Developing and Testing an Approach for Assessing Histological Sections of the Pyloric Stomach in Mature and Kelt Steelhead

Trout

Veatasha H. Dorsey CRISSP REU 2011 Fisheries

Prepared for the CRISSP REU Symposium August 4, 2011

Outline
Steelhead Background 1. Morphology 2. Life Cycle 3. Life History Theory Problem 1. Spawning 2. Feeding Objectives 1. General/Specific Methodology 1. Fish Collection 2. Histology Preparation 3. Measuring Results Discussion Furthering Research Acknowledgements

MATURE MALE

MATURE FEMALE

MALE KELT

FEMALE KELT

Deposit eggs in grave nests and die

Pacific Salmon Life Cycle

Deposit eggs in grave nests and die

Pacific Salmon Life Cycle

Life History Theory of Salmonid Species

Semelparity (Pacific Salmon) Iteroparity (Steelhead)

Emphasis

Juvenile survival

Adult survival

Investment in Spawning

High

Lower

Problem
Spawning Iteroparity Rates < 2% in Snake/Columbia River Historical ratesunknown Physiological Changes?

Problem (con).
Steelhead and Feeding
Mature spawners Stop feeding during final sexual maturation If food is found in stomach; eating out of habit Kelts Feed, rebuilding strength to migrate toward the ocean

Credit: Zach Penney

Project Objectives
General Understand the changes in tissue microstructure of the pyloric stomach in two reproductive phases of steelhead (mature and kelt) Specific

1. Characterize histological profiles by identifying pertinent tissue attributes 2. Develop a method to evaluate tissue architecture in preand post reproductive steelhead 3. Test the method and display preliminary results

Why Histology?
Insight on tissue level organization Disease detection and prevention Static representation of health

Hypothesis
The length of the epithelial folds and the area of the rugae is related to the steelheads ability to feed, digest and assimilate.

Methodology (Fish Collection)

Dworshak (Sexually Mature)
Not feeding
Controlled environment

Steelhead representing sexually mature phase of the adult spawning migration

Lower Granite Dam (Kelts)

Rebuilding energy stores Steelhead in their post-spawning migration back to the Pacific Ocean

Dworshak

Methodology (Histology Prep)

1. Tissue fixed in formalin 2. Sectioned for shipment (Colorado Histo-Prep in Fort Collins, CO.) 3. Hematoxylin and eosin was used for staining of paraffin embedded tissues

Methodology (Measuring)
Measurements (Microscope Software-Calibrated m) Area of Rugae Three measurements per fish Total data points = 18 Length 3 ruga per fish 8 epithelial folds per ruga Length and width measurements Total data points = 144

Measuring Area of Ruga

1. Measure the base of the ruga
(dotted line).

2. Find the midpoint of the line.

3. Calculate length.

4. Calculate the width of the line

horizontally.

Establishing Quadrants
4. Label the quadrants counterclockwise
5. Locate two suitable folds 6. Locate the longest and shortest (max/min) in each quadrant to measure.

Measuring Epithelial Folds

7. Measure the cross sectional area (l x w=a)

The optimum magnification: 100-300.

Length of Epithelial Folds: Mature vs. Kelt

40

Length of Epithelial Folds (microns)

35 30 25 20 15 10 5 0 Mature Kelt

Phase of Reproduction

Length of Epithelial Folds: Mature vs. Kelt (By Sample)

40

Length of Epithelial Folds (microns)

35 30 25 20 15 10 5 0 Mature A Mature B Mature C Kelt A Kelt B Kelt C

Phase of Reproduction

Area of Rugae: Mature vs. Kelt

22000 20000 18000

Area of Rugae (microns)

16000 14000 12000 10000 8000 6000 4000 2000 MATURE A KELT A

Phase of Reproduction

Discussion
Average epithelial fold length in kelts ~ 50% longer than in mature spawners Average rugae area in kelts ~ 55% larger than mature spawners Variation within kelts Migration Distance Spawn Timing Homogeneity within mature spawn Holding tank

Furthering Research
Quantitative histological assessments of entire GI tract Basically, Get to work Zach!!!!

Ethical Implications
Endangered Species and Reconditioning Lethal Sampling Understanding of Iteroparity Tribal Significance

Acknowledgements
Christine Moffitt, Professor-Fisheries Zachary Penney, PhD Candidate-Fisheries

Ann Norton
Moffitt Lab CRISSP REU Program National Science Foundation (NSF)

Questions? Suggestions?
Be nice plueeaazzeee!! K thanks!!!