This study is the first to report on the effects of concurrent IH administration and transport in cattle. A wide variety of outcome measures were evaluated to generate data for development of further studies investigating IH administration during transport. There were drug by transport interactions for the kinetic gait parameter, rear pressure, and IRT and significant drug by transport by time interactions for blood glucose and PGEM. There were transport by time interactions for a variety of outcome variables, including body weight change, accelerometry, complete blood count and serum biochemistry, cortisol, and SAA levels.

There was a significant transport by time interaction for body weight change, with TRANS steers not yet returning to their baseline (-24 or 0 h) body weight by the final collection point at 48 h. These results are consistent with previous transportation literature: cattle tend to lose 1% body weight per hour for the first three to four hours of transport and less thereafter (Coffey et al. 2001), lose the most body weight within the first 12 hours of initiation of transport (Knowles 1999; Knowles et al. 1999), and generally return to baseline body weight within 24 to 72 h following transport (Knowles et al. 1997). While the TRANS steers in the present study had not returned to baseline body weight by 48 h, there was an upward trend in body weight change (less negative change, or increased body weight) from 8 to 48 h.

The accelerometric results support prior research that has observed increased lying times in transported heifers compared to controls (Theurer et al. 2013). Conversely, studies in bulls have reported decreased lying times (Earley et al. 2013), potentially in favor of interacting with other animals (Cafazzo et al. 2012). While TRANS steers laid down more compared to baseline, similar to the findings from Theurer et al. (2013), in the present study, CNTL steers were also observed to have increased lying times. This is likely an allelomimetic response on the part of the CNTL steers, as cattle are known to synchronize lying times within a herd (Stoye et al. 2012), although this has not been specifically in non-transported cohorts. All steers in the present study had been housed together for a prolonged period prior to this study; it is possible that the stable social dynamics between the steers in the present study contributed to the changes observed in the CNTL steers following return of the TRANS cohort. While CNTL steers did follow a similar pattern to TRANS steers regarding motion index, step count, standing ratio, and lying ratio, CNTL steers had more lying bouts than TRANS steers. Thus, TRANS steers appear to stay in recumbency longer, whereas CNTL steers appear to shift between standing and recumbency. The increased lying bouts in CNTL compared to TRANS steers suggests that CNTL steers may have been more comfortable or less fatigued, and thus more willing to expend energy moving between standing and recumbent positions.

To the authors’ knowledge, there are no reports of MNT outcomes in transported cattle. The results of the present study suggest that cattle acclimate to MNT measurement over time. The observed temporal pattern of decreasing sensitivity over time is consistent with habituation to the stimulus (Grandin 1997). While sensitivity generally decreased with time (MNT values increased), left front limb MNT values for TRANS steers at 8 h were not different from baseline. One explanation for this delayed habituation in TRANS steers is the possibility of tissue damage caused by transport, which has been previously reported (Jarvis et al. 1996; Huertas et al. 2010). Tissue damage and subsequent release of prostaglandins and other inflammatory mediators may initiate a state of hyperalgesia or sensitize nerve endings in the skin or deeper tissues (Grandin et al. 2023). Further work would be needed to distinguish the temporal pattern in MNT values from the effects of transport, due to the fact that the transport period was early in the study period, when MNT outcomes were already lower than later timepoints.

This is also the first report of kinetic gait analysis being used for evaluation of transport effects. Kinetic gait analysis has previously been used to evaluate the efficacy of analgesics in cattle with induced lameness. Schulz et al. (2011) observed that cattle treated with meloxicam placed greater force on both lame and non-lame limbs compared to placebo animals. In the present study, there was a time main effect for most parameters. One explanation for the time effects is that steers were becoming habituated or more resistant to handling at 48 h than earlier in the study, resulting in shorter, slower strides with less force. Introducing cattle to the pressure mat system prior to baseline collection would allow a more direct comparison of values throughout the study. Further work is needed to determine the utility of kinetic gait analysis subsequent to transportation.

Infrared thermography has been used previously to evaluate the effects of transportation. Lei et al. (2023) investigated non-invasive markers of stress in Arouquesa cattle and found increased mean and maximum eye IRT values immediately after transport, but temperatures subsequently returned to baseline following a short rest. Brief elevations in IRT values could also be caused by elevated ambient temperatures within the transport vehicle. In the present study, left eye maximum temperatures were greatest at 8 h, which is likely due to that timepoint occurring in the afternoon. The authors believe that the drug by transport interaction observed for left eye maximum temperature percent change from baseline is largely due to the transport effect. No other IRT parameters had a significant drug main effect or interaction and the percent change from baseline was not different between HEMP-TRANS and PLBO-TRANS steers. The interaction with IH is likely due to random variation in treatment groups.

