While rodents are the most prevalently used animal in preclinical animal research, high rates of translational failure concerning drug development have brought into sharp focus the need to study mammalian species that are physiologically more similar to humans, particularly in relation to the aspects of the diseases being modeled (Public Law 89-544, 1966; Public Law 91-579, 1970; Public Law 94-279, 1976; Ioannidis, 2005, 2006; Goodman and Greenland, 2007; Chavalarias and Ioannidis, 2010; Paul et al., 2010; Bailoo et al., 2014b; Gaire et al., 2021). Pigs are an important model in preclinical biomedical research, historically accounting for approximately 6% of all the United States Department of Agriculture (USDA) species protected under the Animal Welfare Act (Public Law 89-544, 1966; Public Law 91-579, 1970; Public Law 94-279, 1976). Pigs share numerous physiological and neuroanatomical similarities with humans (e.g., Lee et al., 2013; Schomberg et al., 2017) and, consequentially, are used in the research setting for the study of oncology, cardiovascular diseases, pediatric disorders, regenerative medicine, transplantation, medical imaging, genomic and reproductive biotechnology, neurological diseases, surgical innovation, gene therapy/immunotherapy, infectious diseases, effects of microbiota, metabolism, nutrition, and education (Vodicka et al., 2005; Lunney et al., 2021).

There is a relative dearth of reliable, validated means of behavioral assessments for pigs within a biomedical setting (Gieling et al., 2011; Murphy et al., 2014). One of the commonly evaluated behavioral phenotypes in the laboratory pig is impaired motor coordination. The apparatuses used to evaluate motor coordination differences in pigs include the open field, the balance/inclined beam, and the treadmill. It has been argued that analysis of pig behavior within these apparatuses permits researchers to evaluate motor behavior within a controlled environment. However, these behavioral assessments are derived from rodent models with little emphasis on the degree to which rodents and pigs differ and the consequences of such differences on the validity of derived experimental results (Carter et al., 2001; Vodicka et al., 2005; Murphy et al., 2014; Shultz et al., 2020). For example, the inclined beam test, which is very similar to the balance beam test for rodents, can also be potentially dangerous to a pig with impaired motor or cognitive function if it were to fall. Moreover, at baseline/unimpaired levels, some animals cannot walk at least halfway up the beam, calling into question the reliability and validity of such assessments (Sullivan et al., 2013). Similarly, in the open-field test, the ability to measure locomotor behavior while inducing conflict in the emotional/arousal domains of behavior often yields conflicting results across replicate studies (Gieling et al., 2011; Murphy et al., 2014). Additionally, the typical open field arena for pigs is orders of magnitude smaller than the typical foraging distance observed within an ethological setting, calling into question the ethological validity of this test (Murphy et al., 2014). Assessment of gait using a treadmill often requires advanced equipment and facilities, which are generally cost-prohibitive and for which infrastructure is sorely lacking (Boakye et al., 2020; Shin et al., 2021). Therefore, inexpensive apparatuses that are safe to use, and protocols which are easy-to-train, and which produce reliable and repeatable evaluations of motor behavior are critically needed for assessing motor impairment in pigs.

To bridge this knowledge gap, an apparatus modeled after a horizontally placed ladder was designed. While this apparatus design may appear to be similar to the “elevated ladder beam” test employed with rats (Soblosky et al., 1997) and mice (Cummings et al., 2007), the design of the Horizontal Ladder Test (HLT) used here drew inspiration from the human standardized field sobriety test (FST), specifically the ‘walk and turn’ test (WAT) utilized for detecting driving under the influence (DUI) of alcohol (Cole and Nowaczyk, 1994; Burns, 2003). In the DUI detection test, individuals are instructed to take nine heel-to-toe steps along a straight line, followed by turning on one foot and returning in the opposite direction (Downey et al., 2016). Given the unique physical characteristics of pigs—short legs, substantial weight, and an ungainly gait (Vodicka et al., 2005; Gieling et al., 2011; Lunney et al., 2021)—the apparatus and protocol were customized to minimize physical contact with the apparatus during crossing; the operational definition of motor incoordination used here related to the degree of contact with the apparatus (see Behavioral Ethogram, Table 1). The modular design of the apparatus allowed for easy adjustment of rung height from 3 to 6 inches in 1-inch increments. The straight, linear ladder with a restricted width parallel to the ground simulated the straight line in the WAT, considering the physical and behavioral differences of pigs. The concept of “walk and turn” was incorporated into the strategy for pigs to perform double-crossings. The number and height of the rungs presented varying difficulty levels, limiting the steps the pigs could take. This approach allowed the pigs to learn to “pick up their feet” and permitted for adjustment of difficulty for testing varying hypothesis while avoiding ceiling and floor effects in terms of the number of errors made. Furthermore, the ethogram that was developed based on the eight indicators of impairment used from the walk and turn test (c.f., Table 1). These indicators included the inability to maintain balance during instructions, starting too soon, stopping while walking, failure to touch heel-to-toe, stepping off the line, using arms for balance, executing an improper turn, or taking an incorrect number of steps. It was hypothesized that: (1) by increasing the rung height to 6 inches during the initial training phase, the pigs would be forced to lift their feet in order to be able to cross the ladder to learn the task, and (2) the pigs would make significantly fewer errors as the rung height was lowered from 6 to 3 inches, in 1-inch increments across training sessions.

