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Introduction
The general acceptance and regulatory outlook concerning to Cannabis spp. crops have changed in the past few years. This plant is no longer treated as a solely source of psychotropic agents; biological compounds have been focused on therapeutically approaches successfully1. In fact, the cannabidiol (CBD) and tetrahydrocannabidiol (THC) are the main bioactive compounds contained in the plant; these compounds are synthetized mostly in the flower and leafs2. Due to the latter, pharmaceutical industry has been attempting in updating and becoming purer the extractive compounds of Cannabis spp. for their use in medicines and drugs 1,3. In fact, some crops of Cannabis sp. have been used as a supplier of hemp for textile industry4, for production of biofuel5 and as a component in the automotive industry6. In addition, some livestock farmers use seeds as additives in animal feeding7. However, the extractive methods of bioactive compounds of the plant generate agricultural residue which may contain minimal concentrations of cannabinoids but considerable contents of fiber, cellulose and hemicellulose; these contents may arise as an important forage source in animal feeding, mainly ruminants. Worldwide information about production and plantations of Cannabis sp. is limited due to legal traits. Nevertheless, reports from the USDJ affirmed that the hemp production reached 10,000 t in Mexico in 2006; even though plantations above 31,000 ha were eradicated8. Accordingly, an increasing production of Cannabis sp. for extraction of cannabinoids with medical purposes may represent a substantial expansion of agricultural residue that may potentially be used as a forage source in ruminants feeding 9. However, published information about the nutritional value of by-products of hemp in animal nutrition is limited. Furthermore, this study aimed to determine the concentration of crude protein (CP), ether extract (EE), non-structural carbohydrates (NSC), fibers, total phenolic compounds (TPC), condensed tannins (CT), cannabidiol (CBD), delta-9-tetrahydrocannabinol (THC), in vitro digestibility of dry matter and rumen fermentation parameters of agro-industrial residues of Cannabis sativa L. from two extractive processes of cannabinoids as a potential source of forage in the ruminants feeding.
Material y methods
Study area
This study was carried out in the Faculty of Veterinary Medicine and Husbandry of the Durango State Juarez University, located in Durango, Mexico.
Ingredients and feedstuffs
All vegetative material was donated by the IIAC (Institute for Research and Exploitation of Cannabis) located in Durango, Mexico. The flower of Cannabis sativa L. was processed by two methods of extraction, which were performed by the IIAC. Briefly, samples of the flower of Cannabis sativa L. were cold-pressed for oil obtaining; the process did not exceed 35 ºC and no solvents were used. The obtained residues after cold-pressing were named CPC due to the cold-presses Cannabis sativa L. flower cake obtained. On the other hand, other samples of the flower of Cannabis sativa L. were exposed to an alcoholic extraction, which was carried out at room temperature for 5 h. Later, the by-product or residue was obtained by filtering the solvent; this treatment was named AEC (alcoholic extraction of Cannabis sativa L. flower cake). Thus, using raw flower of Cannabis sativa L. (RFC) as a control, it was compared to agro-industrial residues obtained after extractive processes. Afterwards, samples of agro-industrial wastes of Cannabis sativa L. were dried in a forced air oven (Felisa, Model FE-294AD) at 55 ºC for 48 h and were ground in a miller (Thomas Wiley Miller Lab, Model 4) at a 1 mm particle size. Consequently, samples were stored for further analyses.
Chemical composition
Samples were analyzed for dry matter (DM), ether extract (EE), crude protein (CP) and ashes according to standardized procedures10. Cellulose, hemicellulose, neutral detergent fiber (NDF), acid detergent fiber (ADF) and acid detergent lignin (ADL) were evaluated according to proposed by Van Soest et al11. Non-structural carbohydrates (NSC) were estimated with the following equation:
NSC= 100 - (CP+EE+NDF+A); where NSC= non-structural carbohydrates (% DM); CP= crude protein (% DM); EE= ether extract (% DM); NDF= neutral detergent fiber (% DM); A= ashes (% DM).
