This research was supported by Alltech and USDA Hatch funds through the Center for Food Animal Health at UC Davis School of Veterinary Medicine.

All methods, protocol and studies described here were approved by the Institutional Animal Care and Use Committee (IACUC) of University of California, Davis.
1. Obtaining a Liver Biopsy from a Holstein Dairy Cow
NOTE: A liver biopsy should be performed by a licensed veterinarian. Liver biopsies can be performed on the dairy site where the cows are located. Lactating dairy cows can continue to be milked normally and milk does not need to be withdrawn from the food supply before or after the procedure. It is recommended that at least 4 people are needed to perform the liver biopsy on a dairy cow: a veterinarian to perform the biopsy, an animal handler to stand at the cow&#39;s hip to protect the biopsy area and veterinarian, a lab technician on the outside of the pen to transfer tools, materials and biopsy sample to and from the veterinarian and maintain the clean area, which can be in the back of a vehicle (Figure 1), and a technician to retrieve the liver sample and begin mitochondrial isolation.
<ol>
	<li>One month prior to liver biopsies, give cows a clostridia vaccination. Create surgical packs by autoclaving surgical towels, biopsy instrument, scalpel holders and surgical equipment.</li>
	<li>One day before the liver biopsy, inject the cow with Ceftiofur Hydrochloride 0.044 mL/kg bodyweight subcutaneously in the neck. Monitor cow temperature, intake and fecal scores to use as a baseline for normal function.</li>
	<li>Create mitochondria isolation media (MIM) containining 220 mM mannitol, 70 mM sucrose, 20 mM HEPES, 1 mM EDTA, and 0.1% (w/v) fatty acid free BSA, pH 7.4 at 4 &#176;C. Approximately 30 mL per sample will be needed.</li>
	<li>Restrain cow physically utilizing a headlock with a halter as needed (Figure 2). Using the halter, tie her head to the left side of the stanchion. If necessary, a chemical restraint (Xylazine hydrochloride 100 mg/mL IV at 0.010-0.015 mg / kg bodyweight) can be used.</li>
	<li>The area of the biopsy is found at the right 10 - 11th intercostal space (Figure 3). Draw a straight line from the right tuber coxae to the point of the right shoulder. The biopsy site is where this line intersects with the 10-11th intercostal space. Sterilize the area of the cow to be biopsied by shaving a 10 cm square area (Figure 4). Wash area with 10% providone scrub (Figure 5) using a circular motion. Spray area with 70% ethanol solution (Figure 6). Repeat providone and ethanol washes. 
	NOTE: The liver is in a slightly different position in Holstein dairy cattle compared to beef cattle.</li>
	<li>Inject 2% lidocaine HCl (10-15 mL) locally to the area to provide anesthesia of the skin and underlying muscle and connective tissue (Figure 7). Repeat providone and 70% ethanol washes. 
	NOTE: The nerve endings are in the skin and muscle, but not internal organs, so only a local anesthestic is needed. At most, the cow should only feel some pressure and no pain during the biopsy procedure.</li>
	<li>Make a 1-2 cm stab-incision through the skin of the 10-11th intercostal space (Figure 8). Pass a Schackelford-Courtney bovine liver biopsy instrument through the skin and direct the biopsy instrument in a slight cranial direction while continuing through the diaphragm and into the liver (Figure 9, Figure 10). Obtain a 1 g sample of the liver and remove the instrument (Figure 11). Close the skin with suture placement (Figure 12).</li>
	<li>Place liver sample in a conical tube with enough MIM to cover sample, on ice for immediate mitochondria isolation</li>
	<li>Check incision for any redness, swelling, heat, or pain within 24 hours of biopsy and inject the cow with Ceftiofur Hydrochloride 0.044 mL/kg bodyweight subcutaneously in the neck once a day for the next 3 days (Figure 13). Monitor the cow&#39;s temperature, intake and fecal scores daily for 1 week after liver biopsy. If a fever develops, continue antibiotics at the discretion of the veterinarian. 
	NOTE: If a cow is exhibiting signs of pain, such as kicking at the incision site, recumbancy, redness, heat, or reaction to touch within 1 h after the liver biopsy, a 1 mg/kg bodyweight IV injection of flunixin meglumine can be used to alleviate pain and inflammation. A second injection can be administered if necessary.</li>
	<li>Remove sutures 7 days after biopsy.</li>
</ol>
2. Isolating Mitochondria from Dairy Cow Liver
<ol>
	<li>As soon as possible after the liver sample is removed from the cow, wash the liver sample in MIM (step 1.3) to remove red blood cells and finely mince the sample with scissors. The liver should be minced in a chilled beaker containing enough isolation media to keep the tissue moist.</li>
	<li>Place the minced liver into a 30 mL glass vial with a teflon pestle of 0.16 mm clearance incubated in ice and containing MIM (1:4 w/v).</li>
	<li>Homogenize liver sample in teflon pestle at 500 rpm for a min with 4 strokes/min. 
	NOTE: The liver homogenate is kept in an ice-packed beaker in MIM during the entire process, and all of the following centrifugation steps are completed at 4 &#176;C</li>
	<li>Centrifuge homogenate at 500 x g for 10 min, discard pellet, transfer supernatant to a chilled centrifuge tube and then centrifuge the resulting supernatant at 10,000 x g for 10 min to obtain the mitochondrial pellet.</li>
	<li>Resuspend and wash the pellet in 10 mL of MIM with fatty acid free BSA and centrifuge at 8100 x g for 10 min. Discard supernatant.</li>
	<li>Resuspend and wash the pellet in 10 mL of MIM without fatty acid free BSA and centrifuge at 8100 x g for 10 min. Discard supernatant.</li>
	<li>Suspend the pellet in 200 &#181;L of isolation media and place on ice until used for oxygen consumption and proton leak kinetic assays.</li>
	<li>Determine protein concentration of the pellet suspension (1/100 dilution) using Bicinchoninic acid (BCA) kit per manufacturer&#39;s protocol with BSA as the standard. All protein is considered to be mitochondrial protein.</li>
</ol>
3. Measuring Mitochondrial Oxygen Consumption (State 3 and State 4)
<ol>
