*2.3. Dietary Intervention*

The intervention study was designed to assess the efficacy of daily Atlantic farmed smoked salmon intake intervention on serum 25(OH)D level. All participants were enrolled in and finished the study at the same time. The fish intake intervention lasted from October 24, 2018 to December 18, 2018. The period of intervention was planned because from other studies it is known that in countries such as Poland, skin synthesis of vitamin D, which is the major source of the vitamin, is only possible from April to October [20], so it was decided to conduct the study directly after this period.

Atlantic farmed smoked salmon was chosen in the intervention as a source of vitamin D that is possible to be applied in practice, for numerous reasons. Salmon contains significant amounts of vitamin D (17.1 μg/100 g for smoked salmon, based on United States Department of Agriculture—USDA food composition tables [31]) and is widely available on the Polish market, independently from the region of the country [32]. Moreover, Atlantic farmed salmon is a species that contains very little mercury (≤ 0.1 μg/g) and dioxins (0.5–4 pg Toxic Equivalent (TEQ)/g), so it is classified to the first and second group respectively when it comes to the smallest content of those contaminants [33]. The very high content of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) (> 15 mg/g) also contributes to the Food and Agriculture Organization (FAO) of the United Nations and the World Health Organization (WHO) [33] recommending salmon as a fish species having much more benefits than risks.

The Atlantic farmed smoked salmon that was used for the study was obtained from one producer, one of the leading salmon sellers in Poland (Suempol Polska Ltd.), and for all the participants, the provided salmon was obtained from the same batch, in identical sliced tray modified atmosphere packaging.

Studies show a large variation in the content of vitamin D in salmon [34], therefore its content was measured in the smoked Atlantic salmon used in the study by a leading vitamin laboratory in Europe – Eurofins Vitamin Testing Denmark (EN 12821: 2009-08, LC-DAD, accredited methodology no. 581). The measured content of vitamin D was 21.3 ± 5.55 μg/100 g, which is higher than the value typical for smoked salmon, like the one shown in the Polish Food Composition Tables [22].

The daily intake was planned as 50 g, so it was attributed to around 10.65 μg of vitamin D, which covers the recommended 10 μg [11,25,26]. Atlantic farmed smoked salmon was packed in 50 g portions to facilitate consuming 1 portion (50 g) each day. Participants were given 7 packages (7 × 50 g) of smoked salmon once a week, for 8 weeks of the study duration. To increase adherence to intervention, participating women were asked to report their intake daily. Participants were asked to incorporate the given salmon in their daily food intake substituting it for other products such as meat, cheese, or eggs. They were not recommended to exclude other fish products from their diet, so if they had previously consumed them, they were recommended not to change this habit.

Participants had their serum 25(OH)D level measured three times: before the dietary intervention (at baseline (t0)), after 4 weeks of dietary intervention (t4), and after 8 weeks of intervention (after the study (t8)). The study course is shown in Figure 1.

**Figure 1.** Study design and number of participants.
