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We Know Next to Nothing About Vitamin D in Horses!
Journal of Equine Veterinary Science 35 (2015) 785–792
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Review Article
We Know Next to Nothing About Vitamin D in Horses! Lone Hymøller*, Søren Krogh Jensen Department of Animal Science, Aarhus University, Tjele, Denmark
a r t i c l e i n f o
a b s t r a c t
Article history: Received 20 February 2015 Received in revised form 5 May 2015 Accepted 11 June 2015 Available online 18 June 2015
Very few references on vitamin D in horses exist, but the limited research available suggests that the vitamin D physiology of horses may be very different from other species. Horses can obtain vitamin D both through endogenous synthesis in the skin during sunlight exposure and through dietary sources either from synthetic vitamin D supplements or the natural vitamin D content of roughages. However, regardless of the source of vitamin D, circulating levels of vitamin D metabolites in plasma are generally reported to be very low in horses and vitamin D appears less involved in maintaining normal calcium and phosphorus homeostasis in horses than in other species. Current recommendations on the vitamin D supplementation of horses are based on a scarce amount of more or less outdated literature. Very little research has been carried out regarding the vitamin D physiology of horses and even less regarding the efficiency of different sources of vitamin D in the nutrition of horses. Furthermore, the use and management of horses has changed dramatically during the last 25 to 50 years. Hence, research in the vitamin D physiology and nutrition of modern riding horses is highly necessary, before a much needed update on the recommended vitamin D supplementation of horses can be carried out. Ó 2015 Elsevier Inc. All rights reserved.
Keywords: Ergocalciferol Cholecalciferol Nutrition Physiology Roughage Horse
1. Introduction Vitamin D has become one of the most discussed nutrients in the press and among researchers in human nutrition and physiology because it was discovered to be involved in a vast amount of physiological and immunologic processes [1,2], which stretch beyond the classically recognized effects on skeletal health and calcium and phosphorus homeostasis in the body [1,3]. How to secure a sufficient supply of vitamin D in humans has been of particular interest in parts of the world where sunlight is only a significant source of endogenous vitamin D for a limited time of the year. Hence, in Northern latitudes, advice on sun exposure and the necessity of vitamin D supplementation through the diet has been under heavy scrutiny during the later years [4]. This interest in vitamin D
* Corresponding author at: Lone Hymøller, Department of Animal Science, Aarhus University, Blichers Allé 20, DK-8830 Tjele, Denmark. E-mail address: [email protected] (L. Hymøller). 0737-0806/$ – see front matter Ó 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jevs.2015.06.010
has found its way into the nutrition of companion and production animals. For horse owners, consultants, and so forth, a large number of computer programs, Web services, and so forth for ration formulation are now commercially available. These provide easy access to current nutrient requirements of horses under different housing and exercise conditions, including the current recommendations for covering the vitamin D requirements of horses [5,6]. But, where do these recommendations come from, when no recent experimental data on the vitamin D requirements and physiology of horses exists? It appears that the current minimum requirement of vitamin D in horses of 0.17 mg (6.6 International Unit [IU]) per kg body weight per day is mainly based on older literature and extrapolations from other species [7]. However, the limited amount of literature that does exist on the subject suggests that horses may have a very different vitamin D physiology than other species [8]. The aim of the present review was to provide an overview of the data available regarding the vitamin D status and supply of horses and point to areas of future research within vitamin D supply, nutrition, and physiology
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of horses, which can support a well-founded update of the current recommendations on the vitamin D supplementation of horses. 2. Vitamin D Vitamin D exists in two forms important for the vitamin D status and supply of horses: vitamin D2 (ergocalciferol, D2), which is produced by fungi growing on plant material used as roughage for horses [9], and vitamin D3 (cholecalciferol, D3), which is either provided orally as synthetic additives or synthesized endogenously in the skin during exposure to sunlight (UV-B spectre 295–315 nm) [10,11]. To become physiologically active, both D2 and D3 must first undergo enzymatic hydroxylation in the liver to synthesize 25-hydroxyvitamin D2 (25OHD2) and 25-hydroxyvitamin D3 (25OHD3), respectively. Not all analytical methods distinguish between 25OHD2 and 25OHD3 when assessing vitamin D status in plasma but report the combined status of 25OHD2 and 25OHD3 as 25OHDx. In most species, 25OHD2 and 25OHD3 are the main vitamin D metabolites which circulate in plasma bound to vitamin D–binding protein (VDBP) and measured as indicators of physiological vitamin D status in the body. The second step of the physiological activation of vitamin D takes place in almost all organ systems in the body but mainly in the kidneys facilitated by the enzyme 25OHDx-1a-hydroxylase, which hydroxylates 25OHD2 and 25OHD3 to form 1a,25-dihydroxyvitamin D2 (1,25(OH)2D2) and 1a,25dihydroxyvitamin D3 (1,25(OH)2D3), respectively. These are hormonally active metabolites of D2 and D3 responsible for binding to the vitamin D receptor (VDR) in different organs and carrying out the physiological functions of vitamin D in the body [1,12]. In horses, the vitamin D status in the body, measured as plasma content of both 25OHD2 and 25OHD3, is generally reported to be very low (<10 ng/mL regardless of season and latitude) (Table 1) compared with other species, for example, cattle (w30 ng/mL during summer in Scandinavia) [27], but the VDBP circulating in horses has the same binding affinity for 25OHDx as in other species [19]. Rickets, a disease caused by vitamin D deficiency, is, however, rare in horses and difficult to induce experimentally [28], and horses have high serum calcium (2.75–3.25 mmol/L) and low serum phosphorus levels (0.7–1.7 mmol/L) compared with other species. Serum levels of these minerals do appear regulated, but vitamin D control of the calcium uptake is not as effective as in other species and VDR expression is low in target organs [19,29]. In vitro studies on 25OHDx-1a-hydroxylase activity revealed no enzyme activity in the kidneys of horses, but it is unknown if this was due to down regulation of the enzyme due to high serum calcium levels or a lack of 25OHDx-1a-hydroxylase enzyme all together [19]. Vitamin D has many important functions in the body, best known are probably the classical functions of maintaining calcium and phosphorus homeostasis through controlling their uptake from the gastrointestinal tract and excretion through the kidneys together with the kinetics of bone mineralization of the skeleton [3] described in many species, whereas some more recently discovered
