Does Rancidity, As Measured by Peroxide Value, Affect Animal Performance?

C. R. Hamilton, Ph. D.1 and D. Kirstein, M.S.2
Darling international Inc.

Rancidity refers to the oxidative state of fats, which is a characteristic that may have nutritional relevance and, in extreme cases, affect the well being of poultry and livestock. However, these effects are poorly understood. Most tolerances and standards used to evaluate animal feeds and ingredients for rancidity are derived from human thresholds for detecting off-flavors associated with rancidity. Therefore, it is assumed that feeding fats that have undergone severe oxidation may reduce feed consumption and growth rate in animals.

Oxidative rancidity is a complex process that is thought to occur in phases: (1) initiation, (2) auto-oxidation and (3) termination. During each phase, the formation of products increase and decrease over time, as shown in Figure 1. Hydroperoxides form when oxygen and unsaturated fatty acids combine in the presence of a catalyst (such as iron, copper, heat, light, enzymes, etc.) during the initiation phase. These peroxides are reactive and can combine with other fats to form additional reactive products during auto-oxidation. Hydrocarbons, aldehydes and ketones are formed during the termination or final phase. These compounds are volatile, but relatively unreactive.

The best in vitro indicator of rancidity for feeds and feed ingredients has not yet been defined. Rancidity is a qualitative term or state that is not chemically defined and is not quantifiable. As a result, a number of different methods have been used to test for various intermediates or products of oxidation. However, these products are “moving targets”, so it is not possible to predict the best indicator of fat oxidation.

N-PAL (2001) described various methods used to predict or interpret the rancidity of fats. Their review was used in developing the following list:

Active Oxygen Method (AOM) attempts to predict the stability of a fat by bubbling air through the sample under specific conditions for flow rate, temperature and concentration. Peroxides are measured at time intervals. The number of hours required to reach 100 milliequivalents (meq) of peroxide per kg of fat is reported as the AOM for the fat. This method is time consuming and seldom used any more.

The thiobarbaturic acid (TBA) test measures aldehyde formation as an indicator of the stability or rancidity of fats. The test was originally developed to detect malonaldehyde, but other aldehydes also react with 2-thiobarbituric acid. However, aldehydes are volatile - low molecular weight compounds and may be lost when fats are heated for extended periods of time as is necessary to facilitate handling and processing in a feed mill. The TBA test is most often used to determine the stability or antioxidant status of animal tissues, such as plasma, serum, organs and other tissues.

p-Anisidine Value is another indicator method. Anisidine reacts with the non-volatile portion of fatty acids left behind when hydroperoxides break down to aldehydes. The aldehydes are volatile and follow the same formation-degradation curve as other oxidation products. Even if aldehydes are no longer detectable with the TBA test, the Anisidine value may still indicate that the fat has been oxidized because the residues detected are not volatile. While this method may provide some indication of the oxidative history of a fat, it is not suitable for the detection of fat oxidation.

Oxidative Stability is a predictive test that measures resistance of a fat to oxidation and gives some indication of shelf-life. Heat, oxygen and catalytic metals accelerate the rate of oxidation. Saturated (harder) fats are more resistant to oxidation than unsaturated (soft) fats. The cost of this analysis is approximately two times that of the AOM test.

Peroxide Value (PV) is the most widely used indicator of fat oxidation. A peroxide value is required only for USDA certified edible animal fats, such as tallow or lard. However, the feed industry also uses PV to assess the stability or rancidity of fats used as feed ingredients, by measuring lipid peroxides and hydroperoxides formed during the initial stages of oxidation. Values are reported as meq of peroxide per kg of fat. It should be noted that difficulties in the titration end point are common for low PV levels, which may account for a portion of the high variability generally associated with the method.

Peroxides (O2) are intermediate products of fat oxidation and breakdown rapidly to aldehydes, ketones and other products. An example of a peroxide production curve is shown in Figure 2 using choice white grease exposed to heat and oxygen for 11 days (DeRouchey et al., 2000). The peroxide values peaked on day 7 at about 105 meq/kg of fat and subsequently declined to baseline values.

