WO2020212600A1 - Meal fraction of brassica carinata oilseed - Google Patents

Abstract

A meal fraction of Brassica carinata oilseed is provided. The meal fraction has a higher protein and lower fibre content than meal fractions isolated from oilseed of other Brassica species and related oilseeds such as camelina. The meal fraction of Brassica carinata oilseed contains from about 0 to about 45 micromoles per gram of glucosinolate, of which 70% or more is sinigrin (2- propenyl glucosinolate). Also provided is a feed ration comprising the meal fraction of Brassica carinata oilseed. Such feed ration is suitable for feeding ruminant livestock, monogastric livestock, poultry livestock, camelid livestock, or farmed fish.

Description

Meal Fraction of Brassica carinata oilseed

FIELD

The present invention is in the field of meal fractions from oilseeds.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of and priority to United States Provisional

Application Serial Number 62/835,754 filed on April 18, 2019, which is incorporated herein by reference in its entirety.

BACKGROUND

[0002] Brassica carinata is a member of the Brassicaceae (formerly Cruciferae) family, commonly known as the mustard family. The genus Brassica is a member of the tribe Brassiceae in the mustard family. In addition to B. carinata, the Brassica genus includes several economically important oilseed crop species: B.juncea (L). Czern. (brown mustard), B. napus L. (rape, Argentine canola), B. nigra (L.) W.D.J. Koch (black mustard), and B. rapa L. (field mustard, Polish canola). The genus Brassica also includes B. oleracea L. food crops, including cabbage, broccoli, cauliflower, Brussels sprouts, kohlrabi and kale. The six Brassica species are closely related genetically, as described in the Triangle of U (Nagaharu, 1935, Japan J. Bot. 7:389-452, reviewed in Branca and Cartea, 2011 , Brassica Wild Crop Relatives: Genomic and Breeding Resources, Oilseeds. C. Kole. Berlin Heidelberg Spring er-Verlag 17-36). Brassica carinata is an amphidiploid (BBCC, 2n=34) thought to be derived from interspecific

hybridization of the diploid species B. nigra L. (BB, 2n=16) and B. oleracea L. (CC, 2n=18; Prakash et al, 2012 In: Plant Breeding Reviews . Janick J (ed). Vol. 35.19-84).

[0003] In terms of economic value, Brassica carinata’s greatest potential as a crop resides in its prolific yields of oil and protein rich seed. In southern Europe, carinata seed oil has been investigated for its potential as a feedstock for biofuel and as a bio-industrial feedstock with applications in production of lubricants, paints, cosmetics, plastics (Cardone, et al., 2002.

Environ. Sci. Technol. 36:4656-4662.; Cardone, et ah, 2003 Biomass and Bioenergy 25(6):623- 636; Bouaid, et al, 2005, Catalysis Today 106(1-4):193-196; Gasol, et ah, 2007, Biomass and Bioenergy 31 :543-555; Gasol, et al, 2009, Biomass and Bioenergy 33: 119-129). In North America, carinata has been shown to be a suitable renewable feedstock crop for biofuel production (Gesch, et al, 2015, Industrial Crops and Products 75b: 2-7; Seepaul, etal, 2015, Carinata, the Jet Fuel Cover Crop: 2016 Production Recommendations for the Southeastern United States. Agronomy Department, IF AS Extension and U. o. Florida, University of Florida. SS-AGR-384: 1-8), and oil extracted from B. carinata seed has been used to produce green bio diesel and bio-jet fuel (Drenth, et al, 2015, Fuel 153: 19-30). In October 2012, experimental aviation flights by the National Research Council of Canada using the world’s first 100% bio-jet fuel were successful (“ReadiJet 100% biofuels flight - one of 2012's 25 most important scientific events”, Popular Science Magazine, 2012(12). Carinata production can be adapted to diverse regions, climate zones, and soil types, and the low carbon intensity of carinata production and processing into end products makes carinata grain, oil, meal and the downstream products of carinata oil and meal highly sustainable from an overall greenhouse gas reduction standpoint (WO2019/046498A1).

[0004] While the oils produced by Brassica carinata and other novel oilseed crops are of great value largely because of their utility as an industrial feedstock, the meal produced as part of the oil extraction process is also a potentially valuable co-product.

[0005] To meet an ever-increasing demand for protein in human diets, global meat, poultry, egg, dairy and fish consumption is rising and consequentially, to ensure supply of these commodities, an increasing amount is farmed. This in turn, necessitates an increased reliance on animal feeds to provide the essential nutrients for farmed livestock production. Although precise nutritional requirements for farmed meat, dairy, poultry, egg and fish production may differ somewhat, they all require protein as a key component in their diets.

[0006] Much of the protein in diets of farmed animals is sourced from plant sources, such as oilseed and cereal grains. Currently, the meal fraction of oilseeds such as canola, double low rapeseed, cottonseed, sunflower seed and soybean are used as additives in animal feed rations to provide sources of protein and metabolic energy. Other plant derived feed constituents may include whole grains or meals of cereals such as wheat, barley, corn as well as by-products of the distilling industry such as dried distillers’ grains. In the case of oil seed derived meals, the meal fractions may represent by-products of processes to extract a primary commodity such as edible oil (i.e., canola), industrial feedstock oil (i.e., brassica carinata), fiber (in the case of cotton), or the meal fraction itself may represent the primary product (i.e., in the case of soybeans).

[0007] The selection of a meal as an additive in an animal feed application may be dictated by factors such as local availability, relative cost, meal quality and suitability (in terms of nutrient content, freedom from pathogens or anti-nutritional factors), as well as regulatory approval for use at recommended inclusion rates in specific animal feed applications.

[0008] While oilseed meal may be a rich source of crude protein, factors such as protein composition and solubility may influence how much of that protein may be metabolically available to meet nutritional requirements in feed applications. This is particularly evident in ruminants where, depending on their physical and chemical properties, crude proteins may either be broken down in the rumen (rumen degraded protein or RDP) or may bypass the rumen (rumen undegraded protein or RUP) to be metabolized predominantly in the small intestine, where it can be absorbed and utilized by the animal. The RDP digested in the rumen by contrast is incorporated into microorganisms that colonize the rumen, although some of these can be flushed out of the rumen and their protein can then supplement the RUP digested in the small intestine (reviewed in Andrade-Montemayor et al., 2009, R. Bras. Zootec., 38, .277-291).

[0009] As well, other components of an oilseed meal can influence a meals overall nutritional benefit as a feed additive. For example, meals may contain insoluble fibrous forms of carbohydrate such as lignins, cellulose, hemicellulose and tannins that cannot be broken down readily in the digestive tract of monogastric livestock types and can contribute to the sensation of satiety and a reduction in over all feed intake and nutrient uptake. In poultry, insoluble fiber is thought to decrease the transit time of nutrient material in the gut, resulting in reduced time for digestion and absorption of nutrients (reviewed in Khajali and Slominski 2012, Poult. Sci.

91 ,2564-2575). By contrast, ruminant livestock, by virtue of their unique digestive system, can digest cellulose via the process of ruminal fermentation (Castillo-Gonzalez et al.,2014, Arch Med Vet 46, 349-361).

[0010] Meals may also contain antinutritional compounds which can adversely affect feed nutrition and/or health, depending on the type of feed application. For example, the use of mustard meal as a feed additive has been limited due to its the relatively high content of anti- nutritional compounds, chiefly glucosinolates.

[0011] Glucosinolates constitute a large family of over 100 related molecules with a common sulfur containing core structure and with side chains of varying size and chemistry (Fahey et al, 2001, Phytochemistry 56: 5-51, Halkier and Gershenzon, 2006, Annual Review of Plant Biology 57(1): 303-333). Glucosinolates are found in many species of plants, particularly those within the order Brassicales but also among plants of the of the Putranjivaceae family. The predominant glucosinolate species in Brassica carinata seed is sinigrin (2- propenylglucosinolate, also known as allyl glucosinolate) comprising more than 90% of the total glucosinolate content (Xin, et al., 2014, J. Agric. Food Chem. 62(32): 7977-7988). This is quite distinct from other commercial Brassica oilseeds such as canola type Brassica napus which, although lower in overall seed glucosinolate levels ( 5-12 mihoΐ/g) than Brassica carinata, has a very different chemical profile, with progoiterin (2-(R)-2-Hydroxy-3-butenylglucosinolate), gluconapin (3 -Butenylglucosinolate) and 4 hydroxyglucobrassicin (4-hydroxyindole methylglucosinolate) being the predominant species and having little or no sinigrin (Xin, et al., 2014, J. Agric. Food Chem. 62(32): 7977-7988).

[0012] Glucosinolates can accumulate to high concentrations in the seed of Brassica oilseeds (Bellostas et al, 2004, Agroindustria 3(3): 5-10.). These compounds and their metabolites can impact the taste of the meal, reducing its palatability and, in some cases (depending on the type of glucosinolate and glucosinolate metabolites present), may impact the animal’s health. For example, hydrolysis products of beta hydroxyalkenyl glucosinolates (such as progoiterin found seeds of Brassica napus and Brassica rapa) have been shown to possess goitrogenic activity in animal models (reviewed in Fahey et al, 2001, Phytochemistry 56: 5-51). This is particularly an issue in monogastric animals such as swine, but poultry and cattle can be susceptible to varying degrees.

[0013] In the presence of myrosinase enzyme and water, glucosinolates breakdown to form active compounds. A comprehensive European study on glucosinolates in animal nutrition (Alexander et al, 2008 EFSA J. 590, 1-76) determined that the main breakdown product formed from hydroxybutenyl glucosinolate (progoiterin), one of the main glucosinolates in canola and rapeseed ( Brassica napus and Brassica rapa), is 5 -vinyl-2 -oxazolidinethione (OZT), an important goitrogenic compound (Bell et al., 1971 Can. J. Anim. Sci. 51, 259-269) implicated in cases of endemic goiter in Finland (Fenwick and Heaney, 1983, Food Chem.l 1, 249-271). Conversely, the main breakdown product of allyl glucosinolate (sinigrin) is allyl isothiocyanate (AITC), which is responsible for the“hot” taste of mustard. Both sinigrin and AITC have been shown to have therapeutic capabilities (Tanaka et al., 1992, Jpn. J. Cancer Res.83, 835-42; Fahey et al., 2001, Phytochem. 56, 5-51). As pointed out in the same European study cited above, while much work has focused on the adverse effects of the glucosinolates and glucosinolate breakdown products of rapeseed meal, there has not as yet been similar systematic study of the meals of other Brassica oilseeds with glucosinolate profiles that are chemically distinct from those of rapeseed meal, such as Brassica carinata meal.

[0014] With the increased human demand for animal derived protein there is a concomitant demand for on animal feeds and animal feed ingredients and additives. In particular, the identification of a novel and renewable source of high protein, low fiber, low glucosinolate content oilseed meal would be particularly beneficial in this regard.

[0015] It is the object of the present invention to provide a meal fraction of Brassica carinata oilseed having a high protein content, a low fibre content, and a low glucosinolate content, suitable for inclusion in feed rations for ruminant livestock, monogastric livestock, poultry livestock, camelid livestock, and fish.

SUMMARY OF INVENTION

[0016] In one aspect, the present invention provides a meal fraction of Brassica carinata oilseed comprising as a percentage of dry matter, from about 35% to about 55% crude protein, from about 15% to about 30% neutral detergent fibre, from about 8% to about 15% acid detergent fibre, from about 1% to about 8% lignin, and from 0 to about 45 pmol glucosinolate per gram of meal. In one embodiment, the meal fraction of Brassica carinata oilseed further comprises, as a percentage of dry matter, from about 1% to about 5% oil and from about 6% to about 10% ash. In other embodiments, the meal fraction of Brassica carinata oilseed further comprises, as a percentage of dry matter, from about 0% to about 1 % erucic acid. [0017] In another aspect, the present invention provides a meal fraction of Brassica carinata oilseed comprising as a percentage of dry matter, from about 42% to about 50% crude protein, from about 20% to about 26% neutral detergent fibre, from about 8% to about 12% acid detergent fibre, from about 1% to about 5% lignin, and from 0 to about 30 pmol glucosinolate per gram of meal.

[0018] In some embodiments, the meal fraction of Brassica carinata oilseed further comprises, as a percentage of dry matter, from about 1% to about 5% oil and from about 6% to about 10% ash. In other embodiments, the meal fraction of Brassica carinata oilseed further comprises, as a percentage of dry matter, from about 0% to about 1 % erucic acid. In other embodiments, the meal fraction of Brassica carinata oilseed further comprises, as a percentage of dry matter, from about 0.1% to about 3% erucic acid.

[0019] In other embodiments, the meal fraction of Brassica carinata oilseed further comprises, as a percentage of dry matter, from about 0.1% to about 0.5% oleic acid. In other embodiments, the meal fraction of Brassica carinata oilseed further comprises, as a percentage of dry matter, from about 0.1% to about 1% linoleic acid. In other embodiments, the meal fraction of Brassica carinata oilseed further comprises, as a percentage of dry matter, from about 0.1% to about 1% linolenic acid. In other embodiments, the meal fraction of Brassica carinata oilseed further comprises, as a percentage of dry matter, from about 0.1% to about 0.5% eicosenoic acid.

[0020] In some embodiments, the meal fraction of Brassica carinata oilseed as described herein is produced from seeds of a Brassica carinata variety comprising at least 18% protein, on a dry weight basis. In other embodiments, the meal fraction of Brassica carinata oilseed as described herein is produced from seeds of a Brassica carinata variety comprising at least 20% protein, on a dry weight basis.

[0021] In other embodiments, the meal fraction of Brassica carinata oilseed as described herein is produced from seeds of a Brassica carinata variety that produces yellow or dark yellow seed. In other embodiments, the meal fraction of Brassica carinata oilseed is produced from seeds of a Brassica carinata variety that produces yellow or dark yellow seed comprising from about 4.5% to 7.5% acid detergent fibre and from about 8% to about 13% neutral detergent fibre.

