α-Casozepine supplementation shows no effect on foals’ growth and cortisol levels before and after weaning

Moriah K. Wood Department of Equine Science, Otterbein University, Westerville, OH

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Rocky M. Mason Lexington Equine Medical Group, Lexington, KY

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Steffanie V. Burk Department of Equine Science, Otterbein University, Westerville, OH

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Sheri S. W. Birmingham Department of Equine Science, Otterbein University, Westerville, OH

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Abstract

OBJECTIVE

The goal of this study was to determine whether oral administration of α-casozepine (Zylkene) supplementation decreases the risk of disease in foals, as measured through salivary cortisol levels, temperature, clinical signs of disease, and body weight before and after weaning.

ANIMALS

20 Thoroughbred foals were observed over 10 weeks, beginning 14 days prior to the time of weaning.

METHODS

Experimental foals were randomly selected and given α-casozepine daily for 10 weeks, while the controls received a placebo with the same frequency as the experimental group. The average daily height and weight gain were calculated to examine the effects of α-casozepine before and after weaning. Salivary cortisol levels were also compared before versus after weaning.

RESULTS

There was no statistically significant difference in average daily height/weight gain or cortisol levels between the α-casozepine group and the control before and after weaning. However, the α-casozepine group had numerically less of a decline in height and growth compared to the control. This suggests that with further study Zylkene may decrease losses in foal growth rate.

CLINICAL RELEVANCE

The weaning process is one of the largest stressors in a young horse’s life. This abrupt process can contribute to an increased risk of disease and even morbidity. Other major stressors including separation from the mare, diet change, and new social groups can also lead to animal health and welfare concerns, as well as economic losses in weanling foals. However, further study is required to determine the benefits of α-casozepine on equine stress and health.

Abstract

OBJECTIVE

The goal of this study was to determine whether oral administration of α-casozepine (Zylkene) supplementation decreases the risk of disease in foals, as measured through salivary cortisol levels, temperature, clinical signs of disease, and body weight before and after weaning.

ANIMALS

20 Thoroughbred foals were observed over 10 weeks, beginning 14 days prior to the time of weaning.

METHODS

Experimental foals were randomly selected and given α-casozepine daily for 10 weeks, while the controls received a placebo with the same frequency as the experimental group. The average daily height and weight gain were calculated to examine the effects of α-casozepine before and after weaning. Salivary cortisol levels were also compared before versus after weaning.

RESULTS

There was no statistically significant difference in average daily height/weight gain or cortisol levels between the α-casozepine group and the control before and after weaning. However, the α-casozepine group had numerically less of a decline in height and growth compared to the control. This suggests that with further study Zylkene may decrease losses in foal growth rate.

CLINICAL RELEVANCE

The weaning process is one of the largest stressors in a young horse’s life. This abrupt process can contribute to an increased risk of disease and even morbidity. Other major stressors including separation from the mare, diet change, and new social groups can also lead to animal health and welfare concerns, as well as economic losses in weanling foals. However, further study is required to determine the benefits of α-casozepine on equine stress and health.

Introduction

Weaning is defined by the change of nutritional source from the mother’s milk to another species-specific food source.1 This abrupt process is a major stressor for foals as they adapt to the new situation.2 Unfortunately, weaning is not the only stressing factor foals face: separation from the mare, diet change, new social groups, change in location, and change in management procedures can also contribute to foal morbidity.2

These major stressors can lead to stress-induced adrenal steroidogenesis, which can result in a release of endogenous cortisol, suppressing immune function and the inflammatory response.3 This impairs the ability to fight infection and results in an increased risk of disease. Economic losses are associated with the treatment of illness, inability to sell the foals, and occasionally death of the foal attributed to primary or secondary bacterial pneumonia.4 Therefore, reduction of the stress response to environmental factors in weanling foals may result in improved immune function during the weaning period and prevent economic losses. Benefits of reducing stress include decreased stress-related behaviors, better social cohesion, and decreased physiological symptoms of stress including improved immune response and decreased heart rate, fecal cortisol levels, and glucocorticoid metabolites.5,6

