A comprehensive description of oocyte developmental stages in Pacific halibut, Hippoglossus stenolepis

Abstract Accurate characterization of oocyte development is essential to understanding foundational aspects of reproductive biology and successful management of Pacific halibut (Hippoglossus stenolepis). Here this study provides complete histological descriptions for eight oocyte developmental stages in addition to postovulatory follicles and demonstrates the potential for oocyte size frequency distribution to act as a proxy for ovarian developmental stage and future maturity assessments. Importantly, it provides the first histological evidence that Pacific halibut have a group‐synchronous ovarian developmental pattern with determinate fecundity and support for their batch‐spawning strategy.

developmental stage, histology, maturity, oocyte, Pacific halibut, reproduction Understanding the species-specific components of fish reproductive biology (e.g., age at maturity, fecundity, spawning strategy) is foundational for effective stock management. These indices vary by species (Kennedy et al., 2014;Núñez et al., 2015;TenBrink & Wilderbuer, 2015) and can dramatically alter our perception of stock status (Morgan, 2008). This is especially true for long-lived fish species, such as Pacific halibut (Hippoglossus stenolepis), as lifetime contributions to stock recruitment continue for many seasons after reproductive maturity is reached. For example, changes in reproductive performance, as deduced from female maturity estimates, exert a strong influence on spawning biomass estimates and, consequently, on the stock assessments of the Pacific halibut (Stewart & Hicks, 2020).
Currently, assessments of female Pacific halibut reproductive maturity involve visual macroscopic inspection of ovaries in the field (Stewart & Hicks, 2020). While convenient, this approach has yet to be corroborated by more definitive analysis methods and lacks the specificity required to provide information on many species-specific components of reproductive biology. As accurate characterizations of reproductive development are essential to fisheries management, an evaluation of the reliability of the current macroscopic staging methods using more precise assessment techniques is of utmost importance.
Histological analysis of oocyte developmental stages represents an important initial step to evaluating current maturity assessment methods (West, 1990). Moreover, when providing oocyte size-frequency distributions, histological analyses may also offer alternative methods for characterizing fish reproductive phases and offer additional information on reproductive parameters, such as fecundity type and spawning pattern. Histological examinations have successfully characterized ovarian development in many flatfishes, including California halibut Paralichthys californicus (Lesyna & Barnes, 2016) and Atlantic halibut Hippoglossus hippoglossus (Neilson et al., 1993). Furthermore, framing this characterization using universally descriptive terminology for teleost oogenesis (Grier et al., 2009) will facilitate future comparative examinations (Brown-Peterson et al., 2011).
Pacific halibut represent an important economic and cultural resource in the Gulf of Alaska and the rest of the northeastern Pacific Ocean. In this region, spawning occurs between November and March along the continental slope and in depressions on the continental shelf (St-Pierre, 1984). Spawning is generally thought to occur annually after fish reach reproductive maturity (Stewart & Hicks, 2020;St-Pierre, 1984;Thompson, 1914), but this assumption has received mixed support in the literature (Bell, 1981;Novikov, 1964;Seitz et al., 2005;Vernidub, 1936). Early investigations of Pacific halibut oocyte size (Kolloen, 1934;Thompson, 1914Thompson, , 1916 documented a developing cohort of oocytes within the ovary immediately after spawning, thus supporting the premise of annual spawning. Nonetheless, a detailed histological characterization of ovarian development in female Pacific halibut to be used in histology-based reproductive phase maturity classification has not been conducted to date. Here, this study presents comprehensive histological descriptions of oocyte developmental stages and documents postovulatory follicles (POFs) in Pacific halibut. In addition, it details the range of oocyte diameters among developmental stages and explores differences in the size-frequency distributions of oocytes in ovarian tissue at different developmental stages to investigate the relation between oocyte size and oocyte developmental stages. This work provides the most precise assessment of developmental stage for the species to date, documents the spawning strategy and offers a foundation for more specific assessments of Pacific halibut reproductive biology in the future.
T A B L E 1 Description of oocyte developmental stages of Pacific halibut, Hippoglossus stenolepis, associated growth phases (modified from Brown-Peterson et al., 2011 andGrier et al., 2009) (Clark et al., 1999;Loher & Seitz, 2008). Once fish were on-board the fishing vessel, approximately 1 cm 3 tissue was excised from the central area of the ovary and fixed in 10 ml of 10% buffered formalin from each fish.
Ovarian tissue samples were sent to an independent laboratory to be processed for histology where two series of 4 μm thick Paraffin sections, separated by approximately 500 μm, were mounted on two slides and stained with haematoxylin and eosin.
Oocyte developmental stages were categorized following the univer-   Explorations of oocyte size distributions in fish at different female developmental stages showed that Pacific halibut follow a pattern typical of fish species with group-synchronous ovarian development with determinate fecundity (Ganias, 2013;Lubzens et al., 2010). Fish in early developmental stages displayed unimodal distributions of oocyte diameters, which became increasingly right-skewed from PGpn to CA (Figure 1b). This modegenerally up to 500 μm in diametershares similar morphological characteristics to corresponding PG oocytes described in other fish species (Grier et al., 2009;Selman et al., 1993) and was present in all female developmental stages These observations suggest that, despite the lack of information of the temporal progression of these events and the relatively small sample size in the PO developmental stage, GVM oocytes may be recruited for final maturation, hydration and subsequent ovulation in batches, supporting the notion that Pacific halibut, like its Atlantic congener (Haug & Gulliksen, 1988), is a batch spawner, as suggested by a previous report of the spawning behaviour of one tagged Pacific halibut female (Seitz et al., 2005).
The histological description of oocyte developmental stages produced by this work provides an important and necessary

CONFLICT OF INTEREST
All authors declare that they have no conflict of interest.

AUTHOR CONTRIBUTIONS
T.F. contributed to the study design, collected the samples, analysed and interpreted the results, and was the primary manuscript author.
N.W. assisted with analysis, interpretation and presentation of the results and assisted T.F. in writing the manuscript. B.P.H. assisted with the presentation of the results and edited the manuscript.
J.V.P. conceived the study, supervised data collection, analysis and interpretation, and edited the manuscript. All authors read and approved the final manuscript.

COMPLIANCE WITH ETHICAL STANDARDS
Pacific halibut collections were conducted under a letter of acknowledgement from National Oceanographic and Atmospheric Administration (NOAA) Fisheries and biological samples were obtained following guidelines for the euthanasia of finfish from the American Veterinary Medical Association (AVMA, 2020).