read the full study

Local Poultry Feed Options from Hawaii By-Products

Final Report

PREPARED FOR: ULUPONO INITIATIVE

April 8, 2013

Authored by: Kristin Mack DBA Farm n’ Forages

[email protected]

Poultry Feed Research Report

I. Table of Contents II. Table of Figures ............................................................................................................................... 4

III. Executive Summary ......................................................................................................................... 5

IV. Introduction .................................................................................................................................... 5

V. Project Layout.................................................................................................................................. 6

A. Phase I: Evaluation and Analysis of Local Options ......................................................................... 6

B. Phase II: On-farm Feed Trials........................................................................................................ 6

VI. Phase I ............................................................................................................................................. 7

A. Identification of By-products ........................................................................................................ 7

1. Algae Co-products .................................................................................................................... 7

2. Brewer’s Spent Grains .............................................................................................................. 7

3. Wheat Millrun .......................................................................................................................... 7

4. Cassava .................................................................................................................................... 7

5. Black Soldier Fly Larvae ............................................................................................................ 7

6. Papaya Processing Waste ......................................................................................................... 8

B. Analysis of Ingredients ................................................................................................................. 8

1. Amino Acid Profiles .................................................................................................................. 8

2. Proximate Analyses and Nutrient Digestibility ........................................................................ 12

3. Pigment Analysis .................................................................................................................... 14

VII. Phase II .......................................................................................................................................... 14

A. Ration Development .................................................................................................................. 14

B. Feed Trials ................................................................................................................................. 15

1. Hen Health ............................................................................................................................. 16

2. Egg Quality ............................................................................................................................. 17

VIII. Results and Conclusions ................................................................................................................. 24

A. Research Results ........................................................................................................................ 24

B. Business Analysis ....................................................................................................................... 24

1. Algae ...................................................................................................................................... 24

2. Wheat Millrun ........................................................................................................................ 25

3. Cassava .................................................................................................................................. 25


4. Summary of Ingredient Replacement ..................................................................................... 25

C. Recommendations ..................................................................................................................... 26

IX. Works Cited ................................................................................................................................... 27

X. Appendix ....................................................................................................................................... 28

A. Balanced Research Ration- 20% Algae Ration ............................................................................. 28

B. Research Ration-Control ............................................................................................................ 29

C. Potential Feed Trial Ration-with Cassava .................................................................................... 30

D. Statistical Analysis of Production Data ....................................................................................... 31

E. Complete Yolk Analysis Lab Results ............................................................................................ 33


II. Table of Figures Figure 1: KA19 algae (left) and C323 (right) research ingredients ............................................................. 8

Figure 2: Algae amino acid profiles with digestibility .............................................................................. 10

Figure 3: KA19 algae amino acid profile ................................................................................................. 11

Figure 4: Cassava amino acid profile ...................................................................................................... 11

Figure 5: Digestibility of phosphorus, energy, protein and calcium in co-products ................................. 12

Figure 6: Fatty acid profile of KA19 algae provided by Cellana ............................................................... 12

Figure 7: Cassava proximate analysis ..................................................................................................... 13

Figure 8: KA19 Pigment Lab Report ........................................................................................................ 14

Figure 9: KA19 (treatment 2) mixed ration (left) and C323 (treatment 3) mixed ration........................... 15

Figure 10: Hens in research cages, eating research diet. ........................................................................ 16

Figure 11: Egg production over the trial period and average daily production ........................................ 17

Figure 12: TTEst results for egg production ............................................................................................ 18

Figure 13: Egg mass over the trial period and average egg size (grams).................................................. 19

Figure 14: Resulkts of the TTest on egg weights showing a significant difference between the Control and

Treatment groups. ................................................................................................................................. 19

Figure 15: ALA fatty acid content of egg yolks ........................................................................................ 20

Figure 16: DHA fatty acid content of egg yolks ....................................................................................... 20

Figure 17: EPA fatty acid content of egg yolks ........................................................................................ 21

Figure 18: Crude moisture percent in research egg yolks ....................................................................... 21

Figure 19: Crude lipid % in research egg yolks ........................................................................................ 21

Figure 20: Comparison of percent ALA omega-3 fatty acids in one egg of treatments ............................ 22

Figure 21: Comparison of percent EPA+DHA fatty acids in eggs from treatments ................................... 22

Figure 22: Comparison of Omega-3 content in research and commercial eggs ....................................... 23

Figure 23: Omega 3 fatty acid concentrations of samples versus USDA standard and normal marketed

omega 3 eggs. ....................................................................................................................................... 23

Figure 24: Yolk color differences in the Group 1 (Control), Group 2 (KA19) and Group 3 (C323) diets. .... 23

Figure 25: Percentage of local ingredients included in diets ................................................................... 25

Figure 26: Estimate of amount (tons) of local ingredients needed per year to supply the needs of current

Hawaii layer industry (Ag Census 2007) ................................................................................................. 26

Figure 27: Comparison of percentage of imported ingredients replaced by sample diet formulations and

estimated ration costs ........................................................................................................................... 26


III. Executive Summary Poultry production in Hawaii is limited by the high price and risk of imported feed from the mainland.

This study set out to build upon the work of past studies, by focusing on by-products of industry that

were currently available in Hawaii. Wheat millrun, brewer’s spent grain, algae co-products and cassava

were identified as the most available by-products and samples were sent to the lab for further analysis.

Based on the lab results, algae and wheat millrun were selected to be fed in the research rations.

Cassava tested favorability, but was included later in the trial and enough material was not available for

a feeding trial at the time of the study.

Rations were developed based on the analyses and added to base mixes. Three diets were developed

and fed, a control (1), 20% KA19 algae (2), and 20% C323 algae (3). Research birds were fed the diets

and data was collected over a 124 day period. The production of eggs in the research groups did not

vary significantly from the control group although there was a depression of production with the C323

treatment. The egg weight was significantly less in the research groups compared to the control group,

but was still within the same weight class for egg sizing. Bird health and weight was not affected by the

diet. Egg yolk color was considerably darker in the KA19 algae treatment. The Eicosapentaenoic acid

(EPA) omega-3 fatty acid content was highest in the control diet and the Docosahexaenoic acid (DHA)

omega-3 fatty acid content was highest in the KA19 algae treatment. The Alpha-Linolenic (ALA) omega-3

fatty acids were slightly higher in the C323 algae treatment.

