Sexy Horses

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Facial expressions in animals have mainly been studied using Facial Action Coding Systems (FACSs) and Grimace Scales (GSs). In FACSs, all possible facial muscle movements and resulting expressions are systematically catalogued as Action Units or Action Descriptors ( 4). Originally developed for humans, FACSs have now been adapted to different animal species, including primates [orangutans ( 5), macaques ( 6), chimpanzees ( 7, 8), gibbons ( 9)], dogs ( 10), cats ( 11), and horses ( 12). Besides their application in comparative psychology [e.g., ( 13)] and research on the evolution of emotional communication [e.g., ( 14)], FACSs have more recently also been used to associate facial expressions with emotional states ( 11). In a statistical model, collinearity of explanatory variables can affect model interpretation and increase the standard errors of the coefficients ( 43). To explore the relationship between the different explanatory variables (“age”, “sex”, “Body Condition Score”, “breed type”, “coat colour”), each combination was tested for independence. For two continuous variables a Pearson's product-moment correlation coefficient was computed with 0.7 as the cut-off value for a “strong correlation” ( 44). When testing two categorical variables (e.g., “sex” and “breed type”), a Crammer's V test was performed ( 45). If V was above 0.7, only one of the two associated variables was included for further analyses. When testing one categorical and one continuous variable (e.g., “sex” and “age”), a Kruskal-Wallis test was run; if it reached statistical significance ( p≤ 0.05), a pairwise Wilcoxon rank sum test was performed as a post-hoc test to identify the levels of the categorical variable that differed from each other. If the results of the Wilcoxon rank sum test were statistically significant at all levels ( p≤ 0.05), only one of the associated variables was chosen for further analyses. This was the case for the association between “BCS” and “breed type”, as the mean “BCS” differed significantly across “breed types” (Kruskal-Wallis test: χ 2 2 = 145.99, p< 0.001; pairwise Wilcoxon rank sum test: all three p-values < 0.01) with coldbloods having the highest BCS ( M = 6.00, SD = 0.70), followed by warmbloods ( M = 5.10, SD = 0.70), and thoroughbreds ( M = 4.80, SD = 0.90) having the lowest BCS. Additionally, “BCS” and “sex” were associated (Kruskal-Wallis test: χ 2 2 = 95.18, p< 0.001; pairwise Wilcoxon rank sum test: all three p-values < 0.01) with stallions ( M = 5.90, SD = 0.90) having a greater “Body Condition Score” than both mares ( M = 5.20, SD = 0.80) and geldings ( M = 5.10, SD = 0.70). Since “BCS” had been assessed by two experimenters without testing for inter-observer agreement, we kept “breed type” and “sex” as the more reliable variables and excluded “BCS” from all further analyses. Association Between Outcome Measures From all pictures we excluded blurry pictures and pictures in which the eye area was not fully visible from further assessment. Pictures were defined as blurry if wrinkles were not clearly detectable or the beginning and/or end of wrinkles was not visible ( 1). From the remaining pictures ( n = 2,259), two or three pictures per horse and eye (left, right) were randomly selected for scoring (62 horses × 4 pictures and one horse with one eye × 2 pictures, data collection in summer 2014; 118 horses × 6 pictures, data collection in spring/summer 2015 and spring 2016) using the “sample” function in R (R Version 3.5.1, R Studio Version 1.1.453) and resulting in a total of 958 pictures. The selected pictures were cropped to only show the eye area needed for scoring and picture size was standardised using Microsoft Picture Manager (version 2018.18051.17710.0). Eye Wrinkle Assessment Scale

In conclusion, do not have sex with horses unless you are a professional, have the proper training, don't care what other people will think if they find out, and don't mind the possibility of extreme bodily injury and/or death, and breaking the law. Assessing emotional states in animals is a critical goal in animal welfare science, but it is generally agreed that the subjective experience of an emotion cannot be assessed directly [but see ( 2) for a different point of view]. Emotional states are multifaceted, including not only the subjective experience but also behavioural, physiological, and cognitive components, which can be assessed objectively and could therefore serve as indicators to infer animals' subjective experience [e.g., ( 3)]. Ideally, such indicators can be assessed non-invasively as well as reliably across various contexts, and do not require the animals to be trained. Spontaneous behaviour, including facial expressions, are promising examples of indicators to assess animals' emotional states. The problems with this are many, since it's still your perogative if you want to have sex with horses or not, hey, ain't nobodys buisness if you do, I will not pass judgement on this practice, other than the medical problems I will now mention. Top form Malin has been on many Swedish teams, including the silver-medal winning teams in the Athens Olympics (2004) and the World Games (2002). LS, KK, and SH conceived the study. LS and SH developed the methodology, collected the data, and wrote the manuscript. LS scored all pictures and performed the data analysis. All authors edited the manuscript, contributed to manuscript revision, read, and approved the submitted version. Funding

