Sexual maturation in triploid male plaice (Pleuronectes platessa) and plaice x flounder (Platichthys flesus) hybrids

Triploid male plaice, Pleuronectes platessa , and plaice x flounder, Platichthys flesus , hybrids produced from cold-shocked eggs were reared to maturity at three years of age. The testes of triploid hybrids were similar in macroscopic appearance and size to those from control fish but in histological section spermatocyte and spermatid cell formation was grossly abnormal. In two fish the majority of cells appeared to be aborted spermatids and spermatocytes although there were some spermatozoa with enlarged heads and thick bent flagellae.
The testes of mature triploid plaice were similar to controls both in shape and size and in histological section. Spermatocyte and spermatid cell formation was normal, apart from an increase in cell and nuclear size, and fully motile spermatozoa were produced from several fish. Plaice eggs fertilized with semen from triploids showed a slight reduction in fertility compared with controls but developed normally to gastrulation with little mortality. Beyond gastrulation there was increasing mortality associated with abnormalities in the appearance of embryos. Few hatched and none of these survived longer than 48 h. The value of triploidy in fish farming is briefly discussed.     

Sexual maturation in triploid rainbow trout, Salmo gairdneri Richardson

This paper compares some morphological and endocrinological characteristics of diploid and triploid rainbow trout.
Significant differences were found between diploid and triploid females in GSI, condition factor, gut weight, liver weight and percentage dress-out, and between diploid and triploid males in GSI, condition factor and gut weight.
Diploid females had large, well-developed ovaries containing yolk-filled secondary oocytes whereas the triploids had only string-like ovaries containing nests of oogonia. No primary oocytes were present.
All the diploid males produced copious quantities of milt but it was possible to express a thin, watery milt containing motile spermatozoa from only two of the 12 triploid males. Testes weights in triploids were similar to those of diploids but, while the diploid testes were packed with spermatozoa, those of the triploids consisted mainly of spermatocytes and spermatids with few spermatozoa present. Measurements of the heads of spermatozoa revealed that those from triploids were larger and had a wider size range than those from diploids.
Levels of testosterone and 11-ketotestosterone in triploid and diploid males were not significantly different. However, levels of testosterone and 17β-oestradiol in diploid females were considerably higher than those of triploid females.     

Gross and histological observations of ovarian development and related condition changes in American plaice

The largest increase in gonadosomatic index of American plaice Hippoglossoides platessoides occurred between January and February. Hepatosomatic index was lowest in the spawning season, suggesting cessation of feeding activity during spawning, but rose immediately in July when condition factor recovered from its low spawning level. Increases in white muscle water content during the spawning season suggests protein depletion. Conservation of some white muscle fibres is suspected, but no regular pattern in area conserved was found. Gonad development was advanced in December and in April some gonads were showing evidence of spawning. Ovaries from spawning females showed hydration patterns and histological detail consistent with batch or serial spawning strategy. Evidence of recent spawning activity, including the presence of postovulatory follicles, was found in ovaries that also contained oocytes undergoing exogenous vitellogenesis. Oocyte size frequency distributions show a gap in size between cortical alveoli and vitellogenic oocytes prior to spawning which does not exist during the spawning season. This may represent the ability of plaice to push oocytes through vitellogenesis from a previtellogenic condition during the spawning period. The increased white muscle moisture during June and July may reflect the use of muscle protein to bring oocytes through vitellogenesis for the current spawning season.     

Induced triploidy in carp, Cyprinus carpio L.

A method for mass production of triploid carp was developed by the cold treatment of eggs after fertilization. Triploidy was demonstrated by cytophotometry and chromosome preparation techniques. Of the haploid embryos, 100% showed morphological abnormalities and died soon after hatching. Haploid embryos of maternal and paternal origin occurring in the course of cold treatment were distinguished by using a genetic marker. Among morphologically normal larvae, the frequency of triploidy was 100%, and the viability of the triploid embryos, larvae and the juvenile fish did not differ from the diploid control. Sex differentiation started in the triploid fish, but was greatly retarded. Gonad size and the small number of growing oocytes suggest that most of the triploid individuals will be sterile. Triploid carp grew at the same rate as diploid controls in the laboratory test.     

