The only source of cholecalciferol (vitamin D3, D3) in grazing herbivores is endogenous D3 produced in the skin during exposure to UV light from sunlight, since common grassland plants do not contain D3. Endogenous D3 is derived from 7-dehydrocholesterol (7DHC) produced in epithelial cells from acetate through the cholesterol synthesis pathway(Reference Gaylor and Sault1, Reference Stryer2). Irradiation with UV light between 290 and 315 nm cleaves the C9 and C10 bonds of 7DHC, rendering pre-cholecalciferol (preD3), which as a result of its thermodynamically unstable cis–cis configuration, spontaneously isomerises into D3 by a rearrangement of its double bonds catalysed by heat at body temperature(Reference Stryer2, Reference MacLaughlin, Anderson and Holick3), a process completed in 2–4 d(Reference McDowell4). If preD3 is exposed to excessive amounts of UV light it will, instead of being isomerised into D3, be turned into different inactive metabolites of D3 catalysed by UV light(Reference MacLaughlin, Anderson and Holick3, Reference Webb, Kline and Holick5).
Even though the biochemical and physiological processes behind endogenous D3 synthesis in the skin are well described, it is still heavily debated as to how much UV light or sunlight is necessary to secure and maintain a sufficient D3 status in plasma, measured as 25-hydroxycholecalciferol (25(OH)D3), which is the major physiological metabolite of D3 circulating in plasma and indicative of the physiological D3 status of living organisms(Reference Olds, McKinley and Moore6). Solar UV light intensity is affected by latitude, time of day and season; and Engelsen et al. (Reference Engelsen, Brustad and Aksnes7) showed that cutaneous D3 synthesis could not be sustained throughout the year at latitudes higher than 50°. In addition, clouds, aerosols and thick ozone events reduce the duration of D3 synthesis and can induce a ‘vitamin D winter’ even at the equator(Reference Engelsen, Brustad and Aksnes7).
In studies on rat and pig skin subjected to UV light, between 15 and 30 % of the 7DHC present in the skin samples was turned into D3, resulting in a total endogenous production of D3 between 0·125 and 0·375 μg/cm2 in rat skin and between 0·75 and 2·25 μg/cm2 in pig skin(Reference Bekemeier8, Reference Bekemeier and Pfennigsdorf9). Studies with human skin samples, expressing varying degrees of pigmentation and exposed to different UV light intensities, showed that the formation of preD3 from 7DHC was non-linear and reached a plateau when 10–15 % of the 7DHC present in the skin was photoisomerised. This was irrespective of the degree of pigmentation, but the necessary exposure time increased as pigmentation darkened(Reference Holick10, Reference Holick, MacLaughlin and Doppelt11). An in vitro method, utilising the exposure of solutions of pure 7DHC in ethanol to UV light and subsequent measurement of the amount of D3 produced, also revealed a non-linear relationship between the UV light dose given and the resulting D3 synthesis(Reference Olds, McKinley and Moore6).
However, none of these in vitro methods allowed for prediction of the impact of UV light or sunlight irradiation on the 25(OH)D3 status of living organisms. If a direct link between UV light or sunlight exposure and 25(OH)D3 status in plasma in vivo should be established, it would involve completely restricted access to UV light sources and sunlight during the experimental period, a diet void of D3 and a possibility for easy blood sampling. Such a study would be difficult to perform in wild herbivores. Furthermore, the relationship between sunlight exposure and the resulting 25(OH)D3 status is of interest to both wild herbivores and domestic livestock reared outside; especially livestock in organic production, where the use of artificial feed additives, including synthetic vitamins, is restricted to fulfil the principles of sustainable organic production(12).
Recently we showed that cows, in contrast to common notion, synthesise D3 in their entire skin surface when exposed to summer sunlight(Reference Hymøller and Jensen13). Furthermore, commercial dairy cows are easy to control with respect to D3 supply in contrast to wild herbivores, because their daily routines vary extremely little and they do not obtain any D3 from sources other than the sun at pasture or synthetic feed additives in the barn. Hence, the present study was carried out in black and white Danish Holstein cows.
The aim of the present experiment was to investigate the necessary time-interval that large herbivores, exemplified by dairy cows, must have during access to pasture in mid-summer at 56°N in order to maintain a constant 25(OH)D3 status in plasma, and to investigate the suggested non-linear relationship between sunlight exposure time and 25(OH)D3 status in plasma in vivo.
Materials and methods
Animals and management
The study complied with the Danish Ministry of Justice Law no. 1306 (23 November 2007) concerning experiments with animals and care of experimental animals and was under the supervision of the Danish Animal Experiments Inspectorate. A total of twenty dairy cows of black and white Danish Holstein breed in first lactation with an average yield of 27·5 (sem 0·8) kg energy-corrected milk per d(Reference Sjaunja, Baevre and Junkkarinen14) were used. The animals were divided into five treatment groups according to their daily energy-corrected milk yield and dominant coat colour (black or white), and let out for pasture daily during different time intervals at daytime. In the remaining time, the animals were housed in tie stalls without access to sunlight. In the barn, the feed consisted of an ad libitum maize- and grass clover silage-based total mixed ration without added D3. The total mixed ration was given once daily at 09.00 hours and milking was carried out twice daily at 06.00 and 17.00 hours. The study was conducted from 3 June to 30 June 2010 at the Department of Animal Science, Aarhus University in Tjele, Denmark at 9·3°E/56·3°N. Median, minimum and maximum values of global radiation in the geographical area during the study period are shown in Fig. 1. The data were obtained from the meteorological database at Aarhus University.