[0022] In another aspect, the present invention relates to a feed ration comprising the meal fraction of Brassica carinata oilseed as described herein. In some embodiments, from about 5% to about 25% of dry matter in the feed ration is the meal fraction of Brassica carinata oilseed.

In other embodiments, the feed ration comprises up to 10 pmol glucosinolate per gram of feed.

In other embodiments, from about 10% to 100% of the crude protein in the feed ration is provided by the meal fraction of Brassica carinata oilseed.

[0023] In some embodiments, the feed ration comprising a meal fraction of Brassica carinata oilseed is for feeding ruminant livestock, monogastric livestock, poultry livestock, camelid livestock, or farmed fish. In some embodiments, the ruminant livestock is cattle, sheep, goats, bison, or buffalo. In other embodiments, the monogastric livestock is swine. In other embodiments, the poultry livestock is chicken, turkey, geese, or duck. In other embodiments, the farmed fish are salmonids, carp, tilapia, and catfish. In other embodiments, the camelid livestock is alpaca, camel, or llama.

[0024] The invention provides for, without limitation, the following numbered embodiments:

1. A meal fraction of Brassica carinata oilseed comprising, on a percentage of dry matter,

(a) from about 35% to about 55% protein;

(b) from about 15% to about 30% neutral detergent fibre;

(c) from about 8% to about 15% acid detergent fibre;

(d) from about 1% to about 8% lignin; and

(e) from 0 to about 45 mhioΐ total glucosinolate per gram of meal

2. A meal fraction of Brassica carinata oilseed comprising, on a percentage of dry matter,

(a) from about 42% to about 50% protein;

(b) from about 20% to about 26% neutral detergent fibre;

(c) from about 8% to about 12% acid detergent fibre;

(d) from about 1% to about 5% lignin; and

(e) from 0 to about 30 mhioΐ total glucosinolate per gram of meal.

3. The meal fraction of Brassica carinata oilseed of embodiment 1 or 2, further comprising, on a percentage of dry matter:

(a) from about 1% to about 5% oil; and

(b) from about 6% to about 10% ash.

4. The meal fraction of Brassica carinata oilseed of any one of embodiments 1 to 3, further comprising, on a percentage of dry matter, from about 0.1% to about 3% erucic acid. The meal fraction of Brassica carinata oilseed of any one of embodiments 1 to 3, further comprising, on a percentage of dry matter, from about 0.1% to about 0.5% oleic acid. The meal fraction of Brassica carinata oilseed of any one of embodiments 1 to 3, further comprising, on a percentage of dry matter, from about 0.1% to about 1% linoleic acid. The meal fraction of Brassica carinata oilseed of any one of embodiments 1 to 3, further comprising, on a percentage of dry matter, from about 0.1% to about 1% linolenic acid. The meal fraction of Brassica carinata oilseed of any one of embodiments 1 to 7, comprising up to 45 glucosinolate per gram of meal.

The meal fraction of Brassica carinata oilseed of any one of embodiments 1 to 7, comprising up to 40 glucosinolate per gram of meal.

The meal fraction of Brassica carinata oilseed of any one of embodiments 1 to 7, comprising up to 35 glucosinolate per gram of meal.

The meal fraction of Brassica carinata oilseed of any one of embodiments 1 to 7, comprising up to 30 glucosinolate per gram of meal.

The meal fraction of Brassica carinata oilseed of any one of embodiments 1 to 7, comprising up to 25 glucosinolate per gram of meal.

The meal fraction of Brassica carinata oilseed of any one of embodiments 1 to 7, comprising up to 20 glucosinolate per gram of meal.

The meal fraction of Brassica carinata oilseed of any one of embodiments 1 to 7, comprising up to 15 glucosinolate per gram of meal.

The meal fraction of Brassica carinata oilseed of any one of embodiments 1 to 7, comprising up to 10 glucosinolate per gram of meal.

The meal fraction of Brassica carinata oilseed of any one of embodiments 1 to 7, comprising up to 5 glucosinolate per gram of meal.

The meal fraction of Brassica carinata oilseed of embodiments 8 to 16, wherein at least 70% of the glucosinolate is sinigrin.

The meal fraction of Brassica carinata oilseed of embodiments 8 to 16, wherein at least 80% of the glucosinolate is sinigrin.

The meal fraction of Brassica carinata oilseed of embodiments 8 to 16, wherein at least 90% of the glucosinolate is sinigrin. The meal fraction of Brassica carinata oilseed of embodiments 8 to 16, wherein at least 95% of the glucosinolate is sinigrin.

The meal fraction of Brassica carinata oilseed of any one of embodiments 1 to 20, wherein the meal fraction is produced from seeds of a Brassica carinata variety comprising at least 18% protein, on a dry weight basis.

The meal fraction of Brassica carinata oilseed of any one of embodiments 1 to 20, wherein the meal fraction is produced from seeds of a Brassica carinata variety comprising at least 20% protein, on a dry weight basis.

The meal fraction of Brassica carinata oilseed of embodiment 22, wherein the Brassica carinata variety is selected from the group consisting of: AAC-A120, AGR044-312D- HP11, AGR044-M01, AGR044-M06, AGR159-4A1A, AGR159-4A1D-Y, DH-18.047, DH-069.485, DH-157.715, DH-129.B036, DH-146.047, DH-146.194, DH-146-214, and DH-146.842.

The meal fraction of Brassica carinata oilseed of any one of embodiments 1 to 22, wherein the meal fraction is produced from seeds of a Brassica carinata variety that produces yellow or dark yellow seed.

The meal fraction of Brassica carinata oilseed of embodiment 24, wherein the meal fraction is produced from seeds of a Brassica carinata variety that produces yellow or dark yellow seed comprising from about 4.5% to 7.5% acid detergent fibre and from about 8% to about 13% neutral detergent fibre.

A feed ration comprising the meal fraction of Brassica carinata oilseed of any one of embodiments 1 to 25.

The feed ration of embodiment 26, wherein from about 5% to about 25% of dry matter in the feed ration is the meal fraction of Brassica carinata oilseed.

The feed ration of embodiment 26, wherein up to 5% of dry matter in the feed ration is the meal fraction of Brassica carinata oilseed.

The feed ration of embodiment 26, wherein up to 10% of dry matter in the feed ration is the meal fraction of Brassica carinata oilseed.

The feed ration of embodiment 26, wherein up to 15% of dry matter in the feed ration is the meal fraction of Brassica carinata oilseed. The feed ration of embodiment 26, wherein up to 20% of dry matter in the feed ration is the meal fraction of Brassica carinata oilseed.

The feed ration of embodiment 26, wherein up to 25% of dry matter in the feed ration is the meal fraction of Brassica carinata oilseed.

The feed ration of any one of embodiments 26 to 32, wherein from about 10% to 100% of protein in the feed ration is provided by the meal fraction of Brassica carinata oilseed. The feed ration of any one of embodiments 26 to 32, wherein from about 25% to 100% of protein in the feed ration is provided by the meal fraction of Brassica carinata oilseed. The feed ration of any one of embodiments 26 to 32, wherein from about 50% to 100% of protein in the feed ration is provided by the meal fraction of Brassica carinata oilseed. The feed ration of any one of embodiments 26 to 32, wherein from about 75% to 100% of protein in the feed ration is provided by the meal fraction of Brassica carinata oilseed. The feed ration of any one of embodiments 26 to 36, wherein the feed ration comprises up to 20 mihoΐ glucosinolate per gram of feed.

The feed ration of any one of embodiments 26 to 36, wherein the feed ration comprises up to 15 mhioΐ glucosinolate per gram of feed.

The feed ration of any one of embodiments 26 to 36, wherein the feed ration comprises up to 10 mhioΐ glucosinolate per gram of feed.

The feed ration of any one of embodiments 26 to 36, wherein the feed ration comprises up to 8 pmol glucosinolate per gram of feed.

The feed ration of any one of embodiments 26 to 36, wherein the feed ration comprises up to 6 pmol glucosinolate per gram of feed.

The feed ration of any one of embodiments 26 to 36, wherein the feed ration comprises up to 4 pmol per glucosinolate per gram of feed.

The feed ration of any one of embodiments 26 to 36, wherein the feed ration comprises up to 2 mhioΐ glucosinolate per gram of feed.

The feed ration of any one of embodiments 37 to 43, wherein at least 70% of the glucosinolate is sinigrin.

The feed ration of any one of embodiments 37 to 43, wherein at least 80% of the glucosinolate is sinigrin. The feed ration of any one of embodiments 37 to 43, wherein at least 90% of the glucosinolate is sinigrin.

The feed ration of any one of embodiments 37 to 43, wherein at least 95% of the glucosinolate is sinigrin.

The feed ration of any one of embodiments 26 to 47, wherein the feed ration is for feeding ruminant livestock, monogastric livestock, poultry livestock, camelid livestock, or farmed fish.

The feed ration of embodiment 48 wherein the ruminant livestock is cattle, sheep, goats, bison, or buffalo.

The feed ration of embodiment 49, wherein the cattle is beef cattle, dairy cattle, lactating cattle, or heifers.

The feed ration of embodiment 48, wherein the monogastric livestock is swine.

The feed ration of embodiment 48, wherein the poultry livestock is chicken, turkey, geese, or duck.

The feed ration of embodiment 52, wherein the chicken are broilers or layers.

The feed ration of embodiment 48, wherein the camelid livestock is alpaca, camel, or llama.

The feed ration of embodiment 48, wherein the farmed fish is a salmonid, carp, tilapia, and catfish.

The feed ration of embodiment 55, wherein the salmonid is trout or salmon.

The feed ration of any one of embodiments 26 to 56, further comprising one or more of: silage, fodder, corn cobs, corn or wheat germ, corn or wheat gluten, grain, bran, blood meal, bone meal, brewers grain, distiller’s grain, distiller’s dry grain solids, seed hulls (cotton, peanut, flaxseed), milk, buttermilk, molasses, vegetables, fruits whey, yeast, beans, beets, urea, soybean meal, canola meal, rapeseed meal, cottonseed meal, linseed meal, amino acids, fatty acids, oil, glycerol, vitamins, minerals, salts, and crustacean shells. DETAILED DESCRIPTION

[0025] Definitions

[0026] In the following description and tables, many terms are used. To aid in a clear and consistent understanding of the specification, the following definitions and evaluation criteria are provided.

[0027] Acid detergent fiber (ADF). The ADF content of a feed/meal samples is measured as follows: samples to be analyzed are digested in a solution containing the detergent cetyl trimethyl ammonium bromide (CTAB) and sulfuric acid that is brought to the boiling point; after digestion, the insoluble residue remaining contains predominantly cellulose and lignin which is collected by filtration and quantitated gravimetrically. ADF is expressed as a percentage of starting sample weight, either on an as received basis or on a DM basis. ADF content is a particularly descriptive parameter of feed/ meal quality since feed/meal digestibility has been generally found to correlate inversely to ADF content

[0028] Acid detergent lignin (ADL; Lignin): Analysis for ADL is carried out sequentially after ADF determination as follows: the acid detergent insoluble residue is further extracted with concentrated sulfuric acid (72% H2SO4) and insoluble material remaining after the acid extraction, representing predominantly lignin, is collected by filtration and determined gravimetrically. The lignin is expressed as a percentage of starting sample weight, either on an as received basis or on a DM basis

[0029] Anti-nutritional, as used herein, is a general description of a number of compounds found in Brassica and other seed meals that reduce the nutritional benefit of animal feed products in which the meal is used as an additive. Glucosinolates and isothiocyanates are classified as anti-nutritionals since, when present in high enough concentrations, they impart a bitter and pungent taste to the feed ration, potentially reducing its palatability and adversely affecting the livestock’s intake of the meal.

[0030] Ash, or Crude Ash, as used herein, refers to the mineral components of the

food/feed/meal sample. Ash content of a food/feed/meal sample is determined by igniting the sample in a muffle furnace at 600°C for 2 h. Under these conditions, only mineral content of the initial sample remains and is directly weighed to estimate Ash (mineral) content of the food/feed/meal sample. Ash can be expressed as a percentage of the original weight of sample on an as received basis or on a DM basis

[0031] Average, as used herein, refers to the arithmetic mean. “Substantially equivalent” or “statistically equivalent” refers to a value or measurement, when compared, does not show a statistically significant difference from the mean. In contrast,“statistically different” or “statistical significance” refers to a value or measurement that, when compared, shows statistically significant differences from means of the same value or measurement of another group or groups. Most often, statistical significance of differences is measured at levels of P < 0.05 using standard tests to compare Least Square Means, such as Tukey’s HSD or Student’s t- test.

[0032] Camelid livestock, or camelids, as used herein, refers to herbivorous animals with slender necks, long legs, and a three-chambered stomach. Examples of camelids include, but are not limited to, alpacas, camels, llamas, vicunas, and guanacos.

[0033] Carbohydrate (calculated), as used herein, refers to the carbohydrate fraction of food feed and meal substances is compositionally diverse. As a first approximation, if it is assumed that the only constituents of food/ feed /meal substance comprise ash, protein, fat, moisture and carbohydrate and, of these, the first four are directly determined by the methods described herein, then the carbohydrate content can be estimated as the difference between the measured weight of the food/ feed /meal substance and the sum of the determined weights of Ash, Crude Protein, Crude Fat, and moisture in said sample. The calculated carbohydrate content is typically expressed as a percentage of the original weight of sample on an as received basis or on a DM basis. In practice, the carbohydrate (calculated) value, as described herein, and provided in a proximate analysis, is of limited utility since it does not distinguish between forms of carbohydrate with differing solubility, digestibility and nutritive value.