nonclassical functions of vitamin D are regulation of inflammatory markers, immune system control, and antiproliferative effects [1,2]. Although the effect of vitamin D on the calcium and phosphorus metabolism of horses appears limited, its effect on other physiological processes is unknown. Traditionally, the biological response variables measured in animals when determining quantitative requirements of vitamins have been clear cut signs of deficiency and physical performance, for example, osteomalacia and rickets in case of vitamin D. However, currently measuring immunocompetence has become the method of choice when assessing vitamin requirements which usually gives rise to greater requirements than the traditional biological measures [30]. 3. Endogenous Synthesis of Vitamin D3 in Horses Endogenous synthesis of D3 is facilitated by UV-light in the UV-B spectre of sunlight, which penetrates the skin and cleaves 7-dehydrocholesterol (7DHC) in the epithelial cells rendering pre-D3, which at body temperature spontaneously isomerizes into D3 [10] and enters the blood stream bound to VDBP. The efficiency of the endogenous D3 synthesis depends on the intensity of the sunlight and is therefore affected by latitude and time of year. This is because the zenith angle between the sun and the earth increases during winter, causing sunlight with wavelengths in the UV-B spectre, to be reflected away from the earth in the atmosphere [31,32]. Hence, above 51 N, the conversion of 7DHC to pre-D3 in the skin is not possible during winter months [23], that is, between September and April in the Northern Hemisphere, even in horses that are not covered with rugs. This endogenous route of obtaining D3 is considered the natural way of obtaining D3 in humans and most other mammals. However, not all species are able to obtain D3 from endogenous synthesis. For instance, the 7DHC levels in the skin of polar bears are only 10% of the levels found in humans, probably due to evolutionary adaptation to heavy fur coverage and a life at very northern latitudes lacking in sunlight [33]. Mole rats have no access to sunlight due to their subterranean life [34]. Hence, the natural source of vitamin D in species like these appears to be through dietary sources either as D2 or D3. It has been heavily debated whether hair-coated animals, for example, horses and cattle, are able to synthesize D3 in the skin or if their hair coating prevents sunlight from reaching the skin surface [35] like clothes in humans [36]. However, in cattle, a direct positive correlation between the percentage of the body area exposed to sunlight and the 25OHD3 status in plasma has recently been established [27] and the amount of time cattle are exposed to sunlight during the day correlates directly to their 25OHD3 status in plasma [37], but it is unknown if these direct positive correlations can also be applied in horses. In studies on horses and ponies, limited response in 25OHD3 status to sunlight exposure has generally been reported. In Finland, Mäenpää et al [14] found all over very low 25OHDx levels in mares with very small seasonal variation. In January, the plasma concentration of 25OHDx was 4.20 0.34 ng/mL and in June 6.20 0.36 ng/mL,
Table 1 Reported plasma or serum contents of 25OHD2 and 25OHD3 (25OHDx) in horses. n
Breed
Location
Month
Feed Additive (mg/d)
Sunlighta (Yes/No)
Mare Mare Mare d Adult Adult Adult Mare Mare Mare Adult d Adult Adult Adult Mare Mare Adult d Adult
30 20 30 111 2 40 40 30 30 20 40 78 20 17 40 30 30 40 42 16
Finnhorse Finnhorse Finnhorse Coldblooded and trotter Danish Warmblood Finnhorse and half bred Finnhorse and half bred Finnhorse Finnhorse Finnhorse Finnhorse and half bred Coldblooded and trotter Thoroughbred Thoroughbred Finnhorse and half bred Finnhorse Finnhorse Finnhorse and half bred Thoroughbred and riding type Thoroughbred, coldblood and warmblood Thoroughbred Standardbred Thoroughbred Finnhorse Finnhorse Thoroughbred Thoroughbred Finnhorse Finnhorse Finnhorse Finnhorse Finnhorse Pony Pony d d d d d
Finland Finland, 60 N Finland Finland Denmark, 56 N Finland Finland Finland Finland Finland, 60 N Finland SW Finland Thailand, 14 N USA, 38 –40 N Finland Finland Finland Finland S England Germany
January January January January January March May June June June June July July–September July–September September October October December d d
50–100c 50–100c 37.5–75c d NRCd 0–337.5c 0–337.5c d d d 0 d 0 0 0–337.5c 50–100c 37.5–75c 0–337.5c d d
N N N d Y N N Y Y Y Y d Y Y N N N N d d
Italy Germany Thailand, 14 N Finland Finland Thailand, 14 N USA, 38 –40 N Finland Finland Finland Finland Finland Germany Thailand, 19 N USA USA USA USA USA
d d July–September January January d May–June June June August October October d July–September Day 14 Day 26 Day 1 Day 14 Day 26
d NRCd 0 125c 50–100c 0 0 d d d 125c 50–100c d 0 825 mg D2/kg 825 mg D2/kg 825 mg D3/kg 825 mg D3/kg 825 mg D3/kg
N N Y N N Y Y Y Y Y N N d Y N N N N N
Mare Mare Yearling Foal Foal Foal Foal Foal Foal Foal Foal Foal Adult Adult Yearling Yearling Yearling Yearling Yearling a b c d e f
Stallion Stallion Stallion Stallion Stallion
5 2 10 30 30 13 10 30 30 15 30 30 8 21 1 1 1 1 1
BW/dc BW/dc BW/dc BW/dc BW/dc
25(OH)D2 (ng/mL)
25(OH)D3b (ng/mL)
2.75 0.28 4.20 0.34 2.66 0.36 2.14 0.13 1.90 0.23 0.6 0.1 0.1 0.03 6.77–9.00 5.23–7.54 3.21 0.20 2.74 0.23 6.20 0.36 5.78–7.32 2.16 0.14 2.43 0.09 18.4–30.5e e 14.3–37.2 4.34–7.65 3.39 0.28 3.59 0.33 4.18–7.46 4.6–5.1 <2.0 <1.9–18.0 6.0 0.2 0.6 18.1–31.1e 1.73 0.21 2.16 0.25 9.3–22.0e 9.5–19.2e 2.22 0.24 1.76 0.13 2.2–7.2 2.24 0.24 2.54 0.30 <2.5–36.0 5.7–13.1e 43.1 117.5 18.9 201.0 182.0
Reference [13] [14] [13] [15] [16] [17] [17] [13] [13] [14] [17] [15] [8] [8] [17] [13] [13] [17] [18] [19] [20] [21] [8] [13] [13] [8] [8] [13] [13] [13] [13] [13] [19] [8] [22],f [22],f [22],f [22],f [22],f
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Animal
At latitudes above 51 N sunlight is not a significant source of UV-B light during winter months [23]. In most references reported as 25OHDx which is the total plasma content of 25OHD2 and 25OHD3 combined. Estimated from International Units (IU) of vitamin D, 1 IU vitamin D w0.025 mg vitamin D [24]. National Research Council (NRC): 7.5 mg (300 IU)/kg feed dry matter or 0.17 mg (6.6 IU)/kg body weight (BW) [7,25]. The enzyme immunoassay used crossreacts with 1,25(OH)2D3 (1000%) and 24,25-dihydroxyvitamin D3 (50%) [26]. Toxicological study with pharmacological doses of 825 mg vitamin D/kg body weight (BW)/d, no 25OHD2 or 25OHD3 detected on day 0 and no 25OHD2 detected on day 1 of the study, results not shown. 787
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which is a statistically significant (P .001) but numerically very small difference of 47.6%. Mäenpää et al [15] found that 25OHD2 levels were the same in serum samples obtained in January (2.14 0.13 ng/mL) and June (2.16 0.14 ng/mL), whereas 25OHD3 was 28% greater (P .05) in June (2.43 0.09 ng/mL) than in January (1.90 0.23 ng/ mL); however, the numerical difference is extremely narrow. Also Saastamoinen and Juusela [17] found no effect of access to sunlight during summer grazing on the serum concentration of 25OHDx in a study with adult Finnhorse and half bred warmblood horses. A more recent study by Pozza et al [8] in Thoroughbred horses in both Thailand and, Kentucky and Ohio, USA confirmed an effect of season and latitude on the 25OHDx status of horses and found higher plasma 25OHDx contents in their horses (15–40 ng/ mL) than previously reported by others [14,15,17] (Table 1). This may be related to the studies by Pozza et al [8] being performed at lower latitudes than the previously mentioned studies. However, the enzyme immunoassay used for measuring 25OHD3 in the study by Pozza et al [8] is reported to crossreact with both 1,25(OH)2D3 (1000% crossreactivity) and 24,25-dihydroxyvitamin D3 (50% crossreactivity) [26], and it is currently unknown if these metabolites are an important part of the peculiar vitamin D physiology of horses instead of 25OHD3 as in other species. It appears that horses do synthesize D3 in the skin during exposure to sunlight but that the response with respect to increasing plasma contents of 25OHD3 may be limited compared with other species, for example, cattle [27] at least at high latitudes. This should be thoroughly researched before well-founded recommendations on the vitamin D requirements of horses at different latitudes, during different seasons, and under different management conditions (access to outside areas and use rugs and so forth) can be made. 4. Vitamin D Supplementation of Horses Through Dietary Sources In many different species, it has been reported that ingested D2 and D3 enter systemic circulation through the lymph system after being absorbed with the fat fraction of the feed from the distal part of the small intestine [38–40]. Digestion of the fat fraction of feed in the small intestine is accomplished through emulsification of monoglyceride, diglyceride, and triglycerides by bile salts, forming a micellar solution of fatty acids, which aids the absorption of the fat into the lymph system. The anatomical peculiarity that horses do not have a gall bladder was previously believed to make horses relatively inefficient at digesting fat, particularly if the fat content of meals was substantial [41]. This has previously led to traditionally feeding horses rations relatively low in fats and oils, which may have affected the uptake of some fat soluble vitamins in horses [42]. Resent research has shown that horses, contrary to common belief, are able to use and digest fat from the diet very efficiently [42,43]. Although the importance of presence of fatty acids and bile for the uptake of D3 has not been unambiguously proven in other species [44], the effect of the fat content of the diet on the vitamin D uptake in horses should be investigated.
