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Attempted triploid induction in Atlantic salmon (Salmo salar) using cold shocks
Aquaculture, 4(1974)287-297 o Elsevier Scientific Publishing
Company,
Amsterdam
ATTEMPTED TRIPLOID INDUCTION USING COLD SHOCKS
-
Printed
IN ATLANTIC
in The Netherlands
SALMON (SAL&IO
SALAR)
R.F.
LII\ICOLN*,
D. AULSTAD**
and A. GRAMMELTVEDT**
*Ministry of Agn’culture, **Agricultural University (Norway)
Fisheries and Food, Fisheries Laboratory, of Norway, Institute of Animal Genetics
(Received
revised
May lst,
1974;
July
2nd,
Lowestoft (GrFat Britain) and Breeding, As-NHL
1974)
ABSTRACT Lincoln, R.F., Aulstad, D. and Grammeltvedt, A., 1974. Attempted triploid induction Atlantic salmon (Salmo salar) using cold shocks. Aquaculture, 4: 287-297.
in
Radiation gynogenesis was used to investigate the effect of sub-zero cold shocks in inhibiting the second meiotic division of eggs of Atlantic salmon (Salmo salar). If cold shocks were successful in giving rise to diploid gynogenesis they could be used to produce triploids from eggs fertilized with non-irradiated spermatozoa. Different concentrations of saline and glycerol solution were used to depress the freezing point of water, enabling eggs to be cold-shocked at temperatures below 0°C. Control experiments using eggs fertilized with unirradiated spermatozoa were made to assess the effect of saline and glycerol on normal egg development. A 4 h immersion in saline solution following dry fertilization was lethal to eggs, and haploid development was observed in some experiments when eggs were pre-treated for short periods in fresh water before immersion in saline. No such anomalies in development were observed using glycerol. Eggs fertilized with sperm receiving 60Co r-irradiation at a dose of lo5 rad were coldshocked at between -0.2 and -1.7”C, at varying time intervals up to 40 min after fertilization, for a duration of 4 h. No treatments were particularly successful in inducing diploid gynogenesis. These findings are discussed in relation to studies in which cold shocks are highly successful in inducing diploid gynogenesis and triploidy in eggs of plaice (Pleuronectes platessa).
INTRODUCTION
The #current rise in fish farming has highlighted the need to produce faster growing strains of fish suitable for high-intensity commercial cultivation. One of the rnajor problems in rearing salmon (Salmo salar) at present is the reduced growth rates observed among fish at the onset of sexual maturation and the subsequent death of a proportion of the stock during spawning. This problem can be partly overcome by culling fish early, before sexual maturity checks growth rate, but this is an unacceptable solution where larger, more highly priced fish are needed to satisfy market requirements. Sterility would
288
not only remove those problems associated with sexual maturity but might promote an increase in growth (as flesh production) at a time when food would normally be utilized in the formation of sexual products. Surgical castration has been carried out in salmon with some success (N. Johansson, personal communication), but the method is time consuming and inappropriate in commercial fish farming. Chemical castration, however, may be possible in the future. ,4 genetical approach which lends itself well to massrearing techniques is that of induced triploidy. At present all experimentally induced triploids in sexually reproducing animals have been sterile for the female and partially or completely so for the male. Triploids have been produced for a number of animal species, mostly amphibia (see Fankhauser, 1945, for general review), using thermal shocks applied to eggs soon after fertilization. The effect of the shock is to inhibit metaphase II of the second meiotic division, and subsequent fusion of the diploid egg nucleus with the haploid fertilizing spermatozoan produces a triploid zygote. For successful induction of triploidy the timing, duration and intensity of the shock are all critical (Purdom and Lincoln, 1973). Similar procedures have been used to produce triploids in fish. Swarup (1959) obtained up to 25% triploid sticklebacks (Gasterosteus aculeatus) from eggs cold-shocked for more than 1 h. In plaice (Pleuronectes platessa) and its hybrid with the flounder (Platichthys pesus) cold shocks delivered up to 20 min after fertilization for durations of 3 h or more produced 100% triploids in hatched larvae (Purdom, 1972). Svardson (1945), working with salmonids, cold-shocked eggs of the gwyniad Coregonus lauaretus in a Thermos flask of melting ice 10 min after fertilization for a total duration of 13 h. Cytological examination of young embryos showed that of the 30 eggs examined one was found to be triploid, and the rest were diploid, haploid or mosaics. In a second and less controlled experiment than the first, eggs from the hybrid produced by crossing salmon (Salmo salar) with brown trout (Salmo trutta) were cold-shocked at a temperature of between 0.3 and 1.2”C at varying times after fertilization, ranging from lo-120 min, for durations of l-3 h. Chromosome analysis of 161 embryos showed one to be triploid. Neither of these salmonid triploid eggs was reared beyond the blastula stage. Previous attempts to induce triploidy in rainbow trout (Salmo gairdneri) (C.E. Purdom and R.F. Lincoln, unpublished), using cold shocks of 0°C delivered at 15-min intervals up to 8 h after fertilization, produced only diploid embryos. Evidence of spindle disruption was found in eggs cold-shocked 8 h after fertilization when aneuploid and highly polyploid cells were present in 3.5-dayold embryos, but this was thought to be due to disturbance of the first mitotic division. To explain the lack of effectiveness of the cold shock in these experiments three possibilities may be put forward: the shock may not have been intense enough, the shock may have been delivered too late after fertilization, or the arrested stage of the second meiotic division in the salmonid egg may be particularly insensitive to cold shocks. Ginsburg (1968) has demon-
289
