note : for in situ hybridization or oil - red - o staining , 1-phenyl-2-thiourea ( ptu ) at a final concentration of 0.2 mm is added to inhibit pigment formation , but is not necessary for seahorse analysis . for pharmacological studies ,
add the required chemical at an appropriate final concentration to developing zebrafish embryos at around 26 hr post fertilization ( hpf ) with an appropriate vehicle - only control .
note : for light - sensitive pharmacological inhibitors , embryos can be allowed to develop in the dark prior to analysis .   before each run , the seahorse xf 24 analyser cartridge that houses the o2 and h fluorophores is calibrated through an automated process performed by the analyser .  
the following experimental sequence is employed : since oxygen consumption is sensitive to temperature fluctuations , four wells are used to control for possible temperature fluctuations across the plate .
these wells do not contain embryos , but are filled with 700 l of e3 medium ( figure 2a ) .
the remaining 20 wells ( see part 2.2 ) are filled with 700 l of e3 medium and one embryo each ( figure 2b).for each experiment , 10 embryos are treated with vehicle - only ( control ) and 10 treated with chemical inhibitor ( treated ) .
control and treated embryos are alternated ( e.g. well a2 : control embryo ; well a3 : treated embryo : well a4 : control embryo ; well a5 treated embryo , etc ) .
an islet capture screen is added on the top of each well to ensure that the specimen remains in the measurement chamber throughout the assay ( figure 2b ) .
note : chemically - treated embryos remain with their original chemical solution throughout the entire procedure , with no need to wash out the chemical prior running the seahorse analyser program .
once finished , the plate is loaded into the seahorse analyser and the assay is started . 
 
since oxygen consumption is sensitive to temperature fluctuations , four wells are used to control for possible temperature fluctuations across the plate .
these wells do not contain embryos , but are filled with 700 l of e3 medium ( figure 2a ) .  
the remaining 20 wells ( see part 2.2 ) are filled with 700 l of e3 medium and one embryo each ( figure 2b ) . for each experiment ,
10 embryos are treated with vehicle - only ( control ) and 10 treated with chemical inhibitor ( treated ) .
control and treated embryos are alternated ( e.g. well a2 : control embryo ; well a3 : treated embryo : well a4 : control embryo ; well a5 treated embryo , etc ) .  
an islet capture screen is added on the top of each well to ensure that the specimen remains in the measurement chamber throughout the assay ( figure 2b ) .
note : chemically - treated embryos remain with their original chemical solution throughout the entire procedure , with no need to wash out the chemical prior running the seahorse analyser program .  
once finished , the plate is loaded into the seahorse analyser and the assay is started .
two different assays are routinely performed . basal respiration / maximum respiration program ( duration approx .
alterations in basal respiration underpin metabolic dysfunction , while maximal respiration is a measure of total energy production capacity and alterations in this parameter associated with a number of both pathological and physiological states .
the spare respiratory capacity , or the capacity for system to further increase atp production , is also calculated from this test by the subtraction of basal from maximal respiration .
three repeats of each mix ( 2 min)/wait ( 1 min)/measurement ( 2 min ) cycle is performed to first establish basal respiration . at the end of the third cycle , a final concentration of 2.5 m of fccp ( mitochondrial protonophore / uncoupler )
respiration due to atp turnover represents the major function of mitochondria in the form of atp production , while respiration due to proton leak , or uncoupled respiration , is inextricably linked with other parameters of mitochondrial function including basal respiration and reactive oxygen species formation .
respiration due to atp turnover is represented by the difference in respiration following the addition of 25 m oligomycin ( an inhibitor of atp synthase ) compared to basal respiration .
uncoupled respiration , or respiration due to proton leak , is determined by calculating the difference between oligomycin - mediated respiration and respiration following the addition of 25 m rotenone ( a complex i inhibitor ) .
measurement cycles are repeated 8 times after addition of each mitochondrial inhibitor . 
  basal respiration / maximum respiration program ( duration approx .
alterations in basal respiration underpin metabolic dysfunction , while maximal respiration is a measure of total energy production capacity and alterations in this parameter associated with a number of both pathological and physiological states .
the spare respiratory capacity , or the capacity for system to further increase atp production , is also calculated from this test by the subtraction of basal from maximal respiration .
