Lipids Lipids are commonly added to diets

As an energy source to improve feed efficiency

Reduce dust

Provide essential fatty acids

Supply fat soluble vitamins

Milling efficiency/pellet quality

Lipids Variable composition and quality

Food processing facilities and restaurants (e.g. yellow grease, brown grease) or agricultural co-products (e.g., animal fats & proteins, DDGS) Subjected to heat for variable lengths and temperatures

Limited information about state of lipid peroxidation and subsequent animal impacts

Why interest in lipid quality? Improved genetics have made pigs leaner

Leaner pigs are affected more by dietary fat consumed

More sources of unsaturated fats are being added to swine diets DDGS Contains 10% crude fat (5.9% linoleic acid)

Bakery by-product Contains 11% crude fat (5.7% linoleic acid)

Wheat midds Contains 4.2% crude fat (1.7% linoleic acid)

Are supplemental fat sources (i.e. CWG)becoming more unsaturated

Alternative fat sources Yellow or Brown grease Soap stock/lecithin, glycerin bottoms

Impact on shelf-life and further processing

Energy of lipids in swine (NRC, 2012)

DE values were calculated (Jayne Powles/Julian Wiseman, 1995):

DE (kcal/kg) = (36.898 – (0.005 × FFA) – (7.330 × e-0.906 × U:S)/4.184FFA = free fatty acid content in g/kg U:S = ratio of unsaturated to saturated fatty acids

FFA concentrations of all fats were assumed to be 50 g/kg (5%), [% FFA have been reported to range from 1.6 to 19.1% in tallow, 2.6 to 61.0% in AV blends]

ME = DE × 0.98 (van Milgen et al., 2001)

NE = ME × 0.88 (van Milgen et al., 2001)

Energy of Lipids in Poultry (NRC, 1994)

Ketels and DeGroote, 1989 MEn = 8,227 – 10.318(-1.1685[Unsaturated:Saturated ratio])

Huyghebaert et al., 1988 MEn = 28,119 – 235.8(C18:1 + C18:2) –

6.4(C16:0) – 310.9(C18:0) + 0.726(IV × FR1) –(0.0000379(IV[FR1 + FFA])2

IV = iodine value; FR1 = first fraction from a column chromatography separation that contains the practically unaltered triglycerides plus other apolor components.

U:S Ratio and FFA on Energy ValueWiseman et al., 1998 (AR Nutr. Conf. 2003)

•Increase unsaturation = increased energy value.•Increase FFA, decreased energy value.•Largest impact in young birds, least in older pigs.

•Main effect is on lipid digestibility, not utilization.

Low FFA High FFA

Lipid source 10:0 12:0 14:0 16:0 18:0 16:1 18:1 18:2 18:3 20:4 22:6 U:S IV DE CalcCoconut 6.7 43.8 16.8 8.4 2.5 5.9 1.7 0.09 8 7180Palm Kernel 3.7 47.0 16.4 8.1 2.8 11.4 1.6 0.16 13 7246Tallow 0.1 0.9 3.7 24.9 18.9 4.2 36.0 3.1 0.6 0.91 42 7825Palm 1.1 44.0 4.5 0.1 39.2 10.1 0.4 1.00 52 7880Lard 0.1 0.2 1.3 23.8 13.5 2.7 41.2 10.2 1.9 1.46 61 8128Menhaden 1.0 10.0 18.0 5.0 10.5 14.5 2.2 1.5 5.0 10.0 1.87 163 8304Poultry 0.1 0.9 21.6 6.0 5.7 37.4 19.5 1.0 0.1 2.27 75 8443Soybean 0.1 10.3 3.8 0.2 22.8 51.0 6.8 5.47 126 8936Flaxseed 5.3 4.1 20.2 12.7 53.3 9.17 179 9036Canola 4.0 1.8 0.2 56.1 20.3 9.3 15.21 110 9053

The energy prediction equation may need modification based upon the degree of saturation in relation to its chain length—and modification relative to FFA and chain length (degree of hydrogenation).

