Performance and health of broiler chickens fed low erucic acid, lower fiber pennycress (CoverCress^TM) grain

Abstract

CoverCress (low erucic acid, lower fiber pennycress) is being developed as a cover crop to be planted in the fall after corn and harvested in the spring prior to planting soybeans. Two experiments were conducted to evaluate 2 lines of the whole grain (CCWG-1: natural mutation and mutation breeding; CCWG-2: gene edited) and the whole grain pretreated with the potential palatability agent copper sulfate (CCWG-1-CuSO₄; CCWG-2-CuSO₄) as an ingredient for broilers. In Experiment 1, CCWG-1-CuSO₄ was included in the diet at 0, 4, and 6% for 41 d. Feed intake, body weight gain, feed conversion, processing characteristics, organ weights, serum thyroid, macropathology and histology data were collected. In Experiment 2, broilers were fed diets containing Control, 2% CCWG-1, 4% CCWG-1, 4% CCWG-2, and 4.35% CCWG-1-CuSO₄ for 42 d. Feed intake, body weight gain, feed conversion, organ weights, serum thyroid, blood chemistries, macropathology, and histology data were collected. In Experiment 1, feed intake and body weight were diminished with no effect on feed conversion for the birds consuming diets containing CCWG-1-CuSO₄. In Experiment 2, feed intake and body weight were lower with no difference in feed conversion in birds fed diets containing greater than 2% CoverCress grain during d 0 to 28. During d 28 to 42 no difference in feed intake, body weight and an improvement in feed conversion was observed in birds fed all of the CoverCress grain products. In both experiments no significant negative effects were observed in processing, liver, kidney, and thyroid weights, T3, T4, blood chemistries, macropathology, and histopathology between the control and any of the CoverCress grain treatments. No difference in performance was observed in birds fed the mutant (4% CCWG-1) and gene-edited (4% CCWG-2) products. Pretreating CoverCress grain with copper sulfate did not have a significant effect on improving palatability. In conclusion, CoverCress grain can be safely fed to broilers when included at a target rate of 4% in diets and with total glucosinolate levels not to exceed 4.9 µmoles g^-1.

INTRODUCTION

Field pennycress (Thlaspi arvense L.) is an annual weed that grows over the winter in Eurasia and North America. Pennycress is relatively high in oil (∼24–39%) that is a potential source of biodiesel (Fan et al., 2013). Mousavi-Avval and Shah (2021) concluded that pennycress production requires less nitrogen, pesticides, and farm operations as compared to other fuel feedstocks such as canola, soybeans, and lignocellulosic biomass, resulting in a reduction in environmental impacts and energy use in the production of hydroprocessed renewable jet fuel. To become commercially viable as a fuel feedstock, field pennycress has been domesticated and cultivated as a winter annual for use in a corn/pennycress/soybean rotation where pennycress is sown and harvested as a cash winter cover crop (Phippen and Phippen, 2012; Sedbrook et al., 2014; Chopra et al., 2020).

In addition to use as an ultra-low carbon fuels feedstock, field pennycress may also be an energy source for animals. Pennycress is a member of the Brassicacae family, commonly known as the mustard family. Other members of this family include oilseed rape (Brassica rapa and Brassica napus varieties), canola (rapeseed variants producing edible oil), carinata (Brassica carinata), and camelina (Camelina sativa). Brassicas contain glucosinolates, which are considered as antinutrients and can prevent the absorption of iodine and may affect thyroid function in target species (Schöne et al., 1993). Glucosinolates can also impart a bitter flavor which may affect feed consumption (Bischoff, 2021). The literature on glucosinolates and animal nutrition have been previously reviewed (Tripathi and Mishra, 2007; EFSA, 2008).

A significant consideration when evaluating the possible utility of CoverCress for use as an energy source in broiler diets was the presence of high concentrations of sinigrin, a glucosinolate, in the whole grain product (Al-Sheihbaz and Al-Shammary, 1987). A careful analysis of previous data, including broiler studies done on wild-type field pennycress hexane-extracted meal and on oilseed rape and canola whole grain and/or meal led to the conclusion that the No Observed Effect Level (NOEL) of total glucosinolates in broiler diets was ∼1 to 4 µmol/g finished diet (Summers et al., 1982; Alhotan et al., 2017; Yadav et al., 2022). At somewhat higher incorporation levels, the main effects at levels from ∼4 to 10 µmol/g finished diet were an immediate, but transient, reduction in feed intake and associated reduced body weight gain without negative changes to the Feed:Gain ratio or significant evidence of toxicity. Glucosinolates are known to reduce diet palatability in several species including broiler chickens. The reduced feed intake was observed to result in lowered body weight gain with no change in Feed:Gain ratios or evidence of toxicity. Therefore, the palatability effect has not been considered adverse. At higher total glucosinolate levels in finisher diets, adverse effects associated with glucosinolate metabolites include toxicity to the thyroid, liver and kidney (EFSA, 2008). As a result, the evidence indicated that the No Observed Adverse Effect Level (NOAEL) for total glucosinolates for broiler chickens >6 µmol glucosinolates/g finished diet (Tripathi and Mishra, 2007). Based on the published literature, it was reasonable to conclude that incorporation of CCWG in the broiler diets over a range of 2 to 4% would not result in adverse effects associated with glucosinolate toxicity. It was on this basis that experiments described herein were designed, conducted and interpreted. One additional consideration was that copper sulfate treatment has been suggested to overcome the deleterious effects of glucosinolates for high glucosinolate grains (Schöne et al., 1993; Payvastegan et al., 2013); thus, pretreatment with copper sulfate was also investigated for its utility.

Like conventional oilseed rape, Field pennycress oil typically contains >35% erucic acid (Evangelista et al., 2012; Isbell et al., 2015; McGinn et al., 2019). High dietary intake of erucic acid has been associated with myocardial lipidosis and heart lesions (Bremer and Norum, 1982). Reduction of erucic acid through the disruption of Fatty Acid Elongation 1 (FAE 1) with a subsequent increase in oleic (C18:1) has been achieved in pennycress (McGinn et al., 2019). Field pennycress is also high in fiber, which can negatively impact digestibility as a feed ingredient. A mutation of pennycress resulting in lower fiber content has been observed (Chopra et al., 2018). Reduction of erucic acid and fiber levels in pennycress has been achieved through conventional breeding and gene editing for loss of function in the associated gene pathways. A low-erucic acid, lower-fiber pennycress has been developed under the trade name of CoverCress.

The objectives of the study were to assess the safety and efficacy of the mutant and gene-edited lines when fed as whole grain treated with or without copper sulfate to broiler chickens over 41 or 42 d. Two experiments were conducted. In Experiment 1, dietary inclusion (0, 4 and 6%) of low erucic acid, lower fiber pennycress grain developed by natural mutations and mutation breeding (CCWG-1) and treated with copper sulfate (CCWG-1-CuSO₄) was investigated in a 41-d floor pen study. In Experiment 2, CCWG-1, CCWG-2 (developed through gene editing) and CCWG-1-CuSO₄ were investigated in a 42-d floor pen study.

MATERIALS AND METHODS

General Procedures

Test Material

CoverCress grain (CCWG-1 and CCWG-2) was grown and harvested by CoverCress Inc. (St. Louis, MO). Grain was cleaned using a hand screen and transported to CoverCress's warehouse in St. Louis where it was dried to approximately 8 to 10% moisture and stored in cloth bags.

CCWG-1-CuSO₄ was prepared by CoverCress Inc. by spraying a ratio of 0.15 L of 83 mM copper sulfate pentahydrate to 1 kg of CCWG-1. Pretreatment with a copper sulfate slurry results in approximately 32 ppm copper in the final diet when included at 4%. Product was prepared and shipped to Colorado Quality Research Inc (CQR) (Wellington, CO) approximately two weeks prior to the manufacture of each of the starter, grower and finisher diets. Samples of each batch of CCWG-1-CuSO₄ for Experiment 1 were sent to EPL Bio Analytical Services (EPL BAS) (Niantic, IL) for sinigrin and copper analysis. In Experiment 2, sinigrin analyses were conducted by CoverCress Inc. (St. Louis, MO). Nutritional analyses of grains were conducted by Dairyland Laboratories (Arcadia, WI) to facilitate formulation of starter, grower and finisher phase diets based on current industry practices.

Diet Formulation

The sources of dietary protein used in both studies were primarily from maize and soybean meal. Diets were formulated to meet or exceed the nutritional requirements as stated in Nutrient Requirements of Poultry: Ninth Revised Edition (1994), Washington D.C., National Academies Press (NRC, 1994) and/or based on industry practices. Diets were isocaloric, and isodigestible for lysine and methionine and contained added Phytase. Iodine levels were ∼1 ppm in all diets as recommended for Cobb 500 birds (Cobb 500 Broiler Performance & Nutrition Supplement, Cobb-Vantress.com, April 2018, https://cobbstorage.blob.core.windows.net/guides/5a171aa0-6994-11e8-9f14-bdc382f8d47e). Accessed October 12, 2020). CoverCress grain is being considered primarily as an energy source replacing more expensive sources of oil in the broiler diet. The commercial target inclusion rate for CoverCress grain is 2 to 4%. Experiment 1 contains the 4 and 6% inclusion levels of copper sulfate applied CoverCress grain including the target inclusion level of 4%. Experiment 2 contains the target range (2–4%) of inclusion levels of CoverCress grain with and without copper sulfate application. In addition, CoverCress grain developed through conventional breeding and gene editing were compared at the target inclusion level. In Experiment 1, starter, grower and finisher broiler diets containing 0, 4, and 6% CCWG-1-CuSO₄ were formulated. In Experiment 2, diets were formulated to contain 0, 2% CCWG-1, 4% CCWG-1, 4% CCWG-2, or 4.35% CCWG-1-CuSO₄ in the diet, in separate, but similar diet formulations. Diets were formulated similar to commercial conditions where about 100 ppm of copper was added to each diet during starter and grower phases and removed during the finisher phase (d 28–42).

Manufacturing of Diets/Feed Processing

Starter, grower and finisher diets were manufactured one to two weeks prior to the initiation of each phase. A sample of each diet manufactured by CQR in Experiment 1 was analyzed by EPL BAS for nutrient content. A sample of each diet manufactured by CQR in Experiment 2 was sent to Dairyland Laboratories (Arcadia, WI) for nutrient analysis and to University of Missouri Agriculture Experimental Station Chemical Laboratory (Columbia, MO) for amino acid analysis.

Control (no CoverCress grain) and treatment diets were prepared at the CQR feed mill. The same formulation for the control diet was used in all studies. Diets were prepared using a 226.8 kg capacity vertical mixer, or a 1814.4 kg capacity vertical mixer and pelleted and crumbled using a California Pellet Mill. The test material was blended in as whole grain and the complete diet augered from the mixer directly to the pellet machine. Feed was pelleted through a ∼5-mm die with live steam addition at ∼65ºC. The starter diets were further processed into crumbles. Samples of the finished feed were taken for nutrient analysis. Feed was stored in 22.7 kg capacity feed sacks and/or bulk storage bins labeled with treatment number.

Broilers

The broiler experiments were conducted at CQR using day-old Cobb 500 male chicks obtained from a Cobb Vantress Hatchery (Siloam Springs, AR). The Guide for the Care and Use of Agricultural Animals in Research and Teaching (Ag Guide, 2020) was followed. Birds were vaccinated for Marek's at the hatchery. Upon receipt (study d 0), birds were vaccinated for Newcastle and Infectious Bronchitis (1⁄2X dose) and coccidiosis using Advent (1X dose) by spray application using a spray cabinet.

Birds were housed within an environmentally controlled facility with concrete floor pens (∼1.22 m × 1.22 m minus 0.21 m² of feeder space) providing floor space for an initial bird density of 0.067 m²/bird and 0.075 m²/bird from d 7 through the remainder of the study.

Temperature, humidity, lighting, feeder and water space were similar for all test groups. Birds were placed in pens containing about 15 cm of clean wood shavings to provide a comfortable environment for the chicks. In order to prevent bird migration, each pen had a solid wood or plastic divider between each pen for approximately 0.3 m. Each pen was checked to ensure no openings greater than 2.54 cm existed. Water was provided ad libitum throughout the study via one Plasson bell drinker per pen. Drinkers were checked twice daily and cleaned as needed to ensure a clean and constant water supply to the birds. Feed was provided ad libitum throughout the study via one hanging, ∼43 cm diameter tube feeders per pen. A chick feeder tray was placed in each pen for approximately the first 4 d. Birds were placed on their respective treatment diets upon receipt (d 0) according to the Experimental Design. Feed added and removed from pens from d 0 to study end was weighed and recorded weekly on a pen basis.

The test facility, pens, and birds were observed at least twice daily for general flock condition, lighting, water, feed, ventilation, and unanticipated events. If abnormal conditions or abnormal behavior was noted at any of the twice-daily observations, they were documented, and documentation was included with the study records. The minimum-maximum temperature and humidity of the test facility was recorded once daily.