As an initial validation of construct validity, changes in motor coordination as a consequence of voluntary consumption of escalating concentrations of alcohol (ethanol) in water was evaluated. It was predicted that, similarly to humans, as the percentage of ethanol in water consumed increased from 2.5 to 10% during voluntary drinking sessions, a significantly greater number of motor coordination errors would be observed when performing the HLT task. By conducting training and testing on flat ground, the risk of injury to the pigs, even when they were heavily intoxicated, was minimized. The HLT protocol can therefore be used to evaluate motor coordination across a wide array of disease models in pigs. This includes applications in the pre-and post-treatment evaluation for substance use disorders including AUD, neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and Parkinson’s disease (PD), motor impairments resulting from conditions including multiple sclerosis (MS), strokes, traumatic head injuries, and ataxic disorders of balance, vestibular function and proprioception.

All materials and equipment used in this protocol are listed in Table 2.

As predicted, the motor coordination score in the HLT decreased as the rung height was decreased from 6 to 3 inches [χ²(4) = 14.04, p = 0.0014, Figure 4].

A sequential training approach was implemented as detailed in Section 2 of the HLT protocol, where the rung height of the ladder gradually decreased from 6 inches to 3 inches. Figure 4 illustrates results of the final day of training at each specific rung height. Initially, the motor coordination score in the HLT was higher (more errors) and more variable at 6 and 5 inches. However, as training progressed, each pig’s performance became more consistent, with lower variability and a lower score (fewer errors), at the 3-inch rung height. On the last training day at the 3-inch height, the median score was low, at 5 points from the total of 3 double crossings. These data suggested that the specified training strategy was effective—the pigs completely lifted their legs when crossing which, in turn, minimized the number of motor coordination errors.

The pigs drank a larger amount of alcohol as alcohol concentration increased [χ²(4) = 13.56, p = 0.0001, Figure 5A] and drank to intoxication levels (BAC > 0.8 mg/mL).

As shown in Figure 5A, a significant increase in alcohol consumption was observed at alcohol concentrations of 7.5% [Z = 3.184, p = 0.0087] and 10% [Z = 2.939, p = 0.0197], relative to 2.5% concentration levels. Two pigs drank to intoxicating levels, 0.8 mg/mL (0.08 g/dL), as defined by National Institute on Alcohol Abuse and Alcoholism (NIAAA) (NIAAA, 2023), already at 2.5% alcohol concentration levels. As shown in Figure 5B, as alcohol concentration increased, four pigs reached the intoxication criterion at 5% alcohol concentration levels, and all pigs reached criterion at 7.5 and 10% alcohol concentration levels [t(4) = 3.008, p = 0.0396 and t(4) = 3.993, p = 0.0162, respectively]. Thus, motor coordination deficits as a consequence of consuming alcohol to intoxicating levels was evaluated next.

The number of motor coordination errors increased with increased alcohol consumption.

All the animals showed good recollection of the HLT task, 1 year later, with the median score being lowest at baseline (i.e., no alcohol exposure). During testing, as the concentration of alcohol increased, the score in the HLT also increased (Figure 6). Consistent with our BAC data, the highest motor coordination scores at 3 inches were observed at 7.5 and 10% alcohol concentrations, with a statistically significant difference being observed between baseline and the 10% alcohol concentration [Z = 2.8, p = 0.0204]. A similar pattern of performance was also observed at 6 inches (the highest rung height), with a statistically significant difference relative to baseline being observed at 7.5% [Z = 2.7, p = 0.0277] and 10% [Z = 3.9, p = 0.0004] alcohol concentrations. The median baseline score, already high at 6 inches, increased further with the escalation of alcohol, suggesting that excessive alcohol consumption exacerbated the already challenging task.

A positive linear relationship between motor coordination and escalating alcohol consumption was observed at both the 3- and 6-inch rung height ([F(1,3) = 30.28, p = 0.0118 and F(1.3) = 39.55, p = 0.0081], respectively; Figure 7); as alcohol concentration increased, the motor coordination score also increased. The proportion of variance (r²) accounted by the fit model was 91 and 93% at 3 and 6 inches, respectively. Notably, a steeper slope was observed when comparing the relationship between motor coordination and alcohol concentration at 6 inches compared to the 3 inches [β = 15.057 vs. 7.916]. This result highlights that crossing the ladder at higher rung heights is more difficult than at lower rung heights.