	<li>Create oxygen consumption media (OCM) from 120 mM KCl, 5 mM KH2PO4, 5 mM MgCl2, 5 mM Hepes and 1 mM EGTA, pH 7.4 at 30 &#176;C with 0.3% defatted BSA. Approximately 3 mL per sample will be needed. Also prepare a solution of 8 &#956;g/mL oligomycin in ethanol.</li>
	<li>Incubate OCM at 30 &#176;C. Set up respiration chamber, pump and oxygen electrode according to manufacturer directions (oxygraph system). The oxygraph software should already be installed on the computer.</li>
	<li>Place 1 mL of OCM into the respiration chamber and stir vigorously. This will help insure that the solution becomes saturated with air.</li>
	<li>Add 0.35 mg protein of mitochondrial protein to the respiration chamber and maintain temperature at 30 &#176;C.</li>
	<li>Record oxygen consumption for approximately 5 min. The oxygraph system records oxygen concentration so as respiration increases, oxygen concentration decreases. When oxygen consumption becomes constant (a decreasing straight line), record oxygen consumption (slope of line = concentration of oxygen/time). This is baseline oxygen consumption.</li>
	<li>Add 1.25 &#181;L of 4 mM rotenone solution to inhibit Complex I and then add 5 &#181;L of 1 M succinate solution to reach a final concentration in the respiratory chamber of 5 mM succinate. This is state 4 respiration.</li>
	<li>Add 1 &#181;L of 100 mM ADP solution to reach a final concentration in the respiration chamber of 100 &#956;M. Oxygen concentration will decrease (increased respiration) and then after approximately 5 min becomes a straight line. Record oxygen consumption (slope of line = concentration of oxygen/time). This is State 3 respiration.</li>
	<li>Optional: At the end of the run, add FCCP (0.2 &#956;M total volume) to induce maximal respiration. Record respiration for about 5 min (approximately). When oxygen consumption becomes constant, record oxygen consumption. This is maximal oxygen consumption.</li>
	<li>Calculate Respiratory Control Ratio (RCR) using the equation State 3 oxygen consumption / State 4 oxygen consumption.</li>
	<li>Aspirate all of the solutions out of the respiration chamber. Rinse the chamber several times with double deionized water.</li>
</ol>
4. Measuring Mitochondrial Membrane Potential (MMP) and Proton Motive Force (PMF)
<ol>
	<li>Prepare solution of 80 ng/mL nigericin in ethanol. 
	NOTE: These chemicals are dissolved in ethanol, and every effort should be made to limit the amount of ethanol that is added to less than 1 &#956;L, since ethanol can uncouple the electron transport system and cause mitchondrial dysfunction.</li>
	<li>After thoroughly rinsing the chamber with double deionized water, place 1 mL of the OCM into the respiration chamber and stir vigorously with a magnetic stir bar. This will help insure that the solution becomes saturated with air. Add methyl-triphenyl-phosphonium (TPMP+) sensitive electrode to chamber setup. TPMP+ electrode should be connected to a pH meter and values are read from the pH meter.</li>
	<li>Add 0.35 mg of mitochondrial protein to the respiration chamber.</li>
	<li>Add 1.25 &#181;L of 4 mM rotenone solution to inhibit Complex I. Record respiration for 2-5 min (approximately). When oxygen consumption becomes constant, record oxygen consumption.</li>
	<li>Add 0.56 &#956;L of 8 &#956;g/mL oligomycin solution for a final concentration of 2.8 &#181;g oligomycin /0.35 mg of mitochondrial protein to inhibit ADP utilization. Record respiration for 2-5 min (approximately). When oxygen consumption becomes constant, record oxygen consumption.</li>
	<li>Add 0.112 &#956;L 80 ng/mL nigericin solution to abolish the pH gradient across the mitochondrial inner membrane. Record respiration for 2-5 min (approximately). When oxygen consumption becomes constant, record oxygen consumption. 
	NOTE: Rotenone and oligomycin are used to block electron transport chain at Complex I and ATP synthase, respectively. Nigericin is added to convert transmembrane H+ gradient to a K+ gradient that can be measured with an electrode.</li>
	<li>Prepare a standard curve for TPMP+ by adding 5 &#181;L of 10 mM TPMP+ solution to the mitochondrial incubation. Repeat this step four more times until a total concentration of 2.5 &#956;M TPMP+ has been added.</li>
	<li>Initiate respiration by adding 5 &#956;L of 1M succinate to the chamber.</li>
	<li>Record respiration until you have achieved a stable trace, and then titrate the system by adding malonate. Additions of malonate should be 0.5 &#181;L, 1 &#181;L, 1.5 &#181;L, 3.0 &#181;L, 6.0 &#181;L, 9.0 &#181;L, then 12.5 &#181;L of 0.1 mM Malonate solution to achieve successive additions of malonate concentrations in the incubation chamber of 0.1, 0.2, 0.3, 0.6, 1.2, 1.8, and 2.5 mM.</li>
	<li>Collect data from the two electrodes (oxygen and TPMP+). Data acquisition software from the oxygraph system can be used to collect simultaneous measurements of mitochondrial oxygen consumption and mitochondrial membrane potential and observe changes in oxygen consumption in real time. Figure 14 shows how the oxygraph system records oxygen consumption as the experiment progresses.</li>
	<li>Calculate MMP in mV based on the Nernst equation: 
	MMP = 61.5 log ([TPMP+] added &#8211; external [TPMP+]) x TPMP+ binding correction/ (0.001 x mg of protein/mL x [TPMP+]) 
	A TPMP+ binding correction of 0.4 &#181;L/mg of mitochondrial protein-1 is used. 
	Example calculation based on concentrations in protocol: 
	MMP = 61.5 x log (5 &#181;M &#8211; 2 &#181;M) x 0.4/ (0.001 x 0.35 mg mitochondrial protein/mL x 2 &#181;M) 
	MMP = 198.9 mV</li>
	<li>Estimate PMF by plotting a graph of MMP vs. oxygen consumption (Figure 15). PMF is reported as oxygen consumption at a membrane potential of 165 mV. 
	NOTE: Titrating the electron transport chain with malonate (0.1 to 2.5 mM) shows the kinetic response of proton leak to MMP. Then, plotting MMP against oxygen consumption determines proton leak kinetics. PMF is determined by calculating oxygen consumption at a common membrane potential (165 mV).</li>
	<li>At the end of the last run of the sample, add FCCP (0.2 &#956;M total volume) to induce maximal respiration and release TPMP+ for baseline correction.</li>
	<li>Aspirate all of the solutions out of the respiration chamber. Rinse the chamber several times with double deionized water. At the end of the day, the chamber should also be rinsed a few times with ethanol.</li>
</ol>