Birds that developed clinically significant concurrent disease unrelated to the test procedures were euthanized in accordance with site Standard Operating Procedures (SOPs). In addition, moribund/morbid or injured birds were also euthanized upon authority of a site veterinarian or a qualified technician. The reason for withdrawal was documented. If an animal died, or was removed and euthanized for humane reasons, it was recorded on the mortality sheet for the pen and a necropsy performed and filed to document the reason for removal. If euthanasia was deemed necessary, animals were euthanized by cervical dislocation, with exception to day 41/42, in which case animals were euthanized by carbon dioxide administration in order to preserve the thyroid for necropsy and histopathology. From d 0 to study end, any bird that was found dead or was sacrificed was weighed and necropsied. The weight and probable cause of death and necropsy findings were recorded. If sex-slips were noted at any time during the study they were removed, weighed, and necropsied to confirm sex and recorded on the pen mortality record.

Experiment 1

Objective

The objective of the study was to determine the effect of including CoverCress CCWG-1-CuSO₄ at 0, 4, and 6% in the diet on performance, processing characteristics, and health (morbidity and mortality) when fed to broilers for 41 d.

Experimental Design

The experimental design was a randomized complete block design with 16 blocks and three treatments (Control (0% CCWG-1-CuSO₄), 4% CCWG-1-CuSO₄, and 6% CCWG-1-CuSO₄). Treatments were assigned to pens using a complete randomized block design. One-day old Cobb 500 male birds were assigned to the pens randomly according to facility SOP. There were 16 pens per treatment and 19 birds per pen. Birds were fed starter (d 0–14), grower (d 14–28) and finisher (d 28–41) diets containing the 0, 4 or 6% CCWG-1-CuSO₄. On d 7 of age, the birds were counted and adjusted to 17 birds/pen to allow for more uniform bird number and density per pen following any potential early mortality.

The test facility was divided into 16 blocks of 3 pens per block.

Body Weights and Feed Intake

Birds were weighed by pen, on d 0, 14, 28, and 41. The feed remaining in each pen was weighed and recorded on d 7, 14, 21, 28, 35, and 41. The feed intake during d 0 to 7, 0 to 14, 0 to 21, 0 to 28, 14 to 28, 0 to 35, 0 to 41, and 28 to 41 was calculated. The feed intake per bird during d 0 to 7, 0 to 14, 0 to 21, 0 to 28, 14 to 28, 0 to 35, 0 to 41, and 28 to 41 was calculated by dividing the amount of feed consumed in a period, by the number of birds that survived in a period.

Weight Gain and Feed Conversion

Average bird weight, on a pen basis, on each weigh day was summarized. Bird weight gain by pen d 0 to 14, 0 to 28, 14 to 28, 0 to 41, and 28 to 41 was calculated. The average feed conversion was calculated on study d 14, 28, and 41 (d 0 to 14, 0 to 28, 14 to 28, 0 to 41, and 28 to 41) using the total feed consumption for the pen divided by the total weight of surviving birds. Adjusted feed conversion was calculated using the total feed consumption in a pen divided by the total weight of surviving birds and weight of birds that died or were removed from that pen.

Macropathology

On day 41, after final body weight data was collected, one bird from each pen was randomly selected for blood sample collection and macropathology evaluation at necropsy, which included examination and gross evaluation (determined to be normal or abnormal) of the basic systems (i.e., digestive, respiratory, reproductive, circulatory), including the GI tract, heart, lungs, skin, kidneys, liver and thyroid, and any abnormalities noted. Thyroid, liver and kidneys were removed, weighed, and recorded, and placed into 10% neutral buffered formalin for histopathological examination. The samples were shipped to Texas Veterinary Medical Diagnostic Laboratory (College Station, Texas) for histological examination by a Board Certified Pathologist.

Sufficient blood was withdrawn from each bird prior to euthanasia to provide approximately 2 mL of serum. Whole blood was collected into gold top serum-separating tubes (SST) tubes. Blood collection tubes were centrifuged and ∼1.5 mL of serum was placed into a single Eppendorf tube for a standard thyroid profile (code 20015) at Michigan State University Veterinary Diagnostic Laboratory (Lansing, MI). Additionally, ∼0.5 mL of serum was placed into a second Eppendorf tube and retained at CQR at ∼2 to 4°C.

Processing

After the final weight data was collected on D 41, the respective feed was returned to the pens. Feed was removed from the pens approximately 12 h prior to the scheduled processing time. On d 42, all surviving birds in each pen were euthanized and processed. The individual bird carcass weight was obtained, and then the bird was deboned, and the individual parts were weighed and recorded. Yield data included: live weight, hot carcass weight, breast meat weight (skinless, boneless), wing weight (bone in, skin on), thigh weight (bone in, skin on) and drum weight (bone in, skin on). Calculations were conducted to express weights on a percentage basis. Percentage of parts weights was calculated by dividing the part by the hot carcass weight of the bird and multiplying by 100 (i.e., percent breast yield = breast weight/hot carcass weight × 100). Percent of hot carcass weight was calculated by dividing the hot carcass weight by the live weight per bird and multiplying by 100.

Statistical Analyses

The experimental design is a randomized complete block design with 16 blocks and three treatments (Control (0% CCWG-1-CuSO₄), 4% CCWG-1-CuSO₄, and 6% CCWG-1-CuSO₄). Blocks were considered a random effect and treatments a fixed effect. An analysis of variance for each measurement (performance, thyroid hormone level, organ weights, mortality, and processing yield) was conducted using a randomized complete block model with pen as the experimental unit. SAS version 9.4 (SAS Institute, Cary, NC) was used for the analyses. Post-tests included all pairwise comparisons. All tests used a 0.05 level of significance. PROC MIXED was used to compare the three treatment means for all data sets except for mortality. Since mortality is binomially distributed (2 results–number dead, number alive) PROC GLIMMIX was used.

Experiment 2

Objective

The objective of the study was to characterize the performance (i.e., utility of the feed) and tolerance (i.e., safety of the feed) of broilers fed CCWG-1 at 2 and 4% of the diet, CCWG-2 at 4% of the diet and CCWG-1 treated with copper sulfate, CCWG-1-CuSO₄, at 4.35% of the diet for 42 d. The rationale for this study was to confirm the utility and tolerance of both CCWG-1 and CCWG-2 for the full 42-d broiler treatment period including assessment for target organ toxicity of glucosinolates. Further, the study was designed to confirm earlier studies suggesting that pretreatment with a copper sulfate slurry has no significant impact on palatability of CCWG (data not shown).

Experimental Design

The experimental design is a randomized complete block design with 8 blocks and five treatments (Control, 2% CCWG-1, 4% CCWG-1, 4% CCWG-2, and 4.35% CCWG-1-CuSO₄). One-day old Cobb 500 male birds were randomly distributed among 5 treatment groups with eight pens per treatment and 19 birds per pen. Birds were fed starter (d 0–14), grower (d 14–28) and finisher (d 28–42) diets. On d 7 of age, the birds were counted and adjusted to 17 birds/pen. The test facility was divided into 8 blocks of 5 pens per block. Treatments were assigned to pens using a complete randomized block design. Birds were assigned to the pens randomly according to facility SOP.

Body Weights and Feed Intake

Birds were weighed, by pen, on d 0, 14, 28, and 42. The feed remaining in each pen was weighed and recorded on d 7, 14, 21, 28, 35, and 42. The feed intake during d 0 to 7, 0 to 14, 0 to 21, 0 to 28, 14 to 28, 0 to 35, 0 to 42, and 28 to 42 was calculated. The feed intake per bird during d 0 to 7, 0 to 14, 0 to 21, 0 to 28, 14 to 28, 0 to 35, 0 to 42, and 28 to 42 was calculated by dividing the amount of feed consumed in a period, by the number of birds that survived in a period.

Weight Gain and Feed Conversion

Average bird weight, on a pen basis, on each weigh day was summarized. Bird weight gain by pen d 0 to 14, 0 to 28, 14 to 28, 0 to 42, and 28 to 42 was calculated. The average feed conversion was calculated on study days 14, 28, and 42 (d 0 to 14, 0 to 28, 14 to 28, 0 to 41, and 28 to 42) using the total feed consumption for the pen divided by the total weight of surviving birds. Adjusted feed conversion was calculated using the total feed consumption in a pen divided by the total weight of surviving birds and weight of birds that died or were removed from that pen.

Macropathology

On d 42, after final body weight data was collected, one bird from each pen was randomly selected for blood sample collection and macropathology evaluation at necropsy, which included examination and gross evaluation (determined to be normal or abnormal) of the basic systems (i.e., digestive, respiratory, reproductive, circulatory), including the GI tract, heart, lungs, skin, kidneys, liver and thyroid, and any abnormalities noted. Thyroid, liver and kidneys were removed, weighed, and recorded, and placed into 10% neutral buffered formalin for histopathological examination. The samples were shipped to Texas Veterinary Medical Diagnostic Laboratory (College Station, Texas) for histological examination by a Board Certified Pathologist.

Sufficient blood was withdrawn from each bird prior to euthanasia to provide approximately 2 mL of serum. Whole blood was collected into gold top SST tubes. Blood collection tubes were centrifuged to affect the separation of serum, and serum was placed into two Eppendorf tubes for a standard thyroid profile (code 20015) and blood chemistry including at a minimum protein, albumin, globulin, glucose, alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatine kinase (CK), calcium, phosphorus, sodium, potassium and uric acid analysis by MSU Veterinary Diagnostic Laboratory.

Statistical Analyses

The experimental design is a randomized complete block design with eight blocks and five treatments (Control, 2% CCWG-1, 4% CCWG-1, 4% CCWG-2, and 4.35% CCWG-1-CuSO₄). Blocks were considered a random effect and treatments a fixed effect. An analysis of variance for each measurement (performance, thyroid hormone level, organ weights, mortality and blood chemistries) was conducted using a randomized complete block model with pen as the experimental unit. SAS version 9.4 (SAS Institute, Cary, NC) was used for the analyses. Post-tests included all pairwise comparisons. All tests used a 0.05 level of significance. PROC MIXED was used to compare the three treatment means for all data sets except for mortality. Since mortality is binomially distributed (two results – number dead, number alive) PROC GLIMMIX was used.

RESULTS AND DISCUSSION

Experiment 1

Test Material

The nutrient and glucosinolate content for CCWG-1-CuSO₄ is presented in Table 1. The high moisture (17.8%) and high copper (587 ppm) are a result of adding the copper sulfate solution. The copper level in the test material should have been about 800 ppm. The difference may have been due to difficulty in obtaining a representative sample from the mixture Based on the copper levels analyzed in the finished diets (Table 3) the CCWG-1-CuSO₄ had about 765 ppm of copper.

Table 1.

Experiment 1–Nutrient and sinigrin content of CCWG-1-CuSO₄ (as is basis).

Analyte
Dry matter, %	82.8
ME, kcal kg-1 calculated1	3804
Crude protein, %	20.6
Crude fat,2 %	26.8
Crude fiber, %	15.2
Acid detergent fiber, %	20.8
Neutral detergent fiber, %	21.1
Carbohydrates, %	30.6
Ash, %	4.3
Calcium, %	0.69
Phosphorus, %	0.66
Magnesium, %	0.30
Potassium, %	0.65
Sodium, %	0.00
Sulfur, %	0.82
Chloride, ppm	159
Iron, ppm	85
Copper, ppm	587
Manganese, ppm	25
Zinc, ppm	31
Indispensable Amino Acids
Arginine	1.22	1.133
Histidine	0.55	0.483
Isoleucine	0.82	0.673
Leucine	1.44	1.243
Lysine	1.08	0.833
Methionine	0.35	0.303
Phenylalanine	0.92	0.803
Threonine	0.91	0.723
Tryptophan	0.32	0.283
Valine	1.06	0.863
Dispensable Amino Acids
Alanine	0.96	0.793
Aspartic acid	1.65	1.403
Cysteine	0.29	0.213
Glutamic acid	3.15	2.843
Glycine	1.23	NA4
Proline	1.03	0.853
Tyrosine	0.49	0.413
Serine	0.82	0.673
Glucosinolates, µmoles g-1	82.2

¹Value was calculated from the ME value of the defatted meal (precision-fed cecetomized rooster assay – Parson, University IL) times the proportion of the defatted meal in the seed plus the proportion of oil in the seed times the ME value of canola oil. Fatty acid composition of CCWG is similar to canola oil.

²Fatty acids as a percent of total fatty acids: C14:0, 0.1; C16:0, 4.04; C16:1, 0.15; C18:0, 1.03; C18:1, 40.1; C18:2; 34.5; C18:3, 18.6, C20:1, 0.15; C20:1, 0.77; C20:2, 0.13; C22:1, 0.26; C24:1, 0.21.

³Calculated digestible amino acids using digestibility coefficients from cecectomized rooster assay (Parson University IL).

⁴Not available.

Table 3.

Experiment 1 - Analyzed nutrient composition of starter diets (d 1–14), grower diets (d 14–28), and finisher diets containing 0, 4, or 6% CCWG-1-CuSO₄ fed from d 28 to 41 (as is basis).