Horses are generally reported to respond evenly limited to PO supplementation with D3 as they do to sunlight, with respect to increasing the plasma content of 25OHD3. In the study by Saastamoinen and Juusela [17], they found no effect of different levels of D3 supplementation between 0 and 337.5 mg D3/d in adult Finnhorses and half bred warmblood horses. In Finnhorses with no access to sunlight during winter and supplemented between 37.5 and 125 mg D3/d, Mäenpää et al [13,14] reported plasma levels of 25OHDx below 5 ng/mL. In a survey on horses and ponies in the UK, Smith and Wright [18] found serum levels of 25OHD2 between 4.6 and 5.1 ng/mL and in general 25OHD3 levels below 2 ng/mL, which are comparable to plasma levels of horses reported by others [16]. The reason for these low levels of circulating 25OHDx in horses compared with other species is unknown. It is, however, not the capacity of the liver-based Dx-25-hydroxylase enzyme that prevents plasma levels of 25OHDx from increasing because D2 or D3 intoxicated horses or horses treated with pharmacologic doses of 825 mg D2 or D3/d have been reported to develop very high serum levels of 25OHDx over time (Table 1) [22]. When determining horses’ need for supplemental vitamin D in the feed, an attempt of taking into consideration the existing content of vitamin D in various roughages is often made. However, very little research has been done regarding the vitamin D content of different roughages, and the literature on the subject is generally outdated and to a large extend based on biological rat assays, which at present are almost completely abolished in favor of modern chemical methods for assessing the vitamin D content of various sample matrixes. Early studies on the fate of vitamin D in plant material used as feed for animals showed increasing vitamin D activities of plant material with increasing sunlight exposure during wilting in the field [45–48]. This is because the vitamin D present in roughages is D2, which is synthesized as a response to sunlight exposure in the cell membranes of fungi growing on withering plant material [9]. Very little recent information exists on the actual vitamin D content of plant material used as roughage (Table 2). Classical research on the vitamin D activity of plant material used biological assays with rachitic rats, where the vitamin D activity was calculated in IUs according to the amount of healing a given feed gave rise to in rachitic rat bones when fed to the rats [66]. Biological assays of this kind reveal the total vitamin D activity of a given type of plant material in concert with a basic ration but do not provide the true content of different metabolites of vitamin D. For this purpose, chemical methods must be applied. Continuing to refer back to the classical estimated vitamin D activities for estimating actual content of vitamin D metabolites in modern roughages is hazardous due to differences in agricultural production intensity, and methods together with changes in plant genetics and use of fungicides over time from the assays were carried out and until the present day. Furthermore, during the last 50 years, the crop harvest, wilting, and storage methods for roughage have changed considerably. Another issue with the biological assays is that they originally were developed for determination of the vitamin D activity of high-potency
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Table 2 Reported vitamin D content of various roughages. Plant Material
Curing Method
Analytical Method
Perennial ryegrass Perennial ryegrass Perennial ryegrass Perennial ryegrass Lucerne hay Lucerne hay Lucerne hay Lucerne hay Lucerne hay Lucerne hay Lucerne hay Lucerne bud stage Lucerne half bloom hay stage Lucerne mature seed stage Lucerne dead leaves Lucerne green leaves Lucerne leaves Lucerne stems Lucerne Lucerne Lucerne Lucerne Lucerne second cut Lucerne second cut Lucerne second cut Lucerne Lucerne Lucerne Lucerne Lucerne Brome Brome Brome Brome Brome Brome Timothy Timothy Timothy 10% heads Timothy early bloom Timothy seed stage Timothy first cut Timothy silage first cut Timothy hay first cut Timothy hay first cut Timothy hay Alsike clover Alsike clover silage Alsike clover hay Alsike clover hay Red clover Red clover silage Red clover hay Red clover hay Lucerne and Brome hay Lucerne and Brome hay Lucerne and Brome hay Lucerne and Brome hay Lucerne and Brome hay Lucerne and Timothy hay Alpine pasture Alpine pasture Lowland pasture Lowland pasture Lowland hay Highland hay Mixed hay Mixed hay
Field grown, Juneb Field grown, Julyb Field grown, Septemberb Field grown, Novemberb Barn dried Barn dried Field cured Field cured d Sun cured Sun cured d d d Mow dried Mow dried Field cured Field cured Field cured Sun cured 0 hr Sun cured 24 hr Sun cured 70 hr Sun cured 0 hr Sun cured 28 hr Sun cured 57 hr Field grown sun cured Artificially UV irradiated Field grown sun cured Artificially UV irradiated Dried and pelleted Sun cured 0 hr Sun cured 6 hr Sun cured 24 hr Sun cured 44 hr Sun cured 48 hr Sun cured 70 hr Barn cured Field cured Field cured 51 hr Field cured 55 hr Field cured 7 hr Green Wilted Mow cured Field cured Sun cured Fresh Wilted Mow cured Field cured Fresh Wilted Mow cured Field cured Sun cured Sun cured 0 hr Sun cured 12 hr Sun cured 29 hr Sun cured 51 hr Sun cured Early summer Late summer Early summer Late summer Second cut Barn cured Ground cured Tripod cured
LC-MS/MS LC-MS/MS LC-MS/MS LC-MS/MS Rat assayc d Rat assay d Rat assay Rat assay Rat assay Rat assay Rat assay Rat assay d d Rat assay Rat assay Rat assay Rat assay Rat assay Rat assay Rat assay Rat assay Rat assay HPLC-UV/GC-MS HPLC-UV/GC-MS HPLC-UV/GC-MS HPLC-UV/GC-MS Rat assay Rat assay Rat assay Rat assay Rat assay Rat assay Rat assay Rat assay Rat assay Rat assay Rat assay Rat assay Rat assay Rat assay Rat assay Rat assay Rat assay Rat assay Rat assay Rat assay Rat assay Rat assay Rat assay Rat assay Rat assay Rat assay Rat assay Rat assay Rat assay Rat assay Rat assay HPLC HPLC HPLC HPLC HPLC Rat assay Rat assay Rat assay
Vitamin D Activity (IU/kg)
470–580 473 880–970 978 1100 444–1533d 150–1460 213 322 647 7111 0 10,450 1720 1644 250d 820d 360d 70d 280d <2000d
<89–213 100d 420d 870d 1190d 2780d 520d 510–2330d 610–2000d 576 656 796 51 80 162 231 2067–2667d 51e 333–373e 422–504e 322–433e 51e 80–171e 162–251e 182–231e 2533d 110d 200d <1000d 1600d 1044–2022d
500–1080e 340–1630e 190–590e
Vitamin D Content (mg/kg)a
Reference
0.1–0.4 (D2) 1.0–4.7 (D2) 2.9–6.4 (D2) 0.4–2.0 (D2) 12–15 12 22–24 24 28 11–38 4–37 5 8 16 178 0 261 43 41 6 21 9 2 7 <50 40 (D2) 2 (D2) 0.6 (D3) 1 (D3) 2–5 3 11 22 30 70 13 13–58 15–50 14 16 20 1 2 4 6 52–67 1 8–9 11–13 8–11 1 2–4 4–6 5–6 63 3 5 <25 40 26–51 20 (D2)e 32 (D2)e 52 (D2)e 74 (D2)e 63 (D2)e 13–27 9–41 5–15