Gatlin et al., 2005/JAS 83:1890

Energy value of lipids in swine and poultry

Energy value of specialty lipids in nursery pigsKerr & Shurson, 2017 / PAS 33:127

Distillers Corn Oil Fatty Acid Profile

Similar to refined corn oil

Primary Quality Factors Moisture, Insolubles, Unsaponifables Free Fatty Acids

Secondary Quality Factors Degree of Lipid Peroxidation Tocopherols/tocotrienols (lipid-soluble

antioxidants) Caroteinoids ← Xanthophylls (lutein and

zeaxanthin) Feurlic acid (phenolic acid)

0

1,000

2,000

3,000

4,000

5,000

6,000

7,000

8,000

9,000

10,000

RBD-0.04 DCO-4.9 DCO-12.8 DCO-13.9 DCO-93.8 SO-0FFA SO-90FFA

DE,

kca

l/kg

GE DE DE-p

Energy Content of FFA in SwineKerr et al., 2016 / JAS 94:2900

Kerr & Shurson, 2017 / PAS 33:127

Proposed Palm Fractionation Research C10:0 FA (capric)

C12:0 FA (lauric)

C14:0 FA (myristic)

C16:0 FA (palmitic)

C18:0 FA (stearic)

C18:1 FA (oleic)

PKO lipid PKO-olein PKO-stearin

PO lipid PO-olein PO-stearin

Lipid sources high in unsaturated fatty acids are most susceptible to peroxidation

Most vulnerable fatty acids in typical lipids fed to animals Oleic acid Linoleic acid Linolenic acid

Rate of peroxidation increases with the degree of unsaturation (Holman, 1954*) Oleic = 1x rate Linoleic = 40x rate Linolenic = 80x rate Arachidonic = 160x rate

*Peroxidizability Index (PI) = [(0.025 × % monoeniocs) + (1 × % dienoics) + (2 × % trienoics) + (4 × % tetraenoics) + (6 × % pentaenoics) + (8 × % hexaenoics)]. Holman, 1954.

Lipid peroxidation ‐ a free radical chain reaction

Lipid peroxidationproducts

Chain cleavage

AldehydeKetoneAcid

Polymer

(Vickers et al., 2001)

Free radical

TemperatureOxygenOxidizing minerals

Co, Cu, Fe, Mn, NiTimeLightDegree of unsaturation

Many peroxidation products are produced and decomposed at different rates and time points

What Peroxidation Compounds Are Measured in Each Test?

Initiation phase (1°) Peroxide value – peroxides and hydroperoxides Conjugated dienes – hydroperoxides

Propagation phase (2°) TBARS – measures compounds similar to malondialdehyde Anisidine value – high MW saturated and unsaturated aldehydes,

carbonyl groups of peroxidized lipids

Termination phase (3°) Hexanal – volatile compound formed from peroxidation of linoleic

acid and n-6 fatty acids 2,4-decadienal (DDE) – by-product of peroxidized linoleic acid 4-hydroxynonenal (HNE) – α,β-unsaturated lipophilic aldehydes

formed from n-6 PUFA, linoliec acid Acrolein- a unsaturated aldehyde from the breakdown of glycerin Triacylglycerol dimers and polymers

Predictive Tests Measure the Potential of a Lipid for Peroxidation

Active oxygen method (AOM) time (hours) required to reach a PV of 100 mEq lipid

Oxygen stability index (OSI) measures time (hours) to reach a predetermined value of

water conductivity due to decomposition of volatile acids formed by artificial peroxidation

Oxygen bomb method (OMB) measures reduction in oxygen pressure which is

proportional to amount of lipid peroxidation

Which peroxidation measures should we use?? Measures of peroxidation vary with heating

conditions (time, temperature)

Rate and amount of production of peroxidation products vary with lipid fatty acid composition

How do various peroxidation measures relate to changes in growth performance?

MIXED FEED:  How hot and how long in the summer?

