Materials and Methods

All procedures involving the use of animals received approval from the University Committee on Animal Care and Supply at the University of Saskatchewan and were conducted in accordance with the guidelines established by the Canadian Council on Animal Care.

Seventeen registered horses (6 Clydesdales, 3 Percherons, and 8 Quarter horses) of known age (median, 5.73 years; interquartile [25% to 75%] range, 2.0 to 12.9 years; range, 2 to 21.2 years) that had been donated to the study for reasons other than weight loss or dental disease were used. Horses had been maintained solely on 1 of 3 different hay diets fed ad libitum for not less than 14 days to ensure that the gastrointestinal tract and feces contained nothing other than this feed prior to euthanasia. They were euthanatized by means of IV administration of a mixture of pentobarbital^a and embutarate^b administered as a bolus into the left jugular vein.

Heads were disarticulated at the atlanto-occipital joint, and the mandibles were separated from the cranium by sectioning through the vertical ramus of the mandibles with a band saw. Teeth and oral mucosa were cleaned in running water and dried. Oral examinations were performed in a standard fashion. Incisors were examined for evidence of malocclusions and missing teeth. The cheek teeth were examined, and the presence of malocclusions (ie, hooks, ramps, excessive transverse ridges, and stepped or cupped teeth), missing teeth, diastemata, gingivitis, or periodontal disease (defined as gingival recession with hyperemia and feed packing) was recorded. On the basis of these findings, an overall oral pathology score was assigned as described.¹⁷

Maxillary and mandibular cheek teeth were photographed with a digital camera.^c For all photographs, a scale was positioned at the level of the occlusal surface to account for any size distortion of the image. A computer-based photographic program^d linked to a personal computer with an attached graphic pad^e was used to trace the outlines of the teeth and their internal morphology, resulting in a black-and-white replica of the occlusal surface. Morphologic variables that were measured and calculated from these images included the total surface area, the inner surface area (ie, the area of each tooth bounded by the outer enamel ridge), the outer surface area (calculated as total surface area minus inner surface area), the infundibular enamel ridge perimeter distance, the outer enamel ridge perimeter distance, and the total enamel ridge perimeter distance (calculated as infundibular enamel ridge perimeter distance plus outer enamel ridge perimeter distance). Morphologic variables were determined separately for the maxillary and mandibular arcades and then summed to obtain an overall value for each horse; overall values for each horse were used in all analyses. Methods for measuring these morphologic variables have been published previously.¹⁸

Gastrointestinal tract samples were collected from each horse within 30 minutes of euthanasia. The entire contents of the stomach were emptied into a plastic bucket. This was then agitated to ensure thorough mixing of the ingesta, and a 1-L plastic container was used to collect a representative sample of the stomach contents. Entire fecal balls were removed from the small colon or rectum. Stomach and small colon or rectum samples were immediately frozen at −20°C.

Small colon or rectum samples and samples of each of the 3 hay diets were submitted to a commercial laboratory^f for digestibility analysis, including determination of dry matter, crude protein, acid-detergent fiber, neutral-detergent fiber, energy, and acid insoluble ash contents. Apparent digestibility was calculated by means of the acid insoluble ash method.^19–21 In brief, apparent indigestibility of a nutrient was calculated by multiplying the concentration of acid insoluble ash in the feed by concentration of the nutrient in the feces and dividing by the concentration of acid insoluble ash in the feces multiplied by the concentration of the nutrient in the feed. Apparent digestibility was then calculated as 1 minus apparent indigestibility.

Ten-liter samples of each hay diet were used for feed particle size analysis. Mean feed particle size was measured with a purpose-built particle separator. A stack of screens (diagonal pore sizes of 26.9, 18.0, 8.98, 5.61, and 1.65 mm) was shaken for 2 minutes in accordance with published standards.²² Stems > 26.9 mm in length were left on the top tray. One hundred of these were randomly chosen and measured with a tape measure graduated in millimeters.

