Fecundity & Histology

  • Atlantic Menhaden Ovaries
    Atlantic Menhaden Ovaries  Laboratory processing of each fish includes: fork length, whole & eviscerated weight, sex, macroscopic maturity classification, paired ovary weight, scale and otolith removal.  
  • Embedding
    Embedding  Ovarian tissue sections in paraffin wax  
  • Embedding ovary sections
    Embedding ovary sections  on the HistoStarâ„¢ Embedding Workstation. After the sections are mounted in molten paraffin they are placed on a cooling plate. Once they have fully cooled the samples are popped out of the plastic molds.  
  • Sections form a ribbon
    Sections form a ribbon  Ideally 5-6 good sections are stuck together. The sections are removed using forceps and a wet paint brush and carefully laid on top of 42° C water bath. Choose the best 1-2 sections (depending on the size of the sample) and position them on a microscope slide. Place in a slide drying rack.  
  • Drying
    Drying  Once the tissues are mounted on the slides they are placed on a heated slide drying rack overnight.  
  • Staining
    Staining  Once the slides have been dried overnight they are put in racks and placed in the automatic stainer, and ran through a routine staining procedure which takes ~1 hour. Once complete the stained slides are coverslipped and put in the drying oven for ~48 hours.  
  • Stained and cover-slipped slides
    Stained and cover-slipped slides    
  • Ova counts
    Ova counts  Conduct ova counts based on results of the histological slide interpretation.  
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Why is the Multispecies Research Group studying the fecundity of Atlantic Menhaden?

Understanding the fecundity of fish species is a vital part of fish stock management. Estimates of population-level fecundity offer improved measures of reproductive potential relative to spawning stock biomass.

Objectives
  • Collect female Atlantic Menhaden over all seasons & from throughout the Mid-Atlantic Bight
  • Process ovaries using standard histological techniques. Information on maturity stages, spawning mode, seasonality & frequency.
  • Count ova based on histological findings
  • Model fecundity as a function of size & age.
Sample Area

Sample Area - The Chesapeake Bay and Mid-Atlantic Bight

Laboratory Processing & Supplies:

Laboratory processing of each fish includes: fork length, whole & eviscerated weight, sex, macroscopic maturity classification, paired ovary weight, scale and otolith removal.

 Laboratory Processing Supplies:Female Atlantic Menhaden ready for ovary removal and workup. Image credit: Joel Hovanesian

  • Analytical Balance - minimum of 0.001 g to a maximum of 410 g
  • Fish Measuring Board
  • Hubco labels
  • Soil sample bags – ovary preservation
  • Screw top vials – otolith storage
  • Sample envelopes – Fish Scales
  • Scalpel + blades
  • Tissue Processing Embedding Cassettes
  • Tissue-Tek Marking Pencils
  • Plastic 1 gallon jugs for sample storage
  • Chemicals used for preservation:
    • Normalin
    • 10% Neutral Buffered Formalin
    • 70% Ethanol
Histology Laboratory Supplies:
  • Lab tissue processor - Thermo Excelsior ES
    • Ethanol
    • Xylene
    • Paraffin Wax
  • HistoStar™ Embedding Workstation
    • Paraffin Wax
  • Histology embedding molds
  • Razor scraper
  • Tanner Scientific TN6000 Manual Precision Microtome 
  • High Profile disposable Microtome Blades
  • Sharps container
  • Lighted tissue flotation water bath
  • Gelatin
  • Styrofoam Tray
  • Crushed Ice
  • Frosted microscope slides
  • Forceps
  • Paint brushes
  • Slide drying rack
  • Slide warmer
  • Kimtech wipes
  • Microscope slide boxes
Staining Supplies:

Stained Atlantic menhaden Ovary

  • Lab Coat
  • Nitrile Gloves
  • Shandon Varistain Gemini automatic stainer
  • Chemicals for staining:
    • Xylene
    • 100% ethanol
    • 95% ethanol
    • Hematoxylin
    • Acid Alcohol
    • NaHCO2
    • Eosin Y
  • Laboratory Fume Hood
  • Glass coverslips
  • Permount Mounting Medium
  • Kimtech wipes
  • Cotton Swabs
  • Aluminum slide trays
  • Drying Oven
Histological Interpretation Supplies:
Compound Microscope and software
  • Analytical Balance - minimum of 0.001 g to a max. of 410 g
  • Spoonula
  • Custom made grid for counting / measuring eggs
  • Glycerin to separate eggs
  • Compound microscope
  • NIS Elements software