	1–14 d			14–28 d			28–41 d
Item	01	4	6	0	4	6	0	4	6
Dry matter, %	86.9	87.0	86.5	86.7	87.1	86.5	87.0	86.6	86.3
Crude protein, %	22.2	22.5	22.5	19.9	20.1	20.2	18.6	18.5	18.6
Crude fat, %	3.7	5.1	3.8	4.2	4.3	5.0	4.1	4.2	4.6
Crude fiber, %	2.2	2.6	2.9	2.5	2.5	2.6	2.0	2.3	2.4
Acid detergent fiber, %	6.6	6.8	7.2	6.6	5.9	7.2	5.7	5.9	6.0
Neutral detergent fiber, %	8.6	9.6	9.5	9.8	9.1	9.7	9.0	9.4	9.0
Carbohydrates, %	56.6	54.6	55.9	58.2	58.3	57.1	60.5	59.8	59.3
Ash, %	4.5	4.7	4.3	4.4	4.4	4.2	3.9	4.1	3.9
Minerals
Calcium, %	0.90	0.96	0.82	0.89	0.88	0.88	0.79	0.83	0.87
Phosphorus, %	0.69	0.71	0.68	0.67	0.67	0.66	0.60	0.63	0.64
Magnesium, %	0.21	0.21	0.21	0.19	0.19	0.19	0.17	0.18	0.18
Potassium, %	1.07	1.07	1.06	0.98	0.96	0.91	0.84	0.83	0.82
Sodium, %	0.18	0.17	0.15	0.19	0.19	0.18	0.18	0.24	0.18
Sulfur, %	0.31	0.33	0.34	0.29	0.30	0.33	0.29	0.31	0.32
Chloride, ppm	1380	987	1121	1678	1772	1915	1439	1152	1262
Iron, ppm	158	178	150	150	163	156	150	144	149
Copper, ppm	17	41	64	15	46	63	13	41	59
Manganese, ppm	77	78	71	74	72	78	67	73	77
Zinc, ppm	75	77	67	76	76	76	66	67	75
Amino Acids
Arginine %	1.18	1.22	1.20	1.17	1.11	1.16	1.09	1.02	0.98
Histidine, %	0.68	0.68	0.67	0.66	0.65	0.65	0.66	0.62	0.61
Isoleucine, %	0.91	0.94	0.93	0.88	0.86	0.86	0.85	0.77	0.77
Leucine, %	1.76	1.80	1.78	1.75	1.71	1.70	1.71	1.58	1.57
Lysine %	1.36	1.40	1.41	1.28	1.26	1.30	1.17	1.10	1.10
Methionine, %	0.62	0.67	0.68	0.68	0.57	0.57	0.66	0.60	0.66
Phenylalanine, %	1.05	1.06	1.06	1.03	1.00	1.00	1.04	0.94	0.91
Threonine, %	0.96	0.97	0.96	0.91	0.89	0.90	0.90	0.86	0.85
Trptophan, %	0.24	0.23	0.26	0.19	0.20	0.19	0.18	0.18	0.17
Valine, %	1.03	1.05	1.06	1.01	0.99	0.99	0.97	0.89	0.89
Alanine, %	1.13	1.17	1.15	1.14	1.12	1.12	1.09	1.03	1.03
Aspartic acid, %	2.12	2.21	2.22	2.02	1.97	1.94	1.82	1.72	1.70
Cysteine, %	0.23	0.31	0.31	0.32	0.26	0.26	0.28	0.21	0.23
Glutamic acid, %	3.82	3.96	3.94	3.71	3.63	3.61	3.40	3.27	3.24
Glycine ,%	1.08	1.12	1.11	1.12	1.09	1.15	1.08	1.01	0.97
Proline, %	1.27	1.30	1.27	1.31	1.26	1.25	1.26	1.16	1.15
Tyrosine, %	0.60	0.61	0.59	0.62	0.58	0.63	0.64	0.54	0.53
Serine, %	1.03	1.07	1.05	1.01	0.97	0.97	1.06	0.93	0.89

¹Percent CCWG-1-CuSO₄ in the diet.

Diets

Ingredient and calculated nutrient compositions of the diets are presented in Table 2. All diets within the starter, grower and finisher phases were isocaloric and calculated to be similar in other nutrients except for sulfur and copper which are increased with the inclusion of CCWG-1-CuSO₄. Glucosinolates were 0, 3.3, and 4.9 µmoles g^-1 of diet for the 0, 4, and 6% CCWG-1-CuSO₄ inclusion level, respectively. Table 3 contains the analyzed nutrient and amino acid values of the diets. In general, the nutrient contents among treatment groups within the starter, grower and finisher phases were similar.  The sulfur and copper levels are increased in a dose dependent manner due to the inclusion of CCWG-1-CuSO₄.  Amino acids were similar across diets.  Dietary fatty acids are presented in Table 4. The changes in fatty acids reflect the addition of the fatty acids from CCWG-1-CuSO₄ with a decrease in C16:0, C18:0, and C18:2 and an increase in C18:1 and C18:3. These differences are all within the range of typical broiler diets and meet minimum industry standards for growth. Erucic acid (C22:1) was not detectable in the final diets.

Table 2.

Experiment 1 - Ingredient and calculated nutrient composition of starter diets (d 1–14), grower diets (d 14–28), and finisher diets containing 0, 4, or 6% CCWG-1-CuSO₄ fed from d 28 to 41 (as is basis).

	1–14 d			14–28 d			28–41 d
Item	01	4	6	0	4	6	0	4	6
Ingredients
Corn, %	55.07	53.24	52.26	60.55	58.73	57.81	64.79	62.96	62.03
Soybean meal, %	32.23	30.79	30.09	26.28	24.77	24.01	22.09	20.58	19.85
Corn DDGS, %	5.00	5.00	5.00	5.00	5.00	5.00	5.00	5.00	5.00
Meat & bone meal, %	4.43	4.36	4.33	4.50	4.50	4.50	4.70	4.70	4.69
CCWG-1-CuSO4, %	0.00	4.00	6.00	0.00	4.00	6.00	0.00	4.00	6.00
Soybean Oil, %	1.43	0.79	0.50	1.85	1.21	0.88	1.71	1.06	0.75
Limestone, %	0.45	0.42	0.39	0.47	0.43	0.41	0.49	0.44	0.42
DL-Methionine, %	0.38	0.38	0.39	0.32	0.32	0.32	0.28	0.28	0.29
Salt, %	0.28	0.28	0.28	0.29	0.28	0.28	0.30	0.29	0.29
L-Lysine HCL, %	0.20	0.21	0.22	0.19	0.20	0.21	0.18	0.20	0.20
Choline Chloride,%	0.12	0.13	0.13	0.10	0.10	0.11	0.01	0.02	0.02
L-Threonine 98%, %	0.11	0.10	0.10	0.09	0.08	0.08	0.09	0.09	0.09
Trace minerals2, %	0.10	0.10	0.10	0.10	0.10	0.10	0.10	0.10	0.10
Vitamins3, %	0.10	0.10	0.10	0.10	0.10	0.10	0.10	0.10	0.10
Phytase4, %	0.05	0.05	0.05	0.05	0.05	0.05	0.05	0.05	0.05
Sodium bicarbonate, %	0.05	0.06	0.06	0.12	0.13	0.13	0.11	0.12	0.12
Organic iodine 405, %	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00
Calculated nutrients
ME, kcal kg-1	2988	2988	2990	3076	3076	3076	3116	3116	3116
Crude protein, %	22.7	22.7	22.7	20.4	20.5	20.5	19.0	19.0	19.0
Crude fat, %	4.6	5.0	5.3	5.1	5.6	5.8	5.0	5.5	5.8
Calcium, %	0.95	0.95	0.95	0.93	0.94	0.94	0.94	0.94	0.95
Total phosphorus, %	0.55	0.55	0.56	0.53	0.53	0.54	0.52	0.53	0.53
Available phosphorus, %	0.45	0.45	0.45	0.44	0.45	0.45	0.45	0.45	0.45
Dig Methionine, %	0.68	0.68	0.68	0.59	0.59	0.59	0.54	0.54	0.54
Dig TSAA, %	0.98	0.98	0.98	0.86	0.86	0.86	0.79	0.79	0.79
Dig Lysine, %	1.25	1.25	1.25	1.10	1.10	1.10	1.00	1.00	1.00
Dig Threonine, %	0.81	0.81	0.81	0.72	0.72	0.72	0.67	0.67	0.67
Glucosinolates, μmoles g-1	0.0	3.3	4.9	0.0	3.3	4.9	0.0	3.3	4.9

¹Percent CCWG-1-CuSO₄ in the diet.

²Trace mineral mix provided per kg of complete diet: 66 mg manganese from manganese oxide; 44 mg zinc from zinc oxide; 10 mg magnesium from magnesium oxide; 88 mg iron from ferrous sulfate; 8.8 mg copper from copper sulfate; 0.385 mg iodine from ethylenediamine dihydroiodide; 0.15 mg Se from sodium selenite; 173 to 211 mg calcium from calcium carbonate.

³Vitamins provided per kg of complete diet: 9,262 IU Vitamin A; 4190 IU Vitamin D₃; 33 IU Vitamin E; 0.014 mg Vitamin B₁₂; 2.60 mg menadione; 8.7 mg riboflavin; 13 mg d-pantothenic acid; 2.1 mg of thiamine; 48 mg niacin; 2.9 mg Vitamin B₆; 0.95 mg folic acid; 0.120 mg biotin.

⁴Phyzyme 2500 TPT premix (NutrBlend LLC, Neosho, MO) provided 1250 FTU per kg of diet.

⁵Organic Iodine 40 (International Nutrition) was included at 0.001% adding 0.6 mg I per kg of complete diet.

Table 4.

Experiment 1 - Analyzed dietary fatty acids (% of total fatty acids) of starter diets (d 1–14), grower diets (d 14–28), and finisher diets containing 0, 4 or 6% CCWG-1-CuSO₄ fed from d 28 to 41.

	1 – 14 d			14 to 28 d			28 to 41 d
Fatty Acid1	02	4	6	0	4	6	0	4	6
	—————% of Total Fatty Acids—————
Myristic (C14:0)	0.23	0.22	0.20	0.21	0.20	0.21	0.21	0.21	0.20
Palmoitic (C16:0)	14.0	12.0	11.0	13.5	12.0	10.9	13.5	11.6	10.8
Palmitoleic (16:1)	0.36	0.34	0.34	0.35	0.34	0.35	0.33	0.35	0.34
Heptadecanoic (C17:0)	0.13	0.11	0.10	0.13	0.11	0.10	0.13	0.11	0.11
Heptadecenoic (C17:1)	0.08	0.09	0.08	0.08	0.08	0.08	0.08	0.09	0.08
Stearic (C18:0)	4.42	3.62	3.14	4.42	3.63	3.43	4.31	3.63	3.26
Oleic (C18:1)	26.4	30.5	32.1	26.0	29.7	31.7	26.3	30.2	31.8
Linoleic (C18:2)	49.1	44.7	43.3	49.7	45.8	43.5	49.7	45.4	43.7
Linolenic (C18:3)	4.06	7.06	8.24	4.19	6.79	8.36	4.08	7.09	8.30
Arachidic (C20:0)	0.36	0.31	0.28	0.36	0.32	0.28	0.37	0.31	0.29
Eicoenoic (C20:1)	0.31	0.44	0.49	0.30	0.43	0.50	0.30	0.44	0.48
Eicoadienoic (20:2)	0.14	0.15	0.15	0.13	0.14	0.15	0.12	0.14	0.14
Behenic (22:0)	0.26	0.21	0.18	0.26	0.22	0.20	0.26	0.21	0.18
Tricosanoic (C23:0)	0.07	0.06	0.96	0.07	0.06	0.06	0.07	0.06	0.06
Lignoceric (C24:0)	0.20	0.17	0.16	0.20	0.18	0.16	0.20	0.17	0.16
Nervonvic (C24:1)	0.00	0.00	0.07	0.00	0.00	0.06	0.00	0.00	0.06

¹The following fatty acids were measured and were not detected: Caproic (C6:0), Caprylic (C8:0), Capric (C10:0), Lauric (C12:0), Pentadecanoic (C15:0), Pentadecenoic (C15:1), Gamma Linolenic (C18:3), Nonadecanoic (C19:0), Nonadecenoic (C19:1), Eicosatrienoic (20:3), Arachidonic (C20:4), Heneicosanoic (C21:0), Erucic (C22:1), Docosadienoic (C22:2).

²Percent CCWG-1-CuSO₄ in the diet

Feed Intake

Feed consumption of birds fed diets containing 0, 4, and 6% CCWG-1-CuSO₄ is presented in Table 5. In all feeding phases, the control group consumed more feed than either the birds fed the 4 or 6% CCWG-1-CuSO₄.  Cumulative feed consumption values per pen from d 0 to 41 were decreased (P = 0.001) in the 4 and 6% inclusion CCWG-1-CuSO₄ groups and were 95.6 and 94.5% of control, respectively. Feed intake per pen for the birds fed 6% CCWG-1-CuSO₄ was lower (P ≤ 0.001) than the 4% group for the first 14 and 21 d but was not different in subsequent periods nor d 0 to 41 overall, indicating that there is no dose dependent effect on intake due to CCWG-1-CuSO₄.  These results suggest there was a significant negative effect on palatability that was not different between the 4 and 6% inclusion rates. Over d 0 to 41, feed intake of birds fed the CCWG-1-CuSO₄ consumed ∼95% and ∼96% of control when analyzed on per pen and per bird basis, respectively. The controls consumed 2.2% more and the 4% and 6% CCWG-1-CuSO₄groups consumed 1.8 and 1.6%, respectively, less than the target of 4.776 kg for male Cobb 500 birds (Cobb-Vantress, 2018).

Table 5.