Determining Peroxide Value

Peroxide value is most often determined using the AOCS Official Method Cd 8-53 (same as AOAC Official Method 965.33). An alternative procedure that is sometimes used is AOCS Official Method Cd 8b-90, which uses isooctane instead of chloroform as a solvent. These procedures all measure the milliequivalents of peroxide per kilogram of fat. In order to determine the PV on a sample of protein meal (such as meat and bone meal) it is necessary to first extract the 5 grams of fat needed for the procedure.

Unfortunately, there is no standard extraction procedure for obtaining fat from protein meals on which to subsequently perform a PV test. One area of disagreement is the solvent used for the extraction. Some of these solvents include hexane, hexane:methanol, and pet ether. One could argue that the hexane:methanol solvent provides the best mixture to most efficiently extract tri-, di-, and mono-glycerides as well as more polar free fatty acids. However, the use of a standard solvent has not been established. PV results can be expected to vary when comparing fats extracted from meals using different solvents.

Another issue involving the extraction step involves how the solvent is evaporated from the fat. It is critical to evaporate the solvent without using elevated temperatures and under a stream of nitrogen gas. Overlooking this step in the extraction process will itself cause a rise in the PV. Laboratories in the United States have not universally adopted this step. The American Oil Chemists’ Society (AOCS) notes that the official PV method is “highly empirical, and any variation in the test procedure may result in variation of the results” (AOCS, 1997). Variation in the sample size of extracted fat appears to be a good example of this. Although the method calls for a standard 5 grams of fat, when labs run short on meal volume, they may run the PV test on whatever amount of fat they were able to extract. Preliminary results comparing different sample sizes appear to confirm AOCS’s warning that doing so will cause variation in the results (National By-Products Analytical Lab, 2003). In addition, Fiebig (2003) reported that sample size strongly influences the PV obtained. He further suggested variability in the quantity of sample to be the most likely cause of poor reproducibility limits.
Given the empirical nature of the PV test and no official method for extracting fat from protein meals, it is important that buyers and sellers agree upon the methodology. Prior agreements should be made to have involved laboratories follow identical protocols. Arrangements should also be made for a referee lab to follow the same procedure in order to arbitrate any conflicting results.

Interpreting Peroxide Value Results

Nutritionists and buyers have arbitrarily established maximum initial PV levels of between 5 and 20 meq O2/kg of fat as acceptable. The origin of these standards is unknown. Carpenter et al. (1966) suggested the source to be from the footnote to a table in a paper published in 1941 (Gray and Robinson). The footnote gave analytical data for a series of meat meal samples and included the comment: “a fat with a peroxide value of more than 20 is definitely rancid”, even though they concluded later in the paper that such “rancid” meat meals may be fed to animals without harm.

Poultry and livestock are relatively resistant to all but excessively oxidized fats. Peroxide values below 100 meq O2/kg of fat did not depress feed consumption or growth rate in broiler chickens (L’Estrange et al., 1966; Cabel and Waldroup, 1988) or turkeys (Lea et al., 1966). This is illustrated in Table 1. Peroxide Values below 40 meq/kg of fat did not affect growth rate or the digestibility of nutrients, including unsaturated and saturated fatty acids, in young pigs (DeRouchey et al., 2000). This is shown in Figure 3.

Pesti et al. (2002) fed diets containing either 3 % or 6 % of poultry fat, restaurant grease, choice white grease, an animal/vegetable blend, palm oil, yellow grease or edible soybean oil to broiler chickens. All fat sources had similar initial PV levels, but their oxidative stability, measured by the Active Oxygen Method, varied from 2 to 370 meq O2/kg of fat. Growth rate, feed consumption and feed conversion ratio were not affected by fat source or oxidative stability. The authors further concluded that rancidity indicators, such as initial PV and AOM, were not correlated (P > 0.60) with broiler chick performance.

Because animal proteins can contain between 8 and 14 % fat, some nutritionist and regulatory officials have been concerned about their oxidative status. Only the fat portion is subject to oxidation. Therefore, any PV determined on the extracted fat must be diluted accordingly before making inferences about the protein meal. In addition, as with using pure fat, animals are quite resistant to the presence of peroxides in animal protein meals. Growth rate, feed consumption and feed efficiency were not affected when broiler chickens were fed poultry byproduct meal (Kirkland and Fuller, 1971) or fish meal (Lea et al., 1966) containing oxidized fat having peroxide values of from 32 to 110 meq O2/kg of fat. Carpenter et al. (1966) reported that performance was not affected when pigs were fed oxidized (105 meq O2/kg of fat) meat meal (Table 2).

