Materials and methods
All used chemicals and reagents were at least of analytical grade
and purchased from Sigma-Aldrich (Bornem, Belgium), unless
otherwise specified.
2.1. Production of alfalfa and flax sprouts
Alfalfa (Medicago sativa L.) and flax (Linum usitatissimum L.) seeds
were germinated on a substrate consisting of moistened tissue
paper lying on a layer of silica sand sterilised at 105 °C in aluminium
trays (22 cm × 30 cm for alfalfa and 30 cm × 36 cm for
flax). In each tray, the sand (600 g for the alfalfa and 1 kg for
the flax) was distributed to create a uniform layer on the bottom
of the tray and moistened with demineralised water.The trays
were placed in a temperature-controlled room at 20 °C in the
dark and kept in these conditions for three days. Water was
added periodically to compensate for sand water loss due to
evaporation. In contrast to the usual sprouting procedure used
for alfalfa in which sand water content is restored once a day,
flaxseed requires several separate additions of water, as the
seeds tend to produce a glue-like mucilaginous exudate in the
presence of high water content that would hamper seedling
development. For each species, the sprouts obtained on the third
day from different trays were combined to prevent a possible
tray effect and stored at 4 °C in plastic bags until use (i.e., within
three days).
2.2. Animals and diets
The experimental protocol was devised according to the Italian
directives (Gazzetta Ufficiale, 1992) on animal welfare and the
research was carried out at the experimental farm of the Department
of Agricultural, Food and Environmental Science of the
University of Perugia (Italy) from November 2013 to February 2014.
Thirty White Leghorn hens, 30 wk. of age at the start of
experiment, were randomly assigned to one of the following
conditions:
• Standard diet (C);
• Standard diet + 40 g/d of alfalfa sprouts (A);
• Standard diet + 40 g/d of flax sprouts (F).
The hens of each group were kept in indoor pens under standard
housing conditions with an artificial photoperiod of 16 h
per day of light was applied.The building was under a controlled
ventilation regime (10 m3/hen/h): the temperature ranged from
23 to 27 °C, and the relative humidity ranged from 50 to 80%.
Standard feed and water were provided with manual bell
feeders and automatic drinkers. Feed and water were provided
ad libitum (Table 1) to all the groups and the daily residues
were weighed for evaluation of voluntary feed intake. Fresh
sprouts were placed daily near the feeders.
The overall experimental period lasted 66 days. Egg deposition
was recorded daily; in particular the deposition rate was
evaluated the wk. before the experiment started (baseline day
0) and at the end of sprout supplementation.
2.3. Eggs and blood sampling
For each dietary treatment, 10 pools of 10 egg yolks/per group
were collected at day 0 and day 66 stored at 5 °C until the analyses
(maximum 2 days after) that were performed in the
laboratory of the department.The blood was sampled at 0 and
66 day from the brachial vein of 10 hens per group, in
heparinised vacutainers and centrifuged at 1500 × g for 15 min
at +4 °C, to measure the plasma cholesterol concentration