An analysis of the percentage replacement of imported ingredients showed the algae research rations

replacing approximately 36% of imported ingredient. If utilized by the entire layer industry in Hawaii,

this could replace about 6000 tons of imported ingredients per year or over 263 container loads per year

of bulk ingredients. A balanced ration containing about 11% cassava was also formulated. This ration

could replace about 41% of imported ingredients (over 6800 tons or about 300 container loads per

year).

IV. Introduction Poultry production in Hawaii continues to be limited by the high price of feed rations imported from the

mainland. As reported by the Hawaii Poultry Task Force Report in July 2007, imported feed costs are one

of the top areas of concern to industry representatives doing business in the state. The lack of a local

ration for the production of poultry in Hawaii is a hindrance to the expansion of the business on the

islands. Past studies focused on compiling information on the potential ingredients, whether currently

available of not, and their potential for inclusion in a research ration. This study focused on the available

by-products of industry that could be used in a local feed ration and established detailed nutritional

analyses on those possible ingredients, such as poultry digestibility information. Also, the objective of

this study was to create feed rations containing these by-products and feed them to a flock to obtain

production data.


V. Project Layout

A. Phase I: Evaluation and Analysis of Local Options The focus of this project was on the by-products of industry, therefore only the products of current

industries that are in commercial production or in the research and development stages were evaluated.

Phase 1 of this trial involved the identifying and sourcing of these by-products and sending them for

analysis.

Several businesses and industries on the islands were contacted to better understand the waste stream

from their production lines and the possibility of obtaining by-product materials for testing as a

component of a poultry diet. The industries focused on included the brewing, biofuel, and grain milling

industries because of their potential for suitability in a poultry ration and the larger scale of these

industries in Hawaii.

The Oceanic Institute in Waimanalo, Oahu, was crucial in the evaluation of local ingredients as well as

insight into the by-products which are currently available on the island. Farm n’ Forages consulted with

Dr. Warren Dominy from Oceanic Institute on several occasion throughout the course of this study in

order to better utilize time and resources in focusing on ingredients with the best potential for inclusion

in a commercial ration.

Cellana, LLC was helpful in submitting algae material for analysis and for use in the feed trial. Because

they are still mostly in the research stage, they were not able to produce large enough quantities for a

multi-island study, but were able to submit enough for an on-farm feeding trial on Maui. Their variety

development included interest in the value-added potential of inclusion of beneficial omega-3 fatty

acids in their algaes, therefore omega 3 fatty acid analyses were included and reported on a part of the

egg yolk analyses.

Farm n’ Forages was in discussion with a biofuel company which is growing and processing research

plots of local oilseeds into biofuel and by-products, but the company was unable to submit and samples

for use in the feed trials at this time.

B. Phase II: On-farm Feed Trials The feed trial portion of this project involved the development of a local ration utilizing the ingredients

tested and feeding them to laying hens over a three month period in a research trial format to gain data

of effects of the feed on various production factors. The ration developed utilized ingredients that could

be stored in fed in a commercial feed mill setting, in order to focus on creating rations for the poultry

industry as a whole.


VI. Phase I

A. Identification of By-products

1. Algae Co-products

Cellana, LLC was willing to submit samples of their research algaes to the trial and were able to provide

enough material to feed to the research birds over an extended period. Cellana operates out of Kona,

Hawaii where it has a research unit producing algae with the focus on biofuel production and alternative

markets. They produce both whole and defatted material and were researching several varieties. Their

long term goals included the construction of a larger commercial biofuel production facility; therefore

larger quantities of this material could be available in the coming years. Also, many other companies in

Hawaii are studying the production of algae for biofuel, so there is potential of this being an available

feedstock in the near future.

2. Brewer’s Spent Grains

Spent brewer’s grain was obtained from Maui Brewing Company in Lahaina, Maui. MBC currently offers

their spent brewing grains to farmers and livestock producers from their brewing facility and will be

expanding to a larger facility in Kihei, Maui in late 2013.

3. Wheat Millrun

Wheat Millrun is a by-product of the flour industry and was sold by Hawaii Flour Mill (HFM) in Oahu.

Millrun is the most common by-product of flour manufacturing, available for livestock feeding. It

consists of the bran, aleurone, germ and pollard fractions. Millrun is commonly fed by many hog and

cattle producers on the island and is easily available in 60 lb. bags and loose containers. Wheat Millrun

was digestible and was able to be included in the research diet at a 16% rate and control in the control

ration at a 26% rate, replacing a portion of the soybean meal and corn products.

4. Cassava

Cassava was being cultivated by Phycal on Oahu for research into the production of bioenergy. They

were able to submit some samples of Cassava at a later stage in the trial. These samples were sent to

Oceanic Institute for analysis and included in the Phase I results. Although Phycal did not have the

volume to do a feeding trial, a ration was developed including the Cassava into the ration, along with

other local ingredients based on the information from the nutritional analysis.

5. Black Soldier Fly Larvae

While not necessarily directly a by-product of industry, Black Soldier Fly Larvae can be a grown on by-

products of industries such as the offal from livestock slaughter or fruit and vegetable waste. Although

no BSF were analyzed or fed in this study, it was important to note it in this study as the technology to

efficiently grow this protein source develops. Through connections made as part of this project, further

grant funding has been obtained to research this ingredient more thoroughly as a feed source for

poultry.


6. Papaya Processing Waste

Papaya processing waste can be obtained on the Big Island and Oahu, and can potentially be used as a

portion of the ration or to ferment and grow a higher protein microorganism. Communication was made

with Dennis Gonsalves of USDA PBARC, who was looking into the processing of papaya waste for the

production of biofuel and feeds, however they did not have enough material at the time of the trial to

do any actual feeding trials. Dr. Dominy and the Oceanic Institute published a study using the papaya

was as a medium for growth of yeast and used it in successfully feeding trials of shrimp. Farm n’ Forages

did not pursue papaya waste as part of this study, because it would have been duplication of efforts in

analyzing the ingredients, and there was not sufficient material to supply for a feeding trial at that point.

