Partially substituting alfalfa hay with hemp forage in the diet of goats improved feed efficiency, ruminal fermentation pattern and microbial profiles

2.2. Experimental animals and diets

Forty growing Xiangdong black goats (a local breed of Hunan province, China; mean BW = 7.82 ± 0.57 kg) were grouped by BW and randomly assigned into 1 of 4 experimental diets (10 goats/treatment). Diets were control (CON; 60% AH and 40% concentrate), 55% AH and 5% HF (HF5), 50% AH and 10% HF (HF10), and 40% AH and 20% HF (HF20). The diets were formulated according to the recommendation of NRC (2007), and contained 60% forage and 40% concentrate (dry matter [DM] basis; Table 1). The substitution rates of HF for AF were mainly based on the favorable nutrient contents of HF versus AF (shown as footnotes of Table 1) and consideration of the potential detrimental effect of cannabinoids in HF on meat quality. The hemp was grown in June and harvested manually in October, 2019 in Derong County, Sichuan Province, China and the HF was prepared by crushing hemp stems and leaves into powder. The HF contained 4.35% CBD, 0.11% THC, and 0.28% cannabidiolic acid (analyzed on a wt/wt basis).

2.3. Animal management, feed sampling, and chemical analysis

The experiment lasted 70 d, with 10 d of adaptation and 60 d of data and sample collection. Goats were housed in individual pens and fed a total mixed ration (TMR) ad libitum twice daily at 08:00 and 18:00, with free access to clean water. Daily feed offered and orts were recorded for each goat throughout the collection period to calculate DM intake (DMI). Goats were weighed at the beginning and end of the experiment before morning feeding and the average daily gain (ADG) was calculated by dividing the BW gain (final BW − initial BW) by the number of days on feed. The feed conversion efficiency (F:G) was estimated by dividing DMI by ADG. The AH, HF, and TMR samples were collected once weekly, whereas orts were collected daily, pooled, and subsampled weekly for each goat. All samples were air dried in an oven at 55 °C for 48 h and ground through a 1-mm screen (standard model 4, Arthur Thomas Co., Philadelphia, PA, USA) for chemical composition determination.

Analytical DM was determined by drying at 135 °C for 2 h (AOAC, 2005; method 930.15); the ash concentration was determined by combustion at 550 °C for 5 h and the OM concentration was calculated as 100 minus the ash concentration (AOAC, 2005; method 942.05). Neutral detergent fiber (NDF) was determined using heat-stable α-amylase (Termamyl 120 L, Novo Nordisk Biochem, Franklinton, NC, USA) and sodium sulfite (AOAC, 2005; method 2002.04). Ash-free acid detergent fiber (ADF) was determined according to AOAC (2005; method 973.18). Starch was analyzed via enzymatic hydrolysis of α-linked glucose polymers (Rode et al., 1999), and the total ether extract concentration was determined using method 920.39 (AOAC, 2005). Total N was analyzed using flash combustion and thermal conductivity detection (Model 1500, Carlo Erba Instruments, Milan, Italy), and protein concentration was calculated as N × 6.25. The ME was calculated from tabulated feed values according to NRC (2007).

2.4. Carcass characteristics and meat quality

At the end of the experiment, the goats were slaughtered in a commercial slaughterhouse and carcass traits and meat quality were measured. Dressing percentage was calculated individually as hot carcass weight divided by final live BW × 100. Organs including liver, lung, spleen, heart and kidney were weighed, and the organ index (OI) was calculated as percentage of live BW. Longissimus dorsi samples, kept at 4 °C, were collected to determine meat quality, including pH, eye muscle area, drip loss, cooking loss, shear force, and meat color. The pH of longissimus dorsi was determined 24 h after slaughter using a pH indicator (S210, Mettler Toledo, USA). The width and height of the cross section of longissimus 6 to 7 ribs were determined with a Vernier caliper, and the eye muscle area was calculated as width × height × 0.7. Analysis of the drip loss, cooking loss and shear force was conducted according to guidelines of 1995. Meat color was measured using a Minolta CR-410 color-meter (Minolta Camera Co., Ltd., Osaka, Japan), where L∗ represents lightness, a∗ redness, and b∗ yellowness.