[49] [49] [49] [49] [50] [51] [50] [51] [52] [53] [54] [55] [55] [55] [51] [51] [56] [56] [55] [57] [57] [57] [57] [57] [57] [58] [58] [58] [58] [59] [57] [57] [57] [57] [57] [57] [57] [57] [60] [60] [60] [60] [60] [60] [60] [53] [60] [60] [60] [60] [60] [60] [60] [60] [53] [57] [57] [57] [57] [53] [61] [61] [61] [61] [61] [62] [62] [62]
(continued on next page)
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Table 2 (continued ) Plant Material
Curing Method
Analytical Method
Vitamin D Activity (IU/kg)
Vitamin D Content (mg/kg)a
Reference
Mixed Mixed Mixed Mixed
Fresh Sun cured Barn cured Artificially dried
Rat Rat Rat Rat
40–320e 320–2300d 180–2330d 120–1140d
1–8 5–58 5–58 5–29
[62] [63] [63] [63]
grass hay hay hay
assay assay assay assay
Abbreviations: GC, gas chromatography; HPLC, high pressure liquid chromatography; LC, liquid chromatography; MS, mass spectrometry, UV, ultra violet detector. a In rat assays, vitamin D content (mg/kg) is estimated from International Units (IUs), 1 IU vitamin D w0.025 mg vitamin D [24]. b Denmark 55 200 N, 12 230 E [49]. c Rat assay in principle: Rats suffering from induced rickets were fed the test material for given number of days and euthanized. A bone, usually left tibia, was dissected and stained in silver nitrate to reveal the size of areas with resent calcium embedment (bone regrowth). Bone regrowth in test rats was then visually compared with regrowth in rachitic control rats fed pure vitamin D supplements for the same number of days. The vitamin D activity of the test material was subsequently extrapolated from the visual comparisons of the bone regrowth [64,65]. d US Pharmacopeia units (USP), 1 USP ¼ 1 IU. e Dry matter basis.
pure oils and not of complex structures like feed samples [67]. In complex feeds the biological effect can be due to any number of compounds, for example, calcium and phosphorus that increase or inhibit the effect on bones of the vitamin D metabolites present in the sample material [68]. It should also be noted that roughages may not be a particularly good source of vitamin D in horses because D2 has in many species been shown to be much less physiologically efficient than D3 [69,70], but this has not been studied in horses. There is a great lack of experimental information about how much vitamin D horses can obtain through natural sources like sunlight and roughage and the efficiency of the vitamin D obtained. Also, research is needed on how to efficiently and safely supply horses with supplemental synthetic vitamin D in the feed to maintain a physiologically optimal vitamin D status in plasma, particularly during periods where sunlight is not a significant source of endogenous D3. 5. Factors Affecting the Vitamin D Requirements of Horses The use and management of horses and the breeding practices surrounding them have changed immensely during the last 25 to 50 years. For instance, modern riding horses spend far more time indoor, both during rest and during exercise, than horses working in the fields ever did. Furthermore, many riding horses today are covered in blankets to protect them from the weather. In both cases, endogenous synthesis of D3 in the skin of the horses is consequently prevented, increasing the need for dietary supplementation with synthetic vitamin D, as previously shown in cattle [27]. The priorities when breeding modern riding horses have also changed dramatically during the later years. Riding horses are now bred larger and taller than before and with specific emphasis on ground-covering gaits. Rapid growth and large gaits increase the strain on the bones and tendons of the skeleton, which in turn increases the physiological need for vitamin D [1,3]. Not only the knowledge about how modern riding horses obtain vitamin D through different sources, and how efficient the different sources are, is lacking; even less is known about how much vitamin D horses actually need to
support their normal physiology. Because blood sampling of equines in the wild is difficult, it is unknown if the peculiarities of the vitamin D status of modern horses, compared with other species, are general for equines or related to the housing and management of modern riding horses, in contrast to equines in the wild. Discrepancies between the vitamin D status of wild and captive animals of the same species, for example, rhinoceros and deer, have previously been described by other authors [71,72]. Modern horses may be compromised with respect to vitamin D supply and physiology compared with their wild relatives or ancestors and research into what is “normal” in equines could provide valuable information regarding the normal vitamin D physiology of modern horses. However, the modern horse, or at least the working or eventing horse, is a very different animal from wild equines and under a very different set of strains and challenges, particularly with respect to longevity and performance and so forth. Therefore, the requirements of modern horses may be very different from equines living in the wild, again emphasizing the need for research in the vitamin D physiology and nutrition of modern riding horses. 6. Where to Go From Here? In conclusion, We know next to nothing about vitamin D in horses! An update of the current recommendations on the vitamin D supplementation of horses is very much needed, but to form a reasonable basis for making any adjustments to the recommendations, there are several areas of the vitamin D nutrition and physiology of horses that need to be studied. First of all, because of the low circulating plasma levels of 25OHDx, it must be established if 25OHDx is the major vitamin D metabolite circulating in plasma of horses or if other vitamin D metabolites are better suited for assessing the vitamin D status of horses. Second, to establish when to supplement horses with synthetic vitamin D in the feed, the efficiency of sunlight under different conditions versus dietary sources of vitamin D must be established. Furthermore, the D2 content of roughage, together with a comparison of the physiological efficiency of D2 compared with D3 in horses, must be investigated. Last but not least, before drawing up guidelines for the vitamin D supplementation of horses is
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possible, biological measures relevant to horses under modern management conditions for sufficient and insufficient vitamin D supply must be established. Also, studies must be carried out to assess the vitamin D requirements of horses under different management conditions and of different breed, age, and gender.
Acknowledgments This work was supported by Aarhus University, Department of Animal Science. The authors declare no conflicts of interest.
