Characterization of the Nutritional and Safety Properties of Hemp Seed Cake as Animal Feed Ingredient

Full text

Vol. 12(2), pp. 53-63, April-June 2021
DOI: 10.5897/IJLP2020.0750
Article Number: 6BD8EF266388
ISSN 2141-2448
Copyright © 2021
Author(s) retain the copyright of this article
http://www.academicjournals.org/IJLP
International Journal of Livestock
Production
Full Length Research Paper
Characterization of the Nutritional and Safety
Properties of Hemp Seed Cake as Animal
Feed Ingredient
Rajasekhar Kasula1, Fausto Solis1*, Byron Shaffer2, Frank Connett2, Chris Barrett2,
Rodney Cocker2 and Eric Willinghan3
1Wenger Animal Nutrient and Technology Innovation Center, The Wenger Group, 101 West Harrisburg Ave, Rheems,
PA 17570, USA.
2Kreider Farms, 1145 Colebrook Rd, Mount Joy, PA 17552, USA.
3Winfield Veterinary Consulting, Inc., Lake Worth, Florida, USA.
Received 25 October, 2020; Accepted 4 March, 2021
Although the nutrient composition of hemp products provides evidence that these potentially serve as
valuable livestock feed ingredients and may enhance human health, the cultivation of hemp was
prohibited due to the high content of the Δ-9 tetrahydrocannabinol (THC). Recently, regulatory changes
by several countries allowed the cultivation of industry hemp under a license that permits plants and
plant parts of the genera Cannabis with a THC lower than 0.3%. The concern of a higher THC value still
remains; thus, it is justified to test the nutritional and safety properties of Hemp Seed Cake (HSC) in
animal feed. The objectives of this study were to determine the nutritional (proximate principles,
minerals, amino acids and fatty acids), and safety properties (mycotoxin, heavy metals and cannabinoid
profiles) of HSC and feed manufactured with the ingredient for use in animal feed. Three replicate
samples of HSC and two replicate samples of each feed manufactured with 0, 10%, 20 and 30% of HSC
were analyzed by reference laboratories for parameters identified under study objectives. The results of
the nutritional values were consistent with published results. Similarly, the safety parameters were
below the detectable levels and maximum legal levels. The results of this study confirm that HSC can
safely be used as animal feed ingredient.
Key words: Hemp, Δ-9 tetrahydrocannabinol, cannabinoids, safety, heavy metals, hemp seed cake.
INTRODUCTION
The Food and Agriculture Organization (FAO) forecasts
that the human population will increase by 30% by 2050
(FAO, 2019) with corresponding increase in demand for
food. Animal protein, the largest component of human
food is entirely dependent on livestock production
channels as its source. Over 70% of the cost of livestock
production is feed, and the second largest component
and cost of feed is the crude protein, a segment that has
been challenged for its sufficiency for decades forcing the
commercial and scientific communities to be innovative
and creative. Several unconventional and less
conventional ingredients have been explored as
*Corresponding author. E-mail: fausto.solis@thewengergroup.com. Tel: 800-692-6008, 717-917-7545/717-361-4211.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution
License 4.0 International License

54 Int. J. Livest. Prod.
alternative protein sources for livestock in the last several
decades.
Hemp (Cannabis sativa L.) is an annual herbaceous
plant belonging to the family Cannabinaceae (Turner et
al., 1979), traditionally grown for fiber and seed
production. Whole hemp seed contains approximately
25% crude protein, 33 to 35% oil, and 34% carbohydrate,
in addition to a broad range of vitamins and minerals
(Darshan and Rudolph, 2000; Callaway, 2004; House et
al., 2010). Hemp seed oil contains 75 to 80%
polyunsaturated fatty acids (PUFA), including 60%
linoleic acid and 17 to 19% α-linolenic acid (ALA) (Parker
et al., 2003). After the extraction of the oil from the seed
with a cold press; then the cakes are run through a
hammer mill to produce hemp seed cake with a
consistent particle size. The nutrient composition of hemp
products provides evidence that these products may
serve as potentially valuable livestock feed ingredients.
In the past, the cultivation of hemp was prohibited due
to the high content of Δ-9 tetrahydrocannabinol (THC) a
psychoactive substance present in the hemp plant. In the
recent decades, regulatory changes undertaken by
several countries across the globe allowed for the legal
cultivation of industry hemp under a license that permits
plants and plant parts of the genera Cannabis, the leaves
and flowering heads of which do not contain more than
0.3% THC, and includes the derivatives of such plants
and plant parts. The nutritional profile, in addition to the
increase in production and availability of hemp and hemp
products create opportunities to use them in livestock
diets (Gakhar et al., 2012). Significant research across
the globe that has gone into evaluating the safety of the
ingredient showed that including hemp in animal feed is
safe and offers benefits for improved animal performance
and human health (Gakhar et al., 2012; Jing et al., 2017;
Kasula et al., 2021c). Initial research indicates hemp
products in layers, in addition to the protein contribution,
are valuable sources of linoleic acid which is important to
improve egg weight (Parker et al., 2003; Silversides and
LeFranc, 2005) and linolenic acid and omega fatty acids,
which have proven beneficial effects on human health
(Lewis et al., 2000; Erasmus, 1993; Silversides et al.,
2002; Kasula et al., 2021a). Hemp products are also
shown to be excellent sources of yolk pigmentation, lutein
and fatty acid enrichment of eggs. Genetic improvements
to limit Δ-9 tetrahydrocannabinol to less than 0.3% (w/w)
in hemp leaves and flowering heads of the genera
Cannabis, have made them safer as a feed ingredient.