strated metaphase chromosomes of the arrested second meiotic division in sections of pre-fertilized eggs of the lake trout (Salvelinus namaycush), and this is thought to be the normal arrested stage of eggs at ovulation and fertilization in fish. There is no reason to suppose that the arrested stage of the eggs at the time o:t ovulation, as seen in lake trout, would be any different in other salmonid species, and it may be expected that the salmon egg would be most susceptible to suppression of the second meiotic division by cold shocks at or immediately after fertilization. The simultaneous delivery of a cold shock with fertilization is possible in salmonids since water is required to activate the egg before gametic fusion can occur following “dry fertilization” (Ginsburg, 1963). The ability of the salmonid egg to withstand sub-zero temperatures is unknown, but work on plaice eggs has shown that temperatures of -1.7”C (the freezing point depression of sea water at 32°/00 salinity) can be tolerated for up to 6 h or longer immediately after fertilization. The choice of medium with which to depress the freezing point during the cold shock may be important to successful egg development. It is known that eggs of salmon will tolerate up to 20°/oo salinity to hatching, although with some mortality, and that eggs of brown trout, pike (Esox lucius) and roach (Rutilus ru tilus) can develop in liquid paraffin to the blastomere stage (Parry, 1966). It seemed possible, therefore, that salmon eggs would tolerate short periods of immersion in fluids other than fresh water, and experiments reported here investigated the use of sub-zero cold shocks, using saline and glycerol solutions, delivered at or soon after fertilization. The work was carried out during October and November, 1973 at the Fish Breeding Experimental Station, Sunndalsdra, Norway. MATERIALS
AND METHODS
Radiation gynogenesis was used as the experimental technique to investigate the efficiency of the cold shock in inhibiting the second meiotic division of the egg (Purdom, 1969). This method was preferred since in previous experiments difficulty had been experienced in coun%ing the large number of chromosomes present in salmonid fish. The chromosome number of Salmo salar is reported to be 58 (Rees, 1967) and it was felt easier to distinguish between 29 and 58 chromosomes in haploid and diploid gynogenetic eggs than between 58 and 87 chromosomes in diploid and triploid eggs. Sperm was irradiated with 6oCo -y-rays for a total dose of 10’ rad given over a 15 min period. This dose has been found sufficient to inactivate completely all the genetic material in the sperm head (Purdom, 1969). During irradiation and transport, sperm was held in small stoppered glass vials on ice in a Thermos flask. Freshly stripped eggs from wild and reared fish, held in 10 x 1 m circular concrete ponds, were used throughout the experiments. Reared fish (in sea water) were acclimatized to fresh water over a 3 week period just before spawning was expected to begin and held, together with wild fish, at ambient temperatures which varied between 3.0 and 7.O”C throughout the experimental period. Prior to stripping, fish were anaesthetized in chlorobutanol, washed off
290
in fresh water and the vent wiped dry. Eggs were collected and fertilized dry in accordance with normal salmonid hatchery procedure. Chromosome analysis was carried out on cells from the blastodisc removed from 3.5-day-old eggs. Blastodiscs were dissected from living eggs in 1% NaCl, stained on glass slides in two drops of 2% aceto-orcein in 45% glacial acetic acid, and squashed under a cover slip. The cover slip was ringed with nail varnish and the slides were stored in a refrigerator until scored, usually on the following day. Where possible, ten preparations were made from each experimental batch of eggs. At least half the blastodiscs was scored for chromosome spreads and no difficulties were experienced in distinguishing between haploid and diploid preparations. Where haploid and diploid chromosome numbers regularly occurred in the same preparation, then the egg was scored as a mosaic. Where no individual chromosomes could be counted, a subjective assessment could usually be made as to the ploidy of the preparation from the size of the metaphase plate. In addition, eggs could be scored for haploids and diploids after 11-14 days fertilization at lO.O”C when the embryo had reached post-gastrulation. At this stage haploid embryos appeared short and thickened with a wide open blastopore, whereas diploids were typically long and thin with little or no trace of a blastopore present. To make the embryo visible, eggs were cleared in glacial acetic acid-methanol (1:3, v/v) for 1 h in a refrigerator at approx. 4°C. Initial trials were made to assess if control eggs (no cold shock) fertilized with unirradiated sperm would develop normally after immersion for 4 h in full sea water ( 32°/oo ) and in three dilutions of it, and in two glycerol solutions made up ir river water. The freezing point depression of these solutions as observed in the cold shock apparatus are given in Table I. Eggs were transferred to these solutions (at lO.O”C) directly from dry TABLE
I
The freezing point depression of full sea-water (32”/,,) and three dilutions of it and of two glycerol solutions in river water as observed in a the cold shock apparatus ~~.. Solution
Freezing depression (“C)
Sea water 32’/,, (full) 24O I,, 1601,,0 5”lOO
-1.7 -1.3 -0.9 -0.2
Glycerol in river water 5% 8%
-1.2 -1.5
291
fertilization, or after periods of between 2 and 10 min fresh-water treatment following dry fertilization. Development of these control eggs was followed up to post-gastrulation and then discarded. Cold shocks were applied to eggs, using the apparatus shown in Fig.1 which utilized the melting point of ice made from the above solutions (frozen in a cold store at -3O.O”C) to depress the freezing point of water below 0°C. During the cold shock small batches of approx. 100-150 eggs were held in open mesh nylon bags which fitted into compartments of the egg boxes. This arrangement facilitated the addition and removal of eggs to and from the apparatus without causing a disturbance to eggs already being cold-shocked. A diffuser plate, mounted in the top of the bucket, was found necessary to promote uniform melting of the ice during water circulation. The water tended to become heavily charged with air bubbles during its passage through the ice which collected on the surface of the eggs in the boxes, causing them to float to the surface. A glass jar placed beneath the discharge pipe from the bucket, effectively deflected the bubbles to the surface, where they dispersed and calsed no disturbance to the eggs.
Fig.1. Diagram of cold-shock apparatus.