Experiment 1 - Feed consumption of birds fed diets containing 0, 4, or 6% CCWG-1-CuSO₄.

Variable	01	4	6	SEM	P value
Days 0–7
Feed consumed, kg pen-1	3.180a	3.060b	2.926c	0.0427	< 0.001
Feed consumed, kg bird-1	0.187a	0.180b	0.172c	0.0025	< 0.001
Days 0–14
Feed consumed, kg pen-1	9.259a	9.058b	8.769c	0.0751	< 0.001
Feed consumed, kg bird-1	0.551a	0.535b	0.520c	0.0042	< 0.001
Days 0–21
Feed consumed, kg pen-1	20.854a	20.042b	19.612c	0.1762	< 0.001
Feed consumed, kg bird-1	1.255a	1.197b	1.180b	0.0105	< 0.001
Days 14–28
Feed consumed, kg pen-1	28.594a	27.430b	27.179b	0.2417	< 0.001
Feed consumed, kg bird-1	1.748a	1.651b	1.661b	0.0139	< 0.001
Days 0–28
Feed consumed, kg pen-1	37.853a	36.488b	35.948b	0.2863	< 0.001
Feed consumed, kg bird-1	2.314a	2.196b	2.198b	0.0190	< 0.001
Days 0–35
Feed consumed, kg pen-1	60.109a	57.518b	56.451b	0.5979	< 0.001
Feed consumed, kg bird-1	3.719a	3.577b	3.561b	0.0346	0.002
Days 28–41
Feed consumed, kg pen-1	41.039a	38.916b	38.629b	0.7209	0.017
Feed consumed, kg bird-1	2.537a	2.418b	2.432b	0.0321	0.001
Days 0–41
Feed consumed, kg pen-1	78.891a	75.404b	74.576b	0.9409	0.001
Feed consumed, kg bird-1	4.881a	4.689b	4.701b	0.0494	0.002

¹CCWG-1-CuSO₄ dietary inclusion.

^a,b,cMeans with different superscripts are different at P < 0.05.

Body Weight and Feed Conversion

BW, BW gain, Feed:Gain, and adjusted Feed:Gain (adjusted for dead and culled birds) results are presented in Table 6.  Though statistically different, the small numerical difference (0.001 kg) between the control (0% inclusion) and the two treatment groups on d 0 is not biologically significant.  BW when expressed on a pen basis (the experimental unit) was not different between the control and the 4% CCWG-1-CuSO₄ inclusion throughout the 41-d study. Birds consuming the 6% CCWG-1-CuSO₄ consistently weighed less than the control for 41 d and the 4% inclusion level for the first 14 d, thereafter, there was no significant difference between the 4 and 6% treatments. According to Cobb-Vantress (2018) Cobb 500 males should weigh 3.0 kg at day 41. The control and CCWG-1-CuSO₄ treatment groups met or exceeded this target weight.

Table 6.

Experiment 1 - BW, BW gain, and feed conversion of birds fed diets containing 0, 4, or 6% CCWG-1-CuSO₄.

Variable	01	4	6	SEM	P value
Day 0
BW, kg pen-1	0.767a	0.776b	0.778b	0.0024	0.003
BW, kg bird-1	0.040a	0.041b	0.041b	0.0001	0.003
Day 14
BW, kg pen-1	7.964a	7.804a	7.568b	0.0824	0.001
BW, kg bird-1	0.474a	0.461b	0.448c	0.0042	< 0.001
Days 0–14
BW gain, kg pen-1	7.197a	7.028a	6.789b	0.0827	0.001
BW gain, kg bird-1	0.433a	0.420b	0.408c	0.0043	< 0.001
Feed:Gain	1.287	1.290	1.293	0.0075	0.854
Adjusted Feed:Gain	1.232	1.238	1.240	0.0062	0.508
Day 28
BW, kg pen-1	27.534a	26.898a,b	26.266b	0.2804	0.004
BW, kg bird-1	1.682a	1.618b	1.605b	0.0121	< 0.001
Days 14–28
BW gain, kg pen-1	19.570	19.094	18.699	0.2587	0.051
BW gain, kg bird-1	1.208a	1.158b	1.156b	0.0104	< 0.001
Feed:Gain	1.463	1.438	1.456	0.0119	0.301
Adjusted Feed:Gain	1.440a	1.418b	1.423b	0.0061	0.007
Days 0–28
BW gain, kg pen-1	26.767a	26.121a	25.488b	0.2813	0.004
BW gain, kg bird-1	1.642a	1.577b	1.564b	0.0121	< 0.001
Feed:Gain	1.415	1.397	1.411	0.0075	0.218
Adjusted Feed:Gain	1.382a	1.368b	1.373a,b	0.0044	0.021
Day 41
BW, kg pen-1	48.820a	46.380a,b	45.561b	1.0518	0.040
BW, kg bird-1	3.113a	2.995b	3.013b	0.0412	0.011
Days 28–41
BW gain, kg pen-1	21.286	19.482	19.295	0.8920	0.130
BW gain, kg bird-1	1.431	1.377	1.408	0.0364	0.391
Feed:Gain	1.960	2.055	2.054	0.0742	0.498
Adjusted Feed:Gain	1.818	1.827	1.803	0.0352	0.861
Days 0–41
BW gain, kg pen-1	48.053a	45.604a,b	44.783b	1.0522	0.040
BW gain, kg bird-1	3.073a	2.954b	2.972b	0.0412	0.010
Feed:Gain	1.647	1.661	1.674	0.0230	0.665
Adjusted Feed:Gain	1.576	1.569	1.564	0.0117	0.669

¹CCWG-1-CuSO₄ dietary inclusion.

^a,b,cMeans with different superscripts are different at P < 0.05.

Cumulative BW gains per pen from d 0 to 41 were statistically decreased in the 6% group only and gains were 94.9 and 93.2 % of control in the 4 and 6% inclusion groups, respectively. BW gain (pen basis) was not different between the control and 4% treatment groups at any time over the 41-d period. BW gains per pen were lower (P < 0.001) at d 0 to 14 in the 6% CCWG-1-CuSO₄ group whereas gains from d 14 to 28 and d 28 to 41 were not lower (P = 0.051 and P = 0.130), respectively) in either treatment group.

Overall, Feed:Gain and adjusted Feed:Gain were not different (P > 0.05) between the birds fed the control or the 4% or 6% CCWG-1-CuSO₄ inclusions. Thus, the differences observed in BW gain were due to the difference in feed consumption. Adjusted Feed:Gain met the target of 1.57 for Cobb 500 males (Cobb-Vantress, 2018) for all treatment groups.

Mortality and Culls

The overall mortality was greater (P = 0.028) for the birds consuming the 6% CCWG-1-CuSO₄ diet (Table 7). However, there was no significant difference among treatments when combining the mortality with the culls (Table 7). Reasons for mortality and culls by treatment and growing phase were similar and included common causes such as sudden death, bacterial infection and ascites. Overall d 1 to 41 mortality rates for all three groups were within the historical control range of 3 to 10% for the facility.

Table 7.

Experiment 1 - Mortality, mortality plus culls, an ascites incidence of birds fed diets containing 0, 4 or 6% CCWG-1-CuSO₄ for 41 d.

Variable	01	4	6	P value
Mortality, %	3.6a	5.6a,b	9.2b	0.028
Mortality + Culls, %	7.9	8.8	12.8	0.118
Ascites incidence, %	2.57	4.04	5.51	0.239

¹CCWG-1-CuSO₄ dietary inclusion.

^a,b,Means with different superscripts are different at P < 0.05.

The apparent main contributors to the statistical difference in overall mortality observed in the 6% inclusion group appeared to be two main factors: an apparent increase in bacterial infections during d 0 to 7 (2, 3, and 6 birds cumulatively in the 0, 4, and 6% groups, respectively) and a numerically higher rate of ascites, primarily during d 28 to 41 (5, 11, and 12 birds at 0, 4, and 6% inclusion rates, respectively). Bacterial infections and sudden death syndrome are common in young birds after transport which may not be treatment related.

During d 28 to 41, the major contributor to higher mortality appeared to be ascites. Ascites was observed in all treatment groups but numerically higher in the CCWG-1-CuSO₄ treatments (Table 7). However, these differences were not statistically different (Table 7). Singh et al. (2013) provides a review of the factors affecting ascites. High altitude, fast growing and cool temperatures are all contributing factors to ascites. Colorado Quality Research is located near Wellington, Colorado with an elevation of 1585.9 m above sea level. During the last 2 wk of the study, outdoor temperatures during the last 6 d on study reached a high of 0ºC. Though the facility had controlled temperature, pens near the door to the outside had higher incidence of ascites.

Given that the two apparent contributors to the increased overall mortality rate were a fast onset of apparent bacterial infection and later appearance of ascites, and that the incidence of overall mortality fell in the historical control ranges for the test facility, the observed increase in mortality at 6% was determined to be unrelated to treatment.

Processing

Processing data is presented in Table 8. There were no differences between CCWG-1-CuSO₄ treatments on any processing or carcass parameters. There were no statistical differences in hot carcass weight when expressed as a percentage of live weight among treatments. Breast meat and wings when expressed as percent of hot carcass weight were statistically more and the thighs and drums less in the birds fed CCWG-1-CuSO₄ as compared to the control. These differences are small (∼2%), unexplained and may not have any biological relevance.

Table 8.

Experiment 1 - Processing yield data from birds fed diets containing 0, 4, or 6% CCWG-1-CuSO₄.

Variable	01	4	6	SEM	P Value
Live weight kg bird-1	3.06a	2.94b	2.95b	0.038	0.003
Hot carcass weight, kg bird-1	2.19a	2.11b	2.11b	0.027	0.005
Hot carcass weight, % of live weight	71.8	71.8	71.6	0.18	0.500
Breast, kg bird-1	0.68	0.68	0.67	0.0120	0.674
Breast, % of hot carcass weight	31.1a	32.0b	31.8b	0.19	0.001
Wing, kg bird-1	0.22	0.22	0.22	0.002	0.109
Wing, % of hot carcass weight	10.2a	10.4b	10.4b	0.05	0.008
Thigh, kg bird-1	0.35a	0.34b	0.33b	0.004	0.002
Thigh, % of hot carcass weight	16.0a	15.9a,b	15.8b	0.07	0.037
Drums, kg bird-1	0.30a	0.28b	0.28b	0.003	< 0.001
Drums, % of hot carcass weight	13.6a	13.4b	13.4b	0.07	0.017

¹CCWG-1-CuSO₄ dietary inclusion.

^a,bMeans with different superscripts are different at P < 0.05.

Organ Weights and Serum Thyroid

Liver, kidney, thyroid, and serum thyroid hormone levels are presented in Table 9. No significant differences in liver or thyroid weights were observed.  Kidneys expressed as % of live weight weighed 10.7% more in the CCWG-1-CuSO₄ birds as compared to the control. There were no effects on actual kidney weights in either group compared with control.

Table 9.

Experiment 1 - Liver, kidney, and thyroid weights and serum thyroid concentrations from birds fed diets containing 0, 4 or 6% CCWG-1-CuSO₄.

Variable	01	4	6	SEM	P Value
Liver, g bird-1	70.0	66.8	65.3	2.38	0.351
Liver, % of live weight	0.0221	0.0220	0.0214	0.00055	0.569
Kidney, g bird-1	17.9	19.0	19.0	0.68	0.330
Kidney, % of live weight	0.0056a	0.0062b	0.0062b	0.00015	0.015
Thyroid, g bird-1	0.4	0.4	0.5	0.04	0.432
Thyroid, % of body weight	0.0001	0.0002	0.0001	0.0001	0.345
Total thyroxine
TT4, nmol L-1	9.25	10.88	11.31	0.715	0.111
Free, FT4, pmol L-1	5.38a	7.44b	8.75b	0.493	< 0.001
Total triiodothyronine
TT3, nmol L-1	0.99	0.91	0.99	0.086	0.635

¹CCWG-1-CuSO₄ dietary inclusion.

^a,bMeans with different superscripts are different at P < 0.05.

Feeding birds diets containing 4 or 6% CCWG-1-CuSO₄ had no effect on T4 or T3 serum concentrations. However, free thyroxine levels were statistically elevated in birds fed CCWG-1-CuSO₄ as compared to the control. If there is a glucosinolate effect, a lower free thyroxine would be expected; however, higher values were observed. Therefore, these results are not considered to be treatment related.

Macropathology and Histopathology

No abnormalities were noted during gross examination during necropsy. Histopathological analysis of livers, kidneys, and thyroids showed tissues to be normal for all three treatment groups. Overall, no treatment related findings were observed.

Experiment 2

Test Material

The nutrient and glucosinolate content of CCWG-1, CCWG-2, and CCWG-1-CuSO₄ are presented in Table 10. CCWG-1 and CCWG-2 were similar in nutrient and antinutrient content. CCWG-1-CuSO₄ was higher in moisture and copper due to the addition of copper sulfate solution.

Table 10.

Experiment 2 - Analyzed nutrient composition of CCWG-1, CCWG-2, and CCWG-1-CuSO₄ (as is basis).