The use of HSC has not been approved in diets for any
class of livestock in the USA due to a lack of research in
support of its safety and efficacy. The current study is
designed to determine the feeding potential and safety to
be used as animal feed ingredient.
Objectives of the study
The objective of the study is to characterize the nutritional
and safety properties of HSC as animal feed ingredient
as determined by the nutritional (proximate principles,
minerals, amino acids and fatty acids) and safety (heavy
metal, mycotoxin and cannabinoid) profiles of HSC and
its inclusion at increasing levels in finished diets.
MATERIALS AND METHODS
Processing of hemp HSC and location
After the hemp seed is harvested, it is delivered to the processing
plant. Upon arrival, it is tested to make sure that it meets quality
standards. From there, it is sent through a cleaner to remove
foreign material, weed seeds, and other unwanted material. After it
is cleaned, it is placed in a storage bin where it is kept until
processing. From the storage bin, the hemp seeds move to a cold
press to extract the oil. This is a mechanical process so hexane, or
other chemicals, are not used in the extraction of the hemp seed oil.
The cold press produces a solid cake which is then run through a
hammer mill to produce Hemp Seed Cake (HSC) with a consistent
particle size. After the product runs through the hammer mill, it is
placed in a storage bin for shipment or sent to a bagging facility to
be bagged before shipping.
The HSC used in the current study was processed in the lot
number 005-2019 and originated from seeds collected from a hemp
plant variety CRX-1 which was grown locally, procured and
processed by Susquehanna Mills, 349 Village Rd, Pennsdale, PA,
USA, with the geographical latitude 40.899938,-77.570296. The
feed was manufactured at the Wenger Feed mill located at the 101
West Harrisburg Ave, Rheems, PA 17552.
Study design
In order to determine the nutritional and safety properties of HSC
and its increasing levels in finished diets, the study was designed to
analyze the HSC and iso-caloric, and iso-nitrogenous diets
containing 16% crude protein, as follows : 1. Control diet – regular
diet with no HSC (C0), 2. Regular diet with 10% HSC (H10), 3.
Regular diet with 20% HSC (H20) and 4. Regular diet with 30%
HSC (H30).
Study parameters
The study parameters were classified under two categories as
nutritional and safety. Three samples of every batch of HSC and
two samples of each of the four types of finished feed manufactured
for the study were analyzed for nutritional parameters comprising of
proximate principles including moisture, crude protein, crude fat,
crude fiber, total ash, minerals, amino acids and fatty acids
analyzed at the New Jersey Feed Lab, Trenton, NJ, and Eurofins
Laboratories, (Food Integrity and Innovation-Madison, Madison,
WI). The safety parameters included mycotoxins, heavy metals
analyzed at the New Jersey Feed Lab, Trenton, NJ, and
cannabinoid profile, at Eurofins Laboratories, (Food Integrity and
Innovation-Madison, Madison, WI).
Test and analytical methods
Nutritional parameters
Moisture and dry matter (method 925.09 (AOAC, 2000):
(i) Set the oven at 80°C before entering the sample

(ii) Weigh 1 g of the ground sample and weigh the paper that will
contain the sample.
(iii) Leave samples in the oven for 48 h; weigh the sample at 24 h
(iv) Leave the sample in a desiccator for 10 minutes until the
sample reach constant weight,
(v) Use the following formula to calculate moisture and dry matter
(vi) % moisture: ((wet weight-dry weight)/wet weight) × 100
(vii) % dry matter: 100-% moisture
Crude protein (method 990.03 (AOAC, 2000)
(i) Weigh and place 0.5 g of the feed sample in a filter paper
(ii) Transfer the sample in a crystal bottle
(iii) Add 3.5 g of the catalytic mix into the bottle
(iv) Add 8.5 ml of sulfuric acid and then turn on the stove to heat up
the bottle with the sample and the catalytic mix for about 90
minutes or until the sample has a green color
(v) Allow the sample to cool down for 5 min;
(vi) Add 25 ml of distilled water and gently move the bottle to
spread the water around the bottle (inside).
(vii) Add 30 ml of boric acid and 2 drops of indicator
(viii) Turn on the distiller and open the water to recirculate this
(ix) Transfer sample into the distiller and pour sodium hydroxide into
the boiling chamber until it takes a brown coffee color.