2.5. Rumen fermentation and ruminal microbiota

Rumen fluid was collected from each goat immediately after slaughter, and was passed through four layers of cheesecloth. Ruminal pH was measured immediately using a portable pH meter (Model S210 SevenCompact pH; Mettler-Toledo Instruments Ltd., Shanghai, China). The filtrates were mixed and sub-sampled for measuring rumen fermentation parameters and microbial communities. Five milliliters of filtrate were preserved with 1 mL of 25% (wt/vol) HPO₃ for analysis of total volatile fatty acids (VFA) and another 5 mL was preserved with 1 mL of 1% (wt/vol) H₂SO₄ for NH₃-N analysis. The prepared samples were stored at −20 °C until analysis. Two additional ruminal filtrate samples (1.5 mL) were collected in 2-mL polypropylene centrifuge tubes, frozen in liquid nitrogen, and stored at −80 °C for analysis of rumen microbial community and metabolites, respectively.

The concentrations of VFA and NH₃-N were determined using gas chromatography (GC7890A, Agilent, California, USA) and a spectrophotometer (UV-2600, Shimadzu Global Laboratory Consumables Co., LTD, Shanghai, China), respectively. For ruminal microbiota determination, the filtrate samples were thawed at 4 °C overnight and total DNA was extracted using a QIAamp Fast DNA stool mini kit (Qiagen, Hilden, Germany). After quality checking, the extracted DNA was used for 16S amplicon high-throughput sequencing analysis as previously reported (Ran et al., 2021a). In short, the V3-V4 hypervariable region of the 16S rRNA gene was amplified using the universal primers 341F and 806R (Zhu et al., 2018), followed by paired-end library preparation, cluster generation, and sequencing on Illumina MiSeq platform (Illumina, Inc., San Diego, CA, USA) according to the manufacturer’s instructions. Raw sequencing data were processed using QIME2 (Bolyen et al., 2019) and the R-package DADA2 (version 1.4) denoising method. Richness (number of operational taxonomic units [OTUs]) and diversity (Shannon index) were calculated and principal coordinates analysis (PCoA) and non-metric multidimensional scaling (NMDS) were performed based on Bray-Curtis similarity distances using the R packages vegan (version 2.4.4) and phyloseq (version 1.20.0).

2.6. Blood regular indices, immune and antioxidant activity

Blood samples were collected from the jugular vein into vacuum tubes with and without clot activator (Shandong Aosaite Medical Devices Co., LTD, Heze, China) before the morning feeding on d 60. The samples were maintained at room temperature for 15 min. Then serum samples were obtained by centrifuging at 2,000 × g for 15 min at 4 °C and plasma samples were obtained by centrifuging at 3,000 × g for 20 min at 4 °C. Both serum and plasma were subsampled and frozen at −20 °C until analysis. Serum samples were used for analysis of blood regular indices, immune and antioxidant activity, while the plasma samples were used for analysis of cannabinoids and their metabolites.

The serum concentrations of regular blood metabolites including total protein (TP), albumin (ALB), glucose, triglyceride (TG), total cholesterol (TC), blood urea N (BUN), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) and antioxidant indices including malondialdehyde (MDA), superoxide dismutase (SOD), total antioxidant capacity (T-AOC), and glutathione peroxidase (GSH-PX) were measured using commercial kits (CUSABIO Biotech Co., Ltd, Wuhan, China). Blood immune indices including IgG, IL-1β, IL-2, IL-4, and interferon-γ (IFN-γ) were analyzed using ELISA kits (Nanjing Jiancheng Biology Co., LTD, Nanjing, China) according to the supplier’s instructions.

2.7. Hemp-related cannabinoid metabolism and residues in meat

Cannabinoids and their metabolites in the rumen fluid, plasma, and meat (longissimus dorsi) were determined using ultra-high-performance liquid chromatography (UHPLC) tandem high-resolution mass spectrometry (MS; Thermo Fisher, USA), as reported previously (Jamwal et al., 2017; Kleinhenz et al., 2020a). Raw data were subjected to baseline filtering, peak identification, integration, retention time correction, peak alignment, and normalization using the metabolomics processing software Progenesis QI (V2.3 software; Nonlinear Dynamics, Newcastle, UK). Metabolite information was obtained from the Human Metabolome Database (http://www.hmdb.ca/), Metlin Database (https://metlin.scripps.edu/), and Bovine Metabolome Database (http://www.cowmetdb.ca/) (Artegoitia et al., 2017).