Effects of exogenous estradiol-17β on early growth and gonadal development of diploid and triploid female rainbow trout (Oncorhyncus mykiss)

Triploidy is a viable condition in teleosts. However, in many salmonids, the triploid condition in the female results in sterility as gametogenesis appears to be disrupted. Although the underlying mechanisms regulating the gonadal development of teleosts have not been clearly elucidated, the reversal of phenotypic sex by the administration of the appropriate exogenous steroid during early development supports the argument that gonadal steroids play a pivotal role in sexual differentiation and subsequent gonad development in these fish. To determine whether the failure of normal ovarian development in triploid female rainbow trout (Oncorhynchus mykiss) is due to an absence or reduction of endogenous sex steroids, ovarian morphology was compared between diploid and triploid juvenile rainbow trout treated with exogenous estradiol-17β (E₂). The ovaries of both untreated and E₂ treated diploid fish, at 145 days post-fertilization, contained synchronously developing oocytes in the perinucleolar stage, whereas ovaries from untreated and estradiol-treated triploid fish of the same age were considerably smaller and devoid of developing oocytes. No differences in the ovaries of triploid untreated fish and triploid fish treated with E₂ were observed. It is reported that exposure to exogenous E₂ during the period of gonadal differentiation is not sufficient to induce oocyte development in triploid rainbow trout. © 1996 Wiley-Liss, Inc.     

Predation by sprat and herring on pelagic fish eggs in a plaice spawning area in the Irish Sea

Predation on planktonic fish eggs was examined by stomach content analysis of sprat and herring sampled in a plaice spawning area to the east of the Isle of Man in March 1993. Plankton samples were taken to examine prey selection. The clupeids showed a selection for the later developmental stages of plaice eggs. Plaice eggs had a refuge in size from predation by sprat <80 mm total length which selected smaller non-plaice eggs. However, herring and sprat >80 mm total length showed a strong selection for plaice eggs, presumably due to their large size.     

Prey resources before spawning influence gonadal investment of female, but not male, white crappie

In this study, an outdoor pool experiment was used to evaluate the effect of prey resources during 4 months before spawning on the gonadal investments of male and female white crappie Pomoxis annularis , a popular freshwater sportfish that exhibits erratic recruitment. Fish were assigned one of three feeding treatments: starved, fed once every 5 days (intermediate) or fed daily (high). All measurements of male testes ( i.e. wet mass, energy density and spermatocrit) were similar across treatments. Conversely, high-fed females produced larger ovaries than those of intermediate-fed and starved fish, and invested more energy in their ovaries than starved fish. Compared to pre-experiment fish, starved and intermediate-fed females appeared to increase their ovary size by relying on liver energy stores ('capital' spawning). Conversely, high-fed females increased liver and gonad mass, implying an 'income'-spawning strategy (where gonads are built from recently acquired energy). Fecundity did not differ among treatments, but high-fed fish built larger eggs than those starved. Females rarely 'skipped' spawning opportunities when prey resources were low, as only 8% of starved females and 8% of intermediate-fed females lacked vitellogenic eggs. These results suggest that limited prey resources during the months before spawning can limit ovary production, which, in turn, can limit reproductive success of white crappies.     

Comparative morphology and histology of the ovaries of the spined loach Cobitis taenia L. and natural allopolyploids of Cobitis (Cobitidae)

The maturity of ovaries, gonado-somatic index and size of oocytes in particular developmental stages during the reproductive period of spined loach Cobitis taenia and allotriploid and allotetraploid Cobitis were examined. The ploidy levels of all the loaches were determined according to karyotypes. All the investigated females spawned multiple times and the spawning period was from the end of May (water temperature c. 18·5° C) to mid- or the end of July. Some of the triploids had a longer spawning period which lasted until the end of August. Each female could lay several portions of eggs which differed in size and number during the spawning season. The relative proportion of oocytes of each stage in the ovaries during the reproductive period changed. The sizes of oocytes at the particular stages of triploids were statistically significantly larger than oocytes in the respective stages in C. taenia (diploid). The sizes of the oocytes (each stage) of C. taenia from both populations were not significantly different.     

Plasma concentrations of ovarian steroids in relation to oocyte final maturation and ovulation in female plaice sampled at sea

Blood plasma concentrations of free 17 β -oestradiol, free testosterone and glucuronidated testosterone were strongly positively related to the percentage of vitellogenic oocytes remaining in the ovaries of plaice Pleuronectes platessa caught at sea–being at their highest in pre-spawning (stage IV) females (i.e. those in which the oocytes were close to fully grown, but had not yet entered the stage of final maturation). In contrast, the concentrations of free and sulphated 17,20 β -P, 3α aL ,17, 20 β -P-5 β , and 3 α ,17,21-P-5 β were at their lowest in stage IV females. Free 17,20 β -P (the putative maturation-inducing steroid) became only slightly elevated (less than twofold) during spawning (i.e. in stage V and VI females with hydrated and/or ovulated eggs). Sulphated 17,20 β -P and 3 α ,17,21-P-5 β became slightly more elevated (three- to fourfold). However, sulphated 3 α , 17,20 β -P-5 β concentrations increased 30-fold and were at their highest in fish in which only 40% of vitellogenic oocytes remained in the ovaries. Sulphated 17,20 β -P, 3 α , 17, 20 β -P-5 β and 3 α ,17,21-P-5 β concentrations were significantly positively related to hyaline oocyte batch size; and sulphated 17,20 β -P and sulphated 3 α , 17,20 β -P-5 β were significantly negatively related to the degree of hydration of the hyaline oocytes. None of the steroid concentrations, however, was related to the time of capture. More ovulated females were found in the afternoon than at any other time of the day.     

