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Anaerobic

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Submitted By prsexton
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Introduction Most vertebrates use aerobic metabolism for their energy needs. Although, some animals use anaerobic metabolism, in which food molecules are oxidized incompletely to lactic acid, lactate (Randall et al., 2002). There are particular times when animals cannot use aerobic metabolism for their energy needs. Such times include when oxygen is low in the environment. Also, when animals exercise, their increase in muscle contraction raises ATP demand resulting in the capacity for oxygen delivery to tissues and the aerobic energy production to be exceeded. When oxygen is very low or unavailable, pyruvate and NADH accumulate and NAD+ will deplete. Pyruvate is converted into lactate by the enzyme lactate dehydrogenase. The conversion of NADH to NAD+ allows for glycolysis to continue. Based on the information obtained from lab, most animals cannot sustain anaerobic glycolysis and intense exercise for a long period of time because there is a limit in carbohydrate fuels and end products accumulate. The goal of this experiment was to use blood lactate concentrations as an indicator of the amount of anaerobic metabolism used. Also, this experiment is conducted to observe the speed of recovery after the use of anaerobic metabolism.

Materials and Methods Blood samples were collected from 63 individuals of the species Fundulus grandis. The fish were subject to different levels of rest and exercise. Some had very minimal handling from an aerated tank, while others were exercised for five minutes, had sixty minutes of recovery after exercise and one hundred and eighty minutes for recovery. From each tank, a fish was obtained by each group and placed in a bowl of MS-222 until the fish lay belly up in the solution. Immediately following, blood samples from a total of 15, 17, 15 and 16 individuals were collected and added to perchloric acid to stop metabolic reactions for the fish at rest, exercise (5 minutes), 60 minutes recovery, and 180 minutes recovery respectively. These samples were neutralized with KOH. To measure lactate concentrations a dilution factor was determined using the following equation:
DF= (200 + BV) x (150 + 2 + KOH) BV 150
The following biochemical reaction produces a molecule that can be directly measured:
Lactate + NAD+ ←→ Pyruvate +NADH
Lactate is transformed to pyruvate and in the process, the coenzyme NAD+ is reduced to NADH. The equilibrium in the reaction is in favor of lactate formation so pyruvate must be removed from the solution to force the reaction to the right. The absorbance of NADH is directly proportional to the lactate concentration and is read using a spectrophotometer. Once the standard curve was used to calculate the lactate concentrations, the results were multiplied by the dilution factor previous determined to obtain the blood lactate from each fish. The lab procedures were followed as described in the protocol.
The statistical tests used in this experiment were descriptive analysis to find the means and standard deviations of each treatment and a T-test, unpaired two-tail assuming equal variance of rest versus exercise, exercise versus 60 minutes of recovery, exercise versus 180 minutes of recovery, and rest versus 180 minutes of recovery by using the p values to determine if lactate concentrations differ between treatments.

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