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[60] Keener HA. The effect of various factors on the vitamin D content of several common forages. J Dairy Sci 1954;37:1337–45. [61] Kohler M, Leiber F, Willems H, Merbold L, Liesegang A. Influence of altitude on vitamin D and bone metabolism of lactating sheep and goats. J Anim Sci 2013;91:5259–68. [62] Henry KM, Kon SK, Thompson SY, McCallum JW, Steward J. The vitamin D activity of pastures and hays. Br J Nutr 1958;12:462–9. [63] Newlander JA. Vitamin D content of roughages. J Dairy Sci 1948;63: 455–60. [64] McCollum EV, Simmons N, Shipley PG, Park EA. Studies on experimental rickets. XVI: a delicate biological test for calcium depositing substances. J Biol Chem 1922;51:41–51. [65] Norman AW, Henry HL, Vitamin D. In: Zempleni J, Rucker RB, McCormick DB, Suttie JW, editors. Handbook of vitamins 4th edition. London UK: CRC Press; 2007. p. 41–110. [66] Bills CE. Vitamin D assaydline test and chemical methods. Biol Symposia 1947;12:409–34. [67] Weits J. Preparation of roughage samples for biological determination of vitamin D content. J Dairy Sci 1958;41:1088–93. [68] Gridgeman NT, Lees H, Wilkinson H. The biological vitamin D assay of low-potency materials with special reference to the role of the mineral content of the diet. Biochem J 1939;33:645–54. [69] Horst RL, Napoli JL, Littledike ET. Discrimination in the metabolism of orally dosed ergocalciferol and cholecalciferol by the pig, rat and chick. Biochem J 1982;204:185–9. [70] Hymøller L, Jensen SK. Vitamin D2 impairs utilization of vitamin D3 in high-yielding dairy cows in a cross-over supplementation regimen. J Dairy Sci 2011;94:3462–6. [71] Clauss M, Jessup DA, Norkus EB, Chen TC, Holick MF, Streich WJ, et al. Fat-soluble vitamins in blood and tissues of free-ranging and captive rhinoceros. J Wildl Dis 2002;38:402–13. [72] Waters WR, Nonnecke BJ, Gibbs SEJ, Yabsley MJ, Schmitt SM, Cosgrove MK, et al. Serum 25-hydroxyvitamin D concentrations in captive and free-ranging, White-tailed deer (Odocoileus virginianus). Int J Vitam Nutr Res 2009;79:180–7.
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Review Article
We Know Next to Nothing About Vitamin D in Horses! Lone Hymøller*, Søren Krogh Jensen Department of Animal Science, Aarhus University, Tjele, Denmark
a r t i c l e i n f o
a b s t r a c t
Article history: Received 20 February 2015 Received in revised form 5 May 2015 Accepted 11 June 2015 Available online 18 June 2015
Very few references on vitamin D in horses exist, but the limited research available suggests that the vitamin D physiology of horses may be very different from other species. Horses can obtain vitamin D both through endogenous synthesis in the skin during sunlight exposure and through dietary sources either from synthetic vitamin D supplements or the natural vitamin D content of roughages. However, regardless of the source of vitamin D, circulating levels of vitamin D metabolites in plasma are generally reported to be very low in horses and vitamin D appears less involved in maintaining normal calcium and phosphorus homeostasis in horses than in other species. Current recommendations on the vitamin D supplementation of horses are based on a scarce amount of more or less outdated literature. Very little research has been carried out regarding the vitamin D physiology of horses and even less regarding the efficiency of different sources of vitamin D in the nutrition of horses. Furthermore, the use and management of horses has changed dramatically during the last 25 to 50 years. Hence, research in the vitamin D physiology and nutrition of modern riding horses is highly necessary, before a much needed update on the recommended vitamin D supplementation of horses can be carried out. Ó 2015 Elsevier Inc. All rights reserved.
Keywords: Ergocalciferol Cholecalciferol Nutrition Physiology Roughage Horse
1. Introduction Vitamin D has become one of the most discussed nutrients in the press and among researchers in human nutrition and physiology because it was discovered to be involved in a vast amount of physiological and immunologic processes [1,2], which stretch beyond the classically recognized effects on skeletal health and calcium and phosphorus homeostasis in the body [1,3]. How to secure a sufficient supply of vitamin D in humans has been of particular interest in parts of the world where sunlight is only a significant source of endogenous vitamin D for a limited time of the year. Hence, in Northern latitudes, advice on sun exposure and the necessity of vitamin D supplementation through the diet has been under heavy scrutiny during the later years [4]. This interest in vitamin D
* Corresponding author at: Lone Hymøller, Department of Animal Science, Aarhus University, Blichers Allé 20, DK-8830 Tjele, Denmark. E-mail address: [email protected] (L. Hymøller). 0737-0806/$ – see front matter Ó 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jevs.2015.06.010
has found its way into the nutrition of companion and production animals. For horse owners, consultants, and so forth, a large number of computer programs, Web services, and so forth for ration formulation are now commercially available. These provide easy access to current nutrient requirements of horses under different housing and exercise conditions, including the current recommendations for covering the vitamin D requirements of horses [5,6]. But, where do these recommendations come from, when no recent experimental data on the vitamin D requirements and physiology of horses exists? It appears that the current minimum requirement of vitamin D in horses of 0.17 mg (6.6 International Unit [IU]) per kg body weight per day is mainly based on older literature and extrapolations from other species [7]. However, the limited amount of literature that does exist on the subject suggests that horses may have a very different vitamin D physiology than other species [8]. The aim of the present review was to provide an overview of the data available regarding the vitamin D status and supply of horses and point to areas of future research within vitamin D supply, nutrition, and physiology
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of horses, which can support a well-founded update of the current recommendations on the vitamin D supplementation of horses. 2. Vitamin D Vitamin D exists in two forms important for the vitamin D status and supply of horses: vitamin D2 (ergocalciferol, D2), which is produced by fungi growing on plant material used as roughage for horses [9], and vitamin D3 (cholecalciferol, D3), which is either provided orally as synthetic additives or synthesized endogenously in the skin during exposure to sunlight (UV-B spectre 295–315 nm) [10,11]. To become physiologically active, both D2 and D3 must first undergo enzymatic hydroxylation in the liver to synthesize 25-hydroxyvitamin D2 (25OHD2) and 25-hydroxyvitamin D3 (25OHD3), respectively. Not all analytical methods distinguish between 25OHD2 and 25OHD3 when assessing vitamin D status in plasma but report the combined status of 25OHD2 and 25OHD3 as 25OHDx. In most species, 25OHD2 and 25OHD3 are the main vitamin D metabolites which circulate in plasma bound to vitamin D–binding protein (VDBP) and measured as indicators of physiological vitamin D status in the body. The second step of the physiological activation of vitamin D takes place in almost all organ systems in the body but mainly in the kidneys facilitated by the enzyme 25OHDx-1a-hydroxylase, which hydroxylates 25OHD2 and 25OHD3 to form 1a,25-dihydroxyvitamin D2 (1,25(OH)2D2) and 1a,25dihydroxyvitamin D3 (1,25(OH)2D3), respectively. These are hormonally active metabolites of D2 and D3 responsible for binding to the vitamin D receptor (VDR) in different organs and carrying out the physiological functions of vitamin D in the body [1,12]. In horses, the vitamin D status in the body, measured as plasma content of both 25OHD2 and 25OHD3, is generally reported to be very low (<10 ng/mL regardless of season and latitude) (Table 1) compared with other species, for example, cattle (w30 ng/mL during summer in Scandinavia) [27], but the VDBP circulating in horses has the same binding affinity for 25OHDx as in other species [19]. Rickets, a disease caused by vitamin D deficiency, is, however, rare in horses and difficult to induce experimentally [28], and horses have high serum calcium (2.75–3.25 mmol/L) and low serum phosphorus levels (0.7–1.7 mmol/L) compared with other species. Serum levels of these minerals do appear regulated, but vitamin D control of the calcium uptake is not as effective as in other species and VDR expression is low in target organs [19,29]. In vitro studies on 25OHDx-1a-hydroxylase activity revealed no enzyme activity in the kidneys of horses, but it is unknown if this was due to down regulation of the enzyme due to high serum calcium levels or a lack of 25OHDx-1a-hydroxylase enzyme all together [19]. Vitamin D has many important functions in the body, best known are probably the classical functions of maintaining calcium and phosphorus homeostasis through controlling their uptake from the gastrointestinal tract and excretion through the kidneys together with the kinetics of bone mineralization of the skeleton [3] described in many species, whereas some more recently discovered