Item	CCWG-1	CCWG-2	CCWG-1-CuSO4
Dry matter, %	94.1	93.8	85
ME, Kcal kg-1 calculated1	3804	4006	3557
Crude protein, %	23.0	24.2	19.5
Crude fat, %	35.3	33.3	31.9
Crude fiber, %	16.0	16.0	10.3
Acid detergent fiber, %	14.4	14.4	17.9
Neutral detergent fiber, %	18.2	18.4	23.8
Ash,%	5.2	5.2	4.8
Calcium, %	1.13	0.96	1.10
Phosphorus, %	0.63	0.62	0.56
Magnesium, %	0.26	0.34	0.23
Potassium, %	0.82	0.90	0.80
Sodium, %	0.02	0.02	0.01
Sulfur, %	0.84	0.96	0.77
Chloride, %	0.11	0.09	0.15
Iron, ppm	102	136	94
Copper, ppm	5	3	848
Manganese, ppm	37	36	40
Zinc, ppm	38	41	25
Indispensable Amino Acids
Arginine, %	1.63	1.72	1.30
Histidine, %	0.51	0.51	0.43
Isoleucine, %	0.81	0.88	0.67
Leucine, %	1.64	1.81	1.40
Lysine %	1.20	1.26	1.02
Methionine, %	0.39	0.39	0.33
Phenylalanine, %	0.96	1.07	0.84
Threonine, %	1.10	1.11	0.95
Tryptophan, %	0.39	0..40	0.42
Valine, %	1.32	1.26	1.03
Dispensable amino acids
Alanine, %	1.03	1.08	0.90
Aspartic acid, %	2.01	2.10	1.71
Cysteine, %	0.45	0.44	0.36
Glutamic acid, %	3.52	3.75	3.02
Glyine, %	1.48	1.54	1.29
Proline, %	1.17	1.42	1.20
Tyrosine, %	0.70	0.76	0.62
Serine, %	0.99	1.02	0.85
Fatty Acids (% of Total Fatty Acids)2
C16:0 Palmitic Acid	4.46	4.17	4.40
C18:0 Stearic Acid	0.00	0.84	0.33
C18:1 Oleic Acid	43.17	45.82	42.65
C18:2 Linoleic Acid	34.99	31.99	34.57
C18:3 Linolenic Acid	17.38	17.8	18.05
Anti-nutrient
Glucosinolates, µmoles g-1	97.3	103.3	93.9

¹Value was calculated from the ME value of the defatted meal (precision-fed cecetomized rooster assay – Parson, University IL) times the proportion of the defatted meal in the seed plus the proportion of oil in the seed times the ME value of canola oil. Fatty acid composition of CCWG is similar to canola oil.

²Fatty acids that were not detected in either product. C:12,C:14, Lauric and Mystic acid; C14:1, Myristoleic acid; C16:1, Palmitoleic acid; C17:0, Margaric acid; C19:0, C20:0, Nonadecanic acid, Arachidic acid; C20:1, Gadoleic; C20:2, C20:3, Eicosadienoic acid, Eicisatrienoic acid; C20:4, Arachidonic acid; C20:5, Eicapentaenoic acid; C22:1, Erucic acid; C22:6, Docosahexanoic acid; C24:0, Lignoceric acid; C24:1, Nervonic acid.

Diets

Ingredient and calculated nutrient compositions of the diets are presented in Table 11, Table 12, Table 13, Table 14, Table 15, Table 16. All diets within the starter, grower and finisher phases were isocaloric and calculated to be similar in other nutrients except for sulfur and copper which are increased with the inclusion of CCWG-1-CuSO₄. During the starter and grower phases (d 0–28) all diets had 100 ppm of supplemental copper except that the CCWG-1-CuSO₄ had an additional 34 ppm of copper. Following commercial practice, the 100 ppm of supplemental copper was removed from all diets for the finisher phase (d 28–42). Glucosinolates were 0, 1.9, 3.9, 4.1, and 4.2 µmoles g^-1 of diet for the 0, 2% CCWG-1, 4% CCWG-1, 4% CCWG-2, and 4.35% CCWG-1-CuSO₄ inclusion level, respectively.

Table 11.

Experiment 2 -Ingredient composition of starter diets (d 1–14) containing 0% (Diet 1), 2% CCWG-1 (Diet 2), 4% CCWG-1 (Diet 3), 4% CCWG-2 (Diet 4) and 4.35% CCWG-1-CuSO₄ (Diet 5) (as is basis).

	Diet 1	Diet 2	Diet 3	Diet 4	Diet 5
Ingredient	Broiler starter
	%	%	%	%	%
Corn, Yellow	55.00	54.33	53.97	53.68	52.95
Soybean Meal	32.22	31.40	30.16	30.52	30.63
DDGS	5.00	5.00	5.00	5.00	5.00
Meat & Bone	4.43	4.35	4.28	4.35	4.30
Soybean oil	1.43	1.09	0.75	0.60	0.93
Limestone	0.44	0.42	0.41	0.40	0.40
Methionine, DL	0.38	0.38	0.39	0.39	0.39
Salt	0.28	0.27	0.27	0.27	0.27
L-Lysine HCL	0.20	0.21	0.21	0.22	0.21
Choline CL-60%	0.12	0.12	0.13	0.13	0.13
Threonine 98.5%	0.11	0.11	0.11	0.11	0.11
Trace Mineral Premix #21	0.10	0.10	0.10	0.10	0.10
Broiler Vitamin Premix2	0.10	0.10	0.10	0.10	0.10
Phytase 2500 Premix3	0.04	0.04	0.04	0.04	0.04
Sodium Bicarbonate	0.06	0.06	0.07	0.07	0.07
TBCC Copper Chloride	0.09	0.02	0.01	0.02	0.02
Organic Iodine 404	0.00084	0.00084	0.00084	0.00084	0.00084
CCWG-1	0.00	2.00	4.00	0.00	0.00
CCWG-2	0.00	0.00	0.00	4.00	0.00
CCWG-1-CuSO4	0.00	0.00	0.00	0.00	4.35
Total	100.00	100.00	100.00	100.00	100.00

¹Trace mineral mix provided per kg of complete diet: 66 mg manganese from manganese oxide; 44 mg zinc from zinc oxide; 10 mg magnesium from magnesium oxide; 88 mg iron from ferrous sulfate; 8.8 mg copper from copper sulfate; 0.385 mg iodine from ethylenediamine dihydroiodide; 0.15 mg Se from sodium selenite; 173 to 211 mg calcium from calcium carbonate.

²Vitamins provided per kg of complete diet: 9,262 IU Vitamin A; 4190 IU Vitamin D₃; 33 IU Vitamin E; 0.014 mg Vitamin B₁₂; 2.60 mg menadione; 8.7 mg riboflavin; 13 mg d-pantothenic acid; 2.1 mg of thiamine; 48 mg niacin; 2.9 mg Vitamin B₆; 0.95 mg folic acid; 0.120 mg biotin.

³Phyzyme 2500 TPT premix (NutrBlend LLC, Neosho, MO) provided 1250 FTU per kg of diet.

⁴Organic Iodine 40 (International Nutrition) was included at 0.001% adding 0.6 mg I per kg of complete diet so the final diet is ∼1 ppm iodine.

Table 12.

Experiment 2 - Ingredient composition of grower diets (d 14–28) containing 0% (Diet 6), 2% CCWG-1 (Diet 7), 4% CCWG-1 (Diet 8), 4% CCWG-2 (Diet 9) and 4.35% CCWG-1-CuSO₄ (Diet 10) (as is basis).

	Diet 6	Diet 7	Diet 8	Diet 9	Diet 10
Ingredient	Broiler grower
	%	%	%	%	%
Corn, Yellow	59.58	58.83	58.08	58.19	57.48
Soybean Meal	27.09	26.18	25.31	25.30	25.36
DDGS	5.00	5.00	5.00	5.00	5.00
Meat & Bone	4.50	4.50	4.47	4.50	4.49
Soybean oil	2.01	1.67	1.32	1.18	1.50
Limestone	0.46	0.44	0.42	0.42	0.41
Methionine, DL	0.33	0.33	0.33	0.33	0.33
Salt	0.28	0.28	0.27	0.28	0.28
L-Lysine HCL	0.19	0.20	0.21	0.21	0.21
Choline CL-60%	0.09	0.10	0.10	0.10	0.10
Threonine 98.5%	0.09	0.09	0.10	0.10	0.10
Trace Mineral Premix #21	0.10	0.10	0.10	0.10	0.10
Broiler Vitamin Premix2	0.10	0.10	0.10	0.10	0.10
Phytase 2500 Premix3	0.04	0.04	0.04	0.04	0.04
Sodium Bicarbonate	0.13	0.13	0.14	0.14	0.14
TBCC Copper Chloride	0.01	0.01	0.01	0.01	0.01
Organic Iodine 404	0.00084	0.00084	0.00084	0.00084	0.00084
CCWG-1	0.00	2.00	4.00	0.00	0.00
CCWG-2	0.00	0.00	0.00	4.00	0.00
CCWG-1-CuSO4	0.00	0.00	0.00	0.00	4.35
Total	100.00	100.00	100.00	100.00	100.00

¹Trace mineral mix provided per kg of complete diet: 66 mg manganese from manganese oxide; 44 mg zinc from zinc oxide; 10 mg magnesium from magnesium oxide; 88 mg iron from ferrous sulfate; 8.8 mg copper from copper sulfate; 0.385 mg iodine from ethylenediamine dihydroiodide; 0.15 mg Se from sodium selenite; 173 to 211 mg calcium from calcium carbonate.

²Vitamins provided per kg of complete diet: 9,262 IU Vitamin A; 4190 IU Vitamin D₃; 33 IU Vitamin E; 0.014 mg Vitamin B₁₂; 2.60 mg menadione; 8.7 mg riboflavin; 13 mg d-pantothenic acid; 2.1 mg of thiamine; 48 mg niacin; 2.9 mg Vitamin B₆; 0.95 mg folic acid; 0.120 mg biotin.

³Phyzyme 2500 TPT premix (NutrBlend LLC, Neosho, MO) provided 1250 FTU per kg of diet.

⁴Organic Iodine 40 (International Nutrition) was included at 0.001% adding 0.6 mg I per kg of complete diet so the final diet is ∼1 ppm iodine.

Table 13.

Experiment 2 - Ingredient composition of finisher diets (d 28–42) containing 0% (Diet 11), 2% CCWG-1 (Diet 12), 4% CCWG-1 (Diet 13), 4% CCWG-2 (Diet 14) and 4.35% CCWG-1-CuSO₄ (Diet 15) (as is basis).

	Diet 11	Diet 12	Diet 13	Diet 14	Diet 15
Ingredient	Broiler finisher
	%	%	%	%	%
Corn, Yellow	64.72	63.94	63.19	63.32	62.60
Soybean Meal	24.44	23.61	22.79	22.73	22.84
DDGS	5.00	5.00	5.00	5.00	5.00
Meat & Bone	2.42	2.35	2.27	2.35	2.29
Soybean oil	1.66	1.32	0.98	0.83	1.16
Limestone	0.54	0.53	0.51	0.51	0.50
Methionine, DL	0.27	0.28	0.28	0.28	0.28
Salt	0.31	0.31	0.30	0.30	0.30
L-Lysine HCL	0.17	0.18	0.19	0.19	0.19
Choline CL-60%	0.01	0.01	0.02	0.02	0.02
Threonine 98.5%	0.09	0.09	0.09	0.09	0.09
Trace Mineral Premix #21	0.10	0.10	0.10	0.10	0.10
Broiler Vitamin Premix2	0.10	0.10	0.10	0.10	0.10
Phytase 2500 Premix3	0.04	0.04	0.04	0.04	0.04
Sodium Bicarbonate	0.13	0.14	0.14	0.14	0.14
TBCC Copper Chloride	0.00	0.00	0.00	0.00	0.00
Organic Iodine 404	0.00084	0.00084	0.00084	0.00084	0.00084
CCWG-1	0.00	2.00	4.00	0.00	0.00
CCWG-2	0.00	0.00	0.00	4.00	0.00
CCWG-1-CuSO4	0.00	0.00	0.00	0.00	4.35
Total	100.00	100.00	100.00	100.00	100.00

¹Trace mineral mix provided per kg of complete diet: 66 mg manganese from manganese oxide; 44 mg zinc from zinc oxide; 10 mg magnesium from magnesium oxide; 88 mg iron from ferrous sulfate; 8.8 mg copper from copper sulfate; 0.385 mg iodine from ethylenediamine dihydroiodide; 0.15 mg Se from sodium selenite; 173 to 211 mg calcium from calcium carbonate.

²Vitamins provided per kg of complete diet: 9,262 IU Vitamin A; 4190 IU Vitamin D₃; 33 IU Vitamin E; 0.014 mg Vitamin B₁₂; 2.60 mg menadione; 8.7 mg riboflavin; 13 mg d-pantothenic acid; 2.1 mg of thiamine; 48 mg niacin; 2.9 mg Vitamin B₆; 0.95 mg folic acid; 0.120 mg biotin.

³Phyzyme 2500 TPT premix (NutrBlend LLC, Neosho, MO) provided 1250 FTU per kg of diet.

⁴Organic Iodine 40 (International Nutrition) was included at 0.001% adding 0.6 mg I per kg of complete diet so the final diet is ∼1 ppm iodine.

Table 14.