Global radiation per hour (median, minimum and maximum values) during the time dairy cows spent at pasture each day of the study period in June 2010 at 56°N (data from the meteorological database at Aarhus University).
Treatments
Treatments consisted of five different durations of daily access to pasture: 15, 30, 75, 150 and 300 min. Animals with 300 min daily access to pasture were let out at 10.45 hours followed by the other treatment groups in reverse access time order, so that all animals were at pasture at sun zenith at 13.23 hours. Then, the animals returned to the barn in the opposite order in which they were let out for pasture, with animals in the 300 min treatment group returning to the barn last at 15.45 hours (Fig. 2). From the onset of the study, synthetic D3 was omitted from the total mixed feed ration used in the barn, rendering the animals with endogenous D3 from the sunlight as their only source of D3.
Study design. 
, Animals at pasture.
Blood samples
Blood was collected from the tail vein in Na-heparin-coated Vacuette® tubes (Greiner Bio-One GmbH, Kremsmünster, Austria). Samples were collected on days 0, 1, 3, 5, 7, 9, 12, 14, 16, 19, 21, 23, 26 and 28 of the study between 08.00 and 09.00 hours, except for the day-1 sample, which was taken at 16.00 hours after the animals returned from pasture on the first day of the study. Samples were centrifuged in a Thermo Scientific SL40 centrifuge (Thermo Scientific, Asheville, NC, USA) for 10 min at 1500 g and plasma was transferred to sample tubes (Sarstedt Group, Nümbrecht, Deutschland) and stored at − 18°C until analysis.
Chemical analysis
In the laboratories at the Department of Animal Science, Aarhus University in Tjele, Denmark, the plasma samples were analysed for content of 25(OH)D3, as described by Hymøller & Jensen(Reference Hymøller and Jensen15).
Statistical analysis
ANOVA was performed in the MIXED models procedure of SAS® (SAS Institute, Inc., Cary, NC, USA) using the model: Y ijk = μ+αi+βj+(αβ)ij+C ijk+ɛijk, where Y ijk is the plasma concentration of 25(OH)D3, μ is the overall mean, αi is the fixed effect of i minutes daily access to pasture (15, 30, 75, 150, 300), βj is the fixed effect of sampling day j (0, 1, 3, 5, 7, 9, 12, 14, 16, 19, 21, 23, 26, 28), (αβ)ij is the effect of the interaction between i minutes daily access to pasture and sampling day j, C k is the random effect of animal k and ɛijk is the random residual error. To account for the covariance of repeated measures during consecutive days within animals, the covariance structure was modelled using the repeated statement of the MIXED procedure of SAS®(Reference Littell, Milliken and Stroup16). The best model fit was obtained from the autoregressive first-order covariance structure (AR(Reference Gaylor and Sault1)). To test for fit of a second-degree polynomial, a quadric term of αi2 was added to the model; however, as the effects of the quadric term were non-significant and the model fit statistics worsened, a linear model was maintained. Because the animals were grouped according to their dominant coat colour, the effects of coat colour on the repeated measures of plasma 25(OH)D3 were tested by adding the fixed effect of coat colour and the interaction between coat colour and minutes of daily access to pasture to the model. Random effects were assumed normally distributed with mean value zero and constant variance C ijk approximately N(0, σc2) and ɛijk approximately σ2. Results are presented as means with their standard errors and the differences considered statistically significant if P ≤ 0·05.
Results
The average concentration of 25(OH)D3 in plasma at the beginning of the study across all treatment groups was 44·9 (sem 2·4) nmol/l. At the end of the study at day 28, the 25(OH)D3 concentrations were: 15 min: 36·2 (sem 6·4) nmol/l, 30 min: 26·7 (sem 2·8) nmol/l, 75 min: 43·9 (sem 8·5) nmol/l, 150 min: 67·4 (sem 8·6) nmol/l and 300 min: 95·9 (sem 6·4) nmol/l. There was a significant effect of duration of daily pasture access (P ≤ 0·001) and day of sampling (P ≤ 0·001), as well as a significant interaction between duration of daily pasture access and day of sampling (P ≤ 0·001). The developments in plasma concentrations of 25(OH)D3 within the five different treatments during the 28 d of study are shown in Fig. 3. Animals allowed 15, 30 or 75 min of daily access to pasture were unable to maintain their initial plasma status of 25(OH)D3 and showed a declining linear relationship between plasma 25(OH)D3 concentration and day of sampling in contrast to animals allowed 150 or 300 min of daily sunshine access, which showed an increasing relationship between plasma 25(OH)D3 and day of sampling (Fig. 3). All slopes of plasma concentration curves, except for the 75 min treatment group (slope − 0·01), were significantly different from zero in linear regression (P ≤ 0·05).