Both the processing waste and yeast grown on processing waste are possible feed sources that are

worthy of continued research focus.

B. Analysis of Ingredients The ingredients selected to send for digestibility analysis depended on the current availability of the

ingredients at the time of the research and potential for application to a poultry ration. The three

varieties of algae, brewer’s grain, and wheat millrun were sent Dr. Jeff Firman at the University of

Missouri for analysis. This included a standard proximate analysis as well as an in-vivo digestibility

analysis. An in-vivo digestibility analysis involves feeding the ingredient to poultry and collecting the

feces to determine the actual utilization of the nutrient by the animal. This information was important

because it provided insight into how the nutrients from each ingredient metabolized and the efficiency

of the individual ingredients. This aided in the rations being developed more accurately to better meet

the balanced nutritional needs of poultry.

Figure 1: KA19 algae (left) and C323 (right) research ingredients

1. Amino Acid Profiles

Amino acid profiles were performed on all ingredients to understand the levels of essential amino acids

in each ingredient in order to better balance research diets for the feeding trials. Profiles for each of the

selected by-products are provided in Figure 2 to Figure 4. In poultry, 22 amino acids are necessary; some

can be synthesized by the body (non-essential) while others must be supplied through the diet

(essential). Special consideration must be made to properly balance the amino acids in a diet in order to

meet the bird’s specific requirements without exceeding them and decreasing the efficiency of the

production system.


Wheat millrun

Amino acid % in Feed % digestibility Digestible content

Threonine 0.52 0.77 0.40

Serine 0.61 1.00 0.61

Glycine 0.88 0.24 0.21

Alanine 0.77 0.76 0.59

Cysteine 0.34 0.84 0.29

Valine 0.78 0.78 0.61

Methionine 0.26 0.81 0.21

Isoleucine 0.55 0.80 0.44

Leucine 1.06 0.83 0.88

Tyrosine 0.44 0.81 0.36

Phenylalanine 0.69 0.85 0.59

Lysine 0.68 0.82 0.56

Arginine 1.07 0.87 0.93

Tryptophan 0.21 0.92 0.19

Brewer's grain


Threonine 0.56 0.79 0.44

Serine 0.58 1.03 0.60

Glycine 0.68 0.76 0.52

Alanine 0.77 0.79 0.61

Cysteine 0.31 0.84 0.26

Valine 0.86 0.80 0.69



Leucine 1.18 0.84 0.99

Tyrosine 0.47 0.84 0.40


Lysine 0.70 0.81 0.57

Arginine 0.80 0.85 0.68


Algae C323 Whole biomass


Threonine 1.02 0.84 0.85

Serine 0.81 0.97 0.79

Glycine 1.34 0.79 1.06

Alanine 1.85 0.86 1.59

Cysteine 0.28 0.79 0.22

Valine 1.36 0.82 1.12



Leucine 1.88 0.85 1.59

Tyrosine 0.74 0.84 0.62


Lysine 1.19 0.84 1.00

Arginine 1.28 0.89 1.14


Amino Acid Profiles


Figure 2: Algae amino acid profiles with digestibility

Algae C046 High Lipid


Threonine 0.65 0.89 0.58

Serine 0.54 1.09 0.59

Glycine 0.73 0.63 0.46

Alanine 0.83 0.90 0.75

Cysteine 0.20 0.80 0.16

Valine 0.74 0.89 0.66



Leucine 1.01 0.91 0.92

Tyrosine 0.42 0.90 0.38


Lysine 0.61 0.89 0.54

Arginine 0.65 0.93 0.60


Algae C323 Defatted biomass


Threonine 0.82 0.83 0.68

Serine 0.72 0.96 0.69

Glycine 1.08 0.53 0.57

Alanine 1.44 0.85 1.23

Cysteine 0.20 0.67 0.13

Valine 1.01 0.82 0.83



Leucine 1.49 0.85 1.27

Tyrosine 0.58 0.83 0.48


Lysine 1.05 0.84 0.89

Arginine 0.94 0.89 0.84



Figure 3: KA19 algae amino acid profile

Figure 4: Cassava amino acid profile

KA19 Amino Acid Profile

AA Total % Digestibility Digestible content (%) Required in Complete Diet (NRC)

Threonine 1.02 0.78 0.8 0.72

Serine 0.81 0.95 0.77

Glycine 1.34 - --

Alanine 1.85 0.83 1.53

Cysteine 0.28 0.69 0.19 0.59 (Methionine and Cysteine)

Valine 1.36 0.78 1.05 0.8

Methionine 0.37 0.76 0.28 0.59 (Methionine and Cysteine)


Leucine 1.88 0.78 1.46 1.2

Tyrosine 0.74 0.79 0.58


Lysine 1.19 0.77 0.91 0.68

Arginine 1.28 0.81 1.04 0.4

Tryptophan 0.35 0.91 0.32 0.239

Non-essential AA g/100g

Ala 0.12

Asp+ASN 0.17

CYS 0.03

Glu+Gln 0.19Gly 0.01

Pro 0.07

Ser 0.04

Tyr 0.15

Taurine 0

Essential AA g/100g

Arg 0.14

His 0.03

Lle 0.14

Leu 0.15

Lys 0.16

Met 0.01

Phe 0.22

Thr 0.03

Val 0.25

Subtotal of Non Essential AA 0.78

Subtotal of Essential AA 1.12

Total 1.90


2. Proximate Analyses and Nutrient Digestibility

Figure 5: Digestibility of phosphorus, energy, protein and calcium in co-products

KA19 Algae Proximate Analysis:

Moisture ~5%

Opal ~15%

Lipid ~30%

Protein ~30%

Carbs~15%

+/-5% error

Fatty acids profile (error bars represent standard deviation of analytical triplicates)