In operation the tank was half filled (30 1) with the saline or glycerol solution required, and the pump was adjusted to complete approx. 1 circulation/min. Small portions of ice made from the same concentration saline or glycerol solution as that in the tank were then packed in the bucket and the diffuser plate was wedged in place. Initially the bucket required recharging every few minutes until the main body of water in the tank had reached the melting point of the ice used, but thereafter topping up every 30 min was found adequate. As long as ice was present in the bucket the water temperature in the tank never fluctuated more than O.l”C. Excess water added to the system from the melting ice was allowed to drain off via the overflow pipe and refrozen as required. Eggs were held at lO.O”C before cold shock (duration 4 h)
292
and incubated in standard hatchery trays in running water of the same temperature. All experiments in which eggs were cold-shocked in saline solutions are summarized in Table II where the original code numbers have been retained. RESULTS
Salinity effects
on egg development
(1) Eggs fertilized dry. Normally fertilized eggs transferred directly to the four salinities investigated were not activated. They remained soft and sticky throughout the period in saline and the perivitelline space was not formed. On transfer to fresh water 4 h later activation changes began to take place in all groups except those from 32°/00 saline, which began to turn white (yolk coagulation) within a few minutes and were all obviously dead 30 min later. The remaining groups showed irregular cleavage, which was delayed by approx. 4 h compared with controls, which confirmed that activation had not taken place in any of the saline solutions used. No development was observed beyond blastulation and all eggs began to die 6 days later, when control eggs had reached gastrulation. (2) Eggs fertilized dry followed by water activation. Eggs were fertilized dry and then activated with fresh water for durations of 2, 4, 6, 8 and 10 min before immersion in various saline solutions. Five experiments were carried out using the three highest salinities, 32, 24 and 16’/,, salt. Eggs activated with fresh water for 8 and 10 min survived the 32’/,, salt treatment, but those activated for 2, 4 and 6 min showed mortalities of 100, 93 and 4%, respectively, as indicated by whitening of the yolk during the first 30 min on transfer back into fresh water. Yolk coagulation was not observed in any eggs treated with 24 and 16’/,, and these eggs continued to show progressive changes associated with activation during the 4 h immersion in saline from all pre-treatments in fresh water. Cleavages were highly irregular in the two surviving groups from the 32’/,, salt-treated eggs and also from those groups which received 2 min water activation prior to immersion in 24 and 16’/,, saline. Cleavages were more normal in all the remaining groups and, as expected, no evidence of a cleavage delay was found for any eggs that had first been activated with fresh water before saline treatment. Development was monitored to post-gastrulation, when closure of the blastopore was completed, and all eggs reached this stage before being discarded. In one experiment where eggs were treated with 16°/oo saline ll-dayold embryos from the 2 min fresh-water activated group appeared identical to known haploid embryos of the same age produced from eggs fertilized with spermatozoa which had received 10” rad 6oCo y-rays. It was thought possible that the superficial haploid appearance of these embryos may have been caused by some abnormal ontogenic development caused by the saline treatment
II activation,
10 10 10 3 5 5 5 10 10 10 10 9
1 5 10 10 15 20 40 10 15 20 30 40
-1.3
-1.7
-1.7
32
32
s2
s4
-0.2
5.3
24
6 5 10 4
1 5 10 15
(“C)
Number of haploid gynogenetic embryos 3
~~
of diploid
Number of diploid gynogenetic embryos
on the production
(“1”“)
Number of blastodisc preparations scored
water
Minutes after water activation cold shock given
after
Temperature of cold shock
times
Saline concentration
applied to salmon eggs at varying of cold shock, 4 h
s13
S6
Experiment number
Effect of sub-zero cold shocks, gynogenetic embryos. Duration
TABLE
.~___
294
per se. Another experiment, not specifically comparable since eggs were wateractivated following dry fertilization for 1, 5, 10 and 15 min before immersion in 16°/oo saline, nevertheless confirmed the possibility that these embryos were haploid; in this experiment chromosome analysis of blastodiscs from 3.5-dayold eggs revealed seven haploids, two mosaics and one diploid from ten preparations of the 1 min activated group. A further two haploids and two mosaics from ten preparations were also observed in the 5-min water-activated group. the remaining groups were all diploid. From these two experiments it would appear that 16°/oo saline treatment soon after water activation is detrimental to the formation of a diploid zygote, possibly through destruction of the fertilizing spermatozoan after it has activated the egg to develop (haploid gynogenesis). Although no such phenomenon was observed in eggs treated with 24°/00 saline, all embryos appearing normal 14 days after fertilization, it became apparent that saline solutions were highly unsuitable media in which to cold-shock eggs. Egg development
in glycerol
The anomalies in egg development observed after treatment with saline solution led to the search for a more suitable medium in which two cold-shock eggs. The use of glycerol in many cryogenic solutions to deep-freeze sperm suggested that this would be a suitable alternative. Two solutions containing 5 and 8% glycerol were made up in river water and trials were carried out with control eggs, as for the saline solutions. Eggs added directly from dry fertilization activated in both 5 and 8% glycerol, as seen from direct observation on the eggs and by comparisons made with untreated controls of the times to first cleavage. All cleavages looked normal and analysis of blastodiscs from 3.5day-old eggs revealed only diploid chromosome numbers. The appearance of 14-day-old embryos was identical with those of untreated eggs. Similar results were obtained with eggs pretreated with water before immersion in the glycerol solutions and no abnormal development was observed as with the saline solutions. Glycerol thus appears to be a very suitable medium in which to cold-shock eggs. Effect of cold shocks, using saline, in inducing diploid gynogenesis The comparatively short season over which eggs can be obtained, coupled with the long development time of the salmon egg, necessitated the early evaluation of the effect of cold shocks using saline solutions before its haploidinducing effect in some of the control experiments was discovered. Nevertheless, the effect of the cold shock itself, using saline, in inhibiting the second meiotic division could still be evaluated using the technique of radiation gynogenesis. Table II lists the results of four experiments using three different cold shock temperatures. The experiment using 32’/,, saline was repeated because of the small number of blastodiscs preparations which gave countable chromo-
295