In vitro dry matter digestibility (IVDMD) was estimated using the DAISYII® equipment (ANKOM Technology Corp., Macedon, NY) and according to manufacturer procedures12. Metabolizable energy was calculated according to the following equation13:
ME = [2.20 + 0.136(GP24) + 0.0057(CP) +0.0029(EE)2]/4.184; where ME= metabolizable energy (MCal/kg); GP24= in vitro gas production at 24 h (ml/g); CP= crude protein (g/kg); EE= ether extract (g/kg).
Secondary metabolites
Dry samples for each treatment were exposed to alcoholic extraction (0.5 g dissolved in 45 ml of 70% ethanol-water solution) during overnight. Afterwards, samples were filtered and vacuum-evaporated (at 40 ºC) until total removal of ethanol solution and leave them dry overnight. Yields of concentrated extractions were calculated based on dry matter. Dry samples were stored for further secondary metabolites analyses.
Analysis of condensed tannins (CT)
Briefly, samples for each treatment were diluted (0.5 g dissolved in 45 ml of 70% ethanol-water solution) and let them extract during overnight. Later, 50 µL aliquots were mixed with a 4% solution of vainillina-methanol and concentrated HCl according to Heimler et al14. Absorbance was measured at 500 nm using catequine as standard. Yield of CT was estimated with the final concentration in solution and yield in dry matter.
Analysis of total phenolic compounds (TPC)
Briefly, samples for each treatment were diluted (0.5 g dissolved in 45 ml of 70% ethanol-water solution) and let them extract during overnight. Total phenolic compounds were estimated through the Folin-Cioucalteau method adapted by Dewanto et al15 using gallic acid as standard and measuring absorbance at 760 nm for every diluted sample. Yield of TPC was estimated with the final concentration in solution and yield in dry matter.
Analysis of cannabinoids
For the detection of cannabinoids (specifically CBD and THC) was used the method of thin layer chromatography (TLC), according to procedures proposed by Novak et al16. This trial was conducted at the facilities of the IIAC.
In vitro dry matter degradability (IVDMD)
For this analysis, 1 g (DM) of sample from each experimental treatment was placed in nylon bags (F57, ANKOM Technology, Corp., Macedon, NY) into glass modules equipped with electronic transducers for pressure measuring according to manufacturer’s procedures (ANKOM, USA) and incubated in triplicate with buffer solutions (CaCl2 13.2% w/v; MnCl2 10% w/v; CoCl2 1% w/v; FeCl3 8% w/v; NaHCO3 39% w/v) and ruminal inoculum in a 2:1 ratio, according to Theodorou et al17. Ruminal inoculum was obtained from two fistulated Angus steers fed with alfalfa hay based diet before the morning feeding; ruminal liquor was extracted from rumen and immediately placed into a thermal container preheated at 39 ºC and then it was transported to the lab for further analysis. The Nylon bags were incubated individually for each fermentation time (0, 3, 6, 12, 24, 36, 48, 72 and 96 h). Bags were removed from modules and washed until attaining crystalline water. They were placed in the oven at 55 ºC for 48 h. Changes in dry matter were registered and digestibility data was fitted into the Gompertz function for kinetics parameters estimation according to Murillo et al18.
Where: Deg= dry matter degradability (% DM); Ad= maximum degradability (% DM); kd= degradability constant rate (h-1); Ld= latency time before degradability begins (h).
In vitro gas production and ruminal fermentation parameters
For in vitro gas production, 1 g of sample from each treatment previously dried was placed into glass modules equipped with pressure transducer (ANKOM, USA) with 120 mL a mix of buffer solutions (CaCl2 13.2% w/v; MnCl2 10% w/v; CoCl2 1% w/v; FeCl3 8% w/v; NaHCO3 39 % w/v) and ruminal inoculum in a 2:1 and were incubated at 39 ºC for 24 h by triplicate according Theodorou et al17; ruminal inoculum was obtained from two fistulated Angus steers fed with alfalfa hay based diet before the morning feeding; ruminal inoculum was extracted from rumen and immediately placed into a thermal container preheated at 39 ºC and then it was transported to the lab for further analysis. Thus, changes in gas volume were registered at 0, 3, 6, 12, 24, 36, 48, 72 and 96 h and data was fitted into the Gompertz function for kinetics parameters estimation according to Murillo et al18.