Total lipids percentage in biomass: 30%

Figure 6: Fatty acid profile of KA19 algae provided by Cellana

Phosphorus

Feedstuff P % Feed Digestibility

Wheat Millrun 0.92 0.34

Brewer's Grains 0.56 0.32

Algae C323 Whole Biomass 0.47 0.56

Algae C046 High Lipid Biomass 0.69 0.74

Algae C323 Defatted Biomass 0.6 0.42

Energy, Protein, Calcium

AMEn

2333

2210

1832

3085

2282Algae C323 Defatted Biomass

Feedstuff

Wheat Millrun

Brewer's Grains

Algae C323 Whole Biomass

Algae C046 High Lipid Biomass


Figure 7: Cassava proximate analysis

PHYCAL

Cassava

ANALYSIS As Is

Dry Matter % 96.20

Moisture % 3.80

Ash % 0.78

Crude Protein % 2.37

Crude Lipid % 0.26

Crude Fiber % 2.90

NFE % - Carbohydrate 89.89

100.00

Gross Energy cal/g 3920.97

Minerals

P % (Phosphorus) 0.21

K % (Potassium) 1.53

Ca % (Calcium) 0.17

Mg % (Magnesium) 0.09

Na % (Sodium) 0.04

B ppm (Boron) 7

Cu ppm (Copper) 2

Fe ppm (Iron) 15

Mn ppm (Manganese) 5

Mo ppm (Molybdenum)

Zn ppm (Zinc) 34

Se ppm (Selenium)


3. Pigment Analysis

Oceanic institute tested the KA19 algae product for pigmentss in the feed that might affect the egg yolk color. This report showed high levels of yellow pigments (highlighted in yellow) which could contribute to darker yellow color of egg yolks or meat (in broiler chickens).

Analysis Report on Pigment Content of KA19 Algae Sample

Pigment mg/g dry sample

Chlorophylls Chlorophyll a Equivalent

Chlorophyll a 2.6992

Chlorophyll b 0.2072

Chlorophyll c1+c2 0.0672

Chlorophyllide a 0.0422

Chlorophyll a allomer Chlorophyll a epimer 0.6412

Sub-total 3.6568

Carotenoids Astaxanthin Equivalent

Astaxanthin 0.0631

a-Carotene

b-Carotene 3.4524

Alloxanthin 1.6044

Diadinoxanthin pH dependent yellow to

brown 6.0284

Diatoxanthin 2.5508

Fucoxanthin

Peridinin

Prasinoxanthin 0.0476

Violaxanthin 0.9520

Zeaxanthin 7.4564

Lutein 3.4272

Neoxanthin 0.1316

Crocoxanthin

Monadoxanthin

Lycopene

Phodopin

Sub-total 25.7139

Total 29.3707 Figure 8: KA19 Pigment Lab Report

VII. Phase II

A. Ration Development Rations were developed based on the balanced rations from the digestibility analyses. The program used

for ration balancing was the Windows User –Friendly Feed Formulation Program with assistance from Dr.


Jeff Firman from the University of Missouri. The trial rations were developed using the best suited

ingredients as according to the digestibility analyses, Wheat Millrun and the Algae co-products.

Appendices A through C include the feed rations developed including the control and research rations

used in the feeding trial and the sample ration developed with the inclusion of Cassava.

The developed rations were then sent to Jeff Peterson from Land O’ Lakes, who was crucial to the

production of rations for use in this trial. The base ingredients were mixed on the mainland and the

partial rations were shipped to Maui. On Maui, the appropriate rates of local ingredients were added to

the rations and mixed.

B. Feed Trials Feed trials were conducted on 23 week old laying hens. The hens were housed in movable cages which

were rotated on a regular basis. To intensively monitor the initial response of the layers to the research

rations, 30 birds have been divided into 3 groups. Each group contained 3 Rhode Island Reds, 4 Golden

Comet, and 1 Siler Wyandotte hen. The birds were placed in 3 separate 5’X7’ research pens, which were

moved two times per week. The birds were placed in the cages on September 5th and fed their same

layer ration (Land O’ Lakes Mid Lay Mash) in order to adapt them to the research situation. The groups

consisted of the Control Group, C323 Group, and KA19 Group.

The first stage of the feeding trials involved feeding a 5% rate of the algae in the ration. On September

12, the C323 and KA19 Groups were fed the research rations (5% algae starter diet) while the Control

group continued with the same diet. The 5% rate was used initially in order to adapt the birds to the

new diet gradually and to observe them closely for any negative effects. The birds were switched fully to

the 20% algae research rations on October 16, 2012, and were fed through February 13, 2013. Birds

were fed 0.25 lbs. per birds per day or 40 ounces per group per day. Feed was weighed out at each

feeding to provide for trial accuracy and to account for the differences in texture of the feeds.

Figure 9: KA19 (treatment 2) mixed ration (left) and C323 (treatment 3) mixed ration


Figure 10: Hens in research cages, eating research diet.

1. Hen Health

a) Birds Health

The overall bird health was good for all three groups during the trial. One bird was removed from the

C323 group on December 18, 2012, due to an injury which was not associated with the diet. Group 2

(KA19) exhibited some loose and dark stools throughout the course of the trial, but it did not seem to

affect their overall health or production. This would be something important to note if diet is fed in a

production system where hens had contact with their eggs, as eggs could become more soiled.

b) Bird Weights

The bird group weights were collected on September 12, 2012, at the beginning of the trial, and again at

the conclusion of the trial on Feb 27, 2013. Overall bird weights dropped slightly in the Control and C323

treatments and increased in the KA19 treatment, although weight changes were not significant.

Hen Weights (ounces)

Treatment 1 2 3 10/24/2012 698 700 722 2/27/2013 666 716 711

69.8 70 72.2 Starting Weight per Bird

66.6 71.6 71.1 Ending Weight per Bird

-32.0 16.0 -11.0 Treatment Group Gain or Loss (oz)

-3.2 1.6 -1.1

Per bird average weight change (oz)


2. Egg Quality

a) Egg Production

Egg production was measured on a daily basis by collecting the eggs and recording the quantity from

each treatment. If any egg damage occurred, it was recorded also, although this was rare in the trial.

According to the analysis, there was no significant difference in the egg production of the research

treatments at the 95% significance level over the trial period. However, the C323 treatment egg

production was outside of the standard error for the control treatment, and therefore it should be

noted that this depression in production could be related to the diet. The production of the KA19

treatment was within the standard error for the control treatment. The results of the TTest are available

in Figure 12. The statistical analysis of the data is available in Appendix D.