some spreads in experiment S.2. Eggs could not be cold-shocked sooner than 10 min after water activation using 320/00 saline, because of the complete mortality of eggs observed from the initial control experiments during this period. The results show that none of the treatments was effective in producing diploid gynogenetic embryos, except those eggs cold-shocked at -0.2”C 1 min after activation in experiment S6 (Table II). All other embryos from this group, together with all those from the other experiment.s, revealed only haploid-looking embryos when scored 14 days after fertiliza-Lion. Effect
of cold shocks using glycerol solutions
in inducing diploid gynogenesis
An initial experiment was carried out in which eggs were cold-shocked in l-l glass jars containing 500 ml of 5% glycerol made up in river water. The jars were p;.aced in the egg containers in the cold shock apparatus containing 32O/,, saline, which reduced the temperature of the glycerol to -1.7”C. Approx. 100 eggs were added to each jar direct from dry fertilization and at intervals of 1, 2, 5 and 10 min after water activation, and cold-shocked for a duration of 4 h. Im.mediately after the addition of eggs to each jar the temperature rose to -1.5”C but returned to -1.7”C after approx. 10 min and thereafter remained at this temperature throughout the cold-shock period. It was found that at this temperature 5% glycerol was super-cooled but no ice crystal formation was observed in any of the jars containing eggs. After the cold shock the eggs were rinsed ;.n water at 10°C and returned to the incubation trays for rearing on. From ten blastodisc preparations made for each group, two diploid eggs were found from those water-activated for 1 min: all the remaining groups were haploid. All the surviving eggs were scored 14 days later but no diploid embryos were observed in any of the cold-shocked groups. Two more experiments were carried out using 5 and 8% glycerol, which was frozen down and used in the cold-shock apparatus as for previous experiments using saline. The temperatures achieved using these two solutions were -1.2 and -1.5”C, respectively. The difficulty of obtaining irradiated sperm at this period necessitated the use of normal sperm to fertilize eggs. Eggs in both experiments were cold-shocked from dry fertilization and after 1 min water activation only. Again, no evidence of inhibition of the second meiotic division was observed, either from chromosome analysis of blastodiscs or from the appearance of later embryos. DISCUSSION
AND
CONCLUSIONS
Cold shocks below 0°C applied to salmon eggs directly from dry fertilization with irradiated sperm, and after short periods in water following fertilization, were not successful in inducing diploid gynogenesis. Some evidence of inhibition of the second meiotic division was obtained from eggs cold-shocked 1 min s.fter water activation following fertilization. Blastodisc preparations
296
from 3.5-day-old eggs revealed that diploid chromosome numbers were present in three out of nine eggs cold-shocked at -0.2”C (in saline) and two out of ten eggs cold-shocked at -1.7”C (in glycerol). A later examination of eggs from the same groups revealed embryos showing the typical characteristics of the haploid syndrome, which include short thickened bodies and open blastopores. The failure of cold shocks to inhibit metaphase separation at the second meiotic division strongly contrasts with similar experiments on eggs of the plaice (Pleuronectes platessa), where cold shocks have been highly successful in inducing both diploid gynogenesis and triploidy. The contrasting sensitivity of the eggs of the two species in duplicating the maternal chromosome set may be a function of slight differences in the maturation stage of the egg at the time of fertilization and cold shock. Plaice eggs are thought to be ovulated and fertilized at the stage of prometaphase of the second meiotic division. No direct evidence for this is available since the plaice egg is small, making direct cytological examination extremely difficult, but the high frequency of triploid induction (100% in hatching larvae) following cold shocks, a unique phenomenon in the field of experimental polyploidy, implies that an earlier stage in the second maturation division of the egg is involved. Metaphase chromosomes of the second meiotic division have been demonstrated in sections of prefertilized eggs of the lake trout, and it is generally supposed that this is the arrested stage for all salmonid eggs at the time of fertilization. The apparent insensitivity of salmon eggs to cold shocks suggests that at metaphase the second meiotic division has already progressed to a stage where thermal shocks cannot easily disrupt chromosome separation. The uniformity of response of plaice eggs to cold shocks in inducing inhibition of the second meiotic division has been attributed to the method of ovulation in this species (Purdom, 1969). Here ovulation occurs serially, in batches of a few thousand eggs at a time every 2 or 3 days, until the ovary is depleted and spawning ceases. Any over-ripe eggs fail to fertilize, leaving the remainder as a uniform sample. In contrast, salmonid eggs are shed progressively from the ovary into the body cavity, where they may remain for a considerable period of time before release. The period over which the eggs are held in the body cavity may be a prerequisite for the final ripening process in which the eggs mature to metaphase of the second meiotic division before release. This may explain the lack of any uniform effect of cold shocks in the experiments reported here and may represent a natural adaptation in salmon and other salmonids to low temperatures, which may be frequently encountered on the spawning beds. It may be expected that artificially stripped eggs would include a small proportion that had recently been ovulated into the body cavity and which had not matured to the stage of metaphase of the second meiotic division but were nevertheless potentially capable of fertilization and further development. These young eggs may be of a stage in which cold shocks could be effective in inducing diploid gynogenesis or triploidy. Although suppression of the second meiotic division, using cold shocks, was
297
not achieved in the present experiments, the reported case of one triploid rain bow trout amongst 18 hatchery reared fish (Cuellar and Uyeno, 1972) indicates that polyploidy is possible in salmonids. REFERENCES Cuellar, 0. and Uyeno, T., 1972. Triploidy in rainbow trout. Cytogenetics, 2(6): 508-515 Fankhauser, G., 1945. The effect of changes in chromosome numbers on amphibian development. Q. Rev. Biol., 20: 20-78 Ginsbmg, A.S., 1963. Sperm-eggs association and its relationship to the activation of the egg in Salmonid fishes. J. Embryol. Exp. Morphol., 11: 13-33 Ginsburg, A.S., 1968. Fertilisation in Fishes and The Problem of Polyspermy. “Nauka” Publ. House (Jerusalem, Israel Program for Scientific Translation, National Marine Fisheries Service, 1972), 366 pp., TT 71-50111 (translation Russian) Parry, G., 1966. Osmotic adaptation in fishes. Biol. Rev., 41(3): 392-444 Purdom, C.E., 1969. Radiation-induced gynogenesis and androgenesis in fish. London, 24(3): 431-444 Purdom, C.E., 1972. Induced polyploidy in plaice (Pleuronectes platessa) and with the flounder (Plutichthys pesus). Heredity, London, 29(l): 11-24 Purdom, C.E. and Lincoln, R.F., 1973. Chromosome manipulation in fish. In: J.H. Schroder (Editor), Genetics and Mutagenesis of Fish. Springer Verlag, pp.8:3--89 Rees, H., 1967. The chromosomes of Salmo salar. Chromosoma, 21: 472-474 Svardson, G., 1945. Chromosome studies of Salmonidae. Meddn St. Unders-o. SottvattFisk., 23: l-151 Swarup, H., 1959. 129--142
Production
of triploidy
in Gasterosteus
aculeatus
(L.).