Experiment 2 - Calculated dietary nutrient content of starter diets (d 1–14) containing 0% (Diet 1), 2% CCWG-1 (Diet 2), 4% CCWG-1 (Diet 3), 4% CCWG-2 (Diet 4) and 4.35% CCWG-1-CuSO₄ (Diet 5) (as is basis).

		Diet 1		Diet 2		Diet 3		Diet 4		Diet 5
Nutrient	Units	Broiler starter
ME, Poultry	Kcal/kg		2,988		2,988		2,988		2,988		2,988
Moisture	%		12.86		12.82		12.78		12.80		13.08
Crude Protein	%		22.68		22.72		22.76		22.75		22.75
Crude Fat	%		4.56		4.77		4.99		4.95		5.13
Linoleic Acid	%		2.17		2.29		2.40		2.32		2.55
Crude Fiber	%		2.60		2.73		2.85		2.86		2.84
Ash	%		5.11		5.12		5.13		5.11		5.13
Calcium	%		0.95		0.95		0.95		0.95		0.95
Total Phosphorus	%		0.55		0.55		0.56		0.55		0.56
Avail Phosphorus	%		0.45		0.45		0.45		0.45		0.45
Salt	%		0.32		0.32		0.31		0.32		0.32
Sodium	%		0.16		0.16		0.16		0.16		0.16
Potassium	%		0.89		0.89		0.89		0.88		0.89
Chloride	%		0.30		0.30		0.30		0.30		0.30
Na+K-Cl	mEq/kg		212.17		212.29		212.42		210.46		212.28
Choline	mg/kg		1741.95		1741.95		1741.95		1741.95		1741.95
Phytate	%		0.25		0.24		0.24		0.24		0.24
Arginine	%	1.43	1.311	1.43	1.311	1.43	1.311	1.43	1.311	1.43	1.311
Lysine	%	1.40	1.251	1.40	1.251	1.41	1.251	1.41	1.251	1.41	1.251
Methionine	%	0.71	0.681	0.71	0.681	0.71	0.681	0.71	0.681	0.71	0.681
Met + Cys	%	1.07	0.981	1.07	0.981	1.07	0.981	1.07	0.981	1.07	0.981
Tryptophan	%	0.27	0.231	0.27	0.231	0.27	0.231	0.27	0.231	0.27	0.231
Threonine	%	0.93	0.811	0.93	0.811	0.93	0.811	0.93	0.811	0.93	0.811
Isoleucine	%	0.97	0.871	0.96	0.871	0.96	0.871	0.96	0.871	0.96	0.871
Leucine	%	1.90	1.741	1.89	1.741	1.88	1.731	1.89	1.731	1.88	1.721
Valine	%	1.08	0.961	1.08	0.961	1.08	0.961	1.08	0.961	1.08	0.961
Cystine	%	0.36	0.301	0.36	0.301	0.36	0.301	0.36	0.291	0.36	0.301

¹Digestible amino acid value.

Table 15.

Experiment 2 - Calculated dietary nutrient content of grower diets (d 14–28) containing 0% (Diet 6), 2% CCWG-1 (Diet 7), 4% CCWG-1 (Diet 8), 4% CCWG-2 (Diet 9) and 4.35% CCWG-1-CuSO₄ (Diet 10) (as is basis).

		Diet 6		Diet 7		Diet 8		Diet 9		Diet 10
Nutrient	Units	Broiler Grower
ME, Poultry	Kcal/kg		3076		3076		3076		3076		3076
Moisture	%		12.97		12.93		12.88		12.90		13.18
Crude Protein	%		20.71		20.76		20.80		20.79		20.79
Crude Fat	%		5.21		5.43		5.65		5.60		5.79
Linoleic Acid	%		2.56		2.67		2.78		2.71		2.93
Crude Fiber	%		2.49		2.62		2.75		2.75		2.73
Ash	%		4.84		4.86		4.88		4.85		4.88
Calcium	%		0.94		0.94		0.95		0.94		0.95
Total Phosphorus	%		0.53		0.54		0.54		0.54		0.54
Avail Phosphorus	%		0.45		0.45		0.45		0.45		0.45
Salt	%		0.32		0.32		0.32		0.32		0.32
Sodium	%		0.18		0.18		0.18		0.18		0.18
Potassium	%		0.79		0.79		0.79		0.78		0.79
Chloride	%		0.30		0.30		0.30		0.30		0.30
Na+K-Cl	mEq/kg		195.83		195.6		195.55		195.80		195.41
Choline	mg/kg		1543.5		1543.5		1543.5		1543.5		1543.5
Phytate	%		0.23	0.23	0.22	0.22			3076		0.22
Arginine	%	1.28	1.181	1.28	1.181	1.28	1.181	1.28	1.181	1.28	1.181
Lysine	%	1.26	1.121	1.26	1.121	1.26	1.121	1.26	1.121	1.26	1.121
Methionine	%	0.63	0.601	0.63	0.601	0.63	0.601	0.63	0.601	0.63	0.601
Met + Cys	%	0.96	0.871	0.96	0.871	0.96	0.871	0.96	0.871	0.96	0.871
Tryptophan	%	0.24	0.211	0.24	0.211	0.24	0.211	0.24	0.211	0.24	0.211
Threonine	%	0.93	0.811	0.93	0.811	0.93	0.811	0.93	0.811	0.93	0.811
Isoleucine	%	0.87	0.781	0.86	0.781	0.86	0.771	0.86	0.771	0.86	0.771
Leucine	%	1.76	1.621	1.75	1.611	1.74	1.601	1.75	1.611	1.74	1.601
Valine	%	0.99	0.871	0.99	0.871	0.98	0.871	0.98	0.871	0.98	0.871
Cystine	%	0.33	0.281	0.34	0.271	0.34	0.271	0.33	0.271	0.34	0.271

¹Digestible amino acid value.

Table 16.

Experiment 2 - Calculated dietary nutrient content of finisher diets (d 28–42) containing 0% (Diet 11), 2% CCWG-1 (Diet 12), 4% CCWG-1 (Diet 13), 4% CCWG-2 (Diet 14) and 4.35% CCWG-1-CuSO₄ (Diet 15) (as is basis).

		Diet 11		Diet 12		Diet 13		Diet 14		Diet 15
Nutrient	Units	Broiler Finisher
ME, Poultry	Kcal/kg		3,109		3,109		3,109		3,109		3,109
Moisture	%		13.37		13.33		13.29		13.30		13.58
Crude Protein	%		18.78		18.82		18.86		18.85		18.84
Crude Fat	%		4.82		5.04		5.26		5.21		5.40
Linoleic Acid	%		2.45		2.56		2.67		2.60		2.82
Crude Fiber	%		2.44		2.57		2.69		2.70		2.68
Ash	%		4.20		4.21		4.29		4.20		4.22
Calcium	%		0.76		0.76		0.76		0.76		0.76
Total Phosphorus	%		0.42		0.43		0.43		0.43		0.43
Avail Phosphorus	%		0.36		0.36		0.36		0.36		0.36
Salt	%		0.34		0.34		0.33		0.33		0.33
Sodium	%		0.18		0.18		0.18		0.18		0.18
Potassium	%		0.74		0.74		0.74		0.73		0.74
Chloride	%		0.30		0.30		0.30		0.30		0.30
Na+K-Cl	mEq/kg		182.19		182.32		182.44		180.49		182.31
Choline	mg/kg		1102.5		102.5		1102.5		1102.5		1102.5
Phytate	%		0.23		0.23		0.22		0.22		0.22
Arginine	%	1.04	1.051	1.04	1.051	1.04	1.051	1.04	1.051	1.04	1.051
Lysine	%	1.12	1.001	1.13	1.001	1.13	1.001	1.13	1.001	1.13	1.001
Methionine	%	0.55	0.531	0.55	0.531	0.55	0.531	0.56	0.531	0.55	0.531
Met + Cys	%	0.87	0.791	0.87	0.791	0.87	0.791	0.87	0.791	0.87	0.791
Tryptophan	%	0.22	0.191	0.21	0.191	0.21	0.191	0.21	0.191	0.21	0.191
Threonine	%	0.77	0.671	0.77	0.671	0.77	0.671	0.77	0.671	0.77	0.671
Isoleucine	%	0.80	0.721	0.79	0.721	0.79	0.721	0.79	0.721	0.79	0.721
Leucine	%	1.65	1.521	1.64	1.511	1.64	1.511	1.64	1.511	1.63	1.501
Valine	%	0.90	0.801	0.90	0.801	0.9	0.801	0.90	0.801	0.90	0.801
Cystine	%	0.31	0.261	0.32	0.261	0.32	0.261	0.32	0.261	0.32	0.261

¹Digestible amino acid value.

Table 17, Table 18 to 19 contain the analyzed nutrient values of the diets. In general, the nutrient contents among treatment groups within the starter, grower and finisher phases were similar.  As expected, the sulfur levels are higher in diets containing the CoverCress grain due to higher sulfur content in the ingredient. Copper levels in the starter and grower diets were over 100 ppm and about 32 ppm even higher in the CCWG-1-CuSO₄ diet as expected. This study followed industry practices whereby the starter and grower diets were formulated to contain approximately 100 ppm with the finisher diet containing less than 20 ppm except for the diet containing CCWG-1-CuSO₄ which contained more copper originating from the copper treated CoverCress grain. Amino acids were similar across diets. Erucic acid (C22:1) was not detectable in the final diets.

Table 17.

Experiment 2 - Analyzed nutrient content of starter diets (d 1–14) containing 0% (Diet 1), 2% CCWG-1 (Diet 2), 4% CCWG-1 (Diet 3), 4% CCWG-2 (Diet 4) and 4.35% CCWG-1-CuSO₄ (Diet 5) (as is basis).

	Diet 1	Diet 2	Diet 3	Diet 4	Diet 5
Nutrient	Broiler Starter
Moisture, %	11.99	12.59	11.77	11.48	12.20
Crude Protein, %	22.80	23.90	23.42	23.09	23.15
Crude Fat, %	4.57	4.88	5.36	4.86	5.16
Linoleic Acid, %	2.16	1.84	1.90	1.73	1.83
Crude Fiber, %	2.40	2.40	2.70	2.50	2.70
Acid Detergent Fiber, %	5.00	4.53	4.46	3.90	3.41
Neutral Detergent Fiber, %	6.34	6.51	6.71	7.53	7.27
Ash, %	5.03	5.15	5.11	4.90	4.89
Calcium, %	0.96	1.01	1.02	0.93	0.89
Total Phosphorus, %	0.68	0.71	0.70	0.64	0.61
Magnesium, %	0.22	0.22	0.23	0.22	0.21
Potassium, %	1.16	1.20	1.18	1.13	1.05
Sulfur, %	0.33	0.0.36	0.38	0.37	0.35
Sodium, %	0.21	0.21	0.21	0.21	0.19
Chloride, %	0.34	0.31	0.34	0.32	0.33
Zinc ppm	106	96	105	106	92
Iron, ppm	200	213	225	192	183
Manganese, ppm	99	104	91	104	125
Copper, ppm	126	137	141	132	203
Arginine, %	1.39	1.48	1.45	1.52	1.48
Lysine, %	1.40	1.46	1.48	1.51	1.47
Methionine, %	0.68	0.72	0.71	0.71	0.71
Met + Cys, %	1.05	1.12	1.12	1.11	1.10
Tryptophan, %	0.24	0.25	0.26	0.25	0.26
Threonine, %	0.90	0.96	0.94	0.96	0.95
Isoleucine, %	0.99	1.03	1.04	1.04	1.04
Leucine, %	1.87	1.96	1.93	1.96	1.94
Valine, %	1.08	1.14	1.14	1.16	1.14
Cysteine, %	0.37	0.40	0.41	0.40	0.39

Table 18.

Experiment 2 - Analyzed nutrient content of grower diets (d 14–28) containing 0% (Diet 6), 2% CCWG-1 (Diet 7), 4% CCWG-1 (Diet 8), 4% CCWG-2 (Diet 9) and 4.35% CCWG-1-CuSO₄ (Diet 10) (as is basis).

	Diet 6	Diet 7	Diet 8	Diet 9	Diet 10
Nutrient	Broiler Grower
Moisture, %	12.22	11.79	11.83	11.57	12.15
Crude Protein, %	21.04	20.84	21.18	20.81	20.93
Crude Fat, %	5.10	5.46	5.55	5.30	5.59
Linoleic Acid, %	2.02	2.11	1.93	1.96	2.09
Crude Fiber, %	2.10	2.30	2.10	2.40	2.40
Acid Detergent Fiber, %	3.16	4.14	3.47	3.89	3.05
Neutral Detergent Fiber, %	6.53	7.16	7.41	7.55	7.58
Ash, %	4.79	4.84	4.96	4.79	5.62
Calcium, %	1.11	1.18	1.01	1.23	0.97
Total Phosphorus, %	0.69	0.67	0.70	0.76	0.65
Magnesium, %	0.20	0.19	0.21	0.20	0.20
Potassium, %	1.03	0.99	1.05	0.98	1.02
Sulfur, %	0.31	0.30	0.34	0.33	0.33
Sodium, %	0.23	0.20	0.23	0.20	0.24
Chloride, %	0.31	0.33	0.34	0.30	0.31
Zinc ppm	108	81	92	98	86
Iron, ppm	201	168	217	202	188
Manganese, ppm	99	87	108	95	89
Copper, ppm	115	94	111	95	190
Arginine, %	1.28	1.33	1.27	1.27	1.26
Lysine, %	1.30	1.34	1.26	1.27	1.28
Methionine, %	0.65	0.62	0.58	0.66	0.59
Met + Cys, %	1.01	1.01	0.91	1.03	0.94
Tryptophan, %	0.22	0.23	0.23	0.20	0.21
Threonine, %	0.83	0.86	0.82	0.87	0.82
Isoleucine, %	0.89	0.92	0.86	0.87	0.88
Leucine, %	1.72	1.76	1.71	1.71	1.71
Valine, %	0.98	1.01	0.97	0.97	0.98
Cysteine, %	0.36	0.36	0.33	0.37	0.35

Table 19.

