We identified changes in the ruminal, nasopharyngeal, vaginal, and uterine microbiota in beef heifers fed a finishing diet containing 20% hempseed cake relative to changes in heifers fed 20% DDGS over the course of a 16-week feeding trial. All heifers appeared healthy throughout feeding trial and heifers fed hempseed cake consumed a similar amount of feed and exhibited similar feeding behaviours as heifers fed a DDGS [29]. However, hempseed cake inclusion led to a decrease in average daily gain (ADG), ultimately resulting in a lighter final body weight in hempseed cake-fed heifers than control heifers. Several factors may be contributing to the reduced ADG observed in the hemp group. As stated in our previous publication, this may include the greater acid detergent fiber concentration that was present in the hempseed cake compared to DDGS (16 vs. 11% on DM basis) [29]. Another factor associated with the lower feed to gain ratio observed in hempseed cake-fed heifers could be the altered ruminal microbiota and subsequent ruminal fermentation due to the ingestion of hempseed cake which contains anti-nutrient and antimicrobial agents [18, 19, 39]. This is evident by the significant alterations observed in the ruminal microbiota following hempseed cake feeding (Figs. 2 and 3).
Alterations in the ruminal microbiota was observed within a week of initiating hempseed cake ingestion despite the ruminal microbiota in these mature (19-month old) heifers being more resilient and robust compared to younger calves [40]. Starting from day 42 onward, the impact of hempseed cake ingestion on the ruminal microbiota became more evident as reflected by the significant differences in the microbial community structure, diversity, and composition between the hemp and DDGS-fed heifers (Figs. 2 and 3). Hempseed cake ingestion resulted in increased rumen microbial diversity (Shannon diversity index). Although alpha diversity metrics for the ruminal microbiota was reported to be the same between beef cattle with high or low feed efficiency [41, 42], the increased ruminal microbial diversity measured in the hemp group may be negatively associated with feed efficiency given the reduced ADG observed in hempseed cake-fed heifers. The compositional changes induced by hempseed cake ingestion were characterized by an increase in the relative abundance of six bacterial genera (Eubacterium nodatum group, Lachnospiraceae UCG-002, Oribacterium, Prevotellaceae UCG-001, Prevotellaceae UCG-004, and Rikenellaceae RC9); most of which have been reported to have associations (positive or negative) with feed efficiency in beef cattle [41–43]. For example, Liu and colleagues reported that there was a greater relative abundance of Lachnospiraceae in Angus heifers with low residual feed intake (RFI) (more feed efficient) compared with high RFI heifers [41]. Members of the Rikenellaceae RC9 gut group, Lachnospiraceae, and Prevotellaceae have also been reported to be present in greater abundance in ruminal fluid of Nellore steers with low feed efficiency compared to high feed efficient steers [42]. The low feed efficient Nellore steers also had greater abundance of Succinivibrio taxa than high feed efficient steers. Similarly, the relative abundance of Rikenellaceae RC9 gut group, Prevotella and Succinivibrio taxa were negatively associated with feed efficiency in Nellore beef cattle (male and female) [43]. Thus, the 10 genera (Fig. 3) that were differentially abundant in the rumen microbiota between the hemp and control cattle may be associated with reduced feed digestion and nutrient absorption given that most of these genera are known to be associated with feed efficiency in cattle. Therefore, further research is warranted to evaluate the effect of hempseed cake inclusion in the diet on the members of these genera, and fermentation parameters in vitro.
The significant alteration in ruminal microbiota community structure, diversity and composition observed in hemp heifers may be attributed to several antimicrobial components contained within the hempseed cake. The oil content (7%) may have influenced the ruminal microbial composition as the antibacterial activity of hempseed oil against a wide range of Gram-positive bacterial species has been well documented [18, 44, 45]. In addition, the psychoactive components and cannabinoid derivatives in hempseed cake, have known antimicrobial activity against a large panel of Gram-positive and Gram-negative pathogens [46], and may have similar activity in the rumen. Overall, the results of our study indicate the need for further research to investigate the impact of feeding hemp by-products on the gut microbiota and fermentation parameters.