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The authors have nothing to disclose.

The most critical point in the protocol is obtaining a representative liver tissue sample and beginning the isolation of mitochondria as soon as possible after biopsy. Variation in respiration measurements is low (Table 1) due to a short transport time from cow to laboratory. To reduce transport time, a small laboratory was set up in the office of the dairy, and liver samples were driven to the office laboratory as each was collected so that mitochondria were isolated within 10 min of biopsy. Setup and t...

Mitochondria are very sensitive to stress and their cellular environment can contribute to a wide variety of metabolic diseases. Oxygen consumption and proton leak in mitochondria are indicators of mitochondria health. The methods described in this paper estimate mitochondrial energy efficiency using RCR based on oxygen consumption with and without proton leak. These results can account for 30% of the energy lost in nutrient utilization1. Changes in oxygen consumption and proton leak can identify mitochondrial dysfunction which contributes to metabolic disease and results in decreased energy efficiency. These methods can also be used to examine the effect of different treatments on mitochondrial respiration. The overall goal of measuring mitochondrial oxygen consumption and proton leak kinetics is to assess mitochondrial function and energetic efficiency.
Hepatic mitochondrial dysfunction has been associated with several diseases in dairy cattle. The ability of cellular metabolism to switch between carbohydrate and lipid fuels when faced with an energy deficit in early lactation is influenced by the number and function of mitochondria in the cell2. Defects in the ability of mitochondria to adapt to an increased demand for energy and increased &#946;-oxidation can lead to accumulation of intracellular lipid associated with insulin resistance and may lead to the formation of fatty liver in early lactation dairy cows. Mitochondria, as the site of ketone body production and use, can play a key role in ketosis in dairy cows3. A lack of mitochondria or mitochondrial dysfunction will impact fuel availability to the periphery and be reflected in changes in oxygen consumption or RCR.
Mitochondrial oxygen consumption changes in response to inflammation. Seven-day-old broilers were randomly assigned to a group infected with Eimeria maxima and a control group4. Broilers that did not undergo coccidiosis challenge had lower oxygen consumption due to proton leak and higher RCR indicating that liver mitochondria respond to an immune challenge by increasing proton leak. While proton leak and reactive oxygen species production was once considered a sign of mitochondrial membrane dysfunction and detrimental to energetic efficiency, now it is known that it is important for import of proteins and calcium into mitochondria5, and for the generation of heat1.
Electron leak from the respiratory chain makes mitochondria susceptible to reactive oxygen species production and oxidative damage to mitochondrial membrane proteins, lipids and mitochondrial DNA. As mitochondria age, damage can accumulate especially to mtDNA causing further dysfunction in mitochondrial metabolism6 and greater susceptibility of the cow to disease. In practice, many livestock animals are fed high levels of supplements such as Cu, Zn and Mn to boost antioxidant function. However, feeding high levels of Cu, Zn and Mn decreased milk production and increased oxygen consumption due to proton leak (State 4 respiration)7.
Previous research on the role of mitochondrial function in energy efficiency in cattle has focused on changes in mitochondrial oxygen consumption and proton leak. Very few studies have been published in dairy cattle and most papers compare production efficiency in the form of residual feed intake (RFI) to mitochondrial function in beef cattle. Variability in mitochondrial respiration rates were examined by measuring state 3, state 4 and RCR in livers from both lactating Holstein cows and lactating beef cows (Angus, Brangus and Hereford)8. The researchers did not find any correlation in mitochondrial respiration with growth or milking traits for beef cattle but did report a correlation between mitochondrial respiration and milking traits for Holsteins. In two studies, RFI was compared in beef cattle to mitochondrial respiration rates (state 3, state 4 and RCR) in muscle mitochondria9,10. Mitochondrial respiration rates changed in response to DMI and low rates were associated with less efficient beef steers. In another study, RFI of steers from high or low RFI bulls were compared with mitochondrial respiration rates and proton leak kinetics between the two groups of progeny11. Differences were due to gain confirming the conclusion that gain does not impact mitochondrial respiration in beef cattle.
In this paper, an experiment examining liver RCR in response to feeding 3 antioxidant minerals to lactating dairy cattle illustrates the use of methods to measure oxygen consumption during State 4 and State 3 respiration and PMF.

