Amphibians: Guidelines for the breeding, care, and management of laboratory animals

VII Breeding

A. ANURANS

1. General Comments

Species that normally mate immediately upon emergence from hibernation can be readily artificially inseminated any time during hibernation. Laboratory-reared or laboratory-bred specimens may be in a physiological state allowing ovulation at any time. Artificial insemination using species that normally mate some weeks after hibernation has been accomplished only in the days immediately before the time of natural breeding. Laboratory. maintained specimens of other species, such as Xenopus, B. orientalis (The University of Michigan Amphibian Facility information sheet), and Engystomops pustulosus (Davidson and Hough, 1969), may be ovulated at any time depending on the physiological cycling regimens followed.

a. Sexing

See Chapter II.

b. Readiness for Reproduction

See Chapter VI, especially Sections B.1 .a(2) and c.

c. Identification of Individuals

See Chapter VIII.

2. Artificial Induction of Ovulation

The following description is based on R. pipiens, whose ovulation has been most routinely conducted under laboratory conditions (Rugh, 1965;

TABLE 8 Pituitary and Hormone Doses for Inducing Ovulation

Month	Pituitaries Alone	OR Pituitaries	+ Progesterone (mg)
Sept-Oct	10-12	2	5.0
Nov-Dec	6-8	2	2.5
Jan-Feb	4-5	1	2.5
Mar-Apr	1	1	-

Di Berardino, 1967). Kits including pituitary preparations plus directions for fertilizing eggs are available from commercial dealers. However, pituitaries are readily collected following the technique described by Rugh (1965). These may be used immediately or following storage at 4 ^oC (39.2 ^oF) in absolute (not 95 percent) ethanol or following lyophilization. Since seasonal changes in pituitary potency and in female sensitivity occur, Table 8, based on the use of pituitaries collected at the seasons indicated, will serve as a guide to dosages (see Bagnara and Stackhouse, 1973). The technique was developed by Wright and Flathers (1961). Masui (1967) and Schiietz (1967) discuss the pertinent physiological mechanisms.

Doses shown in Table 8 are calculated as female pituitaries; two male pituitaries are equivalent to one female pituitary. Both the pituitary and progesterone* should be injected into the coelomic cavity about 48 h before scheduled fertilization.

3. Amplexus

If breeding is to be accomplished by amplexus, both male and female should be treated. Follow the above table for pituitary dosage for females. Males will respond to half the pituitary dose; progesterone is not needed. The injected animals should be placed in an aquarium with 200-250 mm (8-10 in.) of dechlorinated water at 20-22 ^oC (68-72 ^oF) (see Chapter V, Section B.2.a). The animals should be placed above plastic screening with a coarse mesh 9.5 mm (3/8 in.) held approximately 50 mm (2 in.) above the floor of the aquarium to protect the egg masses. Depending on the season, fertile eggs should be available in 1848 h if the aquarium is placed in an undisturbed area. Note that the precise time of fertilization is not known when this technique is used.

*Progesterone may be obtained from organizations such as Nutritional Biochemical Corporation and Sigma Chemical Company.

4. Artificial Insemination

Eggs may be obtained for insemination following the technique described above for artificial induction of ovulation. Sperm should be prepared when eggs are observed at the cloaca following the application of slight pressure to the abdomen of the female. The male need not be injected with hormone for this purpose.

To obtain the sperm, pith a male frog, remove both testes, dissect away adhering tissues, and wash the testes free of blood. Macerate them in 10-20 ml of medium consisting of pond water, dechlorinated water, or 10 percent Steinberg's solution [see Chapter VI, Section B.1 .a(l)]. A convenient way to do this is to force the testes through a syringe with an 18-gauge needle. Large clumps may be broken up by aspiration. Wait 15-20 min for full sperm activity, which can be determined by examination under a compound microscope. This preparation containing sperm from two testes may be brought to 100 ml prior to inseminating the eggs. Higher concentrations may be used but young (October for wild-caught frogs) or old (artificially prolonged hibernation) eggs are susceptible to polyspermy when dense sperm preparations are used.