There were several changes to complete blood count and serum biochemistry parameters that are consistent with previous transportation literature, including an elevation in total white blood cell, neutrophil, and monocyte concentrations and a decrease in lymphocyte concentrations. Alterations in leukocyte numbers and function following transport result in transient changes in immune system responses (Van Engen and Coetzee 2018). Concentrations of white blood cells, neutrophils, and monocytes in all steers in the present study remained within the reference interval for our laboratory. Thus, the changes observed in the present study likely had little biological significance in regards the immunocompetence of the enrolled steers. Numerous publications have reported transport-induced neutrophilia, which is due to elevation in glucocorticoids causing decreased margination and expression of extravasation proteins (Murata et al. 1987; Earley et al. 2006; Earley and O’Riordan 2006; Buckham Sporer et al. 2008; Hulbert et al. 2011; Van Engen and Coetzee 2018). Similar to neutrophils, monocyte concentrations were also increased in TRANS steers at 8 h in the present study. Reports on monocyte concentrations following transport vary. Masmeijer et al. (2019) reported monocytosis in lightweight, but not “normal” weight, calves following transport. In a study evaluating the effects of meloxicam on stress biomarkers in transported bulls, both control and meloxicam groups exhibited elevated monocyte concentrations following transport compared to baseline (Van Engen et al. 2014). Other reports observed no change in monocyte levels (Gupta et al. 2007; Ishizaki and Kariya 2010). It is possible that age, breed, or handling differences could be responsible for the disparity in reports on monocyte concentrations. Lymphocyte concentrations were reduced in TRANS steers in the present study. Previous work has also demonstrated this reduction, which is glucocorticoid-mediated (Murata et al. 1987; Earley et al. 2006; Earley and O’Riordan 2006). As the present study was a pilot study, only immune cell concentrations were measured. Evaluation of the effects of transport on immunological function after IH administration would be an area of future interest, considering the potential anti-inflammatory effects of some cannabinoids (Pellati et al. 2018; Cosentino et al. 2023).

Not only do glucocorticoids alter immune cell concentrations, but they have also been shown to transiently increase blood glucose concentrations in transported cattle (Kenny and Tarrant 1987; Warriss et al. 1995; Browning and Leite-Browning 2013). In the present study, all steers exhibited mild hyperglycemia, with PLBO-TRANS steers at 8 h having the greatest levels. While the trends we observed do match previous transport literature [eg Kenny and Tarrant (1987)], the small numerical differences observed here are likely biologically insignificant and reflect the fact that these steers were well-accustomed to people and handling. Although there was a drug by transport by time interaction, the lack of biological significance suggests blood glucose is likely of low interest for future IH research.

While the elevations in immune cells and glucose point to transport-mediated stress, the increases observed in total protein support existing data on dehydration during transport (Jarvis et al. 1996). The transport distance used in the present study was chosen to be an intermediate transport period. Jarvis et al. (1996) found that cattle transported less than 80 mi had lower packed cell volume and total protein than those transported more than 80 miles. It has also been observed that cattle lose more body weight in high ambient transport temperatures than cooler conditions due to thermal stress (Coffey et al. 2001; Theurer et al. 2013). Total protein did not fall outside the reference interval for any steer in the present study. It is possible that, despite the prolonged transport distance and time without water, the changes in total protein were mitigated by the cooler temperatures.