MOSCOW, for U.S. from the
Heredity, its hybrid
Berlin,
FiirsAnst.
J. Genet.,
56:
Company,
Amsterdam
ATTEMPTED TRIPLOID INDUCTION USING COLD SHOCKS
-
Printed
IN ATLANTIC
in The Netherlands
SALMON (SAL&IO
SALAR)
R.F.
LII\ICOLN*,
D. AULSTAD**
and A. GRAMMELTVEDT**
*Ministry of Agn’culture, **Agricultural University (Norway)
Fisheries and Food, Fisheries Laboratory, of Norway, Institute of Animal Genetics
(Received
revised
May lst,
1974;
July
2nd,
Lowestoft (GrFat Britain) and Breeding, As-NHL
1974)
ABSTRACT Lincoln, R.F., Aulstad, D. and Grammeltvedt, A., 1974. Attempted triploid induction Atlantic salmon (Salmo salar) using cold shocks. Aquaculture, 4: 287-297.
in
Radiation gynogenesis was used to investigate the effect of sub-zero cold shocks in inhibiting the second meiotic division of eggs of Atlantic salmon (Salmo salar). If cold shocks were successful in giving rise to diploid gynogenesis they could be used to produce triploids from eggs fertilized with non-irradiated spermatozoa. Different concentrations of saline and glycerol solution were used to depress the freezing point of water, enabling eggs to be cold-shocked at temperatures below 0°C. Control experiments using eggs fertilized with unirradiated spermatozoa were made to assess the effect of saline and glycerol on normal egg development. A 4 h immersion in saline solution following dry fertilization was lethal to eggs, and haploid development was observed in some experiments when eggs were pre-treated for short periods in fresh water before immersion in saline. No such anomalies in development were observed using glycerol. Eggs fertilized with sperm receiving 60Co r-irradiation at a dose of lo5 rad were coldshocked at between -0.2 and -1.7”C, at varying time intervals up to 40 min after fertilization, for a duration of 4 h. No treatments were particularly successful in inducing diploid gynogenesis. These findings are discussed in relation to studies in which cold shocks are highly successful in inducing diploid gynogenesis and triploidy in eggs of plaice (Pleuronectes platessa).
INTRODUCTION
The #current rise in fish farming has highlighted the need to produce faster growing strains of fish suitable for high-intensity commercial cultivation. One of the rnajor problems in rearing salmon (Salmo salar) at present is the reduced growth rates observed among fish at the onset of sexual maturation and the subsequent death of a proportion of the stock during spawning. This problem can be partly overcome by culling fish early, before sexual maturity checks growth rate, but this is an unacceptable solution where larger, more highly priced fish are needed to satisfy market requirements. Sterility would
288
not only remove those problems associated with sexual maturity but might promote an increase in growth (as flesh production) at a time when food would normally be utilized in the formation of sexual products. Surgical castration has been carried out in salmon with some success (N. Johansson, personal communication), but the method is time consuming and inappropriate in commercial fish farming. Chemical castration, however, may be possible in the future. ,4 genetical approach which lends itself well to massrearing techniques is that of induced triploidy. At present all experimentally induced triploids in sexually reproducing animals have been sterile for the female and partially or completely so for the male. Triploids have been produced for a number of animal species, mostly amphibia (see Fankhauser, 1945, for general review), using thermal shocks applied to eggs soon after fertilization. The effect of the shock is to inhibit metaphase II of the second meiotic division, and subsequent fusion of the diploid egg nucleus with the haploid fertilizing spermatozoan produces a triploid zygote. For successful induction of triploidy the timing, duration and intensity of the shock are all critical (Purdom and Lincoln, 1973). Similar procedures have been used to produce triploids in fish. Swarup (1959) obtained up to 25% triploid sticklebacks (Gasterosteus aculeatus) from eggs cold-shocked for more than 1 h. In plaice (Pleuronectes platessa) and its hybrid with the flounder (Platichthys pesus) cold shocks delivered up to 20 min after fertilization for durations of 3 h or more produced 100% triploids in hatched larvae (Purdom, 1972). Svardson (1945), working with salmonids, cold-shocked eggs of the gwyniad Coregonus lauaretus in a Thermos flask of melting ice 10 min after fertilization for a total duration of 13 h. Cytological examination of young embryos showed that of the 30 eggs examined one was found to be triploid, and the rest were diploid, haploid or mosaics. In a second and less controlled experiment than the first, eggs from the hybrid produced by crossing salmon (Salmo salar) with brown trout (Salmo trutta) were cold-shocked at a temperature of between 0.3 and 1.2”C at varying times after fertilization, ranging from lo-120 min, for durations of l-3 h. Chromosome analysis of 161 embryos showed one to be triploid. Neither of these salmonid triploid eggs was reared beyond the blastula stage. Previous attempts to induce triploidy in rainbow trout (Salmo gairdneri) (C.E. Purdom and R.F. Lincoln, unpublished), using cold shocks of 0°C delivered at 15-min intervals up to 8 h after fertilization, produced only diploid embryos. Evidence of spindle disruption was found in eggs cold-shocked 8 h after fertilization when aneuploid and highly polyploid cells were present in 3.5-dayold embryos, but this was thought to be due to disturbance of the first mitotic division. To explain the lack of effectiveness of the cold shock in these experiments three possibilities may be put forward: the shock may not have been intense enough, the shock may have been delivered too late after fertilization, or the arrested stage of the second meiotic division in the salmonid egg may be particularly insensitive to cold shocks. Ginsburg (1968) has demon-
289
