Each jenny was teased daily from Day 15 after the previous
ovulation with a fertile and mature jackass for at least
3 to 4 minutes [20] to verify the presence of behavioral
signs of estrus. The day of the behavioral willingness to
mating (heat) was considered the Day 1 of the estrus (Ed1).
The day when the jenny refused the jackass was considered
the Day 1 of the diestrus (Dd1). Thus, the estrus time was
defined as the time, in days, between Ed1 to Dd1-1; the
diestrus time was defined as the days between Dd1 to the
subsequent Ed1-1. At Ed1, the behavioral characteristics of
the heat were recorded and the jennies in estrus
were submitted to gynecological and ultrasonographic
examination. The ultrasound examinations of the reproductive
tract were performed transrectally using an ultrasound
Concept 2000 (Dynamic Imaging Limited,
Livingston, Scotland, UK) equipped with a 7.5-MHz linear
transducer. Uterus was monitored for the diameter of both
horns at Ed1. Jennies with anechoic content of the uterus
were excluded from the trial. The follicular dynamics was
followed by ultrasonographic examinations repeated every
12 hours between Ed1 and the visualization of a follicle of
30-mm diameter, then every 6 hours until ovulation. The
day at which the dominant follicle disappeared on the
ovarywas defined as the day of ovulation, and the diameter
of the dominant follicle at the last examination before
ovulation was recorded. Estrous cycle was considered as
the time elapsing between two consecutive ovulations,
detected using ultrasonography.
Blood samples were daily collected from Ed1 until Ed1
of the following estrus in each jenny by venipuncture in
heparinized tubes, centrifuged at 1000 g for 20 minutes
and plasma stored at 20 C until E2 and P4 analysis. This
complete monitoring protocol was used for each animal
and repeated during the four year-round seasons.