Oocyte density was estimated by microscopic counts (3 × 50 μL in
10 mL to 2 L, depending on individual oocyte concentration). Total
number of oocytes collected from each oyster was calculated (gamete
density × seawater volume). Furthermore, morphological characteristics
of oocytes (Feret diameter, perimeter, area and circularity ranging from
0 to 1, where a value of 1 indicates a perfect circle) were assessed
30 min after gonad stripping, using Image J (n= 30 oocytes).
Triplicate batches of 25,000 oocytes each were then fertilized
according to the following crosses (female × male): diploid × diploid
(n = 5), diploid × triploid (4), triploid × diploid (2), and triploid ×
triploid (3). Oocytes were fertilized using a non-limiting sperm
to egg ratio (500). Oocyte lots were incubated in 2 L beakers and
the D-larval yield was estimated 48 h post-fertilization: (number of
D-larvae / 25,000) ∗ 100.