The importance of feeding oxidized fats to animals may be of less importance today than at the time that Gray and Robinson (1941) made their comments about rancid fats because of advances made in commercial antioxidants and fat soluble vitamins (vitamins A, D and E), which function as natural antioxidants. Adding 125 ppm ethoxyquin alleviated the effects on performance that occurred when broilers were fed fat with 175 meq O2/kg (Cabel and Waldroup, 1988; Table 1). Some growth reductions observed in commercial feeding that are attributed to fat stability issues, may actually be caused by insufficient supplementation of fat soluble vitamins and/or over supplementation with iron or copper. However, because these vitamins are excellent antioxidants, attention must be given to the levels added to commercial feeds whenever fat is used, especially when complete feeds fortified with trace minerals are stored for prolonged periods of time in hot climates.
Establishing Acceptable Tolerances for Peroxides in Fat and Ingredients

The effects of increased peroxide concentration on broiler growth were predicted (Figure 4) using broiler performance data. The curve clearly illustrates that growth rate is not affected at concentrations up to 100 meq O2/kg of fat, but may sharply decline at concentrations greater than about 150 meq O2/kg of fat.

The growth rate of broilers was reduced when fed diets containing 5 % poultry fat with a PV of 175 meq O2/kg of fat (Cabel and Waldroup, 1988; Table 1) and 11 % vegetable oil with a PV of 156 meq O2 /kg of fat (Engberg et al., 1996). Based on these data, a negative response occurred when broilers were fed from 8.75 to 17.2 meq O2/kg of complete feed. Peroxide levels of 5.0 (Cabel and Waldroup, 1988) and 5.45 meq/kg of complete feed (L’Estrange at al., 1966) did not affect broilers and can be considered to be safe.

Studies conducted with monogastric animals suggest that 4 meq O2/kg or less in the complete feed will not affect performance. Using this as a maximum acceptable PV will provide a safety factor of at least 25 %. A fat source making up 5% of the diet could have a PV of up to 80 meq O2/kg of fat in order to reach 4/meq O2/kg in the complete feed.

Even though a standardized method for determining the PV of fat in meat and bone meal has not been developed, it would be helpful to establish a maximum PV tolerance for meat and bone meal as well as feeding fats. We recommend a tolerance of 80 meq O2/kg of fat in MBM and 40 meq O2/kg of feeding fats. These levels will provide adequate safety margins as shown in the examples below:

For Fat In Meat and Bone Meal:
|Tolerance in complete feed, meq O2/kg of feed |4.0 |
|Level of meat and bone meal in complete feed, % |5 |
|Level of fat in meat and bone meal, % |12 |
|Level of fat from meat and bone meal, % |0.6 |
|PV of fat in meat and bone meal, meq O2/kg of feed |80 |
|PV of complete feed, meq O2/kg of feed: |0.48 |
|(fat level * PV of fat) = (0.6% * 80) | |
|Safety factor (4.0/0.48 meq O2/kg of feed), % |833 |

For Feeding Fat:
|Tolerance in complete feed, meq O2/kg of feed |4.0 |
|Level of fat in complete feed, % |5 |
|PV of fat, meq O2/kg of feed |40 |
|PV of complete feed, meq O2/kg of feed: |2.0 |
|(fat level * PV of fat) = (5.0% * 40) | |
|Safety factor (4.0/2.0 meq O2/kg of feed), % |200 |

Summary

• Reliable and accurate indicators of rancidity in fats have not been developed. Most laboratory methods either attempt to predict the oxidative status of fats or measure intermediary products of oxidation as indicators of rancidity.

• Peroxide Value is the most commonly used indicator of fat rancidity. Peroxides are not static and may not accurately predict the oxidative status of fats.

• Standard methods have been developed for measuring peroxides in pure fats, but not for protein meals. These methods are “highly empirical, and any variation in the test procedure may result in variation of the results”.