strated metaphase chromosomes of the arrested second meiotic division in sections of pre-fertilized eggs of the lake trout (Salvelinus namaycush), and this is thought to be the normal arrested stage of eggs at ovulation and fertilization in fish. There is no reason to suppose that the arrested stage of the eggs at the time o:t ovulation, as seen in lake trout, would be any different in other salmonid species, and it may be expected that the salmon egg would be most susceptible to suppression of the second meiotic division by cold shocks at or immediately after fertilization. The simultaneous delivery of a cold shock with fertilization is possible in salmonids since water is required to activate the egg before gametic fusion can occur following “dry fertilization” (Ginsburg, 1963). The ability of the salmonid egg to withstand sub-zero temperatures is unknown, but work on plaice eggs has shown that temperatures of -1.7”C (the freezing point depression of sea water at 32°/00 salinity) can be tolerated for up to 6 h or longer immediately after fertilization. The choice of medium with which to depress the freezing point during the cold shock may be important to successful egg development. It is known that eggs of salmon will tolerate up to 20°/oo salinity to hatching, although with some mortality, and that eggs of brown trout, pike (Esox lucius) and roach (Rutilus ru tilus) can develop in liquid paraffin to the blastomere stage (Parry, 1966). It seemed possible, therefore, that salmon eggs would tolerate short periods of immersion in fluids other than fresh water, and experiments reported here investigated the use of sub-zero cold shocks, using saline and glycerol solutions, delivered at or soon after fertilization. The work was carried out during October and November, 1973 at the Fish Breeding Experimental Station, Sunndalsdra, Norway. MATERIALS
AND METHODS
Radiation gynogenesis was used as the experimental technique to investigate the efficiency of the cold shock in inhibiting the second meiotic division of the egg (Purdom, 1969). This method was preferred since in previous experiments difficulty had been experienced in coun%ing the large number of chromosomes present in salmonid fish. The chromosome number of Salmo salar is reported to be 58 (Rees, 1967) and it was felt easier to distinguish between 29 and 58 chromosomes in haploid and diploid gynogenetic eggs than between 58 and 87 chromosomes in diploid and triploid eggs. Sperm was irradiated with 6oCo -y-rays for a total dose of 10’ rad given over a 15 min period. This dose has been found sufficient to inactivate completely all the genetic material in the sperm head (Purdom, 1969). During irradiation and transport, sperm was held in small stoppered glass vials on ice in a Thermos flask. Freshly stripped eggs from wild and reared fish, held in 10 x 1 m circular concrete ponds, were used throughout the experiments. Reared fish (in sea water) were acclimatized to fresh water over a 3 week period just before spawning was expected to begin and held, together with wild fish, at ambient temperatures which varied between 3.0 and 7.O”C throughout the experimental period. Prior to stripping, fish were anaesthetized in chlorobutanol, washed off
290
in fresh water and the vent wiped dry. Eggs were collected and fertilized dry in accordance with normal salmonid hatchery procedure. Chromosome analysis was carried out on cells from the blastodisc removed from 3.5-day-old eggs. Blastodiscs were dissected from living eggs in 1% NaCl, stained on glass slides in two drops of 2% aceto-orcein in 45% glacial acetic acid, and squashed under a cover slip. The cover slip was ringed with nail varnish and the slides were stored in a refrigerator until scored, usually on the following day. Where possible, ten preparations were made from each experimental batch of eggs. At least half the blastodiscs was scored for chromosome spreads and no difficulties were experienced in distinguishing between haploid and diploid preparations. Where haploid and diploid chromosome numbers regularly occurred in the same preparation, then the egg was scored as a mosaic. Where no individual chromosomes could be counted, a subjective assessment could usually be made as to the ploidy of the preparation from the size of the metaphase plate. In addition, eggs could be scored for haploids and diploids after 11-14 days fertilization at lO.O”C when the embryo had reached post-gastrulation. At this stage haploid embryos appeared short and thickened with a wide open blastopore, whereas diploids were typically long and thin with little or no trace of a blastopore present. To make the embryo visible, eggs were cleared in glacial acetic acid-methanol (1:3, v/v) for 1 h in a refrigerator at approx. 4°C. Initial trials were made to assess if control eggs (no cold shock) fertilized with unirradiated sperm would develop normally after immersion for 4 h in full sea water ( 32°/oo ) and in three dilutions of it, and in two glycerol solutions made up ir river water. The freezing point depression of these solutions as observed in the cold shock apparatus are given in Table I. Eggs were transferred to these solutions (at lO.O”C) directly from dry TABLE
I
The freezing point depression of full sea-water (32”/,,) and three dilutions of it and of two glycerol solutions in river water as observed in a the cold shock apparatus ~~.. Solution
Freezing depression (“C)
Sea water 32’/,, (full) 24O I,, 1601,,0 5”lOO
-1.7 -1.3 -0.9 -0.2
Glycerol in river water 5% 8%
-1.2 -1.5
291
fertilization, or after periods of between 2 and 10 min fresh-water treatment following dry fertilization. Development of these control eggs was followed up to post-gastrulation and then discarded. Cold shocks were applied to eggs, using the apparatus shown in Fig.1 which utilized the melting point of ice made from the above solutions (frozen in a cold store at -3O.O”C) to depress the freezing point of water below 0°C. During the cold shock small batches of approx. 100-150 eggs were held in open mesh nylon bags which fitted into compartments of the egg boxes. This arrangement facilitated the addition and removal of eggs to and from the apparatus without causing a disturbance to eggs already being cold-shocked. A diffuser plate, mounted in the top of the bucket, was found necessary to promote uniform melting of the ice during water circulation. The water tended to become heavily charged with air bubbles during its passage through the ice which collected on the surface of the eggs in the boxes, causing them to float to the surface. A glass jar placed beneath the discharge pipe from the bucket, effectively deflected the bubbles to the surface, where they dispersed and calsed no disturbance to the eggs.
Fig.1. Diagram of cold-shock apparatus.