The data regarding increases in white blood cells and glucose are consistent with the increased cortisol concentrations observed in TRANS steers. Cortisol exposure, as measured by AUC, was also greater for TRANS steers compared to CNTL. This stress response to transport is well-established in the literature. Buckham Sporer et al. (2008) reported a 321% increase in cortisol concentration 4.5 h into a 9 h transport period; cortisol returned to baseline by 24 and 48 h following transport. In a multiple experiment trial evaluating the effects of transport, novel environments, and loading, authors reported cortisol increased in response to transport, introduction to a novel environment, and loading (Browning and Leite-Browning 2013). That study demonstrated the multifactorial nature of transport stress, in that many aspects of typical cattle processing (loading, transport, new environment), all induced a cortisol response. The authors did not find a difference in cortisol response between cattle that were loaded and unloaded versus transported, suggesting those events are similarly stressful. In the present study, steers were returned to their home environment, so they were subjected only to the stress of loading and transport. The logarithmic mean concentrations observed in the present study study for -24, 32, and 48 h were less than 1 ng/mL, and the greatest mean concentration (averaged across treatments) was observed at 8 h (3.06 ng/mL, 95% CI: 1.83 to 5.12). Past transport studies have generally reported baseline values between roughly 2 and 15 ng/mL, with substantial increases following transport (Kenny and Tarrant 1987; Gupta et al. 2007; Buckham Sporer et al. 2008; Browning and Leite-Browning 2013). Other studies have reported reduced cortisol levels with long-distance transport and with increasing exposure to transportation (Lay et al. 1996; Van Engen et al. 2014) reported a reduction in cortisol following long-distance (16 h) transport and postulated that the decrease was due to acclimation to the stressor. While the steers in the present study were not acclimated to transport, the frequent prior exposure to handling and people could have impacted the magnitude of cortisol response. The consistently low cortisol concentrations in the present study may reflect the habituation of the steers to people and manipulation. (The authors also believe that the low cortisol concentrations observed in this study may be responsible for the large intra-assay CV, as CV increases with decreasing mean values, if SD stays the same.) Burdick et al. (2010) reported on the effects of temperament of Brahman bulls on physiologic responses to transport and found that bulls scored as ‘calm’ had lower pre- and post-transport cortisol concentrations than bulls scored as ‘temperamental’. Considering that cortisol values from the previous study by Kleinhenz et al. (2022) were also low (albeit those cattle did not undergo transport), it is possible that there is some breed or bloodline temperament effect on cortisol levels consistent across the animal sources used for that study and the present one.

In a previous study evaluating the effects of short-term IH feeding (Kleinhenz et al. 2022), hemp-fed Holstein steers had lower mean cortisol concentrations (1.59 ng/mL) than controls (5.97 ng/mL; P = 0.001). The hemp group had cortisol concentrations as low as 0.76 ng/mL on the last day of feeding. The authors concluded that additional work was needed, as some work has shown increased cortisol following CBD administration; however, these effects may be offset by the anxiolytic properties of some cannabinoids. There was not a significant effect of IH on cortisol in the present study; steers in the HEMP group had numerically lower AUC values than PLBO steers. Considering the previous findings from Kleinhenz et al. (2022), the handling exposure of the steers in the present study, and the small sample size of this study, the authors think it would be worthwhile to evaluate the effects of IH on cortisol with a larger cohort or in other stressful management scenarios.

To the authors’ knowledge, this is the first report of PGEM data from transported cattle. There was an overall negative change in PGEM concentration in HEMP-CNTL steers compared to a positive change in PLBO-CNTL steers at 48 h. This is consistent with a previous study, in which cattle fed IH for 14 d had an 8.8% reduction in PGEM concentrations compared to baseline versus a 10.2% increase for control cattle (Kleinhenz et al. 2022). Previous work has shown that CBD and an IH water extract have inhibitory effects on PGE₂ production in animal and in vitro models of inflammation (Di Giacomo et al. 2021; Costa et al. 2004). Furthermore, cannabinoids, including CBD, CBDA, THCA, CBG, CBGA, and IH leaf and inflorescence extracts have been shown to decrease cyclooxygenase production in ex vivo and in vitro inflammatory models (Ruhaak et al. 2011; Costa et al. 2004; Shin et al. 2024). Specifically, THCA, CBG, CBGA have been shown to have COX-2 inhibitory properties (Ruhaak et al. 2011); results for CBDA have been mixed, with some studies showing inhibition (Takeda et al. 2008) and others showing an increase in COX-2 (Ruhaak et al. 2011). Bartkowiak-Wieczorek et al. (2025) showed dose-dependent increases in COX-1 expression in rat brain following administration of two IH extracts and variable changes in COX-2 expression based on brain region. The effect observed in this study, complicated by the three-way interaction, suggests that further work is needed to confirm the effects of PGEM in cattle.