Sperm may also be obtained from R. pipiens without the death of the donor by injecting males with 100 IU of human chorionic gonadotropin (HCG). Following this injection, the animal should be held in a quiet area and any manipulation should be gentle to avoid premature urination. Sperm of maximum activity may be recovered by stimulating urination after 1-3 h (McKinnell, 1962). The urine that contains the sperm should be used as described below.

Where sperm of rare or genetically defined males are to be used, destruction of the male is to be avoided. Where hormone stimulation of sperm release has been ineffective, single testes or portions of testes may be surgically removed. Using the appropriate anesthesia (see Chapter IX, Section F), a testis may be exposed through a 5-mm (0.2 in.) dorsolateral incision and part of or the whole testis may be removed. No procedures for hemostasis are needed and the incison may be closed with simple surgical stitches.

After sperm activity has been confirmed, eggs are stripped from the female (Figure 23) by holding her in such a manner that pressure is applied to her abdomen with the force directed toward her cloaca, thus squeezing the eggs from the ovisacs into a dry Petri dish. This is accomplished by bringing the legs of the female forward parallel to the abdomen. This assures that pressure is not dissipated laterally. The third and fourth fingers are applied firmly over the throat and thoracic region to avoid dissipation of pressure anteriorly. The middle finger, index finger, and thumb

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are used to "milk" the abdomen toward the cloaca. Initially, rather sharp pressure may be needed to open the cloacal sphincter muscle; subsequently, gentle pressure will suffice to aid the natural peristalsis. This method requires the use of only one hand; the other is free to wipe moisture from the cloaca so that eggs do not stick to the skin, to manipulate the collecting dish, or to assist in the "milking" action. If the tongue of the female protrudes during this process, pressure is being applied incorrectly.

"Milking" eggs from large frogs, such as bullfrogs, may be aided by wrapping the animal with an "Ace bandage." Start the wrap snugly at the throat and wind spirally, loosening the wrap as it progresses over the abdomen.

The eggs expressed into the Petri dish are inseminated by pipetting the sperm suspension over them, seeing that each egg comes in contact with the sperm preparation. Just moistening the egg strings with the sperm suspension conserves sperm. This application of sperm must be done before the egg jelly has started to swell. After 10-15 mm flood the eggs with medium. Use of a dry Petri dish is recommended because the eggs stick to its surface. This facilitates changing the medium. A spiral pattern is formed in the Petri dish to prevent the eggs from clumping. After an additional 15 mm pour off the medium plus sperm and replace with fresh medium. Set the eggs aside for 30-40 mm. They may then be scraped free from the Petri dish and transferred to culture bowls or trays. A single. edge razor blade, tissue section lifter, scalpel, or glass slide should be used for this: Do not attempt to pull the eggs from the Petri dishes with fingers or forceps as they may be distorted and thus damaged. About 1 h after fertilization those eggs successfully fertilized will rotate so the black animal hemisphere is uppermost.

Using scissors, cut the clutch of eggs into masses of 10-25 eggs each. This allows escape of waste products and sufficient surface for gas exchange. In nature the volume and movement of water in the ponds adequately handle these functions. The eggs are now ready to be treated as described for the stages of early development (Chapter VI, Section B.1 .a).

The procedure for artificial insemination described here is applicable to any type of mating involving biparental reproduction (e.g., random mating lines, heterozygous marked lines, mutant lines) (see Chapter III, Section C).

