A new method of oocyte separation 

 and preservation for fish 

 reproduction studies* 



Susan K. Lowerre-Barbieri 

 Luiz R. Barbieri 



The College of William & Mary, School of Marine Science 

 Virginia Institute of Marine Science 

 Gloucester Point, Virginia 23062 



Studies on the reproduction of 

 multiple-spawning fishes often in- 

 volve estimates of batch fecundity 

 and oocyte size (Hunter & Goldberg 

 1980, DeMartini & Fountain 1981, 

 Hunter et al. 1985, Brown-Peterson 

 et al. 1988). Because data collection 

 and laboratory analysis are rarely 

 concurrent, oocytes which are pre- 

 served and hardened are generally 

 used for these analyses. It is criti- 

 cal, therefore, to have a method of 

 oocyte preservation which does not 

 damage or destroy oocytes and has 

 a determinate effect on oocyte size. 

 The preferred oocyte preservative 

 (Bagenal & Braum 1978, Snyder 

 1983, Cailliet et al. 1986) has been 

 a modified Gilson's solution: 

 100 mL 60% ethanol or methanol, 

 880 mL water, 15 mL 80% nitric 

 acid, 18 mL glacial acetic acid, and 

 20g mercuric chloride (Snyder 

 1983). The benefit of using Gilson's 

 is its ability to harden oocytes while 

 chemically separating them from 

 ovarian tissue. However, a number 

 of problems are associated with 

 this procedure, including degenera- 

 tion of hydrated oocytes ( Hunter et 

 al. 1985, Schaefer 1987, Brown- 

 Peterson et al. 1988); substantial 

 and continuous oocyte shrinkage, 

 reported to range from 15% to 24% 

 (DeMartini & Fountain 1981, 

 Schaefer 1987, Witthames & Greer 

 Walker 1987); a relatively long fixa- 

 tion period of several days to a few 

 weeks (Cailliet et al. 1986); and the 



extreme toxicity of mercuric chlo- 

 ride (West 1990). 



Formalin solution (4-10%) has 

 also been used to preserve whole 

 fish ovaries (Bagenal & Braum 

 1978, Hunter 1985, Cailliet et al. 

 1986). It is recommended by Hunter 

 et al. (1985) as the only preserva- 

 tive appropriate for use with the 

 hydrated oocyte method. This is be- 

 cause the hydrated oocyte method 

 estimates batch fecundity by calcu- 

 lating the number of hydrated, 

 unovulated oocytes in gravid ova- 

 ries, and Gilson's destroys hydrated 

 oocytes (Hunter et al. 1985). 



Formalin preservation has the 

 advantages over Gilson's of ( 1 ) pre- 

 serving hydrated as well as other 

 oocytes over long periods of time, 

 (2) having a short fixation period 

 and (3) low shrinkage rates, 

 varying from to 7% (Hiemstra 

 1962, Fleming & Ng 1987, Hislop 

 & Bell 1987), and (4) relative ease 

 of handling (Hunter et al. 1985, 

 Cailliet et al. 1986, Schaefer 1987, 

 West 1990). Its greatest disadvan- 

 tage is that oocytes and ovarian tis- 

 sue may become fixed into a hard 

 mass, making it extremely difficult 

 and tedious to separate oocytes 

 without damage (Schaefer & Or- 

 ange 1956, Bagenal & Braum 1978, 

 Cailliet et al. 1986). 



In this paper we propose a new, 

 two-step method to obtain hard- 

 ened, separated oocytes for fish re- 

 production studies. Oocytes are 



physically separated before being 

 preserved in formalin, thus main- 

 taining the advantages of formalin 

 fixation and preservation while also 

 providing well-separated oocyte 

 samples. The objectives of this pa- 

 per are to (1) describe this new 

 method and evaluate its effective- 

 ness, (2) determine the shrinkage 

 rates of weakfish Cynoscion regalis 

 oocytes separated and preserved by 

 this method, after 3-4 and 6-7 mo 

 preservation, and (3) assess the ap- 

 propriateness of this method for use 

 with the hydrated oocyte method of 

 estimating batch fecundity ( Hunter 

 et al. 1985). 



Methods 



Twenty-eight weakfish Cynoscion 

 regalis, with ovaries in the hydrated 

 but unovulated developmental 

 stage, were collected in the sum- 

 mer of 1991. Fresh (unpreserved) 

 oocytes were removed from the right 

 ovary of each fish and spread onto 

 a microscope slide. Twenty hydrated 

 oocyte diameters were then mea- 

 sured, after a minimum sample size 

 of 15 oocytes was determined using 

 the iterative method described in 

 Sokal & Rohlf (1981) (S=0.05, 

 a=0.05; P=0.90, 5=0.06 mm). An 

 ocular micrometer in a dissecting 

 microscope was used to measure 

 oocyte diameters to the nearest 

 0.038 mm (1 micrometer unit at a 

 total magnification of 24 x). Mea- 

 surements were taken along the 

 median axis of the oocyte, parallel 

 to the horizontal micrometer gra- 

 dations (Macer 1974, DeMartini & 

 Fountain 1981). 



Ten of these fish were also used 

 to estimate batch fecundities gravi- 



*Contribution 1780 of the College of Wil- 

 liam & Mary, School of Marine Science, 

 Virginia Institute of Marine Science 



Manuscript accepted 28 January 1993. 

 Fishery Bulletin, U.S. 91:165-170 (1993). 



165 