Gonadal morphology of normal and sex-reversed triploid and gynogenetic diploid coho salmon (Oncorhynchus kisutch)

In this study, the gonadal morphology of untreated and sex-reversed juvenile triploid and gynogenetic diploid coho salmon was compared with that of diploids. Testes of triploids were of the same size as those of diploids. Spermatogonia, however, were significantly bigger than those of diptoids in both diameter (P<0·001) and volume (P<0·01), suggesting that this characteristic can be a useful indicator of ploidy in the early stages of gonadal development. In females, induction of triploidy did not affect the lamellar structure of the ovaries but reduced their size considerably. Further, these ovaries had no oocytes. Treatment of triploids with oestrogen resulted in the feminization of genotypic males, which had ovaries similar to those found in tripioid females. However, gonads of triploid males partially sex-re versed into females were identified by their enlargement, the presence of remnants of the male vascular system, and by the appearance of ovarian lacunae and germinal and somatic cells typical of triploid females, Induction of gynogenesis resulted in 100% females, of which 34% had ovaries of reduced size with areas devoid of oocytes. However, and contrary to what has been found in cyprinids, no male germ cells were observed in these ovaries. This discrepancy may reflect differences, in the mechanisms of sex determination between salmonids and cyprinids. Treatment of gynogenetics with androgen increased the number of fish with abnormal ovaries but also resulted in the production of phenotypic-male gynogenetic diploids, of which 11% had testes indistinguishable from those of untreated control diploids.     

The interrelation of the size of fish eggs, the date of spawning and the production cycle

The variability in size of pelagic and demersal marine and freshwater fish eggs is examined. The difference between the smallest and largest volumes, based on published figures for the diameters, is large in many species. In marine species with planktonic eggs, the median percentage difference is just over 100%, and this is similar in species with demersal eggs and in freshwater fish.
The available evidence suggests that geographical differences in egg size are small, but in marine fish there is a well-known seasonal decline in egg size. In herring it has previously been shown that egg size in different spawning groups can be correlated with the timing of the production cycle. A similar correlation can be seen in the seasonal shift in time and locality of spawning, and egg size, of the plaice. Sufficient data on seasonal freshwater fish egg variations are not available, but the time of spawning does appear to be linked with the availability of food for the larvae in both lake and stream species.     

Sexual dimorphism in the energetics of reproduction and growth of North Sea plaice, Pleuronectes platessa L.

The chemical composition and energy content of North Sea plaice during the spawning period were examined in mature males and females and in immature fish, to study differences in the allocation of energy over reproduction and somatic growth between the sexes. At the beginning of the spawning period mature males and females had equal dry weights of lipid that were 70% higher than in immatures. Protein content in mature males was equal to that in immatures but was 23 % higher in mature females. Immature males and females did not differ in chemical composition. At the end of the spawning period, spent and immature fish had equal lipid contents, but protein content in spent females was 10% lower than in spent males, and 17% lower than in immatures. Gross energy content of the body decreased by 44% (65·2 to 36·3 J cm^-3) in mature females, 27% (55·0 to 40·OJ cm^-3) in mature males, and 9% (48·7 to 44·2J cm^-3) in immatures. Energy content of plaice eggs was estimated at 6·60 kJ per 1000 eggs. Reproductive investment was estimated from the energy loss during the spawning period and included the energy of sex products and spawning metabolism. Somatic growth comprised the annual increase in energy content of fish. The pattern of energy allocation over reproduction and somatic growth differed between males and females. Males started their reproduction at a smaller length and a younger age and allocated a higher proportion of the available energy into reproduction than females. Available energy resources for somatic growth and reproduction (surplus production) were equal between the sexes up to a length of about 30 cm. Beyond this length male surplus production levelled off whereas female surplus production continued to increase. The differences in surplus production and the allocation patterns are discussed. For female plaice the energy allocated into egg production was estimated as between 48 and 64% of the total amount of energy lost during spawning. The remaining energy is used for metabolism during the spawning period, yielding an estimate of the metabolic rate of mature females of between 6·4 and 9·1 kJ day^-1. A maximum estimate of the metabolic rate of mature males was 7·4 kJ day^-1.     