<table>
	<tbody>
		<tr>
			<td>Liver Biopsy</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Schackelford-Courtney bovine liver biopsy instrument</td>
			<td>Sontec Instruments Englewood CO</td>
			<td>1103-904</td>
			<td></td>
		</tr>
		<tr>
			<td>Suture</td>
			<td>Fisher Scientific</td>
			<td>19-037-516</td>
			<td></td>
		</tr>
		<tr>
			<td>Suture needles</td>
			<td>NA</td>
			<td>NA</td>
			<td>Included with Suture</td>
		</tr>
		<tr>
			<td>Scalpels</td>
			<td>Sigma - Aldrich</td>
			<td>S2896 / S2646</td>
			<td># for handle and blades</td>
		</tr>
		<tr>
			<td>Surgery towels</td>
			<td>Fisher Scientific</td>
			<td>50-129-6667</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon tubes 50 mL</td>
			<td>Fisher Scientific</td>
			<td>14-432-22</td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezers</td>
			<td>Sigma - Aldrich</td>
			<td>Z168750</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL syringes</td>
			<td>Fisher Scientific</td>
			<td>22-314387</td>
			<td></td>
		</tr>
		<tr>
			<td>Injection needles (22, 2 1/2)</td>
			<td>VWR</td>
			<td>MJ8881-200342</td>
			<td></td>
		</tr>
		<tr>
			<td>Cow halter</td>
			<td>Tractor Supply Co.</td>
			<td>101966599</td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton swabbing</td>
			<td>Fisher Scientific</td>
			<td>14-959-102</td>
			<td></td>
		</tr>
		<tr>
			<td>cotton gauze squares (4x4)</td>
			<td>Fisher Scientific</td>
			<td>22-246069</td>
			<td></td>
		</tr>
		<tr>
			<td>Medical scissors</td>
			<td>Sigma - Aldrich</td>
			<td>Z265969</td>
			<td></td>
		</tr>
		<tr>
			<td>Chemicals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Coccidiosis Vaccine 0.75 bottle/cow</td>
			<td></td>
			<td></td>
			<td>Provided by Veterinarian</td>
		</tr>
		<tr>
			<td>Clostridia Vaccine</td>
			<td></td>
			<td></td>
			<td>Provided by Veterinarian</td>
		</tr>
		<tr>
			<td>Liver biopsy antibiotics excenel 2 cc/100 lbs for 3 days</td>
			<td></td>
			<td></td>
			<td>Provided by Veterinarian</td>
		</tr>
		<tr>
			<td>Providone Scrub</td>
			<td>Aspen Veteterinary Resources</td>
			<td>21260221</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 70%</td>
			<td>Sigma - Aldrich</td>
			<td>793213</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine hydrochloride 100 mg/mL IV at 0.010-0.015 mg/kg bodyweight</td>
			<td></td>
			<td></td>
			<td>Provided by Veterinarian</td>
		</tr>
		<tr>
			<td>2% lidocaine HCl (10-15 mL)</td>
			<td></td>
			<td></td>
			<td>Provided by Veterinarian</td>
		</tr>
		<tr>
			<td>1 mg/kg IV injection of flunixin meglumine</td>
			<td></td>
			<td></td>
			<td>Provided by Veterinarian</td>
		</tr>
		<tr>
			<td>Isolation of Mitochondria (liver)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Wheaton vial 30 mL with a Teflon pestle of 0.16 mm clearance</td>
			<td>Fisher Scientific</td>
			<td>02-911-527</td>
			<td></td>
		</tr>
		<tr>
			<td>Homogenizer Motor</td>
			<td>Cole Parmer</td>
			<td>EW-04369-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Homogenizer Probe</td>
			<td>Cole Parmer</td>
			<td>EW-04468-22</td>
			<td></td>
		</tr>
		<tr>
			<td>Auto Pipette (10 mL)</td>
			<td>Cole Parmer</td>
			<td>SK-21600-74</td>
			<td></td>
		</tr>
		<tr>
			<td>Beaker (500 mL) with ice</td>
			<td>Fisher Scientific</td>
			<td>FB100600</td>
			<td></td>
		</tr>
		<tr>
			<td>Refrigerated microfuge</td>
			<td>Fisher Scientific</td>
			<td>75-002-441EW3</td>
			<td></td>
		</tr>
		<tr>
			<td>Microfuge tubes (1.5 mL)</td>
			<td>Fisher Scientific</td>
			<td>AM12400</td>
			<td></td>
		</tr>
		<tr>
			<td>Chemicals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bicinchoninic acid (BCA) protein assay kit (microplates for plate reader)</td>
			<td>abcam</td>
			<td>ab102536</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma - Aldrich</td>
			<td>S7903-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>Sigma - Aldrich</td>
			<td>T1503-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma - Aldrich</td>
			<td>EDS-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA (fatty acid free)</td>
			<td>Sigma - Aldrich</td>
			<td>A7030-50G</td>
			<td></td>
		</tr>
		<tr>
			<td>Mannitol</td>
			<td>Sigma - Aldrich</td>
			<td>M4125-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Deionized water</td>
			<td>Sigma - Aldrich</td>
			<td>38796</td>
			<td></td>
		</tr>
		<tr>
			<td>Hepes</td>
			<td>Sigma - Aldrich</td>
			<td>H3375-500G</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">Use to create mitochondria isolation media: 220 mM mannitol, 70 mM sucrose, 20 mM HEPES, 20 mM Tris-HCl, 1 mM EDTA, and 0.1% (w/v) fatty acid free BSA,&#160; pH 7.4 at 4 &#176;C, will last 2 days in refrigerator</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mitochondrial Oxygen Comsuption</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Oxygraph Setup + Clark type oxygen electrode</td>
			<td>Hansatech (PP Systems)</td>
			<td>OXY1</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermoregulated Water Pump</td>
			<td>ADInstruments</td>
			<td>MLE2001</td>
			<td></td>
		</tr>
		<tr>
			<td>Clark type Oxygen electrode</td>
			<td>NA</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Autopipette (1 mL)</td>
			<td>Cole Parmer</td>
			<td>SK-21600-70</td>
			<td>Included with Oxy1</td>
		</tr>
		<tr>
			<td>Small magnetic stir bar</td>
			<td>Fisher Scientific</td>
			<td>14-513-95</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette (10 &#956;L)</td>
			<td>Cole Parmer</td>
			<td>SK-21600-60</td>
			<td></td>
		</tr>
		<tr>
			<td>pH meter</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Chemicals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma - Aldrich</td>
			<td>P9333-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Hepes</td>
			<td>Sigma - Aldrich</td>
			<td>H3375-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Sigma - Aldrich</td>
			<td>P5655-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma - Aldrich</td>
			<td>M1028-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma - Aldrich</td>
			<td>E3889-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Use to make mitochondrial oxygen consumption media: 120 mM KCL, 5 mM KH2PO4, 5 mM MgCl2, 5 mM Hepes and 1 mM EGTA,&#160; pH 7.4 at 30 &#176;C with 0.3% defatted BSA</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Rotenone (4 mM solution)</td>
			<td>Sigma - Aldrich</td>
			<td>R8875-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Succinate (1 M solution)</td>
			<td>Sigma - Aldrich</td>
			<td>S3674-250G</td>
			<td></td>
		</tr>
		<tr>
			<td>ADP (100 mM solution)</td>
			<td>Sigma - Aldrich</td>
			<td>A5285-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Oligomycin (solution of 8 &#956;g/mL in ethanol)</td>
			<td>Sigma - Aldrich</td>
			<td>75351</td>
			<td></td>
		</tr>
		<tr>
			<td>FCCP</td>
			<td>Sigma - Aldrich</td>
			<td>C2920</td>
			<td></td>
		</tr>
		<tr>
			<td>Mitochondrial Membrane Potential and Proton Motive Force</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TPMP electrode</td>
			<td>World Precision Instruments.</td>
			<td>DRIREF-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Chemicals-solutions do not need to be fresh but they do need to be kept in a freezer between runs</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Malonate (0.1 mM solution)</td>
			<td>Sigma - Aldrich</td>
			<td>M1296</td>
			<td></td>
		</tr>
		<tr>
			<td>Oligomycin (8 &#956;g/mL in ethanol), keep in freezer</td>
			<td>Sigma - Aldrich</td>
			<td>75351</td>
			<td></td>
		</tr>
		<tr>
			<td>Nigericin (80 ng/mL in ethanol), keep in freezer</td>
			<td>Sigma - Aldrich</td>
			<td>N7143</td>
			<td></td>
		</tr>
		<tr>
			<td>FCCP</td>
			<td>Sigma - Aldrich</td>
			<td>C3920</td>
			<td></td>
		</tr>
		<tr>
			<td>TPMP</td>
			<td>Sigma - Aldrich</td>
			<td>T200</td>
			<td></td>
		</tr>
		<tr>
			<td>TPMP solution: 10 mM TPMP, 120 mM KCL, 5 mM Hepes and 1 mM EGTA,&#160; pH 7.4 at 30 &#176;C with 0.3% defatted BSA</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