A 50-g aliquot of each small colon or rectum sample was used for fecal particle size analysis. Mean fecal particle size was measured with a Tyler sieve shaker.^g Analyses were performed in 2 stages. Standard sieve sizes 3, 4, 5, 6, 8, 14, and 20 (diagonal pore sizes of 6.7, 4.75, 4.00, 3.36, 2.36, 1.41, and 0.85 mm, respectively) were used in the first stage, and this sieve stack was shaken for 6 minutes.²³ The second stage included standard sieve sizes 20, 30, 45, 100, 200, and 270 (diagonal pore sizes of 0.85, 0.589, 0.355, 0.15, 0.075, and 0.053 mm, respectively). This sieve stack was shaken for 12 minutes. Between each sample, sieves were cleaned with a 5-inch coarsehair paintbrush and reweighed to ensure that the entire sample had been removed. The same methodology was used to determine mean particle size for each sample of stomach contents.

Statistical analysis—Descriptive statistics were calculated. The Shapiro-Wilk test was used to determine whether data were normally distributed. The Wilcoxon rank sum test was used to examine whether there were significant differences in morphologic variables between adult horses with complete dentition (ie, 24 permanent cheek teeth) and juvenile horses. The Spearman rank correlation method was used to test for correlations between age and morphologic variables in the subset of horses with complete dentition. One-way ANOVA was used to determine whether there were significant differences in apparent digestibility among diet groups and to determine whether diet had an effect on feed, stomach content, and fecal particle sizes. Linear regression was used to examine the effect of oral pathology score on stomach and fecal particle sizes before and after controlling for diet group. The Spearman rank correlation was used to examine the effect of morphologic variables on feed digestibility.^24,25 All analyses were performed with standard software.^h Values of P < 0.05 were considered significant.

Results

Morphologic variables were summarized (Table 1). Ten of the 17 horses were found to have complete dentition (ie, 24 permanent cheek teeth). The remaining 7 horses were all < 3.5 years old and were missing both mandibular and both maxillary third molars (ie, 20 permanent cheek teeth). Morphologic variables (ie, inner surface area, outer surface area, total surface area, infundibular enamel ridge perimeter distance, outer enamel ridge perimeter distance, and total enamel ridge perimeter distance) were significantly (P < 0.001) different between horses with complete dentition and horses with incomplete dentition. Median oral pathology score was 4.5 (interquartile range, 0.0 to 8.5; range, 0.0 to 17). We did not detect a significant (P = 0.25) linear correlation between oral pathology score and age.

Nutrient analysis did not reveal any important differences in nutrient contents among the 3 hay diets (Table 2), and apparent digestibility did not differ significantly among diets. Mean feed particle size differed significantly (P < 0.001) among the 3 diets (diet 1, 43.09 mm; diet 2, 50.62 mm; diet 3, 74.40 mm). However, particle size in stomach contents and fecal particle size did not vary significantly among diet groups, and fecal particle size (mean ± SD, 1.36 ± 0.16 mm; range, 1.15 to 1.72 mm) was not significantly different from particle size in stomach contents (1.29 ± 0.26 mm; range, 0.69 to 1.76 mm).

We did not detect a significant linear correlation between oral pathology score and particle size in stomach contents (P = 0.22) or between oral pathology score and fecal particle size (P = 0.99). There was also no significant linear correlation between oral pathology score and the difference between feed and stomach content particle sizes, even when controlling for diet. There was no correlation between any of the morphologic variables and apparent feed digestibility (P = 0.32 to 0.99).

Discussion

Results of the present study did not provide any evidence of associations between cheek tooth morphology, fecal particle size, and apparent digestibility in horses. Specifically, we did not detect significant correlations between morphologic variables that were measured and feed digestibility, indicating that there were no differences in the ability of these horses to digest their feed, as determined by apparent feed digestibility, despite the wide range of values obtained for size of the chewing surface and amount of enamel upon which to shear feed material. In addition, fecal particle size was not significantly associated with oral pathology score.

Values of all morphologic variables measured were significantly higher for horses with complete dentition (ie, 24 permanent cheek teeth) than for horses with incomplete dentition in the present study, indicating that horses with complete dentition had a greater surface area for chewing and greater amounts of enamel. There was an association between body size and age in the present study, in that all 7 horses that were < 3.5 years old were Quarter Horses, and 9 of the 10 horses that were > 3.5 years old were Clydesdales or Percherons. Therefore, we could not examine the effect of body size on morphologic variables. No attempts were made to obtain horses of a similar age or size for the present study because our intention was to investigate the effect of a wide range of cheek tooth morphologic measurements on apparent feed digestibility.

