Supplementary Material: Spent hemp biomass as a feed ingredient for beef steers: effects on performance, blood parameters, behavior, cannabinoid residues, and consumer exposure levels

Prior studies in ruminants demonstrated the safety of feeding spent hemp biomass (SHB) to lambs and dairy cows. However, the potential accumulation of cannabinoids in animal products, particularly the psychoactive compound ∆⁹-tetrahydrocannabinol (Δ⁹-THC), remains a significant barrier to the legalization of SHB as a feed ingredient for livestock. In this study, we investigated the inclusion of SHB in the diet of beef stockers to assess its effect on animal health, performance, and cannabinoid accumulation in meat. For this purpose, 24 Angus steers (BW = 263±22.5 kg and BCS = 4.0±0.5) were enrolled in the study to receive either CON (basal diet + 15% alfalfa meal) or HEMP (basal diet + 15% SHB) diet for eight weeks of intervention period (IP) followed by 4 weeks of a withdrawal period (WP), where both groups consumed the basal diet. The steers fed SHB had higher feed intake than CON animals, indicating no palatability issue with SHB, but their feed efficiency was decreased. Consuming SHB enhanced ruminal acetate production and reduced the propionate proportion. Consuming SHB increased plasma concentration of cholesterol, globulin (and circulating lymphocytes), ceruloplasmin, oxidative and antioxidative biomarkers, alkaline phosphatase, and Na. Consuming SHB did not affect the behavior of the steers but lowered water consumption. We detected a high concentration of cannabidiol (CBD) and CBD-acid (CBDA) in the liver (averaged 462±207 and 2,256±492 ng/g), muscle (271±73 and 35±17 ng/g), and adipose tissues (48.8±5.1µg/g and 80.7±52.1 ng/g) of steers fed SHB and very low Δ⁹-THC in the muscle (1.44±0.86 ng/g). With the exception of CBD in the adipose tissue (detected at 382±245 ng/g), cannabinoids were undetectable in all tissues after 33 days of withdrawal from SHB. We estimated the exposure of consumers to total THC for all population groups below the acute reference dose of 1 µg/kg BW when consuming meat from cattle, according to surveillance of meat intake data from the EU and USA populations. Overall, our data indicated that SHB is safe to be used as a feed ingredient in steers with minimal effects on rumen fermentation and the physiology of the animals. Furthermore, the risk of THC exposure from consuming the meat of these animals is expected to be minimal.