Aixio imager m2 microscope

Ovaries were processed as described in Forner-Piquer et al.^{65 (link)}. Briefly, after fixation in formaldehyde-glutaraldehyde, samples were dehydrated in a series of alcohol baths, cleared in Xylene and finally embedded in paraffin. Sections 5 µm thick were cut with a microtome and stained with Mayer’s haematoxylin – eosin. The histological slides were observed under a Zeiss Aixio Imager M2 microscope and microphotographed with a high-resolution camera Zeiss Axiocam 105 color. Female samples were classified as mature or immature depending on the most abundant oocyte developmental stage (see Supplementary Fig. S2). Immature female size and gutted weight were 111 ± 3.4 cm (LJFL) and 14.8 ± 2.1 Kg, respectively, while size and gutted weight of mature female were 154.6 ± 9.6 cm (LJFL) and 49.2 ± 6.3 Kg, respectively.

Upon fixation, small ovary pieces of about 3–4 mm were dehydrated through graded ethanol of increasing concentration, cleared in xylene and embedded in paraffin. Sections of 5-µm thick were cut with a microtome Leica RM2125 RTS (Leica Biosystems, Milan, Italy), stained with Mayer’s haematoxylin-eosin and mounted on a glass slide with SafeMount^® (Bio-Optica, Milan, Italy). Mounted slides were observed under the Zeiss Aixio Imager M2 microscope and photographed with a high-resolution camera (ZEISS Axiocam 105 colour). The reproductive stages of ABFT females were classified according to [43 ]. This classification consists of 4 stages, based on the observed histological features, as follows: active non-spawning (ANS), active spawning (AS), inactive mature (IM) and resting (R). The frequency of late vitellogenic oocytes and the rate of α-atresia was calculated according to [44 (link)]. The mean diameter of oocytes for each specimen was manually calculated as the mean value of the minor and major axis taken randomly from 30 oocytes cross-sectioned through the nucleus. The image processing and measurements were carried out with Fiji [45 (link)].