Where: GP= gas production (ml); Ag= maximum gas production (ml); kg= gas production constant rate (h-1); Lg= latency time before gas production begins (h). Meanwhile, two 10 mL aliquots of in vitro ruminal fermentation were destined for determination of in vitro ruminal parameters after 24 h of fermentation time, were processed with metaphosphoric acid (25% w/v) and sulfuric acid (50% v/v) for volatile fatty acids (VFA) and nitrogen-ammonia (N- NH3), respectively and according to Galyean19.
Likewise, the same number of experimental treatments were incubated in glass modules (ANKOM, USA) until 24 h of fermentation time17. Once the time was elapsed, modules pressure release valve was opened and released gas was measured for methane and CO2 compositions according to procedures proposed by Herrera Torres et al20 using the portable gas analyzer GEM5000 (Landtec, USA).
Determination of rumen bacteria and protozoa
This assay was determined by weighing 1 g of sample from each treatment previously dried and placed into glass modules equipped with pressure transducer (ANKOM, USA) with 120 mL a mix of buffer solutions and ruminal inoculum in a ratio 2:1, and were incubated at 39 ºC for 24 h by triplicate17; ruminal inoculum was obtained from two Angus steers fed with alfalfa hay based diet. Afterwards, concentrations of total and cellulolytic bacteria were determined according to Dehority21, protozoa were analyzed according to Ogimoto and Imai22, whereas fungi were determined according to Joblin23 for each treatment. Briefly, culture mediums were prepared in sterile Petri dishes with nutritive agar (BD, Bioxon, USA) for total bacteria determinations and nutritive agar (BD, Bioxon, USA) plus carboxymethylcellulose (SIGMA, USA), as a cellulose source for cellulolytic bacteria determinations. Inoculum was obtained from in vitro ruminal fermentations of treatments and dilutions were carried out until 10-6 was reached. After, dilutions with inoculum were placed in previously labeled Petri dishes and incubated under CO2 atmosphere at 39 ºC. Total bacteria dishes were incubated for 48 h whereas cellulolytic bacteria dishes were incubated for 72 h. Once the incubation time was elapsed, Petri dishes were opened and microorganisms were measured using the most likely number technique 24. For fungal determination, culture medium was prepared with PDA agar (3 %) and placed into plates under sterile conditions. Later, 10 mL of in vitro ruminal inoculum was mixed with peptone solution (1:9 ratio) and dilutions were prepared with 1 ml of inoculated solution until 10-5 was reached and placed in plates previously labeled. Immediately, a sample of each dilution is flushed with CO2 and incubated under CO2 atmosphere at 39 ºC for 120 h for identification. For protozoa determination, in vitro ruminal inoculum was filtered through four layers of cheese clothes and obtained filtered was placed in a separation funnel during 15 min approximately, until protozoa precipitated (a whitish ring appeared at the bottom of the funnel). Later, 1 mL of the obtained sample with protozoa was mixed with 4 mL of ruminal inoculum (previously incubated at 39 ºC and flushed under CO2 conditions) in 18 x 150 mm culture tubes and placed one more time in the incubator; this last step was repeated five times for each dilution until 10-5 was reached. Consequently, protozoa number was determined through direct quantification in Neubauer chamber using a contrasting microscope (Collegiate 400) and an amplification of 400x.
Statistical design
Obtained data for all agroindustrial residues were analyzed through a completely randomized design using the GLM procedure in SAS25. Means comparison was evaluated with the Tukey multiple range test declaring significances at P<0.05.