Experiment 2 - Analyzed nutrient content of finisher diets (d 28 to 42) containing 0% (Diet 11), 2% CCWG-1 (Diet 12), 4% CCWG-1 (Diet 13), 4% CCWG-2 (Diet 14) and 4.35% CCWG-1-CuSO₄ (Diet 15) (as is basis).

	Diet 11	Diet 12	Diet 13	Diet 14	Diet 15
Nutrient	Broiler finisher
Moisture, %	12.14	11.90	11.28	11.81	12.43
Crude Protein, %	18.61	19.24	18.58	19.01	19.07
Crude Fat, %	4.79	5.45	5.27	4.19	5.00
Linoleic Acid, %	2.03	1.95	2.16	1.86	1.99
Crude Fiber, %	2.00	2.10	2.30	2.50	2.00
Acid Detergent Fiber, %	2.61	3.26	3.55	3.37	3.21
Neutral Detergent Fiber, %	7.31	8.51	7.86	7.90	7.47
Ash, %	4.31	4.24	4.37	4.41	4.21
Calcium, %	0.77	0.74	0.84	0.66	0.80
Total Phosphorus, %	0.55	0.55	0.59	0.49	0.56
Magnesium, %	0.20	0.20	0.19	0.19	0.20
Potassium, %	1.03	1.01	0.90	0.90	0.96
Sulfur, %	0.28	0.30	0.30	0.32	0.32
Sodium, %	0.22	0.22	0.25	0.22	0.21
Chloride, %	0.32	0.32	0.31	0.33	0.30
Zinc ppm	95	95	98	88	100
Iron, ppm	187	206	195	208	201
Manganese, ppm	89	95	98	86	97
Copper, ppm	15	17	19	18	63
Arginine, %	1.20	1.17	1.16	1.23	1.10
Lysine, %	1.18	1.18	1.18	1.21	1.13
Methionine, %	0.57	0.57	0.54	0.59	0.55
Met + Cys, %	0.92	0.92	0.89	0.94	0.89
Tryptophan, %	0.20	0.20	0.20	0.20	0.19
Threonine, %	0.80	0.80	0.76	0.81	0.76
Isoleucine, %	0.85	0.83	0.82	0.87	0.80
Leucine, %	1.66	1.65	1.58	1.68	1.59
Valine, %	0.93	0.93	0.91	0.96	0.89
Cysteine, %	0.35	0.34	0.35	0.35	0.34

Feed Intake

Feed consumption of birds is presented in Table 20.  Cumulative 0 to 42 d feed consumption values per pen were statistically different (P < 0.05) from control in the 4% CCWG-1 group only; no significant differences were found in the 2% CCWG-1, 4% CCWG-2 or 4% CCWG-1-CuSO₄ groups. For comparison, the cumulative feed consumption values as a percent of control were 100, 93.3, 97.6 and 96.8% in the 2% CCWG-1, 4% CCWG-1, 4% CCWG-2 and 4% CCWG-1-CuSO₄ groups, respectively. All treatment groups except for the 4% CCWG-1 consumed more than the target of 4.776 kg for male Cobb 500 birds (Cobb-Vantress, 2018). The 4% CCWG-1 fed birds consumed 99% of the target value. Pretreatment with copper sulfate did not significantly impact the performance of CCWG-1; there was also no significant difference between 4% CCWG-1 and CCWG-2.

Table 20.

Experiment 2 - Effect of feeding diets containing a control, 2% CCWG-1, 4% CCWG-1, 4% CCWG-2, and 4.35% CCWG-1-CuSO₄ to broilers for 42 d on feed consumption.

	Treatment
Variable	Control	2% CCWG-1	4% CCWG-1	4% CCWG-2	4.35% CCWG-1-CuSO4	SE	P value
Feed consumed pen-1 (kg)
D 0–14	8.16c	7.79bc	7.10a	7.42ab	7.56b	0.100	<0.001
D 0–21	19.30c	18.84c	16.73a	17.77b	17.73b	0.243	<0.001
D 0–28	36.96c	36.48c	32.36a	34.13b	34.35b	0.478	<0.001
D 0–35	58.48c	58.18bc	52.83a	55.62abc	55.47ab	0.863	<0.001
D 0–42	82.08b	82.43b	76.60a	80.11ab	79.46ab	1.209	0.003
D 0–7	2.86b	2.71ab	2.60a	2.60a	2.69a	0.043	0.001
D 14–28	28.80c	28.69c	25.26a	26.72ab	26.78b	0.409	<0.001
D 28–42	45.13	45.95	44.24	45.98	45.12	0.867	0.452
Feed consumed bird-1 (kg)
D 0–14	0.49c	0.46b	0.42a	0.44ab	0.45b	0.006	<0.001
D 0–21	1.19c	1.11b	1.01a	1.05ab	1.05ab	0.016	<0.001
D 0–28	2.28c	2.15b	1.98a	2.02a	2.04a	0.024	<0.001
D 0–35	3.63c	3.47b	3.23a	3.32a	3.34a	0.036	<0.001
D 0–42	5.22c	5.00bc	4.72a	4.78ab	4.91ab	0.070	<0.001
D 0–7	0.17b	0.16ab	0.15a	0.15a	0.16a	0.003	0.001
D 14–28	1.77c	1.69b	1.54a	1.58a	1.59a	0.018	<0.001
D 28–42	2.87	2.79	2.73	2.74	2.78	0.043	0.069

^a,b,cMeans with different superscripts are different at P < 0.05.

There was no difference (P < 0.05) in feed consumption between the pens that were fed the control and the 2% CCWG-1 at any timepoint. When the inclusion level of CCWG-1 was increased to 4%, whether as the grain (CCWG-1) or treated with copper sulfate solution (CCWG-1-CuSO₄), a decrease in feed intake was reported in the first few weeks of treatment. The reduction in feed intake was observed with the gene-edited version (CCWG-2) as well. During the last 2 wk of the study no differences in feed consumption per pen were noted among any of the treatments. In other words, the effect on feed intake diminished as the birds acclimated to the diet such that no differences in feed intake were detected among all treatment groups for the last 2 wk (d 28–42) of the study. The initial decrease in feed intake appears to be a palatability effect.

Body Weight and Body Weight Gain

BW and BW gain results are presented in Table 21. BW at d 42 and cumulative 0 to 42 d BW gain values per pen were not statistically different from control for any treatment group.

Table 21.

Experiment 2 - Effect of feeding diets containing a control, 2% CCWG-1, 4% CCWG-1, 4% CCWG-2, and 4.35% CCWG-1-CuSO₄ to broiler for 42 d on BW and BW gain.

	Treatment
Variable	Control	2% CCWG-1	4% CCWG-1	4% CCWG-2	4.35% CCWG-1-CuSO4	SE	P value
BW pen-1 (kg)
D 0	0.757	0.759	0.761	0.758	0.766	0.0037	0.463
D 14	7.325d	7.080cd	6.273a	6.695b	6.815bc	0.0927	<0.001
D 28	27.265c	27.752c	24.520a	26.085b	26.087b	0.3836	<0.001
D 42	51.982	53.185	50.297	52.862	51.075	1.0788	0.240
BW bird-1 (kg)
D 0	0.040	0.040	0.040	0.040	0.040	0.0002	0.463
D 14	0.437d	0.416cd	0.374a	0.394b	0.404bc	0.0049	<0.001
D 28	1.678b	1.632b	1.498a	1.546a	1.546a	0.0163	<0.001
D 42	3.302c	3.222bc	3.099a	3.156ab	3.145ab	0.0388	<0.001
Body gain pen-1 (kg)
D 0–14	6.57d	6.32cd	5.51a	5.94b	6.05bc	0.092	<0.001
D 0–28	26.51cd	26.99d	23.76a	25.33b	25.32bc	0.383	<0.001
D 0–42	51.23	52.43	49.54	52.10	50.31	1.078	0.238
D 14–28	19.94bc	20.67c	18.25a	19.39a	19.27ab	0.335	<0.001
D 28–42	24.72	25.43	25.78	26.78	24.99	0.922	0.491
Body gain bird-1 (kg)
D 0–14	0.40d	0.38c	0.33a	0.35b	0.36bc	0.005	<0.001
D 0–28	1.64b	1.59b	1.46a	1.51a	1.51a	0.016	<0.001
D 0–42	3.26c	3.18bc	3.06a	3.12ab	3.10ab	0.039	<0.001
D 14–28	1.24b	1.22b	1.12a	1.15a	1.14a	0.014	<0.001
D 28–42	1.62	1.59	1.60	1.61	1.60	0.033	0.881

^a,b,c,dMeans with different superscripts are different at P < 0.05.

Body weights of birds fed the control and 2% CCWG-1 diets were not different (P >0.05) at any time point when expressed on a per pen or per bird basis. At d 14 and 28, birds fed the 4% CCWG-1, 4% CCWG-2 and 4.35% CCWG-1-CuSO₄ diets weighed less (P < 0.001) than the controls when expressed on a pen basis. However, pen weights were not different (P = 0.240) among treatment groups at d 42. CCWG-2 body weights were slightly higher than CCWG-1 on a pen basis on d 14 and 28, but not significantly different at d 42.

BW gains were not different (P > 0.05) between the birds fed the control and 2% CCWG-1 diets at any timepoint. For d 28 to 42 no differences in BW gain/pen (P = 0.491) or BW gain/bird (P = 0.881) among treatment groups were observed. During this period about 50% of the bird weight gain takes place. When expressed as BW gain/bird for D 0 to 42, birds fed 4% CCWG-1, 4% CCWG-2 and 4.35% CCWG-1-CuSO₄ were not different among each other or birds fed 2% CCWG-1 and gained 94, 96, and 95% of the birds fed the controls. All treatment groups either met or exceeded the target body weights for male Cobb 500 birds (Cobb-Vantress, 2018).

Feed Conversion (Feed:Gain) and Adjusted Feed Conversion

Feed conversion, (Feed:Gain) and adjusted Feed:Gain (adjusted for dead and culled birds) results are presented in Table 22.  Feed:Gain and adjusted Feed:Gain for all CoverCress treatment groups were improved (P < 0.05) over the controls for the periods of d 0 to 28 and d 14 to 28. For the d 0 to 42 period no differences (P = 0.067) were observed for Feed:Gain; however, birds fed the 4% CCWG-1 and 4% CCWG-2 diets had a 3% improvement (P < 0.001) in adjusted Feed:Gain over the controls. Adjusted Feed:Gain met the target of 1.57 or less for Cobb 500 males (Cobb-Vantress, 2018) for all treatment groups.

Table 22.

Experiment 2 - Effect of feeding diets containing a control, 2% CCWG-1, 4% CCWG-1, 4% CCWG-2, and 4.35% CCWG-1-CuSO₄ to broilers for 42 d on feed conversion (Feed:Gain).

	Treatment
Variable	Control	2% CCWG-1	4% CCWG-1	4% CCWG-2	4.35% CCWG-1-CuSO4	SE	P value
Feed conversion (Feed:Gain)
D 0–14	1.24a	1.23a	1.29b	1.25a	1.25a	0.010	0.001
D 0–28	1.39b	1.35a	1.36a	1.35a	1.36a	0.007	<0.001
D 0–42	1.60	1.58	1.55	1.54	1.58	0.019	0.067
14–28	1.45b	1.39a	1.38a	1.38a	1.39a	0.009	<0.001
D 28–42	1.84	1.83	1.72	1.72	1.82	0.050	0.172
Adjusted Feed conversion (Feed:Gain)
D 0–14	1.19a	1.20ab	1.23b	1.21ab	1.21ab	0.008	0.026
D 0–28	1.37b	1.34a	1.34a	1.33a	1.35a	0.006	0.001
D 0–42	1.57b	1.54ab	1.52a	1.52a	1.54ab	0.009	0.001
D 14–28	1.43b	1.39a	1.37a	1.37a	1.39a	0.007	<0.001
D 28–42	1.77b	1.75ab	1.69a	1.71ab	1.74ab	0.022	0.021

^a,bMeans with different superscripts are different at P < 0.05.

Mortality

A summary of the bird mortality and cull data are presented in Table 23. No differences were seen in mortality (P = 0.663) or mortality plus culls (P = 0.977) among any of the treatment groups.

Table 23.

Experiment 2 - Effect of feeding diets containing a control, 2% CCWG-1, 4% CCWG-1, 4% CCWG-2, and 4.35% CCWG-1-CuSO₄ to broilers for 42 d on mortality and mortality and culls.

