scientificprotocols authored about 6 years ago
Authors: Yara Bernaldo de Quirós, Óscar González-Díaz, Manuel Arbelo, Marisa Andrada & Antonio Fernández
Gas sampling in stranded marine mammals can now be performed in situ using the appropriate vacuum tubes, insulin syringes and an aspirometer. Glass vacuum tubes are used for extraction of gas from cavities such as the intestine, pterigoyd air sacs, pneumothorax or subcapsular emphysema as well as for storage of the gas sample at room temperature and pressure. Insulin syringes are used for extraction of bubbles found in veins, then immediately injecting its content into the vacuum tubes for storage. Finally an aspirometer is used to extract and separate the gas mixed with blood inside the heart. We have found that these are reliable tools for in situ gas sampling, storage and transportation without appreciable loss of gas and without compromising the accuracy of the analysis. Gas analysis is conducted by gas chromatography in the laboratory.
Gas-bubble lesions have been described in cetaceans stranded in spatio-temporal concordance with military maneuvers (1,2). These authors suggested decompression like sickness as an explanation for the observed lesions.
Decompression sickness is the disease caused by bubble formation due to gas phase separation in the body. Gas phase may arise from supersaturated gas tissues after decompression when the sum of the dissolved gas tensions (oxygen, carbon dioxide, nitrogen, helium) and water vapor exceeds the local absolute pressure (3,4). According to Bert (1878), the main gas “which would threaten life on being liberated would be exclusively the one the proportion of which was considerably increased in the blood”: nitrogen (5).
Gas chromatography has been demonstrated as a valid method to discriminate putrefaction gases from air embolism (6,7), and has been used as a forensic tool in humans for this purpose (8). Indeed putrefaction gas is one of the problems that we might face when dealing with stranded marine mammals.
We describe the step by step protocol that has been experimentally tested and verified for the reliable and consistent collection, storage and analysis of gas from different body compartments in stranded cetaceans. Furthermore, we have demonstrated by using this protocol that gases in acute and chronic gas embolism-affected cetaceans that were minimally decomposed had high or very high nitrogen contents in bubbles (9).
Equipment set up
Aspirometer set up: A tygon tube of approximately 1 meter long must connect the aspirometer and the aspirator bottle. Another tygon tube of 50 cm should connect the aspirometer with the needle. At the top of the burette another needle must be placed. All joints have to be silicon and parafilm sealed. The whole system is filled with distilled water (Figure 1).
Gas chromatograph set up: TCD temperature at 80°C, filament temperature at 160°C. The temperature for the FID is fixed at 230°C. Samples are run for 25 minutes with an isothermal temperature of 45°C and an electronically controlled flux with a fixed pressure of 13.1 psi on the head column. Helium is used as the carrier gas.
Dissection
^1 Gas sampling from bubbles in veins
CRITICAL STEP: place the vein under water whenever possible to avoid atmospheric air contamination.
^2 Gas sampling from cavities (intestine, pterigoyd air sacs) and gas associated lesions (pneumothorax and subcapsular emphysema)
^3 Gas sampling from the heart cavities using the aspirometer
Storage and transport
Gas analyses and calculations
Time to collect samples varies depending on the presence or absence of bubbles, amount of bubbles and studied species. For a dolphin, maximum estimated sampling time is 30 minutes if the aspirometer is used. If it is not used, sampling should take less time.
The evacuated tubes contain some atmospheric air, which we correct from our samples using the detection limit. We strongly recommend the use of BD 5ml additive-free vacutainer, because they contain low levels of atmospheric air and are made of break-resistant glass. If you don’t have this tube available, use the smallest glass evacuated tube you can find, and take many blanks with your samples to correct for the standard deviation.
Evacuated tubes do not resist too changes in pressure very well. If tubes need to be transported in a plane they should go into the passenger cabin. If you need to ship them, you should use a pressure resistant housing.
Very small bubbles, won’t give you signals higher than the detection limit. Sample the largest bubbles you find. Usually, bubbles larger than 0.5 mL give goods results.
Gas embolism found in fresh animals might be composed of high or very high nitrogen, while gases produced by putrefaction might be composed of a mixture of nitrogen, hydrogen and carbon dioxide^9.
We would like to thank all colleagues who contributed to this work and, especially, the different stranding networks and governments: Canary Islands, Andalusia, United Kingdom and Italy, along with the hyperbaric medicine division of the NTNU (Norway) for its scientific contribution.
This work was supported by the Spanish Ministry of Science and Innovation with two research projects: (AGL 2005-07947) and (CGL 2009/12663), as well as the Canary Islands government with the project: SolSub C200801000288. The Spanish Ministry of Education contributed with a PhD fellowship (the University Professor Formation fellowship).
First author current affiliation is Woods Hole Oceanographic Institution, Biology Department, MS#50, Woods Hole, MA 02543, United States of America. The Woods Hole Oceanographic Institution Marine Mammal Centre and Wick and Sloan Simmons provided funding for the latest stage of this work.
Figure 1: Aspirometer display
Schematic figure showing the different components of the aspirometer.
Figure 1: Aspirometer display
Schematic figure showing the different components of the aspirometer.
Figure 2: Sampling position
At this position, the differences in heigh between the flasks provokes a difference in pressure. Liquid is moved from the aspirator bottle to the aspirometer and form here to the sampling needle. Liquid is coming out through the sampling needle. There is no atmospheric gas even in the sampling needle.
Figure 2: Sampling position
At this position, the differences in heigh between the flasks provokes a difference in pressure. Liquid is moved from the aspirator bottle to the aspirometer and form here to the sampling needle. Liquid is coming out through the sampling needle. There is no atmospheric gas even in the sampling needle.
Figure 3: Gas extraction position
At this position, the differences in heigh between the two flasks provokes a negative pressure, suctioning whatever is found inside the heart cavities.
Figure 3: Gas extraction position
At this position, the differences in heigh between the two flasks provokes a negative pressure, suctioning whatever is found inside the heart cavities.
Protocol Full Version: PROTOCOL FOR GAS SAMPLING AND ANALYSIS IN STRANDED MARINE MAMMALS
Download Protocol Full Version
Methodology for in situ gas sampling, transport and laboratory analysis of gases from stranded cetaceans. Yara Bernaldo de Quirós, Óscar González-Díaz, Pedro Saavedra, Manuel Arbelo, Eva Sierra, Simona Sacchini, Paul D. Jepson, Sandro Mazzariol, Giovanni Di Guardo, and Antonio Fernández. Scientific Reports 1 () 14/12/2011 doi:10.1038/srep00193
Yara Bernaldo de Quirós, Manuel Arbelo, Marisa Andrada & Antonio Fernández, Institute of Animal Health, University of Las Palmas de Gran Canaria
Óscar González-Díaz, Physical and Chemical Instrumental Center for the Development of Applied Research Technology and Scientific estate, University of Las Palmas de Gran Canaria.
Correspondence to: Yara Bernaldo de Quirós ([email protected]), Antonio Fernández ([email protected])
Source: Protocol Exchange (2012) doi:10.1038/protex.2012.002. Originally published online 31 January 2012.