Temperature-dependent development rates of plaice (Pleuronectes platessa L.) eggs from the Irish Sea

Experimentally derived temperature-dependent development ratesfor planktonic fish eggs are required for developing IndividualBased Models (IBMs) of early life history stages and for estimatingdaily egg production in ichthyoplankton surveys. Developmentrates of Irish Sea plaice eggs were experimentally determinedfor temperatures between 4.5 and 12° C. Differences in developmentrates were apparent when these results were compared with literaturedata for eggs from North Sea parents. On average, plaice eggsfrom the Irish Sea developed more rapidly and hatched up to2 days earlier compared with North Sea eggs incubated at thesame temperature. In terms of parental effects, larger femalestended to produce larger eggs but egg size decreased over successivebatches from the same female within a year. Temperature-dependentegg development rates were influenced by egg size in a non-linearmanner for eggs incubated at 6° C, but egg size had no effectat higher temperatures. Since most plaice eggs sampled fromthe Irish Sea during ichthyoplankton surveys in 1995 and 2000were found in waters cooler than 8° C, such effects maymodify egg development rates in the wild.     

Reproductive capacity of triploid loaches obtained from Hokkaido Island, Japan

Reproductive capacity was investigated in naturally occurring triploid individuals of the loach Misgurnus anguillicaudatus collected from Memanbetsu Town, Abashiri County, Hokkaido Island, Japan. These triploids have been considered to appear by accidental incorporation of the haploid sperm genome from normal diploid into unreduced diploid eggs from the clonal lineage that usually reproduces unisexually. By fertilization with sperm from the normal male, one triploid female gave many inviable aneuploid (2.1–2.7n) and very few tetraploid progeny, whereas the other produced both diploid and triploid progeny. The results suggest that at least four different types of eggs can be formed in triploid females in this locality. In contrast, no progeny hatched when eggs of the normal female were fertilized with sperm or sperm-like cells obtained from triploid males. These gametes exhibited inactive or no motility after adding ambient water. They had larger head sizes than those of normal haploid sperm and had a short or no tail. Although their ploidy was triploid or hexaploid, a small number of haploid cells were detected in the semen by flow cytometry. Thus, triploid males were generally sterile, but they have a little potential for producing very few haploid sperm.     

Comparison of the gonadal development and plasma levels of sex steroid hormones in diploid and triploid sea bass, Dicentrarchus labrax L

The goal of this study was to compare the reproductive physiology of triploid and diploid European sea bass (Dicentrarchus labrax L.). Gonads of diploid and triploid fish (males and females) were examined both microscopically and macroscopically, together with the plasma levels of the major sex steroids produced (testosterone and estradiol-17beta) when fish were adults. Prior to sexual maturation, the gonadosomatic index (GSI) of triploid males was similar to that of diploids. However, the GSI in 4-year-old adult triploid males was 1.8 times lower than that of diploids (P < 0.05). All diploid males exhibited normal gonadal development. In contrast, in triploid males spermatogenesis was impaired during late meiosis, affecting severely spermiogenesis. This was achieved by an increasing imbalance in the amount of DNA present in daughter cells of the same type as spermatogenesis progressed, as demonstrated by abnormal cell sizes, culminating in inviable spermatids. Thus, no spermiating triploid fish were observed during 4 years, which included three full consecutive maturation cycles. Furthermore, the germ cells from triploids were significantly larger than those from diploids (P < 0.001). Seasonal profiles of plasma levels of testosterone in 4-year-old males were essentially similar in both ploidies. On the other hand, triploid females had rudimentary ovaries containing oogonia and primary oocytes that were arrested during meiotic prophase I, while diploid females exhibited all stages of ovarian development. Diploid females showed levels of testosterone and estradiol-17beta significantly higher than those of triploids (P < 0.05), in which no endocrine signs of maturation were observed at all. Regarding sex ratios, triploids had 10% more females than diploids (P < 0.05) but in both ploidies males predominated, as is usually found in this species under culture conditions. These results show that triploidy blocked the initial phases of meiosis in females and the latter ones in males, resulting in the absence of or reduced gonadal development, respectively. In conclusion, we provide an explanation for the lack of gonadal development in triploid male fish, and, to the best of our knowledge, we report for the first time a case in which induced triploidy completely blocks meiosis in both sexes, thus conferring functional sterility in the sea bass.     