(x) Hold the beaker under the distiller and collect not less than 20
ml
(xi) Pour 0.1 N Chloridric acid into the sample until it turns to pink
color
(xii) Write down the amount of 0.1 N Chloridric acid spent
(xiii) Calculate the % protein with the following formula:
% protein= spent ml × 0.28× 6.25
Crude fat (method 963.15 (AOAC, 2000)
(i) Weigh 5 g of the sample and weigh the cartridge where the
sample will be in
(ii) In a beaker previously weighed add 70 ml of petroleum ether
(iii) Put the beaker in the extractor-digester
(iv) Heat up the extractor until the sample start boiling
(v) Have the sample boing for 6 h and the condensation should be
at 60 drops per minute
(vi) After the 6 h, put the beaker to evaporate at room temperature
(vii) After the evaporation, put the beaker in the oven to dry for 1 h
and temperature of 80°C
(viii) After 1 hour drying, put the sample in the desiccator and then
have a constant weight
(ix) Calculate the % of fat with the following formula:
% fat= (fat net weight/sample weight) × 100
Crude fiber
The crude fiber was determined according to AOAC (2000); briefly
two grams of defatted sample were treated successively with boiling
solution of H2SO4 of 0.26 N and KOH of 0.23 N. The residue was
then separated by filtration, washed and transferred into a crucible
then placed into an oven adjusted to 105°C for 18-24 h. The
crucible with the sample was weighed and ashed in a muffle
furnace at 500°C and weighed. The crude fiber was calculated
using the following equation:
CF=W2-W1W3 × 100
Where:
CF= Crude fiber
W1= Weight of crucible with sample before ashing
W2= Weight of crucible with sample after ashing
Kasula et al. 55
W3= Weight of sample
Total ash (method 942.05 (AOAC, 2000)
(i) Weigh the container where the sample will be in
(ii) Take 3 g of the ground sample and put into the container
(iii) Put the sample plus the container into the oven at 500°C for 3 h
(iv) After the 3 h, put the sample in the desiccator until the sample
reach constant weight
(v) Calculate the % of ash with the following formula:
% ash= (ash weight-weight of the container)/weight of the sample)
× 100
Minerals and heavy metals
Mineral Elements by ICP Emission Spectrometry (ICP-OES)
(Methods of Analysis of AOAC International, Method 984.27) has
been cited by Applegate et al. (2009). The minerals analyzed with
the procedure are: Calcium, Ca, Iron, Fe, Sodium, Na, Copper, Cu,
Magnesium, Mg, Manganese, Mn, Phosphorous, P, Potassium, K,
Zinc, Zn, Aluminum, Al, Barium, Ba, Boron, B, Chromium, Cr,
Molybdenum, Mo, Strontium, Sr, Beryllium, Be, Cadmium, Cd,
Cobalt, Co, Nickel, Ni, Vanadium, V.
Samples are either dry ashed, wet ashed, or read directly. If dry
ashed, the sample is placed in a muffle furnace set to maintain
500°C until ashing is complete. The resulting ash is treated with
concentrated hydrochloric acid, dried and re-dissolved in
hydrochloric acid solution. If wet ashed, the sample is digested in a
microwave or on a hot plate with nitric acid, hydrochloric acid,
and/or hydrogen peroxide. The amount of each element is
determined with an ICP spectrometer by comparing the emission of
the unknown sample against the emission of each element from
standard solutions.
The official methods of analysis of AOAC International method
984.27, 985.01, and 2011.14, AOAC International, Gaithersburg,
MD, USA (Modified). For the general analysis of component
minerals, samples are mineralized by an ashing process at 600°C.
The resulting ash is dissolved in mixed acids, diluted as required,
and analyzed via ICP-OES (Inductively Coupled Plasma Optical
Emission Spectrometry) at (New Jersey Feed Laboratory, Inc.,
Trenton, NJ).
Amino acids
The samples are hydrolyzed in 6 N Hydrochloric acid for 24 h at
approximately 110°C. Phenol is added to the 6N Hydrochloric acid
to prevent halogenation of tyrosine. Cystine and Cysteine are
converted to S-2-carboxyethylthiocysteine by the addition of
dithiodipropionic acid. Tryptophan is hydrolyzed from proteins by
heating at approximately 110°C in 4.2 N Sodium Hydroxide. The
samples are analyzed by HPLC after pre-injection derivitization.
The primary amino acids are derivitized with o-phthalaldehyde
(OPA) and the secondary amino acids are derivitized with
fluorenylmethyl chloroformate (FMOC) before injection (Schuster,
1988; Henderson et al., 2000).
Fatty acids
The fatty acid composition was determined using standard gas
chromatographic techniques of the fatty acid methyl esters (AOAC,
2000, method 969.33), using C17:1 fatty acid (Nu-Chek Prep, Inc.,
Elysian, MN) as an internal standard. Total lipids were extracted
from the HSC and test diets by homogenization in
chloroform/methanol (2:1, v/v) according to the methods of Folch et

56 Int. J. Livest. Prod.
al. (1957). After centrifugation, the organic phase was collected and
evaporated under a N2 stream. The all lipid extracts obtained were
trans-esterified with methanolysis (1% (v/v) H2SO4 in methanol) for
3 h at 70°C. After cooling, the resulting fatty acid methyl esters
(FAMEs) were extracted with hexane and transferred into gas
chromatography (GC) vials. All solvents contained 0.005% (v/v)
butylated hydroxyanisole (BHA) as an antioxidant. FAMEs were
then separated and quantified with a Varian450-GC with CP-8400
autosampler, equipped with a flame ionization detector and a GC
column (length 30 m, inner diameter 0.25 mm and film thickness
0.25 μm, DB-225MS) (Agilent Technologies, Mississauga, ON,
Canada). Nitrogen was the carrier gas at a column flow rate of 1
ml/min. The inlet split ratio was set at 10:1. The oven temperature
programming was as follows: 60°C for 1.5 min, raised to 180°C at
20°C/min, 205°C at 6°C/min, 220°C at 2°C/min for 4 min, and
240°C at 10°C/min for 3 min. The injector and detector temperature
were set at 260 and 290°C, respectively. FAMEs were identified by
comparison of retention times to known lipid standards (Nu-Chek
Prep, Inc., Elysian,Mn) (Folch et al., 1957; Jing et al., 2017).
Safety parameters
Mycotoxins
Mycotoxin concentration was determined by ELISA at the New
Jersey Feed Laboratory with the following procedure: The
mycotoxin–protein conjugates were adsorbed in separate
microplate wells and washed. Samples in solution (50 μl) with a
methanol content of 25%, and 50 μl of specific antibodies in PBST
(at concentrations of 100, 100, and 500 ng/ml for AFB1, OTA, and
ZEA, respectively) were added into the wells and incubated for 8
min with vigorous stirring at room temperature. After washing, a
diluted solution of the streptavidin–polyperoxidase conjugate
(1:4000 in PBST) was added at 100 μl per well and incubated for 6
min at 37 °C with vigorous stirring. The microplate was then
washed four times with PBST, and following 8 min incubation with
the substrate solution and the formed immune complexes were
detected and quantitatively characterized.