Incidence and severity of dental disease in horses have been previously reported to increase with age.^2–4 As horses age, the amount of residual crown is reduced, until it equals the amount of exposed crown. Further attrition will result in loss of occlusion and, eventually, tooth loss. Despite this tooth loss, it is our clinical impression that we do not see clinically apparent weight loss associated with loss of occlusion in most older horses, nor do we see evidence of insufficient mastication or poor apparent feed digestibility. Similarly, while there are many influences on growth of young horses from weaning to 4 years of age (during which time the number of cheek teeth doubles from 12 to 24), such as management, seasonality, and nutrition, there is no clinically apparent saltatory increase in weight or condition associated with the eruption of more cheek teeth and the associated increase in enamel or surface area for mastication. Findings of the present study agree with these observations, in that there was no difference in apparent feed digestibility between immature horses with incomplete dentition and mature horses with all 24 cheek teeth.

Increases in bite force, masticatory time, and voluntary intake; decreases in feed passage rate; and selection of more nutritious or more biologically available feeds to reduce energy expenditure needed to obtain nutrients from ingested material have been reported in other herbivorous animals in which chewing effectiveness was reduced because of dental wear.^11,13 Masticatory time was not recorded in the present study, and it may be that horses with an incomplete dentition require longer masticatory times for the particle size to be reduced to the same size as in horses with complete dentition.

In the present study, even though particle size varied substantially among the 3 hay diets that were fed, there was surprisingly little variation in the size of particles in the stomach contents. In addition, fecal particle size was not significantly different from particle size of the stomach contents, indicating that there was little further reduction in particle size as the ingesta passed through the gastrointestinal tract. This would suggest that mastication was the most important determinant of feed particle size reduction in these horses, which is similar to findings for ruminants.¹¹

We have previously reported that dental floating does not affect mean fecal particle size or feed digestibility in healthy horses.⁸ On the basis of results of that study, we theorized that any change in the process of mastication and feed breakdown in the oral cavity that occurred as a result of dental floating was compensated for by the gastrointestinal tract, such that fecal particles were uniform at the end of the digestive process. In the present study, however, we found that particles in the stomach contents were the same size as particles in contents from the small colon or rectum, indicating that mastication resulted in physical breakdown of the ingested feed, and little or no further changes occurred after gastric digestion. Importantly, we did not detect any significant linear association between oral pathology score and stomach content or fecal particle size, even when controlling for diet, indicating that horses with high scores were just as efficient at breaking down ingested feed as were horses with low scores.

The acid insoluble ash method is a common method of calculating apparent digestibility and is used in situations when measuring total fecal production or administering chromic oxide is not practical.¹⁹ In studies^20,21 comparing digestibility values obtained with the total fecal collection method, which is considered the most accurate, with values obtained with the chromic oxide and acid insoluble ash methods, the latter method was considered to be more accurate.

In a previous study¹³ involving red deer in which morphologic variables of the first molar were measured, animals with a low outer surface area had a greater mean number of chews per gram of dry matter than did animals with a high outer surface area. Enamel ridge perimeter distance in koalas has been found to be directly related to chewing effectiveness in this species.¹⁶ More research is needed to determine the role of dentition on mastication and gastrointestinal tract processing of ingested feed in horses. Presently, there is no evidence that dental abnormalities contribute to maldigestion or weight loss. One horse in the present study, for instance, was 30 years old and had an oral pathology score of 17 because of missing teeth, diastemata, and advanced periodontal disease. However, stomach content and fecal particle sizes in this horse were not substantially different from values for other horses in the study, and apparent digestibility in this horse was within the range for other horses.

There obviously is a critical point at which oral pain related to dental disease is sufficient to limit voluntary feed intake, and it may be that the adverse effect of dental disease on voluntary intake has a greater impact on feed performance than does any effect dental disease has on digestibility. In addition, there must be some minimum amount of tooth necessary for efficient mastication, such that below this limit, the reserve capacity of the gastrointestinal tract is exceeded and maldigestion and weight loss result. However, this morphologic limit is currently unknown.