Related FAQs: Hormonal Manipulation of Fishes,  Related Articles:  (A Review of the Literature On) Hormonal Manipulation of Fishes As An Aquacultural Technique
Bob Fenner	Good injection technique

I. Introduction, Definitions, Purpose & History

II. Methods of Induced Breeding

A. Introduction

B. Environmental Factors & Diet

1. Temperature

2. Light

3. Other

4. Diet

C. Hormonal Manipulation

1. Criteria: Biological & Chemical Assay

2. Problems in Standardized Dosage

3. Maturity of Recipient Fishes

4. Variability in Pituitary Gonadotropin Related to Phylogeny, Sex & Season

5. Use of Mammalian Hormones

6. Pituitary Extracts, Use & Preservation

7. Gonadal Steroids

III. Discussion

IV. Bibliography

Note: The following is an outline of a scientific paper I produced for a college class. Though somewhat technical, this information is of interest and utility to advanced aquarists seeking to understand fishes better through their physiology and endocrinology, as well as the breeder of "difficult" species. Much work is done around the world producing food and ornamental fishes through hormonal manipulation. For many species, commercial numbers of stock would not be available or cost-effective without employing these techniques. It may come as a surprise to you how similar "fish" biochemistry and our own are.

Introduction:

Aquaculture is the controlled reproduction and growing of aquatic organisms for food or ornament. Current trends in human population growth, habitat and natural stocks destruction versus advances in world agricultural and fisheries production clearly show the basic need for developing and implementing aquacultural technology.

Widespread use of hypophysation (use of the pituitary gland, a part of the brain and endocrine system of humans and fishes) has permitted breeding of a number of species otherwise not spawned in confinement (e.g. kuhli loaches, Acanthopthalmus kuhlii, dolphinfish, Coryphaena hippurus). Many of the important cultured pet and food fishes fall into this category. Previously fingerling-stock had to be collected from the wild. This supply was seasonal and unpredictable. By manipulating hormonal and environmental factors seed can be produced all year long; reducing cost, disease, and incidence of trash fish (undesireable contaminants) and predators. (Hickling 1968, 1971; Jhingran 1969).

Hypophysation involves the injection of gonadotropins (hormones=chemical messengers produced by the pituitary, that stimulate the reproductive organs) from one animal into another. Among vertebrates (fishes, amphibians, reptiles, birds and mammals) these are not very species-specific. Sometimes it has been found better to mix mammalian pituitary extracts and steroids in varying concentrations with fish gonadotropin. There is increased success in the use of prepared mammalian hormones in breeding procedures, preservation of fish sperm, maintenance of brood stock; yielding reliable sources of fish seed and acceleration of breeding research.

Problems relating to standardization of dosage, determination of maturity of recipient fish and nutritional requirements particularly need to be further explored. Current knowledge on the use of pituitary hormones in fish culture and technology regarding collecting, processing & storing of glands & methods of application have been covered comprehensively in the literature.

S. Aschheim & B. Zondek are credited with the first experiments regarding reproductive behavior (in female mice) and replacement therapy using pituitary implants. The first use in fishes was in 1930 by B.A. Houssay of Argentina. He induced premature birth in a viviparous (livebearing, like guppies) fish by injecting pituitary glands recently removed from other fish. Other South Americans, like von Ihering, a Brazilian, used this technique for aquaculture extensively beginning in 1934.

Soviets attempted to induce captive sturgeons to spawn in 1932 by means of mammalian hormones. They were unsuccessful until 1937, when N.L. Gerbil'skii produced eggs and sperm in a number of the sturgeon, Acipenser stellatus that had been intracranially injected with 1-2 fresh pituitaries of conspecifics (the same species). As of 1957 the Soviets have obtained all their sturgeon eggs for culture from pituitary treated fishes, very important to their caviar industry. As of 1963 the Chinese have been able to supply all the seed used in carp culture (big-head, silver, common & other food species) by means of hypophysation.

II. Methods of Induced Breeding:

A. Introduction:

Fish are induced to spawn by altering environmental parameters, particularly temperature and photoperiod, and hormonal constitution. These variables are generally constant for the species but vary widely among cultured food fishes.

B. Environmental Factors & Diet:

Environment and diet are the principal sources of phenomena determining hormonal secretion. Several factors, photoperiod, temperature, metabolites, pheromones, light strength and temperature shock control release of gonadotrophins, which in turn induce gamete (eggs & sperm) production and concomitant reproductive behavior. Reproduction may occur out of season by manipulation of temperature and photoperiod (how long the light is on daily) alone.