Heavy metals
The levels of heavy metals were determined in HSC and finished
feed with the procedure outlined in the mineral procedure section.
Cannabinoids
The levels of cannabinoids in HSC and feed were determined with
the following procedure: HSC samples by triplicate and feed
samples by duplicate were shipped overnight for the analysis of the
residues of various hemp cannabinoids to Eurofins Laboratory,
Madison, WI, method 2018.11, by the procedures described in the
publication "Quantification of Cannabinoids in Cannabis Dried
Plant Materials, Concentrates, and Oils Liquid Chromatography-
Diode Array Detection Technique with Optional Mass Spectrometric
Detection (Lukas et al., 2018).
Statistical analysis
The safety parameters, mycotoxins, and heavy metals were
analyzed with the General Linear Model Procedure (PROC GLM) of
SAS (SAS, 2012). The treatment mean separation was carried out
with the Tukey Multiple Range test with a probability of error of 5%
(P<0.05). The cannabinoids data were not subjected to statistical
analysis because all results below the detectable levels by
chromatographic methods in the laboratory.
RESULTS AND DISCUSSION
Nutritional composition of HSC and finished Feed
The analysis of nutritional composition of HSC and feeds
formulated with HSC are presented in Table 1. In
general, the nutritional composition results were within
the expected levels and in agreement with the results in
available published literature.
Moisture
The average moisture level of the HSC was 7.53%. This
moisture value is in the vicinity of those reported in
previous researches, 8.6% in hemp seed meal by
Silverside and Lefrancois (2005), 8.8% in HSC (Halle and
Schone, 2013) and 9.7% in HSC (Mierliță, 2019). The
analyzed moisture levels in the feed were at 12.12% in
the control compared to 11.21, 10.03 and 8.40% in the
H10, H20 and H30, respectively. This tendency of
decreasing moisture level with increasing levels of HSC
in finished feed may be attributed to higher dry matter
contributed by HSC while replacing corn and soybean
meal with soy oil.
Protein
The average analyzed crude protein content of HSC was
32.06%, which is within the range of previous reports,
31.22% reported by Mierliță (2019), and 28.1% by Halle
and Schone (2013). The analyzed protein levels of the
finished feeds were at 14.81% in the control, 16.31, 16.75
and 16.57% in the H10, H20 and H30, respectively. The
analytical variance in crude protein levels in the feed was
found to be closer in trends to those reported by
Silverside and Lefrancois (2005) who reported crude
protein levels of 17.5% in all the control, 5, 10 and 20%
HSC treatments in layer feeds when using hemp seed
meal, while Halle and Schoene (2013) reported 15.9,
16.5 and 16.9% when HSC was included in the feed at 5,
10 and 15% of the diets, respectively.
Crude fat
The average crude fat levels of HSC were at 9.02%,
lower than 12.35% reported by Mierliță (2019) and 11%
reported by Halle and Schone (2013). The analyzed
levels of crude fat levels in finished feed were at 2.70% in
the control, 5.57, 8.78 and 11.47% in the H10, H20 and
H30, respectively (Table 1). The higher levels of crude fat
with increasing levels of HSC in feed may be attributed to
the high level of fat (9.02%) of the HSC compared to

Kasula et al. 57
Table 1. Hemp seed cake and Feed nutritional analysis (% as is basis).
Nutrients
Hemp seed cake (HSC) and treatments
HSC
SD
C0
SD
H10
SD
H20
SD
H30
SD
Moisture
7.53
0.31
12.12
0.01
11.21
0.38
10.03
0.47
8.40
0.20
Protein (Crude)
32.06
0.30
14.81
0.51
16.31
0.19
16.75
0.06
16.57
0.25
Fat (Crude)
9.02
0.03
2.70
0.00
5.57
0.05
8.78
0.26
11.47
0.16
Fiber (Crude)
32.21
0.44
1.79
0.11
4.92
0.87
7.07
0.18
9.82
0.11
Ash
5.38
0.05
11.27
0.21
11.48
0.28
12.71
0.04
12.21
0.55
Minerals (%)
Ca
0.17
0.01
3.38
0.03
3.18
0.08
3.61
0.24
3.45
0.14
P
0.71
0.47
0.50
0.06
0.50
0.01
0.56
0.04
0.57
0.01
Na
0.01
0.00
0.14
0.01
0.14
0.01
0.16
0.01
0.15
0.01
Mg
0.48
0.01
0.17
0.01
0.21
0.00
0.26
0.01
0.28
0.00
Mn (ppm)
133.00
0.58
78.50
3.54
93.55
1.77
135.00
9.90
145.00
7.07
Fe (ppm)
133.67
2.01
283.50
38.89
260.00
7.07
261.50
13.44
244.00
12.21
Zn (ppm)
