Measuring the respiration rates of eggs and embryos
Researchers and fertility clinics need to know the quality of embryos for successful fertilisation and reproduction. Not only is this important in human fertility but also for economically significant species such as cattle (bovine).
Ideally, a method needs to be quantitative and non-destructive yet many methods available to researchers and clinicians are qualitative (that is, the quality of an embryo is based on the judgement of an observer) or can potentially damage the organism.
The Unisense NanoRespiration System has been demonstrated to measure the quality of embryos with a quantitative, non-destructive methodology. Scientists had discovered that embryo quality could be determined by its metabolic rate. Oxygen consumption, or organism respiration, is one parameter that infers metabolic rate. Therefore, the higher the respiration rate the better the quality of an embryo.
The Unisense NanoRespiration System has been used to measure the respiration of embryos in a wide variety of animals such as cattle, crabs and amphibians (Lopes et al 2005, Seymour 1999, Simoni et al 2013). The NanoRespiration System has also measured the respiration rate of eggs of organisms such as copepods and zebrafish (Bang et al 2004).
In this case study, we examine the research of Lopes et al (2005) to see how they measured the respiration rates of bovine embryos and how the results were used to determine embryo quality.
The Unisense NanoRespiration System has been designed to specifically measure the respiration rates of eggs, embryos and small organisms. The NanoRespiration System consists of an oxygen microsensor connected to the Unisense Multimeter or OXY-Meter. The oxygen microsensor is moved by a micromanipulator and motor controller that is supported by a lab stand. The software, SensorTrace Respiration, collects, stores and analyses all of the measured data on a computer:
The Nanorespirometer Unit, depicted in the figure above, is the unique design that allows for the measurement of small organisms. This unit consists of 7 wells, configured as a rosette, with the 6 outside wells containing the samples to be measured (the central well cannot be reached by the micromanipulator). The wells are 0.68mm in diameter and 3mm in depth. The rosette wells are placed on a rosette stand that is then held firmly to the lab stand at a pre-determined depth below the oxygen microsensor. In this way, the oxygen microsensors are able to precisely move into each well without damaging the microsensor or organisms. The oxygen microsensor is moved up and down with the micromanipulator and motor controller, and each well is moved around the rosette manually for each measurement profile:
The rosette well fixed to a rosette stand. Individual embryos can be placed inside the wells for respiration measurements.
Even though there are 6 sample wells around the rosette it is always important to leave at least one of the wells empty and without an organism. The empty well actually contains the same fluid as the wells with organisms and it acts as a control measurement. This way, any oxygen consumption that is measured is known to come from the egg or embryo rather than some other biological organisms (that is, it is a way to control for contamination).
A single egg or embryo is placed inside a well and the well is then filled with fluid. The system is left to rest for at least an hour in order to create a diffusive oxygen gradient. The oxygen microsensor starts a measurement profile from near the top of the well and measures at 250μm steps to a depth of 2.5mm – a point above the egg or embryo so as not to damage the organism (or break the sensor), but deep enough to measure a diffusion curve:
The slope of the curve above is then entered into Fick’s Law of Diffusion and the respiration rate can be calculated. This calculation is performed in the SensorTrace Respiration Software.
The experiment by Lopes et al (2005) found that the control well had a consistent oxygen concentration at approximately 19% yet a steady consumption of oxygen with varying rates depending on the embryo (Figure 3, Lopes et al, 2005).
The researchers also compared a qualitative embryo health score, based on definitions by the International Embryo Transfer Society, against respiration rate for 7 day old blastocyts. They found that a high quality score (a score of 1 or 2) was correlated with a relatively high respiration rate and that this observation was consistent for male and female embryos (Figure 7, Lopes et al, 2005).
Lopes et al (2005) concluded that microsensor technology can be used to accurately and rapidly assess embryo quality. Each assessment in their experiment only took 8 minutes to complete and may possibly lead to greater efficiencies for researchers and clinicians.
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