85C105C

35C/50h

85C

Air425C

100C

Steam175C

60C

Animal Rendering115‐145C; 45‐90 minutes

50‐65C

Fat tanks

• Fat tanks at feed mills hold up to 70,000L (20,000 gal; 10,000 gal/TL); mainly 1 tank/mill but some have 2 tanks/mill

• New fat addition: 1TL/d to 1TL/4 wks• Insulated tanks held at 50 to 65°C (120‐150°F)

– (10°C > MP—depends upon fat source and time of year)

• Clean out frequency– Flat bottom—never, only when there is a problem, 1x‐4x/year; cone bottom—not cleaned

65C hold temp

≥ 85C initial temp and allowed to cool to 65C; may last 6 hours

UCO has been thermally stressed at 190C during the frying process over the coarse of several h or d

Getting the most from lipids? Need more precision in predicting caloric value as

‘composition’ varies among sources. “Nutritional tools” are needed to manage nutrient value variability

that are fast, accurate, and inexpensive.41 samples, van Kempen and McComas, 2002

Central IA feed mill: MIU, 0.8‐3.7%; AOM, 8‐332h; PV, 0.4‐7.3 mEq/kg; FFA, 5.8‐51.6%

History Research by UMN scientists on HNE

(Csallany/Chen/Shurson, 180C cooking oil (round bottom flasks)

Lit review (originally these were abstracts) KSU 2004: CWG @ 80C up to 11d (0.85L O2/min; 208L barrel @

65% full; barrel heaters) UIUC/Novus 2012: CO @ 95C up to 72H (80L air/min; 208L barrel @

65% full; barrel heaters) VT/Novus 2014: SO @ 95C up to 72h (80L air/min; 208L barrel @

65% full; barrel heaters) NCSU 2015: SO @ 80C up to 12 d (1 L O2/min; 208L barrel @ 65%

full; immersion heaters)

2009: proposed lipid peroxidation project proposed to FPRF with 190C, they liked but said the 190C did not reflect the rendering industry (115-145C for 45-90 min); we came up with 95C for 72h

MN (Liu PhD) 2014: canola oil-corn oil-poultry fat-tallow ×22.5C-0h vs 95C-72h vs 185C-7h (12 L air/min; 135L Al pot @ 65% full)

Effects of Dietary Peroxidized Lipids

↓ Growth performance (feed intake) Rat (Kimura et al., 1984; Behniwal et al., 1993; Nwanguma et al., 1999)

Broiler (Cabel et al., 1988; Dibner et al., 1996; Engberg et al., 1996; Wang et al., 1997)

Pig (DeRouchey et al., 2004; Fernandez-Duenas et al., 2009; Harrell et al., 2010)

Lipid metabolism effects in rats (Chao et al., 2001, 2004, 2005; Sülzle et al., 2004)

↑ activation of peroxisome proliferator-activated receptor-α (PPARα)

↑ expression of PPARα target genes involving in fatty acid oxidation

↓ liver and serum triglyceride concentration

↑ → Oxidative stress (Engberg et al., 1996; Ringseis et al., 2006; Vazquez-Anon et al., 2008; Fernandez-Duenas et al., 2009; McGill et al., 2011)

→ Immune responses (Kimura et al., 2006, Yun et al., 2009)

↓ Intestinal integrity & gut barrier function (Dibner et al., 1996)

Pigs Metabolic effect of peroxidized lipids???

Effects of Dietary Peroxidized Lipids

Literature summary of the effects of feeding peroxidized lipids on broiler and swine growth performance

(Andrea Hanson @ UMN)

Data compiled from 43 comparisons of feeding peroxidizedlipids vs. unoxidized lipids published studies (1963-2012) n = 26 for broilers n = 17 for swine

Evaluated only supplemental feed fats and oils Excluded lipids from feedstuffs (e.g. meat and bone meal, fish meal,

rice bran, etc.) Diets were isocaloric

Variables of interest included: Diet PV meq/kg Diet MDA meq/kg (TBARS) ADG, ADFI, and G:F