nonclassical functions of vitamin D are regulation of inflammatory markers, immune system control, and antiproliferative effects [1,2]. Although the effect of vitamin D on the calcium and phosphorus metabolism of horses appears limited, its effect on other physiological processes is unknown. Traditionally, the biological response variables measured in animals when determining quantitative requirements of vitamins have been clear cut signs of deficiency and physical performance, for example, osteomalacia and rickets in case of vitamin D. However, currently measuring immunocompetence has become the method of choice when assessing vitamin requirements which usually gives rise to greater requirements than the traditional biological measures [30]. 3. Endogenous Synthesis of Vitamin D3 in Horses Endogenous synthesis of D3 is facilitated by UV-light in the UV-B spectre of sunlight, which penetrates the skin and cleaves 7-dehydrocholesterol (7DHC) in the epithelial cells rendering pre-D3, which at body temperature spontaneously isomerizes into D3 [10] and enters the blood stream bound to VDBP. The efficiency of the endogenous D3 synthesis depends on the intensity of the sunlight and is therefore affected by latitude and time of year. This is because the zenith angle between the sun and the earth increases during winter, causing sunlight with wavelengths in the UV-B spectre, to be reflected away from the earth in the atmosphere [31,32]. Hence, above 51 N, the conversion of 7DHC to pre-D3 in the skin is not possible during winter months [23], that is, between September and April in the Northern Hemisphere, even in horses that are not covered with rugs. This endogenous route of obtaining D3 is considered the natural way of obtaining D3 in humans and most other mammals. However, not all species are able to obtain D3 from endogenous synthesis. For instance, the 7DHC levels in the skin of polar bears are only 10% of the levels found in humans, probably due to evolutionary adaptation to heavy fur coverage and a life at very northern latitudes lacking in sunlight [33]. Mole rats have no access to sunlight due to their subterranean life [34]. Hence, the natural source of vitamin D in species like these appears to be through dietary sources either as D2 or D3. It has been heavily debated whether hair-coated animals, for example, horses and cattle, are able to synthesize D3 in the skin or if their hair coating prevents sunlight from reaching the skin surface [35] like clothes in humans [36]. However, in cattle, a direct positive correlation between the percentage of the body area exposed to sunlight and the 25OHD3 status in plasma has recently been established [27] and the amount of time cattle are exposed to sunlight during the day correlates directly to their 25OHD3 status in plasma [37], but it is unknown if these direct positive correlations can also be applied in horses. In studies on horses and ponies, limited response in 25OHD3 status to sunlight exposure has generally been reported. In Finland, Mäenpää et al [14] found all over very low 25OHDx levels in mares with very small seasonal variation. In January, the plasma concentration of 25OHDx was 4.20 0.34 ng/mL and in June 6.20 0.36 ng/mL,
Table 1 Reported plasma or serum contents of 25OHD2 and 25OHD3 (25OHDx) in horses. n
Breed
Location
Month
Feed Additive (mg/d)
Sunlighta (Yes/No)
Mare Mare Mare d Adult Adult Adult Mare Mare Mare Adult d Adult Adult Adult Mare Mare Adult d Adult
30 20 30 111 2 40 40 30 30 20 40 78 20 17 40 30 30 40 42 16
Finnhorse Finnhorse Finnhorse Coldblooded and trotter Danish Warmblood Finnhorse and half bred Finnhorse and half bred Finnhorse Finnhorse Finnhorse Finnhorse and half bred Coldblooded and trotter Thoroughbred Thoroughbred Finnhorse and half bred Finnhorse Finnhorse Finnhorse and half bred Thoroughbred and riding type Thoroughbred, coldblood and warmblood Thoroughbred Standardbred Thoroughbred Finnhorse Finnhorse Thoroughbred Thoroughbred Finnhorse Finnhorse Finnhorse Finnhorse Finnhorse Pony Pony d d d d d
Finland Finland, 60 N Finland Finland Denmark, 56 N Finland Finland Finland Finland Finland, 60 N Finland SW Finland Thailand, 14 N USA, 38 –40 N Finland Finland Finland Finland S England Germany
January January January January January March May June June June June July July–September July–September September October October December d d
50–100c 50–100c 37.5–75c d NRCd 0–337.5c 0–337.5c d d d 0 d 0 0 0–337.5c 50–100c 37.5–75c 0–337.5c d d
N N N d Y N N Y Y Y Y d Y Y N N N N d d
Italy Germany Thailand, 14 N Finland Finland Thailand, 14 N USA, 38 –40 N Finland Finland Finland Finland Finland Germany Thailand, 19 N USA USA USA USA USA
d d July–September January January d May–June June June August October October d July–September Day 14 Day 26 Day 1 Day 14 Day 26
d NRCd 0 125c 50–100c 0 0 d d d 125c 50–100c d 0 825 mg D2/kg 825 mg D2/kg 825 mg D3/kg 825 mg D3/kg 825 mg D3/kg
N N Y N N Y Y Y Y Y N N d Y N N N N N
Mare Mare Yearling Foal Foal Foal Foal Foal Foal Foal Foal Foal Adult Adult Yearling Yearling Yearling Yearling Yearling a b c d e f
Stallion Stallion Stallion Stallion Stallion
5 2 10 30 30 13 10 30 30 15 30 30 8 21 1 1 1 1 1
BW/dc BW/dc BW/dc BW/dc BW/dc
25(OH)D2 (ng/mL)
25(OH)D3b (ng/mL)
2.75 0.28 4.20 0.34 2.66 0.36 2.14 0.13 1.90 0.23 0.6 0.1 0.1 0.03 6.77–9.00 5.23–7.54 3.21 0.20 2.74 0.23 6.20 0.36 5.78–7.32 2.16 0.14 2.43 0.09 18.4–30.5e e 14.3–37.2 4.34–7.65 3.39 0.28 3.59 0.33 4.18–7.46 4.6–5.1 <2.0 <1.9–18.0 6.0 0.2 0.6 18.1–31.1e 1.73 0.21 2.16 0.25 9.3–22.0e 9.5–19.2e 2.22 0.24 1.76 0.13 2.2–7.2 2.24 0.24 2.54 0.30 <2.5–36.0 5.7–13.1e 43.1 117.5 18.9 201.0 182.0
Reference [13] [14] [13] [15] [16] [17] [17] [13] [13] [14] [17] [15] [8] [8] [17] [13] [13] [17] [18] [19] [20] [21] [8] [13] [13] [8] [8] [13] [13] [13] [13] [13] [19] [8] [22],f [22],f [22],f [22],f [22],f
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Animal
At latitudes above 51 N sunlight is not a significant source of UV-B light during winter months [23]. In most references reported as 25OHDx which is the total plasma content of 25OHD2 and 25OHD3 combined. Estimated from International Units (IU) of vitamin D, 1 IU vitamin D w0.025 mg vitamin D [24]. National Research Council (NRC): 7.5 mg (300 IU)/kg feed dry matter or 0.17 mg (6.6 IU)/kg body weight (BW) [7,25]. The enzyme immunoassay used crossreacts with 1,25(OH)2D3 (1000%) and 24,25-dihydroxyvitamin D3 (50%) [26]. Toxicological study with pharmacological doses of 825 mg vitamin D/kg body weight (BW)/d, no 25OHD2 or 25OHD3 detected on day 0 and no 25OHD2 detected on day 1 of the study, results not shown. 787