3.2. Ruminal fermentation and microbial community

The ruminal pH did not differ (P > 0.1) among diets, whereas the NH₃-N concentration linearly (P < 0.01) increased with increasing substitution rate of HF for AF, with greater (P = 0.022) NH₃-N concentration in HF20 than in the other diets (Table 3). Total VFA concentration quadratically (P < 0.01) changed with increasing HF substitution rates, with the greatest concentration observed in the HF10 diet. The molar proportion of acetate quadratically (P < 0.01) changed; whereas the molar proportion of propionate linearly increased (P = 0.024) with increasing HF substitution rates. As a result, the acetate-to-propionate ratio linearly (P = 0.032) decreased with increasing substitution rates of HF. The molar proportion of butyrate tended (P = 0.081) to increase linearly with increasing HF substitution rates. The molar proportion of branched-chain VFA (BCVFA) tended to quadratically (P = 0.061) change with increasing substitution rate of HF, with greater values in the CON than in the HF5 and HF10 diets.

The ruminal microbiota was determined using 16S rDNA sequencing. An average of 62,362 valid tags per sample were obtained, with sufficient coverage of each sample to accurately describe the microbial composition, as indicated by the rarefaction curves (Fig. S1). No difference (P > 0.1) was observed in the numbers of OTUs among treatments (Fig. S2) or among the α-diversity indices including observed species, Shannon, and Simpson indexes (Fig. S3). Furthermore, goats receiving different HF levels could not be separated from each other using PCoA and NMDS analysis, meaning HF5, HF10 and HF20 had similar community structure. Goats in the HF groups showed a much more diffuse distribution pattern as compared to CON (Fig. 1) and no clear clustering of the ruminal microbes, suggesting a greater distance within the groups. The taxonomic composition of the ruminal microbiota by diets was analyzed at the phylum (Fig. 2A) and genus (Fig. 2B) levels by means of relative abundance. Except those lower than 0.1% and unclassified, more than 99.5% of rumen bacteria belonged to the following seven phyla: Bacteroidetes, Firmicutes, Proteobacteria, Spirochaetes, Fibrobacteres, Tenericutes, and Actinobacteria. Bacteroidetes (average of four treatments, 71.26%) and Firmicutes (average of four treatments, 22.07%) were the most abundant, resulting in an Firmicutes-to-Bacteroidetes ratio around 0.30 (Table 4). The relative abundance of Tenericutes increased linearly (P = 0.028) with increasing substitution rate of HF, with a greater relative abundance in HF20 than in the other treatments, whereas the relative abundances of the other phyla were not affected by treatment.

At the genus level, the averaged relative abundances of ruminal bacteria among treatments >0.5% are shown in Table 5. Prevotella_1 (average of four treatments, 23.99%) and Rikenellaceae_RC9_gut_group (average of four treatments, 10.92%) from phylum Bacteroidetes had the first and second highest relative abundances, respectively. The relative abundance of Prevotella_1 linearly (P = 0.040) decreased but that of Rikenellaceae_RC9_gut_group (P = 0.017) increased with increasing substitution rates of HF. Similarly, the relative abundance of Bacteroides decreased linearly as the HF substitution rate increased. However, there were no significant effects (P > 0.1) of HF substitution rates on the relative abundances of the other genera. The treatment effect was observed for the relative abundances of Ruminococcaceae_NK4A214_group and Fibrobacter, which were the highest under HF10 treatment.