After pictures from both eyes had been taken, the body condition of each horse was assessed by visual and tactile evaluation using the scale developed by Henneke et al. ( 32). With this scale, the presence or absence of adipose tissue and the visibility of bone structures is assessed on a nine-point scale (1–9, half points can be given). One person assessed the body condition of horses on all farms except for Farm 7, where the assessment was done by another person with the same scale. Agreement between the two assessors could not be evaluated due to large spatial and temporal distances of data collection. Sixteen horses on Farm 7 were not assessed. The BCS ranged from 2.5 to 8 ( M = 5.3, SD = 0.9). Picture Processing

All outcome measures (“qualitative assessment”, “brow raised”, “number”, “markedness”, “angle”) were tested for association. First a Kruskal-Wallis test, and if statistically significant a pairwise Wilcoxon rank sum test, was run to test for associations between “markedness” and all continuous measures. “Qualitative assessment” ( χ 2 2 = 301.92, p< 0.001), “brow raised” ( χ 2 2 = 333.06, p< 0.001) and “number” ( χ 2 2 = 881.66, p< 0.001) were associated with “markedness”; p-values < 0.01 for all pairwise Wilcoxon rank sum tests. Since “markedness” was a categorical measure on an ordinal scale, it was probably the least sensitive measure, and we therefore dropped it. All remaining outcome measures were continuous variables, and a Pearson's product-moment correlation coefficient was computed. If the correlation coefficient was > 0.7, indicating a “strong correlation” ( 44), only one outcome measure was selected for further analyses. A strong positive correlation between the outcome measures “qualitative assessment” and “brow raised” was found ( r = 0.9, p< 0.001), and we selected “qualitative assessment” for all further analyses. The Effect of the Different Explanatory Variables on the Outcome Measures

Discussion

We found substantial variability within each outcome measure, similar to what Hintze et al. ( 1) described for their presumably neutral control phases before the start of the experimental treatments. This variability may be explained by individual characteristics of horses independent of our tested characteristics, which did not account for the variation we found (with the exception of “breed type” accounting for differences in the “angle”). However, other explanations need to be considered as well. First, individual horses may have reacted differently to the halter, the human handling and/or the photographing. Even though we only took pictures when horses appeared to be calm and relaxed (standing still, head approximately at wither height), we cannot exclude that single individuals were slightly stressed by the procedure. Second, variation in eye wrinkle expression across horses could be caused by differences in underlying mood states, but this explanation is speculative since we did not assess mood in the present study. Variation in “BCS” could be explained by the three “breed types”, with coldbloods having the highest scores, followed by warmbloods and thoroughbreds. This finding is consistent with what has been reported by Giles et al. ( 53), who found that breed was the risk factor most strongly associated with obesity in horses. In line with this, Visser et al. ( 54) found that coldbloods were more prone to develop a higher body condition score compared to thoroughbreds, which was also the case in our study. However, the association between “BCS” and “breed type” in our study could also be explained by confounds in our sample since most coldbloods were from Farm 7 and the management practices on a farm, especially the feeding regime, including the amount of feed and its nutritional value, can influence the body condition of horses. This confound may also explain the strong association between “sex” and “breed type” since most of the stallions on Farm 7 were coldbloods. Interpretation of the Results of the Outcome Measures The ethical guidelines of the International Society for Applied Ethology were respected while carrying out this experiment. For photographing, horses were loosely held on a halter (a normal routine for all horses used in this study) without any further manipulation. Horses from Farm 7 were additionally used in two larger studies, which were approved by the Cantonal Veterinary Office in Vaud, Switzerland (license numbers 2804 and 2804_1). Author Contributions All analyses were performed with the statistical programming language R [R version 3.5.1, ( 37); RStudio version 1.1.453, ( 38)]. The data set can be found in Supplementary Table 2. Intra- and Inter-observer Agreement In the final sample, “qualitative assessment” ranged from 0.25 to 100 on the VAS ( M = 36.63, SD = 31.45), and the “number” of wrinkles varied from 0 to 5 wrinkles ( M = 0.80, SD = 1.10). The “angle” ranged from 5.8° to 50.6° ( M = 13.00, SD = 14.70).

