Both the conventional pigs and the Göttingen minipigs learned the holeboard task. This finding corroborates and extends earlier studies by Arts et al.  and Gieling et al.  and confirms our hypothesis. After intensive training (about 100 trials), the pigs reached nearly errorless asymptotic WM and RM performance. Moreover, all pigs had a higher level of performance than we have ever seen in rats, even after more that 400 training trials . The holeboard performance and motivation of minipigs were not different from those of conventional pigs, showing that both types of pig can be used in cognitive research. This is in marked contrast to the conclusion drawn by Downes  based on a study by Manton , that “the results of testing for learning and memory in minipigs were equivocal and ultimately disappointing”. We found that minipigs could acquire the spatial holeboard discrimination task, suggesting that Downes’ conclusions concerning testing the learning and memory capacity of minipigs, using the holeboard task are premature.
The cholinergic system is involved in spatial discrimination learning , and holeboard-type tasks are sensitive to manipulation of cholinergic neurotransmission with so-called cognition enhancers or cognition impairers (for a recent review see ). Biperiden, an M1 receptor antagonist, is suggested to act as a cognition impairer [7, 17, 19], but we found only marginal effects on RM and WM at very high oral doses (5 to 20 mg.kg-1). The WM performance of the conventional pigs appeared to be unaffected by biperiden, whereas the drug appeared to differentially affect WM performance in the minipigs, with the lowest dose of biperiden marginally improving WM performance and the highest dose marginally impairing WM performance (see Table 1). On the basis of the effects of the cognition impairers scopolamine and MK-108 in well-trained rats , we expected that biperiden would transiently affect WM and RM performance. The lowest dose of biperiden that impaired RM in both groups of pigs was 5 mg.kg-1, which is in the dose range that was found to affect cognition in rats . Thus although we had expected that biperiden would transiently impair spatial memory in conventional pigs and age-matched minipigs, once they have learned the cognitive holeboard task, we cannot unambiguously conclude that this is the case. We can conclude that the effects found on RM are comparable for both breeds, although the highest dose could not be tested in conventional pigs.
It may be difficult to impair memory performance once a task has been learned to almost perfection, but previous studies with the conefield task, a variant of the holeboard task, have shown that the near-maximal, asymptotic WM and RM performance of rats could be decreased with the cognition impairers scopolamine and MK-801. In the drug-free session following each of the scopolamine or MK-801 sessions, the WM and RM performance returned to control level . Although the rats reached an asymptotic performance, they did not reach the maximum performance possible (ceiling level). In the current study, the peak performance of the pigs was close to the maximum performance level possible. In order to detect cognition-impairing effects, it might be appropriate to give biperiden earlier, before pigs reach an asymptotic level of performance, or the task could be made more difficult (e.g. hide 5 instead of 4 rewards). This is supported by the observation that while the performance of laboratory animals in the conefield task can be altered by cognition impairers , performance in other spatial learning and memory tasks, such as the Morris water escape task, is unresponsive to the effects of cognition impairers once the task has been learned to an asymptotic level . In a study involving a well-learned operant task and rats , biperiden at doses of 0.25 and 0.5 mg.kg-1 increased the number of nonreinforced responses and decreased the number of reinforcements obtained. Doses exceeding 0.5 mg.kg-1 already led to long pauses in responding and omissions to respond. In pigs, doses equal to and exceeding 5 mg.kg-1 tended to increase trial duration and the time taken to find the first food reward. The longer trial duration may have been caused by a longer time taken to visit the first hole and an increase of the number of erroneous hole visits before all the baits were found (RM errors).
Our findings suggest that conventional and minipigs differ in their sensitivity to the disruptive effects of biperiden with only the conventional pigs starting to refuse the higher doses. However, this apparent difference might have been caused by differences in body composition. Leanness significantly affects the volume of distribution of a drug and hence its adverse responses. Moreover, biperiden is highly lipophilic, rapidly entering the brain, but slowly entering fat tissue, from where it is slowly cleared . Although we did not measure the proportion of body fat, we presumed that the conventional pigs were leaner than the minipigs. If this is the case, then minipigs are not necessarily less sensitive to the pharmacological effects of the biperiden, but instead have different pharmacokinetics. Although we found that biperiden did not impair retrieval of well-consolidated information from RM, and within-trial WM, it remains to be seen whether biperiden affects the learning process and/or memory consolidation. This can be assessed by administering biperiden before or immediately after the daily training trials during acquisition of the holeboard task. Biperiden has been found to impair consolidation and retrieval in a passive avoidance task with rats [26, 34].
Our data confirm that biperiden is safe, even after administration of very high doses. The pigs were treated orally with up to 20 mg.kg-1: biperiden, a dose approximately 400-times higher than the therapeutic dose usually used in humans. We observed only mild non-cognitive adverse effects at the highest doses tested, such as mild signs of dry mouth , a typical side effect of anticholinergic drugs . Liquid reinforcements could be used to get around the dry-mouth problem. It remains to be determined whether the reduced RM performance observed with the highest dose of biperiden really reflects cognitive deficits caused by a central action of biperiden or noncognitive adverse drug reactions. In the latter case, biperiden would not fulfill the requirements of a pharmacologically active cognition impairer in pigs . A potential limitation of the study is that none of the conventional pigs ate the mixture containing the highest dose of biperiden and one did not did not eat the mixture containing 15 mg.kg-1 biperiden. This reluctance to eat is unlikely to have been due to a dry mouth, because this side effect developed about 30 minutes after drug ingestion. Dry mouth might have diminished the attractiveness or palatability of the M&M rewards. However, all Göttingen minipigs ate the food containing biperiden, even though they showed symptoms of dry mouth. The large volume of the mixture of crushed tablets, pig feed, and honey (the latter needed to stimulate pigs to eat the tablet mixture) may have delayed the absorption of biperiden, such that peak plasma levels, which were expected approximately 1–1.5 hours after drug administration on the basis of published pharmacokinetic data , were reached later. This would have led to underestimation of the ability of biperiden to affect spatial memory. In the future, it might be more effective to administer pure biperiden, so that less “filler” is needed.
The present study did not provide evidence to support the conclusion drawn by Klinkenberg and Blokland  that M1 receptor antagonists can be considered an alternative to scopolamine as cognition impairer, at least if conventional or minipigs are used as subjects. We suggest that biperiden should be evaluated further as putative cognition impairer in pigs, but using a different way to administer the drug. On the basis of our findings, a counterbalanced design could be used in a future study, e.g. involving the doses 1, 3, and 10 mg.kg-1. The learning task could be made more difficult, and RM performance should be monitored daily, so that treatment can be started before performance reaches an asymptotic level, to prevent a ceiling effect. On the basis of our findings, minipigs can be used instead of conventional pigs or can serve as a translational model for other species. The effectiveness of biperiden treatment in conventional and minipigs clearly needs to be evaluated further before definitive conclusions can be drawn about the ability of biperiden to impair cognition in this species.