3.3. Blood biochemistry

Results of blood biochemistry, including regular blood metabolites, antioxidant activity, and immune activity, are shown in Table 6. The serum concentrations of TP, TG, ALT and AST did not differ (P > 0.10) among treatments. However, the concentrations of ALB and TC linearly (P < 0.01) decreased, and that of BUN linearly (P = 0.050) increased with increasing HF substitution rate. Greater serum glucose concentration was detected in HF10 than in other treatments. The serum T-AOC linearly (P = 0.014) decreased with increasing HF substitution rate, whereas neither a linear/quadratic effect nor treatment effect (P > 0.1) was observed for the antioxidant indices measured as MDA, SOD, and GSH-PX among treatments. Regarding the immune indices, serum concentrations of antibody (IgG), pro-inflammatory (IL-1β, IL-2, and IFN-γ), and anti-inflammatory factors (IL-4) were greatly affected (P ≤ 0.05) by dietary HF contents, with the greatest concentration of immune indices consistently observed in goats fed HF5 diet and no differences (P > 0.1) between CON and HF20. The serum concentration of IgG tended to linearly (P = 0.065) decrease, but the concentrations of IL-2, IL-4, and IFN-γ quadratically (P ≤ 0.05) changed with increasing HF substitution rates, with a sharp increase from CON to HF5 and then decreased to the original level as the dietary HF contents increased.

4.1. Effects of substitution of AH with HF on growth performance and carcass traits

In the present study, increasing levels of HF in the diet resulted in a linear decrease in DMI and improvement in the feed conversion rate without decreasing ADG. These results suggest that the feeding value of HF may be higher than that of AH when partially substituted for AH under current experimental conditions. The linear reduction in DMI with increasing HF inclusion rate may be related to the specific odor of hemp-related bioactive compounds. Wagner et al. (2022) also reported a decreased feed intake when corn silage was partially replaced with industrial hemp silage. In contrast, Krebs et al. (2021) reported no effects on DMI when hemp stubble was included in the pelleted diets of sheep. The discrepancy among these studies could be explained by the differences in the composition and form of the hemp used. The HF used in the current study contained mainly stems and leaves and might have greater amounts of hemp-related bioactive compounds than hemp stubble used in the study by Krebs et al. (2021). Furthermore, in Krebs et al. (2021), the hemp stubble was included in the pelleted ration. The pelleting process at high temperature and pressure may have reduced the activity of the bioactive components. In the present study, the improved feed conversion ratio with increasing dietary HF inclusion may have been due to improved nutrient digestibility with HF addition. Krebs et al. (2021) reported greater DM and OM digestibility in hemp stubble than in oat straw. In the present study, although feed digestibility was not measured, the greater ruminal total VFA concentration and higher molar proportion of acetate in the HF10 group suggested an improvement in feed digestibility with HF feeding. The presence of secondary metabolites may favor ruminal microbial activity. This speculation is supported by the enhanced relative abundances of Rikenellaceae_RC9_gut_group and Ruminococcaceae_NK4A214. Similarly, substituting AH with stevia hay, which is also rich in secondary metabolites, increased the digestibility of NDF and ADF (Jiang et al., 2022).

The lack of adverse effects of substituting AH with HF on carcass traits in the present study is consistent with the similar treatment effects on growth rate. Our findings also agree with those of a previous study that fed sheep pelleted rations containing hemp stubble (Krebs et al., 2021). In the current trial, substitution of AH with HF had no impact on meat pH, color, and cooking loss. However, there was greater drip loss when one-third of the AH was replaced by HF, indicating lowered tenderness. A previous study reported that hempseed cake had effects comparable to soybean meal on ruminant carcass and meat quality attributes (Semwogerere et al., 2020); moreover, feeding hempseed cake increased the total antioxidant capacity of sheep milk, perhaps due to the bioactive compounds in the hemp, such as terpenes, CBD, α-tocopherol, and polyphenol (Mierlita, 2018). Although there were no adverse effects of partially substituting AH with HF on meat quality in the current study, further studies are needed to determine whether feeding hemp-based products positively affects meat oxidative stability (Semwogerere et al., 2020).

4.2. Effects of substitution of AH with HF on ruminal pH and fermentation

Ruminal pH reflects the fermentation pattern and internal environment of the rumen and is considered an important determinant of rumen health. In the current study, the substitution of AH with HF in roughage-based diets did not affect ruminal pH. On the current trial, ruminal pH ranged from 6.29 to 6.37 among treatments, which is consistent with the high-forage feeding. Similarly, Krebs et al. (2021) reported that the inclusion hemp stubble did not affect ruminal pH.