5. Parthenogenetic Lines and Haploid Animals

For parthenogenesis, eggs are obtained as described above and, depending on the specific objectives, are inseminated and activated with ultraviolet-irradiated (Nace et al, 1970) or toluidine-blue-treated (Briggs, 1952) sperm of the same species but carrying a dominant mutation as a marker (e.g., Burnsi sperm on wild-type eggs) or of species whose normal sperm would produce lethal hybrids (e.g., R. clamitans sperm on R. pipiens eggs) Moore, 1955); such eggs can also be activated by pricking with a needle (Shaver, 1953). After 20 min at 18 ^oC (64 ^oF), the inseminated eggs are exposed to 37 ^oC (99 ^oF) for 4 mm and returned to 18 ^oC (65 ^oF). In a significant percentage of eggs this heat shock results in retention of the second polar body and produces diploid animals whose genome is of maternal origin. This particular form of parthenogenesis is called gynogenesis because it utilizes only the maternal genome. It is used for the production of the inbred and gynogenetic diploid lines (see Chapter III, Sections C.1.c and f). It is modified for haploid animals (Chapter III, Section C. I .h) by eliminating the heat shock.

The irradiation used to treat the sperm is from a 15-W Westinghouse Sterilelamp (G15T18) at a distance of 0.84 m (2.76 ft) for 5 min. Sperm thus treated retain their motility and are capable of insemination; only their nuclei are inactivated. Toluidine-blue-treated sperm are similarly inactivated. Should sperm inactivation be ineffective, the dominant phenotype will appear among the progeny when the homologous mutant sperm are used, or death of the progeny will occur as a result hybrid incompatibility when foreign sperm are used. Should the heat shock have been ineffective in suppressing meiosis II, the progeny will develop the haploid syndrome and die.

The technique just described is suitable for those species that lay their eggs in cold water shortly after emerging from hibernation. For those species that lay their eggs after a period of activity and when water in the breeding sites has warmed, cold shock should be substituted for the heat

shock. The conditions for ultraviolet irradiation and the precise timing and the temperature of either the heat or cold shock is dependent on the species and perhaps also on geographic variance. Thus the investigator should confirm or evaluate these values in each laboratory and for each species.

The earliest studies of parthenogenesis were of eggs stimulated to develop by agents other than sperm (Loeb, 1899). The cleavage-initiating factor responsible for the success of parthenogenesis in R. pipiens is a protein (Frazer, 1971).

6. Homozygous Lines and Androgenesis

Because of crossing-over, three generations of gynogenesis are required to attain 99 percent homozygosity (Nace et al., 1970). Absolute homozygosity may be attained in one generation, however, by using the insemination techniques described for gynogenesis and eliminating the diploidization step. Thus development is started as though haploid animals were to be produced. However, when the first cleavage furrow becomes evident, the eggs are exposed to 5,000 psi for approximately 4 min using a hydrolic press. This step inhibits cytokinesis but allows karyokinesis to continue, thus diploidizing the eggs. Since the entire diploid genome derives from the post-meiosis II haploid set, total homozygosity is attained. Animals obtained by this procedure are poorly viable unless derived from a selected or inbred or gynogenetic line.

Androgenesis is development utilizing only the paternal genome. The maternal nucleus of an egg is destroyed by irradiation (Gurdon, 1960; McKinnell et al, 1969) or by mechanical removal after insemination by sperm of the same species porter, 1939) but before karyogamy. Upon the initiation of the first cleavage, pressure is applied resulting in homozygous androgenetic development. Such animals are as labile as maternally derived homozygous animals.

Homozygous diploids can be produced by the nuclear transplantation method (see Chapter III, Section C.1.g and Chapter VII, Section A.9). These homozygous diploids, as other homozygous individuals, show reduced viability (Subtelny, 1958).

7. Polyploid Animals

Polyploid animals either occur spontaneously (Fankhauser, 1955; Kawamura and Nishioka, 1963, 1967) or are produced by following normal biparental insemination or gynogenetic insemination by first cleavage inhibition (see Chapter III, Section C.1.i). Triploid animals are most

frequently produced by inhibiting the second meiotic division following normal biparental insemination.

Polyploid animals occur in two types of nuclear transplantation experiments. In the first type, a somatic nucleus is fused with the maternal gamete nucleus or with the zygote nucleus (Sambuichi, 1959; Subtelny and Bradt, 1963; McKinnell, 1964). In the second type of experiment, spontaneous delay of one cytoplasmic cleavage interval, accompanied by karyo. kinesis at its proper time, results in the production of embryos of double the expected chromosome number (Gurdon, 1959).

