TITLE: Sablefish Reproductive Life History and Genetics
DATES: August 1, 2012- July 31, 2014
There are three primary components of this project described below.

1) Sablefish Reproductive Life History Study

The portion of this study that is included in the grant is the analysis of the samples
that are being collected in the field. The samples will include blood, ovaries, testes,
pituitaries and livers. The analysis of these samples would include what is detailed
below. The funds in the grant for this section cover supplies and partial salary for the
technician(s) in Penny Swanson’s lab to do analysis of steroids in the blood, gonadal
histology and fecundity.

• Blood Hormone Analysis – Blood will be centrifuged on-shore to obtain plasma
and stored at -20C until analysis. Plasma samples will first be extracted with
dichloromethane (males) or ether (females) and the dried extracts will be assayed
for estradiol (females) by radioimmunoassay and 11-ketotestosterone (males)
using an enzyme immunoassay (citations as in Goetz et al. 2011).
• Gonadal Histology – Fixed gonad samples will be further processed for histology
to produce 2 slides/gonad with 5 sections per slide, stained with hematoxylin/
eosin as described previously (Campbell et al. 2003, 2006). Gonad sections will
be viewed under a compound microscope and stages of oogenesis and
spermatogenesis described. As far as we know, there has never been a published
complete description of gametogenesis in male and female sablefish and, therefore,
we will characterize stages in this species relative to those described for other fish
(Nagahama 1983; Campbell et al. 2003, 2006).
• Fecundity – Fecundity will be analyzed in females sampled at the peak of
oogenesis when GSIs reach a maximum as described in Mason et al. (1983). In
this case, one of the ovaries will be used for fecundity while the other ovary will
provide samples for normal histology.
• Data Analysis – Lengths (L) and weights (W) of fish will be analyzed by ANOVA
to test for significant difference in L or W across sex and sampling sites. GSI,
HSI, E2 and 11-KT will be analyzed by ANOVA followed by Tukey’s to test for
differences across time and between sites. Fish will be categorized as maturing
(females- with vitellogenic oocytes; males with meiotic germ cells) or immature
(females with only primary oocytes; males with premeiotic germ cells) based on
gonadal histology. The significance level will be adjusted by the Bonferroni
correction to reduce type I error because multiple Student t-tests will be used.
2) Technologies to preserve and extend the milt of sablefish for aquaculture

This portion of the grant covers the analysis described in the paragraph below for
cryopreservation of sablefish sperm. Funding in the grant covers salary/tuition for Doug
Immerman, the graduate student working on this project. It also covers the supplies
necessary to do the research.

Approach: Successful cryopreservation requires sperm dilution in a physiological
medium (e.g., Mounib’s) containing a cryoprotectant (e.g., propylene glycol, DMSO)
followed by freezing in these solutions under controlled rates. Based on the results of
studies on other marine species, we will test the effects of two diluents, two
cryopreservants and three freezing rates on sablefish sperm. To conduct these
experiments and to routinely cryopreserve sperm once a protocol has been established,
we will need a controlled freezing system and a liquid nitrogen storage vessel, both of
which are being requested in this proposal. The success of the various cryopreservation
approaches will be assessed initially using a computer aided sperm analysis (CASA)
system and software that was designed to quantify physical parameters of sperm motion,
and adapted for the unique features of movement of fish spermatozoa (i.e., short period of
motility after activation and high frequency of flagellar beat; Rurangwa et al., 2004;
Aquaculture 234:1-28). CASA systems utilize microscopy in conjunction with
videography and analysis of digitized data to quantify sperm movements. They have
been used widely to evaluate motility of spermatozoa in humans and livestock in both
fresh and cryopreserved semen. We have previously used a CASA system developed for
fish (Wilson-Leedy and Ingermann, 2006; Theriogeniology 67:661-672) to analyze
effects of pH on fish sperm motility so we have all of the equipment and software (Lab
View and NIH Image) on hand. In the proposed work, cryopreservation protocols that
produce the best results with these quantitative cytology-based assays will be followed up
by actual fertilization assays with eggs.
The development of these protocols and the acquisition of cryopreservation equipment
will give us the ability to bank sperm so that it can be utilized by industry partners in the
future, especially when mature males are limiting. This ability has not been possible in
the past and has limited larval production and research.


3) Genetic Analysis of Wild Sablefish in the Eastern Pacific Ocean for Broodstock

This project involves the collection of finclips from the entire range of sablefish from
Mexico to Japan and the collections are being done as part of the normal sampling of fish
by the FRAM, RACE and MESA surveys of NOAA. So that part is not on the grant. As
indicated below, there will be construction of RAD libraries and sequencing of them. A
year of graduate student (Andy Jasonowicz) salary/tuition is included on the grant as is
supplies for library construction and sequencing. As with the cryo grant there is some
equipment being purchased separately and is not on the grant budget.

Approach: Samples will be obtained from at least 7 regions: California, Oregon,
Washington, Canada, Bay of Alaska, Bering Sea, and the Aleutian Islands. From each
region, we will sequence restriction-site associated DNA (RAD) tags from 50-100
individuals for SNP genotyping and further development of SNP panels that will be
transferred to the aquaculture industry. Briefly, fin tissue samples will be extracted to
obtain genomic DNA, which is then used for RAD-tag library construction and Illumina
sequencing as described by Miller et al. (2007, Genome Res. 17: 240-248). At the time of
collection, size and sex will be recorded, and otoliths taken for aging. RAD-tag
sequencing generates millions of 100 base sequence reads for each individual, which will
be processed to identify SNP genotypes that are used for genetic analyses. SNPs will be
analyzed to evaluate the genetic relationship of sablefish across their range and to
identify possible SNPs under selection (showing unusually high or low patterns of
diversity) in these populations. These analyses will reveal the genetic relationships and
diversity within and among sampling regions, the potential number of unique genetic
groups, and the SNPs that might be most useful in genetic selection programs in sablefish

Project Summary: Sablefish Reproductive Life History and Genetics
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