An example of improved culture just through manipulation of environmental factors may illustrate the point. The Ayu, Plecoglossus altivelus, a smelt-like fish cultured in Japan during the summer months, normally spawns in October-November., after which most of the fish die. Due to lower water temperatures that time of year, it's difficult to maintain live food organisms in adequate densities in rearing ponds to feed fry. Adjustment of photoperiod resulted in successful acceleration of sexual maturation (August), enabling rearing of fry when natural food was abundant. Spawning was also delayed to prolong the life of adults. The light period was extended to 18 hours per day with artificial light (August through October). Reproduction was retarded & adults were marketed out of season in February.

1. Temperature

The relative importance of temperature varies per species, investigator and experiment, but all agree a certain range and lowering or elevating optimizes results. Smiglieski (1975) stated that temperature was the controlling factor in his work with flounders. Yamamoto et. al (1966) observed spermatogenesis is goldfish, Carassius auratus below 14 degrees C & accelerated spermiation above 20 degrees C. Often, if much temperature fluctuation occurs, reproductive behavior will cease.

Haydock (1971) has observed temperature threshold below which gulf croaker will not hydrate or ovulate. For each species or sometimes race (a sub-specific classification) there are limits, both high and low, which eliminate reproduction physiologically and behaviorally.

2. Light

As with temperature, there are optimal amounts of strength, quality and duration of light in relation to reproductive behavior. On work with bluegill sunfishes it has been found that a longer photoperiod 16light/8dark versus 8L/16D induced a better gonosomatic index (G.S.I.= ovary weight divided by total body weight times 100). Males were more aggressive, dug more nests and fertilized more eggs.

Hoar (1965b) sums up the effect of longer light periods by stating that they stimulate secretory activity of anterior pituitary, inducing pre-sexual behavior. The pituitary LH, Lutenizing Hormone (one of the gonadotropic hormones produced in the brain) activates interstitial tissue of the gonads which produce gonadal steroids that in turn dominate sexual phases, taking complete control during prenatal phases. Several investigators have shown that elevated temperature and prolonged photoperiod increased gonad maturation in the green sunfish, Lepomis cyanellus.

3. Other

Photoperiod and temperature have received most attention & are generally considered to be of greatest importance in inducement of sexual maturity and spawning. Other factors such as the effects of other environmental stimuli; meteorological (rain, floods, etc.) & water conditions (pH, Ammonia, C02, turbidity) on controlled breeding of food and ornamental fishes has been investigated. In several species, the presence of con- (the same species) and/or hetero- (other) specifics is important. In bluegills and some cichlids it has been found that when males are present, the most aggressive female has greaater ovarian development. Another application of environmental control is employed in mullet culture in Israel. By keeping the adults in freshwater ponds and not allowing access to the sea where they migrate to spawn, fish-culturists are able to extend the spawning season.

4. Diet

Researchers have demonstrated gonadal regression induced by restricted diets accompanied by an inversion (opposite) of the lasophil/acidophil (cell types distinquished by their staining characteristics) ratio of the mesoadenohypophyis (an anterior part of the pituitary gland in ours and fishes brains) and a reduction in gonadotrophin content. These conditions were reversed with adequate food. Some authors have stressed the interaction between photoperiod and diet and pointed out that optimum benefit from photoperiod adjustment can be over-rided by poor diets. Hmmm.

C. Hormonal Manipulation

1. Criteria: Biological and Chemical Assay

Several methods are applied towards determining sex and sexual maturity of spawners. Externally there may be sexual dimorphism (structural differences between the sexes) in the species and/or differences that occur during sexual ripeness. Such is the case with the channel catfish, Ictalurus punctatus. Females assume a dropsical appearance with raised and reddened genitalia. The males develop swollen head musculature and well defined genital papilla.

A practical and precise way to measure oocyte (egg cell) development is through inserting a sterile polyethylene or glass catheter in the oviduct and removing egg samples. The eggs are cleared in successive concentrations of ethyl (grain, drinking) alcohol (30, 50, 70, 90, 100%) then xylene (an organic solvent) for 5 minutes each. Eggs are removed by catheter before each hormone injection to determine gonadal development.

Physiological parameters are also valuable as sources for assays. Elevated plasma (the fluid portion of blood, the same as for humans) calcium levels have been reported during gonad development in many species. Peterson & Shehadeh (1971) reported a four time increase in calcium and two time plasma copper in striped mullet, Mugil cephalus, under methyl testosterone treatment.