8. Mosaic Animals

Application of the pressure technique to inhibit cytokinesis at the second or third or combination of cleavage stages can produce mosaic animals. Depending on which of the previously described techniques were used to initiate development1 a variety of mosaic types can be produced.

9. Nuclear Transplantation

Nuclear transplantation was first successfully accomplished in vertebrates by Briggs and King (1952). The procedure has been described for the leopard frog by King (1966, 1967), X. laevis by Elsdale et al. (1960), and urodeles by Signoret et al. (1962).

Activated eggs are enucleated by manual removal porter, 1939), ultraviolet irradiation (Gurdon, 1960), or laser irradiation (McKinnell et al., 1969). Donor cells are dissociated in an electrolyte medium that may contain a chelating agent and/or a protoeolytic enzyme (King and Briggs, 1955). A dissociated cell is drawn into a micropipette in such a manner that its plasma membrane is broken. This liberates the undamaged nucleus. The micropipette containing the nucleus and some protective cytoplasm is then inserted into the previously activated and enucleated host egg. Low temperature and a polycationic amine enhances certain nuclear transfer experiments (Hennen, 1970). As stated elsewhere, these procedures may be used to produce isogenic groups (see Chapter III, Section C.1.b), polyploids (see Chapter III, Section C.1.i), and homozygous diploids (see Chapter III, Section C.1.g).

10. Xenopus

Gurdon (1967) has described breeding and husbandry methods for X. laevis. Eggs may be obtained from female Xenopus, judged to be gravid by abdomens distended with eggs and by their somewhat reddened cloacal

lips. Though not absolutely necessary, sperm are best obtained from males with dark "nuptial pads" on the underside of their forearms. In laboratory-maintained colonies ovulation may be induced once a week for several weeks (Wolf and Hedrick, 1971). Both female and male are stimulated to breed with chorionic gonadotropin, which is usually dissolved in physiological saline at 500 or more IU/ml. Injection is into the dorsal lymph sac. The dose administered depends on the size of the animal; unless the animals are quite small, however, a total of 500 IU per female and 250 per male is usually satisfactory. Best results are obtained when infrequently ovulated females are administered 100-unit "primer" doses about 5 h before administering the remainder of the quantity. Two males should be prepared for each female and be kept separate between the "primer" and final injection. After amplexus has been established, the unsuccessful male should be removed.

The fully injected animals are placed in an amplexus chamber as described in Section A.3 above for R. pipiens. The temperature of the medium should be 20-25 ^oC (68-77 ^oF). Below 20 ^oC (68 ^oF) the likeliness of success is lessened. The amplexus chamber should be covered and placed in a quiet location to minimize distraction. Spawning should begin within 12 h and continue through the next 24 h. The parents are then removed and the larvae separated from the screen of the amplexus chamber; hatching occurs between 2 and 3 days after the spawning. The larvae should be left in the amplexus chamber until after they have fed for several days. They may then be transferred to containers as described in Chapter VI, Section B.2.a(l).

Xenopus eggs may also be artificially inseminated. Wolf and Hedrick (1971) developed the technique quoted below:

Oviposition began 6-12 hours after the administration of hormone, and eggs were obtained by stripping laying females in a manner similar to that described for Rana pipiens (Rugh, 1965). Due to the limited capacity of the ovisac of Xenopus laevis, only 400-1000 eggs could be stripped from the animal at a time; however, additional eggs could be obtained upon repeated stripping every I to 2 hours.