77.83
0.56
86.15
7.85
89.60
4.53
123.50
10.61
128.00
2.83
Cu (ppm)
18.83
0.46
19.40
0.28
17.55
0.35
17.95
0.07
19.20
3.54
K
0.95
0.02
0.73
0.05
0.72
0.01
0.73
0.04
0.62
0.00
Amino acids (%)
Methionine
0.51
0.12
0.42
0.10
0.42
0.01
0.44
0.10
0.52
0.01
Cysteine
0.34
0.05
0.24
0.04
0.23
0.00
0.22
0.02
0.24
0.01
Lysine
1.13
0.02
0.86
0.05
1.04
0.05
1.00
0.05
0.97
0.16
Phenylalanine
1.24
0.01
0.72
0.02
0.81
0.01
0.71
0.00
0.75
0.00
Leucine
1.93
0.02
1.34
0.03
1.45
0.03
1.25
0.01
1.29
0.00
Isoleucine
0.91
0.01
0.52
0.02
0.69
0.02
0.52
0.01
0.61
0.01
Threonine
1.18
0.03
0.59
0.07
0.72
0.01
0.67
0.02
0.66
0.06
Valine
1.13
0.02
0.57
0.03
0.77
0.01
0.61
0.02
0.76
0.01
Histidine
0.73
0.02
0.41
0.02
0.50
0.01
0.41
0.00
0.48
0.00
Arginine
4.00
0.05
0.93
0.06
1.26
0.01
1.39
0.02
1.82
0.04
Aspartic acid
1.37
0.03
1.60
0.13
1.63
0.02
1.76
0.00
1.56
0.11
Serine
3.55
0.03
0.82
0.07
0.87
0.05
0.82
0.02
0.77
0.05
Glutamic acid
1.45
0.02
2.73
0.23
2.70
0.01
2.75
0.03
2.46
0.23
Proline
4.94
0.03
1.07
0.06
1.03
0.02
0.99
0.01
0.98
0.06
Hydroxyproline
1.35
0.04
0.13
0.01
0.08
0.01
0.17
0.01
0.14
0.00
Alanine
1.16
0.01
0.78
0.05
0.84
0.01
0.70
0.04
0.78
0.01
Tyrosine
0.89
0.01
0.51
0.01
0.54
0.01
0.50
0.01
0.51
0.01
Tryptophan
0.27
0.00
0.10
0.01
0.11
0.01
0.19
0.01
0.13
0.01
Data are the mean of three replicates (n=3) of HSC and two replicates (n=2) of each feed type, HSC= hemp seed cake, C0= Control no
HSC, H10:10% HSC, H20:20%HSC, H30:30HSC. SD= standard deviation.
the fat content of the major ingredients corn (3.39%) and
soybean meal (1.88%) replaced and drawing of soy oil
into the formulation as a result of the inclusion of HSC
(Table 2).
Crude fiber
The crude fiber content of HSC was 32.21%, a value
found to be higher than 25.14% reported by Mierliță
(2019). The analyzed crude fiber content results of
finished feeds were at 1.79 and 4.92% in H10, 7.07% in
H20 and 9.82% in H30. This trend of higher crude fiber
content with the increasing level of HSC was due to high
level of crude fiber (32.21%) of the HSC. These trends of
crude fiber agree with those reported by Silverside and
Lefrancois (2005) who reported 2.29% in the control feed,
4.22, 6.15 and 10% in layer rations with 5, 10 and 20%
hemp seed meal, respectively.
Ash
The ash content of HSC was at 5.38%. Available

58 Int. J. Livest. Prod.
Table 2. Study diets formulated by treatment (% in an as is basis).
Ingredient
Hemp seed cake levels
C0
H10
H20
H30
Corn
65.24
59.40
53.34
45.96
Soybean meal- solvent
23.15
16.70
10.30
5.10
Calcium chip
4.90
4.85
4.90
4.90
Limestone
4.90
4.85
4.90
4.90
Monocalcium phosphate 21%
1.02
0.91
0.79
0.67
Salt
0.25
0.26
0.26
0.26
Methionine, DL
0.20
0.20
0.20
0.19
Sodium sesquicarbonate
0.18
0.18
0.18
0.18
Vitamin premix
0.05
0.05
0.05
0.05
Trace minerals premix
0.05
0.05
0.05
0.05
Choline, Liq. 70%
0.03
0.07
0.11
0.15
Alphagal 280 P
0.02
0.02
0.02
0.02
Phytase
0.01
0.01
0.01
0.01
HSC
0.00
10.00
20.00
30.00
Soybean oil
-
2.20
4.50
6.95
Lysine sulfate 60%
-
0.17
0.35
0.46
Tryptophan
-
0.02
0.05
0.07
Threonine
-
0.02
0.05
0.05
Ingredient total
100
100
100
100
Calculated Nutritional composition (%)
Moisture
11.57
13.32
16.13
17.06
Crude protein
15.86
15.88
15.90
16.34
Fat (Ether extract)
2.65
5.39
8.20
11.16
Crude fiber
1.99
5.01
8.01
11.04
Ash
12.34
11.80
11.79
10.79
Minerals
Available Ca
Available P
0.44
0.44
0.44
0.44
Na
0.17
0.17
0.17
0.17
Cl
0.195
0.195
0.195
0.195
Poultry ME (MJ/kg)
11.90
11.90
11.90
11.90
Amino acids
Lysine, digestible
0.75
0.764
0.777
0.787
Methionine, dig
0.43
0.43
0.43
0.42
Met & Cys, dig
0.65
0.65
0.64
0.63
Tryptophan, dig
0.17
0.17
0.17
0.16
Threonine, dig
0.53
0.53
0.52
0.52
Glycine, dig
0.59
0.58
0.56
0.57
Phenylalanine, dig
0.74
0.69
0.64
0.61
Leucine, dig
1.32
1.22
1.12
1.05
Histidine, dig
0.40
0.37
0.35
0.34
HSC= hemp seed cake, C0= Control no HSC, H10:10% HSC, H20:20%HSC, H30:30HSC.
published literature showed at 7.2% (Silverside and
Lefrancois (2005) in hemp seed meal, 6.81% (Mierliță, 2019) and 7.2% (Halle and Schone, 2013) in HSC. The
analyzed ash in the feed was at 11.27% in the control,

Kasula et al. 59
Table 3. Hemp seed cake fatty acid profile (% as is basis).