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which is a statistically significant (P .001) but numerically very small difference of 47.6%. Mäenpää et al [15] found that 25OHD2 levels were the same in serum samples obtained in January (2.14 0.13 ng/mL) and June (2.16 0.14 ng/mL), whereas 25OHD3 was 28% greater (P .05) in June (2.43 0.09 ng/mL) than in January (1.90 0.23 ng/ mL); however, the numerical difference is extremely narrow. Also Saastamoinen and Juusela [17] found no effect of access to sunlight during summer grazing on the serum concentration of 25OHDx in a study with adult Finnhorse and half bred warmblood horses. A more recent study by Pozza et al [8] in Thoroughbred horses in both Thailand and, Kentucky and Ohio, USA confirmed an effect of season and latitude on the 25OHDx status of horses and found higher plasma 25OHDx contents in their horses (15–40 ng/ mL) than previously reported by others [14,15,17] (Table 1). This may be related to the studies by Pozza et al [8] being performed at lower latitudes than the previously mentioned studies. However, the enzyme immunoassay used for measuring 25OHD3 in the study by Pozza et al [8] is reported to crossreact with both 1,25(OH)2D3 (1000% crossreactivity) and 24,25-dihydroxyvitamin D3 (50% crossreactivity) [26], and it is currently unknown if these metabolites are an important part of the peculiar vitamin D physiology of horses instead of 25OHD3 as in other species. It appears that horses do synthesize D3 in the skin during exposure to sunlight but that the response with respect to increasing plasma contents of 25OHD3 may be limited compared with other species, for example, cattle [27] at least at high latitudes. This should be thoroughly researched before well-founded recommendations on the vitamin D requirements of horses at different latitudes, during different seasons, and under different management conditions (access to outside areas and use rugs and so forth) can be made. 4. Vitamin D Supplementation of Horses Through Dietary Sources In many different species, it has been reported that ingested D2 and D3 enter systemic circulation through the lymph system after being absorbed with the fat fraction of the feed from the distal part of the small intestine [38–40]. Digestion of the fat fraction of feed in the small intestine is accomplished through emulsification of monoglyceride, diglyceride, and triglycerides by bile salts, forming a micellar solution of fatty acids, which aids the absorption of the fat into the lymph system. The anatomical peculiarity that horses do not have a gall bladder was previously believed to make horses relatively inefficient at digesting fat, particularly if the fat content of meals was substantial [41]. This has previously led to traditionally feeding horses rations relatively low in fats and oils, which may have affected the uptake of some fat soluble vitamins in horses [42]. Resent research has shown that horses, contrary to common belief, are able to use and digest fat from the diet very efficiently [42,43]. Although the importance of presence of fatty acids and bile for the uptake of D3 has not been unambiguously proven in other species [44], the effect of the fat content of the diet on the vitamin D uptake in horses should be investigated.
Horses are generally reported to respond evenly limited to PO supplementation with D3 as they do to sunlight, with respect to increasing the plasma content of 25OHD3. In the study by Saastamoinen and Juusela [17], they found no effect of different levels of D3 supplementation between 0 and 337.5 mg D3/d in adult Finnhorses and half bred warmblood horses. In Finnhorses with no access to sunlight during winter and supplemented between 37.5 and 125 mg D3/d, Mäenpää et al [13,14] reported plasma levels of 25OHDx below 5 ng/mL. In a survey on horses and ponies in the UK, Smith and Wright [18] found serum levels of 25OHD2 between 4.6 and 5.1 ng/mL and in general 25OHD3 levels below 2 ng/mL, which are comparable to plasma levels of horses reported by others [16]. The reason for these low levels of circulating 25OHDx in horses compared with other species is unknown. It is, however, not the capacity of the liver-based Dx-25-hydroxylase enzyme that prevents plasma levels of 25OHDx from increasing because D2 or D3 intoxicated horses or horses treated with pharmacologic doses of 825 mg D2 or D3/d have been reported to develop very high serum levels of 25OHDx over time (Table 1) [22]. When determining horses’ need for supplemental vitamin D in the feed, an attempt of taking into consideration the existing content of vitamin D in various roughages is often made. However, very little research has been done regarding the vitamin D content of different roughages, and the literature on the subject is generally outdated and to a large extend based on biological rat assays, which at present are almost completely abolished in favor of modern chemical methods for assessing the vitamin D content of various sample matrixes. Early studies on the fate of vitamin D in plant material used as feed for animals showed increasing vitamin D activities of plant material with increasing sunlight exposure during wilting in the field [45–48]. This is because the vitamin D present in roughages is D2, which is synthesized as a response to sunlight exposure in the cell membranes of fungi growing on withering plant material [9]. Very little recent information exists on the actual vitamin D content of plant material used as roughage (Table 2). Classical research on the vitamin D activity of plant material used biological assays with rachitic rats, where the vitamin D activity was calculated in IUs according to the amount of healing a given feed gave rise to in rachitic rat bones when fed to the rats [66]. Biological assays of this kind reveal the total vitamin D activity of a given type of plant material in concert with a basic ration but do not provide the true content of different metabolites of vitamin D. For this purpose, chemical methods must be applied. Continuing to refer back to the classical estimated vitamin D activities for estimating actual content of vitamin D metabolites in modern roughages is hazardous due to differences in agricultural production intensity, and methods together with changes in plant genetics and use of fungicides over time from the assays were carried out and until the present day. Furthermore, during the last 50 years, the crop harvest, wilting, and storage methods for roughage have changed considerably. Another issue with the biological assays is that they originally were developed for determination of the vitamin D activity of high-potency
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Table 2 Reported vitamin D content of various roughages. Plant Material
Curing Method
Analytical Method
Perennial ryegrass Perennial ryegrass Perennial ryegrass Perennial ryegrass Lucerne hay Lucerne hay Lucerne hay Lucerne hay Lucerne hay Lucerne hay Lucerne hay Lucerne bud stage Lucerne half bloom hay stage Lucerne mature seed stage Lucerne dead leaves Lucerne green leaves Lucerne leaves Lucerne stems Lucerne Lucerne Lucerne Lucerne Lucerne second cut Lucerne second cut Lucerne second cut Lucerne Lucerne Lucerne Lucerne Lucerne Brome Brome Brome Brome Brome Brome Timothy Timothy Timothy 10% heads Timothy early bloom Timothy seed stage Timothy first cut Timothy silage first cut Timothy hay first cut Timothy hay first cut Timothy hay Alsike clover Alsike clover silage Alsike clover hay Alsike clover hay Red clover Red clover silage Red clover hay Red clover hay Lucerne and Brome hay Lucerne and Brome hay Lucerne and Brome hay Lucerne and Brome hay Lucerne and Brome hay Lucerne and Timothy hay Alpine pasture Alpine pasture Lowland pasture Lowland pasture Lowland hay Highland hay Mixed hay Mixed hay
Field grown, Juneb Field grown, Julyb Field grown, Septemberb Field grown, Novemberb Barn dried Barn dried Field cured Field cured d Sun cured Sun cured d d d Mow dried Mow dried Field cured Field cured Field cured Sun cured 0 hr Sun cured 24 hr Sun cured 70 hr Sun cured 0 hr Sun cured 28 hr Sun cured 57 hr Field grown sun cured Artificially UV irradiated Field grown sun cured Artificially UV irradiated Dried and pelleted Sun cured 0 hr Sun cured 6 hr Sun cured 24 hr Sun cured 44 hr Sun cured 48 hr Sun cured 70 hr Barn cured Field cured Field cured 51 hr Field cured 55 hr Field cured 7 hr Green Wilted Mow cured Field cured Sun cured Fresh Wilted Mow cured Field cured Fresh Wilted Mow cured Field cured Sun cured Sun cured 0 hr Sun cured 12 hr Sun cured 29 hr Sun cured 51 hr Sun cured Early summer Late summer Early summer Late summer Second cut Barn cured Ground cured Tripod cured