Sperm suspensions were prepared by macerating excised testes in 0.05 DeBoers solution with a glass rod in 12-mi conical centrifuge tube. DeBoers solution (DB) consists of 0.11 M NCl, 0.0013 M KCl, 0.00044 M CaCl2 with the addition of NaHCO3 to pH 7.2 (Katagiri, 1961). The concentration of DB employed is expressed in decimal form. Other standard salt solutions such as Amphibian Ringers (for composition see Rugh, 1965) can be used in place of DB. This solution is approximately the same ionic strength as DB, one of the most important parameters affecting sperm motility and viability (see results). The administration of human chorionic gonad- tropin to mature males resulted in the appearance of black nuptial pads on the inner aspects of the forelimbs but did not noticeably affect the viability or fertilizability of the resulting sperm suspensions and, therefore, was not routinely employed. Sperm

counts and motility were determined at 100-fold magnification with a light microscope. Only active swimming spermatozoa were classified as motile. Estimates of sperm concentration were made from hemacytometer counts and the average value of 25 x 106 cells/ml was obtained for sperm suspensions resulting from the maceration of one pair of testes in 10 ml of buffer.

Artificial insemination was conducted in watchglasses (34 inch diameter) by strip-ping eggs (50-100) directly into sperm suspensions prepared in 0.05 DB or by adding a concentrated sperm suspension (0.05--0.1 ml) to eggs (10-500) in 0.05 DB. The insemination mixture was agitated manually for several minutes to ensure uniform exposure, and 10 minutes later samples were flooded with 0.05 DB. The sperm suspension should be used immediately after preparation at room temperature or it should be iced if it is to be held longer than about 10 minutes.] Fertilized eggs rotated 15-20 minutes after insemination and fertilization were measured by scoring eggs in morula or late blastula stage 3.5-7 hours after insemination.

B. URODELES

1. Mating

Laboratory-bred axolotls reach sexual maturity at about i year of age, unless retarded by insufficient food or improper care. Mature males show a marked enlargement of the lateral margins of the cloacal opening, resulting from the increased size of the cloacal glands as they reach a functional state. The corresponding glands of the female enlarge only slightly, but enough to distinguish a normal female from a castrated or an immature animal. Maturity in the female is usually well indicated by the plumpness of the body resulting from the increased size of the ovaries and oviducts.

The usual breeding season for axolotls under laboratory conditions extends from November or December to the following June. If the animals are maintained between 20 and 22 ^oC (68-72 ^oF), spawnings may be obtained even in summer months, especially if animals just reaching the age of 1 year are mated. The cloacal margins of males that have been in breeding condition through the winter and spring are usually reduced in size for several weeks during the summer and fall. During this period the ducti deferentes are devoid of spermatozoa. Similarly, the ovaries of females that have spawned once or more during the spring do not contain mature ova, but only immature growing eggs and degenerating old ones. Such females, if of the white strain, will probably lack the black toe tips noted on animals in good breeding condition.

An aquarium about 0.30 X 0.46 m (12 X 18 in.) or a 0.38-m (15-in.)-diameter dishpan are good mating enclosures. The enclosure should contain dechlorinated water that need be no deeper than 0.10-0.15 m (4-6 in.);

the bottom should be covered with a thin layer of fine gravel or very coarse sand. This serves to anchor the spermatophores deposited by the male and to hold them upright; this is essential to permit the female to make proper contact for insemination. Spermatophores do not adhere to smooth glass or enamel and hence are swept aside by the movements of the animals in such enclosures.

When in the proper breeding conditions, no preliminary "conditioning" is necessary to ensure successful axolotl mating. The water in the mating enclosure should, however, be at a temperature no higher than that in the animals' own bowls or aquaria. During the summer months, cooling the water with ice may be advantageous.

When mating axolotls, it is advisable to place a single male and female together in the early evening and leave them undisturbed through the night. The mating enclosure should be darkened by appropriate covering and be placed in a quiet location; bright lights or extraneous sounds may terminate their courtship behavior (Arnold, 1972).