three repeats of each mix ( 2 min)/wait ( 1 min)/measurement ( 2 min ) cycle is performed to first establish basal respiration . at the end of the third cycle , a final concentration of 2.5 m of fccp ( mitochondrial protonophore / uncoupler )
the measurement cycle is then repeated 5 to 8 times ( figure 3 ) .  
respiration due to atp turnover represents the major function of mitochondria in the form of atp production , while respiration due to proton leak , or uncoupled respiration , is inextricably linked with other parameters of mitochondrial function including basal respiration and reactive oxygen species formation .
respiration due to atp turnover is represented by the difference in respiration following the addition of 25 m oligomycin ( an inhibitor of atp synthase ) compared to basal respiration .
uncoupled respiration , or respiration due to proton leak , is determined by calculating the difference between oligomycin - mediated respiration and respiration following the addition of 25 m rotenone ( a complex i inhibitor ) .
measurement cycles are repeated 8 times after addition of each mitochondrial inhibitor .   at the end of the runs , mean values of the 10 control and 10
. embryos at 50 hpf are dechorionated using fine forceps and fixed in 4% pfa overnight at 4 c .  
oil - red - o staining is performed , as previously described ( see figure 4 ) .
note : for in situ hybridization or oil - red - o staining , 1-phenyl-2-thiourea ( ptu ) at a final concentration of 0.2 mm is added to inhibit pigment formation , but is not necessary for seahorse analysis . for pharmacological studies ,
add the required chemical at an appropriate final concentration to developing zebrafish embryos at around 26 hr post fertilization ( hpf ) with an appropriate vehicle - only control .
note : for light - sensitive pharmacological inhibitors , embryos can be allowed to develop in the dark prior to analysis .
before each run , the seahorse xf 24 analyser cartridge that houses the o2 and h fluorophores is calibrated through an automated process performed by the analyser .  
the following experimental sequence is employed : since oxygen consumption is sensitive to temperature fluctuations , four wells are used to control for possible temperature fluctuations across the plate .
these wells do not contain embryos , but are filled with 700 l of e3 medium ( figure 2a ) .
the remaining 20 wells ( see part 2.2 ) are filled with 700 l of e3 medium and one embryo each ( figure 2b).for each experiment , 10 embryos are treated with vehicle - only ( control ) and 10 treated with chemical inhibitor ( treated ) .
control and treated embryos are alternated ( e.g. well a2 : control embryo ; well a3 : treated embryo : well a4 : control embryo ; well a5 treated embryo , etc ) .
an islet capture screen is added on the top of each well to ensure that the specimen remains in the measurement chamber throughout the assay ( figure 2b ) .
note : chemically - treated embryos remain with their original chemical solution throughout the entire procedure , with no need to wash out the chemical prior running the seahorse analyser program .
once finished , the plate is loaded into the seahorse analyser and the assay is started . 
 
since oxygen consumption is sensitive to temperature fluctuations , four wells are used to control for possible temperature fluctuations across the plate .
these wells do not contain embryos , but are filled with 700 l of e3 medium ( figure 2a ) .  
the remaining 20 wells ( see part 2.2 ) are filled with 700 l of e3 medium and one embryo each ( figure 2b ) . for each experiment ,
10 embryos are treated with vehicle - only ( control ) and 10 treated with chemical inhibitor ( treated ) .
control and treated embryos are alternated ( e.g. well a2 : control embryo ; well a3 : treated embryo : well a4 : control embryo ; well a5 treated embryo , etc ) .  
an islet capture screen is added on the top of each well to ensure that the specimen remains in the measurement chamber throughout the assay ( figure 2b ) .
note : chemically - treated embryos remain with their original chemical solution throughout the entire procedure , with no need to wash out the chemical prior running the seahorse analyser program .  
once finished , the plate is loaded into the seahorse analyser and the assay is started .
alterations in basal respiration underpin metabolic dysfunction , while maximal respiration is a measure of total energy production capacity and alterations in this parameter associated with a number of both pathological and physiological states .
the spare respiratory capacity , or the capacity for system to further increase atp production , is also calculated from this test by the subtraction of basal from maximal respiration .
three repeats of each mix ( 2 min)/wait ( 1 min)/measurement ( 2 min ) cycle is performed to first establish basal respiration . at the end of the third cycle , a final concentration of 2.5 m of fccp ( mitochondrial protonophore / uncoupler )
respiration due to atp turnover represents the major function of mitochondria in the form of atp production , while respiration due to proton leak , or uncoupled respiration , is inextricably linked with other parameters of mitochondrial function including basal respiration and reactive oxygen species formation .