• A standard procedure has not been developed for determining the peroxide value of animal proteins. Variation in sample size, oxidation of the fat while evaporating the solvent and methodology variation among laboratories suggest that peroxide values determined on animal protein meals may be inaccurate, unreliable and highly variable.

• The buyer and seller must agree upon the methodology used to determine the peroxide value of animal proteins. All parties should also agree on a third-party lab for arbitration.

• Peroxide levels of 100 meq/kg of fat can be fed to poultry without affecting performance, while young pigs can tolerate levels up to 40 meq/kg of fat.

• Fats should be stabilized with an antioxidant and the nutritionist should insure that diets are fortified with adequate levels of fat soluble vitamins.

• If is becomes necessary to establish a tolerance for peroxides in animal proteins, a maximum of 80 meq O2/kg of fat is recommended. Because of their potential for higher dietary inclusion, we recommend a maximum of 40 meq O2/kg of feeding fats.

Literature Cited

AOCS. 1997. The Official Methods and Recommended Practices of the American Oil Chemists’ Society, 4th edition. AOCS Official Method Cd 8-53: Peroxide Value – Acetic Acid-Chloroform Method, Re-approved 1997.

Cabel, M. C. and P. W. Waldroup. 1988. Poultry Science. 67:1725-1730.
Carpenter, K. J., J. L. L’Estrange and C. H. Lea. 1966. Proc. Nutr. Soc. 25:25-31.
DeRouchey, J. M. et al. 2000. Kansas State University Swine Research Report pp 83-86
Engberg, R. M., C. Lauridsen, S. K. Jensen and K. Jakobsen. 1996. Poultry Science 75:1003-1011.
Fiebig, Hans-Jochen. 2003. Inform 14 (10):651-652.
Gray, R. E. and H. E. Robinson. 1941. Poultry Science. 20:36.
Kirkland, W. M. and H. L. Fuller. 1971. Poultry Science. 50:137-143
L’ Estrange, J. L., K. J. Carpenter, C. H. Lea and L. J. Parr. 1966. British Journal of Nutrition 20:113-122.
Lea, C. H., L. J. Parr, L’Estrange and K. J. Carpenter. 1966. British Journal of Nutrition 20:123
National By-Products Analytical Laboratories, 2003. Des Moines, IA. (Personal communication).
N-PAL. 2001. N. P. Analytical Laboratories. http://www.ralstonanalytical.com/npal2/
Pesti, G. M., R. I. Bakalli, M. Qiao and K. G. Sterling. 2002. Poultry Science. 81:382-390.

Figure 3. Effects of Lipid Peroxides on Weanling Pig Performance

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Table 1. Effects of PV on Final Weight (kg) of Broilers Fed Poultry Fat With or Without Ethoxyquin for 49 Days. a
|Ethoxyquin |Peroxide level (PV, meq/kg) |Average |
|added |0 |50 |100 |175 |ethoxyquin |
|0 ppm |1.63 bc |1.63 bc |1.61 c |1.53 d |1.60 |
|62.5 ppm |1.64 bc |1.64 bc |1.64 b |1.56 cd |1.62 |
|125 ppm |1.65 bc |1.64 bc |1.60 c |1.61 bc |1.63 |
|Peroxide level average: |1.64 x |1.64 x |1.61 x |1.56 y | |

a Cabel and Waldroup (1988), 5% poultry fat added to corn-soybean meal diets. bcd Treatment means without a common superscript differ (P< .005). xy Main effect means without a common superscript differ (P < 0.05).

Table 2 . Effects of Feeding Fresh or High-Peroxide Meat Meal to Pigs .a
| |Fresh |High-Peroxide Meat Meal |
|Item |Meat Meal |Without Vitamin E |With Vitamin E |
|Gain, kg/head |40.37 |38.10 |41.27 |
|Feed Conversion Ratio |2.55 |2.57 |2.53 |
|Vitamin A in liver |29 |27 |26 |
|(i.u x 10-3) | | | |

a Carpenter et al., 1966. Diets contained 10 % meat meal, which contained 17% fat. Peroxide value of the fat in oxidized meat meal = 105. There were no treatment effects (P > 0.10).

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Figure 1. Progression of Fat Oxidation

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Figure4. Predicted Effects of Peroxide Value of Fat on Broiler Growth.

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