Figure 11: Egg production over the trial period and average daily production

779

731

705

660

680

700

720

740

760

780

800

Total Eggs Produced Over Research Period

Control Ration KA19 20% Ration C323 20% Ration

6.28 5.9

5.4

4.50

5.00

5.50

6.00

6.50

Egg Production per Treatment Group (Average/Day)



Figure 12: TTEst results for egg production

b) Egg Weights

The size of the eggs is an important factor, because nutrition can contribute to the increase or decrease

in egg size. The eggs from each group were collected on a daily basis and weighed to determine if there

was any change in egg size over time or in comparison to the other diets. The average weights per eggs

were significantly less in the Group 2 and 3 treatment groups as compared to the control diet. It is

important to note that average egg size remained within the medium weight class with all treatment

groups. This factor may not be commercially significant since eggs are sold on a count basis per weight

classes and not on a gross weight basis. Because the birds in this trial were relatively young (23 weeks at

trial start) it was expected that the egg sizes would be smaller at first and become larger over the trial

period, which is why they are averaging in the medium-size weight class.

Control to KA19 Control to C323 KA19 to C323

TTest 0.748 1.180 0.423 t=1.96, p<.05

NS NS NS

Egg Production Statistics

5889.8

5811.5 5790.7

5700.0

5750.0

5800.0

5850.0

5900.0

1

Total Egg Weights Over Research Period (grams)

Control KA19 C323


Figure 13: Egg mass over the trial period and average egg size (grams)

Figure 14: Resulkts of the TTest on egg weights showing a significant difference between the Control and Treatment groups.

c) Omega-3 Content

Omega-3 fatty acids are polyunsaturated fatty acids with a double bond (C=C) starting after the third

carbon atom from the end of the carbon chain and are considered essential because they cannot be

synthesized by the human body. Recent studies have found a variety of benefits to higher levels of

omega-3 intake on human health. Particularly this is true of Docosahexaenoic (DHA) fatty acids

compared to Alpha-Linolenic (ALA) fatty acids (C, March 2003). High levels of DHA were associated with

a reduced risk of breast cancer (Pala, et al., July 2001), increased cognitive and behavioral performance

(Van De Rest, et al., August 2008), lowered blood pressure and heart rate (Mori, Bao, Burke, Puddey, &

Beilin, 1993), and a variety of other positive benefits. Although ALA can be converted to the longer chain

fatty acids (DHA and Eicosapentaenoic acid (EPA)) studies have found that the process is very inefficient

(Brenna, March 2002), therefore they are best obtained as part of the diet.

The omega-3 content of the eggs was tested by Oceanic Institute in Oahu. Ten eggs from each treatment

were gathered randomly over several days, and the egg yolks were collected, frozen, and sent to

Oceanic Institute for analysis. The complete fatty acid analysis is provided in Appendix E.

The omega-3 contents of the egg yolks of all of the research groups were higher than a standard

commercial egg and a standard advertised elevated omega-3 egg. However, the KA19 contained the

56.20

55.0

55.5

54.00

54.50

55.00

55.50

56.00

56.50

Average Egg Weight (grams/egg)


Control to KA19 Control to C323 KA19 to C323

TTest 3.285 2.148 0.153 t=1.96, p<.05

Significant Significant NS

Egg Weight Statistics


highest levels of DHA fatty acids, each egg supplying an average of 60% the recommended daily value of

DHA and EPA. The complete comparison with a breakdown of the three most important omega-3 fatty

acids to human health, ALA, DHA, EPA, is shown in Figure 23.

Figure 15: ALA fatty acid content of egg yolks

Figure 16: DHA fatty acid content of egg yolks

71.57 73.1 78.03

18

48

0

50

100

Linolenate (ALA) mg per Egg

Control KA19 C323 USDA Standard Omega 3 Egg

299.54

579.87

415.82

19 150

0

200

400

600

800

1

Docosahexaenoate (DHA) mg per Egg



Figure 17: EPA fatty acid content of egg yolks

Figure 18: Crude moisture percent in research egg yolks

Figure 19: Crude lipid % in research egg yolks

45.9

20.74

12.41

2 2

0

10

20

30

40

50

1

Eicosapentaenoate (EPA) mg per Egg


55.24 55.6

54.14

52.31

50

52

54

56

Moisture % in Egg Yolks

Control KA19 C323 USDA Standard

54.13 52.88 53.33

26.54

0

20

40

60

Crude Lipid % in Egg Yolks

Control KA19 C323 USDA Standard


Figure 20: Comparison of percent ALA omega-3 fatty acids in one egg of treatments

Figure 21: Comparison of percent EPA+DHA fatty acids in eggs from treatments

7% 7% 7%

2% 4%

0%

10%

Percent Recommended Daily Value of ALA Fatty Acids in One

Egg

Control KA19 C323 USDA Omega 3 Eggs

35%

60%

43%

2% 15%

0%

20%

40%

60%

80%

Percent Recommended Daily Value of EPA + DHA Fatty Acids in

One Egg

Control KA19 C323 USDA Omega 3 Eggs


Figure 22: Comparison of Omega-3 content in research and commercial eggs

d) Yolk Color

The yolk color of eggs is easily altered based on the diet of the birds. A darker colored yolk is generally

associated with higher levels of pigmentation. The yellow color is due to lutein and zeaxanthin and beta

carotene, which are yellow or orange carotenoids known as xanthophylls. Carotenoids have been

associated with better health and lower mortality (A. T. Diplock1, 1998). The yolk color in the KA19

treatment (2) was actually so dark that it might actually take some additional consumer education to

ensure them that there is not a problem with the egg. The differences in the color of the control and

C323 algae group were minimal.

Figure 24: Yolk color differences in the Group 1 (Control), Group 2 (KA19) and Group 3 (C323) diets.

e) Egg Flavor

The flavor of the eggs was evaluated in studies using a panel to randomly taste the eggs and note any off

flavors detected. No off flavors were detected in any of the egg samples, although the KA19 eggs were

reported to be richer in texture and flavor.