	Treatment
Variable	Control	2% CCWG-1	4% CCWG-1	4% CCWG-2	4.35% CCWG-1-CuSO4	P value
Mortality (%)
D 0–42	4.6	9.2	7.2	7.2	7.2	0.663
Mortality + Culls (%)
D 0–42	8.5	9.2	7.8	7.2	8.5	0.977

Organ Weights, Serum Thyroid, and Serum Blood Chemistry

Liver, kidney, and thyroid weights as well as serum thyroid hormone levels are presented in Table 24. No differences at P < 0.05 in liver or thyroid weights were observed when expressed as % of live weight or actual weight. Kidneys (% of BW) of the 4% CCWG-1 fed birds weighed 19.9% more than the controls but was not significantly different from the controls when expressed as actual weight. No statistical differences in kidney weight (actual or % of BW) between 4% CCWG-1 and CCWG-2 groups were observed.

Table 24.

Experiment 2 - Effect of feeding diets containing a control, 2% CCWG-1, 4% CCWG-1, 4% CCWG-2, and 4.35% CCWG-1-CuSO₄ to broilers for 42 d on organ weights and thyroid hormones.

	Treatment
Variable	Control	2% CCWG-1	4% CCWG-1	4% CCWG-2	4.35% CCWG-1-CuSO4	SE	P value
Organ weights (% of BW)
Kidney	0.433a	0.495ab	0.519b	0.476ab	0.469ab	0.0172	0.008
Liver	2.036	2.081	2.003	2.066	2.092	0.0710	0.863
Thyroid	0.013	0.015	0.012	0.013	0.012	0.0012	0.600
Organ weights (g)
Kidney	14.55ab	16.51b	16.51b	15.33ab	13.90a	0.636	0.014
Liver	68.58	69.47	63.90	66.46	61.94	2.836	0.183
Thyroid	0.42	0.49	0.39	0.40	0.37	0.036	0.213
Thyroid hormones
Free Thyroxine (pmol/L)	9.62	12.5	14.00	12.75	11.75	1.074	0.065
Total Thyroxine (mmol/L)	9.62	11.75	11.50	12.00	10.50	0.769	0.177
Total Triiodothyronine (mmol/L)	1.04	1.12	0.94	1.10	0.98	0.070	0.228

^a,bMeans with different superscripts are different at P < 0.05.

There were no differences at P < 0.05 in serum T4 (Total Thyroxine), T3 (Total Triiodothyronine) and free thyroxine concentrations. Though free thyroxine approached significance, one would expect a lower free thyroxine level with a glucosinolate effect; however, higher values were observed. Therefore, these numerical differences are not considered to be treatment related.

There were no differences at P < 0.05 between the control and treatment groups in serum chemistry values except for inconsistent differences in iron and alkaline phosphatase (ALP) (Table 25). Iron levels were statistically significantly higher in the 4% CCWG-1 group only and values were not statistically significantly higher in the 4% CCWG-1-CuSO₄ or CCWG-2 groups despite similar dietary glucosinolate levels. Compositional analyses of the iron concentrations of the finished diets showed no meaningful differences. Given the lack of reproducibility across the 4% treatment groups this difference in serum iron was not considered CCWG related. ALP was statistically significantly higher compared to control in the 4% CCWG-2 group only. All ALP values for all treatments were within reported literature ranges for broiler chicken. Therefore, these differences were not considered to be CCWG-1 or CCWG-2 treatment related.

Table 25.

Experiment 2 - Effect of feeding diets containing a control, 2% CCWG-1, 4% CCWG-1, 4% CCWG-2, and 4.35% CCWG-1-CuSO₄ to broilers for 42 d on serum blood chemistry.

	Control	2% CCWG-1	4% CCWG-1	4% CCWG-2	4.35% CCWG-1-CuSO4	SEM	Pr > F	Reference values
Analyte	TRT 1	TRT 2	TRT 3	TRT 4	TRT 5
Urea Nitrogen (mg/dL)	2.1	2.1	2.3	2.3	2.0	0.14	0.623
Creatinine (mg/dL)	0.2	0.2	0.2	0.2	0.2	0.02	0.800
Sodium (mmol/L)	161.1	163.9	162.9	163.6	161.8	1.40	0.590	138–1662
Potassium (mmol/L)	5.4	5.6	5.5	5.3	5.5	0.16	0.431	6.07–12.092
Chloride (mmol/L)	117.0	119.3	119.3	120.5	118.4	1.26	0.400	1083
TCO2 (mmol/L)	22.8	23.0	22.2	22.1	21.8	0.93	0.823	24.93
Na/K ratio	30.3	29.4	29.7	31.3	29.8	0.90	0.468
Anion Gap (mmol/L)	26.7	27.3	27.0	26.5	27.0	0.96	0.964	16.23
Osmolarity (mmol/L)	337.1	341.7	340.7	342.3	338.0	2.91	0.653
Calcium (mg/dL)	7.5	7.3	7.2	8.3	7.7	0.34	0.182	4.70–16.251
Phosphorus (mg/dL)	5.7	6.0	5.8	6.2	6.0	0.19	0.467	3.59–11.401
Magnesium (mg/dL)	2.1	2.1	2.0	2.2	2.1	0.05	0.209	1.8–2.82
Iron (µg/dL)	104.3b	129.0ab	139.7a	113.3ab	123.1ab	6.59	0.008
Total Protein (g/dL)	3.1	3.5	3.4	3.3	3.3	0.13	0.177	2.58–5.221
Albumin (g/dL)	1.3	1.5	1.4	1.3	1.3	0.06	0.134	1.17–2.741
Globulin Calculated (g/dL)	1.8	2.0	1.9	2.0	2.0	0.12	0.612	1.99–2.942
Glucose (mg/dL)	254.7	252.8	254.3	256.6	246.3	5.47	0.560	117–2292 2353
Amylase (U/L)	474.4	512.3	482.4	479.1	504.6	44.76	0.964
Total Bilirubin (mg/dL)	0.3	0.2	0.3	0.2	0.2	0.02	0.106
Direct Bilirubin (mg/dL)	0.0	0.0	0.0	0.0	0.0	0.0
Indirect Bilirubin (mg/dL)	0.3	0.2	0.3	0.2	0.2	0.02	0.106
ALP (U/L)	3165.1b	3819.8b	4095.9ab	6684.0a	3691.4b	683.44	0.008	568–88311
ALT (U/L)	3.1	3.5	3.3	3.5	2.9	0.31	0.561
AST (U/L)	516.5	507.8	466.5	445.1	423.0	58.92	0.741	70–2201 3933
CK (U/L)	48333.	54065.	46387.	46246.	37169.	10246.7	0.817	43593
Cholesterol (mg/dL)	125.4	135.1	124.6	120.3	121.0	4.76	0.187	87.0–1921
Uric Acid (mg/dL)	6.8	6.4	6.3	5.5	5.8	0.43	0.255	4.41–11.152
Hemolysis Chem4
Icterus Chem	Normal	Normal	Normal	Normal	Normal
Lipemia Chem	Normal	Normal	Normal	Normal	Normal

¹Meluzzi, et al. (1992). Determination of blood constituents reference values in broilers. Poultry Science 71:337-345.

²95% confidence limits. Ross et al. (1976). Determination of haematology and blood chemistry values in six-week old btoiler hybrids. Avian Pathology 5:273-281.

³Livingston et al. (2020). Effect of broiler genetics, age, and gender on performance and blood chemistry. Heliyon 6:e04400.

⁴Hemolysis: TRT 1 to 5 slight, 2 normal, 1 moderate; TRT 2 to 4 slight, 2 normal, 2 moderate; TRT 3 to 4 slight, 0 normal, 4 moderate; TRT 4 to 2 slight, 5 normal, 1 moderate; TRT 5 to 3 slight, 4 normal, 1 moderate.

^a,bMeans with differing superscripts are statistically different at P < 0.05.

Macropathology and Histopathology

No abnormalities were noted during gross examination during necropsy. Histopathological analysis of kidneys and thyroid was completed for all three treatment groups. Liver samples were lost in transit to the laboratory. In all kidney and thyroid tissues, no significant changes of active degeneration, necrosis, inflammation, atrophy, hypertrophy, or hyperplasia were apparent. The appearance of small lymphocytic aggregates were observed in various tissues (including kidney and thyroid) are common and considered incidental findings, not indicative of any disease process, and not treatment related. In many instances, the thyroid gland is in close association with the thymus and some of these lymphoid aggregates may be normal thymus tissue within the body of the thyroid.

Overall, collected samples were considered within the normal range for all treatment groups with no treatment related findings.

Discussion of Results Across the Two Studies

Two studies were conducted feeding CoverCress whole grain up to 6% inclusion in broiler diets. The studies contained treatments with CCWG-1 or CCWG-2, two versions of CoverCress derived from mutations to the fatty acid synthesis pathway and seed coat fiber pathway. In addition, pretreatment with copper sulfate was utilized to understand its role as a palatability agent.

Previously reported results established the compositional and nutritional similarities between CCWG and canola, and five lot analyses of either CCWG-1-CuSO₄ or CCWG-2-CuSO₄ showed adequate product consistency (Hartnell et al., 2023). Based on those analyses and an extensive literature of Brassica composition, safety and benefits, the only nutrient, antinutrient or contaminant detected in CCWG of possible toxicologic concern is sinigrin, the only glucosinolate detected in pennycress. Sinigrin is found in many generally recognized as safe human food ingredients such cruciferous vegetables, brown mustards and wasabi.

Two studies previously by Alhotan et al. (2017) fed pennycress meal provided by CoverCress Inc and demonstrated it could be fed to broilers up to 8.5% in the diet, with a glucosinolate level of 5 to 5.8 µmol g^-1 diet. In the current studies, CoverCress grain was included in broiler diets at levels between 2 and 6%, at a maximum glucosinolate level of 4.9 µmol g^-1 diet.

Overall, a small decrease in feed intake and body weight with no effect on feed conversion was initially observed with the feeding of CoverCress grain to broilers. As the studies progressed, the negative effect on feed intake diminished with no significant differences observed by the end of the 6-wk studies. This effect on feed intake was not observed with 2% CCWG-1. In all studies birds fed the CoverCress grain met or came very close to meeting the target body weights, feed consumption and Feed:Gain standards for male Cobb 500 birds (Cobb-Vantress, 2018). The pattern of reduced feed palatability and BW gains without changes in Feed:Gain ratios at similar finished diet total glucosinolate levels is common in extensive previous studies done with canola whole grain, canola meal or oilseed rape meal. This is illustrated well by studies with canola seed conducted by Summers et al. (1982) and Yadav et al., 2022. In the first case, there were clear changes in BW at the lower dose of 3.15 µmol g^-1 diet, which were also present at the higher dose of 6.3 µmol g^-1, but without a negative impact on Feed:Gain on the overall test period of 7 wk. In Yadav et al., 2022, the lower dose of 2.95 µmol g^-1 diet was not different from controls while the higher dose of 9.66 µmol g^-1 diet resulted in reduced BW and food intake compared to the control but without a change in Feed:Gain.

In the current studies, there were no findings of toxicity in any of the treatment groups, including on organ weights, macro or micro histopathology, serum thyroid hormones or clinical chemistry parameters. Based on these findings, and sinigrin concentrations in finished broiler diets, the No Observed Adverse Effect Level for CoverCress in broiler chickens was determined to be greater than or equal to 6% inclusion, achieving a NOAEL of greater than or equal to 4.9 µmol sinigrin g^-1 finished broiler diet, which is similar to the NOEL value reported for wild-type field pennycress hexane-extracted meal (and low erucic acid) by Alhotan et al. (2017) of 5.0 to 5.8 µmol sinigrin g^-1 finished broiler diet. The intended use of CCWG in broiler diet at levels of 2 to 4% is below the 6% inclusion NOAEL established herein.

CONCLUSION

From the results of these studies it can be concluded that: bird performance was similar whether CoverCress grain at 4% inclusion was pretreated with copper sulfate or not; there was no difference between the mutant and gene-edited varieties of CoverCress (CCWG-1 and CCWG-2) fed at 4% inclusion; inclusion of 4% CoverCress grain may lead to transitory reduction in feed intake and BW with no negative effect on feed conversion or health of the bird during the first four weeks; birds appear to acclimate to the diet overtime; performance was not different from the control when CoverCress grain was included at 2% of the diet; and no negative effects on feed conversion or health were observed when CoverCress grain was included up to 6% of the diet. CoverCress grain (low fiber, low erucic acid pennycress) can be safely fed to broilers when the dietary glucosinolates levels do not exceed 4.9 µmoles g^-1.

ACKNOWLEDGMENTS

The authors acknowledge CoverCress Inc. for providing the test materials and funding for these studies and Jerry Hjelle (Hjelle Advisors LLC, St. Louis, MO) for his toxicology expertise. The authors thank EPL Bio Services (Niantic, IL), Dairyland Laboratories Inc. (Arcadia, WI), University of Missouri Agriculture Experiment Station Chemical Laboratories (Columbia, MO), Texas A&M Veterinary Medical Diagnostic Laboratory (College Station, TX) and Michigan State University Veterinary Diagnostic Laboratory (Lansing, MI) for their analytical services.

DISCLOSURES

Hartnell and Lemke are CoverCress Inc. paid consultants. Liu and Aulbach are employees of CoverCress Inc. CoverCress Inc. paid for the statistical services of Nemeth and the studies conducted by Colorado Quality Research Inc. where Moore and Matthews are employees. Brister has no conflict of interest.