[0034] Carbohydrates, as used herein, refers to soluble and readily digestible intracellular components are found in meal samples such as sugars and their polymeric forms, starches.

[0035] Carinata, as used herein, refers to seeds or plants of the species Brassica carinata containing both the B genome from Brassica nigra and the C genome from Brassica oleracea (Nagahuru, 1935, Japanese J. Botany 7: 389-452). [0036] Cattle, or cattle livestock, as used herein, refers to domesticated bovine farm animals that are raised for their meat, milk or hides, or for draft purpose. Examples of cattle include, but are not limited to, cows, beef cattle, dairy cattle, lactating cattle, heifers, yak, bison, buffalo, and water buffalo.

[0037] Cellulose, as used herein, refers to a linear homopolymer of D-glucose monomers covalently bound to one another via a b 1-4 glycosidic bonds. Individual cellulose polymers can self-associate via hydrogen boning to form higher order bundles or“microfibrils”. Many mammals lack the specific enzymes to process cellulose fibers in their gut. Ruminants on the other hand can process cellulose fibers by virtue of the bacteria populating their ruminal cavities.

[0038] Crude fat, as used herein, refers to the fat content of a food/feed/meal substance determined by exhaustive diethyl ether extraction of the dried and ground food/feed/meal substance. Diethyl ether solvent is then removed by evaporation and the extracted fraction comprising crude fat fraction of the food/feed/meal sample is quantitated gravimetrically. Crude fat can be expressed as a percentage of the original weight of sample on an as received basis or on a dry matter (DM) basis.

[0039] Crude fiber, as used herein, refers to the portion of insoluble carbohydrate remaining after extraction of a food/feed/meal sample with 1.25% H2SO4 and subsequent hydrolysis of the acid insoluble material.

[0040] Crude protein, as used herein, refers to the protein content of a food/feed/meal substance determined indirectly by measuring its total nitrogen (N) content either by via the Kjeldahl method (Kjeldahl, J. 1883. Neue Methods zur Bestimmung des Stickstoffs in

Organischen Korpern, Z. Anal. Chem. 22:366) or by the method of Dumas (Dumas, J.B.A. 1831. Procedes de l’Analyse Organique, Ann. Chim. Phys. 247:198; Jung. S. et al. 2003. Comparison of Kjeldahl and Dumas Methods for Determining Protein Contents of Soybean Products. J. Ass. Offic. Anal. Chem.80: 1169), which is then used to calculate the total crude protein content. Two assumptions are made, that all protein is 16% nitrogen by weight and that proteins are only source of nitrogen in the food/feed/meal substance. The determined amount of nitrogen is thus multiplied by 100/16 (or 6.25) to arrive at a crude protein estimate. Crude protein is usually expressed as a percentage of the initial weight of the sample“as received” or, preferably, it can be expressed as a percentage of the total weight of sample after removal of moisture (DM or dry matter basis).

[0041] Fatty acid content, as used herein, refers to the typical percentages by weight of fatty acids present in the endogenously formed oil of the mature whole dried seeds, as determined by Near Infrared Spectroscopy at less than 6% seed moisture. The NIR is calibrated using a large array of samples whose fatty acid profile is determined by American Oil Chemists Society (AOCS) Official Method Cel -66 Fatty Acid Composition by Gas Chromatography. This is one of the official methods recommended by the Western Canada Canola/Rapeseed Recommending Committee (WCC/RCC). Aside from erucic acid (C22:l), the most significantly occurring fatty acids in oil extracted from Brassica carinata oilseed are oleic acid (C18:l), linoleic acid (08:2), linolenic acid (08:3), and eicosenoic acid (C20: l).

[0042] Feed ration, or feed, or animal feed, as used herein, refers to those processed formulations that are fed to livestock, as opposed to those sources of nutrition that animals may forage for themselves. Animal feeds are formulated to provide optimum nutrition for particular applications, such as maximizing weight gain and meat quality of beef cattle in the feed lot or maximizing milk production of lactating dairy cattle. For poultry, feeds may be formulated to provide a consistent nutritional source to supplement or even replace the variable nutritional quality obtained through foraging. While the composition of feeds may vary greatly based on animal, application and geography, a constant requirement is the addition of supplements to the base feed to improve its protein content.

[0043] Fish, or farmed fish, as used herein, refers to fish raised commercially in tanks, ponds, or hatcheries using the methods of aquaculture or mari culture. Farmed fish include, but are not limited to, salmonids (salmon and trout), carp, tilapia, and catfish.

[0044] Genotype, as used herein, refers to the genetics or DNA sequence of individual carinata lines, as opposed to their actual appearance, which is called the phenotype.

[0045] Glucosinolate, as used herein, is a b-thioglucoside N-hydroxysulfate with a variable side chain (R) and a sulfur-linked b-d-glucopyranose moiety, representing a large and heterogeneous family of naturally occurring compounds— more than 120 varieties are known to occur in nature (Fahey, J. W., et al., Phytochem. 56, 5-51). Glucosinolates have the following chemical structure:

[0046] Grain, as used herein, refers to the seed produced by carinata crops that are intended for processing for oil or feed uses. This is in contrast with parent seed or planting seed, which is intended for growth of another generation of plants.

[0047] Hemicellulose, as used herein, refers to a polysaccharide of plant cell walls and seed husks, composed of hexose monomers connected via b 1 -4 glycosidic bonds. Hemicellulose differs from cellulose in that it is a heteropolymer of diverse hexose sugars, which, in addition to glucose, may include galactose, xylose, mannose, arabinose and others. Hemicellulose chains are often much shorter than those of cellulose and can be branched.

[0048] Hexane extraction, as used herein, refers to a process for extraction of oil from oilseeds that typically includes the following steps: the oilseeds are cleaned, and then crushed in a roller mill to generate flakes of 0.3 -0.38 mm in thickness; the flaked seed is then conveyed to a heated drum where the flakes are cooked at elevated temperatures (from 80-150°C, depending on the source of the seed) for up to 20 min; cooked seed flakes are then pressed in a series of screw presses or expellers which can remove 50-60% of the oil and produce a meal cake that is ideal for solvent extraction; the meal cake is treated with hexane, typically in several cycles of countercurrent extraction, to remove the residual oil from the meal. After the oil has been removed from the flakes or meal cake, the meal will contain approximately 30% of solvent (hexane) content. Typically, the meal would then undergo toasting and desolventizing to remove the hexane solvent and reduce the moisture content to 12% or less using a desolventizer-toaster, where it is heated to remove remaining hexane. Most of the solvent is removed by heating the meal on steam-heated plates. Removal of the final traces of solvent is carried out by injecting steam through the meal (the actual toasting process). In the course of the toasting process (roughly 30 minutes in duration), the meal is exposed to temperatures ranging from 95-115°C and moisture increases to 12-18%. Subsequently, the meal is cooled and dried via forced air circulation until a final moisture content of 12% or less is achieved. The meal is then pelletized or granulated depending on the requirements of the end user.

[0049] Lignin, or lignins, as used herein, refers to a polymeric molecule composed of three monomeric lignol subunits (p-coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol) each a distinct complex alcohol with phenolic sidechain. Monolignol molecules are incorporated into lignin in the form of the phenylpropanoids p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S), respectively.

[0050] Livestock, as used herein, refers to s domesticated animals raised in an agricultural setting to produce labor and commodities such as meat, eggs, milk, fur, leather, and wool.

[0051] Meal, or meal fraction, as used herein, refers to the remaining fraction of the seed content after extraction of the oil and consists mainly of protein. In the case of Brassica carinata, this meal fraction is particularly protein rich relative to other Brassica oilseed species.

[0052] Meal cake, as used herein, refers to the state of the seed meal after it has gone through the flaking and cooking stage and has been mechanically pressed to extrude the bulk of the oil. The term refers to the physical character of the meal at this stage, which has been compressed into a cake-like mass rich in protein and still containing appreciable residual oil. Defatted meal refers to the state of the meal after the meal cake has been solvent extracted to remove the last traces of residual oil.

[0053] Moisture, sometimes referred to as moisture and volatiles (M&V), is determined gravimetrically by weighing the food/feed meal before and after extensive drying in a drying oven, the difference in weights before and after drying representing the moisture content

Dry Matter (DM),% = wt after drying/wt before drying x 100%

% Moisture = 100 - DM,%

[0054] Monogastric livestock, or monogastric animals, as used herein, refers to non-ruminant livestock having a simple single-chambered stomach. Examples of monogastric livestock include, but are not limited to, swine, horses

[0055] Near Infrared Spectroscopy (NIR) refers to a non-destructive, spectrometric method commonly used to assess seed quality parameters in grain producing crops. Estimates of total seed oil content and fatty acid profile, as well as total glucosinolate and protein content on a whole seed basis, are examples of parameters for which NIR is commonly employed.

[0056] Neutral detergent fiber (NDF), as used herein, refers to the fiber fraction that cannot be extracted by neutral detergent solvent. NDF comprises the insoluble components of plant cell walls such as cellulose, hemicellulose and lignin. Samples to be analyzed are digested in a solution containing the anionic detergent sodium dodecyl sulphate (SDS) as well as EDTA, pH7.0 that is brought to boiling. Heat resistant amylase is also added to the extraction solution to reduce starch which may interfere with the assay. After digestion, insoluble material is collected by filtration and is quantitated gravimetrically. NDF is expressed as a percentage of starting sample weight, either on an as received basis or on a DM basis.

[0057] Oilseed, as used herein, refers to any crop species where oil is extracted from the seeds of these grains for food or industrial purposes, and includes Brassicaceae oilseeds such as canola, and non-Brassicaceae oilseeds, such as flaxseed, soybean, safflower, and sunflower. An example of a crop species that produces a seed used primarily for the production of edible oil is Brassica napus. An example of a crop species that is used primarily in the production of industrial feedstock oil is Brassica carinata.

[0058] Pectins, as used herein, refers to a diverse family of structural heteropolysaccharides that are compositionally and structurally distinct from cellulose and hemicelluloses. While they may have diverse monomeric compositions, a common aspect is that they are particularly rich in D- galacturonic acid and the monomeric subunits are linked via al-4 glycosidic bonds.

[0059] Phenotype, as used herein, refers to the outward appearance or manifestation of given traits of varieties, individual plants, or plant parts (such as leaves or seeds).

[0060] Poultry, or poultry livestock, as used herein, refers to domesticated birds raised for their eggs, meat or feathers. Examples of poultry include, but are not limited to, chicken (broilers, layers, fryers), turkey, geese, and duck.

[0061] Proximate analysis of meal, as used herein, refers to the method used to estimate the nutritive value of a food, feed or meal substance prior to engaging in animal feeding trials.

Crude Protein, Crude Fat and Crude Ash and moisture are determined, and the total carbohydrate content can then be calculated by difference: Carbohydrates = amount of total sample - moisture - Crude Protein - Crude Fat.

[0062] Ruminants, or ruminant livestock, as used herein, refers to livestock animals that are able to acquire nutrients from plant-based food by microbial fermentation in a specialized stomach, or rumen, prior to digestion. Typically, the fermented material (or cud, is regurgitated and chewed again. Examples of ruminant livestock include, but are not limited to, cattle, sheep, and goats.

[0063] Salmonids, as used herein, refers to fish of the family Salmonidae, that spawn in fresh water but spend some of the lives in the ocean. Examples of salmonids include, but are not limited to, salmon, trout, char, and freshwater whitefishes.

[0064] Seed oil content, as used herein, refers to the typical percentage by weight (wt%) of oil present in the mature whole dried seeds, at less than 6% moisture, as determined near infrared (NIR) spectroscopy (AOCS Procedure Am 1-92 Determination of Oil, Moisture and Volatile Matter, and Protein by Near-Infrared Reflectance).

[0065] Seed protein content, as used herein, refers to the typical percentage by weight (wt%) of protein in the oil-free meal of the mature whole dried seeds, at less than 6% moisture, analyzed using near infrared (NIR) spectroscopy (AOCS Procedure Am 1 -92 Determination of Oil, Moisture and Volatile Matter, and Protein by Near-Infrared Reflectance).

[0066] Sinigrin is the common name of allyl glucosinolate (or 2-propenyl glucosinolate), where the R position has been substituted with an allyl group. Sinigrin is the predominant glucosinolate species found in Brassica carinata and Brassica nigra seeds, and is also found, in lesser amounts in seeds of other Brassicaceae species. Sinigrin has the following chemical structure:

[0067] Variety, cultivated variety, or cultivar, as used herein, refers to a Brassica carinata line selected for one or more desirable characteristics that are maintained during propagation and may be used for commercial production of oilseed.

[0068] Meal Fraction of Brassica carinata oilseed

[0069] The meal fraction of oilseed is the proteinaceous material remaining after extraction of oil from the seed. Processing of Brassica oilseeds to extract the oil involves multiple steps. Typically, the seeds are cleaned then crushed in a roller mill to generate flakes, followed by cooking of the flakes at elevated temperatures. The cooking helps to reduce the viscosity of the oil to allow for more efficient extraction in subsequent steps. Cooked seed flakes are then pressed in a series of screw presses or expellers to remove the oil and produces a meal cake for solvent extraction. Following solvent extraction of the oil, the meal cake is treated with hexane to remove the residual oil from the meal. Any remaining hexane is removed using a combination of heat and steam, after which the meal is cooled and dried by blowing forced air through it.

[0070] Oilseed can also be processed using a cold press methodology, which is similar to the above process except it does not involve the cooking of the oilseed flakes or hexane extraction to remove residual oil from the oil cake, usually resulting a meal fraction with a higher oil content and a correspondingly lower percentage of crude protein, on a dry weight basis. In addition, cold-press meal produced from Brassica carinata retains very high levels of glucosinolates— up to 168.5 pmol per gram (Ban et al., 2017. J. Agric. Food Chem. 65: 5994-6001). This is significantly higher than the concentration of glucosinolates remaining in the meal fraction of Brassica carinata oilseed produced by a commercial hexane extraction process, as described above and in Example 1.