measuring liver mitochondrial oxygen consumption and proton leak kinetics to estimate mitochondrial respiration in holstein dairy cattle

Oxygen consumption, proton motive force (PMF) and proton leak are measurements of mitochondrial respiration, or how well mitochondria are able to convert NADH and FADH into ATP. Since mitochondria are also the primary site for oxygen use and nutrient oxidation to carbon dioxide and water, how efficiently they use oxygen and produce ATP directly relates to the efficiency of nutrient metabolism, nutrient requirements of the animal, and health of the animal. The purpose of this method is to examine mitochondrial respiration, which can be used to examine the effects of different drugs, diets and environmental effects on mitochondrial metabolism. Results include oxygen consumption measured as proton dependent respiration (State 3) and proton leak dependent respiration (State 4). The ratio of State 3 / State 4 respiration is defined as respiratory control ratio (RCR) and can represent mitochondrial energetic efficiency. Mitochondrial proton leak is a process that allows dissipation of mitochondrial membrane potential (MMP) by uncoupling oxidative phosphorylation from ADP decreasing the efficiency of ATP synthesis. Oxygen and TRMP+ sensitive electrodes with mitochondrial substrates and electron transport chain inhibitors are used to measure State 3 and State 4 respiration, mitochondrial membrane PMF (or the potential to produce ATP) and proton leak. Limitations to this method are that liver tissue must be as fresh as possible and all biopsies and assays must be performed in less than 10 h. This limits the number of samples that can be collected and processed by a single person in a day to approximately 5. However, only 1 g of liver tissue is needed, so in large animals, such as dairy cattle, the amount of sample needed is small relative to liver size and there is little recovery time needed.

Positive results showing RCR and proton leak kinetics are shown in Table 1 and Figure 15, respectively. In this study7, RCR and protein leak kinetics were measured in Holstein dairy cows at 70 days in milk after cows had been fed 1 of 5 different levels of Cu, Zn and Mn for 28 days. State 4, maximum proton leak-dependent respiration, had a tendency to be affected by mineral intake of Cu, Mn and Zn (p &#60; 0.1...

Watch this Scientific Journal Video about Measuring Liver Mitochondrial Oxygen Consumption and Proton Leak Kinetics to Estimate Mitochondrial Respiration in Holstein Dairy Cattle at JoVE.com

Measuring Liver Mitochondrial Oxygen Consumption and Proton Leak Kinetics to Estimate Mitochondrial Respiration in Holstein Dairy Cattle

Here, we share methods for measuring mitochondrial oxygen consumption, a defining concept of nutritional energetics, and proton leak, the primary cause of inefficiency in mitochondrial generation of ATP. These results can account for 30% of the energy lost in nutrient utilization to help evaluate mitochondrial function.

Oxygen consumption, proton motive force (PMF) and proton leak are measurements of mitochondrial respiration, or how well mitochondria are able to convert ...

measuring-liver-mitochondrial-oxygen-consumption-proton-leak-kinetics

Research

JoVE Journal

Biochemistry

11.2K Views.  University of California Davis. Here, we share methods for measuring mitochondrial oxygen consumption, a defining concept of nutritional energetics, and proton leak, the primary cause of inefficiency in mitochondrial generation of ATP. These results can account for 30% of the energy lost in nutrient utilization to help evaluate mitochondrial function.