For examination, gonads are removed from the body cavity, weighed to 0.01 gram, labeled, wrapped in cheese cloth, fixed in 70% ethanol (ethyl alcohol) for 30 minutes and 100% for one hour. Xylene is used for a clearing agent. Infiltration and final embedding is done in fresh paraffin (wax). Typically microscope slides are produced by slicing these specimens on a tool called a microtome at 8 micron thickness. These tissue slides are stained with a compound called Delafields iron hematoxylin and counter stained (for contrast) with eosin. The specimen is permanently covered with a thin glass coverslip and permount.

When you examine these prepared sections under a microscope you can judge the sexual readiness oth your stock. In sexually immature males the lumen (opening) of the seminiferous tubules is unformed. Later primary and secondary spermatocytes develop in the lumen. Still later spermatids and spermatozoa (these are all developmental phases of male reproductive cells) are seen free in the lumen. After spawning the tubules are completely occluded (blocked) and testes become highly vascularized (serviced with blood vessels). In immature females only the oogonia & primary (previtellogenic) oocytes are present. At maaturity secondary oocytes distributed by oolemma and centripedal yolk precursor form in the cytoplasm (the fluid portion of cells).

The pituitary may be examined in sample individuals. They are dissected and promptly fixed in Bouin's or Zenker's for 24 hours. They are subsequently passed through alcohols and stored in 70% ethanol, further processed and sectioned.

In the channel catfish the adult pituitary double from one to two millimeter maximum length at sexual ripeness. The mesoadonhypophysis contains gonadotropic and somatotropic cells; the former enlarge in ripe specimens. In this way the mesoadenohypophysis becomes large during sexual maturity.

2. Problems of Standard Dosage

The endocrine (=cry internally) system acts like a chemical link between an organism and it's environment. Fluctuations in the external environment and diet mediated through the central nervous system (CNS) affect pituitary secretion. As one of several target tissues the gonads are effected. The CNS acts quickly, temporarily and specifically. The endocrine works in concert and antagonistically with the CNS but slower, longer-lasting and more generally.

It is well established that in all vertebrates the pituitary gonadotropins and gonadal steroids are the hormones most directly involved in the regulation of reproductive behavior. Pituitary hormones may act by stimulating secretion of gonadotropic hormones, which in turn regulate behavior. Also, individually these hormones may influence behavior indirectly by their role in the development of secondary sexual characteristics.

In turn the CNS-pituitary-gonad axis is influenced by internal and external factors acting through the sense organs and brain. Studies on birds have emphasized that coordination and instigation of components of reproductive behavior are dependent on a continuous interplay of external and internal factors; hormones effect behavior and behavior effects the endocrine system.

There is some evidence that hormones other than those of the anterior pituitary and gonads play a major role in reproduction. Neurophysiological and thyroid hormones most outstandingly, but other hormones affect reproduction indirectly by their effects on general metabolism and growth. All of the endocrine glands are either directly of indirectly involved, since reproduction requires profound metabolic adjustments, particularly in females.

Injections: Two methods of injection are in wide practice. Intramuscular, in the flank just below the dorsal fin and behind the gill cover. This method is safer but slower working. Interperitoneal injections are made into the body cavity. They are faster acting but involve a greater chance of injury or death.

Dosage, latency period and frequency of injection have not been well determined for any one species; investigators adjust variables according to specific circumstances. This has yielded divergent results, duplication of effort and failure. Problems of determining exact dosages lie in two factors. 1) the largest fish does'nt necessarily need the largest dose; this is more dependent on the hormonal development of the individual. 2) Exact dose of any preparation depends on determination of it's gonadotropic content. Towards the ends of standardization an international standard animal for bioassay has yet to be agreed upon. The Soviets use the Vy'un unit (U.V.) based on the loach, Misgurnis fossilis, & the frog unit (Curimata sp.). The Brazilians use the fish unit (Fontenele 1955). A Wagur unit using Clarias batrachus (the walking catfish!) was advocated by Das & Khan (1962). In the U.S.A. changes in the gonadosomatic index (GSI) of hypophysectomized (with pituitary gland removed) cyprinodontid (killifish family) Fundulus heteroclitus have been often used. More recently, a bioassay assays using seminal fluid viscosity have been used. Goldfish spermiation was used by Yamazaki & Donaldson (1968) in response to purified salmon gonadotropin. Otsuka (1956) monitored uterine and ovarian changes in immature mice. The Galli-mainini reaction and spermiation or hydration in goldfish has been used most frequently. For convenience the wet weight of pituitary or dry weight (acetone dried) of pituitary powder/weight of fish is unfortunately still used versus chemical analysis of hormones to determine which are present, active and their synergistic-antagonistic effect on each other.