α-Casozepine (Zylkene) is a tryptic peptide that has demonstrated calming anxiolytic-like effects in treated horses. This bioactive peptide originates from an α-S1 casein protein in cow’s milk.7 Due to its affinity for GABA receptors in the brain, α-casozepine has been reported to have an anxiolytic effect similar to depressants such as benzodiazepines, without the common side effects of incoordination and disinhibition of aggression.7

This product was patented as a behavioral supplement under the name Zylkene.8 It is suggested that Zylkene be used for loading and travel, veterinary visits, farrier visits, new training procedures, stall rest, new environments, social regrouping, and weaning.9 The active ingredient in this supplement is 8 g of hydrolyzed bovine milk protein, which provides 2,000 mg of α-casozepine.10 The natural, bovine-sourced, lactose-free α-casozepine is believed to promote a sense of calm, relaxation, and mental awareness without sedation with a recommended daily dose of 1 to 2 packets daily for 3 consecutive days.10

Currently, there is an exiguous amount of literature regarding the use of α-casozepine in horses. One study10 of semiferal ponies demonstrated calming anxiolytic-like effects of α-casozepine on treated horses when behavior and training efficiency were analyzed. Ponies were given 1,000 mg of α-casozepine once daily for 5 days prior to being moved to a domestic facility for an introduction to training.10 The ponies supplemented with α-casozepine performed better and retained the most information from the 2-week training period, supporting that α-casozepine could benefit horses experiencing stressful situations caused by domestic management.10 Further study should be conducted on domestic horses. Another study11 found that α-casozepine supplementation improved compliance and apparent comfort in horses known to display mild aversion to routine health-care procedures. Further research could compare the findings from this study to horses without an aversion to health-care procedures to further examine the benefits of Zylkene supplementation.

Similar results have also been observed in cats and dogs. In 1 study,7 cats were fed a diet supplemented with α-casozepine and L-tryptophan and a reduced anxiety response was observed when the cats were placed in an unfamiliar location. Another study8 found that 56 days of α-casozepine supplementation in cats successfully improved anxious disorders and social phobias when compared to the control group. Fear of strangers, contact with familiars, general fears, fear-related aggressions, and autonomic disorders were also significantly improved using α-casozepine.8 In another study,12 dogs supplemented with α-casozepine and L-tryptophan had an improved ability to cope with stress when anxiety-related behaviors and physiological parameters were observed. Findings supporting the impact of Zylkene on stress levels in other species suggest that further study should be conducted on equids.

The purpose of this study was to determine whether oral administration of α-casozepine supplementation influences stress and the risk of disease in weanling foals. This was accomplished by monitoring salivary cortisol levels, temperature, clinical signs of disease (cough, nasal discharge, appetite, attitude), and body weight. It was expected that foals supplemented with α-casozepine before and after weaning would have decreased salivary cortisol levels associated with reduced levels of stress as well as greater height and weight average daily gain following weaning when compared to controls.

Methods

Twenty Thoroughbred foals were observed over a 10-week period, beginning 14 days prior to the time of weaning in the late spring/early summer. These foals were selected for experimentation, as they were from 1 stable with 3 adjacent 50-acre pastures. No more than 8 mare/foal pairs were turned out per pasture. Ten foals were then randomly assigned to receive Zylkene supplementation, and the placebo was delivered to the other 10 foals. Foals were managed according to standard farm protocol, and treatment and control group foals were intermixed in each pasture. Prior to weaning, dams and foals were turned out overnight in groups of a maximum of 8 foal/mare pairs on the basis of date of birth and size. Dams and foals were brought in by 7:00 am daily, administered Zylkene supplementation or a placebo slurry, and fed by 7:30 am. Personnel involved were blinded to each foal’s group status. Zylkene supplementation began 7 days prior to weaning, and foals were administered a dose of one 8-gram packet containing 2,000 mg of α-casozepine once daily. The packets were made into a slurry with water and administered PO via syringe. Control group foals received a placebo containing an 8-gram portion of prepackaged oat flour with the same method and frequency as the experimental treatment. Salivary cortisol samples were taken starting at 9:30 am. The foals and mares resumed turnout into the large pasture at 11:00 am.

As foals got closer to 4 months of age, approximately 3 to 4 foals were chosen to be weaned on the basis of health, size, and weather conditions. Mares of the chosen foals were removed from the group and trailered off the property at the 11:00 am turnout time. Weaned foals were returned into their original turnout group with remaining foal/mare pairs or weaned foals, depending on how many foals had been previously weaned from the group. Foals included in this study were weaned at an average age of 154.47 ± 16.26 days (Table 1). Weaning the foals completely on a given day reduced variability within the study design. Foals would have been excluded from the study if they presented with signs of clinical disease or stress during sampling, but no foals met these criteria. Similarly, if any weanlings were to become injured or too fractious to administer supplementation or take saliva samples from, they would have been excluded from the study; however, no foals were removed on the basis of these criteria.

Table 1

Average foal age (days) ± SD prior to weaning (day –14), at the time of weaning (day 0), and after weaning (day 56).

Weaning status Foal age (d)
Prewean (day –14) 119.47 ± 18.05
Weaned (day 0) 154.47 ± 16.26
Postwean (day 56) 220.53 ± 21.91

Data are reported as mean ± SD.

The swabs used for saliva samples were part of a pediatric kit (SalivaBio Children Swab Method; Salimetrics LLC) validated for equine use. Samples were collected biweekly starting 2 weeks prior to weaning (day –14) for 10 weeks, with the last day of collection at 56 days after weaning. Samples were stored at –20 °C according to the Salimetrics protocol. Cortisol levels were determined using the Salimetrics Salivary Cortisol Assay (Salimetrics LLC). Clinical signs were objective and subjective and included daily physical exams monitoring attitude, appetite, and rectal temperature twice daily. Foals were weighed monthly according to the farm’s routine protocol with an outside commercial weighing service to assess for influence on growth rate. The farm measured the foals roughly every 30 days depending on availability of the commercial weighing service. Weight and height measurements were taken 14 days prior to weaning (day –14) and then 56 days after being weaned (day 56). Since there was no set interval for measuring the foals, there was some variation among the weight, height, and age (in days) at which the 20 foals were measured. Although the foals were weighed on the same day, they differed in age on the basis of the farm’s weaning protocol. Therefore, average foal age prior to weaning was 119.47 ± 18.05 days (Table 1). To account for the inconsistent measurements and varying wean dates, the measurements closest to 56 days after being weaned were used for the postwean data. Data collection ceased 56 days after weaning (day 56). Because of this, the average foal age after weaning was 220.53 ± 21.95 days. The Otterbein University IACUC approved all procedures used in this study (protocol No. 2019121).

Statistical analysis

A mixed ANOVA for the average daily weight gain and average daily height gain was conducted, using one between the subject factor (the treatment of oat or Zylkene) and one within-subjects factor (preweaned versus postweaned foals). Similarly, a mixed ANOVA was conducted to compare the salivary cortisol levels between treatment groups and between preweaned versus postweaned foals. Descriptive statistics and Q-Q plots were used to evaluate normality of data. A value of P < .05 was considered significant. According to the Shapiro-Wilk test, data from the average daily height and weight gain were normally distributed. However, the Q-Q plots and Shapiro-Wilk test were not normally distributed for the salivary cortisol levels, so data were log transformed prior to analysis.

Results

There were no abnormal findings observed upon clinical assessment of attitude, appetite, or rectal temperatures. The log-transformed results from the mixed ANOVA indicated that there was no statistically significant difference between the effects of Zylkene supplementation and the control in salivary cortisol levels in foals before versus after weaning (F[6, 108] = 1.455; P = 0.234). The mean salivary cortisol levels during the weaning period were calculated for both the Zylkene treatment and the oat flour control (Figure 1). There was also no statistically significant difference between the treatment group and the control in the average daily weight gain before and after weaning the foals (F[1, 17] = 0.016; P = .901). The mean average daily weight gain during the weaning period was calculated for the experimental and control groups (Figure 2). Similarly, there was no statistically significant difference between the effects of Zylkene supplementation and the control for the average daily height gain in foals before versus after weaning (F[1, 17] = 0.016; P = .901).The mean average height gain before and after weaning was calculated for the Zylkene and control foals (Figure 3). Average foal age before, after, and during weaning was calculated for the 20 foals.

Figure 1
Figure 1

Comparison of mean salivary cortisol levels (μg/dL) before weaning, at the time of weaning, and after weaning between the treatment (Zylkene) and control (oat) groups including SE. Samples were collected biweekly starting 14 days prior to weaning for a 10-week period.

Citation: Journal of the American Veterinary Medical Association 262, 3; 10.2460/javma.23.09.0545

Figure 2
Figure 2

Comparison of mean average daily weight gain (kg) before and after weaning between the treatment (Zylkene) and control (oat) groups including SE. The average age of preweaned foals was 119.47 ± 18.05 days. Average postweaned foal age was 154.47 ± 16.26 days. Prewean data were collected 2 weeks prior to the time of weaning (day –14), while postwean data were collected 8 weeks after weaning (day 56).

Citation: Journal of the American Veterinary Medical Association 262, 3; 10.2460/javma.23.09.0545

Figure 3
Figure 3

Comparison of mean average daily height gain (cm) before and after weaning between the treatment (Zylkene) and control (oat) groups including SE. The average age of preweaned foals was 119.47 ± 18.05 days. Average postweaned foal age was 154.47 ± 16.26 days. Prewean data were collected 2 weeks prior to the time of weaning (day –14), while postwean data were collected 8 weeks after weaning (day 56).

Citation: Journal of the American Veterinary Medical Association 262, 3; 10.2460/javma.23.09.0545

Discussion

This study did not provide supporting evidence that α-casozepine supplementation reduces stress and decreases the risk of disease in foals before and after weaning. Although there was not a statistically significant difference between Zylkene and the control, there was numerically less of a decline in height and weight for the Zylkene group. It is possible that a difference could be observed with a larger sample size. This finding is noteworthy because maintaining a slow and steady growth rate in foals is vital for preventing osteochondrosis dissecans (OCD). High growth rate due to a high-energy diet has been shown to predispose foals to the development of OCD.11 Both body weight and height are major determining factors when selling foals. In 2020, the median price paid for foals in the top 25% of body weight as yearlings was 5 times greater than the bottom quartile.13 While larger foals are more susceptible to OCD lesions, sesamoiditis occurs less frequently in larger foals compared to average-sized foals.13 Therefore, further studies could examine the influence of α-casozepine supplementation on growth rate and OCD predisposition.

It is possible that the results of this study were impacted by the experimental design. For instance, the 20 foals observed in this study were not all born on the same day, so they were weaned at different times and measured at slightly different ages. The foals were slightly different ages at the time of weaning because they were not weaned solely on the basis of age. Foals were weaned in groups of 3 to 4 depending on overall condition and growth, environmental factors, age, and what was believed to be the best time for each individual foal. The decision to wean was made by farm staff and was not influenced by the design of this study. If the interval at which the measurements were taken for each foal were adjusted to each individual, it may provide more accuracy. For instance, one of the foals from this study was weighed 2 months after weaning (day 28) while another foal was weighed 1 week after weaning (day 7). This means that postwean data were averaged over a 21-day period (from day 7 to day 28). Future studies should take measurements at a consistent interval among individuals to avoid this variation and improve experimental design.

Ijichi et al14 yielded similar results to this study. The effects of α-casozepine on compliance and coping in horses during loading was observed. Horses weighing up to 500 kg were fed 1,000 mg, while horses over 500 kg were given 2,000 mg of Zylkene Equine.14 It was observed that, at the dosage used, Zylkene had no effect on a horse’s ability to cope with loading and confinement.14 Further studies could examine the effectiveness of different dosages of Zylkene.

In this study, α-casozepine (Zylkene) supplementation had no significant effect on salivary cortisol levels, temperature, clinical signs of disease, or average daily weight during the weaning period. However, the Zylkene group had numerically less of a decline in height compared to the control. This suggests that with further study Zylkene may decrease losses in foal growth rate. Further experimentation is required to assess the benefits of Zylkene supplementation on foal stress and health before and after weaning.

Acknowledgments

The authors are extremely grateful to Mt. Brilliant Farm and the staff for providing access to the horses and facilities. The authors would also like to thank Dr. Allison Haspel and Lily Syah for their assistance in the collection and organization of data, respectively.

Disclosures

The authors have nothing to disclose. No AI-assisted technologies were used in the generation of this manuscript.

Funding

Funding was provided by the Faculty Scholars Development Grant through Otterbein University.

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