Abstract
OBJECTIVE
Slow hoof growth poses a clinical challenge when hoof wear exceeds natural growth. Many treatment options have been reported without controlled prospective trials. The objective of this study was to evaluate the effect of transdermal CO2 on the rate of growth in treated hooves.
METHODS
A prospective, randomized, blinded, crossover study of 14 Warmblood mares. Horses were randomly assigned a number and allocated for treatment of the front feet with room air or CO2 for 30 minutes 3 times per week, and groups were switched after 5 weeks. Hoof growth was measured on the dorsal midline and the quarters of each front foot. The distance from coronary band to lines made on the hoof wall was measured at the beginning, middle, and end of the study. The percentage of change in length at each location relative to baseline was evaluated at 5 weeks and 10 weeks using nonparametric analyses.
RESULTS
All treatments were successfully administered. The left front medial (P = .028) and right front lateral (P = .03) sites of the CO2-treated hooves increased growth compared to the room air group at the 5-week point.
CONCLUSIONS
The results of this study suggest that repeated, noninvasive transdermal application of CO2 may accelerate hoof growth in normal horses over a 5-week treatment period.
CLINICAL RELEVANCE
This study suggests that transdermal CO2 may increase the rate of hoof growth in normal horses. The mechanism of action of this treatment is unclear, and further studies are required to fully elucidate the potential effects.
Equine hoof growth is widely variable, with reported rates of 0.19 to 0.28 mm/d.1 The wide variation in growth rate may be due to specific horse-related and environmental factors, including nutrition and activity level as well as environmental conditions. Delayed or slow hoof growth, especially in horses with increased clinical sensitivity, poses a substantial clinical challenge for veterinarians, farriers, and horse owners alike. In more pronounced cases, slow hoof growth may result in morbidity with loss of use or result in decreased welfare due to foot pain from thin sole depth. Numerous commercial products are advertised to aid in hoof growth, including topical salves and oral supplements with variable clinical efficacy. Nutritional supplementation with biotin has been described in a single clinical study2 with some positive effects, but controlled, prospective research studies are lacking. Further medical treatments, such as intracoronary band injection with platelet-rich plasma, have also been evaluated but without measurable clinical improvement between treatment and control groups.3
The paucity of controlled research in this field motivates the evaluation of alternative therapies. Gaesser et al4 anecdotally reported an increase in hoof growth while investigating transdermal CO2 for distal limb wounds in horses. Transcutaneous CO2 has been shown to be effective for wound healing in both human and rodent models and has a positive effect on muscle injury healing in rodent models.5 Increasing oxygen delivery to tissues via CO2 exchange within the vasculature, due to the Bohr effect, has been proposed as the mechanism of action in these studies6 yielding positive results.
The aim of this exploratory pilot study was to investigate the short-term effects of medical-grade transdermal CO2 on the rate of forelimb hoof growth in the healthy horse. We hypothesized that there would be an increased rate of hoof growth in horses treated 3 times per week with transdermal CO2. The outcomes of this study were intended to evaluate additional noninvasive therapeutic options, which may be available to promote hoof growth.
Methods
Study design
A controlled, randomized, crossover study design was used for this 10-week study. This study protocol was reviewed and approved by the Penn Privately Owned Animal Protocol Committee, and owner consent was obtained prior to the start of the study. Fourteen adult Warmblood mares on the same farm undergoing similar management were enrolled. Carbon dioxide treatment was applied using a commercially available system (Dioxyfin; Serendi Medical LLC).
Animals
Enrolled horses were either current (nonpregnant) or retired broodmares that were not in active work. In this study, a normal population of horses was defined as those that were bilaterally forelimb sound and systemically healthy as evaluated by physical examination at the start of the study. Routine hoof trimming was performed on all horses prior to the start of the study and assignment to treatment groups. All horses were barefoot for at least 3 months prior to the start of the study and maintained without shoes or trimming for the duration of the study. Paired front feet of each horse were randomly assigned a number that correlated to 1 of 2 treatment groups, either treatment with room air (control; n = 7) or treatment with transdermal CO2 (treatment; n = 7), and groups were switched after 5 weeks of treatment and treated for an additional 5 weeks.