LC-MS/MS LC-MS/MS LC-MS/MS LC-MS/MS Rat assayc d Rat assay d Rat assay Rat assay Rat assay Rat assay Rat assay Rat assay d d Rat assay Rat assay Rat assay Rat assay Rat assay Rat assay Rat assay Rat assay Rat assay HPLC-UV/GC-MS HPLC-UV/GC-MS HPLC-UV/GC-MS HPLC-UV/GC-MS Rat assay Rat assay Rat assay Rat assay Rat assay Rat assay Rat assay Rat assay Rat assay Rat assay Rat assay Rat assay Rat assay Rat assay Rat assay Rat assay Rat assay Rat assay Rat assay Rat assay Rat assay Rat assay Rat assay Rat assay Rat assay Rat assay Rat assay Rat assay Rat assay Rat assay Rat assay HPLC HPLC HPLC HPLC HPLC Rat assay Rat assay Rat assay
Vitamin D Activity (IU/kg)
470–580 473 880–970 978 1100 444–1533d 150–1460 213 322 647 7111 0 10,450 1720 1644 250d 820d 360d 70d 280d <2000d
<89–213 100d 420d 870d 1190d 2780d 520d 510–2330d 610–2000d 576 656 796 51 80 162 231 2067–2667d 51e 333–373e 422–504e 322–433e 51e 80–171e 162–251e 182–231e 2533d 110d 200d <1000d 1600d 1044–2022d
500–1080e 340–1630e 190–590e
Vitamin D Content (mg/kg)a
Reference
0.1–0.4 (D2) 1.0–4.7 (D2) 2.9–6.4 (D2) 0.4–2.0 (D2) 12–15 12 22–24 24 28 11–38 4–37 5 8 16 178 0 261 43 41 6 21 9 2 7 <50 40 (D2) 2 (D2) 0.6 (D3) 1 (D3) 2–5 3 11 22 30 70 13 13–58 15–50 14 16 20 1 2 4 6 52–67 1 8–9 11–13 8–11 1 2–4 4–6 5–6 63 3 5 <25 40 26–51 20 (D2)e 32 (D2)e 52 (D2)e 74 (D2)e 63 (D2)e 13–27 9–41 5–15
[49] [49] [49] [49] [50] [51] [50] [51] [52] [53] [54] [55] [55] [55] [51] [51] [56] [56] [55] [57] [57] [57] [57] [57] [57] [58] [58] [58] [58] [59] [57] [57] [57] [57] [57] [57] [57] [57] [60] [60] [60] [60] [60] [60] [60] [53] [60] [60] [60] [60] [60] [60] [60] [60] [53] [57] [57] [57] [57] [53] [61] [61] [61] [61] [61] [62] [62] [62]
(continued on next page)
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Table 2 (continued ) Plant Material
Curing Method
Analytical Method
Vitamin D Activity (IU/kg)
Vitamin D Content (mg/kg)a
Reference
Mixed Mixed Mixed Mixed
Fresh Sun cured Barn cured Artificially dried
Rat Rat Rat Rat
40–320e 320–2300d 180–2330d 120–1140d
1–8 5–58 5–58 5–29
[62] [63] [63] [63]
grass hay hay hay
assay assay assay assay
Abbreviations: GC, gas chromatography; HPLC, high pressure liquid chromatography; LC, liquid chromatography; MS, mass spectrometry, UV, ultra violet detector. a In rat assays, vitamin D content (mg/kg) is estimated from International Units (IUs), 1 IU vitamin D w0.025 mg vitamin D [24]. b Denmark 55 200 N, 12 230 E [49]. c Rat assay in principle: Rats suffering from induced rickets were fed the test material for given number of days and euthanized. A bone, usually left tibia, was dissected and stained in silver nitrate to reveal the size of areas with resent calcium embedment (bone regrowth). Bone regrowth in test rats was then visually compared with regrowth in rachitic control rats fed pure vitamin D supplements for the same number of days. The vitamin D activity of the test material was subsequently extrapolated from the visual comparisons of the bone regrowth [64,65]. d US Pharmacopeia units (USP), 1 USP ¼ 1 IU. e Dry matter basis.
pure oils and not of complex structures like feed samples [67]. In complex feeds the biological effect can be due to any number of compounds, for example, calcium and phosphorus that increase or inhibit the effect on bones of the vitamin D metabolites present in the sample material [68]. It should also be noted that roughages may not be a particularly good source of vitamin D in horses because D2 has in many species been shown to be much less physiologically efficient than D3 [69,70], but this has not been studied in horses. There is a great lack of experimental information about how much vitamin D horses can obtain through natural sources like sunlight and roughage and the efficiency of the vitamin D obtained. Also, research is needed on how to efficiently and safely supply horses with supplemental synthetic vitamin D in the feed to maintain a physiologically optimal vitamin D status in plasma, particularly during periods where sunlight is not a significant source of endogenous D3. 5. Factors Affecting the Vitamin D Requirements of Horses The use and management of horses and the breeding practices surrounding them have changed immensely during the last 25 to 50 years. For instance, modern riding horses spend far more time indoor, both during rest and during exercise, than horses working in the fields ever did. Furthermore, many riding horses today are covered in blankets to protect them from the weather. In both cases, endogenous synthesis of D3 in the skin of the horses is consequently prevented, increasing the need for dietary supplementation with synthetic vitamin D, as previously shown in cattle [27]. The priorities when breeding modern riding horses have also changed dramatically during the later years. Riding horses are now bred larger and taller than before and with specific emphasis on ground-covering gaits. Rapid growth and large gaits increase the strain on the bones and tendons of the skeleton, which in turn increases the physiological need for vitamin D [1,3]. Not only the knowledge about how modern riding horses obtain vitamin D through different sources, and how efficient the different sources are, is lacking; even less is known about how much vitamin D horses actually need to
support their normal physiology. Because blood sampling of equines in the wild is difficult, it is unknown if the peculiarities of the vitamin D status of modern horses, compared with other species, are general for equines or related to the housing and management of modern riding horses, in contrast to equines in the wild. Discrepancies between the vitamin D status of wild and captive animals of the same species, for example, rhinoceros and deer, have previously been described by other authors [71,72]. Modern horses may be compromised with respect to vitamin D supply and physiology compared with their wild relatives or ancestors and research into what is “normal” in equines could provide valuable information regarding the normal vitamin D physiology of modern horses. However, the modern horse, or at least the working or eventing horse, is a very different animal from wild equines and under a very different set of strains and challenges, particularly with respect to longevity and performance and so forth. Therefore, the requirements of modern horses may be very different from equines living in the wild, again emphasizing the need for research in the vitamin D physiology and nutrition of modern riding horses. 6. Where to Go From Here? In conclusion, We know next to nothing about vitamin D in horses! An update of the current recommendations on the vitamin D supplementation of horses is very much needed, but to form a reasonable basis for making any adjustments to the recommendations, there are several areas of the vitamin D nutrition and physiology of horses that need to be studied. First of all, because of the low circulating plasma levels of 25OHDx, it must be established if 25OHDx is the major vitamin D metabolite circulating in plasma of horses or if other vitamin D metabolites are better suited for assessing the vitamin D status of horses. Second, to establish when to supplement horses with synthetic vitamin D in the feed, the efficiency of sunlight under different conditions versus dietary sources of vitamin D must be established. Furthermore, the D2 content of roughage, together with a comparison of the physiological efficiency of D2 compared with D3 in horses, must be investigated. Last but not least, before drawing up guidelines for the vitamin D supplementation of horses is
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possible, biological measures relevant to horses under modern management conditions for sufficient and insufficient vitamin D supply must be established. Also, studies must be carried out to assess the vitamin D requirements of horses under different management conditions and of different breed, age, and gender.
Acknowledgments This work was supported by Aarhus University, Department of Animal Science. The authors declare no conflicts of interest.
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