Video: Measuring Liver Mitochondrial Oxygen Consumption and Proton Leak Kinetics to Estimate Mitochondrial Respiration in Holstein Dairy Cattle

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating these studies, reincorporation of detergent-solubilized membrane proteins into liposomes is a stochastic process where protein topology is impossible to enforce. This paper offers an alternative method to these challenging techniques that utilizes a liposome-based scaffold. Protein solubility is enhanced by deletion of the transmembrane domain, and these amino acids are replaced with a tethering moiety, such as a His-tag. This tether interacts with an anchoring group (Ni2+ coordinated by nitrilotriacetic acid (NTA(Ni2+)) for His-tagged proteins), which enforces a uniform protein topology at the surface of the liposome. An example is presented wherein the interaction between Dynamin-related protein 1 (Drp1) with an integral membrane protein, Mitochondrial Fission Factor (Mff), was investigated using this scaffold liposome method. In this work, we have demonstrated the ability of Mff to efficiently recruit soluble Drp1 to the surface of liposomes, which stimulated its GTPase activity. Moreover, Drp1 was able to tubulate the Mff-decorated lipid template in the presence of specific lipids. This example demonstrates the effectiveness of scaffold liposomes using structural and functional assays and highlights the role of Mff in regulating Drp1 activity.

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

This paper describes a method for assessing the interactions and assemblies of integral membrane proteins in vitro with various partner factors in a lipid-proximal environment.

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

Analyses of Mitochondrial Calcium Influx in Isolated Mitochondria and Cultured Cells

Ca2+ handling by mitochondria is a critical function regulating both physiological and pathophysiological processes in a broad spectrum of cells. The ability to accurately measure the influx and efflux of Ca2+ from mitochondria is important for determining the role of mitochondrial Ca2+ handling in these processes. In this report, we present two methods for the measurement of mitochondrial Ca2+ handling in both isolated mitochondria and cultured cells. We first detail a plate reader-based platform for measuring mitochondrial Ca2+ uptake using the Ca2+ sensitive dye calcium green-5N. The plate reader-based format circumvents the need for specialized equipment, and the calcium green-5N dye is ideally suited for measuring Ca2+ from isolated tissue mitochondria. For our application, we describe the measurement of mitochondrial Ca2+ uptake in mitochondria isolated from mouse heart tissue; however, this procedure can be applied to measure mitochondrial Ca2+ uptake in mitochondria isolated from other tissues such as liver, skeletal muscle, and brain. Secondly, we describe a confocal microscopy-based assay for measurement of mitochondrial Ca2+ in permeabilized cells using the Ca2+ sensitive dye Rhod-2/AM and imaging using 2-dimensional laser-scanning microscopy. This permeabilization protocol eliminates cytosolic dye contamination, allowing for specific recording of changes in mitochondrial Ca2+. Moreover, laser-scanning microscopy allows for high frame rates to capture rapid changes in mitochondrial Ca2+ in response to various drugs or reagents applied in the external solution. This protocol can be applied to measure mitochondrial Ca2+ uptake in many cell types including primary cells such as cardiac myocytes and neurons, and immortalized cell lines.

Here, we present two protocols for the measurement of mitochondrial Ca2+ influx in isolated mitochondria and cultured cells. For isolated mitochondria, we detail a plate reader-based Ca2+ import assay using the Ca2+ sensitive dye calcium green-5N. For cultured cells, we describe a confocal microscopy method using the Ca2+ dye Rhod-2/AM.

Ca2+ handling by mitochondria is a critical function regulating both physiological and pathophysiological processes in a broad spectrum of cells. The ...

Measurement of Mitochondrial Oxygen Consumption in Permeabilized Fibers of Drosophila Using Minimal Amounts of Tissue

The fruit fly, Drosophila melanogaster, represents an emerging model for the study of metabolism. Indeed, drosophila have structures homologous to human organs, possess highly conserved metabolic pathways and have a relatively short lifespan that allows the study of different fundamental mechanisms in a short period of time. It is, however, surprising that one of the mechanisms essential for cellular metabolism, the mitochondrial respiration, has not been thoroughly investigated in this model. It is likely because the measure of the mitochondrial respiration in Drosophila usually requires a very large number of individuals and the results obtained are not highly reproducible. Here, a method allowing the precise measurement of mitochondrial oxygen consumption using minimal amounts of tissue from Drosophila is described. In this method, the thoraxes are dissected and permeabilized both mechanically with sharp forceps and chemically with saponin, allowing different compounds to cross the cell membrane and modulate the mitochondrial respiration. After permeabilization, a protocol is performed to evaluate the capacity of the different complexes of the electron transport system (ETS) to oxidize different substrates, as well as their response to an uncoupler and to several inhibitors. This method presents many advantages compared to methods using mitochondrial isolations, as it is more physiologically relevant because the mitochondria are still interacting with the other cellular components and the mitochondrial morphology is conserved. Moreover, sample preparations are faster, and the results obtained are highly reproducible. By combining the advantages of Drosophila as a model for the study of metabolism with the evaluation of mitochondrial respiration, important new insights can be unveiled, especially when the flies are experiencing different environmental or pathophysiological conditions.

In this paper, a method to measure oxygen consumption using high-resolution respirometry in permeabilized thoraxes of Drosophila is described. This technique requires a minimal amount of tissue compared to the classic mitochondrial isolation technique and the results obtained are more physiologically relevant.

The fruit fly, Drosophila melanogaster, represents an emerging model for the study of metabolism. Indeed, drosophila have structures homologous to human ...

