Researchers collect multiple blood samples via an intravenous catheter in order to minimize discomfort and risk of infection associated with multiple venipunctures with a hypodermic needle.

The site of catheterization is shaved and scrubbed with Betadine to remove dirt and pathogens to minimize the risk of infection.

The site of catheterization is washed with a liberal application of 70 % ethanol alcohol to rinse away any remaining Betadine.

Lidocaine, a local anesthetic, is used to numb the site of catheterization.

Approximately 1.0 mL of lidocaine is injected subcutaneously with a 25-gauge needle. Contact between the aseptic area of catheter placement and either the syringe or other non-sterile objects, such as an ungloved hand, is avoided and the area is rescrubbed (as previously described) prior to insertion of the catheter.

Illustrated here is the lidocaine “bleb” at the point of catheterization.

While the aseptic area is being prepared, others get ready to assemble the extension set and place the catheter.

A stopcock is used as a valve to direct the flow of blood samples and heparin-saline flush during the course of sample collection. When the extension set is removed from its sterile packaging and fully assembled, it is flushed and the valve is shut off in order to prevent airborne pathogens from migrating to the exposed surfaces of the sample collection apparatus.

Sterile gloves are worn to minimize exposure of the horse’s cardiovascular system to pathogens.

The catheter and stylet are positioned parallel to the jugular groove, following blood flow in the vein. Notice that the catheter is handled only at the hub.

At the lidocaine bleb, the catheter and stylet are advanced until the stylet enters the vein lumen.

Within the aseptic field, pressure is temporarily applied to the jugular groove so that accurate placement of the catheter and stylet within the jugular can be verified. Positive pressure causes blood to fill the catheter. Observation of blood at the catheter hub confirms that venipuncture has been successful.

The catheter and stylet are stabilized while the catheter is gently advanced down the stylet into the jugular. The catheter should advance with minimal resistance.

An assistant attaches the sterile end of an extension tube to the catheter hub.

A sterile syringe is used to pull blood back into the extension set to confirm that the catheter is placed properly and to verify that all connections are air tight. The line is then cleared with heparin-saline flush. Heparin is included in the flush in order to prevent blood coagulation in the catheter.

The catheter-extension set junction is taped or sutured in place to prevent displacement during the exercise test. The peripheral IV line is taped or braided into the mane to prevent accidental removal due to movement during exercise.

A safety harness (surcingle) is used during the exercise test as a precaution to prevent injuries. In the unlikely event that a horse should stumble while running on the treadmill, a strap attached to the top of the harness will trip an emergency switch shutting off power to the treadmill and allowing for deceleration.

Like humans, horses experience a transient shortening of gait at the start of exercise. Rubberized bell boots are used to protect the coronet band (the point where the hoof wall originates).

An external heart rate monitor is one piece of equipment used during the exercise test. In order to function properly, electrodes must be kept in contact with the body and kept wet using water or electrode gel.

The heart rate monitor transmits data to a receiver that can be worn like a watch. Data are displayed and recorded in real time while horses perform an acute bout of exercise.

Before starting the exercise test, a resting blood sample is taken. Resting heart rate and other data are also recorded.

Two syringes are used during sample collection. One designated “waste” is placed on the stopcock and is used to first remove the flush present in the extension set and to ensure that there is adequate blood flow through the catheter. The stopcock is then opened for the “sample” syringe, so that blood can be collected.

After the blood sample is collected, the “waste” syringe contents are returned and heparin-saline flush is used to clear the extension set of blood and prevent coagulation in the catheter.

Blood samples collected during the exercise test are chilled until the test is completed and samples processed in the laboratory. Blood collection tubes contain different preservatives to prevent metabolism and degradation of substances in the blood.

Once on the treadmill, the safety harness is attached to the strap connected to the emergency switch.

Lead ropes are attached to both sides of the non-breakaway halter so that horses remain balanced and do not back off the treadmill during the test.

The Oxymax-XL is an open-flow indirect calorimeter that measures the rate of oxygen consumption (VO2) and the rate of carbon dioxide production (VCO2) by a horse during exercise. A loose-fitting mask is placed over the horse’s nose and held in a constant position to minimize variability in measurements.

Speed and incline of the treadmill can be adjusted to change exercise intensity.

Heart rate data are transmitted from the monitor on the horse to the “watch.” Data can be downloaded for further analysis.

Horses love to run and often are so eager to perform that they push against the front of the treadmill. The cross-bar on the front of the treadmill is covered with extra padding to ensure their comfort.

Blood samples are obtained at pre-determined time points during the exercise test. For example, during a GXT blood samples are taken prior to the start of the test, during the last 10 seconds of each step of the test, and at various points during recovery.

Blood samples are dispensed into labeled collection tubes. The number of sample tubes needed for each time point depends on the number of variables being measured.

The treadmill is 21 ft long, and can go as fast as 33 miles per hour. Some horses will reach speeds of 25 miles per hour while running up a 6 % grade.

The Equi-Ciser™ is used to examine the adaptive effects of repeated bouts of exercise. Up to six horses can be trained for set periods of time at pre-determined submaximal work intensities specific to the experiment.

Preliminary analysis of blood samples is done in the laboratory adjacent to the treadmill. Following initial analyses, samples are stored at -80°C until further analysis.

Proper disposal of laboratory materials is important. Needles and syringes, suture needles, and stylets are disposed of in specially marked sharps containers.

Sample tubes are spun in high-speed centrifuges to separate red blood cells, white blood cells, and platelets from plasma.

Plasma is aliquoted into cryovials for storage and later analysis.

Whole blood is used to measure hematocrit, or Packed Cell Volume (PCV). The PCV is measured using the microhematocrit technique to determine the ratio of plasma to red blood cells (RBC).

PCV changes during exercise when the horse’s spleen contracts, resulting in the mobilization of its RBC reserve. The increase in circulating RBCs facilitates oxygen delivery to working tissues. Hematocrit also increases due to shifts of fluid out of the blood vessels to provide for sweat production. Both factors result in a greater PCV following exercise.

Capillary tubes are spun on a microhematocrit centrifuge to separate the RBCs from plasma.

PCV is determined by placing the centrifuged capillary tube on a scaled chart, aligning the bottom of the red blood cell column with the 0 % line. The tube is shifted to the right until the top of the plasma column is on the 100 % line. PCV is the value at the interface of the packed red blood cells and plasma, and can be read directly as percent of cell volume.

Plasma total protein concentration is measured using a hand-held refractometer. Total protein concentration is used to assess hydration status and fluid shifts during exercise.

A small amount of plasma is placed on the prism of the refractometer.

The refractometer is held towards a light source. Light travels through the plasma sample, and is projected on a scale displayed through the lens of the refractometer. This provides total protein concentration in g/100 mL of plasma.

Plasma samples from each step of the exercise test are also analyzed for various parameters, such as electrolytes (Na, K, Cl, and Ca), metabolites (glucose and lactate), blood gas (O2 and CO2), and pH.

At the Equine Science Center, we recognize the human-horse bonds that exist physiologically, emotionally, and economically. Our resources are committed to the Center’s mission of Better Horse Care through Research and Education, and service to horse owners around the world.

Thank you for visiting the Rutgers Equine Science Center and the Treadmill Laboratory.