Fatty acids (%)
Hemp seed cake levels
HSC
SD
C0
SD
H10
SD
H20
SD
H30
SD
Total % W6
58.69
0.06
55.30
0.16
55.03
0.23
55.51
0.12
55.72
0.06
Linoleic 18:2 w6
55.26
0.05
55.30
0.16
54.59
0.23
54.80
0.10
54.91
0.04
Linolenic 18:3 w6
3.43
0.02
0.00
0.00
0.45
0.01
0.69
0.02
0.81
0.01
Total % W3
15.34
0.06
2.66
0.15
6.10
0.00
7.63
0.16
8.23
0.12
Linolenic 18:3w3
14.47
0.05
2.66
0.15
6.01
0.00
7.44
0.16
8.00
0.11
LA:ALA
3.82
0.05
20.79
0.15
9.08
0.01
7.36
0.16
6.86
0.11
Oleic 18:1 w7
1.05
0.01
0.80
0.00
1.16
0.01
1.21
0.01
1.26
0.01
Myristic acid
0.07
0.00
0.09
0.00
0.08
0.00
0.08
0.01
0.07
0.00
Palmistic acid
8.01
0.08
12.91
0.06
11.16
0.08
10.52
0.04
10.33
0.03
Palmitoleic
0.20
0.01
0.10
0.01
0.11
0.01
0.09
0.00
0.09
0.00
Heptadecanoic
0.00
0.00
0.00
0.00
0.06
0.00
0.06
0.00
0.07
0.00
Stearic
2.42
0.04
2.12
0.04
2.95
0.00
3.20
0.04
3.325
0.02
Oleic 18:1W9
11.10
0.01
23.70
0.26
21.28
0.08
19.40
0.09
19.025
0.13
Oleic 18:1W7
1.05
0.01
0.80
0.00
1.16
0.01
1.22
0.01
1.26
0.01
Octadecatetraenoic
0.87
0.01
0.00
0.00
0.10
0.00
0.19
0.01
0.22
0.01
Arachidonic
0.79
0.01
0.32
0.06
0.00
0.00
0.38
0.01
0.41
0.00
Eicosanoic
0.34
0.02
0.19
0.01
0.00
0.00
0.20
0.01
0.21
0.00
Behenic
0.36
0.01
0.18
0.00
0.27
0.01
0.27
0.00
0.30
0.01
Lignoceric
0.21
0.01
0.21
0.01
0.17
0.00
0.15
0.00
0.15
0.00
Data are the mean of three replicates (n=3) of HSC and two replicates (n=2) of each feed type. C0= Control no HSC, H10:10% HSC,
H20:20%HSC, H30:30HSC, LA=linoleic acid, ALA=alfa-linolenic acid.
11.48% in the H10, 12.71% in the H20 and 12.21% in the
H30. No published literature was available for comparison
of the ash content of the HSC inclusion in feed with
different levels in the current study.
Minerals
The average analyzed calcium level of HSC was at,
0.17%, found to be lower than 0.28% reported by Halle
and Schoene (2013). The analyzed finished feed had a
calcium level of 3.38% in the control compared to 3.18,
3.61 and 3.45% when HSC was included at 10, 20 and
30%, respectively (Table 1). The analyzed values of other
minerals of HSC and finished feed are presented in Table
1. No published literature was available on these
nutrients for comparison purposes.
Amino acids
The average total lysine level in the HSC was at 1.13%,
methionine at 0.51% and other amino acids as presented
in Table 1. The analysis of finished feeds showed an
average total lysine level of 0.86% in control, 1.04% in
H10, 1.00% in H20 and 0.97% in H30. Halle and Schone
(2013) reported total lysine level of 0.94, 0.87 and 0.85%
in layer feed using 5, 10 and 15% HSC, respectively.
The analyzed values of other amino acids of HSC and
finished feed are presented in Table 1. No published
literature was available on these nutrients for comparison
purposes.
Fatty acids
The HSC analyzed for an average total of Omega 6 fatty
acids at 58.69% that composed of linoleic acid (18:2 w6)
at 55.26% and, linolenic acid (18:3 w6) at 3.43%. The
total Omega 3 fatty acid level was 15.34% of which
linolenic acid (18:3w3) was 14.47% (Table 3).
In the finished feed, a general increase of omega 3
(total w-3) and individual fatty acids was noticed with
increasing levels of HSC; for example, total w-3 values
were 2.66, 6.10, 7.63 and 8.23%. The linoleic acid (LA)
(18:2 w6) did not present a variance between the HSC
treatments; it is at 55.30% in the control feed and 54.595
in the H10, 54.80% in the H20 and 54.91% in the H30.
The alfa Linolenic acids (ALA)(18:3 w6 and 18:3 w3)
were higher with the increasing levels of HSC; ALAin the
control treatment was 2.66% which was increased to
6.01%, 7.44%%, and 8.00% with the H10, H20 and H30,
respectively (Table 3). These results make linoleic acid:
linolenic acid ratios of 20.79, 9.08, 7.36 and 6.86 in the
control and 10, 20 and 30% of HSC. These values are
higher than those reported by Mierliță (2019) who

60 Int. J. Livest. Prod.
reported LA:ALA ratios of 7.98 in the control feed and
3.06 when HSC was included at 20.32% in layer feed.