The spermatophores consist of an almost transparent base, are roughly pyramidal in shape, and are constructed of a gel-like material secreted by certain of the cloacal glands. This base is capped by an opaque white mass of spermatozoa that may freely project in cylindrical form. The spermatozoa are held in a compact arrangement, probably by a substance secreted by more internally situated cloacal glands. From one to 25 or more spermatophores may be deposited at a single mating. If none is seen at first glance, stirring the water should reveal them. If the spermatophores lack the apical white mass of spermatozoa, spawning is unlikely to occur. Although males occasionally emit such spermatophores at the beginning of the mating season, they become fertile within a few weeks when spermatozoa have filled the ducti deferentes. If no spermatophores are found, it is advisable not to remate either animal for at least 2-3 days. In any event, return the two animals to their own enclosures instead of leaving them together.

2. Spawning

Ovulation in the axolotl is rarely spontaneous; it ordinarily occurs only after insemination. Eggs are shed into the peritoneal cavity from which they make their way into the cephalic ends of the oviducts. It is probable that the passage of the eggs through the ducts coincides with a period of considerable activity; the swimming of the female becomes somewhat frantic in the final phases of the process. During the spawning itself, the female may remain quiet for considerable periods punctuated with active swimming.

Although low temperature may delay the onset of spawning by several

hours, the initiation of spawning is normally expected within a few hours of a successful mating, i.e., within 18-30 h after the animals are placed together. Most eggs are usually laid within 24 h, a factor that can also be delayed by lower temperatures. Sometimes all the eggs are laid during the night following the mating.

The number of eggs per spawning, although varying from a few dozen to several hundred, usually ranges from 300 to 600. The percentage fertility is highly variable and probably depends in large part on the number of spermatozoa received at mating and stored in the glandlike spermathecal tubules in the roof of the cloacal chamber. The stored spermatozoa fertilize the eggs as they pass through the cloaca as revealed by the fact that eggs removed from the oviducts are never fertilized. Fertility may be affected also by the condition of the eggs. When egg overripeness is responsible, this may be recognized by the degenerate appearance of the eggs. If the spawning is at all prolonged, the last eggs laid usually are infertile because they are overripe and incapable of further development.

Occasionally, spawning occurs without insemination, for example, when all spermatophores lack spermatozoa. Sometimes many normal spermatophores may be found after a mating yet all the eggs laid may be infertile; ovulation in such cases must have been induced by the stimulus of the courtship or possibly by a mechanical stimulation of the cloaca.

While spawning, the female may be gently transferred to a fresh enclosure with a clean glass, slate, or enameled bottom. Plants, sticks, or glass rods to which eggs may be attached are quite unnecessary. The eggs will be attached singly or in small groups to the bottom of the container. If not detached by the movements of the female, they may be scraped free with a razor blade or the edge of a glass slide and removed with a pipette of suitable diameter equipped with a large rubber bulb.

Spawning females should be disturbed as little as possible. Cover the enclosure during the day. Unless necessary for experimental purposes, remove the eggs only at intervals of several hours and, if possible, without transferring the female to another enclosure. Some females, if handled or frequently disturbed, may discontinue spawning and retain the eggs, sometimes for several days or even weeks. Occasionally, the swelling of the jelly on a large mass of retained eggs may cause the oviduct to rupture, which will kill the female.

Axolotl females that spawn early in the breeding season may be mated again after an interval of 6 or 8 weeks or sooner if the first spawning was fewer than 100 eggs. A third spawning is frequently obtainable from animals 1-2 years of age. Matings of older females are less successful. It is advisable to plan replacement of breeding animals at the end of their third year, although an occasional one may be useful for a year or two longer.

3. Artificial Insemination

Induced ovulation and artificial insemination of axoloti eggs is possible and useful for certain experiments or when off-spring are desired from individuals that cannot be successfully mated. The procedure is as follows:

Induce ovulation by intramuscular injection of 180-200 IU of follicle-stimulating hormone (FS H). The female is held by wrapping the head and upper body in a wet cloth, and the hormone is injected into the muscle dorsal to the hind legs or cloaca.