To assess both intra- and inter-observer agreement, a sample of all pictures was re-scored by the same rater (LS, to assess intra-observer agreement) and by a second rater (SH, to assess inter-observer agreement). To assess intra-observer agreement, ten out of each subset of 50 pictures (in total n = 192 pictures) were randomly selected and scored a second time, at the earliest the day after the first scoring. To assess inter-observer agreement, a sample of 10% of all pictures ( n = 96 pictures) was scored by SH after LS had finished scoring. Both raters were experienced in using the original and adapted eye wrinkle assessment scale. Coat Colour Certain confounding effects (e.g., between “breed type” and “farm”, see below) could not be ruled out since our sample was not fully balanced across all explanatory variables. However, we counteracted this limitation as much as possible under non-experimental conditions by ensuring a relatively large sample for the different categories of “sex” (70 mares, 62 geldings, and 49 stallions), “breed type” (52 coldbloods, 104 warmbloods, and 17 thoroughbreds) and “age” (including young to very old horses). Relationship Between “Body Condition Score” and the Two Explanatory Variables “Breed Type” and “Sex” For one outcome measure (“angle”) and two explanatory variables (“breed type”, “coat colour”), a subset of the full data set was used. For “angle” only pictures with at least one wrinkle (“number”≥ 1) were included since an angle could only be measured if at least one wrinkle was identified, resulting in 427 pictures. For the analyses of “breed type” on the different outcome measures, all ponies ( n = 7) and the horse without known breed were removed from the data set due to the small sample size, leading to a remaining sample of 916 pictures. For 902 pictures a “coat colour” could be assigned and these were therefore used for subsequent analyses. Intra- and Inter-observer Agreement She says 'It seems an awful lot, but that's how I want it. I love trying out new things. I have to have new things going on around me. That's what I thrive on'.

Beside the systematic effect of “breed type” on “angle”, there was no further effect of any of the explanatory variables on our outcome measures. This finding indicates that eye wrinkle expression can be assessed regardless of “age”, “sex”, and “coat colour”, while “breed type” should be considered in future studies. Our study does not give further insight into the relationship between emotion or mood and eye wrinkle expression, but it shows that eye wrinkle expression in horses cannot simply be explained by the investigated characteristics of the horses. Side Effects To our knowledge, this was the first study systematically investigating the effect of individual characteristics on eye wrinkle expression and its assessment in horses. We conclude that our eye wrinkle assessment scale can be used reliably and regardless of horses' age, sex, coat colour, and breed type (here with the exception of the “angle”). Thus, the adapted scale is a promising tool to assess eye wrinkles in horses, but to what extent these are systematically affected by mood or emotion or the interaction of mood and/or emotion with individual characteristics needs further investigation and validation. Data Availability Results are given for all outcome measures including “brow raised” and “markedness” in case these outcome measures will be included in future studies. Comparison of first and second scoring of rater LS (intra-observer agreement) exceeded 0.9 for all continuous outcome measures (“qualitative assessment”: ICC agreement = 0.90, with a 95% CI from 0.87 to 0.93; “brow raised”: ICC agreement = 0.94 with a 95% CI from 0.92 to 0.96; “number”: ICC agreement = 0.97, with a 95% CI from 0.96 to 0.98; “angle”: ICC agreement = 0.97, with a 95% CI from 0.96 to 0.98) and 0.8 for the categorical outcome measure (“markedness”: κ = 0.92) with all p-values being highly significant ( p< 0.001). Inter-observer agreement was slightly lower than within rater LS, but still exceeded 0.75 for all continuous outcome measures (“qualitative assessment”: ICC agreement = 0.80, with a 95% CI from 0.70 to 0.87; “brow raised”: ICC agreement = 0.84, with a 95% CI from 0.77 to 0.89; “number”: ICC agreement = 0.78, with a 95% CI from 0.69 to 0.85; “angle”: ICC agreement = 0.99, with a 95% CI from 0.98 to 0.99) and equalled 0.8 for the categorical outcome measure (“markedness”: κ = 0.80). Again all p-values were highly significant ( p< 0.001). Assessment of the Outcome Measures Trivia She started riding at six - and won a gold medal at the Swedish Championships at the age of 14. Also a model, she became Sweden's face of H&M in 1996 and married Swedish presenter Henrik Johnsson before becoming a TV presenter herself, with her own show. She has appeared in films and owned a restaurant. Her horse H&M Tornesch is an imposing bay stallion who only has one eye, having lost the other following an accident in his youth. 'He trusts me like nothing else and he only sees the jump at the last minute, but he confidently jumps it with no doubt and so little effort.' In the present study we investigated whether age, sex, breed type, body condition, and coat colour systematically affect eye wrinkle expression or its assessment in pictures taken of the left and the right hemiface of horses in a presumably neutral situation. Eye wrinkle expression was assessed using five outcome measures, all of which could be assessed highly reliable with respect to both intra- and inter-observer agreement. Some outcome measures were associated, therefore only “qualitative assessment”, “number”, and “angle” were further analysed. Similarly, “Body Condition Score” was strongly associated with the two explanatory variables “sex” and “breed type” and was thus not included in further analyses. “Breed type” influenced the width of the “angle”: thoroughbreds had a narrower “angle” than warmbloods and coldbloods, and warmbloods had a narrower “angle” than coldbloods. The three other explanatory variables (“age”, “sex”, and “coat colour”) did not affect any of the outcome measures, and eye wrinkle expression did not differ between the left and right eye. Characteristics of the Investigated Sample