[0071] The meal fraction of Brassica carinata oilseed resulting from a typical hexane extraction process is significantly higher in protein and significantly lower in fibre (both neutral and acid detergent fibre) and lignin than the meal fractions from the related Brassicaceae oilseeds, Brassica napus and Camelina sativa (Table 1). Table 1: Compositional analysis of meal fractions oilseeds produced by hexane extraction process

[0072] When produced in a commercial process, such as the hexane extraction process described above, the meal fraction of Brassica carinata oilseed may comprise up to 45 pmol total glucosinolate per gram of meal. For example, the meal fraction of Brassica carinata oilseed may comprise up to 45 pmol total glucosinolate per gram of meal, up to 40 pmol total glucosinolate per gram of meal, up to 35 pmol total glucosinolate per gram of meal, up to 30 pmol total glucosinolate per gram of meal, up to 25 pmol total glucosinolate per gram of meal, up to 20 pmol total glucosinolate per gram of meal, up to 15 pmol total glucosinolate per gram of meal, up to 10 pmol total glucosinolate per gram of meal, or up to 5 pmol total glucosinolate per gram of meal, or any concentration of total glucosinolate, in pmol per gram of meal, therebetween.

[0073] In some embodiments, up to 70% of the glucosinolate in the meal fraction of Brassica carinata oilseed is sinigrin. In other embodiments, up to 80% of the glucosinolate in the meal fraction of Brassica carinata oilseed is sinigrin. In other embodiments, up to 90% of the glucosinolate in the meal fraction of Brassica carinata oilseed is sinigrin. In other embodiments, up to 95% of the glucosinolate in the meal fraction of Brassica carinata oilseed is sinigrin.

[0074] The meal fraction of Brassica carinata oilseed of the present invention comprises, as a percentage of dry weight, from about 35% to about 55% protein, from about 15% to about 30% neutral detergent fibre, from about 8% to 15% acid detergent fibre, from about 1% to about 8% lignin, and from 0 to 45 pmol total glucosinolate per gram of meal. For example, the meal fraction of Brassica carinata oilseed may comprise, as a percentage of dry weight, from about 42% to about 50% protein, from about 20% to about 26% neutral detergent fibre, from about 8% to 12% acid detergent fibre, from about 1% to about 5% lignin, and from 0 to 30 pmol total glucosinolate per gram of meal. For example, the meal fraction of Brassica carinata oilseed may comprise 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, or any percentage therebetween, crude protein; 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, or any percentage therebetween, neutral detergent fibre (NDF); 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, or any percentage therebetween, acid detergent fibre (ADF); 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, or any percentage therebetween, lignin; and from about 0 to about 1, 2, 3,4, 5, 6, 7, 8, 9 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 32, 34, 36, 38, 40, 42, of 45 pmol total glucosinolate per gram of meal.

[0075] In some embodiments of the present invention, the meal fraction of Brassica carinata oilseed comprises, as a percentage of dry matter, at least 42% protein, no more than 26% neutral detergent fibre, no more than 12% acid detergent fibre, no more than 5% lignin, and no more than 30 pmol total glucosinolate per gram of meal

[0076] The meal fraction of Brassica carinata oilseed of the present invention may further comprise, on a percentage of dry weight, from about 1% to about 5% oil and from about 6% to about 10% ash. For example, the meal fraction of Brassica carinata oilseed may further comprise, on a percentage of dry weight, 1%, 2%, 3%, 4% or 5%, or any percentage

therebetween, oil; and 6%, 7%, 8%, 9% or 10%, or any percentage therebetween, ash. In some embodiments, the meal fraction of Brassica carinata oilseed further comprises, as a percentage of dry matter, no more than 5% oil, and no more than 10% ash.

[0077] The oil remaining in the meal fraction of Brassica carinata oilseed has a distinct fatty acid profile the oil remaining in a meal fraction produced from Brassica napus (canola). Fatty acid analysis of the oil extracted from the seeds of a variety of Brassica oilseed species (Sharafi, et al, 2015. Int. J. Food Properties 18: 2145-2154) shows that, relative to canola oil, the oil from Brassica carinata oilseed has significantly lower levels of oleic acid, C18:l (10.08 wt% vs 61.83 wt%) and significantly higher levels of erucic acid, C22: 1 (40.56% vs 1.16%) and linolenic acid, 08:3 (18.37 wt% vs 6.84 wt%). [0078] Therefore, the meal fraction of Brassica carinata oilseed of the present invention, which may comprise up to 5% oil, may further comprise, on a percentage of dry weight, from about 0% to about 3% erucic acid. For example, the meal fraction of Brassica carinata oilseed may further comprise, on a percentage of dry weight, from about 0% to about 3%, from about 0% to about 2.5%, from about 0% to about 2%, from about 0% to about 1.5%, from 0% to about 1%, or from about 0% to about 0.5% erucic acid. In some poultry feeding trials, high levels of erucic acid in poultry diets has been shown to result in cardiac and skeletal muscle abnormalities (Hulan et al, 1982. Poultry Sci. 61 (6): 1154-1166). However, as shown in Example 3, poultry fed with feed rations containing the Brassica carinata meal fraction of the present invention, which contain low levels of erucic acid, do not exhibit such abnormalities.

[0079] Similarly, the meal fraction of Brassica carinata oilseed of the present invention, which may comprise up to 5% oil, may further comprise, on a percentage of dry weight, from about 0.1% to about 0.5% oleic (Cl 8:1) acid. For example, the meal fraction of Brassica carinata oilseed may further comprise, on a percentage of dry weight, from about 0.1% to about 0.2%, from about 0.1% to about 0.3%, from about 0.1% to about 0.4%, or from about 0% to about 0.5% oleic acid.

[0080] Similarly, the meal fraction of Brassica carinata oilseed of the present invention, which may comprise up to 5% oil, may further comprise, on a percentage of dry weight, from about 0.1% to about 1% linoleic (Cl 8:2) acid. For example, the meal fraction of Brassica carinata oilseed may further comprise, on a percentage of dry weight, from about 0.1% to about 0.2%, from about 0.1% to about 0.4%, from about 0.1% to about 0.6%, from about 0.1% to about 0.8%, or from about 0% to about 1% linoleic acid.

[0081] Similarly, the meal fraction of Brassica carinata oilseed of the present invention, which may comprise up to 5% oil, may further comprise, on a percentage of dry weight, from about 0.1% to about 1% linolenic (08:3) acid. For example, the meal fraction of Brassica carinata oilseed may further comprise, on a percentage of dry weight, from about 0.1% to about 0.2%, from about 0.1% to about 0.4%, from about 0.1% to about 0.6%, from about 0.1% to about 0.8%, or from about 0% to about 1% linolenic acid.

[0082] Similarly, the meal fraction of Brassica carinata oilseed of the present invention, which may comprise up to 5% oil, may further comprise, on a percentage of dry weight, from about 0.1% to about 0.5% eicosenoic (C20: 1) acid. For example, the meal fraction of Brassica carinata oilseed may further comprise, on a percentage of dry weight, from about 0.1% to about 0.2%, from about 0.1% to about 0.3%, from about 0.1% to about 0.4%, or from about 0% to about 0.5% eicosenoic acid.

[0083] The meal fraction of Brassica carinata oilseed provided by the present invention may be produced from seeds of a Brassica carinata variety comprising at least 18% protein, on a dry weight basis. For example, the meal fraction of Brassica carinata may be produced from seeds of & Brassica carinata variety comprising at least 18% protein, at least 19% protein, at least 20% protein, at least 21% protein, at least 22% protein, at least 23% protein, at least 24% protein, or at least 25% protein, or at least 25% protein, or at least 26% protein, or at least 27% protein, at least 28% protein, or at least 29% protein, or at least 30% protein, on a dry weight basis. The seed protein content produced by a Brassica carinata variety may vary depending on the environmental conditions under which it is grown as well as the seed oil content. However, after extraction of oil using the commercial process described above, the crude protein content of the meal will always be at least 35% (dry weight), and typically at least 40%.

Table 2: Seed protein content of Brassica carinata varieties

Seed protein content, % of seed dry weight

¹Mean seed protein content for seed harvested from trials conducted in from 2016-2018 across multiple geographies, including Canada and Northern US states, southeast US states, and Uruguay.

²SEM = standard error of the mean.

[0084] In other embodiments, the meal fraction is produced from seeds of Brassica carinata variety selected from the group consisting of: AAC-A120, AGR044-312D-HP11, AGR044- M01, AGR044-M06, AGR159-4A1A, AGR159-4A1D2-Y, DH-18.047, DH-069.485, DH- 157.715, DH-129.B026, DH-146.047, DH-146.194, DH- 146-214, DH-146.842. As shown in Table 2 below, these varieties all produce seeds containing at least 18% protein (lower 95%) and as much as 30% or more protein (upper 95%), on a dry weight basis.

[0085] It has been previously reported (Simbaya et al., 1995), that meal fractions produced from yellow seeded varieties of Brassica carinata, Brassica napus, Brassica juncea, and Brassica rapa have a lower fibre content and higher protein content than meal produced from brown- seeded varieties. A more detailed fibre analysis of yellow and brown Brassica carinata seeds has shown that seeds from yellow-seeded varieties contain lower levels of both acid detergent fibre (ADF) and neutral detergent fibre (NDF), as shown in Table 3.

Table 3: Fibre content of yellow-seeded and brown-seeded Brassica carinata varieties (SEM= std error of the mean)

[0086] In some embodiments of the present invention, the meal fraction of Brassica carinata oilseed as described herein is produced from seed of a Brassica carinata variety that produces yellow or dark yellow seed. In other embodiments, yellow or dark yellow seed comprises from about 4.5% to 7.5% acid detergent fibre and from about 8% to about 13% neutral detergent fibre.

[0087] Feed ration comprising a meal fraction of Brassica carinata oilseed

[0088] The meal fraction of Brassica carinata oilseed as described herein is suitable for use as a source of protein in feed rations and animal feeds. A feed ration comprising the meal fraction of Brassica carinata oilseed as described herein is suitable for ruminant livestock, monogastric livestock, poultry livestock, camelid livestock, of fish.

[0089] Depending on the animal to which the feed ration will be fed, as well as the final glucosinolate content of the meal fraction, which may reduce palatability of the resulting feed ration, the meal fraction of Brassica carinata oilseed as described herein may comprise up to 25%, on a dry weight basis, of the feed ration. For example, the meal fraction of Brassica carinata oilseed, as described herein, may comprise up to 25%, up to 20%, up to 15%, up to 10%, up to 5%, or any percentage therebetween, on a dry weight basis, of the feed ration. As a result, the concentration of glucosinolate, in the feed ration may range from about 1 to about 10 pmol per g of feed ration. For example, the concentration of total glucosinolate, in the feed ration may be 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 pmol per g of feed ration, or any concentration therebetween. Up to 70%, up to 80%, up to 90%, up to 95%, or more of the total glucosinolate in the feed ration may be sinigrin.

[0090] In addition to the meal fraction of Brassica carinata oilseed, as described herein, the feed ration may comprise other sources of protein in addition to that provided by the meal fraction of Brassica carinata oilseed. Other protein sources suitable for use in a feed ration include, but are not limited to, com or wheat germ, blood meal, bone meal, milk, butter milk, whey, yeast, or proteins from other meal sources such as soybean meal, canola meal, rapeseed meal, cottonseed meal, linseed meal, and the like. Thus, the meal fraction of Brassica carinata oilseed, as described herein, may provided from about 25% to about 100% of the protein content of the feed ration. For example, meal fraction of Brassica carinata oilseed, as described herein, may provide about 10% to 100%, about 20% to 100% about 25% to 100%, about 30% to 100%, about 35% to 100%, about 40% to 100%, about 45% to 100%, about 50% to 100%, about 55% to 100%, about 60% to 100%, about 65% to 100%, about 70% to 100%, about 75% to 100%, about 80% to 100%, about 85% to 100%, about 90% to 100%, or about 95% to 100% of the protein content of the feed ration.

[0091] The feed ration comprising the meal fraction of Brassica carinata, as described herein, may further comprise any number of other suitable materials known in the art for inclusion in feed rations as may be required for the animal that will consume the feed ration. For example, the feed ration may further comprise: silage (such as hay, straw, alfalfa), fodder (corn), corn cobs, com or wheat germ, corn or wheat gluten, grain (from corn, oat, spelt, barley, rice wheat), bran, blood meal, bone meal, brewers grain, distiller’s grain, distiller’s dried grain solids (DDGS), seed hulls (cotton, peanut, flaxseed), milk (dry/skim), buttermilk, molasses, vegetables and/or fruits (apples, carrots, potatoes, pumpkins, tomatoes, turnips), whey, yeast, beans, beets, urea, amino acids, fatty acids, oils (including animal fats and glycerol), vitamins, minerals, salts (such as sodium chloride, calcium chloride, potassium chloride, sodium phosphate (mono and dibasic), potassium phosphate (mono and dibasic), ammonium chloride, ammonium sulfate, calcium carbonate, calcium phosphate), and ground or crushed crustacean shells (such as those from shrimp, lobster, crab, or crayfish).

[0092] The feed ration comprising the meal fraction of Brassica carinata oilseed, as described herein, is for feeding for ruminant livestock, monogastric livestock, poultry livestock, camelid livestock, or farmed fish. The feed ration comprising the meal fraction of Brassica carinata oilseed, as described herein, may be a feed ration for

• ruminant livestock, including, but not limited to, cattle (beef cattle, dairy cattle, lactating cattle, and heifers), sheep, goats, bison, and buffalo;

• monogastric livestock, including but not limited to pigs, hogs, sows, and piglets;

• poultry livestock including, but not limited to, chicken (including broilers, layers), turkey, duck, and geese;

• camelid livestock including, but not limited to, alpaca, camel, or llama; and/or

• farmed fish including, but not limited to, salmonid species (for example, trout and salmon) and non-salmonid species such as carp, tilapia, and catfish.