Fatty Acid Code g/kg %

mg per

Egg

% of

RDV* g/kg %

mg per

Egg

% of

RDV* g/kg %

mg per

Egg

% of

RDV*

Linolenate (ALA) C18:3n-3 4.21 0.89% 71.57 7% 4.3 0.94% 73.1 7% 4.59 0.99% 78.03 7%

Eicosapentaenoate (EPA) C20:5n-3 2.7 0.67% 45.9 1.22 0.27% 20.74 0.73 0.160% 12.41

Docosahexaenoate (DHA) C22:6n-3 17.62 3.72% 299.54 34.11 7.43% 579.87 24.46 5.27% 415.82

Total DHA and EPA 20.32 345.44 35% 35.33 600.61 60% 25.19 428.23 43%

Fatty Acid Code %

mg per

Egg

% of

RDV* %

mg

per

Egg

% of

RDV*

Linolenate (ALA) C18:3n-3 0.11% 18 2% 0.28% 48 4%

Eicosapentaenoate (EPA) C20:5n-3 0.01% 2 0.01% 2

Docosahexaenoate (DHA) C22:6n-3 0.11% 19 0.88% 150

Total DHA and EPA 21 2% 152 15%

17 grams weight per egg yolk

*RDV= Recommended Daily Value

Based on RDV of 1100 mg of ALA and 1000 mg of DHA+EPA

Egg Yolk Omega-3 Fatty Acid Content Comparison

USDA Omega 3 Eggs

C323 20% (Group 3)KA19 20% (Group 2)Control (Group 1)


VIII. Results and Conclusions

A. Research Results According to the feed trial research, there was no significant change in egg production over the trial

period of the birds fed the either 20% KA19 algae diet at the 95% confidence level compared to the

control treatment. The depression in production of the C323 group was not significant but was outside

of the standard error of the control group and was notable. A significant difference was noted in the

average egg weights of the algae treatment groups as compared to the control group, although still

within the same USDA egg weight class.

The yolk color was significantly darker in the KA19 (2) treatment as observed in the eggs over the entire

trial period.

The omega-3 composition was higher than conventional eggs in all trial groups, most likely due to the

access of the birds to green pasture in their trial cages. The KA19 (2) treatment produced eggs with the

highest levels of DHA fatty acids while the control (1) treatment eggs contained the highest levels of EPA

fatty acids and the C323 (2) treatment had the highest levels of ALA fatty acids.

The algae diets did not contribute to any off flavors or colors at the 20% inclusion rate in the diets of the

trial birds.

The body weight of the birds did not change significantly and health of the birds remained good for all

trial groups.

Overall, the KA19 algae diet appeared to be a comparable option to the control diet in egg production

and actually exhibited some benefits such as darker yolks and an elevated DHA omega-3 content.

B. Business Analysis

1. Algae

According to a Cellana representative, the potential Maui plant is predicted to produce about 8,000 to

10,000 tons/year of algae. At a 20% inclusion rate in a diet, this would mean about the plant could

supply enough algae to include in about 40,000 to 50,000 tons/year of complete layer feed. This is

estimated to feed over 870,000 hens per year at an intake of 0.25 lbs. of feed per day per hen.

According to the 2007 US Census of Agriculture, the inventory of laying hens in Hawaii was 368,000.

Cellana was not able to provide a price at this point, although this information should be available very

soon. Research into algae prices in the mainland found a price of $0.45 per dried ton, so the price

estimates in this analysis use that number.


2. Wheat Millrun

The current price of wheat millrun is $389/ton. Millrun is very available on the islands and can be

purchased in bagged or bulk quantities. The millrun replaced 26% of the imported ration in the control

group and 16% of the ration in the algae groups. This is comparable in price to soybean meal, which is

the ingredient it would replace in large part in the ration.

3. Cassava

According to a conversation with Larry Trumbore from Phycal, their cassava yield projection is 30 MT/

acre of fresh root. Cost of production was around $48/MT of fresh root which means a cost of $115/ton

of dry matter for production. This does not include peeling, drying and grinding, which would be

necessary to include cassava in a poultry ration. Also, the value of the feed would depend on the variety

produced due to the hydrogen cyanide (HCN) content in the peel and according to Mr. Trumbore, Phycal

is researching about 60 different varieties, some of which are lower HCN varieties. Calculated price of

cassava root meal was $0.16 per pound of dry meal, based on the production costs provided by Mr.

Trumbore ($0.022.lb), and the nutritional value in comparison to corn (about 75%). This price may vary

when more information is gained on processing cassava in Hawaii. A balanced ration for feed trials

including cassava as well as algae and wheat millrun is shown in Appendix C. This diet utilizes 11%

cassava root meal, which was lower than the 25% utilized in a study using laying hens without

depressing egg production (Hennesey, 1986).

4. Summary of Ingredient Replacement

The replacement diets were able to replace about 36% of the imported ingredients in an imported

commercial layer ration while not affecting layer hen production or health. Based on the analyses in this

study, potential algae/cassava diet is estimated to replace about 41% of imported ingredient while still

being nutritionally balanced for optimal layer production.

Figure 25: Percentage of local ingredients included in diets

In Figure 26 is an estimate of the amounts of local ingredients in tons that would be required to supply

the layer feed needs of the current industry (based on the 2007 Ag Census).

Ration Control diet KA19 diet C323 diet Algae/Cassava diet

Millrun 26% 16% 16% 15.0%

KA19 0% 20% 0% 15%

C323 0% 0% 20% 0%

Cassava Root Meal 0% 0% 0% 11%

Total Percent Local

Ingredients Used in Ration 26% 36% 36% 41%

Local Ingredient Inclusion Rates in Research Diets


Figure 26: Estimate of amount (tons) of local ingredients needed per year to supply the needs of current Hawaii layer industry (Ag Census 2007)

A comparison was performed of the relative percentage of imported ingredients replaced by each diet.

From that information, an estimate of ration cost based on ingredients was calculated.

Figure 27: Comparison of percentage of imported ingredients replaced by sample diet formulations and estimated ration costs

C. Recommendations Continued research should be done on algae as a portion of the layer ration. The preliminary work in

this research found that algae is very digestible for poultry and has potential to fit into a complete layer

ration at a 20% rate of inclusion. Also, the positive benefits on DHA fatty acid composition of the eggs

could help provide a higher value product for the island producers and to differentiate them from the

mainland products. Future research should be done on broiler chickens to better understand how the

algae will affect growth rates and carcass quality.