Interestingly, one of the linolenic acids (18:3 w6) was not
detected in the control feed and increased with the
increasing levels of HSC in the feed from 0 in the control
feed to 0.45, 0.69 and 0.81% in the H10, H20 and H30,
respectively.
The world population will increase by 30% in the next
25 years (FAO, 2019) and along with that higher
population, the demand for food will also be higher. The
two most important nutrients in any type of food are
protein and energy and both combined represents more
than 70% of the food cost; thus, any feed ingredient that
can be used to feed animals will contribute to feed the
growing human population. The results of this study show
that HSC is a rich source of protein (32.06%) and very
good source of fat (9.02%) which contribute energy.
Additionally, HSC showed to have essential and non-
essential amino acids such as lysine and methionine to
support egg production and egg weight, respectively.
HSC also has a high level of threonine, a very important
amino acid to support the immune system; threonine is
the highest amino acid found in the mucin amino acid
backbone (Gum, 1992), which is a glycoprotein that
protects the animal from challenges. L-threonine, cannot
be synthesized by humans and animals (Dong et al.,
2011; Li et al., 2017; Fang et al., 2020); therefore, any
ingredient providing this amino acid is important for
animal performance and immunity. Another amino acid
found in high level in HSC is proline (4.94%). Although L-
proline is not considered to be an essential amino acid in
practical poultry diets, chicks fed purified amino acid diets
require 0.5% L-proline for optimal growth and feed
efficiency (Greene et al., 1962) and it has shown to
improve the skin collagen content in poultry (Christensen
et al., 1995); other amino acids such as arginine, serine,
and alanine are also in high levels in the HSC (Table 1)
which are very important for animal feeding and finally to
contribute with food security.
Safety of HSC and finished feed
In this study, owing to the large differences in the
nutritional values of HSC and experimental diets from
published research, the safety of ingredient was
addressed by analyzing for heavy metals, mycotoxins
and hemp cannabinoid levels. The safety data of the
HSC and finished feed represented by mycotoxins, heavy
metals are in Table 3; and the cannabinoids in Table 5.
Mycotoxins
The HSC showed an average of aflatoxin 0.000005%,
zearalenone 0.000025%, fumonisins 0.0001%, T-2
0.00019033%, ochratoxins 0.000002% and vomitoxins
0.00123333% (Table 4). The levels of mycotoxins were
also evaluated in the finished feed with results below the
maximum permissible limits. Aflatoxins were below
0.000005 % across all HSC treatments with no significant
difference among them. Zearalenone did not show any
specific trend with 0.000073% in the control, and
0.000047, 0.000089, and 0.000053% in the H10, H20
and H30, respectively. Fumonisins were recorded at
0.0001% across all treatments. T-2 was 0.000034 % in
the control and 0.00003350%, 0.000055 and 0.000056%
in the H10, H20 and H30 treatments, respectively (Table
4).
The levels of ochratoxins were recorded to be the same
at 0.0000002% in control, H10 and H30 treatments,
significantly different from H20 at of 0.00000003% (Table
4). Although the analyzed levels showed a significant
difference with H20 treatment, the values were below the
permissible limits and did not represent a threat to
animals. The average vomitoxin levels did not show
significant difference among the treatments and the
values ranged from 0.000055 % in the H30 to 0.000125%
in control and H20 and 0.000095% in H10 (Table 4).
The maximum level allowed of aflatoxin in an ingredient
for poultry is 0.000001% for feed intended for mature
animals and not more than 0.000002% if the feed is to be
used in immature animals. The maximum permissible
levels of Fumonisins are up to 0.001% in an ingredient
and not more than 0.0005% in finished feed;
Deoxinivalenol (DON) in an ingredient up to 0.0005%b
and not more than 0.0002% in finished feed as long as
the ingredient is not included at a rate above 50%. No
regulatory levels have been specified for the other
mycotoxins (FDA, 2020).
Heavy metals
The levels of heavy metals, arsenic, cadmium and lead in
HSC and experimental diets are reported in Table 4. The
levels of heavy metals in HSC were below laboratory
detectable levels of 0.000000005% and were below
those reported by several scientists as cited in Table 4.
The control ration showed significantly higher levels of
arsenic and cadmium over HSC diets. Arsenic was at
0.00000002% in the control, significantly higher than the
0.00000001% observed in all the HSC treatments.
Cadmium was recorded at 0.000000009% in the control
and was significantly lower at 0.00000000006% in the
H10 and H20 and 0.0000000005%in the H30. The lead
profiles of experimental rations did not vary significantly.
The heavy metal profile of HSC assessed at
<0.000005%for arsenic, lead and cadmium, was lower
than published literature on hemp seed or its products
and similar conventional processed agricultural
commodities such as soybean meal, sunflower meal,
canola meal and others (Table 5). Heavy metal levels of
various ingredients for animal feeds have been reported

Kasula et al. 61
Table 4. Mycotoxins, heavy metals and CBD/THC in HSC and finished feed (%).