[0093] Feeding studies conducted using a feed ration comprising a meal fraction of Brassica carinata oilseed, as described herein, demonstrate that such feed rations are at least equivalent to, or better than, a control feed ration comprising all of the same ingredients except for replacement of the Brassica carinata meal with another protein source (Examples 2, 3, and 4). No statistically significant differences were observed in weight gain or mortality between animals provided with a feed ration comprising a meal fraction of Brassica carinata oilseed compared to animal provided with a control feed ration.

EXAMPLES

[0094] Example 1: Properties of Brassica carinata meal

[0095] Brassica carinata grain harvested in Canada and US was crushed at commercial scale to obtain carinata oil and meal fractions. Commercial scale crushes were carried out in 2012 at Producers Cooperative Oil Mill (PCOM), Oklahoma City, in 2014 and 2015 at Archer Daniels Midland (ADM) Milling, Redwing MN and in 2016 at Societe Agro Industrielle de Patrimoine Oleagineux S.A.S (SAIPOL) milling plant, Dieppe, France. Commercial crushing of carinata oilseeds was carried out to extract maximal amounts of oil from the seed, resulting in a meal by- product ideally with less than 3% residual oil content by weight. The typical process (described more fully in the description) involves the following steps:

a. Cleaning of carinata grain to remove harvest debris, non-carinata seeds.

b. Preconditioning and flaking, involving a physical treatment of the seed designed to

increase access to the subcellular compartments containing the oil.

c. Cooking fully mobilizes the oil bodies in the flaked seed to make them easier to rupture.

Cooking temperatures can range from 85-115°C. Cooking at elevated temperatures may also inactivate enzymes such as myrosinase which can convert seed GSL to harmful reactive metabolites (WO2017/091891 Al).

d. Flaked and cooked seed is crushed by roller mill, expeller mill or extruder technology, applying pressure to the flaked grain to cause rupture and release of the oil, while leaving a residual oil cake consisting of high protein seed meal with remaining entrained oil. e. Solvent extraction the oil cake with an organic solvent (typically hexane to remove most of the remaining oil and leaving a meal cake with typically less than 3% residual oil). Oil recovered from this step is pooled with the oil removed during the crushing stage.

f. Desolventizing-toasting is carried out to remove residual hexane from the meal cake.

Temperatures used are typically in the range of 95-120°C and residence time of the meal cake in the desolventizer-toaster can be range from 30 to 240 minutes. The process is carried out under negative pressure to allow for full removal of residual hexane.

g. Drying and pelletizing of the meal cake is carried out, producing a final meal with less than 10% residual moisture and which is shaped into uniform pellets to facilitate bulk storage and transport.

[0096] Solvent-extracted and pelleted meal samples obtained for each of the above crushes were subjected to a number of analyses, including proximate analysis to determine percentage content of fiber constituents— specifically, NDF and ADF (Van Soest et al, 1991. J. Dairy Sci. 74, 3583-3597, ANKOM method 5, 6 and 8, ANCHOM Technology Inc, NY, USA; see also Marichal et al., 2011. Animal Feed Sci. Technol. 169, 79- 85), lignin (Official Methods of Analysis of AO AC INTERNATIONAL (2012) 19th Ed., AO AC INTERNATIONAL,

Gaithersburg, MD, USA, Official Method 973.18), crude protein (Official Methods of Analysis of AO AC INTERNATIONAL (2012) 19th Ed., AO AC INTERNATIONAL, Gaithersburg, MD, USA, Official Method 4e-93), minerals as ash (Official Methods and Recommended Practices of the AOCS (2012) 7th Ed., American Oil Chemists' Society, Champaign, Ill. Official method Bc5-49), fat (as oil; Official methods and recommended practices of the AOCS (2012) 7th Ed., American Oil Chemists' Society Champaign, Ill. Official method Am 2-93), moisture (Official methods and recommended practices of the AOCS (2012) 7th Ed., American Oil Chemists' Society Champaign, Ill. Official method Ba 2a-38) and total carbohydrate (calculated as 100 - % ash - % crude protein -% oil - % moisture). To allow for comparison between different meal lots or meal types, values are converted to % of dry matter weight (DM), as summarized in Table 4.

[0097] As can be seen in Table 4, the proximate analysis of meal samples generated from carinata grain harvested over a 4-year period reveals a meal that is consistent in composition and quality across the major meal components: carbohydrate, oil, protein and minerals (ash). The structural carbohydrate fractions (NDF, ADF and lignin) show similar year-to-year consistency.

Table 4: Proximate analysis of Brassica carinata meal produced by commercial scale crush

[0098] Table 5 compares the compositional attributes of the Brassica carinata meal averaged over four years to other commercial oilseed meals such as Brassica napus canola, camelina and soybean meals. Notable among the commercial Brassica oilseed meals, solvent-extracted Brassica carinata meal prepared via commercial scale process has the highest levels of crude protein, approaching those of soybean meal, while its structural and fibrous carbohydrate content is similarly unique in relation to the other commercial brassica oilseed meals in having significantly lower proportions of ADF and lignin. Table 5: Comparing proximate analysis of carinata meal to other commercial oilseed meals

¹ Canola solvent extracted meal (Heuze et al., 2018. Rapeseed meal. Feedipedia, a programme by INRA, CIRAD, AFZ and FAO. https://www.feedipedia.org/node/52 Last updated on June 15, 2018, 15:53)

² Camelina cold pressed (CP) meal (Heuze Vet al., 2017. Camelina (Camelina sativa) seeds and oil meal. Feedipedia, a programme by INRA, CIRAD, AFZ and FAO. https://www.feedipedia.org/node/4254 Last updated on September 11, 2017, 15:19)

³ Extrapolated values of Camelina defatted (DFM) meal from CP meal assuming reduction of lipid content from 15.6% to 3%

⁴ Non-dehulled and ⁵ dehulled Soybean meal values (Heuze et al., 2017. Soybean meal. Feedipedia, a programme by INRA, CIRAD, AFZ and FAO. https://www.feedipedia.org/node/674 Last updated on January 13, 2017, 15:45)

[0099] Previous breeding of oilseed varieties of Brassica napus, Brassica juncea and Brassica rapa have emphasized improving the quality of edible oil that could be obtained, focusing on the removal of erucic acid and increasing the levels of oleic acid in the oil fraction, whereas in the meal a primary focus has been on developing varieties with greatly decreased seed glucosinolate levels. These breeding efforts have resulted in the“double zero” canola-type rapeseed varieties. As a newer oilseed crop developed as a source of industrial feedstock oil, Brassica carinata breeding has not emphasized the same changes in seed quality. On the meal side, due to the distinctive nature of the Brassica carinata genome, and the unique seed glucosinolate profile, it has proven challenging to develop carinata varieties with similar magnitude of seed GSL level reductions to those of the“double zero” canola-type rapeseed varieties.

[0100] Table 6 compares a typical glucosinolate (GSL) profile of whole grain from a commercial carinata variety (AAC A100) versus grain of commercial Brassica napus canola, both determined using the Canadian Grain commission methodology (International Organization for Standardization (1991). Rapeseed - Determination of glucosinolates content - Part 1 :

Method using high-performance liquid chromatography (ISO/DIS Standard No. 9167). Table 6: Glucosinolate content and profile of whole carinata and canola grain

glucosinolates, mihoI/g DM AAC A100 grain Canola grain

2-OH-3-butenyl glucosinolate 0.65b 4.75a allyl glucosinolate 83.40 ND

2-OH-4-pentenyl glucosinolate 0.10 0.15

3-butenyl glucosinolate 0.50 2.25

4-OH-3-CH₃-indolyl glucosinolate 2.35b 4.80a

4-pentenyl glucosinolate ND 0.30

3-CH₃-indolyl glucosinolate 0.10b 0.40a

Ch -thiobutenyl glucosinolate ND 0.15 phenylethyl glucosinolate ND 0.10

CH -thiopentenyl glucosinolate ND 0.10 total glucosinolates 87.0a 12.9b

Table data taken from Xin et al. J. Agric. Food Chem. 2014, 62, 7977. Means with different letters in the same row are significantly different (P< 0.05).

[0101] The amount of total GSL in Brassica carinata whole grain is at least six times higher than in whole grain of Brassica napus canola. However, the chemical profile of glucosinolates found in Brassica carinata whole grain is distinct from that of Brassica napus canola whole grain, with allyl glucosinolate (sinigrin) comprising most of the GSL, while canola grain contains virtually no sinigrin but instead has relatively high amounts of 2-OH-3 -butenyl glucosinolate (progoitrin) , 3-butenyl glucosinolate (gluconapin) and 4-OH-3-CH3-indolyl glucosinolate (4— hydroxyglucobrassicin). It should be noted that, assuming minimum oil content of 40% by weight for Brassica carinata seed, the seed glucosinolate concentration of 87 pmol/g whole seed weight, which represents the typical GSL content for current commercial carinata varieties grown in North America, would be expected to be close to 60% higher in the defatted meal fraction (i.e., 147 pmole/g meal) if no losses occurred during processing to meal.

[0102] Table 7a summarizes amounts and chemical profiles of glucosinolates found in solvent- extracted Brassica carinata meal prepared using the commercial scale process described in Example 1. Data were obtained from analysis of different batches of meal made from grain harvested over the course of four years (four commercial harvests) and processed by three different commercial grain crushers. As in the case of whole seed, sinigrin is the predominant glucosinolate species in all four commercial Brassica carinata meal samples. However, in all meal samples, the total GSL content is considerably lower than that which would be predicted from the whole seed content, ranging between 12 pmol/g DM and 33.2 pmol/g DM, averaging 25.4 mihoΐ/g DM over the four-year period and representing less than 20% of total glucosinolates expected based on extrapolation of glucosinolate content of harvested carinata grain.

Table 7: Anti-metabolites present in solvent-extracted Brassica carinata meal prepared using commercial scale crush (a) Glucosinolate contents of solvent-extracted Brassica carinata meal lots prepared using commercial scale crush process

(b) Other anti-metabolite profiles of solvent-extracted Brassica carinata meal prepared using commercial scale crush (as % of dry matter, % DM)

[0103] Table 7b summarises the amounts of two additional compounds, sinapine and phytic acid, that are found in representative samples acid in solvent-extracted Brassica carinata meal. When present at appreciable levels in feed rations fed to egg laying hens, sinapine was thought to impart a fishy taste to brown shelled eggs (Butler et al., 1992 J. Sci. Food Agric. 33,866-875). This is due to a genetic deficiency in trimethylamine oxidase which has been corrected through breeding (Honkatukia et al, 2005 Genomics. 86, 225-232). Phytic acid is found in meal in a complex with phosphate and exerts an antinutritional effect by forming complexes with other phosphate and other minerals and proteins in feed rations, impeding their absorption in the gut of livestock and poultry. Both phytates and their antinutritional effects can, however, be removed quite efficiently from feed rations by addition of enzyme supplements to the feed rations (Khajali and Slominski, 2012, Poultry Sci., 91 :2564-2575).

[0104] Table 8 compares the average GSL content and profde of solvent-extracted carinata meal with those of commercial canola-type meals as well as meal derived from camelina. As may be expected, carinata meal has a higher GSL content than any of the canola type meals, although the difference is much less than would be expected based on whole grain GSL content (as discussed in the previous paragraph). Among the commercial meals, only camelina has a higher GSL content.

Table 8: Glucosinolate profiles of solvent-extracted Brassica carinata meal prepared using commercial scale crush compared to those of three types of canola meal and camelina meal

Total Glucosinolates 25.36 10.1 13.3 19.6 30.3

[0105] A clearer difference is seen in the GSL profile. In carinata meal, sinigrin accounts for the majority of GSL, while the canola and camelina meal samples contain little or no sinigrin. By contrast, the canola meal samples contain significantly higher amounts of progoitrin (0.9-7.3 versus 0.26 pmol/g), gluconapin (2.3-11.0 versus 0.35 pmol/g), and 4-hydroxyglucobrassicin (0.3-4.3 versus 0.02 pmole/g). The GSL profile of carinata meal is also distinct in relation to that of camelina meal, the latter consisting entirely of complex long chain glucosinolates that are not represented at all in the carinata and canola meal GSL profiles.

[0106] Example 2: Use of Brassica carinata meal as an ingredient in beef cattle feed rations

[0107] Animals: A total of 360 recently weaned calves (shrunk initial average body weight (321.8 ± 1.27 kg) were purchased from commercial sources. The calves were ranked by body weight and calves of similar body weight were assigned randomly to one of twelve pens (30 calves/pen).

[0108] Feeding: Each pen was assigned randomly to one of four treatments (carinata or canola meal at 7.5% or 15% of diet dry matter). Prior to the initiation of the trial, all cattle were fed a receiving diet which consisted of 27.8% rolled barley, 23.9% brome grass/alfalfa hay, 27.0% barley silage, and 5.0% supplement (DM basis). The formulated ingredient composition for the low inclusion level diets (7.5% canola or carinata meal) consisted of, on a dry matter (DM) basis, 30.7% barley silage, 10.0% bromegrass hay, 5.1% vitamin-mineral supplement (DM), 13.0% barley straw, 33.7 % barley grain and 7.5% carinata or canola meal. At the higher inclusion level (15% canola or carinata meal), the extra carinata or canola meal replaced some of the barley grain.