Measuring and Interpreting Oxygen Consumption Rates in Whole Fly Head Segments

Regulated metabolic activity is essential for the normal functioning of living cells. Indeed, altered metabolic activity is causally linked with the progression of cancer, diabetes, neurodegeneration, and aging to name a few. For instance, changes in mitochondrial activity, the cell&#39;s metabolic powerhouse, have been characterized in many such diseases. Generally, the oxygen consumption rates of mitochondria were considered a reliable readout of mitochondrial activity and measurements in some of these studies were based on isolated mitochondria or cells. However, such conditions may not represent the complexity of a whole tissue. Recently, we have developed a novel method that enables the dynamic measurement of oxygen consumption rates from whole isolated fly heads. By utilizing this method, we have recorded lower oxygen consumption rates of the whole head segment in young versus aged flies. Secondly, we have discovered that lysine deacetylase inhibitors rapidly alter the oxygen consumption in the whole head. Our novel technique may therefore aid in uncovering new properties of various drugs, which may impact metabolic rates. Furthermore, our method may give a better understanding of metabolic behavior in an experimental setup that more closely resembles physiological states.

Measuring alterations in metabolic rates is central to understanding the progression of various diseases and aging. Here, we present a novel technique to measure whole head oxygen consumption that more closely resembles the physiological state and may aid in revealing novel drugs that modify mitochondrial activity.

Regulated metabolic activity is essential for the normal functioning of living cells. Indeed, altered metabolic activity is causally linked with the ...

Measuring Mitochondrial Electron Transfer Complexes in Previously Frozen Cardiac Tissue from the Offspring of Sow: A Model to Assess Exercise-Induced Mitochondrial Bioenergetics Changes

The mitochondrial electron transfer complex (ETC) profile is modified in the heart tissue of the offspring born to an exercised sow. The hypothesis proposed and tested was that a regular maternal exercise of a sow during pregnancy would increase the mitochondrial efficiency of offspring heart bioenergetics. This hypothesis was tested by isolating mitochondria using a mild-isolation procedure to assess mitochondrial ETC and supercomplex profiles. The procedure described here allowed for the processing of previously frozen archived heart tissues and eliminated the necessity of fresh mitochondria preparation for the assessment of mitochondrial ETC complexes, supercomplexes, and ETC complex activity profiles. This protocol describes the optimal ETC protein complex measurement in multiplexed antibody-based immunoblotting and super complex assessment using blue-native gel electrophoresis.

Preparation of mitochondria-enriched samples from previously frozen archived solid tissues allowed the investigators to perform both functional and analytical assessments of mitochondria in various experimental modalities. This study demonstrates how to prepare mitochondria-enriched preparations from frozen heart tissue and perform analytical assessments of mitochondria.

The mitochondrial electron transfer complex (ETC) profile is modified in the heart tissue of the offspring born to an exercised sow. The hypothesis ...

High-Throughput Quantification of the Synthesis and Distribution of mtDNA

High-Throughput Image-Based Quantification of Mitochondrial DNA Synthesis and Distribution

The vast majority of cellular processes require a continuous supply of energy, the most common carrier of which is the ATP molecule. Eukaryotic cells produce most of their ATP in the mitochondria by oxidative phosphorylation. Mitochondria are unique organelles because they have their own genome that is replicated and passed on to the next generation of cells. In contrast to the nuclear genome, there are multiple copies of the mitochondrial genome in the cell. The detailed study of the mechanisms responsible for the replication, repair, and maintenance of the mitochondrial genome is essential for understanding the proper functioning of mitochondria and whole cells under both normal and disease conditions. Here, a method that allows the high-throughput quantification of the synthesis and distribution of mitochondrial DNA (mtDNA) in human cells cultured in vitro is presented. This approach is based on the immunofluorescence detection of actively synthesized DNA molecules labeled by 5-bromo-2'-deoxyuridine (BrdU) incorporation and the concurrent detection of all the mtDNA molecules with anti-DNA antibodies. Additionally, the mitochondria are visualized with specific dyes or antibodies. The culturing of cells in a multi-well format and the utilization of an automated fluorescence microscope make it easier to study the dynamics of mtDNA and the morphology of mitochondria under a variety of experimental conditions in a relatively short time.

Author Spotlight: High-Throughput Image-Based Quantification of Mitochondrial DNA Synthesis and Distribution  

A procedure for studying the dynamics of mitochondrial DNA (mtDNA) metabolism in cells using a multi-well plate format and automated immunofluorescence imaging to detect and quantify mtDNA synthesis and distribution is described. This can be further used to investigate the effects of various inhibitors, cellular stresses, and gene silencing on mtDNA metabolism.

The vast majority of cellular processes require a continuous supply of energy, the most common carrier of which is the ATP molecule. Eukaryotic cells ...

Stable Isotope Tracing & LC-MS Analysis to Measure DHODH and SDH Activities

Oxygen-Independent Assays to Measure Mitochondrial Function in Mammals

The flow of electrons in the mitochondrial electron transport chain (ETC) supports multifaceted biosynthetic, bioenergetic, and signaling functions in mammalian cells. As oxygen (O2) is the most ubiquitous terminal electron acceptor for the mammalian ETC, the O2 consumption rate is frequently used as a proxy for mitochondrial function. However, emerging research demonstrates that this parameter is not always indicative of mitochondrial function, as fumarate can be employed as an alternative electron acceptor to sustain mitochondrial functions in hypoxia. This article compiles a series of protocols that allow researchers to measure mitochondrial function independently of the O2 consumption rate. These assays are particularly useful when studying mitochondrial function in hypoxic environments. Specifically, we describe methods to measure mitochondrial ATP production, de novo pyrimidine biosynthesis, NADH oxidation by complex I, and superoxide production. In combination with classical respirometry experiments, these orthogonal and economical assays will provide researchers with a more comprehensive assessment of mitochondrial function in their system of interest.