Results and discussion
The chemical composition and dry matter digestibility for treatments are shown in Table 1. The used method for the extraction of cannabinoids affected mainly contents of ash, EE, and fibers (NDF and ADF). In fact, cannabinoids are separated from the flower in an organic phase, which is removed within the EE fraction3. As a consequence, there is a reduction of 95 % and 40 % in EE for AEC and CPC treatments after extraction, respectively (P<0.05). Otherwise, ash content increased 17 and 8 % in AEC and CPC, respectively when compared to the control (RFC) (P<0.05). These augmentations are positively associated with a redistribution of the chemical components after the extractive process; remained values of NDF, ADF and NSC represent higher fractions in the chemical composition while reducing the EE fraction once it was extracted. On the other hand, fiber content in CPC remains similar to the control. Cold press extraction removed more fiber fractions when compared to alcoholic extraction technique. Cold press is one of the most used methods for oil extraction; this method requires less energy than others and it is considered as an ecofriendly process26. However, the mechanical force applied in the process may remove higher fiber fractions than any other process. Furthermore, others27 presented similar contents of fiber in their study. Likewise, CP content was similar among treatments (P>0.05). However, it is assumed that a little fraction of soluble protein is removed in the extraction process; the remaining protein may be lower in amount but similar in proportion after redistribution of nutrients after the extraction. Similarly, it was observed this behavior in rapeseeds in cold-press extraction processes28. Additionally, another research27 reported similar concentrations of protein in hempseed cake after cold press extraction. Otherwise, NSC increased in AEC and CPC (P<0.05); these augmentations are linked with a dilution effect due to a reduction in fractions of fiber as explained earlier. In fact, Jarrell29 introduced this term related to plant nutrition studies in the early 80s. Moreover, extractive processes reduced TPC and CT (P<0.05). Secondary metabolites as phenolic compounds and condensed tannins are extracted in alcoholic solutions14. Secondary metabolites play a very important role in methane mitigation in ruminants. Thus, different mechanism of action was observed directly or indirectly in ruminants’ fermentation or ruminants’ microorganisms depending on the concentrations of certain metabolites (saponins, condensed tannins, phenolic compounds, etc.)30. Therefore, the importance in evaluating the concentrations of TPC and CT. Fiber fractions (NDF and ADF) increased after alcoholic extraction process (P<0.05). These changes are positively connected to a higher extraction of crude fat in EE. Thus, the more EE is extracted in AEC the higher the fiber compositions are. Additionally, the IVDMD increased about 142 and 97 % in AEC and CPC, respectively when compared to the control (RFC) (P<0.05). Moreover, cannabinoids were also affected by the extractive processes (P<0.05). Both, CBD and THC decreased their concentrations substantially. In this way, concentrations of CBD were reduced 73 and 28 % in AEC and CPC, respectively. Similarly, concentrations of THC decreased 99 and 88 % in AEC and CPC, respectively. Alcoholic extraction is more efficient in the extraction of cannabinoids when compared to a cold-press extraction. Apparently, a polar extraction may be more effective and less expensive when compared to an extractive process using mechanical force, which may remove other nutrients with no interest26. The published information regarding to harmless consumption of CBD and THC in animals is very limited. However, Kleinhenz31 offered industrial hemp to calves as a feed resource. These authors administered 1142 and 120 mg of CBD and THC to calves weighing approximately 215 kg of live weight, respectively; no changes were observed in behavior, feeding intake or chemistry of blood serum (glucose, BUN, creatinine and total protein) in animals. Moreover, THC was not detected in blood plasma; CBD was totally metabolized and was not detected after 48 h. Likewise, Cornette32 administered 5 mg of CBD/ kg live weight and observed no changes in behavior as well. Hence, according to the latter, AEC presented in this study, can be offered harmlessly until 2 kg per animal per day (animals weighting approximately 215 kg live weight) and no effects on any of the chemical parameters or behavior would be expected. Nevertheless, no information on meat safety and hemp consumption in ruminants is available. In agree to the results given in the present study, lower concentrations of cannabinoids (CBD and THC) and higher concentrations of NSC led to a higher IVDMD and ME. In fact, other study16 reported antimicrobial activity of cannabinoids, which may affect directly the digestibility. Consequently, IVDMD increased approximately two-fold when compared to control (RFC). In addition, these changes could not be attributed to variations in TPC and CT since their concentrations are similar among AEC and CPC treatments; changes in IVDMD in AEC and CPC could not be correlated to TPC and CT concentrations in both treatments.