The cassava sample ration with algae showed promise in the ration balancing software and based on

current and past research. This ration replaces

Control KA19 diet C323 diet Algae/Cassava diet

Millrun 4365 2686 2686 2519

KA19 algae 0 3358 0 2519

C323 algae 0 0 3358 0

Cassava Root Meal 0 0 0 1830

Approx. amount (tons) of each ingredient needed per year for Hawaii laying hens (368,000 hd)

Percent Traditional Layer Diet Control Algae Cassava Algae

Corn 58 33.784 26.3 23.3

SBM 20 15 10 9.6

Corn Dist Solubles 10 9.187 10 10

Wheat Millrun 0 26.609 16.266 15

Algae 0 0 20 15

Cassava Root Meal 0 0 0 10.9

Oil 2 6 9 7.5

Limestone 8.8 8.9 7.887 8.115

Other 1.2 0.52 0.547 0.585

Total 100 100 100 100

Calculated Price per Ton 564.00$ 436.00$ 541.33$ 469.98$

Comparison of Ingredients Replaced with Local Ingredients for Sample Rations


The major biofuel and energy production companies should be involved with representatives from the

livestock industries, as they research and develop their strains and varieties for energy production. This

will ensure varieties have a maximum value both as energy producers and livestock feed.

A feed research mill in Hawaii would expedite the process of creating research rations with local

ingredients for use in feed trials. The diets in this study were pre-mixed on the mainland, and shipped

into Maui for the addition of the local ingredients.

IX. Works Cited A. T. Diplock1, J.-L. C.-W.-E.-R. (1998). Functional food science and defence against reactive oxidative

species. Britsh Journal of Nutrition, S77-S112.

Brenna, J. (March 2002). Efficiency of conversion of alpha-linolenic acid to long chain n-3 fatty acids in

man. Curr. Opin. Clin. Nutr. Metab. Care 5 (2), 127-32.

C, v. S. (March 2003). The role of omega-3 fatty acids in cardiovascular disease. Curr. Atheroscler. Rep. 5

(2), 139-45.

Hennesey, S. a. (1986). Evaluation of cooked full fat soybeanmeal and cassava meal in the feeding of

laying hens. Palmira, Colombia: Universidad Nacional de Colombia.

Mori, T. A., Bao, D. Q., Burke, V., Puddey, I. B., & Beilin, L. J. (1993). Docosahexaenoic acid but not

eicosapentaenoic acid lowers ambulatory blood pressure and heart rate in humans.

Hypertension: 34 (2), 253-60.

Pala, V., Krogh, V., Muti, P., Chajès, V., Riboli, E., Micheli, A., et al. (July 2001). Erythrocyte membrane

fatty acids and subsequent breast cancer: a prospective Italian study. Journal of the National

Cancer Institute. 92 (14), 1088-95.

Van De Rest, O., Geleijnse, J. M., Kok, F. J., Van Staveren, W. A., Dullemeijer, C., Olderikkert, M., et al.

(August 2008). Effects of Fish Oil on cognitive performance in older subjects. Neurology 71 (6),

430-8.


X. Appendix

A. Balanced Research Ration- 20% Algae Ration

WUFF DA 4/8/2013 18:06

Diet Name: Layer 16%20%323whole

Feed Cost: 541.33$

Ingredient Cost Min. Amount Max. Lbs. Nutrient Units Min. Amount Max.

$/cwt % % % per ton

Corn Grain 22.00 0.00 26.303 100.00 526.07 Dry Matter % 0.00 81.888 100.00

Poultry BP meal 0.00 0.00 0.000 0.00 0.00 Metabolozable Energy Kcal/kg 2900.00 2900.000 10000.00

Soy 48 30.00 10.00 10.000 100.00 200.00 Protein % 16.00 17.686 100.00

Rend. Meat and Bone Meal0.00 0.00 0.000 100.00 0.00 Ether Extract % 0.00 11.808 100.00

Corn Distillers Solubles 15.00 0.00 10.000 10.00 200.00 Linoleic Acid % 0.00 0.772 100.00

Corn Gluten Meal 60% 30.25 0.00 0.000 0.00 0.00 Crude Fiber % 0.00 1.849 100.00

Wheat Middlings 6.50 0.00 0.000 0.00 0.00 Calcium % 3.50 3.500 100.00

Wheat Bran 6.50 0.00 0.000 0.00 0.00 Total Phosphorus % 0.00 0.506 100.00

Choline Cl 62.50 0.00 0.000 100.00 0.00 Avail. Phosphorus % 0.25 0.262 100.00

Limestone 5.50 0.00 7.887 100.00 157.75 Potassium % 0.00 0.857 100.00

Exportpoultryprmx6071aa129.50 0.20 0.200 100.00 4.00 Chlorine % 0.00 0.210 100.00

Dicalcium Phosphate 34.40 0.00 0.000 100.00 0.00 Manganese mg/kg 0.00 138.592 5000.00

Sodium Bicarbonate 40.00 0.00 0.000 100.00 0.00 Sodium % 0.00 0.152 100.00

Sodium Chloride 8.00 0.25 0.250 0.35 5.00 Zinc mg/kg 0.00 125.103 10000.00

Soy oil 42.00 0.00 0.000 0.00 0.00 Iron mg/kg 0.00 325.775 5000.00

Oil (other) 42.00 0.00 9.000 9.00 180.00 Copper mg/kg 0.00 22.028 5000.00

DL-Methionine 211.70 0.00 0.093 100.00 1.86 Selenium mg/kg 0.00 0.590 5000.00

Lysine HCL 132.00 0.00 0.000 100.00 0.00 Magnesium mg/kg 0.00 0.167 5000.00

Threonine 135.00 0.00 0.000 100.00 0.00 Sulfur % 0.00 3.903 100.00

Wheat millrun 19.00 0.00 16.266 100.00 325.33 Vitamin E mg/kg 0.00 30.051 5000.00

