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Fishery Bulletin 92(4), 1994 



Table 1 



Stages of ovarian development in cobia, Raehycentron canadum. 



Stage 



Characteristics 



1 Previtellogenesis 



2 Vitellogenesis 



3 Final maturation 



4 Postovulation 



1' Sequential previtellogenesis 

 2' Sequential vitellogenesis 



Germinal vesicle develops; evaginations appear in nuclear envelope; cortical alveoli form in 



ooplasm. 



Lipid vacuoles form; uneven dispersal of protein and lipid yolk. 



Clearing of lipid around periphery of oocytes; enlarged size; chromosomes condense. 



Oocytes become distorted and compacted; presence of postovulatory follicles; frothy residual 



lipid vacuoles. 



Sequential development of previtellogenic oocytes after a spawning episode; presence of 



postovulatory follicles and resorbing oocytes in addition to characteristics of stage 1. 



Sequential development of vitellogenic oocytes after a spawning episode; presence of 



postovulatory follicles and resorbing oocytes in addition to characteristics of stage 2. 



Materials and methods 



Sample collection 



Cobia examined in this study were collected from 

 coastal waters of Florida, Alabama, Mississippi, Loui- 

 siana, and Texas, mostly through fishing tourna- 

 ments held along the northern Gulf Coast during 

 April through September of 1991 and 1992, although 

 a few fish were caught by project personnel during 

 that same time period. Fish were stored on ice from 

 the time of capture. Immediately after each fish was 

 weighed and measured (total and fork lengths), the 

 ovaries were removed, placed in plastic resealable 

 bags, and stored on ice for 4 to 20 hours until gonad 

 total weights could be recorded and aliquots of the 

 tissue taken. Separate aliquots of each ovary sample 

 were placed in 10% phosphate-buffered formalin and 

 stored at room temperature until the tissues were 

 processed for microscopic examination (see below). 

 Additional aliquots of each ovary were stored at 

 -80°C until the biochemical analyses (see below) were 

 performed. 



Histology 



Ovaries were processed according to techniques modi- 

 fied from Humason ( 1979). Tissues were dehydrated 

 in ethyl alcohol and embedded in paraffin by means 

 of a Histomatic automatic tissue processor. The em- 

 bedded tissues were sectioned at 4 or 5 um. Sections 

 were stained with Delafield's hematoxylin and eosin 

 (95% ethyl alcohol) (Humason, 1979). 



Aspects of fish ovarian development as described 

 by Blaxter (1969), Wallace and Selman (1981), 

 Overstreet (1983, a and b), Guraya (1986), and 

 Mommsen and Walsh ( 1988) were used to determine 

 the stages of development in cobia ovaries. Four cat- 

 egories of development were observed in this study: 

 stage 1, previtellogenesis; stage 2, vitellogenesis; 



stage 3, final maturation, and stage 4, postovulation 

 (Table 1). Some ovaries appeared to have entered 

 another, sequential round of oocyte maturation. Be- 

 cause we were interested in biochemical differences 

 that might exist between successive clutches of oo- 

 cytes, the following additional categories were stud- 

 ied: stage 1', a sequential previtellogenesis, and stage 

 2', second (or sequential) vitellogenesis. 



Biochemistry 



The frozen tissues were thawed on ice and homog- 

 enized with either a Virtis tissue homogenizer or a 

 hand-held ground glass mortar and pestle. Protein 

 was measured according to Hartree (1972) with bo- 

 vine serum albumin as the standard. Carbohydrate 

 was measured according to Dubois et al. ( 1956 ) with 

 glucose as the standard. Dry weight was determined 

 after drying the samples overnight at 80°C to con- 

 stant weight. The same samples were then com- 

 busted overnight at 500°C to determine ash content. 

 Lipid extraction was performed according to Sasaki 

 and Capuzzo ( 1984) which is a modification of Folch 

 et al. (1957) and Bligh and Dyer (1959); total lipid 

 was measured gravimetrically with a Cahn C-31 

 microbalance. 



Calculations and statistics 



A gonosomatic index (GSI) was calculated as 



GSI = ovary weigh t/( total fish weight 

 - ovary weight) x 100 



(DeVlamingetal., 1982). 



Nonparametric Kruskal-Wallis analysis of variance 

 by ranks (Zar, 1984) was performed with the SPSS- 

 X2.1 statistical software package in order to test the 

 null hypothesis that there were no significant differ- 

 ences among the means being compared. In cases 



