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Crowding factors are those substances released by fish, which, under crowded conditions, inhibit both their growth and reproduction and depress their heart rate. We have partially purified the crowding factor from goldfish and carp by following a heart-rate-depression assay. The heart-rate-depression activity eluted with the diglyceride fraction from both silicic acid pnd Sephedex LH-20. A slightly different activity eluted with the free sterols. The peak activity associated with the diglycerides has a distinctive ultravjolet spectrum distinguishable from that of phthalic acid esters, which arc also active i n the assay sytem and are found in fish tissues and fish water.Crowding factors are species-specific hormoncs secreted by fish and other aquatic animals which have been observed to inhibit their own growth and reproduction when in a small body of water. Greene ('70) has shown that the spawning inhibitor for goldfish could be extracted from the water by chloroform, although he did not purify it further. This inhibitor proved to be species-specific. Similarly, Yu and Perlemutter ('70) extracted a species-specific growth inhibitor from a crowded zebrafish tank using trichloroethane. This inhibitor was observed to cause cardiovascular ailments and high mortality in developing fish embryos, and to shorten the life-span of adult fish in addition to inhibiting growth. It was also observed to depress the heart rate of embryonic fish.We have found that a species-specific growth inhibitor could be extracted from the water of crowded goldfish using either trichloroethane, chloroform, or carbon tettrachloride; that these extracts also contained a species-specific, heart-rate-depressant activity which could be identical or similar to the growth inhibitor; and that this heart-rate-depressor activity could affect juvenile gold6sh as well as embryonic fish (Francis et al., '73). We have set up an assay to measure the heart-rate depression of a juvenile goldfish in water (Francis et al., '73), and have used this heart-rate-depressor assay to follow the purification of this factor from both goldfish and carp.

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