The ruminal NH₃-N concentration reflects the dynamics between protein degradation and microbial protein synthesis in the rumen (Reynolds and Kristensen, 2008). In the present study, the ruminal NH₃-N concentration was within the optimal range suggested by Owens and Bergen (1983), at 0.39 to 29 mg/dL. The higher concentration of NH₃-N with the HF20 diet is likely due to higher dietary CP contents, but the lower DMI than that of CON may have reduced microbial protein synthesis due to low energy availability. Because nutrient digestibility was not measured in the current study, it is unknown whether diets containing HF had greater CP degradability than the CON diet, in which AH was the sole forage source. However, when oat chaff was substituted with hemp stubble in sheep diets, Krebs et al. (2021) reported a greater NH₃-N concentration and CP digestibility.

The ruminal VFA are the main energy source for ruminants, providing up to 70% of their energy requirement (Srinivas and Gupta, 1997). Greater total VFA concentrations, especially of acetate, in the HF diets may indicate an improvement in DM and fiber digestibility. This may explain the improved feed conversion rate with increasing HF substitution rates. Krebs et al. (2021) reported greater total VFA concentrations in diets containing hemp stubble, possibly because of the higher OM digestibility when substituting oat chaff with hemp stubble. We found that the higher molar proportion of acetate with HF10 agreed with the results of Krebs et al. (2021), who also reported an increased concentration of acetate when hemp stubble was included in sheep diets. The results indicated that hemp inclusion may promote acetic acid fermentation in the rumen. The lower BCVFA proportion with HF inclusion could not be explained by the lower proteolytic activity with HF because the lower BCVFA did not influence the total VFA concentration.

4.3. Effects of substitution of AH with HF on ruminal microbial community

The sequencing results showed that the substitution of AH with HF had little effect on the diversity and richness of ruminal bacteria, suggesting no detrimental effects from hemp-related bioactive compounds including cannabinoids (Δ⁹-THC and CBD) and their metabolites; however, substitution with HF altered the microbial community profiles, with greater changes observed under higher HF substitution ratios.

This alteration was mainly reflected in the fluctuations in the relative abundances of several major bacterial genera. More specifically, the relative abundances of Prevotella_1 and Rikenellaceae_RC9_gut_group, both belonging to the phylum Bacteroidetes, were greatly decreased and increased, respectively, with increasing HF substitution rates. Many studies have consistently identified these genera as the core genera in the rumen (Henderson et al., 2015; Xue et al., 2018), suggesting that they play critical roles in rumen fermentation. Bacteria of Prevotella genus are known to be the predominant proteolytic bacteria with a great diversity of extracellular proteolytic activities in the rumen (Griswold et al., 1999); thus, the decreased relative abundance of Prevotella_1 could reduce the degradation of dietary protein in the rumen and increase the amount of protein that enters the small intestine. This suggests that feeding HF may increase by-pass rumen protein. However, this effect was not observed in the present study, likely because AH contains highly degradable non-protein nitrogen, while corn protein is less degradable. Prevotella_1 also plays an essential role in polysaccharide degradation and sugar fermentation (Accetto and Avgustin, 2019) and its relative abundance increases with greater concentrations of dietary hemicellulose and water-soluble carbohydrates when corn silage replaced with sweet sorghum silage (Ran et al., 2021b). Therefore, the reduced relative abundance of Prevotella_1 was likely related to the different concentrations of dietary hemicellulose and water-soluble carbohydrates between AH and HF.

The Rikenellaceae_RC9_gut_group are fiber digesters that increase propionate production and enables more efficient energy recovery by lowering methane emissions through hydrogen scavenging (Conte et al., 2022; Daghio et al., 2021). It was reported that Rikenellaceae_RC9_gut_group was positively correlated with the production of propionate in heat-tolerant dairy cows, providing a more effective energy recovery system than heat-susceptible cows (Wang et al., 2022). Therefore, the enhanced molar proportion of propionate and decreased acetate-to-propionate ratio were largely related to the increased relative abundance of Rikenellaceae_RC9_gut_group when AH was substituted with HF. This suggests that the inclusion of HF in the diet of goats promoted propionic acid fermentation by upregulating the relative abundance of Rikenellaceae_RC9_gut_group. The relative abundance of Rikenellaceae_RC9_gut_group was consistently higher in animals with better growth performances (Conte et al., 2022; Daghio et al., 2021; Liu et al., 2022) and health status (Liu et al., 2022; Wang et al., 2022). We speculate that the inclusion of HF in the diet of goats may improve their health and growth performance. Further research is needed to verify the exact role of Rikenellaceae_RC9_gut_group played in ruminant performance.