respiration due to atp turnover is represented by the difference in respiration following the addition of 25 m oligomycin ( an inhibitor of atp synthase ) compared to basal respiration .
uncoupled respiration , or respiration due to proton leak , is determined by calculating the difference between oligomycin - mediated respiration and respiration following the addition of 25 m rotenone ( a complex i inhibitor ) .
measurement cycles are repeated 8 times after addition of each mitochondrial inhibitor . 
  basal respiration / maximum respiration program ( duration approx .
alterations in basal respiration underpin metabolic dysfunction , while maximal respiration is a measure of total energy production capacity and alterations in this parameter associated with a number of both pathological and physiological states .
the spare respiratory capacity , or the capacity for system to further increase atp production , is also calculated from this test by the subtraction of basal from maximal respiration .
three repeats of each mix ( 2 min)/wait ( 1 min)/measurement ( 2 min ) cycle is performed to first establish basal respiration . at the end of the third cycle , a final concentration of 2.5 m of fccp ( mitochondrial protonophore / uncoupler )
the measurement cycle is then repeated 5 to 8 times ( figure 3 ) .  
respiration due to atp turnover represents the major function of mitochondria in the form of atp production , while respiration due to proton leak , or uncoupled respiration , is inextricably linked with other parameters of mitochondrial function including basal respiration and reactive oxygen species formation .
respiration due to atp turnover is represented by the difference in respiration following the addition of 25 m oligomycin ( an inhibitor of atp synthase ) compared to basal respiration .
uncoupled respiration , or respiration due to proton leak , is determined by calculating the difference between oligomycin - mediated respiration and respiration following the addition of 25 m rotenone ( a complex i inhibitor ) .
measurement cycles are repeated 8 times after addition of each mitochondrial inhibitor .   at the end of the runs , mean values of the 10 control and 10
embryos at 50 hpf are dechorionated using fine forceps and fixed in 4% pfa overnight at 4 c .  
oil - red - o staining is performed , as previously described ( see figure 4 ) .
we are interested in understanding the role specific genes on metabolism , particularly respiration rate and lipid metabolism . therefore , we treated wild type zebrafish embryos from 26 hpf onwards with a specific pharmacological inhibitor of enzyme encoded by one of these genes . at 50 hpf
, half of the vehicle and inhibitor - treated embryos were analysed using the seahorse for mitochondrial function , while the remaining half were fixed in 4% pfa overnight at 4 c and subsequently stained for lipid deposition with oil - red - o .
treatment with the specific enzyme inhibitor led a ~2 fold increase in basal respiration ( figure 4a ) , which was associated with an increase in lipid deposition especially in the telencephalon and underneath the otic vesicle ( figure 4b , c ) .  
( a ) four wells contain embryonic medium only as temperature control ( marked by an x ) and the others contain one embryo / well .
( b ) close - up of one well ( c6 ) showing a 50 hpf wild type embryo treated with vehicle ( dmso ) covered by an islet capture screen ( arrowhead ) .  
( a ) for each measurement , the mean of the 20 individual embryos is presented .
basal respiration ( mean : 299.7 ; sd : 75.7 ; sem : 16.9 ) , maximal respiration ( mean : 387.4 ; sd : 93.3 ; sem : 20.9 ) , spare respiratory capacity ( mean : 87.7 ; sd : 72.0 ; sem : 16.1 ) .
( b ) for each measurement , the mean of the 20 individual results is presented . basal mitochondrial respiration ; mitochondrial respiration due to atp turnover ; mitochondrial uncoupled respiration and non - mitochondrial respiration .
mt : mitochondrial ; src : spare respiratory capacity ; sd : standard deviation , sem : standard error of mean .
( a , b ) 50 hpf embryo stained with oil - red o in lateral view .
the embryo incubated with the selective inhibitor ( b ) displays more oil - red - o staining in the telencephalon ( arrowhead ) and underneath the otic vesicle ( arrow ) compared to a vehicle - treated control embryo .
