Cannabis sativa L. as a Feed Source in Backyard Rabbit Production

Final report for GS20-229

Project Type: Graduate Student
Funds awarded in 2020: $16,419.00
Projected End Date: 08/31/2021
Grant Recipient: Tarleton State University
Region: Southern
State: Texas
Graduate Student:
Major Professor:
Dr. William Smith, Ph.D.
Tarleton State University
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Project Information

Summary:

Recent 2018 Farm Bill hemp legislation may offer an alternative livestock feed protein source. Hemp and marijuana are both derived from the plant Cannabis sativa L., with the sole difference being that hemp must contain less than 0.3% tetrahydrocannabinol, the psychoactive component in the plant. Hemp oil, derived from C. sativa seed, has been gaining popularity over recent years for its use in cosmetics, pharmaceuticals, and human food products. The seed meal that is left after oil extraction is what we will evaluate as a protein source in livestock. Limited research has been conducted on hemp seed meal as a protein feed in livestock, but current publications indicate it may effectively replace other common protein sources in ruminant diets. The objectives of this research are 1) to evaluate variation in nutrient composition and in vitro digestibility of hemp seed meal from various sources, and 2) to evaluate hemp seed meal as a protein replacement in the diet of meat rabbits. Hemp seed meal sample sources will be the sole factor and will be obtained from a minimum of four oil processing factories. Each sample will have four replications from distinct batches at each source. These replications will be the experimental unit. Chemical analysis will be run on each replication and variability among sources will be determined. Once nutritive value variability has been determined, hemp seed meal will be used to replace protein source in meat rabbit diets.

Project Objectives:

The objectives of this project are:

  1. To evaluate variability in nutrient composition and in vitro digestibility of hemp seed meal from various sources
  2. To evaluate the effect of hemp seed meal as commercial pellet ingredients for meat rabbits

Research

Materials and methods:

Objective 1

Hemp processing facilities within the United States of America that used cold pressing as the method of oil extraction were contacted to obtain hempseed meal (HSM) samples. Processors were asked to provide distinct samples from as many batches as possible. All hemp seed was collected from C. sativa plants containing less than 0.3% THC per the 2018 Farm Bill (United States Congress, 2018).

Upon receipt, HSM samples were dried in a forced-air oven at 55oC until weight loss ceased. Samples were then ground to pass through a 2-mm screen in a Wiley mill (Arthur H. Thomas Co., Philadelphia, PA, USA); a subsample was ground to pass thorugh a 1-mm screen.

The experimental design for determining source of variaiton was a completely randomized design. A total of four processing facilities and 15 HSM batches were represented in the experiment. Four laboratory replicates of each batch × source combination were assayed.

Carbon and nitrogen concentrations were determined by combustion using the Dumas total combustion method in a Leco Cornerstone CN 828 (Elementar Americas, Mt. Laurel, NJ, USA; Method 990.09; AOAC, 2000). Nitrogen concentration was used to calculate CP content by multiplying by 6.25 (AOAC, 1984). Neutral detergent fiber (NDF), acid detergent fiber (ADF), and acid detergent lignin (ADL) were measured with the ANKOM200 Fiber Analyzer (ANKOM Technology Corporation, Fairport, NY, USA) using modified methods (Vogel et al., 1999) originally described by Van Soest and Robertson (1980). Acid detergent lignin was measured by the sulfuric acid (Method 973.18; AOAC, 2000). An ANKOM DaisyII incubator (ANKOM Technology Corporation, Fairport, NY, USA) was used to determine in vitro true digestiblity (IVTD) using a modification (Vogel et al., 1999) of the in sacco disappearance method originally described by Lowrey (1969). Rumen fluid was collected from a cannulated steer at the Texas A&M AgriLife Research and Extension Center in Stephenville TX. The steer was offered ad libitum access to ‘Coastal’ bermudagrass (Cynodon dactylon [L.] Pers.) hay with a minumum 120 g CP kg-1 DM.

The experimental design for each of the in vitro dry matter digestibility (IVDMD) experiments in which the effect of HSM inclusion on IVDMD was evaluated was a generalized complete block design. Block was designated as a single rumen fluid collection, and each experiment included two block. Each dietary treatment was replicated three times within each block, with the flask serving as the experimental unit.