Two acute phase proteins (APP), SAA and fibrinogen, were evaluated in the present study. In a study evaluating the effects of transport and commingling on APP in newly weaned calves, Arthington et al. (2003) found a tendency for SAA to be increased in transported calves (48.9 vs. 33.4 µg/mL in nontransported calves). However, these results are not straightforward to interpret, as there was not an unweaned control group. Thus, the relative contribution of transport versus weaning on the APP response in these calves is unknown. In a study by Lomborg et al. (2008), Holstein cows and heifers were subjected to a combination of transport, social isolation, and an uncomfortable environment. The authors reported significantly greater SAA in all animals at 48 h compared to 0 h, with a range of 30 to 482 µg/mL SAA at 48 h. While there was a transport by time interaction in the present study, all SAA values were much lower, with the greatest average concentration in TRANS steers at 24 h (20.8 µg/mL, 95% CI: 8.3 to 52.3). As discussed previously, these lower values could be due to the level of prior human socialization of the steers in the present study. Another possible explanation is that there are differences in baseline APP profiles or the APP response to stress due to age, sex, or breed. There was no effect of IH on SAA levels in the present study, consistent with a previous investigation of stress biomarkers after a 14-d period of feeding IH (Kleinhenz et al. 2022). However, in both the present study and the study by Kleinhenz (2022), animals were free of disease or other diagnosed inflammatory conditions. It is unknown if IH may impact SAA in light of a true inflammatory impetus. A study by Winders et al. (2024) found that cattle fed hempseed cake had lower interleukin 1-α, tumor necrosis factor-α, and interleukin 36 receptor antagonist after LPS challenge than cattle receiving a control diet or a diet with dried distillers grains plus solubles. This suggests that hempseed cake may have anti-inflammatory properties. Further work is needed to evaluate the anti-inflammatory potential of different hemp products.

In the present study, 4 cannabinoids, CBD-7-acid, CBDA, CBDVA, and THCA, were consistently detected above the LLOQ. In a previous study investigating the pharmacokinetics of cannabinoids in cattle following a single oral dose of IH, CBDA, THCA, CBDVA, and CBCA were detected in all samples and CBD in only a few samples (Kleinhenz et al. 2020a). In another study investigating a 14-d IH feeding period on plasma cannabinoid concentrations and effects on behavior and stress in cattle, CBDA, CBD, CBDVA, CBCA, CBGA, and THCA were detected (Kleinhenz et al. 2022). Smith et al. (2023) detected sporadic and low levels of CBDA/THCA, CBDVA, and CBNA in plasma samples from heifers fed hempseed cake for 111 d; the quantification methods used in that study were unable to differentiate between CBDA and THCA. In a recent study evaluating the depletion of cannabinoids in cattle fed IH leaves, Fruge et al. (2025) detected CBD-7-acid, CBD, CBDA, CBDVA, THCA, and THCVA. Irawan et al. (2025) detected CBC, CBCA, CBD, CBDA, CBG, 9-THC, THCA, and THC-11-OH in plasma of cows fed spent hemp biomass.

The modal time of peak CBDA concentrations (24 h) in the present study is later than the time to maximum CBDA concentration previously reported [11.8 h (Kleinhenz et al. 2020a) and 16.7 h (Kleinhenz et al. 2022)]. Fruge et al. (2025) reported maximum observed concentrations of CBD-7-acid at 72 h, as compared to 48 h in the present study. That study involved 14 d of IH leaf administration, but plasma was only collected for the first 72 h. Because the present study was not designed to determine pharmacokinetic parameters and samples were only collected through 48 h, the observed time of maximum cannabinoid concentrations may not be the time of the true peak concentrations.

The varying cannabinoid signatures and pharmacokinetic profiles in the present study compared to previous work could be due to cultivar differences across studies. However, it is also possible that altered rumen pH or other physiological parameters could play a role in changing cannabinoid absorption. Fruge et al. (2025) reported large variability in fecal cannabinoid profiles in cattle fed IH leaves, postulating that there were marked differences in individual animals’ capacity to absorb or metabolize cannabinoids. The different cannabinoid signatures across studies could be due to individual animal differences. In addition, individual variability in metabolic or absorptive capacity could explain why some cannabinoids (including 9-THC) were only detected in one or two steers in the present study. Further investigation is needed to establish pharmacokinetic data for individual cannabinoids and IH forms and to illuminate the cause of the reported variation in individual animals’ cannabinoid profiles.