[0109] The desolventized and toasted carinata meal was supplied as a single lot (Archer Daniel Midland, Red Wing, MN). Desolventized and toasted canola meal was obtained in two separate loads from a local feed supplier. Feed was delivered as a total mixed ration ad libitum, once daily targeting approximately 5% residual feed. The amount of feed provided to each pen was recorded daily. Samples of ingredients and total mixed ration (TMR) were taken every two weeks for measurement of DM content, which was used to adjust each ingredient portion in the diet. The feed ingredients and TMR samples from each pen were dried in a forced air oven at 55°C. Dry matter intake (DMI) was calculated for each pen based on the amount (DM basis) of allotted feed and adjusted for residual feed (orts). All four diets were formulated to meet or exceed NRC requirements (Nutrient Requirements of Beef Cattle: Seventh Revised Edition: Update 2000. Washington, DC, The National Academies Pressjfor crude protein (CP), energy, minerals, and fat-soluble vitamins for 1.1 kg average daily gain (ADG). For analysis of feed compositions, a 0.3 -kg sample (DM basis) from each diet was taken every two weeks for chemical analysis.

[0110] Animal parameter recording: All animals were weighed on two consecutive days at the start (initial body weight, BW) and end of the trial (final BW) and every two weeks throughout the trial. Animal BW was reported as shrunk BW by multiplying BW by a correction factor of 0.96 to account for gut fill (Nutrient Requirements of Beef Cattle: Seventh Revised Edition: Update 2000. Washington, DC, The National Academies Press). The animal’s average daily growth (ADG) was calculated by period for each animal by subtracting the initial shrunk weight from the end shrunk weight and dividing by the number of days on trial and averaged by pen. Simultaneously, cattle feed-grain ratio (G:F) by pen was calculated as ADG/DMI. The net energy for maintenance (NEm), a measure of the ability of the feed to meet the energy requirement for weight maintenance, and net energy for growing (NEg), a measure of the ability of the feed to meet the energy requirements for weight gain, were calculated based on performance data (BW, ADG, and DMI) using the formula for retained energy for large-framed yearling steers as previously described (Gibb et al, 2008. Can. J. Anim. Sci. 88, 659-665).

[0111] Analytical Methods: % Crude protein content of meal was determined using the Dumas combustion technique to determine total nitrogen from which crude protein was extrapolated using formula N X 6.25 (Official Methods of Analysis of AO AC INTERNATIONAL (2012)

19th Ed., AO AC INTERNATIONAL, Gaithersburg, MD, USA, Official Method 990.03).

Soluble protein fraction of meal samples was extrapolated from amount of soluble N extracted from meal sample in a bicarbonate-phosphate buffer (Krishnamoorthy et al., (1982) J. Dairy Sci. 65, 217-225). Ash and fat content of meal samples was determined using standard AO AC methods (for ash: Official Methods of Analysis of AOAC INTERNATIONAL (2012) 19th Ed., AO AC INTERNATIONAL, Gaithersburg, MD, USA, Official method 942.05; for crude fat: Official Methods of Analysis of AOAC INTERNATIONAL (2012) 19th Ed., AOAC INTERNATIONAL, Gaithersburg, MD, USA, Official method 2003.05). Fiber (NDF, ADF and Lignin) content of meal samples was determined using the differential solubility criteria of Von Soest (Van Soest et al. (1991) J. Dairy Sci. 74; 3583-3597; Goering and Van Soest. (1970) Agriculture Handbook. United States Department of Agriculture, Washington DC; Official Methods of Analysis of AO AC INTERNATIONAL (2012) 19th Ed., AO AC

INTERNATIONAL, Gaithersburg, MD, USA, Official method 973.18). Starch content of meal samples was estimated by an enzymatic-colorimetric assay (Hall, (2009) JAOACI 92: 42-49) and sugar content by the phenol-sulfuric acid reaction method (Dubois et al. (1956) Anal. Chem. 28:350-356).

[0112] Table 9 summarizes the proximate nutritional analysis of the canola and carinata meal lots used in this feeding study. Consistent with the values cited in Example 1 , the desolventized and toasted carinata meal had a higher proportion of crude protein on a dry matter basis than the canola meal. The proportion of soluble protein in the carinata meal, on a dry matter basis, was also higher than that of canola meal, although soluble protein levels expressed as percentage of crude protein were similar in both meal samples.

Table 9: The chemical profile of meals derived from canola meal or carinata meal ¹

Item Canola Meal ² Carinata Meal³

Basic chemical profile (%, DM)

Ash 7.7 ± 0.1 9

Crude fat 3.7 ± 0.2 2.2

Crude protein profile (%, DM)

Crude protein 38.0 ± 0.4 42.1

Soluble protein 6.6 ± 1.2 7.3

Soluble protein (%, CP) 17.2 ± 2.9 17.3

Structural carbohydrate profile (%, DM)

Neutral detergent fiber 29.3 ± 0.4 22.6

Acid detergent fiber 21.0 ± 1.7 11.2

Acid detergent lignin 10.3 ± 0.1 3

Non fiber carbohydrate 25.2 ± 0.4 29.8

Non-structural carbohydrate profile (%,

DM)

Ethanol soluble carbohydrate

10.7 ± 0.1 12.7

(sugar)

Starch 1.5 ± 0.1 1.9

Total digestible nutrient, TDN (%, DM) 65.9 ± 0.4 853 Energy value, Mcal/kg DM

Net energy for maintenance 1.51 ± 0.01 2.13

Net energy for gain 0.9 ± 0.01 1.46

¹ Analysis was conducted by Cumberland Valley Analytical Services (Hagerstown, MD)

² Canola meal = Brassica napus (n = 2). ³ Carinata meal = Brassica carinata.

[0113] Similarly, carinata meal had lower levels of NDF and much lower levels of ADF and acid detergent lignin than canola. Both ADF and lignin correlate inversely with digestibility of plant cell wall such that higher levels of these components indicate lower levels digestible nutrients in the feed. This is borne out by the observation that total digestible nutrient in carinata meal is almost 20 percentage points higher than that of canola meal and that carinata meal provides a significantly higher percentage of energy for both weight maintenance and weight gain on a dry meal basis than canola meal.

[0114] Table 10 summarizes the composition of the four diets used. All diets contain similar proportions of hay, barley silage, barley straw and supplemental nutrients, but differ in the amounts of barley grain, canola meal and carinata meal: the 7.5% canola/carinata diet contains 33.7-33.8 % barley grain and nominally 7.5% of canola or carina meals (actual proportions 7.9%-8.0%, differences from nominal values are due to slightly varying moisture contents at the time of preparation) while the 15% canola/carinata diet contains 26.3 % barley grain and nominally 15% of canola or carinata meals (actual proportions 15.4%-15.5%; reasons for differences from nominal values are as described above).

Table 10: Composition of the experimental diets

Canola meal ¹ Carinata meal ²

Item 7.5% ³ 15% ⁴ 7.5% 15%

Ingredients (%, DM)

Barley 3.7 26.3 33.8 26.3 Canola meal 15.5

Carinata meal 7.9 15.4 Hay 0 10 10.1 10

Barley silage (var. Ran 0.2 30.2 30.2 30.2 Barley straw 3 12.9 12.8 12.9 Supplement ⁵

1

5.1

5.1

5.1

[0115] As can be seen in Table 11, despite carinata meal having a significantly higher TDN than canola meal, none of the four diets differ significantly in terms of TDN content due to the relatively lower proportions of canola or carinata meal in the final diet ration. The essential mineral contents of all four diets are similar and meet minimal standard requirements for beef cattle diets.

Table 11: Nutrient profile of experimental diets

Canola Meal¹ Carinata Meal²

[0116] Table 12 summarizes the growth performance of groups of steers fed each of the four diets over the 97-day period. Although a slight increase in shrunk body weight was recorded at 97 days for both 15% canola and carinata diets over that seen for the respective 7.5% diets, this difference was not found to be significant, nor was there any significant difference in body weight seen between the canola and carinata diets at either percentage composition. Similarly, no significant differences were seen between any of the diets in terms of average daily weigh gain or dry matter intake per day and as a result, feed : gain ratios for all diets were virtually identical over the 97-day period. [0117] During the 97-day period, steers on each of the four diets were monitored daily for health issues. Most animals in each feeding group did not encounter visible health issues over the course of the study and there were no significant health issues that could be correlated with diet.

Table 12: The effects of carinata or canola meal fed at one of two inclusion levels on backgrounding performance of weaned steer calves over a 97-day period

[0118] Example 3: Use of Brassica carinata meal as an ingredient in dairy cattle rations

[0119] Eight lactating cows (729 ± 39 kg BW [mean ± SD] that were 107 ± 11 days-in-milk at the start of the experiment) were used in this study. The experimental design was a replicated 4 x 4 Latin square experimental design with 28-day experimental periods (consisting of 18 days of dietary adaptation followed by 10 days of data and sample collection). The four dietary treatments (as per Table 13) consisted of a standard barley silage-based diet containing canola meal as the principal protein supplement and excluding Brassica carinata meal (BCM;

designated 0% BCM) or diets formulated to contain 2 %BCM, 4% BCM and 8% BCM on a dry matter basis. In the 2% BCM, 4% BCM, and 8% BCM diets, BCM replaced an equivalent proportion of canola meal. Single batches of canola meal and BCM were used for the entire study. The batch of BCM was supplied by ADM (Redwing, MN). Chemical compositions of the four experimental diets are presented in Table 14. Cows were fed experimental diets as total mixed rations (TMR) at 0830h and 1600 h for ad libitum intake. Cows had free access to water. Table 13: Ingredient composition of the total mixed rations (TMR) fed to dairy cows

Feed ingredient 0 % BCM 2% BCM 4% BCM 8% BCM

Ingredient composition, % of DM

Barley Silage 28.1 28.1 28.1 28.1

Alfalfa hay 17.6 17.6 17.6 17.6

Brassica carinata meal (BCM) 0 2 4 8.01

Canola meal 17.6 15.6 13.6 9.55

Barley grain 24.2 24.2 24.2 24.2

Soybean hulls 4.04 4.04 4.04 4.04

Cotton seed hulls 3.51 3.51 3.51 3.51

Dairy premix 1.4 1.4 1.4 1.4

Oat hulls 1.4 1.4 1.4 1.4

Molasses dried 1.05 1.05 1.05 1.05

Sodium bicarbonate 0.78 0.78 0.78 0.78

Limestone 0.18 0.18 0.18 0.18

Potassium magnesium sulfate 0.14 0.14 0.14 0.14

Corn gluten Meal 0.04 0.04 0.04 0.04

Soybean meal 0.04 0.04 0.04 0.04

Total 100 100 100 100

Abbreviations: DM, dry matter; BCM, Brassica carinata meal

[0120] Feed intake analysis: Individual feed intake was recorded daily during the last 10 days of each experimental period. TMR samples were collected on days 25, 26, and 27 and stored at - 20°C prior to analysis. TMR chemical analysis was carried out using AO AC procedures (Official Methods of Analysis, 17th edition. 2000. Association of Official Analytical Chemists).

[0121] Milk compositional analysis: Experimental cows were milked three times daily at 0430h, 1230h, and 1900h, and milk weights were recorded. At each milking on days 25, 26, and 27, milk was collected into plastic vials containing 2-bromo-2-nitropropane-l -2-diol as a preservative. Daily milk samples from the 043 Oh, 123 Oh, and 1900h milkings were then pooled proportionally based on milk yield from each milking and pooled milk samples were stored at 4°C before being sent for compositional analyses including fat, protein, lactose, milk-urea nitrogen (MUN) determination (Official Methods of Analysis, 17th edition. 2000. Association of Official Analytical Chemists), as well as somatic cell counts (SCC) using an infrared analyzer (Foss System 4000, Foss Electric, Hillerod, Denmark). [0122] Blood Thyroid hormone levels: On day 28, blood samples were collected at 1030 h from the coccygeal vein of each cow into 10 mL vacutainer tubes containing lithium heparin (Becton Dickinson, Franklin Lakes, NJ). Blood samples were centrifuged at 2,500 x g for 15 min at 4°C and the plasma obtained was stored at - 20°C until analysis was carried out.

[0123] Milk Organoleptic studies: For organoleptic evaluation of milk, 2 L of milk were obtained at each milking (for a total of 6 L per cow) from five cows. Milk samples for were stored at 4°C and tested on the following day after sampling. Milk organoleptic evaluation was conducted with a sensory panel of 13-16 panelists who evaluated the appearance, color, creaminess, flavor, odor, and acceptability of the milk. Sensory evaluation was conducted on a 6-point hedonic scale: a score of 1 = dislike it very much and a score of 6 = like it very much.

[0124] Results and discussion: As expected from the relative proportion of canola/carinata meal to the total dry matter of the four mixed rations, the actual chemical compositions of the rations did not vary significantly among the constituent nutrients listed in Table 14.

[0125] Table 15 summarizes the daily production of milk and milk constituents by lactating cows maintained on each of the four experimental diets during the 28-day test phase. As the table illustrates, there was no significant bias observed between the four diets in terms of the cows’ average daily dry matter intake. Average total daily milk production, as well as milk production normalized for fat content or total energy content, showed a slight trend towards decreased production with increased BCM content of the feed ration, but this was not found to be statistically significant. Similarly, daily production levels of protein, fat, lactose and non-fat solids were not found to vary significantly with increasing BCM content of the feed ration.