The HSM used in rations for the in vitro experiments was a composite of the 15 batches collected from four oil processing factories (336 g CP kg-1 DM). Steam flaked corn (Zea mays L.), bermudagrass hay, alfalfa (Medicago sativa L.), and soybean (Glycine max L.) meal were purchased at a local feed store. All ingredients were ground to pass through a 2-mm screen in a Wiley mill (Arthur H. Thomas Co., Philadelphia, PA, USA). In the first experiment, HSM replaced the concentrate (steam-flaked corn) in a 60:40 forage-to-concentrate ratio ration at 0, 250, 500, 750, and 1000 g kg-1. The forage in this ration was bermudgrass hay. In the second experiment, isonitrogenous rations were formulated in which HSM replaced soybean meal and alfalfa at 0, 250, 500, and 750 g kg-1. Modified methods of the procedure originally described by Tilley and Terry (1963) were utilized. For each experiment, 0.5 g samples were weighed into 125 mL Erlenmeyer flasks with 40 mL of a 1:5 ratio of buffer solution B and buffer solution A (ANKOM Technology, 2017). Ten mL of CO2-flushed rumen fluid collected from a ruminally-fistulated steer was then added to each flask, and flasks were sealed with a topper and placed in a Fisherbrand Isotemp Shaking Water Bath (Thermo Fisher Scientific, Newington, NH, USA) at 39°C and agitated at 30 RPM for 48 h. Then, 2 mL HCl and 0.5 g pepsin were added to each flask and toppers were removed. After 48 h, samples were filtered through P8 filter paper and dried for 2 h at 105°C.

Data were analyzed using SAS v. 9.4 (SAS Institute, Inc., Cary, NC). Prior to analysis, raw data were tested using the NORMAL option of PROC UNIVARIATE to ensure data normality. Normality was assumed when Shapiro-Wilk’s W met or exceeded 0.9 (Shapiro and Wilk, 1965; Royston, 1992).

For the experiment evaluating the source of variation in nutritive value, response variables were analyzed using the linear mixed models procedure (PROC MIXED) in SAS using the COVTEST option for random effects models. Random effects included source (processor), batch within source, and replicate within batch by source (replicate was understood to represent the laboratory replicate and not a statistical replicate).

For the IVDMD experiements, response variables were analyzed using the generalized linear mixed models procedure (PROC GLIMMIX) in SAS. The fixed effect was treatment, and the random effects were block and treatment by block.

For all experiments, denominator degrees of freedom were adjusted using the Kenward-Roger approximation method (Kenward and Roger, 1997). The α-level for mean differences was set at 0.05. When interactions had P < α, the interaction was discussed; otherwise, main effects were discussed. Means separations were performed based on F-protected t-tests using Tukey-Kramer’s HSD.

Objective 2

Hempseed meal samples used in Objective 1 were composited for use in Objective 2 (336 g CP kg-1 DM). Steam flaked corn (Zea mays L.), bermudagrass hay, alfalfa (Medicago sativa L.), and soybean (Glycine max L.) meal were purchased at a local feed store. All ingredients were ground to pass through a 2-mm screen in a Wiley mill (Arthur H. Thomas Co., Philadelphia, PA, USA). Isonitrogenous rations were formulated in which HSM replaced soybean meal and alfalfa at 0, 250, 500, and 750 g kg-1. Rations were pelleted for ease of feeding

Weaned Californian meat rabbits (n = 32) were allocated randomly to eight cages (four animals/cage) in a generalized complete block design. Cages were allocated randomly to one of four levels of protein replacement. Rabbits were fed for 35 d from weaning to harvest. Rabbits were weighed and health checked weekly. Feed samples were collected daily and composited weekly. Orts and feces were collected in the final seven days of each period to calculate measures of digestibility.

Data were analyzed using SAS v. 9.4 (SAS Institute, Inc., Cary, NC). Prior to analysis, raw data were tested using the NORMAL option of PROC UNIVARIATE to ensure data normality. Normality was assumed when Shapiro-Wilk’s W met or exceeded 0.9 (Shapiro and Wilk, 1965; Royston, 1992).

Response variables were analyzed using the generalized linear mixed models procedure (PROC GLIMMIX) in SAS. The fixed effect was treatment, and the random effects were period, treatment by period, animal within cage by period, and sex (for individual animal responses). Denominator degrees of freedom were adjusted using the Kenward-Roger approximation method (Kenward and Roger, 1997). The α-level for mean differences was set at 0.05. When interactions had P < α, the interaction was discussed; otherwise, main effects were discussed. Means separations were performed based on F-protected t-tests using Tukey-Kramer’s HSD.

