*2.2. Experimental Protocols*

The entire offspring from the four groups were weighted and head-to-tail length measured at days 1, 14, and 21 of the lactation periods with an analytical balance (Conecta, Barcelona, Spain) and a digital caliper (Conecta, Barcelona, Spain), respectively. The body weight gain from day 1 to 14 was calculated as Formula (1). A similar formula was used to assess body weight gain during the last week and length gain in both lactation periods. Weigh gain was expressed as g/day and length gain as cm/day.

$$Weight\,\,gain = \frac{\left(\%right\,\,light\_{day\,\,14} - \%right\,\,light\_{day\,\,1}\right)}{number\,\,of\,\,days} \tag{1}$$

Breast milk was collected from MUN and control dams on days 12 and 14 of lactation (see protocol below). On day 21 (weaning), 1–2 males and 1–2 females from each litter were studied and the remaining animals were used for other studies according to the 3Rs rule for experimental animals. Firstly, cardiac structure and function were evaluated by echocardiography. Thereafter, the rats were placed in a CO2 chamber and killed by exsanguination. Then, the thoracic aorta and mesenteric bed were removed and stored in a cold saline solution. The aorta was cleaned, and the aortic arch and immediate region were fixed in 4% paraformaldehyde solution (PFA). Perivascular adipocytes from the mesenteric bed were also dissected and fixed in PFA. Both tissues were stored for later confocal microscopy analysis. Figure 1 shows a summary of experimental protocols.