Table 14: Chemical composition of total mixed rations (TMR) fed to dairy cows

Item 0% BCM 2% BCM 4% BCM 8% BCM

Dry Matter, % 56.5 56.9 56.8 57.2

Crude protein, % of DM 17.3 17 17.4 17.5

Soluble protein, % of DM 5.13 5 5.58 4.94

NDF, % of DM 36.7 36.5 37.1 36.4

ADF, % of DM 26.8 26.2 25.9 25.8

NDICP, % of DM 2.41 2.47 2.64 2.7

ADICP, % of DM 2.17 2.05 2.2 2.11

Lignin, % of DM 5.66 5.83 5.92 5.61

Starch, % of DM 20.2 20.1 20.4 20.3 Crude fat, % of DM 2.49 2.47 2.31 2.27

Ash, % of DM 9.05 9.23 9.18 9.29

Ca, % of DM 1.07 1.02 1.07 1.03

P, % of DM 0.54 0.54 0.55 0.56

Mg, % of DM 0.41 0.41 0.42 0.45

K, % of DM 1.69 1.69 1.67 1.69

Na, % of DM 0.38 0.38 0.38 0.39

S, % of DM 0.32 0.33 0.36 0.39

Cl, % of DM 0.36 0.36 0.36 0.36

Fe, ppm 461 477 474 486

Mn, ppm 76 71 69 67

Zn, ppm 74 72 68 73

Cu, ppm 22 20 21 18

Abbreviations: BCM, Brassica carinata meal; SE, standard error; DM, dry matter; CP, crude protein; NDF,

Neutral detergent fiber; ADF, acid detergent fiber, NDICP, Neutral detergent insoluble CP; ADICP, acid

detergent insoluble CP; Ca, calcium; P, phosphorous; Mg, magnesium; K, potassium; Na, sodium; S,

sulfur; Cl, chloride; Fe, ferrous; Mn, manganese; Zn, zinc; Cu, copper

Table 15: Effects of feeding graded levels of Brassica carinata meal (BCM) in total mixed rations (TMR) on dry matter intake, milk yield and milk component yields in lactating dairy cows¹

Milk and milk component yields, kg/d

^JThe experimental design was a replicated 4 x 4 Latin square consisting of two Latin squares each with four dairy cows ( n = 8). Experimental diets consisted of a standard barley silage-based diet containing canola meal as the principal protein supplement (0% BCM) or diets formulated to contain 2% BCM, 4% BCM, and 8% BCM on a dry matter basis.

¾EM = standard error of mean.

⁴P values indicate overall diet, linear, quadratic and cubic effects. Coefficients for polynomial contrasts: [L: -0.67082; -0.223607; 0.2236068; 0.670820]; [Q: 0.5; -0.5; -0.5; 0.5]; [C: -0.223607; 0.6708204; -0.67082; 0.2236068]

⁵FCM = 3.5% fat-corrected milk. ⁶ECM = energy-corrected milk. ⁷SNF = solids-non-fat.

[0126] Pooled milk samples from cows fed each of the four diets were analyzed to determine whether the overall feed efficiency (total amount of milk corrected for fat content produced during the period per feed intake over the same period) was affected by diet (Table 16). While a slight decrease in feed efficiency was observed in diets with increasing proportion of BCM, this change was not found to be statistically significant. Similarly, no significant changes were observed in total fat, total protein or total fat- or non-fat-solids content with increasing

proportion of BCM in the diet. There was no statistically significant difference in total milk urea nitrogen (MUN) from cows fed the different diets, but it should be noted that milk samples obtained from all of the diets, including the BCM control diet (containing 17% canola meal) showed MUN levels higher than the preferred levels of 12-14 mg/dl. The reason for this is unclear but may reflect an imbalance of nutrients present in each of the experimental diets and is not related to presence or amounts of BCM in the diet. Somatic cell counts were also taken from milk samples obtained from cows fed the different diets. All diets gave rise to milk with SCC well below 250,000 indicating that cows were not infected with significant levels of pathogens (Dohoo and Meek, 1982 Can. Vet. J. 23,119-125) and no statistically significant differences in the SCC of milk samples were observed between any of the dietary treatments.

Table 16: Effects of feeding Brassica carinata meal (BCM) in total mixed rations (TMR) on feed efficiency, milk composition, plasma hormones (T3 and T4), and T3:T4 ratio profiles of lactating cows¹

Experimental Diets P values

MUN⁶, mg/dL 16.9 17.7 18.1 18.2 0.96 0.77 0.34 0.69 0.99

SCC⁷, 10³ cells/ml 178 86 102 164 10.6 0.83 0.94 0.36 0.87

Plasma T3 and T4 profiles

T3, nmol/L 1.3 1.29 1.19 1.32 0.076 0.62 0.93 0.38 0.34

T4, n ol/L 49.7 47.5 45.4 45 3.1 0.69 0.25 0.77 0.91

Ratio of T3:T4 0.027 0.027 0.026 0.03 0.0002 0.52 0.37 0.36 0.45

The experimental design was a replicated 4 x 4 Latin square consisting of two Latin squares each with four dairy cows ( n = 8). Experimental diets consisted of a standard barley silage-based diet containing canola meal as the principal protein supplement (0% BCM) or diets formulated to contain 2% BCM, 4% BCM, and 8% BCM on a dry matter basis.

SEM = standard error of mean.

P values indicate overall diet, linear, quadratic and cubic effects. Coefficients for polynomial contrasts: [L: -0.67082; -0.223607; 0.2236068; 0.670820]; [Q: 0.5; -0.5; -0.5; 0.5]; [C: -0.223607; 0.6708204; -0.67082; 0.2236068]

Eeed efficiency = ratio of dry matter intake to fat-corrected milk yield.

MUN = milk urea nitrogen. SCC = somatic cell count. [0127] When present in animal diets, certain GSL and GSL metabolites have been shown to have effects on thyroid function (reviewed in Tripathi and Mishra, 2007 Anim. Feed

Sci.. Tech.132, 1-27). Therefore, plasma thyroid hormone levels were monitored in the groups of cows fed each of the four dietary treatments (Table 16). No significant differences were observed in serum T3 or T4 levels or in T3:T4 ratio for serum for cows maintained on any of the four diets. Moreover, the range of T3 and T4 levels observed were consistent with those described in (Blum et al., 1983 Anim. Prod. 36, 93-104) for high milk yielding cattle.

[0128] Although a subjective quality, the human perception of milk’s flavor, odor and

appearance are important defining characteristics. To determine whether the use of BCM in dairy cattle diets would result in qualitative changes to milk attributes that would render it less palatable, the milk samples obtained from cows fed each of the four diets were assessed in a taste test carried out as described above. The results of such testing, summarized in Table 17, revealed no statistically significant differences in perception of quality attributes by human subjects for milk obtained from cows fed any of the four diets.

Table 17: Effects of feeding graded levels of Brassica carinata meal (BCM) in total mixed rations (TMR) on organoleptic properties of milk from lactating cows¹

Item⁵ Experimental Diets² P values⁴

0% BCM 2% BCM 4% BCM 8% BCM SEM³ Diet Linear Quadratic Cubic

Appearance 5.19 5.2 5.15 5.18 0.045 0.89 0.85 0.89 0.73

Color 5.27 5.26 5.21 5.26 0.042 0.81 0.85 0.69 0.71

Creaminess 4.95 4.89 4.46 4.86 0.058 0.88 0.56 0.81 0.99

Flavor 4.55 4.51 4.37 4.42 0.069 0.64 0.4 0.71 0.64

Odor 4.62 4.47 4.4 4.46 0.062 0.43 0.33 0.4 0.9

Acceptability 4.68 4.67 4.65 4.57 0.062 0.94 0.52 0.77 0.92

The experimental design was a replicated 4 x 4 Latin square consisting of two Latin squares each with four dairy cows ( n = 8).

Experimental diets consisted of a standard barley silage-based diet containing canola meal as the principal protein supplement (0% BCM) or diets formulated to contain 2% BCM, 4% BCM, and 8% BCM on a dry matter basis.

SEM = standard error of mean.

P values indicate overall diet, linear, quadratic and cubic effects. Coefficients for Chi-square polynomial contrasts: [L: -0.67082; - 0.223607; 0.2236068; 0.670820]; [Q: 0.5; -0.5; -0.5; 0.5]; [C: -0.223607; 0.6708204; -0.67082; 0.2236068]

The sensory evaluation was conducted on a 6-point hedonic scale, 1 = dislike it very much, 6 = like it very much.

[0129] Example 4: Use of Brassica carinata meal as an ingredient in poultry rations

[0130] The experimental design was a 2 x 4 randomized complete block design to evaluate male and female broilers fed diets with one of four different Brassica carinata meal (BCM) inclusion levels: 0% (control), 7%, 14%, and 21% of the total ration. A single source of BCM, manufactured by SAIPOL (Dieppe, France), was used in these experiments. There were six replicate groups for each experimental treatment; one cage was considered the experimental unit.

[0131] A total of 336 male and female broiler (Ross 708) day old chicks were randomly assigned to 48 cages (seven birds / cage; 50 x 50 cm floor space). With removal of birds during sampling (two birds per cage removed at each of days 14 and 28, see below), approved cage densities were maintained throughout the trial. Cages were maintained with optimum temperatures, ventilation and lighting. The birds were monitored twice daily for behavior and for the availability of feed and water; any mortality was recorded, weighed and submitted for necropsy and determination of cause of death. Similarly, any birds that were identified as injured or sick were humanely culled and submitted for examination.

[0132] Diets

[0133] There were two phases of diets fed to the broiler chicks (starter (0 to 21 d); and grow/finish (21 to 35 d)), formulated to meet the nutrient requirements of Ross 708 broilers (Ross 708 Broilers: Nutrition Specifications; 2014, http://en.aviagen.com/tech- center/download/14/Ross-708-Broiler-Nutrition-Specs-2014rl7-EN.pdf Aviagen Group, Huntsville, AL). The study consisted of eight diet treatments (four for each phase) based on four BCM inclusion levels comprising 0%, 7%, 14%, and 21% of the respective diets, fed to male or female broilers. The BCM in the 7%, 14%, and 21% diets replaced soybean meal and wheat such that an equal nutrient profde between diets was maintained. All diets contained 0.8% acid insoluble ash marker for determination of digestibility. The final feed form was a mash for both the starter and grower/finisher diets. Diets were fed ad libitum throughout the trial. Samples of each diet were analyzed for nutrient composition using the following methodologies:

[0134] Percent Dietary Fiber Analysis: Acid detergent fiber (%ADF), neutral detergent fiber (%NDF) and lignin were determined according to the methods originally described by Van Soest (Van Soest et al, 1991. J. Dairy Sci. 74, 3583-3597) adapted for use with an ANKOM fiber analysis apparatus (ANCHOM method 5, 6 and 8, ANCHOM Technology Inc, NY, USA; see also Marichal et al., 2011. Animal Feed Sci. Technol. 169, 79- 85). Crude fiber, which represents the portion of feed resistant to successive extractions with boiling acid and base solution, was determined via a standard procedure for animal feed analysis (Official methods and recommended practices of the AOCS (2012) 7^th Ed., American Oil Chemists' Society Champaign, Ill. Official method Ba6a-05).

[0135] Percent Dietary ash content: Ash % was determined gravimetrically subsequent to dry- ashing of feed samples or excreta samples in a muffle furnace according to the recommended protocol of the American Organization of Analytical Chemists (Official Methods of Analysis of AO AC INTERNATIONAL (2012) 19th Ed., AO AC INTERNATIONAL, Gaithersburg, MD, USA, Official Method 942.05 Acid insoluble ash was determined gravimetrically by treating feed or excreta samples with boiling HC1, collecting the washed insoluble residue by filtration followed by ashing in a muffle furnace (Vogtmann et ah, 1975. Br. Poult. Sci. 16: 531-534).

[0136] Percent Dietary mineral content: Calcium % content and phosphorus % content were determined by use of standard techniques for animal feed analysis (Official Methods of Analysis of AO AC INTERNATIONAL (2012) 19th Ed., AO AC INTERNATIONAL, Gaithersburg, MD, USA, Official Method 968.08; Official Methods of Analysis of AO AC INTERNATIONAL (2012) 19th Ed., AO AC INTERNATIONAL, Gaithersburg, MD, USA, Official Method

935.13A

[0137] Percent Dietary protein content: Total nitrogen content of the feed samples (and in some cases excreta samples) was determined by Kjeldahl analysis and % crude protein then estimated by the formula N x 6.25% (Official Methods of Analysis of AO AC

INTERNATIONAL (2012) 19th Ed., AOAC INTERNATIONAL, Gaithersburg, MD, USA, Official Method 984.13).

[0138] Percent Fat content: Determination of fat content of feed samples was carried out via ether extraction of the samples using a Soxhlet apparatus, evaporation of the extract to dryness and gravimetric analysis of the extracted material (Official Methods of Analysis of AOAC INTERNATIONAL (2012) 19th Ed., AOAC INTERNATIONAL, Gaithersburg, MD, USA, Official Method 920.39).

[0139] Gross energy (GE) content (kcal/kg) of the feed samples and excreta were determined by combustion in a Parr 1281 Bomb Calorimeter (Parr Instrument Company, Moline, IL).

[0140] Percent moisture content of the feed samples was carried out by determining the difference in sample weights before and after extensive oven drying (Official Methods of Analysis of AO AC INTERNATIONAL (2012) 19th Ed., AO AC INTERNATIONAL,

Gaithersburg, MD, USA, Official Method 930.15).

[0141] Metabolizable Energy: Excreta samples collected between 19 d of age and 21 d of age were analyzed to determine apparent metabolizable energy (AME), retained nitrogen and nitrogen corrected AME (AME_N) using the following equations:

where GE = gross energy; marker = % acid insoluble ash in each sample; and N = % crude protein in each sample

[0142] Measurements and sampling

[0143] The performance of each cage of broilers was determined by monitoring body weight (mean body weight, g) and feed intake (g/bird/d for periods) at 0 d, 14 d, 21 d, 28 d, and 31 d of age; this was then used to calculate feed conversion ratio (FCR; g feed/g gain), correcting for any mortality. To maintain optimum bird density in cages, two birds were sampled per cage at 14 d and 28 d, such that there were five birds/cage from 14 - 28 d of age, and three birds/cage from 28 - 31 d of age. Between 19 d and 21 d of age, excreta were collected from each cage for determination of apparent metabolizable energy (AME; kcal/kg) and determination of nitrogen retention based on nitrogen analysis of the diets and excreta. At 31 d of age, birds were sampled for blood measurements (eight birds / dietary treatment). Blood serum was analyzed for thyroxine (T4) and triiodothyronine (T3) hormones along with other mineral and enzymes typically used as diagnostics for poultry health.

