Top ⇑Comparison of Proposed Standard Methods with Prior Methods

This information focuses on determinations of clutch size and oocyte/ovum diameter measurements. Relative clutch mass and oocyte/ovum mass rarely have been determined for minnows and darters.

Methods associated with the single clutch hypothesis did not provide a definition for "mature ova" which was specific enough to limit the use of oocytes or ova in one stage of development. Most aspects of past descriptions appear to indicate that ripe eggs (present terminology) were used for determinations of both clutch size and ovum size. In some cases, there is a possibility that ripening oocytes also were used in obtaining estimates of these parameters.

The methods proposed here advocate counting the oocytes in clutches of MA and MR females before the oocytes are ovulated. Females begin spawning soon after ovulation. Thus, the use of ripe eggs (from RE females) will result in underestimates of clutch size, which may be quite large, and also will yield spurious regression statistics. Diameter measurements of ripe ova alone will be comparable with other similar measurements. If ripe eggs and ripening oocytes were measured the data cannot be compared without error unless it is possible to restage the cells, which were measured. In cases where the specimens and the ovarian material remain in collections, this may be possible.

Methods formerly associated with the multiple clutch hypothesis involved counting all gametic cells, including maturing oocytes (present terminology) and any mature or ripening oocytes or ripe eggs (present terminology). The assumption was that this complement represented all of the eggs to be spawned in one season. Research has shown that individual darters and minnows are capable of producing a much greater number of ova in one season than the number of all gametic cells present at any one time. Thus, counting all gametic cells has very little meaning in relationship to the number of ova spawned within one season. Similarly, measurements of diameters of gametic cells in these stages of ovum formation are likely to have little relevant meaning. Again, the methods advocated here for determining clutch sizes involve making counts of oocytes in a clutch under formation in MA and/or MR females before the clutch is ovulated. Estimates of ovum size ideally should be obtained using the masses of ovulated eggs from RE females. The difficulty is that RE females are not always available when one obtains samples, suggesting that the process of oocyte ripening and ovulation occurs over a relatively short period of time.

In summary, the methods proposed here provide a snapshot of reproduction at one point in time. The more difficult task is to estimate clutch frequency and to factor variations in clutch size into any studies one is conducting. Similarly, variation in ovum size among clutches must be considered. Although both measurements of diameter and mass are useful, measurements of mass are of greater utility. Ideally, measurements should be completed using ripe eggs; however, limitations associated with the availability of ripe females may force one to use mature or ripening oocytes, which vary in the nature of the data they yield.

Top ⇑Fixation and Preservation

The limited information available shows that the methods of fixation and preservation used can have significant effects on the results one obtains. Alcohol can remove lipids from tissues, including oocytes and ova during storage. At present, one is best advised to fix specimens in 10% formalin and to store specimens in either 3%, 5% or 10% formalin.

     © Copyright 1995 by David C. Heins, All rights reserved.

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