In operation the tank was half filled (30 1) with the saline or glycerol solution required, and the pump was adjusted to complete approx. 1 circulation/min. Small portions of ice made from the same concentration saline or glycerol solution as that in the tank were then packed in the bucket and the diffuser plate was wedged in place. Initially the bucket required recharging every few minutes until the main body of water in the tank had reached the melting point of the ice used, but thereafter topping up every 30 min was found adequate. As long as ice was present in the bucket the water temperature in the tank never fluctuated more than O.l”C. Excess water added to the system from the melting ice was allowed to drain off via the overflow pipe and refrozen as required. Eggs were held at lO.O”C before cold shock (duration 4 h)
292
and incubated in standard hatchery trays in running water of the same temperature. All experiments in which eggs were cold-shocked in saline solutions are summarized in Table II where the original code numbers have been retained. RESULTS
Salinity effects
on egg development
(1) Eggs fertilized dry. Normally fertilized eggs transferred directly to the four salinities investigated were not activated. They remained soft and sticky throughout the period in saline and the perivitelline space was not formed. On transfer to fresh water 4 h later activation changes began to take place in all groups except those from 32°/00 saline, which began to turn white (yolk coagulation) within a few minutes and were all obviously dead 30 min later. The remaining groups showed irregular cleavage, which was delayed by approx. 4 h compared with controls, which confirmed that activation had not taken place in any of the saline solutions used. No development was observed beyond blastulation and all eggs began to die 6 days later, when control eggs had reached gastrulation. (2) Eggs fertilized dry followed by water activation. Eggs were fertilized dry and then activated with fresh water for durations of 2, 4, 6, 8 and 10 min before immersion in various saline solutions. Five experiments were carried out using the three highest salinities, 32, 24 and 16’/,, salt. Eggs activated with fresh water for 8 and 10 min survived the 32’/,, salt treatment, but those activated for 2, 4 and 6 min showed mortalities of 100, 93 and 4%, respectively, as indicated by whitening of the yolk during the first 30 min on transfer back into fresh water. Yolk coagulation was not observed in any eggs treated with 24 and 16’/,, and these eggs continued to show progressive changes associated with activation during the 4 h immersion in saline from all pre-treatments in fresh water. Cleavages were highly irregular in the two surviving groups from the 32’/,, salt-treated eggs and also from those groups which received 2 min water activation prior to immersion in 24 and 16’/,, saline. Cleavages were more normal in all the remaining groups and, as expected, no evidence of a cleavage delay was found for any eggs that had first been activated with fresh water before saline treatment. Development was monitored to post-gastrulation, when closure of the blastopore was completed, and all eggs reached this stage before being discarded. In one experiment where eggs were treated with 16°/oo saline ll-dayold embryos from the 2 min fresh-water activated group appeared identical to known haploid embryos of the same age produced from eggs fertilized with spermatozoa which had received 10” rad 6oCo y-rays. It was thought possible that the superficial haploid appearance of these embryos may have been caused by some abnormal ontogenic development caused by the saline treatment
II activation,
10 10 10 3 5 5 5 10 10 10 10 9
1 5 10 10 15 20 40 10 15 20 30 40
-1.3
-1.7
-1.7
32
32
s2
s4
-0.2
5.3
24
6 5 10 4
1 5 10 15
(“C)
Number of haploid gynogenetic embryos 3
~~
of diploid
Number of diploid gynogenetic embryos
on the production
(“1”“)
Number of blastodisc preparations scored
water
Minutes after water activation cold shock given
after
Temperature of cold shock
times
Saline concentration
applied to salmon eggs at varying of cold shock, 4 h
s13
S6
Experiment number
Effect of sub-zero cold shocks, gynogenetic embryos. Duration
TABLE
.~___
294
per se. Another experiment, not specifically comparable since eggs were wateractivated following dry fertilization for 1, 5, 10 and 15 min before immersion in 16°/oo saline, nevertheless confirmed the possibility that these embryos were haploid; in this experiment chromosome analysis of blastodiscs from 3.5-dayold eggs revealed seven haploids, two mosaics and one diploid from ten preparations of the 1 min activated group. A further two haploids and two mosaics from ten preparations were also observed in the 5-min water-activated group. the remaining groups were all diploid. From these two experiments it would appear that 16°/oo saline treatment soon after water activation is detrimental to the formation of a diploid zygote, possibly through destruction of the fertilizing spermatozoan after it has activated the egg to develop (haploid gynogenesis). Although no such phenomenon was observed in eggs treated with 24°/00 saline, all embryos appearing normal 14 days after fertilization, it became apparent that saline solutions were highly unsuitable media in which to cold-shock eggs. Egg development
in glycerol
The anomalies in egg development observed after treatment with saline solution led to the search for a more suitable medium in which two cold-shock eggs. The use of glycerol in many cryogenic solutions to deep-freeze sperm suggested that this would be a suitable alternative. Two solutions containing 5 and 8% glycerol were made up in river water and trials were carried out with control eggs, as for the saline solutions. Eggs added directly from dry fertilization activated in both 5 and 8% glycerol, as seen from direct observation on the eggs and by comparisons made with untreated controls of the times to first cleavage. All cleavages looked normal and analysis of blastodiscs from 3.5day-old eggs revealed only diploid chromosome numbers. The appearance of 14-day-old embryos was identical with those of untreated eggs. Similar results were obtained with eggs pretreated with water before immersion in the glycerol solutions and no abnormal development was observed as with the saline solutions. Glycerol thus appears to be a very suitable medium in which to cold-shock eggs. Effect of cold shocks, using saline, in inducing diploid gynogenesis The comparatively short season over which eggs can be obtained, coupled with the long development time of the salmon egg, necessitated the early evaluation of the effect of cold shocks using saline solutions before its haploidinducing effect in some of the control experiments was discovered. Nevertheless, the effect of the cold shock itself, using saline, in inhibiting the second meiotic division could still be evaluated using the technique of radiation gynogenesis. Table II lists the results of four experiments using three different cold shock temperatures. The experiment using 32’/,, saline was repeated because of the small number of blastodiscs preparations which gave countable chromo-
295
some spreads in experiment S.2. Eggs could not be cold-shocked sooner than 10 min after water activation using 320/00 saline, because of the complete mortality of eggs observed from the initial control experiments during this period. The results show that none of the treatments was effective in producing diploid gynogenetic embryos, except those eggs cold-shocked at -0.2”C 1 min after activation in experiment S6 (Table II). All other embryos from this group, together with all those from the other experiment.s, revealed only haploid-looking embryos when scored 14 days after fertiliza-Lion. Effect