Few effects of feeding hempseed cake were observed in the upper respiratory tract microbiota. Only on day 98 was the community structure of the nasopharyngeal microbiota significantly different between the hemp and DDGS fed heifers. This is a particularly interesting observation as the impact of diet on the respiratory microbiota in cattle has infrequently been investigated. Hall and colleagues reported that feeding selenium-biofortified alfalfa hay for 9 weeks resulted in an altered nasopharyngeal microbiota in weaned beef calves [47, 48]. Vitamin and mineral supplementation during the first six months of gestation has also been reported to induce some compositional changes in the nasopharyngeal microbiota of pregnant beef heifers [31]. Our results here suggest that longer feeding periods may be required to observe dietary alterations of the bovine respiratory tract microbiota in response to diet.
The microbiome-gut-lung axis may be a potential mechanism through which dietary inclusion of hempseed cake may influence the respiratory microbiota [49, 50]. It is also likely that changes in the composition of the rumen gas cap, and potentially the microbes associated with ruminal gas because of hempseed cake ingestion may have affected the respiratory tract microbiota given that about 70–85% of the eructed gases from the rumen are inhaled [51]. Thus, considering the role of the respiratory microbiota in maintaining respiratory health and resilience against bovine respiratory disease, the costliest disease in the modern finishing cattle industry [52–54], the long-term impact of feeding hempseed by-products on the respiratory microbiota and pulmonary health of cattle should be investigated.
Dietary hempseed cake not only affected the gastrointestinal and respiratory tract microbiota but also the microbiota in the reproductive tract, as we observed changes in both the vaginal and uterine microbiota. The vaginal microbiota at slaughter (d 112) in heifers fed hempseed cake was distinct in terms of community structure, microbial richness, and composition in comparison with the control heifers. Microbial richness in the vagina was reduced in the hemp group. In humans, reduced richness in the vaginal microbiota has been reported to be positively associated with reproductive health and pregnancy while increased microbial richness in the vagina was found in women who experienced preterm birth [55] or reproductive infection (bacterial vaginosis) [56, 57]. Thus, the observed decrease in microbial richness in the vagina associated with hempseed cake consumption could be an indicator of a healthy restructuring taking place within the vaginal microbiota. However, the compositional changes observed in the vaginal microbiota at the genus level suggest otherwise as shown by the reduced relative abundance of the most predominant and commensal vaginal genera such as Clostridium [58, 59] and Romboutsia [31]. Conversely, an elevated relative abundance of potentially pathogenic Fusobacterium spp. in heifers fed hempseed cake was observed.
The Fusobacterium genus includes the pathogenic species Fusobacterium necrophorum, and Fusobacterium nucleatum that are often involved in reproductive infections and abortions, as well as liver abscesses in cattle [60–63]. Among the other bacterial genera whose relative abundance was depleted in the vaginal microbiota of hemp heifers, Agathobacter, Negativibacillus and the Ruminococcus gauvreauii group are largely commensal genera that are also dominant members of the bovine [64, 65] and human gut microbiota [66]. The genus Paeniclostridium which contains the pathogenic species Paeniclostridium sordellii (previously known as Clostridium sordellii), is associated with “sudden death syndrome’ in feedlot cattle [67], septic shock in women [68] and enterocolitis in horses [69]. Taken together, however, the observed changes in the vaginal microbiota of hempseed cake-fed heifers are insufficient to make conclusive statements regarding a positive or negative influence of hempseed cake consumption on the vaginal microbiota of heifers. Future studies are needed to verify the impact of hempseed by-product on reproductive microbial composition, particularly on pathogenic species (Trueperella pyogenes, F. necrophorum, F. nucleatum and P. sordellii), and reproductive health and fertility in female cattle.