Author Spotlight: Oxygen-Independent Assays to Measure Mitochondrial Function in Mammals  

Here, we present a compilation of assays to directly measure mitochondrial function in mammalian cells independently of their ability to consume molecular oxygen.

The flow of electrons in the mitochondrial electron transport chain (ETC) supports multifaceted biosynthetic, bioenergetic, and signaling functions in ...

Isolation of Mitochondrial Contact Sites

An Improved Method to Isolate Mitochondrial Contact Sites

Mitochondria are present in virtually all eukaryotic cells and perform essential functions that go far beyond energy production, for instance, the synthesis of iron-sulfur clusters, lipids, or proteins, Ca2+ buffering, and the induction of apoptosis. Likewise, mitochondrial dysfunction results in severe human diseases such as cancer, diabetes, and neurodegeneration. In order to perform these functions, mitochondria have to communicate with the rest of the cell across their envelope, which consists of two membranes. Therefore, these two membranes have to interact constantly. Proteinaceous contact sites between the mitochondrial inner and outer membranes are essential in this respect. So far, several contact sites have been identified. In the method described here, Saccharomyces cerevisiae mitochondria are used to isolate contact sites and, thus, identify candidates that qualify for contact site proteins. We used this method to identify the mitochondrial contact site and cristae organizing system (MICOS) complex, one of the major contact site-forming complexes in the mitochondrial inner membrane, which is conserved from yeast to humans. Recently, we further improved this method to identify a novel contact site consisting of Cqd1 and the Por1-Om14 complex.

Author Spotlight: Unveiling Mitochondrial Contact Sites and Architectural Insights 

Mitochondrial contact sites are protein complexes that interact with mitochondrial inner and outer membrane proteins. These sites are essential for the communication between the mitochondrial membranes and, thus, between the cytosol and the mitochondrial matrix. Here, we describe a method to identify candidates qualifying for this specific class of proteins.

Mitochondria are present in virtually all eukaryotic cells and perform essential functions that go far beyond energy production, for instance, the ...

Analyzing Mitochondrial Physiology and Bioenergetics in Fruit Flies

Analyzing Mitochondrial Function in a Drosophila melanogaster PINK1B9-Null Mutant Using High-resolution Respirometry

Neurodegenerative diseases, including Parkinson&#39;s Disease (PD), and cellular disturbances such as cancer are some of the disorders that disrupt energy metabolism with impairment of mitochondrial functions. Mitochondria are organelles that control both energy metabolism and cellular processes involved in cell survival and death. For this reason, approaches to evaluate mitochondrial function can offer important insights into cellular conditions in pathological and physiological processes. In this regard, high-resolution respirometry (HRR) protocols allow evaluation of the whole mitochondrial respiratory chain function or the activity of specific mitochondrial complexes. Furthermore, studying mitochondrial physiology and bioenergetics requires genetically and experimentally tractable models such as Drosophila melanogaster.
This model presents several advantages, such as its similarity to human physiology, its rapid life cycle, easy maintenance, cost-effectiveness, high throughput capabilities, and a minimized number of ethical concerns. These attributes collectively establish it as an invaluable tool for dissecting complex cellular processes. The present work explains how to analyze mitochondrial function using the Drosophila melanogaster PINK1B9-null mutant. The pink1 gene is responsible for encoding PTEN-induced putative kinase 1, through a process recognized as mitophagy, which is crucial for the removal of dysfunctional mitochondria from the mitochondrial network. Mutations in this gene have been associated with an autosomal recessive early-onset familial form of PD. This model can be used to study mitochondrial dysfunction involved in the pathophysiology of PD.

Author Spotlight: Exploring Mitochondrial Function and Chemical Toxicity Using Drosophila melanogaster 

Here we present a high-resolution respirometry protocol to analyze bioenergetics in PINK1B9-null mutant fruit flies. The method uses the Substrate-Uncoupler-Inhibitor-Titration (SUIT) protocol.

Neurodegenerative diseases, including Parkinson&#39;s Disease (PD), and cellular disturbances such as cancer are some of the disorders that disrupt energy ...

Mitochondrial Fractionation in Small Tissue Samples for Supercomplex Analysis

Isolation of Mitochondria for Mitochondrial Supercomplex Analysis from Small Tissue and Cell Culture Samples

Over the last decades, the evidence accumulated about the existence of respiratory supercomplexes (SCs) has changed our understanding of the mitochondrial electron transport chain organization, giving rise to the proposal of the "plasticity model." This model postulates the coexistence of different proportions of SCs and complexes depending on the tissue or the cellular metabolic status. The dynamic nature of the assembly in SCs would allow cells to optimize the use of available fuels and the efficiency of electron transfer, minimizing reactive oxygen species generation and favoring the ability of cells to adapt to environmental changes. 
More recently, abnormalities in SC assembly have been reported in different diseases such as neurodegenerative disorders (Alzheimer's and Parkinson's disease), Barth Syndrome, Leigh syndrome, or cancer. The role of SC assembly alterations in disease progression still needs to be confirmed. Nevertheless, the availability of enough amounts of samples to determine the SC assembly status is often a challenge. This happens with biopsy or tissue samples that are small or have to be divided for multiple analyses, with cell cultures that have slow growth or come from microfluidic devices, with some primary cultures or rare cells, or when the effect of particular costly treatments has to be analyzed (with nanoparticles, very expensive compounds, etc.). In these cases, an efficient and easy-to-apply method is required. This paper presents a method adapted to obtain enriched mitochondrial fractions from small amounts of cells or tissues to analyze the structure and function of mitochondrial SCs by native electrophoresis followed by in-gel activity assays or western blot.

Author Spotlight: Unveiling Oxidative Phosphorylation System Dynamics and Mitochondrial Roles in Health and Disease

This protocol describes a technique for the analysis of respiratory supercomplexes when only small amounts of samples are available.

Over the last decades, the evidence accumulated about the existence of respiratory supercomplexes (SCs) has changed our understanding of the mitochondrial ...