Outreach Plan

The primary mode of outreach for this project was presentation of research results at the Annual Meeting of the American Society of Animal Science.

Research results and discussion:

Objective 1

Table 1. Random effects estimates of neutral detergent fiber (NDF), acid detergent fiber (ADF), acid detergent lignin (ADL), crude protein (CP), and in vitro true digestibility (IVTD) from 15 hempseed meal samples collected from four oil processing facilities.

Effect

Estimate

SE1

Z-value

P-value

Contribution
to variance2

NDF

Rep (source by batch)

0

Batch (source)

0.000969

0.000434

2.23

0.01

65.08

Source

0

Residual

0.000520

0.000134

3.87

< 0.01

34.92

ADF

Rep (source by batch)

0

Batch (source)

0.000381

0.000172

2.21

0.01

44.82

Source

0.000375

0.000441

0.85

0.20

44.12

Residual

0.000094

0.000024

3.87

< 0.01

11.06

ADL

Rep (source by batch)

0

Batch (source)

0.000287

0.000121

2.38

< 0.01

68.50

Source

0.000096

0.000143

0.67

0.25

22.91

Residual

0.000036

0.000009

3.87

< 0.01

8.59

CP

Rep (source by batch)

0

Batch (source)

0.000964

0.000371

2.60

< 0.01

96.50

Source

0

Residual

0.000035

0.000013

2.74

< 0.01

3.50

IVTD

Rep (source by batch)

0

Batch (source)

0.000050

0000068

0.73

0.23

9.58

Source

0

Residual

0.000472

0.000100

4.74

< 0.01

90.42

 

Source had no contribution to variance for NDF, ADF, ADL, or CP (Table 1; P ≥ 0.20). However, batch within source contributed to variation for NDF (μ = 50.9%; P = 0.01), ADF (μ = 36.8%; P = 0.01), ADL (μ = 12.9%; P < 0.01), and CP (μ = 30.9%; P < 0.01). Regardless of the differences in nutritive value, there was no contribution to variation (P ≥ 0.23) of any measured effect on IVTD (μ = 53.0%). Therefore, while variation existed among hempseed meal samples with respect to nutritive value, digestibility was not affected and the viability of hempseed meal (HSM) as a livestock feed source is strong.

Our findings of variability between batches of HSM were similar to those seen in a study performed in Italy that found variability in CP content among genotypes of hemp grown under the same conditions (Galasso et al., 2016), which indicated cultivar or ecotype may have an effect on this variability. Temperature affects seed formation in hemp with ideal temperatures > 27°C (Suriyong et al., 2012). Protein synthesis may increase when seed filling occurs during periods of high temperatures due to more efficient N transfer to the seeds (Sainio et al., 2009). This was supported by the findings of Russo and Reggiani (2015) in which seeds grown during a year with higher recorded temperatures had greater CP content than seeds grown the year prior with lower recorded temperatures. Therefore, temperature may also have had an effect on variability in nutrient composition between HSM batches; however, this is not consistent with the lack of variability among sources in my study regardless of differing geographical location.

 

Table 2. In vitro dry matter digestibility (IVDMD) of a 60:40 forage-to-concentrate ratio ration in which hempseed meal replaced the concentrate portion at various levels.

 

Hempseed meal replacement of concentrate, g kg-1

 

0

250

500

750

1000

IVDMD, g kg-1 DM

406a

379ab

332ab

285ab

225b

In the first experiment in which HSM replaced the concentrate portion of a 60:40 forage-to-concentrate ratio ration, IVDMD was greatest (P < 0.05) from 0 g HSM kg-1 inclusion (406 g kg-1) and least from 1000 (225 g kg-1), with 250, 500, and 750 intermediate (Table 2). In the second experiment in which HSM was used as a source of CP in isonitrogenous rations, IVDMD was greatest (P < 0.05) at 0 (771 g kg-1), followed by 250 and 500, and least from 750 (643 g kg-1; Table 3).

 

Table 3. In vitro dry matter digestibility (IVDMD) of isonitrogenous rations in which hempseed meal replaced the crude protein portion at various levels.