Hoof wall measurements: weeks 1 through 5
At the start of the study period, following routine trimming, 3 small horizontal lines were made on the dorsal hoof wall of each front foot using a standard handheld rotary power tool (Dremel Rotary Tool; Robert Bosch Tool Corporation). The lines were located 1 cm from the coronary band, with 1 on midline and 1 on each quarter (lateral and medial). Hoof wall markings were made by an individual blinded to the treatment group. The distance from the coronary band to these markings was measured using digital calipers (6-Inch Digital Micrometer Caliper; M MOOCK) (Figure 1). The top of the caliper was placed at the hairline junction for consistent measurement from the coronary band. Measurements were made independently by 2 trained equine professionals blinded to the treatment groups. Two measurements were obtained at each location on the hoof wall, and the averages were recorded. Each examiner was asked to keep the location that the calipers sat within the hoof wall groove consistent between all of their measurements. All horses began enrollment in the study on the same date.
A—A front hoof with horizonal lines formed by the Dremel tool; midline and lateral locations shown in this image. B—Digital caliper tool being used to measure the distance from the coronary band to the horizontal line.
Citation: American Journal of Veterinary Research 85, 11; 10.2460/ajvr.24.06.0161
Description of treatment: weeks 1 through 5
Control group horses had both forelimbs wetted with tap water, and a CO2 product bag was applied to each forelimb and inflated with room air. The bag was maintained for 30 minutes and reinflated as needed. Treatment group horses had both forelimbs wetted with water and the CO2 product bag applied to each forelimb and inflated with medical-grade humidified CO2. The bag was maintained for 30 minutes and reinflated as needed. Bags were secured to the limb with medical elastic adhesive tape (Figure 2). The level of inflation of the product bag was partial relative to the full capacity of the bag. Per manufacturer guidelines, the bags were inflated with 4 pumps of the medical-grade CO2 canister. During treatments, horses were held by a handler, and at the conclusion of the treatments the bags were removed, and the horses resumed their normal husbandry activities. Each horse underwent treatment 3 times a week in equal intervals for a total of 5 weeks.
Bilateral front feet with the Dioxyfin product bags secured to each forelimb, 1 inflated with the medical-grade CO2 therapy and 1 with room air.
Citation: American Journal of Veterinary Research 85, 11; 10.2460/ajvr.24.06.0161
Hoof wall measurements: weeks 6 through 10
After 5 weeks of treatment, each horse had measurements taken at each of the 3 previously marked locations with digital calipers by the 2 independent observers. The distance from the coronary band to the horizontal lines along the axis of hoof growth in each area was recorded. An additional set of horizontal lines was created identical to the previous lines at 1 cm distal to the coronary band at the 3 previously described locations.
Description of treatment: weeks 6 through 10
Following the second set of measurements, the treatment groups were switched, and horses that previously underwent room air treatment (control group) were now enrolled into the CO2 treatment group, and the previous CO2 treatment group (treatment group) was now subjected to room air treatment. Treatments were carried out at even intervals 3 times a week for an additional 5 weeks.
Statistical methods
Raw measurements were transformed into percentage of change from baseline for each observer (ie, 1 and 2) hoof location (ie, medial, midline, lateral) and reported as mean ± SD. Continuous data were evaluated for normality using Shapiro-Wilk tests and visually using quantile-quantile plots. Quantile-quantile plots demonstrated minimal departures from data normality. The Student t test was used to compare growth measurements of hooves treated with CO2 to those without CO2 treatment at a single time point. Growth measurements for each horse hoof location were considered independent observations as observers performed measurements without the other observer present. Observers were blinded to treatment group assignments. A 2-way random-effects model was used to calculate the intraclass correlation coefficient (ICC) to assess interobserver reliability for each limb, timepoint, and location combination between the 2 observers. Measurements from both control and treatment feet were included in the analysis. ICC was categorized as < 0.50 as poor reliability, ICC 0.5 to 0.75 as moderate reliability, 0.76 to 0.9 as good reliability, and 0.91 to 1 as excellent reliability. A level of P < .05 was used for significance. All data were analyzed using R software (version 4.2.2; R Foundation for Statistical Computing) in RStudio (version 2022.12.0 + 353; Posit) and GraphPad Prism (version 9.5.1).
The experimental sample size of 14 hooves per group was calculated using G*Power (version 3.1.9.6).7 Specifically, a priori power analysis was conducted using the relative mRNA expression of hypoxia-inducible factor 1-α in experimentally induced wounds treated with transcutaneous CO2 compared to those without CO2 treatment.8 This power analysis resulted in an effect size of 3.74 and a power of 0.88 using a 95% CI.