○ Dependent variables are reported as a % relative to unoxidizedlipids (control)

A. Hanson, 2014 / Hung et al., 2017 AFST

Composition and peroxidation analysis of thermally processed soybean oils Heating temperature, oC 22.5 45 90 180 Time heated, h1 0 288 72 6 Fatty acids, % of total fat2,3

C14:0, Myristic 0.08 ND 0.08 0.08 C16:0, Palmitic 10.94 11.01 11.99 11.49 C16:1, Palmitoleic 0.08 0.08 0.09 0.09 C17:0, Margaric 0.09 0.10 0.11 0.10 C18:0, Stearic 4.10 4.06 4.41 4.26 C18:1, Oleic 23.27 23.21 24.74 23.82 C18:2, Linoleic 53.09 53.18 50.98 51.98 C18:3, Linolenic 7.38 7.42 6.42 6.85 C20:0, Arachidic 0.31 0.31 0.33 0.32 C20:1, Gadoleic 0.17 0.18 0.19 0.28 C22:0, Behenic 0.33 0.32 0.37 0.36 C24:0, Lignoceric ND ND 0.13 0.11 Other FA 0.15 0.13 0.17 0.26 UFA:SFA4 5.30 5.32 4.73 4.97 IV5 131 132 127 129

Free fatty acids, %2 0.03 0.03 0.20 0.08 Free glycerin, %2 0.77 0.61 0.41 0.62 Moisture, % 0.02 0.02 0.10 0.04 Insoluble impurities, % 0.04 0.06 0.04 0.08 Unsaponifiable matter, % 0.34 0.38 0.32 0.30 Oxidized FA, %2 1.0 1.1 2.7 1.6 OSI @ 110 C, h2,4 4.95 3.35 2.35 2.95 p-Anisidine value2,6 1.92 6.29 149 159 Peroxide value, meq/kg2 7.6 11.5 19.1 13.4 Polar compounds, %2 5.44 8.44 22.80 13.60 PTAG4, % ND ND 4.07 2.66 TBA value2,6 0.05 0.07 0.06 0.07 Aldehydes, mg/kg7

2,4-decadienal 0.40 4.71 835.63 679.65 4-hydroxynonenal 0.60 2.28 159.21 72.23 Acrolein 5.85 5.97 23.23 31.59 2-Decenal 0.10 0.18 46.28 54.96 2,4-Heptadienal 0.15 4.16 238.13 128.12 2-Heptenal 1.52 3.51 220.59 74.41 Hexanal 2.19 2.31 33.07 5.85 2-Octenal 0.45 1.21 166.14 40.26 Pentanal 2.01 0.63 10.86 2.84 2,4-Undecadienal 0.03 0.15 38.97 35.31 2-Undecenal 0.10 0.20 43.42 66.50 Ratio8 0.11 0.20 0.60 0.98

Total tocopherols, mg/kg2 870 821 147 595

Growth performance of pigs fed soybean oil with differing peroxidation levels Processed soybean oil Statistics Parameter 22.5C-0h 45C-288h 90C-72h 180C-6h SEM P value

Nursery 7.1 – 16.6 kg, 21 d trial, 15 reps ADG, kg 0.478a 0.465a 0.406b 0.455a 0.02 0.01 ADFI, kg 0.629a 0.609a 0.548b 0.615a 0.02 0.04 G:F 0.762 0.764 0.745 0.739 0.01 0.37

Grower 25.3 – 70.8 kg, 49 d trial, 14 reps ADG, kg 1.02a 1.05a 0.96b 1.03a 0.02 0.01 ADFI, kg 2.05 2.00 1.94 2.09 0.05 0.19 G:F 0.50b 0.53a 0.49b 0.50b 0.01 0.02

Finisher 46.8 – 131.2 kg, 81 d trial, 14 reps ADG, kg 1.04ab 1.09a 0.98b 1.07a 0.03 0.07 ADFI, kg 2.77 2.77 2.72 2.80 0.07 0.91 G:F 0.38ab 0.39a 0.36b 0.38ab 0.01 0.04