( c ) basal respiration shows an ~ 2 fold increase in chemically treated embryos ( n=10 embryos per group ) .
zebrafish is a well - established genetic model for both forward ( enu mutagenesis ) and reverse ( tilling , zinc finger nuclease targeted knock - out , morpholino knockdown ) genetic approaches , while gene function in zebrafish embryos can also be easily blocked or activated using selective pharmacological compounds specific for the encoded products . due to their external development and small size ,
however , robust measurement of metabolic profile and mitochondrial function in vivo in zebrafish embryos has not been achieved , with only one preliminary description reported .
seahorse analysis was originally designed for cell - based metabolic studies and has been demonstrated to give accurate and reliable results .
the application of this new methodology to zebrafish embryos is significant , and likely to augment the wider usage of this model for metabolic studies . in this study , we demonstrate measurement of a range of respiration parameters in zebrafish embryos using the seahorse analyser , including basal respiration , maximal respiration , spare respiratory capacity , atp turnover and proton leakage .
we also provide an example of how such measurements can be correlated with other physiological parameters , in this case lipid accumulation , and of the use of pharmacological inhibitors in this assay system .
combined with the use of genetically - altered embryos , this provides a powerful experimental platform for understanding factors impacting on metabolism .
there are a variety of applications for this new methodology , with mitochondrial dysfunction is implicated in many human diseases , such as diabetes mellitus , obesity , multiple sclerosis , parkinson 's diseases , alzheimer 's disease and some type of cancers .
importantly , our work is performed in vivo , where all environmental influences - such as cytokines , development - related growth etc - are active , thereby providing a physiologically relevant view in vivo respiration and metabolic profile .
as chemical screens are also routinely performed in wild type and mutant background zebrafish embryos ( figure 1 ) , novel pharmaceuticals that influence respiration , mitochondria function or metabolism could be easily identified using the seahorse analyser .
the results generated using the seahorse analyser could be used in conjunction with other assays to provide additional information .
this can include physiological analysis , such as oil - red staining , or molecular analysis , such as in situ hybridization for specific adipocyte markers such as cebp , ppar , ppar , fas etc .
there remain some limitations to this methodology . although we and others were able to use oligomycin to measure atp production and proton leak on young embryos ( see figure 3 ) , in embryos older than 60 hpf oligomycin treatment is inefficient .
we believe that in older embryos oligomycin can not diffuse as easily than in younger embryos making the results impossible to interpret .
since the oligomycin results are mandatory for the determination of respiration due to atp turnover and uncoupled respiration , we are currently investigating higher concentrations of oligomycin for older embryos .
however , antimycin a treatments remain effective longer , with other measurements , such as basal respiration , maximum respiration and spare respiratory capacity , can be performed in older zebrafish embryos , up to 68 hpf ( not shown ) .  
another limitation in using the current seahorse analyser setup is the physical space within each islet capture plate well , such that they are only suitable for zebrafish embryos and young larvae .
therefore , performing metabolic studies on diet - induced obesity in adult fish , for example , is not yet technically feasible .
however , this study may prompt the development of plates specifically designed for juvenile or adult fish , thereby extending the scope of analyses possible .
 a growing goal in the field of metabolism is to determine the impact of genetics on different aspects of mitochondrial function . 
 understanding these relationships will help to understand the underlying etiology for a range of diseases linked with mitochondrial dysfunction , such as diabetes and obesity . 
 recent advances in instrumentation , has enabled the monitoring of distinct parameters of mitochondrial function in cell lines or tissue explants . here 
 we present a method for a rapid and sensitive analysis of mitochondrial function parameters in vivo during zebrafish embryonic development using the seahorse bioscience xf 24 extracellular flux analyser . 
 this protocol utilizes the islet capture microplates where a single embryo is placed in each well , allowing measurement of bioenergetics , including : ( i ) basal respiration ; ( ii ) basal mitochondrial respiration ( iii ) mitochondrial respiration due to atp turnover ; ( iv ) mitochondrial uncoupled respiration or proton leak and ( iv ) maximum respiration . using this approach embryonic zebrafish respiration parameters 
 can be compared between wild type and genetically altered embryos ( mutant , gene over - expression or gene knockdown ) or those manipulated pharmacologically . 
 it is anticipated that dissemination of this protocol will provide researchers with new tools to analyse the genetic basis of metabolic disorders in vivo in this relevant vertebrate animal model . 
Protocol
Part 1: Chemical Treatment of Zebrafish Embryos
Part 2: Live Metabolic Profile Using the Seahorse XF 24 Analyser
Part 3: Oil-Red-O Staining
Representative Results
Discussion
Disclosures