 

Hempseed meal replacement of concentrate, g kg-1

 

0

250

500

750

IVDMD, g kg-1 DM

771a

716b

693b

643c

 

Our observations of IVDMD in the protein replacement ration were comparable to those of an in vivo feed trial done on sheep where HSM replaced canola meal at 0, 250, 500, 750, and 1000 g kg-1, resulting in DM digestibility coefficients of 0.66, 0.63, 0.64, 0.61, and 0.64, respectively (Mustafa et al., 1998). These data support our findings and encourage the applicability of HSM as a protein replacement in ruminant rations. Digestibility decreased when HSM replaced steam-flaked corn as a concentrate source in a constant forage-to-concentrate ratio, deeming it unsuitable for this purpose. Dry matter digestibility likely decreased due to higher cellulose and lignin content in HSM than corn and SBM. However, all digestibility values for rations with HSM as a protein replacement had digestibility percentages acceptable for ruminant rations. According to Meissner and Paulsmeier (1995), fiber does not begin to depress feed intake or digestibly in ruminants until it reaches a threshold level of 600 g kg-1. However, although there was a slight decrease in digestibility as HSM inclusion increased as a protein source in the ration, the difference was slight and dry matter digestibility remained sufficient to meet ruminant needs at all levels of inclusion. Further studies with 100% HSM inclusion rate and replacement of protein sources more similar in chemical composition are warranted.

 

Objective 2

Initial BW did not differ (P = 0.97; 825 g) among treatments; however, final BW was less (P < 0.05) from 750 g HSM kg-1 (1,662 g) and did not differ among other treatments (2,077 g). Average daily gain was greatest (P < 0.05) from 250 g HSM kg-1 (35 g d-1), followed by 500 g HSM kg-1 (33 g d-1), then 0 g HSM kg-1 (31 g d-1), and was least (P < 0.05) from 750 g HSM kg-1 (21 g d-1). Because of feed consumption differences, feed-to-gain ratios did not differ (P = 0.44) among treatments. However, DM digestibility was greatest (P < 0.05) from 750 g HSM kg-1 (597 g kg-1) and least from 250 g HSM kg-1 (474 g kg-1), with 0 g HSM kg-1 and 500 g HSM kg-1 intermediate (524 g kg-1 and 504 g kg-1, respectively). Results are interpreted to mean that palatability may have impacted animal performance, as evidenced by final BW and ADG, but HSM may be included in the diet of meat rabbits at up to 500 g HSM kg-1 replacement of CP without adverse effects on the animal.

Participation Summary

Educational & Outreach Activities

1 Journal articles
2 Webinars / talks / presentations

Participation Summary:

25 Ag professionals participated
Education/outreach description:

As a result of the funded research, two presentations were given at the 2021 American Society of Animal Science-Canadian Society of Animal Science-Southern Section American Society of Animal Science Annual Meeting and Trade Show. The first presentation, “Sources of variation in the nutritive value of hemp seed meal,” was presented by Ms. Kristen June Jacobson (graduate and grant recipient). The second presentation, “Late-Breaking: Hempseed meal as a protein replacement in the diet of meat rabbits,” was presented by Ms. Emily Verret (undergraduate). Additionally, Ms. Jacobson has prepared a manuscript, “Nutritive value variation and in vitro digestibility of hempseed meal,” that will be submitted to Animals on Friday, September 10, 2021.

Project Outcomes

2 New working collaborations
Project outcomes:

North American hemp industry growth as a result of the 2018 Farm Bill may prove beneficial to the livestock industry. Alternative feed sources are continually being evaluated to advance production efficiency, profitability, and sustainability. While there have been studies on hemp as a livestock feed source, information is limited, especially on U.S.-grown hemp. Our research found nutritive value variability among batches of HSM, which can be attributed to a variety of causes including genetics, environmental factors, and processing techniques. However, IVTD was not affected by this variability, and we found no variability among sources of HSM. Hempseed meal IVDMD only slightly decreased when HSM was included at increasing percentages as a protein replacement in the ration, indicating it should be able to replace other common protein sources in livestock rations. Data from current publications in combination with the results of this research indicate there is promise for the inclusion of HSM in ruminant rations. Ultimately, if HSM viability is widely accepted and its adoption as a feeding option receives interest, sustainability is enhanced by eliminating waste (hempseed byproducts) and promoting holistic agricultural production.

Knowledge Gained:

Over the course of this project, my advisor and I developed a more broadened definition of sustainability. More specifically, we were able to understand how enhancement of agricultural production is not mutually exclusive to sustainability but, rather, can be one-in-the-same objectve.

Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture or SARE.