Brewer's Grains 0.00 0.00 0.000 0.00 0.00 Thiamin mg/kg 0.00 3.979 5000.00

Algae 323 Whole biomass45.00 20.00 20.000 20.00 400.00 Riboflavin mg/kg 0.00 15.911 5000.00

Algae046 High lipid 45.00 0.00 0.000 0.00 0.00 Niacin mg/kg 0.00 127.059 5000.00

Algae 323 Defatted biomass25.00 0.00 0.000 0.00 0.00 Pyridoxine mg/kg 0.00 13.130 5000.00

0 0.00 0.00 0.000 0.00 0.00 Vitamin B-12 mcg/kg 0.00 12.300 5000.00

Biotin mg/kg 0.00 0.556 5000.00

TOTAL 100.000 Choline mg/kg 900.00 1550.260 5000.00

Folate mg/kg 0.00 10.678 5000.00

ARG % 0.00 0.994 100.00

GLY % 0.00 0.821 100.00

SER % 0.00 0.391 100.00

GLY&SER % 0.00 0.912 100.00

HIS % 0.00 0.445 100.00

ILE % 0.00 0.719 100.00

LEU % 0.00 1.405 100.00

LYS % 0.75 0.821 100.00

MET % 0.35 0.376 100.00

CYS % 0.00 0.151 100.00

TSAA % 0.65 0.650 100.00

PHE % 0.00 0.844 100.00

TYR % 0.00 0.315 100.00

TAAA % 0.00 1.393 100.00

THR % 0.50 0.642 100.00

TRP % 0.00 0.227 100.00

VAL % 0.00 0.903 100.00


B. Research Ration-Control

WUFF DA 4/8/2013 18:14

Diet Name: Layer 16%

Feed Cost: 436.00$


$/cwt % % % per ton



Soy 48 30.00 15.00 15.000 100.00 300.00 Protein % 16.00 16.835 100.00












Soy oil 42.00 0.00 0.000 0.00 0.00 Iron mg/kg 0.00 376.741 5000.00










0 0.00 0.00 0.000 0.00 0.00 Vitamin B-12 mcg/kg 0.00 11.476 5000.00

Biotin mg/kg 0.00 0.305 5000.00


Folate mg/kg 0.00 1.244 5000.00

ARG % 0.00 1.046 100.00

GLY % 0.00 0.765 100.00

SER % 0.00 0.282 100.00

GLY&SER % 0.00 0.617 100.00

HIS % 0.00 0.449 100.00

ILE % 0.00 0.721 100.00

LEU % 0.00 1.387 100.00

LYS % 0.75 0.822 100.00

MET % 0.35 0.350 100.00

CYS % 0.00 0.127 100.00

TSAA % 0.65 0.678 100.00

PHE % 0.00 0.824 100.00

TYR % 0.00 0.204 100.00

TAAA % 0.00 1.368 100.00

THR % 0.50 0.595 100.00

TRP % 0.00 0.218 100.00

VAL % 0.00 0.859 100.00


C. Potential Feed Trial Ration-with Cassava

WUFF DA 4/8/2013 18:01

Diet Name: Algae 15% with Cassava 10.9%

Feed Cost: 469.98$


$/cwt % % % per ton



Soy 48 30.00 0.00 9.600 100.00 192.00 Protein % 16.00 15.996 100.00












Soy oil 42.00 0.00 0.000 0.00 0.00 Iron mg/kg 0.00 320.365 5000.00










Cassava 16.00 0.00 10.900 100.00 218.00 Vitamin B-12 mcg/kg 0.00 12.100 5000.00

Biotin mg/kg 0.00 0.482 5000.00


Folate mg/kg 0.00 8.273 5000.00

ARG % 0.00 0.905 100.00

GLY % 0.00 0.801 100.00

SER % 0.00 0.347 100.00

GLY&SER % 0.00 0.895 100.00

HIS % 0.00 0.402 100.00

ILE % 0.00 0.662 100.00

LEU % 0.00 1.268 100.00

LYS % 0.75 0.762 100.00

MET % 0.35 0.374 100.00

CYS % 0.00 0.136 100.00

TSAA % 0.65 0.656 100.00

PHE % 0.00 0.777 100.00

TYR % 0.00 0.288 100.00

TAAA % 0.00 1.355 100.00

THR % 0.50 0.572 100.00

TRP % 0.00 0.223 100.00

VAL % 0.00 0.829 100.00


D. Statistical Analysis of Production Data

Egg Production

Oct 16 to Feb 16 1 2 3

Sum of replicates 779 731 705

N 124 124 124 11.13552873

Mean 6.3 5.9 5.7

sum of squares 6975 6285 5819

Total squared 606841 534361 496711.72

2081.1 1975.6 1813.8

sample variance 16.919683 16.06209022 14.745969

stand dev (s) 4.1133542 4.007753762 3.840048

stand error 0.3693901 0.359906912 0.3448465

mean plus st err 6.6516482 6.255068203 6.0285383

95% confidence limits of the mean 7.0062627 6.600578839 6.3595909

5.5582534 5.189743742 5.0077926

Treatment 2 to 1 0.387

0.138 0.130586099 0.1198859

0.268

0.518

t score 0.748

0.05 probability

1.96 degrees of freedom


0.257

0.507

t score 1.180


0.250

0.500

t score 0.423


Egg Weights

Oct 16 to Feb 16 1 2 3 Sum of replicates 5947.0 5810.3 5819.4938 N 106 106 106 10.29563014

Mean 56.103486 54.81417761 54.900885 sum of squares 334458.1 319373.0 322140.11 Total squared 35366446 33759618.93 33866508

812.41459 886.0591222 2644.7523

sample variance 7.7372818 8.438658306 25.188117 stand dev (s) 2.781597 2.904936885 5.0187765 stand error 0.2701726 0.282152413 0.4874667 mean plus st err 56.373659 55.09633002 55.388352 95% confidence limits of the

mean 56.633024 55.36719634 55.85632

55.573948 54.26115888 53.94545


0.0736884 0.080368174 0.2398868

0.1540566

0.3925004

t score 3.28

0.05 probability

1.96

degrees of freedom


0.3135752

0.5599779

t score 2.15


0.320255

0.5659108

t score 0.15


E. Complete Yolk Analysis Lab Results

read the full study

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