[0144] Results and discussion

[0145] Performance of broilers on starter diets: As is typical in farm settings, broiler chicks are maintained on a starter diet for up to 21 days post hatching. The starter diet is higher in protein content to meet the higher protein requirement of the broiler chicks at this stage.

Brassica carinata meal (BCM) was evaluated as a source of dietary protein during the starter phase of broiler production. Table 18 summarizes the results of incorporating BCM at 0%, 7%, 14%, or 21% of total rations on the performance of broilers at the starter stage.

Table 18: Performance (mortality, body weight gain (g), feed intake (g/b/d), FCR (g feed: g gain), apparent metabolizable energy (AME; kcal/kg diet); nitrogen retention (%); AME_N (AME corrected for nitrogen, kcal/kg diet)) of Ross 708 broilers during the starter phase (0 - 21 d) of Trial CTR1709. n= number of cages of birds/treatment, SEM = standard error of the mean, NS = not significant.

[0146] Over the 21 -day course of the starter diet phase, birds fed the 0% (control), 7%, or 14% BCM diets experienced similar weight gains, while the birds fed the 21% BCM diet had a slightly lower overall weight gain that was statistically significant. This effect correlated to the feed intake of the birds over the 21 -day interval whereby the birds fed the 21% BCM diet also displayed a slight, but statistically significant, decrease in feed intake relative to birds fed 0% or 7% BCM diets. Feed intake of birds fed 0%, 7%, or 14% BCM diets in the starter phase was not found to be significantly different from one another. The feed conversion ratio, i.e., the ratio of feed intake to weight gain for birds fed the four diets over the 21 -day period, was not found to differ significantly in relation to the proportion of BCM in the diet. Overall, male birds displayed a significantly higher weight gain over the 21 -day starter phase than the female birds; however, this was not unexpected based on published growth and feed intake targets for male and female Ross 708 birds in the starter phase (Ross 708 Broilers: Performance Objectives;

2014, http://en.aviagen.com/tech-center/download/600/Ross-708-Broiler-PO-2014-EN.pdf Aviagen Group, Huntsville, AL).

[0147] Protein content and ash content of feed samples and excreta sampled at 21 days were used to calculate the apparent metabolizable energy (AME) and AME corrected for nitrogen (AME_N) of the bird fed the various starter diets. All diets were initially formulated to meet or exceed the target AME requirement for ROS 708 broilers— i.e., 3000 keal/kg diet. As can be seen in Table 1, birds fed the 0%, 7%, and 14% BCM diets all had AME_N in this expected range, but the birds fed the 21% BCM diet had a lower AME_N than the targeted level of 3000 keal/kg diet. In addition, it was observed that the AME_N of birds fed 14% BCM diet was slightly, but significantly, lower than those of birds fed the 0% BCM diet and birds fed the 21% BCM diet had AME_N significantly lower than those fed 0% or 7% BCM diets.

[0148] After completion of the 21 -day starter phase, birds were shifted to grow /finish diets that, like the four experimental starter diets, contained either 0%, 7%, 14%, or 21% BCM. Grow /finish diets typically contain lower overall levels of protein than starter diets but have the same or higher energy content. The growth performance of Ross 708 broilers fed each of the four experimental grow /finish diets from day 21 to 35 post hatching is summarized in Table 19.

[0149] Consistent with the results observed during the starter phase, birds fed grow/ finish diets containing 0%, 7%, or 14% BCM showed virtually identical body weights at 35 days and similar feed intakes over the 21- to 35-day interval. By contrast, birds fed grow/ finish diets containing 21% BCM had significantly lower body weight at 35 days and feed intake during the 21- 35 days than those fed 0% or 14% BCM grow/ finish diets, although it should be considered that the 21 -d weight of this group was also significantly lower than those of birds fed 0%, 7%, or 14% starter diets and so the net weight gain for birds fed the 21% BCM over the 21 to 35 day period was actually comparable to the other groups. Feed conversion ratios for birds fed 0%, 7%, 14%, or 21% BCM grow/ finish diets were not significantly different from one another.

[0150] As observed during the starter phase, male birds achieved an overall significantly higher average body weight at 35 days than corresponding female birds and feed intake for male birds over the 21-35 grow/ finish period was also higher than for female birds. This differential behavior is consistent with observed norms for Ross 708 broilers (Ross Broiler Management Handbook; 2018, http://en.aviagen.com/assets/Tech_Center/Ross_Broiler/Ross- BroilerHandbook2018-EN.pdf ; Aviagen Group, Huntsville, AL).

Table 19: Performance (mortality, body weight (kg), feed intake (g/b/d), FCR (g feed: g gain) of Ross 708 broilers during the grow/finish phase (21 - 35 d) of Trial CTR1709. n= number of cages of

birds/treatment, SEM = standard error of the means, NS = not significant.

[0151] At 35 days post hatching, blood samples were taken from birds that had been fed 0%,

7%, 14%, or 21% BCM-supplemented diets. Samples were analyzed for thyroid hormone levels, blood calcium and phosphorus to assess possible limiting factors for bone development, blood glucose as an indicator of hypoglycemia, urea as an index for dietary protein utilization, the enzyme aspartate aminotransferase as a marker of liver damage, and creatine kinase as marker for potential cardiac and skeletal muscle abnormalities. The results of the analysis are shown in Table 20.

[0152] No significant differences were seen in mean levels of blood thyroid hormones, calcium, glucose, creatine kinase or aspartate aminotransferase among the blood samples taken from birds fed diets containing 0%, 7%, 14%, or 21% BMC. The presence of certain glucosinolates and their metabolites in animal feeds have been associated with both thyroid and liver abnormalities (Tripathi and Mishra, 2007. Animal Feed Sci. Technol. 1321-27; Khajali and Slominski, 2012. Poult. Sci. 91,(10): 2564-2575), while high levels of erucic acid in poultry diets has been shown to result in cardiac and skeletal muscle abnormalities (Hulan et al., 1982. Poultry Sci. 61 (6):

1154-1166). The present data, however, indicate that the residual levels of glucosinolates and erucic acid in BCM meal do not lead to such problems in broilers, even when BCM is incorporated into broiler diets at up to 21% inclusion rates.

Table 20: Blood thyroid hormone levels (nmol/L), blood electrolytes and liver chemistry values on birds at 35 d for Trial CTR1709. One bird sampled/replication. n= number of cages of birds/treatment, SEM = standard error of the mean; NS = not significant.

[0153] In contrast, significant decreases in blood phosphate and uric acid were observed, which correlated with increasing inclusion of BCM in the bird’s diets. In broilers, uric acid is an end product of nitrogen metabolism and thus its levels in blood correlate directly with efficiency of protein and amino acid utilization (Donsbough et al, 2010. Poultry Science: 89 (2): 287-294). Phytates, naturally occurring compounds found in many seeds and grains and which are present at appreciable amounts in BCM, are known to form complexes with minerals such as phosphate and may also complex with amino acids and protein and prevent their absorption in the gut of broilers and other livestock (Selle et al, 2000. Nutrit. Res. Reviews, 13(2), 255-278). Typically, this can be easily addressed by addition of enzymes to the feed ration which break down phytate, a standard industry practice when it is known that a feed ingredient may contain significant amounts of phytate.

[0154] Over the entire start and grow/fmish phases and among 336 birds, only two mortalities were recorded. This represents a very low level of mortality and indicates that levels of up to 21% BCM supplementation are safe for use in broiler diet applications.

[0155] In summary, the present data indicate that BCM incorporated into starter and grow/finish diets for broilers is a well-tolerated and safe nutritional additive even up to 21 % inclusion rate. When incorporated at inclusion rates of up to 14%, BCM supports the growth of broilers at both start and grow finish stages to weights equivalent to the control feed ration. While birds did not achieve the target weights described in the literature for ROS 708 broilers (Ross 708 Broilers: Performance Objectives; 2014, http://en.aviagen.com/tech-center/download/600/Ross-708- Broiler-PO-2014-EN.pdf Aviagen Group, Huntsville, AL), this was also the case for birds fed the control diets which contained no BCM and so was not a result of BCM inclusion. Blood work done on birds after completion of the grow/fmish phase indicated that BCM inclusion did lead to significant changes in markers that would be indicative of thyroid, liver or cardiac abnormalities. While lower blood phosphate and uric acid levels were seen to correlate with increased BCM incorporation, these changes were indicative of nutrient absorption and utilization differences that might be corrected by further adjustment of feed ration components and/or supplementation of the feed rations with specific additives, such as phytase.

[0156] The citation of any publication herein is not an admission that the publication is prior art with respect to the present application.

[0157] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it is readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the scope of the appended claims.

[0158] It is to be understood that any numerical value inherently contains certain errors necessarily resulting from the standard deviation found in the respective testing measurements. Also, as used herein, the term“about” generally means within 10%, 5%, 1%, or 0.5% of a given value or range. Alternatively, the term“about” means within an acceptable standard error of the mean when considered by one of ordinary skill in the art. Unless indicated to the contrary, the numerical parameters set forth in the present disclosure and attached claims are approximations that can vary as desired. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0159] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise. Unless defined otherwise all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs.

[0160] The phrase "and/or," as used herein in the specification and in the claims, should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with "and/or" should be construed in the same fashion, i.e., "one or more" of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the "and/or" clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to "A and/or B", when used in conjunction with open-ended language such as "comprising" can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0161] As used herein in the specification and in the claims, "or" should be understood to encompass the same meaning as "and/or" as defined above. For example, when separating items in a list, "or" or "and/or" shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items.

[0162] As used herein, whether in the specification or the appended claims, the transitional terms "comprising", "including", "carrying", "having", "containing", "involving", and the like are to be understood as being inclusive or open-ended (i.e., to mean including but not limited to), and they do not exclude unrecited elements, materials or method steps. Only the transitional phrases "consisting of' and "consisting essentially of', respectively, are closed or semi-closed transitional phrases with respect to claims and exemplary embodiment paragraphs herein. The transitional phrase“consisting of’ excludes any element, step, or ingredient which is not specifically recited. The transitional phrase“consisting essentially of’ limits the scope to the specified elements, materials or steps and to those that do not materially affect the basic characteristic(s) of the invention disclosed and/or claimed herein.

Claims

Claims:

1. A meal fraction of Brassica carinata oilseed comprising, on a percentage of dry matter,

(a) from about 35% to about 55% protein;

(b) from about 15% to about 30% neutral detergent fibre;

(c) from about 8% to about 15% acid detergent fibre;

(d) from about 1% to about 8% lignin; and

(e) from 0 to about 45 mhioΐ total glucosinolate per gram of meal.

2. The meal fraction of Brassica carinata oilseed of claim 1 comprising, on a percentage of dry matter,

(a) from about 42% to about 50% protein;

(b) from about 20% to about 26% neutral detergent fibre;

(c) from about 8% to about 12% acid detergent fibre;

(d) from about 1% to about 5% lignin; and

(e) from 0 to about 30 mhioΐ total glucosinolate per gram of meal.

3. The meal fraction of Brassica carinata oilseed of claim 1 , further comprising, on a

percentage of dry matter:

(a) from about 1% to about 5% oil; and

(b) from about 6% to about 10% ash.

4. The meal fraction of Brassica carinata oilseed claim 1, further comprising, on a

percentage of dry matter, from about 0.1% to about 3% erucic acid.

5. The meal fraction of Brassica carinata oilseed claim 2, further comprising, on a

percentage of dry matter, from about 0.1% to about 3% erucic acid.

6. The meal fraction of Brassica carinata oilseed of claim 1, further comprising, on a

percentage of dry matter, from about 0.1% to about 0.5% oleic acid.

7. The meal fraction of Brassica carinata oilseed of claim 2, further comprising, on a

percentage of dry matter, from about 0.1% to about 0.5% oleic acid.

8. The meal fraction of Brassica carinata oilseed of claim 1, further comprising, on a

percentage of dry matter, from about 0.1% to about 1% linolenic acid.

9. The meal fraction of Brassica carinata oilseed of claim 2, further comprising, on a

percentage of dry matter, from about 0.1% to about 1% linolenic acid.

10. The meal fraction of Brassica carinata oilseed of claim 1, wherein the meal fraction is produced from seeds of a Brassica carinata variety comprising at least 18% protein, on a dry weight basis.

11. A feed ration for comprising the meal fraction of Brassica carinata oilseed of claim 1.

12. A feed ration for comprising the meal fraction of Brassica carinata oilseed of claim 2.

13. The feed ration of claim 11, wherein from about 5% to about 25% of dry matter in the feed ration is the meal fraction of Brassica carinata oilseed.

14. The feed ration of claim 11, wherein the feed ration comprises up to 10 pmol

glucosinolate per gram of feed.

15. The feed ration of claim 11, wherein the feed ration is for feeding ruminant livestock, monogastric livestock, poultry livestock, camelid livestock, or farmed fish.

16. The feed ration of claim 15, wherein the ruminant livestock is cattle, sheep, goats, bison, or buffalo.

17. The feed ration of claim 15, wherein the monogastric livestock is swine.

18. The feed ration of claim 15, wherein the poultry livestock is chicken, turkey, geese, or duck.

19. The feed ration of claim 15, wherein the camelid livestock is alpaca, camel, or llama.

20. The feed ration of claim 15, wherein the farmed fish are salmonids, carp, tilapia, and catfish.