Results
All horses remained healthy throughout the study period, and no complications or adverse events were reported as a result of the treatments administered. Ages ranged from 6 years old to 25 years old, with a mean of 17 years. None of the horses required sedation for this study. The mares used in this study did not have any negative reaction to the bags placed on their feet.
At the 5-week point, hoof growth was increased in treated feet at 2 sites, the left front medial and right front lateral (Figure 3). Hoof growth was increased (P = .028) at the left medial quarter in CO2-treated hooves (treatment group) (mean ± SD, 27.92% ± 11.81%) compared to hooves treated with room air (control group) (18.18% ± 10.19%). Hoof growth also increased (P = .03) in the right lateral quarter in CO2-treated hooves (25.69% ± 8.36%) compared to hooves treated with room air (17.59% ± 10.06%). Hoof growth was not different between treated and untreated feet in the other locations (Table 1). At the conclusion of the study, the percentage of change between baseline versus 10 weeks and 5 weeks versus 10 weeks showed no significant differences at any of the 6 hoof locations (Table 2). A summary of the data is provided to compare the control and treatment groups using both the raw measurements and percentage of change for both limbs at all 3 measurement locations (Supplementary Table S1; Supplementary Figure S1). Interobserver reliability was evaluated using an interclass correlation coefficient, which was moderate to excellent (range, 0.52 to 0.94) across all 3 hoof wall locations and timepoints (Supplementary Table S2).
Box-and-whisker plots comparing control (air) and treatment (CO2) of the left front (LF) medial (left plot) and right front (RF) lateral (right) locations at the 5-week and 10-week timepoints. The difference in percentage of change after 10 weeks in both locations does not persist, suggesting that the control group growth caught up to the treatment group growth between weeks 5 and 10.
Citation: American Journal of Veterinary Research 85, 11; 10.2460/ajvr.24.06.0161
Percentage of growth change from baseline of the dorsal hoof wall in transdermally treated CO2 (treatment) and air (control) feet of 14 horses.
| Location on hoof wall | Treatment (percentage of change of growth from baseline) | Control (percentage of change of growth from baseline) | P value |
|---|---|---|---|
| Right front midline | 28.43 | 24.86 | .32 |
| Right front medial | 22.98 | 21.14 | .61 |
| Right front lateral | 25.69 | 17.59 | .03 |
| Left front midline | 24.22 | 34.53 | .63 |
| Left front medial | 27.92 | 18.18 | .028 |
| Left front lateral | 21.08 | 19.29 | .622 |
Change in growth increased in treated limbs in the left medial (P = .028) and right lateral (P = .03) quarters.
Comparison of percent change (P values) of hoof growth for 14 horses subjected to both the treatment group (CO2) and control group (air).
| Timepoint | LF lateral | LF midline | LF medial | RF lateral | RF midline | RF medial |
|---|---|---|---|---|---|---|
| Week 0 vs week 5 | .6215 | .0626 | .028 | .028 | .322 | .606 |
| Week 0 vs week 10 | .999 | .062 | .156 | .591 | .853 | .909 |
| Week 5 vs week 10 | .386 | .916 | .916 | .124 | .420 | .250 |
Significant change in growth from baseline to 5 weeks in left front (LF) medial and right front (RF) lateral locations. No changes from baseline to 10 weeks or from 5 weeks to 10 weeks.
Discussion
The results of this study suggest that repeated, noninvasive transdermal application of CO2 may accelerate hoof growth in normal horses over a 5-week treatment period. This crossover study with no washout period includes only healthy adult horses with no known hoof or systemic disease. Since each hoof experiences independent growth, this design compared each front hoof to itself rather than to other subject horses or to a standard measurement. Hoof growth is affected by nutritional, metabolic, and seasonal variability, and the authors attempted to control for these variables by using only adult mares housed in a single location with similar husbandry practices (turnout, nutrition, and routine hoof care prior to the study), with all measurements conducted at the same time to control for seasonal variability.