So it can be seen that due to the variety of test animals as standards, & lack of test for definitive presence and concentration of hormones, comparison of results is difficult, and therefore the data are mostly of local benefit.

More precise bio-physico-chemico-assay methods such as incorporation of radio-phosphorus by gonads is in it's infant stages. (This is a specific labeling and measuring technique)

3. Maturity Of Recipient Fish

Variation in a species in terms of gonad development is intensified in confinement. Variability of results is due to individual variation in sex or season, especially considering crude bioassay techniques. Effectiveness of hypophysation (injection with pituitary hormones) is dependent on the stage of reproductive development of recipients. Aquaculturists recommend that when applying other workers' data; that matching the dosage to the physiological state of the recipient can best be done by administering doses in graded series from above or below those recommended by other investigators. Almost always slightly greater success is achieved with the most intense regime (injecting less hormone more frequently).

If the species has not been spawned before they suggest continuous injections, every 24 hours, with a wide range of doses until spawning does occur. (This is science?)

As has been mentioned, care must be exercised in assaying sexual readiness in spawners. Parameters generally adopted have frequently proven unreliable. An example of this is females with enlarged abdomens, reddish coloration & protrusion of the cloacal region may be due to engorgement of the intestine, or disease, even during the spawning season.

4. Variability In Pituitary Gonadotropin Related to Phylogeny, Sex & Season

In order to determine dosage, effect and success of treatment it is vital that fluctuations in gonadotropic activity related to phylogenetic specificity (basically how closely species are related to each other evolutionarily), sex and season be known for each species. Much controversy exists as to specificity in gonadotropin as it relates to vertebrate phylogeny.

Work with homoplastic (derived from the same species) pituitaries has produced best results. Little correlation exists between phylogenetic affinity and effect of pituitary gonadotropins. Where distantly related donor-recipients are used the general practice has been to increase dosage.

The early literature on sex related differences in the gonadotropic activity of fish pituitaries has been outlined by Pickford & Atz (1957). Much of it and later reports are contradictory. It is more or less agreed that sex-related differences in pituitary extracts are usually minimized by the usual preparation of mixed extracts of both sexes in excess of threshold requirements.

Seasonal fluctuations in gonadotrophic activity are not uncommon in almost all fish species examined. Pituitaries collected for hypophysation are almost always taken from mature fishes during spawning season to be used immediately or dried and prepared for later use. (Kind of hard on your breeders, eh?)

5. Use of Mammalian Hormones

Advantages of using mammalian hormones include:

(1) Reliable source, available all the time.

(2) Easily stored in lab and retain biological activity for a number of years.

(3) More uniform than crude pituitary preparations, permitting controlled experiments and repeatable results.

(4) Cost is about the same.

(5) Saves valuable gravid fish, especially where homoplastic pituitaries are used.

(6) Does away with the tedious task of collecting, preserving, processing & distributing pituitary material.

There is not much in the literature on the use of such hormones for practical breeding of ornamental fishes; quite a bit on food species. Several Indian workers have worked extensively with Heteropneustes and mammalian hypophysial hormones, placental gonadotrophins, gonadal hormones and corticosteroids finding that human chorionic gonadotropin (HCG) and some of the corticoids, especially deoxycorticosteroids could be used profitably to spawn food fishes as a substitute for pituitary extracts. Chorionic gonadotrophin was also found to be an effective ovulating agent in Clarias batrachus. Snead & Clemens (1959) established methods for spawning channel catfish, goldfish, and white crappie, Pomoxis annularis with injections of chorionic gonadotrophins. At the price of $5.65 per 10,000 international units (IU) of APL (Anterior Pituitary Like) they established a cost of 45 cents per pound of fish spawned, comparable to established costs of fresh pituitary preparations.

Other reviews of successful use of mammalian hormones exist detailing one's effectiveness over another, acting singly and together.

Purified fish gonadotropins are available to the fish breeder, especially fractionated salmon pituitary. Much work has been done with these and food fishes; not much reported for pet fish.