The cannabinoid panel used in the present study evaluated CBD-7-acid concentrations. This cannabinoid has only been reported in one other study in cattle, which evaluated cannabinoid depletion in cattle fed IH leaves (Fruge et al. 2025). Although considered an inactive metabolite in people, CBD-7-acid is the major metabolite of CBD and has a long half-life, ranging from 22 to 33 h in humans (FDA CDER 2017; Taylor et al. 2018). In a study investigating the pharmacokinetics of CBD and its metabolites following CBD paste administration in horses, the authors reported the terminal half-life for CBD-7-acid was 79.85 h (Eichler et al. 2023). Fruge et al. (2025) observed the greatest concentrations of CBD-7-acid at 72 h after the first of 14 daily doses; however, they did not sample past 72 h. The FDA has stated an interest in CBD-7-acid, as it is considered a disproportionate metabolite in people (FDA CDER 2023). In the present study, samples were only collected intermittently up to 48 h after IH dosing, so an estimate of time to peak concentration or elimination half-life is impossible. There were detectable levels of CBD-7-acid in baseline samples from all six steers in period 3 and four of six steers in period 4. Steers receiving IH in period 3 had previously received IH in period 2, and steers receiving IH in period 4 had previously received IH in period 1. The baseline CBD-7-acid concentrations from period 3 were greater than period 4, but this difference was not statistically analyzed. All steers underwent at least a 10-d washout period between periods, suggesting that CBD-7-acid has a very prolonged half-life in cattle. Combined with data from Fruge et al. (2025), it appears that the elimination half-life of CBD-7-acid in cattle is long, as in people and horses.

In addition to CBD-7-acid, other cannabinoids were sporadically detected in baseline samples. These other cannabinoids did not follow the temporal trend of the CBD-7-acid baseline samples. For CBC, CBG, CBG, 8-THC, and 9-THC, all positive samples came from one steer from period 1. The samples from this steer were analyzed at the beginning of a UPLC-MS/MS run following analysis of the calibration curve and a methanol wash. While it is possible that these large, neutral cannabinoids were not sufficiently removed from the column following the wash, resulting in false positive results, there were no cannabinoids detected in the methanol wash. The authors feel it is more likely that there were some early-eluting compounds or matrix interference producing these results. However, as previously mentioned, it is possible that individual animal variation in cannabinoid metabolism could explain the positive results from this one steer.

Limitations of this study include the small sample size and the steers having extensive previous handling and human interaction. This prior exposure to people and manipulation during trials may have reduced the steers’ stress response. Another limitation of this study is that the dosing regimen may not have allowed time for cannabinoids to reach effective concentrations during the transport period. In the previous report on the plasma pharmacokinetics of CBDA, time to maximum concentration was 11.8 h (Kleinhenz et al. 2020a). Pharmacodynamic studies evaluating effective cannabinoid concentrations or times to reach effectiveness for specific outcomes in cattle have not been reported. While the dosing regimen chosen may have precluded some pharmacodynamic changes from occurring during the transport period itself, there were complex effects of IH on blood glucose and prostaglandin concentrations. Interaction with transport and time effects makes interpretation of these results difficult. While the single dose can be viewed as a limitation, this regimen was chosen by the authors because it represents the most practical situation for producers, should further research (and approval of IH products for cattle) indicate that IH may have use as a therapeutic agent for alleviating negative effects of transport. Performing a study to evaluate these same outcomes under steady-state conditions would be enlightening, as one of the main goals of IH research in livestock is to pursue IH products as feed ingredients. Animals eating a diet containing IH would presumably be exposed for extended periods and would reach steady-state concentrations of cannabinoids.

As the IH market continues to grow, investigating potential therapeutic applications of IH and IH byproducts in cattle production is of interest to stakeholders. This study evaluated a wide range of outcome variables: body weight, accelerometry, kinetic gait analysis, mechanical nociceptive threshold (MNT), infrared thermography (IRT), complete blood count (CBC), serum biochemistry, blood cortisol, prostaglandin E₂ metabolite (PGEM), and serum amyloid A (SAA). In conjunction with transport and time, IH resulted in reductions in PGEM compared to placebo treatment. Transport resulted in predictable decreases in bodyweight and lymphocyte concentrations and increases in neutrophil, blood glucose, total protein, cortisol, and SAA concentrations. Transported steers had altered activity patterns and transiently increased tissue sensitivity as assessed via MNT when compared to non-transported steers. The results from this study can serve as a basis for the development of future studies regarding transportation and associated stress in cattle.