Variation with age in the timing and duration of spawning in American plaice

Spawning time of female American plaice Hippoglossoides platessoides varied significantly with age. This effect, however, was not consistent across years. Generally, younger fish spawned earlier than older fish, but in the 1990s they spawned later than older fish. Spawning duration also varied with age, with younger fish generally having a longer spawning period than older fish. Changes in depth distribution were related to changes in both spawning time and duration. As depth increased, spawning time became later and duration became shorter. Fish <11 years old showed the largest change in both spawning time and depth distribution.     

Apparent independence of the spawning and moulting cycles in female Antarctic krill (Euphausia superba Dana)

Summary Sixty female Antarctic krill (Euphausia superba Dana) spawned in shipboard experiments and the interval between egg-laying and ecdysis was noted. The number of eggs laid per female ranged from 263–3662, most females produced only one batch of eggs before moulting, and the post-spawn ovaries of all females contained few, if any, mature oocytes. As reported in other studies, the total number of eggs produced per female was not well correlated with body size. Females appeared to spawn at all times during the moulting cycle and although no diurnal rhythm in spawning was observed, moulting occurred mainly at night-time despite the animals being kept in near-constant darkness. No evidence of synchronous moutling was detected.     

Why are triploid zebrafish all male?

Adult triploid zebrafish Danio rerio has previously been reported to be all male. This phenomenon has only been reported in one other gonochoristic fish species, the rosy bitterling Rhodeus ocellatus, despite the fact that triploidy is induced in numerous species. To investigate the mechanism responsible, we first produced triploid zebrafish and observed gonad development. Histological sections of juvenile triploid gonads showed that primary growth oocytes were able to develop in the juvenile ovary, but no cortical alveolus or more advanced oocytes were found. All adult triploids examined were male (n = 160). Male triploids were able to induce oviposition by diploid females during natural spawning trials, but fertilization rates were low (1.0 ± 3.1%) compared with diploid male siblings (67.4 ± 16.6%). The embryos produced by triploid sires were aneuploid with a mean ploidy of 2.4 ± 0.1n, demonstrating that triploid males produce aneuploid spermatozoa. After confirming that adult triploids are all male, we produced an additional batch of triploid zebrafish and exposed them (and a group of diploid siblings) to 100 ng/L estradiol (E2) from 5 to 28 dpf. The E2 treated triploids and nontreated triploids were all male. The nontreated diploids were also all male, but the E2 treated diploids were 89% female. This demonstrates that triploidy acts downstream of estrogen synthesis in the sex differentiation pathway to induce male development. Based on this and the observations of juvenile gonad development in triploids, we suggest that triploidy inhibits development of oocytes past the primary growth stage, and this causes female to male sex reversal.     

Reproduction and the origin of polyploids in hybrid salamanders of the genus Ambystoma

Eggs and larvae produced by diploid, triploid, and tetraploid females collected from breeding ponds on Pelee Island in Lake Erie were studied to examine the reproductive mechanism. No instance of parthenogenesis was found as all examined females required sperm to produce viable progeny. Diploid females produced diploid and triploid larvae, triploid females produced triploid and tetraploid larvae, and tetraploid females produced triploid and tetraploid larvae. The majority of the eggs produced by hybrid females do not develop or do not complete embryogenesis. Electrophoretic examination of females and their offspring demonstrate that the male genome is being incorporated in reduced as well as unreduced eggs produced by all three ploidy classes of females. The elevation of ploidy among Pelee Island Ambystoma is attributed to sperm incorporation in unreduced eggs. Triploid as well as tetraploid individuals are constantly being produced. A critical examination of the literature on parthenogenetic or gynogenetic modes of reproduction in North America Ambystoma hybrids shows no conclusive evidence supporting these modes and it is suggested that the reproductive mechanism found among Pelee Island female hybrids may be more generally applied to other hybrid Ambystoma populations.     

Seasonal changes in serum lipid composition of the plaice, Pleuronectes platessa L.

Seasonal changes in the major lipid classes of plaice serum were examined in fish caught off the north-east coast of Scotland. Both male and female plaice were sampled monthly between June 1983 and May 1984. Total lipid, total cholesterol, free cholesterol, triglycerides, phospholipids and non-esterified fatty acids were measured using commercial kits validated against standard reference methods. There were no significant differences in lipid levels between male and female fish and, although there were real differences between months for each lipid class, there was no regular pattern over the 12-month period. All the lipid class values, however, reached a maximum in September, which could be related to the increase in feeding after spawning. The smaller peak in February could be associated with the mobilization of lipid reserves for gonadal development and to overcome the effect of starvation during spawning.