Plasma concentrations of 25-hydroxycholecalciferol (25(OH)D3) in dairy cows after daily access to pasture for different time durations in June 2010 at 56°N in Denmark. Values are means, with standard errors represented by vetical bars. 
, Linear trend line; 
, 300 min; 
, 150 min; 
, 75 min; 
, 30 min; 
, 15 min.
From the slopes of the average 25(OH)D3 concentration curves within treatments (Table 1), breakeven for maintaining the initial 25(OH)D3 status was determined to be approximately 90 min of pasture per d during mid-summer in Denmark at 56°N (Fig. 4). The dominant coat colour (black or white) of individual animals had no significant effect on the plasma concentrations of 25(OH)D3 within the different treatments.
Linear equations and R 2 values of 25-hydroxycholecalciferol (25(OH)D3) plasma concentration curves (see Fig. 3) from dairy cows after daily access to pasture for different time durations in June 2010 at 56°N in Denmark
x, Daily pasture time (min); y, plasma concentration of 25(OH)D3 (nmol/l).
Relationship between slope of linear 25-hydroxycholecalciferol (25(OH)D3) concentration curves with standard errors (see Fig. 3) from dairy cows and daily time spent at pasture in June 2010 at 56°N in Denmark (intercept with x-axis 90·9 min). y = 0·006x − 0·545; R 2 0·995.
Discussion
In cattle, the plasma status of 25(OH)D3 was shown by Hymøller & Jensen(Reference Hymøller and Jensen13) to be closely related to the percentage of total skin area that was exposed to sunlight; and Barger-Lux & Heaney(Reference Barger-Lux and Heaney17) showed that the size of the exposed skin area was more closely correlated to the plasma status of 25(OH)D3 than the duration of sunlight exposure in human subjects. In the present study, the plasma 25(OH)D3 status of black and white Holstein dairy cows was strongly linearly dependent on the time the animals spent at pasture on a daily basis during summer at 56°N in Denmark. This contradicts previous assumptions based on in vitro studies where the relationship between sunlight or UV light exposure time and the formation of D3(Reference Olds, McKinley and Moore6) and preD3 from 7DHC was shown to be non-linear and to reach a plateau when a fraction of the 7DHC present in the skin was photoisomerised(Reference Holick10, Reference Holick, MacLaughlin and Doppelt11). The plateau phenomenon encountered when studying D3 synthesis during different durations of UV light exposure in vitro, with either skin samples or pure solutions of 7DHC, is probably caused by a photocatalysed degradation of the intermediate metabolite of D3, preD3, when exposed to excessive amounts of UV light, before it had time to be isomerised into D3, catalysed by heat at body temperature(Reference MacLaughlin, Anderson and Holick3, Reference Webb, Kline and Holick5, Reference Webb, DeCosta and Holick18).
The strong linear relationship between plasma 25(OH)D3 and pasture time at 56°N in the present study could be due to non-production of inactivated metabolites of D3 in the skin. There could be different explanations supporting this: The altitude at 56°N could be so far north that the sunlight intensity was significantly reduced compared to lower latitudes(Reference Webb, Kline and Holick5); the hair covering the bodies of the animals could have protected their skin from direct exposure to sunlight; or the blood flow through the skin of living organisms could have transported the produced D3 away from the skin and into circulation in plasma, before it could be photodegraded by excessive sunlight exposure.
Even though it has been shown in both human subjects(Reference Holick10, Reference Holick, MacLaughlin and Doppelt11) and in fur- or hair-covered species like rats(Reference Hess19) and alpacas(Reference Judson, McGregor and Partington20) that dark-coloured individuals respond slower to sunlight than light-coloured individuals, no effect of dominant coat colour was found in the present study with black- and white-coloured animals.
In conclusion, our study showed that plasma concentrations of 25(OH)D3 were linearly correlated to day of sampling within each treatment group allowed daily access to pasture for different time intervals. Animals allowed 15, 30 or 75 min of daily access to pasture were unable to maintain their initial plasma status of 25(OH)D3 and showed a declining relationship between plasma 25(OH)D3 concentration and day of sampling in contrast to animals allowed 150 or 300 min of daily sunshine access, which showed an increasing relationship between plasma 25(OH)D3 and day of sampling. Breakeven for maintaining the initial 25(OH)D3 status of 45 nmol/l in large, hair-covered herbivores was determined to be 90 min of access to pasture per d during summer at 56°N. The dominant coat colour of individual animals (black or white) had no effect on the plasma concentrations of 25(OH)D3.
Acknowledgements
The present work was financed by the Ministry of Food, Agriculture and Fisheries (Copenhagen, Denmark) and the Danish Cattle Federation, Knowledge Centre for Agriculture (Aarhus, Denmark). Both authors contributed equally to the data analysis and the composition of the final manuscript. The authors have no conflicts of interest with any of the founding institutions.