Ruminococcaceae have a dominant role in cellulose and hemicellulose degradation (Pang et al., 2022) and rumen biohydrogenation pathways (Huws et al., 2011). The enhanced abundance of Ruminococcaceae_NK4A214 may have contributed to the greater production of propionate and improved feed efficiency in the current study, ultimately improving animal performance (Wang et al., 2022). As important cellulose and hemicellulose digesters, the abundance of some unclassified Ruminococcaceae could be used as potential biomarkers of subacute ruminal acidosis risk (Zhang et al., 2022). Rikenellaceae_RC9_gut_group and Ruminococcaceae_NK4A214 were negatively associated with residual feed intake (Liu et al., 2022), suggesting that they are beneficial for improving feed efficiency. In brief, the alteration of microbial community profiles by substituting AH with HF was consistent with the effects of promoting propionate fermentation and improving feed efficiency.

4.4. Effects of substitution of AH with HF on blood metabolites and antioxidant capacities

Hemp and its by-products are rich in bioactive compounds (e.g., terpenes, flavonoids, and cannabinoids) and are commonly studied as medicine due to their therapeutic potential in many diseases. Specifically, these bioactive compounds have the potential to alter the blood biochemistry, antioxidant capacity, and immune status. However, few studies have evaluated hemp as a forage source and investigated the effects of hemp bioactive compounds on blood biochemistry in ruminants. Kleinhenz et al. (2020a, 2022) reported no effect on serum biochemistry when feeding industrial hemp to beef cattle and Holstein steers for a short or long term. Similarly, although the differences in some values were observed among treatments in the current study, all values remained within their suitable reference ranges for goats, suggesting the overall safety of HF inclusion in the diet of goats. The linear increase in BUN with increasing dietary HF content was consistent with the linear elevated ruminal NH₃-N concentrations. This is likely due to the numerically greater dietary CP content in the HF.

Hemp-related bioactive compounds have been reported to have antioxidant capacity (Semwogerere et al., 2020); however, no difference in antioxidant indices, including MDA, SOD, and GSH-PX, were observed among treatments in the present study, suggesting that goats did not suffer from oxidative stress during the experimental period. In livestock production, stressful conditions may result from nutrition (e.g., high-grain diets), environmental conditions, and management (weaning, transportation, and feedlot entry; Olagaray and Bradford, 2019). Stress events have been implicated in promoting oxidative stress through excessive production of reactive oxygen species or decreased antioxidant defenses (Olagaray and Bradford, 2019). In the present study, the goats were fed high-forage diets under well-adapted environmental condition. Therefore, stress was minimal. Interestingly, Holstein steers consuming cannabinoids from industrial hemp have lower stress biomarkers and improved lying times, suggesting increased animal welfare (Kleinhenz et al., 2022). Moreover, administration of industrial hemp had little impact on serum amyloid A and haptoglobin (Kleinhenz et al., 2022), which are acute-phase proteins recognized as indicators of inflammation. In the current study, pro-inflammatory (IL-1β, IL-2, and IFN-γ) and anti-inflammatory factors (IL-4) were elevated when AH diets were substituted with low levels of HF, but no such elevations were observed when replacing AH with increasing amounts of HF.

This research was funded by the National Key Research and Development Program of China (2021YFD1300504), “Double First Class” Special Guidance Project, Team Building Funds, Research Startup Funds (561120213), Lanzhou University, Lanzhou, China, High-end Foreign Experts Recruitment Plan (G2023175005L), and The Agricultural Science and Technology Innovation Program, Chinese Academy of Agricultural Sciences (ASTIP-IBFC).