of cold shocks using glycerol solutions
in inducing diploid gynogenesis
An initial experiment was carried out in which eggs were cold-shocked in l-l glass jars containing 500 ml of 5% glycerol made up in river water. The jars were p;.aced in the egg containers in the cold shock apparatus containing 32O/,, saline, which reduced the temperature of the glycerol to -1.7”C. Approx. 100 eggs were added to each jar direct from dry fertilization and at intervals of 1, 2, 5 and 10 min after water activation, and cold-shocked for a duration of 4 h. Im.mediately after the addition of eggs to each jar the temperature rose to -1.5”C but returned to -1.7”C after approx. 10 min and thereafter remained at this temperature throughout the cold-shock period. It was found that at this temperature 5% glycerol was super-cooled but no ice crystal formation was observed in any of the jars containing eggs. After the cold shock the eggs were rinsed ;.n water at 10°C and returned to the incubation trays for rearing on. From ten blastodisc preparations made for each group, two diploid eggs were found from those water-activated for 1 min: all the remaining groups were haploid. All the surviving eggs were scored 14 days later but no diploid embryos were observed in any of the cold-shocked groups. Two more experiments were carried out using 5 and 8% glycerol, which was frozen down and used in the cold-shock apparatus as for previous experiments using saline. The temperatures achieved using these two solutions were -1.2 and -1.5”C, respectively. The difficulty of obtaining irradiated sperm at this period necessitated the use of normal sperm to fertilize eggs. Eggs in both experiments were cold-shocked from dry fertilization and after 1 min water activation only. Again, no evidence of inhibition of the second meiotic division was observed, either from chromosome analysis of blastodiscs or from the appearance of later embryos. DISCUSSION
AND
CONCLUSIONS
Cold shocks below 0°C applied to salmon eggs directly from dry fertilization with irradiated sperm, and after short periods in water following fertilization, were not successful in inducing diploid gynogenesis. Some evidence of inhibition of the second meiotic division was obtained from eggs cold-shocked 1 min s.fter water activation following fertilization. Blastodisc preparations
296
from 3.5-day-old eggs revealed that diploid chromosome numbers were present in three out of nine eggs cold-shocked at -0.2”C (in saline) and two out of ten eggs cold-shocked at -1.7”C (in glycerol). A later examination of eggs from the same groups revealed embryos showing the typical characteristics of the haploid syndrome, which include short thickened bodies and open blastopores. The failure of cold shocks to inhibit metaphase separation at the second meiotic division strongly contrasts with similar experiments on eggs of the plaice (Pleuronectes platessa), where cold shocks have been highly successful in inducing both diploid gynogenesis and triploidy. The contrasting sensitivity of the eggs of the two species in duplicating the maternal chromosome set may be a function of slight differences in the maturation stage of the egg at the time of fertilization and cold shock. Plaice eggs are thought to be ovulated and fertilized at the stage of prometaphase of the second meiotic division. No direct evidence for this is available since the plaice egg is small, making direct cytological examination extremely difficult, but the high frequency of triploid induction (100% in hatching larvae) following cold shocks, a unique phenomenon in the field of experimental polyploidy, implies that an earlier stage in the second maturation division of the egg is involved. Metaphase chromosomes of the second meiotic division have been demonstrated in sections of prefertilized eggs of the lake trout, and it is generally supposed that this is the arrested stage for all salmonid eggs at the time of fertilization. The apparent insensitivity of salmon eggs to cold shocks suggests that at metaphase the second meiotic division has already progressed to a stage where thermal shocks cannot easily disrupt chromosome separation. The uniformity of response of plaice eggs to cold shocks in inducing inhibition of the second meiotic division has been attributed to the method of ovulation in this species (Purdom, 1969). Here ovulation occurs serially, in batches of a few thousand eggs at a time every 2 or 3 days, until the ovary is depleted and spawning ceases. Any over-ripe eggs fail to fertilize, leaving the remainder as a uniform sample. In contrast, salmonid eggs are shed progressively from the ovary into the body cavity, where they may remain for a considerable period of time before release. The period over which the eggs are held in the body cavity may be a prerequisite for the final ripening process in which the eggs mature to metaphase of the second meiotic division before release. This may explain the lack of any uniform effect of cold shocks in the experiments reported here and may represent a natural adaptation in salmon and other salmonids to low temperatures, which may be frequently encountered on the spawning beds. It may be expected that artificially stripped eggs would include a small proportion that had recently been ovulated into the body cavity and which had not matured to the stage of metaphase of the second meiotic division but were nevertheless potentially capable of fertilization and further development. These young eggs may be of a stage in which cold shocks could be effective in inducing diploid gynogenesis or triploidy. Although suppression of the second meiotic division, using cold shocks, was
297
not achieved in the present experiments, the reported case of one triploid rain bow trout amongst 18 hatchery reared fish (Cuellar and Uyeno, 1972) indicates that polyploidy is possible in salmonids. REFERENCES Cuellar, 0. and Uyeno, T., 1972. Triploidy in rainbow trout. Cytogenetics, 2(6): 508-515 Fankhauser, G., 1945. The effect of changes in chromosome numbers on amphibian development. Q. Rev. Biol., 20: 20-78 Ginsbmg, A.S., 1963. Sperm-eggs association and its relationship to the activation of the egg in Salmonid fishes. J. Embryol. Exp. Morphol., 11: 13-33 Ginsburg, A.S., 1968. Fertilisation in Fishes and The Problem of Polyspermy. “Nauka” Publ. House (Jerusalem, Israel Program for Scientific Translation, National Marine Fisheries Service, 1972), 366 pp., TT 71-50111 (translation Russian) Parry, G., 1966. Osmotic adaptation in fishes. Biol. Rev., 41(3): 392-444 Purdom, C.E., 1969. Radiation-induced gynogenesis and androgenesis in fish. London, 24(3): 431-444 Purdom, C.E., 1972. Induced polyploidy in plaice (Pleuronectes platessa) and with the flounder (Plutichthys pesus). Heredity, London, 29(l): 11-24 Purdom, C.E. and Lincoln, R.F., 1973. Chromosome manipulation in fish. In: J.H. Schroder (Editor), Genetics and Mutagenesis of Fish. Springer Verlag, pp.8:3--89 Rees, H., 1967. The chromosomes of Salmo salar. Chromosoma, 21: 472-474 Svardson, G., 1945. Chromosome studies of Salmonidae. Meddn St. Unders-o. SottvattFisk., 23: l-151 Swarup, H., 1959. 129--142
Production
of triploidy
in Gasterosteus
aculeatus
(L.).
MOSCOW, for U.S. from the
Heredity, its hybrid
Berlin,
FiirsAnst.
J. Genet.,
56: