Serum 25(OH) Vitamin D Concentrations in Horses: Effects of Age, Gender, Breed, Skin Colour and Season

ABSTRACT

Background

The optimal amount of vitamin D required for the proper functioning of the immune system differs from the amount necessary for bone homeostasis. Furthermore, vitamin D metabolism varies among horses. Nevertheless, there is a dearth of information regarding reference values for vitamin D in horses, particularly in the Turkmen breed. The primary objectives of this study were to determine the concentrations of 25‐hydroxyvitamin D (25(OH)Vit D) in Turkmen, Thoroughbred and mixed Turkmen × Thoroughbred horses and to explore the effects of various factors on it and its relationship with serum biochemical variables and signalment data.

Methods

For the measurement of 25(OH)Vit D, 90 healthy horses (min age: 6 months; max age: 10 years) that were stable in the north of Iran (37°17′ N, 55°18′ E) were selected for clinical examination and complete blood biochemistry analysis. The horses were categorised into different groups based on sex, season (spring, n = 45; autumn, n = 45), breed (Turkmen, Thoroughbred and mixed Turkmen × Thoroughbred), age (< 2 years, 2–8 years and > 8 years) and skin colour. Sampling was performed only once from each horse.

Results

In this study, the median serum concentration of 25(OH)Vit D in the sampled horses was 17.42 ng/mL (IQR: 9.82–30.85). The serum 25(OH)Vit D concentration was significantly lower in autumn (median: 15.83 ng/mL and IQR: 8.67–54.68) than in spring (median: 18.02 ng/mL and IQR: 13.77–27.54) and was also lower in Turkmen horses (median: 15.83 ng/mL and IQR: 11.63–23.12) than in mixed breed horses (median: 17.72 ng/mL and IQR: 8.94–51.67) (p ≤ 0.05).

Conclusion

According to the results of the present study, the season had a significant effect on the serum 25(OH)Vit D concentration, and this difference was also observed between Turkmen horses and mixed‐bred horses (p ≤ 0.05). It is unclear whether the time of sun exposure or vitamin D content of diet causes the seasonal difference of 25(OH)Vit D concentration.

In this study, the serum concentration of 25(OH)Vit D in the sampled horses exhibited a median of 17.42 ng/mL, with values of 9.82 and 30.85 ng/mL at the 2.5th and 97.5th percentiles, respectively. The serum 25(OH)Vit D concentration was significantly lower in autumn than in spring and was also lower in Turkmen horses compared to mixed breed horses (p ≤ 0.05).

1. Introduction

Vitamin D plays a crucial role in various organ systems, with a primary focus on bone metabolism. However, its significance extends far beyond this domain. Nevertheless, controversy persists regarding the optimal vitamin D levels required to maintain the health of both human and veterinary patients, leading to ongoing debates about recommended daily vitamin D intake (Hurst et al. 2020). Two forms of vitamin D are essential for assessing vitamin D status and supply in horses: ergocalciferol (D2), which is derived from fungi growing on plant material and is acquired by wild/domestic animals through grass consumption (Richardson and Logendra 1997), and cholecalciferol (D3), which is either administered orally as a supplement or synthesised endogenously in the skin upon exposure to sunlight.

Upon absorption in the gastrointestinal tract or synthesis in the skin, vitamin D is stored in adipose tissue or transported to the liver and subsequently to the kidneys, where it is converted into 25‐hydroxyvitamin D (25(OH)Vit D or calcidiol) and 1,25‐dihydroxyvitamin D (1,25(OH)2Vit D or calcitriol), respectively. With a half‐life of 10–21 days, 25(OH)Vit D serves as an indicator for assessing overall vitamin D levels in the body (Mellanby et al. 2005; Weidner and Verbrugghe 2017). Notably, in horses, vitamin D status, assessed through plasma concentrations of both 25(OH)Vit D2 and 25(OH)Vit D3, is generally reported to be very low (Dosi et al. 2023). Horses exhibit elevated serum calcium levels and reduced serum phosphorus levels compared to other species. Rourke et al. (2010) cloned the mRNAs of proteins involved in all three steps of transcellular calcium transport, performed comparative mRNA and protein sequence analyses and quantified their mRNA expression in the equine gastrointestinal tract and kidney. They suggested low VDR expression in the equine small intestine and kidney relative to the large intestine and that epithelial calcium transport in horses is not as dependent on vitamin D as in other species (Rourke et al. 2010). On the other hand, in most recent studies, Azarpeykan, et al. (2016a) and Azarpeykan, et al. (2016) reported that vitamin D receptors are expressed in equine kidneys as in other species, and they also showed that all the hydroxylases needed for the activation and catabolism of vitamin D products are expressed in equine kidneys.

The level of endogenous D3 synthesis is contingent upon the duration and intensity of sunlight exposure and is thus influenced by geographical latitude and seasonal variations. During winter, the zenith angle between the sun and the Earth increases, leading to the reflection of sunlight with UVB wavelengths away from the Earth's surface within the atmosphere (Hymøller and Jensen 2015). Cumulative in vivo (Azarpeykan, et al. 2016; Dosi et al. 2023) and ex vivo (Azarpeykan et al. 2022) studies suggest that horses might not synthesise detectable levels of vitamin D3 in the skin. On the other hand, changes in the UVB index throughout the year influence fungal activity in pastures and, therefore, directly influence the vitamin D2 content of sun‐cured forage and pasture (Richardson and Logendra 1997; Jäpelt and Jakobsen 2013). Comprehensive research is necessary before making well‐informed recommendations regarding vitamin D requirements for horses at various latitudes, during different seasons and under different management conditions, such as access to outdoor areas and the use of rugs, among other factors.

We hypothesised that variables such as age, sex, breed, season and skin colour influence 25(OH)Vit D status in horses. Furthermore, we aimed to explore the relationships between 25(OH)Vit D and serum biochemical parameters, particularly calcium, phosphorus, and magnesium. To the best of our knowledge, few studies have comprehensively investigated the factors influencing vitamin D levels in horses, especially within the Turkmen breed. In addition, this study marks the first report on the levels of 25(OH)Vit D in Turkmen horses in Iran.

2. Materials and Methods

2.1. Study Population

This study involved 90 healthy horses of various breeds (min age: 6 months; max age: 10 years) located in the northern region of Iran (37°17′ N, 55°18′ E). The inclusion criteria for the horses were as follows: they must have passed a physical examination, remained disease‐free for the past six months, not received any medications, including vitamin D supplements, and not been vaccinated in the previous month. In addition, the horses were deemed healthy based on haematological and serum biochemistry analyses. Horses displaying evidence of systemic disease or organ dysfunction were excluded from the study. Owners were requested to complete informed consent forms and questionnaires about the animals' conditions, which included information on age, sex, breed, diet and health measures. All horses were maintained under similar environmental and management conditions. All horses received alfalfa hay as their main diet (4–8 kg/day) throughout the year based on age and physical activity without access to pasture for grazing, but almost all horses had access to sunlight for several hours during rest or training. The exercise protocol was similar for females and males.

In addition, various forms of concentrates, such as commercial and/or homemade (without vitamin and mineral supplements), were used only for horses trained mainly on stallions. Consequently, the 90 horses were categorised as follows: gender (male: 46; female: 44), age (< 2 years: 53; 2–8 years: 34; > 8 years: 3), season (spring: 45; autumn: 45), breed (Turkmen: 23; Thoroughbred: 20; mixed Turkmen × Thoroughbred: 47) and skin colour (dark: 82; light: 8).

The mean temperature of the region was 34.05°C during the year, with a mean of 8.1°C in January being the coolest and 40°C in August as hottest months. The horizontal coordinate system characteristics of the sun during the spring and autumn were as follows: declination: 23.47°, altitude: 75.83°, azimuth: 194.46° and zenith cosine: 0.9696 and declination: −23.55°, altitude: 29.18°, azimuth: 182.87° and zenith cosine: 0.4876, respectively. We sampled the horses at the end of spring (21 May–20 June) and the end of autumn (21 November–20 December) based on the longest and shortest durations of days, respectively.

Subsequently, 10 mL of blood was drawn from the jugular vein before the morning training session (between 8:00 am and 10:00 am). A total of 2.5 mL of the collected blood was transferred to tubes containing EDTA, while the remaining 7.5 mL was transferred to anticoagulant‐free tubes to separate the serum. These samples were stored in refrigerated conditions and transported to the laboratory under appropriate conditions.

Hematological evaluations were conducted within an hour of sample collection. A CBC was performed using a veterinary haematology cell counter (Nihon Kohden, Celltac α, MEK‐6450K, Tokyo, Japan) to assess the haematological status of the horses. To separate the serum, the collected blood was centrifuged at 1800 × g for 10 min. The resulting sera were then stored in a freezer at −20°C until the measurement of serum 25(OH)Vit D.

Furthermore, to verify the health of the studied animals, biochemical parameters were initially assessed using commercial kits (Pars Azmoon, Tehran, Iran) with an autoanalyser (Biotecnica, BT 1500, Rome, Italy). The measured biochemical factors included albumin, blood urea nitrogen, creatinine (CRE), total calcium (Ca), inorganic phosphorus (iP), total magnesium (Mg), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and creatine kinase (CK). Measurement accuracy was ensured using a control serum (Randox Laboratories Ltd., Ardmore, UK).

2.2. Measurement of 25(OH)Vit D

Serum 25(OH)Vit D was assessed using ELISA with commercial vitamin D diagnostic kits previously validated for measuring 25(OH)Vit D in herbivores in our laboratory (Monobind, Inc., Lake Forest, USA). An ELISA washer and reader (BioTek, ELx‐50 and ELx‐800, Winooski, USA) were used for this purpose. The inter‐assay CV, intra‐assay CV and sensitivity of the commercial kits utilised were 9.17%, 4.11% and 1.14 ng/mL, respectively. The kit demonstrated specificity for measuring 25(OH)D3, 25(OH)D2, vitamin D3, vitamin D2, 1,25(OH)2D3 and 1,25(OH)2D2 at 100%, 100%, 0.39%, 0.76%, 115% and 190%, respectively.

2.3. Statistical Analyses

The data were assessed for normality using the Shapiro–Wilk method and were determined to be nonnormally distributed. Consequently, nonparametric methods were applied for analysis. The Mann–Whitney method was used to compare the levels of 25(OH)Vit D for variables, including sex, season and skin colour, between the two groups. The Kruskal–Wallis test was utilised for variables with more than two groups, such as age and breed. Finally, correlations between the levels of 25(OH)Vit D and haematological and serum biochemical indices of blood were established using the Spearman method. The results are presented as median, 2.5–97.5 percentiles and ranges. The statistical software used for analysis was SPSS version 20 (IBM, USA). For all comparisons, a p ≤ 0.05 was considered to indicate statistical significance.

3. Results

In the present study, the median serum concentration of 25(OH)Vit D in the sampled horses was 17.42 ng/mL (9.82–30.85 ng/mL).

Serum 25(OH)Vit D concentrations were significantly lower in autumn (15.83, 8.67–54.68 ng/mL) than in spring (18.02, 13.77–27.54 ng/mL) and were also lower in Turkmen horses (15.83, 11.63–23.12 ng/mL) than in mixed‐bred horses (17.72, 8.94–51.67 ng/mL; p ≤ 0.05). However, no significant difference was observed between 25(OH)Vit D levels and other grouping factors (Table 1).

TABLE 1.

The concentrations of 25(OH)Vit D, calcium, phosphorus and magnesium in total samples and various subgroups (median and 2.5–97.5 percentiles and min–max for horses > 8 years).

Group	Subgroups	Number	25(OH)D (ng/mL)	Ca (mg/dL)	P (mg/dL)	Mg (mg/dL)	Age (mean and median)
Age	< 2 year	53	17.35 (9.13–50.03)	12.9 (12.0–13.5)	4.0 (3.6–4.6)	2.1 (1.8–2.3)	1.73 and 2
	2–8 years	34	17.44 (9.58–29.38)	13.0 (12.3–13.4)	3.6 (3.4–3.8)	2.0 (1.9–2.3)	3.46 and 3
	> 8 year	3	13.77–23.50	9.9–11.8	2.67–3.57	1.73–2.25	9.5 and 10
p value			0.861	0.035 *	0.001 *	0.833
Gender	Male	46	17.38 (9.21–52.51)	13.1 (11.9–13.5)	3.7 (3.4–4.2)	2.0 (1.9–2.3)	2.72 and 2
	Female	44	17.56 (9.69–31.53)	12.8 (12.3–13.4)	3.8 (3.4–4.5)	2.1 (1.8–2.3)	2.56 and 2
p value			0.926	0.774	0.672	0.888
Season	22 May–21 Jun	45	18.02 (13.77–27.54)	13.0 (11.9–13.4)	3.7 (3.4–4.5)	2.0 (1.8–2.2)	2.62 and 2
	22 Nov–21 Dec	45	15.83 (8.67–54.68)	12.9 (12.2–13.4)	3.7 (3.4–4.3)	2.1 (1.9–2.3)	2.66 and 2
p value			0.005 *	0.944	0.831	0.028 *
Breed	Turkmen	23	15.83 (11.63–23.12) a	12.7 (12.0–13.5)	3.5 (3.2–3.9) a	2.3 (1.9–2.6) a	2.67 and 2
	Thoroughbred	20	18.67 (9.58–27.37) ab	12.8 (11.9–13.3)	3.8 (3.4–4.2) ac	2.0 (1.8–2.2) bc	2.61 and 3
	Mix	47	17.72 (8.94–51.67) b	13.0 (12.3–13.5)	3.8 (3.5–4.5) bc	2.1 (1.8–2.3) ac	2.64 and 2
p value			0.076	0.678	0.125	0.088
Skin colour	Dark	82	17.44 (9.67–31.64)	12.9 (12.0–13.4)	3.7 (3.4–4.4)	2.1 (1.8–2.3)	2.69 and 2
	Light	8	17.02 (12.73–20.59)	13.3 (12.2–14.0)	3.6 (3.3–4.5)	2.2 (1.8–2.4)	2.10 and 2
p value			0.940	0.337	0.763	0.386
Total	—	90	17.42 (9.82–30.85)	12.9 (12.2–13.4)	3.7 (3.4–4.4)	2.1 (1.8–2.3)	2.64

Note: a, b, c represent means within column lacking a common superscript differ (p < 0.05).

^*Significant difference between groups (p < 0.05).

The mean and median age (years) of each categorised variable are presented in Table 1.

In all samples, there were no statistically significant correlations between 25(OH)Vit D concentrations and Ca, P, Mg, urea and CRE concentrations, as well as AST, ALP and CK activities.

4. Discussion

The concentration of 25(OH)Vit D in our study differed from previously reported values for horses. For instance, in Arab horses from Yazd Province in Iran, Salehi‐Ardakani et al. (2023) reported a mean concentration of 25(OH)Vit D of 27.33 ng/mL. In contrast, Dosi et al. (2023) reported a total serum 25(OH)Vit D concentration of 6.32 ng/mL in Thoroughbred horses. In New Zealand, the 25(OH)Vit D concentration was less than 4 ng/mL (10 nmol/L) in horses, as reported by Azarpeykan et al. 2016. Similarly, the 25(OH)Vit D concentration in Caspian miniature horses was approximately 2 ng/mL, according to Effati et al. (2018). However, other studies have reported normal 25(OH)Vit D levels in Thoroughbred horses in Thailand (18.4–30.5 ng/mL) and the United States (14.3–37.2 ng/mL), which are similar to the values in our study (Pozza et al. 2014). These variations in 25(OH)Vit D concentrations between studies may be attributed to differences in assessment methods for measuring vitamin D metabolites, vitamin D levels in the diet, geographical latitude and sunlight exposure duration. In the present study, the horses did not have access to the pasture for grazing, but all horses are exposed to sunlight during rest and exercise for different durations in the paddock. Thus, vitamin D variations may be due to human intervention in addition to natural variations. According to the owners' documentation, mineral and vitamin supplementation were not used in the diet. The variation in the amount and content of concentrate used for feeding was another subject that could affect our interpretation. In addition, we did not estimate the vitamin D2/D3 content of the diet. This is one of the limitations of our study.

Effati et al. (2018) observed no significant difference in vitamin D levels between male and female Caspian miniature horses, whereas Salehi‐Ardakani et al. (2023) reported higher 25(OH)Vit D levels in female horses. These authors attributed this difference to the longer duration of sunlight exposure in mares (Salehi‐Ardakani et al. 2023).

Although other authors have reported the effect of age on vitamin D status in horses (Pozza et al. 2014; Salehi‐Ardakani et al. 2023), in the present study, age had no effect on the 25(OH)Vit D concentration. This may be related to age‐grouping criteria in different studies, the number of animals in each group and the dietary content of vitamin D in horses. In our study, we did not have horses less than 6 months of age. It seems that after 6 months of age, when the animal diet is mainly grass/hay, it is unlikely to find the effect of age on 25(OH)Vit D concentration.

In line with our findings regarding the effect of season, Dosi et al. (2023) reported significantly greater 25(OH)Vit D2 concentrations in grazing ponies during long days than during short days (5.77 vs. 3.49 ng/mL), while 25(OH)Vit D3 was undetectable. In another New Zealand study, the concentration of serum 25(OH)Vit D2 was greater in spring and summer (November–March) than in autumn and winter (April–October) (Azarpeykan, et al. 2016a). Caspian miniature horses also exhibited higher levels of 25(OH)Vit D in summer than in winter (2.48 vs. 1.66 ng/mL) (Effati et al. 2018). It has been suggested that the level of UVB radiation reaching the ground primarily influences ergocalciferol concentrations in forage and, subsequently, in the serum of horses (Jäpelt and Jakobsen 2013). In our study, sampling was performed one time from each horse, and the horses in spring differed from the horses in autumn. Repeated sampling from each horse in spring and autumn was better for understanding the effect of season on the amount of 25(OH)Vit D. Unfortunately, we did not have permission for repeated sampling from the owners because of their concerns about blood sampling. This subject could also have reduced the effect of farm, diet and management.

Our results indicate that coat colour had no significant effect on serum 25(OH)Vit D levels in horses, which is consistent with the findings of Salehi‐Ardakani et al. (2023). In addition, a study on the effect of blanketing on serum vitamin D levels in horses revealed that vitamin D production in the horse's skin was low, and the primary source of serum vitamin D was dietary 25(OH)Vit D2 (Azarpeykan, et al. 2016a). In our study, almost all horses had access to sunlight for several hours during rest or training, but according to low number of light colour horses in relation to dark colour, the result is not likely definitive.

Turkmen horses exhibited significantly lower 25(OH)Vit D concentrations than mixed‐bred horses, but the difference between Turkmen and Thoroughbred horses was not significant. In a previous report, Arab horses in Iran had higher 25(OH)Vit D concentrations than Kord horses, possibly due to differences in geographical latitude (Salehi‐Ardakani et al. 2023). Kord horses were kept in the higher latitude Ardabil Province of Iran, whereas Arab horses were kept in Yazd Province at lower latitudes. In a study by Pozza et al. (2014), significant differences in 25(OH)Vit D levels were observed between horses and ponies, which could be explained by breed, location and nutrition (Pozza et al. 2014). The exact reasons for these differences remain unclear.

Contrary to our results, previous studies did not report significant differences in calcium (Ca) levels between horses of different age groups (Pozza et al. 2014; Effati et al. 2018; Salehi‐Ardakani et al. 2023). The lower dry matter intake by older horses in our study may be responsible for this difference, as the calcium content of the diet plays a significant role in calcium absorption in horses. The effect of age on phosphorus (P) levels was consistent with previous reports in horses (Pozza et al. 2014; Effati et al. 2018; Salehi‐Ardakani et al. 2023), as it is related to growth and increased osteoblastic activity. In contrast to our results, magnesium (Mg) concentrations were greater in summer than in winter in Caspian miniature horses in Iran and horses in New Zealand (Azarpeykan, et al. 2016b; Effati et al. 2018). This difference is likely related to the magnesium concentration in the forage. In addition, we did not find significant differences between the longest and the shortest days of the year for total calcium or phosphorus. Azarpeykan, et al. (2016b) reported that the total calcium amount did not significantly differ between the longest and shortest days of the year, although the phosphorus amount was significantly lower on the longest days than on the shortest days of the year. These findings may be related to the concentrations of the active form of vitamin D and parathyroid hormone during different seasons. In our study, the horses were fed alfa alfa hay, which has a high level of calcium; thus, diet and growth stage, rather than hormonal control, might have influenced these ion levels. In addition, our horses did not have access to pasture water, which may be the reason for the consistency of their ion levels throughout the year because their diet was more consistent.

5. Conclusion

According to the results of the present study, the season had a significant effect on the serum 25(OH)Vit D concentration, and this difference was also observed between Turkmen horses and mixed‐bred horses (p ≤ 0.05). It is unclear whether the time of sun exposure or vitamin D content of diet causes the seasonal difference of 25(OH)Vit D concentration. The importance of these differences clinically and biologically needs to be elucidated because the magnitude of these differences is minimal.

Author Contributions

Mohieddin Alemi: investigation, data curation, validation. Mehrdad Mohri: conceptualisation, data curation, formal analysis, funding acquisition, methodology, project administration, resources, supervision, validation, writing–review and editing. Saba Ahmadi Sheikhsarmast: writing–original draft, data curation, formal analysis, writing–review and editing.

Ethics Statement

The authors confirm that the ethical policies of the journal, as noted on the journal's author guidelines page, have been adhered to and the appropriate ethical review committee approval has been received (3/51695). The authors confirm that they have followed EU standards for the protection of animals used for scientific purposes.

Conflicts of Interest

The authors declare no conflicts of interest.

Peer Review

The peer review history for this article is available at https://publons.com/publon/10.1002/vms3.70092.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.