Table 1 Chemical composition and in vitro digestibility of agro-industrial residues of Cannabis sativa L
| Variable | RFC | AEC | CPC | P | SEM |
|---|---|---|---|---|---|
| %, DM | |||||
| Ash | 11.5±0.25b | 13.5±0.25a | 12.5±0.04ab | 0.001 | 1.33 |
| CP | 21.2±0.25a | 20.9±0.29a | 20.2±0.0.28a | 0.10 | 0.12 |
| EE | 12.3±0.06a | 0.5±0.04b | 7.5±0.0b | <0.001 | 0.08 |
| NDF | 27.9±0.39b | 32.2±0.33a | 28.0±0.86b | 0.003 | 0.58 |
| ADF | 16.5±0.02b | 18.9±0.69a | 15.5±0.19b | 0.015 | 1.47 |
| ADL | 2.2±0.15b | 2.5±0.08a | 2.3±0.006ab | 0.002 | 0.07 |
| HEM | 11.5±0.31b | 13.3±2.23a | 12.4±0.28a | <0.001 | 1.06 |
| CEL | 14.4±0.14a | 16.4±0.36a | 13.1±0.11a | 0.009 | 1.24 |
| NSC | 26.5± 0.16b | 32.7± 0.82a | 31.1± 1.0a | <0.001 | 0.77 |
| TPC, mg/g DM | 15.7±0.10a | 14.8±0.08b | 15.4±0.05b | <0.001 | 0.06 |
| CT, mg/g DM | 6.7±0.09a | 4.6±0.16c | 5.4±0.05b | <0.001 | 0.09 |
| IVDMD, % | 23.9±1.11c | 57.9±1.58a | 47.2±2.74b | <0.001 | 1.58 |
| ME, Mcal | 1.4 ± 0.04c | 1.9±0.07a | 1.6 ±0.06b | <0.001 | 0.05 |
| CBD, g/kg | 1.8± 0.01a | 0.5± 0.003c | 1.3± 0.006b | <0.001 | 0.03 |
| THC, g/kg | 10.9± 0.07a | 0.08± 0.001c | 1.4± 0.007b | <0.001 | 0.001 |
RFC= raw flower Cannabis sativa L.; AEC= alcoholic extracted Cannabis sativa L. flower residue; CPC= cold-pressed Cannabis sativa L. flower residue; SEM= standard error of the difference among means; CP= crude protein; EE= ether extract; NDF= neutral detergent fiber; ADF= acid detergent fiber; ADL= acid detergent lignin; HEM= hemicellulose; CEL= cellulose; NSC= non structural carbohydrates; TPC= total phenolic compounds; CT= condensed tannins; IVDMD= in vitro dry matter digestibility; ME= metabolizable energy; CBD= cannabinol; THC= tetrahydrocannabinol.
abc Means with different letters in the same row indicate differences (P<0.05).
In vitro ruminal degradability for treatments is presented in Table 2. Extractive processes affected degradability; AEC and CPC increased maximum degradability (Ad parameter) above of 120 % in both treatments when compared to control (P<0.05). Likewise, extractive processes increased degradability specific rate (kd parameter) about 120 % for both treatments whereas latency period (Ld parameter) increased 140 % for both treatments (P<0.05). Augmentations in the kinetics parameters may be positively associated with a reduction in the cannabinoids content and changes in the NSC; this effects were observed earlier in the IVDMD. Higher values in Ad, kd and Ld are presented in the treatment with lower values of cannabinoids (AEC). According to this, Semwogerere et al33 reported that digestibility of hempseed cake is lower than the digestibility of canola meal and soybean meal. In fact, it was affirmed that hempseed cake increased the rumen retention time which is comparable with the fermentation time in in vitro assays27; this effect was observed in this study. Actually, AEC and CPC presented higher values in degradability specific rate (kd parameter) which led to reach the asymptotic value (Ad) in a shorter time; RFC would need more time to reach Ad which is consistent with previous studies27. Otherwise, more time is necessary for microorganisms to begin degradation of substrate as observed in values of latency time (Ld parameter) for AEC and CPC. The latter may be associated with changes in microorganisms’ populations (protozoa and bacteria) which will be discussed later.
