon dioxide occurs naturally in the blood of horses at concentrations that vary between horses. A number of physiological factors can affect a horse’s TCO2. These factors include the horse’s sex and age, diet, water intake, time of day, climate and ambient temperature and exercise. Pathological conditions such as respiratory disease can also affect TCO2.

Alkalising Substances

Alkalising substances, including bicarbonate and related substances, can increase plasma TCO2. Exercise physiologists advise that the administration of these substances may assist the horse recovering from strenuous exercise. A large number of proprietary oral preparations containing alkalising substances are promoted to the horse industry. Alkalising substances can be used as buffering or bulking agents in other horse treatments.

TCO2 threshold

In the early 1990’s, the practice of ‘milkshaking’ came to the notice of racing authorities. The milkshake consisted of sodium bicarbonate, glucose and a number of other substances and was given to the horse via a stomach tube. Trainers believed that this practice improved the athletic endurance of the horse. In some cases, the ‘milkshake’ could cause colic, dehydration and diarrhoea and therefore be detrimental to the horse’s performance.

There was some concern that ‘milkshaking’ could change the pH of the urine and either inhibit or increase the excretion of certain drugs, reducing the chance of detection in a post race urine sample.

In an attempt to control this practice, a TCO2 threshold was set by the Australian Harness Racing Council. Horses with a TCO2 concentration above this threshold were considered to have received an excess of alkalising agent, whether as a milkshake or by another method, for the purpose of affecting the performance of the horse.

Initially the TCO2 threshold was set at 37mmol/l. After reviewing the actual TCO2 concentrations in racing standardbreds over a three-year period, the threshold was subsequently lowered to 35mmol/l in 1995.

Because of the laboratory method employed for measuring TCO2 concentration, each test result is reported as a range rather than a single result. The limits of the range are determined by the laboratory uncertainty of measurement. All results are reported as TCO2 result +/- uncertainty of measurement. A single uncertainty value is applied to all measurements even though the uncertainty of measurement can increase with the measurement of increasing TCO2 concentrations. The Australian Racing Laboratories set the uncertainty measurement at 1.2mmol/l to 1.4mmol/l.

In the last 18 months, there was concern raised by trainers and their advisers that an increasing number of horses were exceeding the 35mmol/l threshold. The penalty imposed on most violations of the Rule was a 12-month disqualification.

The majority of the violations appeared to be occurring in one Australian State (South Australia). There was considerable debate about the reasons for the TCO2 concentration increase including physiological effects, such as pre race excitement elevating TCO2 concentrations, and doubts about the laboratory testing process for TCO2.

TCO2 Review

The Australian Harness Racing Council created a subcommittee to review the TCO2 situation in Australia under the following Terms of Reference:

  1. Level of laboratory uncertainty factor in the testing process
  2. Integrity of the process
  3. The mean TCO2 level of the harness racing population
  4. The mean level of untreated harness racing population
  5. Has the mean level increased and why?

There were three information-gathering strategies available to the subcommittee.

  1. Submissions from technical experts and the general harness racing community. 30 technical and general submissions were received. A number of published and unpublished papers were submitted.
  2. Biometrical analysis by an independent biometrical service of all pre race TCO2 data since 1995. Approximately 60,000 individual pre race TCO2 results from all Australian states were analysed.
  3. A mail survey of the feeding practices of the Australian Standardbred. A total of 525 trainers returned information on the feeds and additives given to the horses in their stable.

The subcommittee’s findings on TCO2 can be categorised under three main headings:

  1. Sampling influence
  2. Laboratory influence
  3. Trainer influence.

Sampling influence

The collection and storage of the blood sample for TCO2 testing is critical for the accuracy of the test result. Any variation in the collection method can affect the TCO2 concentration. In most cases, adverse handling of the blood sample will decrease the TCO2 concentration. These handling variations that can occur include:

All Australian States now effectively have the same protocol for collection of blood samples at the racetrack. The horses are sampled before they are exercised or ‘warmed-up’ and within 90 minutes before race start.

A Subcommittee has been formed to further review and refine the sampling protocol.

Laboratory influence

The type of equipment used to measure the TCO2 concentration at the laboratory should be consistent between laboratories. At present, all Australian Racing Laboratories use the Beckman Synchron ELISE to measure TCO2 concentration.

The protocol for analysing the blood sample by the equipment should be the similar between laboratories. All Australian laboratories use the Australian Scientific Enterprise Pty Ltd (ASE) standards to calibrate the Beckman to the range of TCO2 concentrations that can occur in the racehorse. A control sample (Verichem) is used to determine the effectiveness of the machine calibration before and during each period of sample testing.

The laboratories are currently reviewing the uncertainty of measurement and it may change in the future.

The Subcommittee is collaborating with the laboratories to further refine the current protocol and make recommendations on any future improvements.

Trainer Influence.

The Diet Survey revealed that ongoing education of the trainer and their advisers on the effective use of feed additives was essential.

The majority of trainers used a variety of products containing alkalising agents yet did not recognise that these products contained substances that could increase the TCO2 concentration of their horses. The multiple use of registered and unregistered products containing these substances in the horse was evident. The effect of these practices on the horse’s resting TCO2 is not known.

The practice of withdrawing water from horses for a period of time before race start was reasonably common. This practice can increase TCO2 concentration. The combined effect of adding alkalising substances to feed and pre-race withdrawal of water could increase the horse’s TCO2 towards the threshold limit.

An equine nutritionist is reviewing the results of the Diet Survey and is developing recommendations that could be incorporated into an education program on the knowledge of feeding practice sand understanding of feed additives for trainers and their advisers.


The Subcommittee’s findings were presented to the Australian Harness Racing Council in April 2001. The Australian Harness Racing Council resolved to raise the TCO2 threshold for harness racing from 35mmol/l to 36mmol/l. It also resolved to carry out an on-going review of all policies and procedures associated with the detection of prohibited substances and to work with the Controlling body in each Australian State to establish educational workshops for all participants.

Click here to view:

* The full text of the TCO2 Subcommittee Review paper
* The Standardbred Diet Survey
* a series of pertinent Questions and Answers on TCO2 in Australian Standardbreds


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