US IV PV AnV OSI Polar OFA HNE DDE HEX ACR Ratio Ttoc0.38 0.37 ‐0.40 ‐0.31 0.34 ‐0.41 ‐0.41 ‐0.40 ‐0.35 ‐0.39 0.410.01 0.01 0.01 0.02 0.01 0.01 0.01 0.01 0.01 0.01 0.010.30 0.29 ‐0.35 0.28 ‐0.34 ‐0.35 ‐0.33 ‐0.26 ‐0.36 0.350.02 0.03 0.01 0.03 0.01 0.01 0.01 0.05 0.01 0.01

US IV PV AnV OSI Polar OFA HNE DDE HEX ACR Ratio Ttoc0.44 0.44 ‐0.33 ‐0.39 ‐0.44 ‐0.33 ‐0.39 ‐0.41 ‐0.36 ‐0.33 0.390.01 0.01 0.01 0.01 0.01 0.02 0.01 0.01 0.01 0.01 0.01

‐0.28 ‐0.280.04 0.04

‐0.32 ‐0.32 ‐0.32 ‐0.34 ‐0.350.02 0.02 0.02 0.02 0.01

US IV PV AnV OSI Polar OFA HNE DDE HEX ACR Ratio Ttoc‐0.32 ‐0.29 ‐0.29 ‐0.33 0.300.02 0.04 0.04 0.01 0.03

‐0.33 ‐0.32 ‐0.32 ‐0.35 0.330.01 0.02 0.02 0.01 0.02

GROWER

ADG

ADFI

GF

FINISHER

ADG

ADFI

GF

ADG

ADFI

GF

NURSERY

What is oxidative stress? A disruption in the balance between free radical

production (electron leakage, heat, sun, lipid, smoking, etc.) and antioxidant defenses (SOD, CAT, GPx, Vit E, Vit C, etc.) in vivo

If antioxidant stores are depleted, free radicals bind to lipids, proteins, and DNA altering cell structure and function (Montuschi et al., 2004)

To date there is no universal marker to determine oxidative balance, so it is essential to study multiple markers

Theoretical summary of oxidative stress

Oxidative Stress

Antioxidants

Reactive Productsaldehydes, ketones

acids, polymers

↓ Growth

Dietary PUFA

Lipid Peroxidation

Tissue Damage: Heart LiverBrainOthers?

Cellular DamageLipid

Protein (e.g. carbonyl adducts)DNA (e.g. 8-OH-deoxyguanosine)

Other Contributing Factors:• Nutrient digestibility?• Nutrient repartitioning?• Reduced health?• Antioxidant deficiencies?

FeedIntake

Peroxidation and oxidative stress Little comprehensive research has been done

in swine and poultry Measured PV only Measured TBARS only

○ Tavarez et al., 2011; Boler et al., 2012○ Liu et al., 2014, Hanson et al., 2014○ Lu et al., 2014○ Rosero et al., 2015

Lindblom MS thesis Swine and poultry Multiple measures

Final thoughts… Do we need different peroxidation measures for:

Different sources of lipids? (In the works!) Different species? (In the works!)

Can we develop a “peroxidation index” of measures to predict if growth reductions will occur when feeding peroxidized lipids? May requires a metabolomic approach? (Thinking!)

What is the threshold of total diet peroxidation content necessary to reduce growth? (New UMN review-need more data-thinking!)

Do changes in biological antioxidants signal depressed growth or compromised health long-term? (Thinking on it.)

How do we relate negative biological effects of peroxidized lipids to price or value? (Thinking on it.)

• There is no single measurement that depicts the overall peroxidation status of oils—thus the need to measure multiple markers of peroxidation.

• There is no single measurement that depicts the overall level of oxidative stress or balance—thus the need to measure multiple markers of oxidative stress.

• Research needs to be analyzed by multiple variant/neural network/??? analysis to accurately describe the relationship between lipid peroxidation and pig performance and oxidative stress.

1984