Table 2 In vitro ruminal degradability parameters of agroindustrial residues of Cannabis sativa L. after two extractive methods
| Parameter | RFC | AEC | CPC | P | SEM |
|---|---|---|---|---|---|
| Ad, % | 24.1±0.29c | 57.6±0.01a | 54.6±0.81b | <0.001 | 0.502 |
| Ld, h | 1.9±0.01b | 4.7±0.04a | 4.6±0.16 a | 0.004 | 0.096 |
| kd, %/h | 0.14±0.005b | 0.31±0.005a | 0.32±0.000 a | <0.001 | 0.002 |
RFC= Raw flower Cannabis sativa L.; AEC= alcoholic extracted Cannabis sativa L. flower residue; CPC= cold-pressed Cannabis sativa L. flower residue; SEM= standard error of the difference among means; Ad: maximum degradability; kd= specific rate of degradability; Ld= latency period before the degradation begins; (lag phase).
abc Means with different letters in the same row indicate differences (P<0.05).
The population of microorganisms after ruminal fermentation of different residues is shown in Table 3. Protozoa increased after the extractive process in CPC when compared to control (P<0.05); no changes were observed in AEC (P>0.05). Contrariwise, no traces of fungi were observed in the control (RFC); nevertheless, fungi increased after the extractive process (P<0.05). Otherwise, total bacteria were reduced after the extractive process in both treatments (P<0.05). However, no changes were registered in cellulolytic bacteria (P>0.05). Novak et al16 affirmed that extracts of hemp exhibited antimicrobial activity in most bacterial habitats from human and animal but these changes were not observed in fungi; conversely, extracts from hemp showed antifungal activity in in vitro assays reported previously34. Additionally, reductions in total bacteria are associated with an increase in the protozoa populations. This is in accordance with others35, who claim that the bacteria predation is caused mainly by protozoa activity.
Table 3 Microorganisms populations in in vitro ruminal fermentation of agroindustrial residues of Cannabis sativa L. after two extractive methods
| Microorganisms | RFC | AEC | CPC | P | SEM |
|---|---|---|---|---|---|
| Protozoa, mL-1 x104 | 7.3±0.07b | 15.8±0.30ab | 26.6±0.37a | 0.007 | 0.22 |
| Cellulolytic bacteria, mL-1 x105 | 142.1±2.88a | 127.6±14.77a | 115.6±7.21a | 0.232 | 7.84 |
| Total bacteria, mL-1 x105 | 187.2±20.20a | 100.6±6.74b | 115.2±6.92b | 0.006 | 10.57 |
| Fungi, CFU | ND | 7.2±0.57a | 6.3±1.15a | 0.001 | 0.60 |
RFC= raw flower Cannabis sativa L.; AEC= alcoholic extracted Cannabis sativa L. flower residue; CPC= cold-pressed Cannabis sativa L. flower residue; CFU: colony forming units; ND= non-detected; SEM= standard error of the difference among means..
abc Means with different letters in the same row indicate statistical differences (P<0.05).
Ruminal fermentation parameters and gas production kinetics are shown in Table 4. No changes were observed in pH (P>0.05). Nevertheless, extractive processes in AEC and CPC affected concentrations of N-ammonia and TVFA (P<0.05). A reduction in the N-ammonia is positively associated with a defaunation of ruminal bacteria due to the presence of protozoa. In fact, Wang et al36 reported a reduction in the protozoa populations when using hempseed oil in ruminal fermentations. Thus, it is expected that RFC promote a reduction of protozoa and an increase in bacteria populations; the latter encourages an increase in the N-ammonia. Furthermore, a reduction in the deamination of protein would be expected as a consequence of an increase of bacteria which would lead to a reduction in the N-ammonia37. These asseverations were observed in this study. Protozoa increased with both treatments and a reduction of total bacteria is observed (P<0.05). Additionally, protozoa play an important role in the synthesis of some volatile fatty acids. As a matter of fact, some others38 affirmed that the ability of protozoa to digest fatty acids could divert more carbon towards volatile fatty acids synthesis; these changes were observed in the present study in AEC and CPC with the higher protozoa populations. Regarding to gas production kinetics, Ag increased above 87 and 77 % in AEC and CPC, respectively (P<0.05). These changes suggest a better nutrients utilization of microorganisms which led to a higher fermentation process and a higher gas production; hence, it is expected a higher gas production rate. However, no changes were observed in latency period (Lg) and gas production specific rate (kg) (P>0.05) but similar trends to those in degradability are observed.