At the first evidence of spawning-usually 18-24 h after injection-keep the female disturbed for a few hours by occasionally prodding, tilting, or shaking the enclosure. This prevents normal expulsion of the eggs and results in their accumulation in the caudal ends of the oviducts. Do not refrigerate: Not only will refrigeration stop spawning but it probably will also stop oviduct activity. Eggs that are spawned may be fertilized if removed from the water at once and placed on absorbent paper in a covered dish. Without this difficult precaution, spawned eggs exposed to water even briefly are useless for insemination and must be removed surgically as described below.

After eggs have accumulated in the oviducts for several hours, apply gentle pressure to strip any eggs from the ducts. This may result in saving many eggs that might otherwise be damaged in later procedures. The female is then decapitated and pithed. The peritoneal cavity is opened, and blood and remaining eggs in the cavity are rinsed out and discarded. The cut is extended caudally to open the cloacal chamber.

Using two pairs of small forceps, tear open the oviduct, starting at its cloacal end. With the forceps, transfer groups of eggs to clean dry Syracuse watch glasses or small Petri dishes. The eggs, which will adhere to the glass, should be spread out in a single layer. Farther cephalad in the oviduct, the eggs may be spaced singly. Remove each one by grasping the jelly with the forceps, avoiding damage to the egg. Eggs in the slender upper end of the duct that lack membranes and jelly and cannot be removed in this fashion are useless because they will not be fertilized. Avoid smearing eggs with blood.

Cover each dish of eggs as they are collected to avoid drying. Eggs so cared for can be successfully inseminated an hour or more after removal from the ducts.

Decapitate and cut away much of the ventral body wall of the male. Rinse the peritoneal cavity to remove any blood. The ducti deferentes are very slender near the cephalic ends of the testes; when transected here, the ducti do not discharge seminal fluid. Grasp a duct at this region and care-

fully free it along its entire length by cutting the supporting membrane with fine scissors. Before transecting the duct at its caudal end, grasp it with forceps to prevent the contents from escaping. If there is blood on the duct, rinse it quickly.

Transfer the duct to a watch glass or stender dish containing 10 ml of 10 percent physiological solution. With forceps tear the duct into short pieces to allow the contents to escape. Stir to ensure a uniform suspension, and pipette this over the surface of the eggs, making sure all are moistened. Run a needle or wire several times beneath the sheet of eggs to permit the suspension to pass beneath them as well.

Both ducts of the male may be used to prepare one suspension, or the second duct may be removed some time later if the eggs of a second female are to be inseminated. Merely avoid drying the duct if it is not being taken out immediately. If removal of the eggs has proved quite time-consuming and only one female is being used, it may be advantageous to remove and inseminate the eggs from one oviduct before proceeding with removal of the eggs from the second. If two females are being used, a second person should assist, although both ova and spermatozoa will survive considerable delay without apparent injury.

Inseminated eggs should be allowed to stand for about 20 min and are then flooded with medium. After another 20 or 30 mm immerse the small dishes containing eggs in a large bowl of medium. Later, the sheet of eggs may be detached from the watch glass with a razor blade or scalpel. By using two needles, somewhat like the blades of scissors, the sheet of eggs may be subdivided without breaking the egg membranes. When the eggs are in early blastula stages, remove those that are infertile or broken, using the needles to detach them from the others.

4. Initial Care of Embryos

Eggs obtained either by artificial insemination or from natural spawning require chlorine-free water. No more than 5O-60 eggs should be cultured per liter medium in a shallow bowl in order to facilitate gas exchange. At 20 ^oC (68 ^oF) cleavage will begin 6-8 h after spawning or insemination; sorting, however, should not be attempted until early blastula stages several hours later. Failure to remove infertile or damaged eggs may contaminate the healthy embryos.

Hatching occurs at about 2 weeks, but may be delayed by keeping the eggs at 10-15 ^oC (50-59 ^oF). Temperatures lower than 10 ^oC (50 ^oF) are inadvisable, and even that temperature may be harmful if exposure is prolonged. The care of later stages has been described in Chapter VI, Section C.1.

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