In this study, a commercial product of medical-grade CO2 was evaluated for its presumptive effect on hoof growth. Transdermal or transcutaneous CO2 has been used in both human and rodent models, as well as in human clinical trials, to improve the healing time of wounds and degree of soft tissue injury healing.8 Transcutaneous CO2 has been shown to have a vasodilatory effect of local tissues in microcapillaries as well as increase blood flow rate.9 The production of hoof wall as it relates to its distal growth is directly dependent on blood supply to the foot.10
The components of the hoof wall that are involved with distal growth include the stratum medium and stratum internum. The stratum internum consists of primary and secondary epidermal laminae. Growth occurs from the horn tubules from the proximal stratum germinativum at the coronary epidermis. Within the horn tubules, the primary epidermal laminae keratinize and grow distally.11 Hoof growth in normal horses has a wide range, from 0.16 mm/d to 0.34 mm/d in front feet,1,12 and is affected by many variables, such as footing surface, time of year, age, and nutrition.2 While this study aimed to control for these variables by enrolling horses in the same herd during the same season with similar management, individual variability likely had an influence on this short-term investigation.
The phenomenon known as the Bohr effect has been postulated to be the mechanism of action for the efficacy of transdermal CO2. The Bohr effect describes the increased pressure of oxygen within tissues following oxyhemoglobin dissociation. In vivo studies using transdermal CO2 on rats and humans showed significantly increased dissociation of oxygen-bound hemoglobin and improved oxygen availability to local tissues.6 Transcutaneous CO2 has also been shown to promote angiogenesis and increase VEGF expression.13,14 VEGF is an essential component of wound healing for stimulating angiogenesis as well as promoting endothelial cell migration.15 In a study by Gaesser et al4 evaluating the effect of transdermal CO2 on equine distal limb wound healing, there was a clinical observation of accelerated hoof growth in feet exposed to the transdermal CO2 therapy. This clincial observation was the basis of this current study.
This pilot exploratory study has limitations warranting discussion. Although the small sample size is a primary limitation of this study, a power calculation was performed to ensure sufficient animals; however, a larger number of horses would provide greater power. The 10-week study period was a direct result of the availability of horses at this time. Carrying out the study over a longer period of time may have yielded even more improved hoof growth and less laterality of the data. However, the accelerated hoof growth shown in the 10-week period indicates that this product may be used in a real clinical setting during a feasible amount of time to implement treatment. Due to the nature of this crossover study, an inherent limitation of switching limbs between groups at 5 weeks could have been reconciled by keeping the limbs within their respective groups for the full 10-week study period.
The study controlled for interobserver variability and bias by blinding the 2 individuals conducting measurements to the treatments and one another and by using digital caliper readings rather than relying on visual measurement reading to reduce human error. Over the study period, the width of the groove in the hoof wall became slightly elongated. The variability of where the digital calipers sat within the groove may have contributed with the inevitability of human error. However, a strong effort was made to keep intraand interobserver measurements as consistent and objective as possible. In the future, the use of alternative measurement strategies, such as artificial intelligence technology, could help diminish the effect of human error in this situation. Of note, the left front midline range is broad, with a large SD. Upon further statistical analysis, if the outlier values in the range are eliminated, the mean percentage of change is almost equivalent between the control and the treatment. Overall, these data may have been lateralized due to human error, or perhaps there is an aspect of equine hoof growth that is asymmetrical and has yet to be elucidated. It is plausible that if this were a longer study period, the left front midline growth may have been more consistent.
Deflation of the bag occurred occasionally in this study, and reinflation was performed according to product guidelines. A more consistent environment may yield more definitive results in the future. This study also does not investigate a potential mechanism of action for how transdermal CO2 may affect the horn tubules, but this should be a future area of study to further validate this treatment.
The focus of this preliminary study was directed to evaluate whether a clinical benefit was observable from a noninvasive and technically feasible treatment. It would have been expected that all locations on the hoof would have demonstrated a positive response to treatment with CO2, so the inconsistent nature of growth mandates further investigation. Although these data suggest that there may be a positive effect of transdermal CO2 on hoof growth, the outcomes of this study should not be overstated, and further research in the area is needed to confirm that this is a consistent and reproducible method, especially if this therapy is to be used in horses of a different demographic, such as those with poor hoof quality or abnormal metabolic status.
Supplementary Materials
Supplementary materials are posted online at the journal website: avmajournals.avma.org.
Acknowledgments
The authors would like to acknowledge Spy Coast Farm LLC and affiliated personnel for their assistance with data acquisition and horse management during the study period.
Disclosures
The authors have nothing to disclose. No AI-assisted technologies were used in the generation of this manuscript.
Funding
Serendi Medical LLC provided the products used in this study free of cost.
ORCID
Meredith J. Rudnick https://orcid.org/0000-0002-5480-2194
Holly L. Stewart https://orcid.org/0000-0002-8655-8069
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