Hemp seed cake levels
P-Value
SD
HSC
C0
H10
H20
H30
Mycotoxins
Aflatoxins
0.000005
0.000005a
0.000005a
0.000005a
0.000005a
0.00
0.00
Zearalenone
0.000025
0.000073a
0.000047a
0.000089a
0.000053a
0.79
45.83
Fumonisin
0.0001
0.0001a
0.0001a
0.0001a
0.0001a
0.00
0.00
T-2 Toxins
0.00019033
0.000034a
0.00003350a
0.000055a
0.000056a
0.05
6.83
Orchratoxins
0.000002
0.0000002b
0.0000002b
0.0000003a
0.0000002b
0.0001
0.00
Vomitoxins
0.00123333
0.000125a
0.000095a
0.000125a
0.000055a
0.08
212.13
Heavy metals
Arsenic
<0.00000005
0.000002b
0.0000001a
0.0000001a
0.0000001a
<0.0001
0.00
Cadmium
<0.00000005
0.00000009a
0.00000006b
0.00000006b
0.00000005c
<0.0001
0.00
Lead
<0.00000005
0.0000002a
0.0000002a
0.00000015a
0.0000002a
0.48
0.04
CBD/THC
<0.005
<0.005
<0.005
<0.005
<0.005
0.00
0.00
Data are the mean of three 3 replicates (n=3) of HSC and 2 samples (n=2) of feed diets. C0= Control no HSC, H10:10% HSC, H20:20%HSC,
H30:30HSC.Means with different superscripts are significantly different (P < 0.05).
Table 5. Levels of heavy metals in agricultural commodities and compounded feed (%).
Material
Heavy metal
References
Lead
Cadmium
Arsenic
Agricultural commodity
Hemp seed
0.00018
0.00011
NR
Linger et al., 2002; Ahmad et al., 2016
Alexieva et al., 2007
Soybean meal
0.000277
0.00004
0.0000193
Soybean cake
ND
0.00005
NR
Adeniji and Okedeyi, 2017
Soybean
0.00006
0.0000084
0.0000109
Khalili et al, 2018
Sunflower meal
0.000313
0.000096
0.0000103
Alexieva et al., 2007
Canola seeds
0.0000109
0.00000495
0.00000035
Yu et al., 2012
Canola plant
0.000074
0.0000028
NR
Abd El Lateef et al., 2013
Linseed
NR
0.000061
NR
Kymalainen and Sjobeg, 2006
Linseed crush
NR
0.000085
NR
Kymalainen and Sjobeg, 2006
Sunflower meal
0.000313
0.000096
0.0000103
Alexieva et al., 2007
Compound feed
Maximum tolerable levels (NRC, 2005)
0.0010
0.0010
0.0030
Deemy and Benjamin, CVM, FDA, 2019
Maximum tolerable levels (AAFCO-OP, 2019
0.0030
0.00005
0.0050
Deemy and Benjamin, CVM, FDA, 2019
EU, Maximum allowed concentration
NR
0.0001
NR
Alexieva et al., 2007
Compound layer feed
0.000777
0.000055
0.0000303
Alexieva et al., 2007
ND: Not Detected, NR: Not Reported.
to be at 0.000277% lead, 0.00004% cadmium,
0.0000193% arsenic in soybean meal (Alexieva et al
2007); and, 0.000313% lead, 0.000096% cadmium, and
0.0000103% arsenic in sunflower meal (Alexieva et al
2007). Other researchers have found heavy metal values
at 0.00018 and 0.0001 mg/kg of lead and cadmium,
respectively in hemp seed (Linger et al., 2002). In
Linseed and linseed crush, cadmium level was recorded
(Kymäläinen and Sjöberg, 2006) at 0.000061 and
0.000085%, respectively.
Hemp cannabinoids
The hemp cannabinoid levels of HSC and finished diets
(Table 4) were reported to be below the detectable levels

62 Int. J. Livest. Prod.
of 0.005% by chromatographic methods in the laboratory
and were under the legal limits of 0.3%. The primary
concern with feeding HSC to animals continues to be the
transfer potential of hemp cannabinoid residues, mainly
cannabidiol (CBD) and delta-9-tetrahydrocannabinol
(THC). Published research states that a level of Δ-9
tetrahydrocannabinol (THC), a psychoactive substance in
the hemp plant (Health Canada, 2012) below 0.3% is
safe for animal feeding (Jing et al., 2017).
Researchers from the European Monitoring Centre of
Drug Addiction have reported that HSC may be fed safely
to about 30% of the diet to hens (EFSA, 2011). It has
been reported that the use of hemp seed up to 30%; up
to 10% and up to 20% did not have adverse effects in
laying hens (Kasula et al., 2021b).
Most of the published literature on this subject and
related areas happens to be with using whole hemp seed
or hemp oil or other hemp products. Given the limited
published research on the safety of feeding HSC in
livestock, the authors are constrained with few supporting
references to quote on the findings. The authors have
attempted to align with the closest possible references.
The current study demonstrates no contribution or
transfer of cannabinoids to finished feed from HSC. The
safety of any feed ingredient is of upmost importance to
prevent potential intoxication, animal health issue, lower
performance and the potential food safety issue by
passing any residues to humans. The three safety
parameters evaluated in this study (mycotoxins, heavy
metals, and cannabinoids/THC) were not detected or
detected at such lower level that do not represent any
threat to livestock or human health. The HSC tested in
this trial was later tested in laying hens for a period of 19
weeks, 3 of adaptation and 16 of the experiment, and the
levels of these three safety factors were not detected or
detected below the legal levels as reported by Kasula et
al. (2021a, b, c); these research used the same HSC
reported in this study.
Conclusions
The current study has sufficiently evaluated and captured
nutritional and safety properties of HSC and finished
feeds thereof, with demonstrated conclusions as follows:
(i) HSC presented a nutritional profile consistent with the
published literature and contained mycotoxin and heavy
metals at levels much lower than permissible.
(ii) HSC did not contain hemp cannabinoids and related
compounds detectable by available laboratory methods
of analysis.
(iii) HSC could be conveniently accommodated in feed
formulations and safely fed up to 30% in commercial
laying hen diets.
(iv) HSC may be included as a safe animal feed
ingredient.
CONFLICT OF INTERESTS
The authors have not declared any conflict of interests.
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