On the other hand, methane production increased 81 and 97 % in AEC and CPC, respectively (P>0.05). Likewise, CO2 increased about 60 % in both treatments when compared to control (P<0.05). Apparently, the reduction of cannabinoids and the increase in the NSC led to an augmentation of the fermentation process, which increased methane production. Whereas, the CH4: CO2 ratio increased 18 % in CPC when was compared to control (P<0.05). This ratio indicates the volume of methane produced divided into the volume of CO2 present in the process; higher values of this variable suggest that more methane is being synthetized through the CO2 reduction pathway18. Thus, these changes in CH4: CO2 ratio may suggest that more methane is being synthetized through the CO2 pathway and that the increases in methane production are not only associated to an improvement in the ruminal fermentation and an extension of the total gas production. In fact, the presence of cannabinoids may result as inhibitors in methanogenesis through the CO2 reduction pathway since lower values of CH4: CO2 ratio are presented with higher values of cannabinoids. The higher the cannabinoids are the lower the protozoa; methane synthesis and protozoa number is positively associated39. Thus, if there is a reduction in protozoa a reduction in methane would expected as well. An increase in the protozoa may affect the bacteria including the methanogens. Regarding to the latter, it was reported33 that hempseed oil is more efficient in the inhibition of methanogens since it has more terpenes, polyphenols and lignin. Similarly, Embaby et al40 found a 10 % reduction in methane production when using hempseed oil and compared to corn oil. However, Patra et al41 affirmed that reductions in methane production are associated to the presence of polyunsaturated fatty acids (PUFA) than the presence of cannabinoids; these fatty acids were not evaluated in this study. According to the findings in this study, raw flower in RFC inhibits not only degradability but methane synthesis and protozoa populations as well. Nevertheless, these affections are highly associated with the nutrients utilization from microorganisms which may lead to a better ruminal fermentation process as exposed earlier in this study.
Table 4 Ruminal fermentation parameters and gas production kinetics of agroindustrial residues of Cannabis sativa L. after two extractive methods
| Variable | RFC | AEC | CPC | P | SEM |
|---|---|---|---|---|---|
| pH | 6.8±0.11a | 6.9±0.10a | 6.8±0.12a | 0.251 | 0.13 |
| N-NH3, mg/dL | 15.2±0.08a | 10.9±0.11c | 13.8±0.11b | <0.001 | 0.29 |
| TVFA, mM | 0.11±0.007b | 0.16±0.003a | 0.16±0.008a | <0.001 | 0.05 |
| Ag, ml/g DM | 41.82±8.89b | 78.46±5.13a | 74.27±2.08a | <0.009 | 4.93 |
| Lg, h | 2.6±0.64a | 3.6±0.35a | 3.24±0.37a | 0.384 | 0.15 |
| kg, %/h | 0.09±0.05a | 0.22±0.01a | 0.24±0.02a | 0.063 | 0.02 |
| CH4, ml/g DM | 3.7±0.15b | 6.7±0.29a | 7.3±0.20a | <0.001 | 0.22 |
| CO2, ml/g DM | 32.5±2.470b | 52.8±0.89a | 52.3±1.52a | <0.001 | 1.75 |
| CH4/CO2 ratio | 0.11±0.004b | 0.12±0.003ab | 0.13±0.005a | 0.023 | 0.004 |
RFC= Raw flower Cannabis sativa L.; AEC= alcoholic extracted Cannabis sativa L. flower residue; CPC= cold-pressed Cannabis sativa L. flower residue; SEM= standard error of the difference among means; N-NH3: N-ammonia; TVFA= total volatile fatty acids; Ag: maximum gas production; kg= specific rate of gas production; Lg= latency period before the gas production begins (lag phase).
abc Means with different letters in the same row indicate differences (P<0.05).
Conclusions and implications
Both residues obtained after the two extractive processes offer acceptable nutritional properties in animal feeding. Residues obtained after the alcoholic extraction offered a better nutrient utilization of microorganisms present in the ruminal fermentation which led to increase in IVDMD and fermentation parameters. Therefore, the agroindustrial residue of Cannabis sativa L. obtained after the extractive processes may arise as a potential forage source in ruminants feeding. However, more in vitro and in vivo assays using these agro-industrial residues as a part of a ration are highly recommended, considering potential secondary effects of cannabinoids on animal health and food safety.