6. Pituitary Extracts, Use & Preservation

In a fish, the pituitary is concerned with regulation of growth, pigmentation, adrenal cortex, thyroid gland and others, as well as reproduction. Cyclical changes in the pituitary are striking, showing increase and decrease in cyanophil cells involved with production of gonad stimulating hormones.

The synergic action of growth hormone, thyrotropic hormone (stimulating the thyroid gland) & sex steroids is indicated by the better results obtained with crude rather than purified gonadotropic hormones.

The pituitary regulates both gametogenesis and steroidogenesis in fishes. Two seperate gonadotropins Follicle-stimulating hormone (FSH) and Lutenizing hormone (LH) are physiologically distinct in the tetrapods (= "four legs", the rest of the vertebrates beyond the fishes, i.e. the amphibians, reptiles, birds & mammals). There is evidence of a single protein in fishes. When fish pituitary extracts are tested in tetrapods, both FSH and LH effects are elicited.

With rare exceptions induced spawning by hypophysation has been restricted to females. This is often due to the availability of male seed.

Collection of pituitaries involves chopping the head off the fish above the eyes, lifting the brain and removing the gland from the base of the cranium. (Ouch! How do you put it back together?)

Preserving the gland is done by freezing immediately with dry ice or dehydrating in acetone. This is done by changing the acetone several times every 12 hours. This is then stored in a desiccator (dryer). It lasts up to two years but the effect is not as good as fresh.

When preparing, care must be taken to not introduce foreign proteins. Combined with pituitary this sometimes causes very strong rejection. Therefore purification is necessary. Whole fish pituitaries may be ground up or injected whole. If ground, they are suspended in physiological saline or distilled water.

7. Gonadal Steroids

In all fishes investigated development of secondary sexual characteristics (are they growing facial hair?) has been shown to depend on gonadal steroids. Several androgens (male sexual hormones like testosterone) and estrogens (the female equivalent) have been identified from the tissues and blood of fishes. Evidence shows that some fish gonadal steroids have the same biochemical pathways as higher vertebrates. Among the steroids found are testosterone, androstenedione, 17 beta estradiol, androsterone, 11 keto testosterone, conjugated testosterone and progesterone. As with higher vertebrates bot males and females have compounds in common but in varying concentrations.

Hoar (1962b) presents evidence that reproductive behavior becomes dependent (in a feed-back loop with the pituitary) on gonadal hormones and less sensitive to pituitary gonadotropic hormones. Acquistion of nuptial coloration & onset of breeding behavior has been correlated with increasing steroidogenesis.

III. Discussion

Even in a paper of this scope it's possible to draw general conclusions about the role of hormones in concert with environmental factors in control of spawning behavior of fishes. In male fishes of several species, secondary sex-morphological changes are dependent on gonadal hormones. In several species, components of reproductive behavior are under gonadal control, yet in other species much of all reproductive behavior persists after castration. (Is this evidence of learned behavior, conditioned response?)

Ovariectomy abolished sexual response in several species examined. In the guppy sexual behavior reappears some time after ovariectomy. There has been no unequivocal demonstation that administration of estrogen or progesterone will induce reproductive behavior in fishes.

Some of the reasons allowing for the differential success in use of hormones to control reproduction in fishes are the phylogenetic differences in protein and peptide hormones of the pituitary. On the other hand, the similarity of steroids suggests there is little problem in phylogenetic specificity in applying steroid therapy.

I'd like to see controlled studies on the use of steroids on color and growth augmentation in sport mutations of oscars, Astronotus, guppies, and discus, Symphysodon among other hormone-treated species from the far east, in terms of related reproductive viability, longevity and vitality. How about you?

Pet Fish Conclusions: 

I hope the following became clearer through this review:

1) The effects of temperature, light (duration, quality & strength), metabolites ("fish wastes"), are very important , and many other factors to a lesser extent, in getting stock up to and increasing success in reproduction.

2) There is an extensive body of knowledge in scientific literature on the behavior and physiology of fishes, in particular food & game species.

3) That you probably "knew" much of this material intuitively from your association with aquarium species, and indeed could write a "scientific" paper of substantial importance concerning your experience, if not a thesis.

IV. Bibliography: On request, send SASE to me or FAMA. It's a long one.

I'd like to dedicate all the work I did towards my Masters on Hormonal Manipulation of Mullets As An Aquacultural Technique to all my friends who have put up with my explanations of the practices of administering hormonal extracts to elicit the behavior and physiology of reproduction; and who did not say "So, you give shots to fish to give them the hots?"