Sixth Quarterly Report (Jan 1-Mar. 31, 1998) on:
Serum and egg vitellogenin measurement in the Atlantic cod Gadus morhua
and its relationship to ovarian development.
Proposal response to CMER NOAA/NMFS RESEARCH TOPICS - 1996:
4. Biochemical indices of maturity and egg quality in Atlantic cod
(contact: Frank Almeida, NMF, Woods Hole Laboratory)
by Joseph G. Kunkel
Biology Department, U. Massachusetts at Amherst

Our project on the Atlantic cod, Gadus morhua, received funding in the Fall of 1996 and I am reporting the progress made toward its goals in its sixth quarter.

Progress Outline:

  1. Egg Yolk Protein Purification: A purified Lipovitellin has been purified from ripe and running ovary eggs.
  2. Antiserum Evaluation Has Continued: Analysis of suitability of the antiserum for different analytical techniques has continued.
  3. Summer Undergraduate Trainee has been  recruited to work .
  4. Lab personnel participates in NOAA Bottom Survey Cruise.
  5. Senior personell to participate in  NOAA Bottom Survey Cruise.
 
  1. Egg Yolk Protein Purification: We have used ripe-and-running ovary eggs to purify the major egg Lipovitellin (Lv) of the cod fish using a combination of fractional heat denaturation, Ammonium Sulfate salting-out and gel-permeation chromatography.  This utilizes a technique, of fractional heat denaturation, which was discovered in a previous CMER/NOAA project on Winter flounder (Hartling et al., 1997).  Purifying the Lv from the ovary, instead of ovulated eggs, however resulted in bringing over some ovarian proteins into the provisionally purified Lv.  To avoid the large number of ovarian proteins included with ovary extracts we changed our source of eggs to the ovary of ripe-and-running females which were collected on the NMF Spring Bottom Survey.
  2. Antiserum Evaluation Has Continued: The first antiserum was harvested and evaluated after its 2nd boost with heat denatured and gel-filtration purified Lv (dn,gf-Lv). Since we see multiple precipitin reaction products with partially purified Lv (i.e. dn,gf-Lv) when tested by Ouchterlony or Rocket Immunoelectrophoresis and since we also have some minor responsiveness from male serum, this antiserum as it stands would not be able to be used in our projected ELISA test.  We have started the immunization of a second rabbit using the more highly purified Lv from a ripe-and-running cod female.
  3. Undergraduate Trainee to work in Lab during the Summer: Ray Moniz a sophomore undergraduate has signed on to do a summer internship for which he will obtain Biology 299 Special Problems credit.
  4. Lab personell participated in two NOAA Bottom Survey Cruises: Lab technician John Bohannon participated in two cruises, starting Feb. 17 with a two week gap between each trip. He collected cod serum, mucous and traditional size statistics (total weight, GSI) for analysis in the laboratory.
  5. Senior personell to participate in  NOAA Bottom Survey Cruise.  PI, Joe Kunkel, will participate in the IV leg of the NOAA Spring Bottom Surveys, the third trip taken by lab personell starting April 13.
While we are still behind schedule due to complications in the Lv purification process, we now have an antiserum which reacts with our tentatively purified Lv protein (along with some other yolk and serum proteins) and have a second antiserum being made in a second rabbit which we hope to be more specific. This will ultimately provide us with the specific antiserum which we need for ELISA. We are also continuing the phase in which we ask if the Lv antigen can be detected in the body surface mucous of reproductive female cod.  This is being done by collecting fresh mucous samples as well as blood samples from male and female cod collected during the Spring Bottom Survey.

Respectfully submitted,

Joseph G. Kunkel

jgk/hs