A remarkably low number of errors was observed after training at the 3-inch height, and the data exhibited uniformity. Even a year post-training, the pigs consistently demonstrated minimal errors at the 3-inch height, surpassing expectations (a minimum of 4 points during a single crossing, resulting in a total score of 24 points with 3 double-crossings; the average baseline performance was 16 points). Furthermore, the pattern of task performance remained consistent across various heights (Figure 6). This suggests that proficiency at the 3-inch level is not solely due to the ease of the task at this rung height, but rather indicates a genuine understanding of the task and retention of the learned information. Additionally, under challenging circumstances where animals may be severely impaired and unable to perform at the 6-inch height, the HLT remains a reliable, easy, and safe measure, height adaptable as needed.

The primary objective of this study was to develop and validate a bespoke HLT apparatus and protocol as a means of evaluating motor coordination deficits in pigs, given their physiological similarities to humans as compared to rodents (Vodicka et al., 2005; Gieling et al., 2011; Lee et al., 2013; Sullivan et al., 2013; Murphy et al., 2014; Schomberg et al., 2017; Lunney et al., 2021). The data, albeit with a small sample size, are consistent with our predictions. During training, the number of errors observed in the HLT decreased as the rung height decreased. This difference while potentially attributable to the test being easier to perform at 3 inches, may also be attributed to the animals understanding the requirements of the task likely due to the forced higher lifting of their legs at the 6-inches rung height. Tentative evidence supporting the latter contention, however, may be gleaned from the observation of a similar pattern of performance, under varying alcohol concentration exposures, at both 3- and 6-inches rung heights (Figure 6). Further research may assist in adjudicating between these two alternative explanations. Given the short legs and heavy body weight of the pigs, training the animals at a rung height of 6 inches, followed by a gradual decrease to 3 inches across training sessions, is sufficient to provide a reasonable baseline measurement of motor coordination.

A secondary objective of this study was to perform an initial evaluation of construct validity of the HLT protocol. Consistent with our predictions, the same animals that were successfully trained on this protocol during the primary objective evaluations had higher motor coordination scores (i.e., made more errors) as ethanol concentration and consumption to intoxication increased. These results are consistent with other studies in mice and rats which demonstrate a greater degree of motor incoordination at high levels of ethanol concentration and/or consumption (e.g., Middaugh et al., 1992; Hanchar et al., 2005; Bohlen et al., 2014). The overall pattern of motor incoordination was consistent at both 3- and 6- inches; as alcohol consumption increased the motor coordination score similarly increased (more errors/contact with the apparatus). Nevertheless, individual differences in motor incoordination were observed, likely attributable to differences in individual pigs’ drinking behavior—both in volume and speed of consumption. It is conceivable, however, that these individual differences in drinking could be eliminated if a fixed volume and concentration of alcohol were administered via gavage. However, this protocol was developed as part of a larger project, which evaluates progression to Alcohol Use Disorder (AUD) at an individual level, and thus, a decision to use a free-choice alcohol consumption paradigm was made (Liu et al., 2023). Together, these results demonstrated that our HLT protocol shows good face and construct validity, as it is both suitable for evaluation of motor coordination in pigs as well as sensitive to deficits in motor coordination as a consequence of ethanol consumption.

The decision to exclusively use female pigs in this study was also influenced by the overarching research question (Liu et al., 2023) and limitations in terms of available space at our vivarium. Given that this was a pilot study focused on method development and prioritizing investigators’ safety, female pigs were chosen for their generally more docile behavior upon reaching puberty. Correspondingly, male pigs can display agonistic behavior at puberty, particularly when housed in close vicinity to female conspecifics (Vodicka et al., 2005; Lunney et al., 2021). While our decision to study female pigs was therefore pragmatic, we do not predict that sex-differences in motor coordination would be observed in subsequent studies.

While using the pig as a model organism has translational advantages; they are costly, require significant space and infrastructure as well as trained personnel in the management and welfare of large mammals. In contrast, a significant benefit of our apparatus is that it is considerably cheaper to make and to perform than many automated rodent devices. The materials for the HLT apparatus can be obtained from any big box hardware store. In addition, it allows adjustments for size and difficulty and represents a low risk of injury even when animals were heavily intoxicated. We, therefore, confidently recommend the HLT protocol for use across a wide array of disease models in pigs and posit its adaptation and use for other large animal species.

The raw data supporting the conclusions of this article will be made available by the authors, upon reasonable request.

The animal study was approved by Institutional Animal Care and Use Committee at Texas Tech Health Sciences Center, Lubbock, TX. The study was conducted in accordance with the local legislation, federal guidelines, and institutional requirements.

XL: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing. AG: Formal analysis, Investigation, Writing – review & editing. AV: Formal analysis, Investigation, Writing – review & editing. JW: Investigation, Writing – review & editing. JD: Investigation, Writing – review & editing. PP: Investigation, Writing – review & editing. JS: Investigation, Writing – review & editing. MA: Investigation, Writing – review & editing. BB: Conceptualization, Resources, Writing – review & editing. JB: Conceptualization, Formal analysis, Methodology, Resources, Supervision, Validation, Visualization, Writing – review & editing. SB: Conceptualization, Funding acquisition, Methodology, Resources, Supervision, Visualization, Writing – review & editing.