The alterations observed in the vaginal and uterine microbiota by hempseed cake intake may be because of several factors. First and most importantly, hempseed cake supplementation induced alterations in microbial population and fermentation parameters in the gut (rumen) that may lead to an altered metabolite profile (e.g. short-chain fatty acids and immunomodulators) in the blood and/or peripheral blood mononuclear cells associated with specific bacteria [70]. Consequently, this may influence the microbial communities along the reproductive tract [50]. Second, hempseed cake ingestion may have affected the production of reproductive hormones, which are known to shape the vaginal microbiota [71, 72]. Third, similar to the respiratory microbiome, the microbiome-gut-reproductive axis may be associated with changes in the reproductive tract microbiome due to hempseed cake consumption [50]. Finally, cannabinoid derivatives in hempseed cake may have altered the immune cell and cytokine profiles in the reproductive tract given the well-documented immunomodulatory properties of cannabinoids [73].
Overall, the results of our study suggest that consumption of hempseed cake can influence the female reproductive microbiome. Also, given that the heifers used in our study were approximately 21 months of age when sampled for vaginal and uterine swabs, the impact of feeding hempseed by-product on the genital microbiota would be expected to be stronger in younger female cattle as they typically have a less robust and resilient reproductive microbiota compared to older cattle [74]). Including hempseed cake in the diet of female cattle used for breeding may impact reproductive microbiome-mediated fertility and pregnancy outcomes. Therefore, more attention should also be given to investigate the impact of feeding hemp by-products in cow-calf operations.
The gastrointestinal tract microbiome is most often the target of dietary interventional studies in both livestock and humans as it is the largest microbial community in the host and plays a central role in host nutrient metabolism and health. Research on the respiratory and reproductive tract microbiome are more frequently focused on infectious microorganisms and diseases as nutrient interventions have been thought to have less impact on these extra-gut microbial ecosystems. However, recent evidence suggests that gut microbiome-mediated health may rely on communication with different organs throughout the body (e.g. lung/reproductive/mammary) [50]. It has also been suggested that the gut microbiome affects distant organs and host metabolic pathways through mediation of endocrine systems [75]. In addition, many bacterial taxa present in the digestive tract are also present in the respiratory and reproductive tracts [31, 75]. Accordingly, we hypothesized that changes in the gut microbiome due to dietary treatment may also affect microbial communities in other organs.
Therefore, we evaluated the impact of hempseed cake inclusion in the diet on not only the ruminal microbiota but also on the respiratory (nasopharyngeal), and reproductive (vaginal, and uterine) microbiota. The alterations observed here in these four microbial communities in response to hempseed cake feeding support the existence of interconnection among these microbiota, potentially through mechanisms involved in the microbiome-gut-respiratory and microbiome-gut-reproductive axes [50]. As expected, the composition, diversity, and structure of the ruminal, nasopharyngeal, vaginal, and uterine microbiota were significantly different from each other due to the physiological and anatomical differences in the mucosal surfaces of these anatomical sites [31]. Although the rumen, nasopharynx, vagina, and uterus have drastically different physiological and anatomical properties, we identified that 28 ASVs were shared by a relatively high proportion (60%) of all samples. Thus, these “core taxa” may be involved in facilitating communication among the gut, respiratory and reproductive microbial communities or could colonize multiple host sites.
Some of the observations reported in this study can potentially be extrapolated to humans as cattle have similar physiological and developmental characteristics and are colonized by microbes that are biogeographically and phylogenetically more similar to those found in the human microbiota when compared with rodent models [28]. The alterations seen here in the bovine gut microbiota, respiratory, and reproductive microbiota due to hempseed cake feeding suggest that the effect of hemp products on the human microbiome and health should also be investigated. In particular, the impact of hempseed cake feeding on the vaginal and uterine microbiota that we observed highlights the need for future research into the impact of feeding hempseed oil and other hemp products on fertility and reproductive health in women.
Although the rumen and nasopharyngeal microbiota were sampled multiple times, the vaginal swabs were collected only at the end of the feeding trial. For logistical reasons, the vaginal samples had to be done over four days, which may have increased inter-individual variation. In addition, only 8 out of 31 uterine swabs collected at necropsy could be sequenced and included in this study. Therefore, the results of uterine microbiota need to be interpreted cautiously. Uterine swabbing of live cattle, especially in virgin yearling heifers, can be invasive, and makes it difficult to collect samples over multiple time points. Nonetheless, we were able to detect differences in the vaginal and uterine microbiota between the two treatment groups.