The reproductive physiology of three age classes of adult female diploid and triploid brook trout (Salvelinus fontinalis)

Triploid female fish show impaired gametogenesis and are unable to produce viable offspring. The reproductive physiology of artificially-induced triploid female salmonids has been well described up until the time of first sexual maturation in diploids, but few reports exist for older triploids. This study reports the influence of triploidy on growth, ovarian development and reproductive endocrinology among three age classes of female brook trout (Salvelinus fontinalis) in comparison to sibling diploids. Triploids were larger than diploids for most of the study period, but the difference was statistically significant only during maturation and spawning of 2⁺ diploids. Plasma estradiol-17 (E₂), testosterone (T) and vitellogenin (VTG) levels in triploids were generally lower than in diploids, and VTG was the only parameter to show seasonal fluctuations resembling those of diploids. Triploids showed significantly lower GSI and total oocyte number than diploids of similar age, and only half of all triploids sacrificed during the study (n=56) had developing oocytes in their ovaries. At age 3⁺, 13 of 19 triploid females had oocytes at various stages of development, including perinucleolar, yolk vesicle and yolk globule stages. In addition, three of these fish had collectively produced 72 mature stage oocytes. Thus, whereas diploid brook trout can produce mature oocytes as two-year-olds, triploids cannot do so until four years of age, with the number of mature oocytes being greatly reduced.     

Plasma levels of Gonadotropin-II and gonadal sex steroids in triploid catfish, Heteropneustes fossilis (Bloch)

Triploid fish have under-developed gonads due to altered reproductive endocrinology. Triploids of Indian catfish (H. fossilis) showed significantly reduced plasma levels of gonadotropin (GtH-II), testosterone (T) and estradiol-17 (E₂) than that of diploids throughout the year, except for the resting phase, irrespective of sex. Plasma levels of GtH-II were significantly different (p<0.001) between diploid and triploid fish during preparatory, prespawning and spawning phase. The plasma testosterone contents in triploids were significantly less (p<0.001) than that of diploids, except during the resting phase. Triploid females showed very low titres of estradiol-17 (<1 ng ml^–1) throughout the annual reproductive cycle in contrast to highly fluctuating levels in diploid females. Thus, this study for the first time provides information on reduced levels of GtH-II and sex steroids in plasma of male triploid fish and additional information on species-specific alteration of sex hormones in female triploids.     

Aquaculture performance of triploid barfin flounder Verasper moseri

ABSTRACT:   To evaluate the aquaculture performance of triploid barfin flounder Verasper moseri , the sex ratio, maturation, growth and the relative proportion of body parts were examined. The sex ratio of triploids was similar to diploids under communal rearing conditions, but the proportion of female diploids was higher than that of triploids under separate rearing conditions. The gonadosomatic index of triploid females was very low even during the spawning season, and the ovaries were rudimentary. These results suggest that triploid barfin flounder females were sterile. In addition, triploid males produced a small quantity of milt containing very few spermatozoa with abnormal shapes. Spermatozoa obtained from triploids were aneuploidies. When normal eggs were fertilized with sperm from triploid males, no fry developed. These results suggest that triploid barfin flounder males were functionally sterile. Triploid males grew more slowly than diploid males, and triploid females showed similar or slower growth than diploid females, whether reared separately (23 months) or communally (35 months). The ratios of visceral weight to the edible parts for triploid males were similar to those for diploid males, but ratios for triploid females were higher than for diploid females during the spawning period. In conclusion, a significant improvement of growth was not found in triploid barfin flounders.     

The breeding potential and biochemical composition of triploid Manila clams, Tapes philippinarum Adams and Reeve

In 1989 and 1990. triploid Manila clam, Tapes philippinarum Adams and Reeve, seed were reared to 15-20 mm at the Fisheries Laboratory, Conwy, and planted out in the Menai Strait, North Wales. In each of the summers of 1992 and 1993, three of these batches, at 2, 3 or 4 years old, were returned to the laboratory to assess ploidy, size, spawning potential and biochemical composition. Percentage triploidy at this time was similar to that in the seed. After 6 and 8 weeks of warmwater conditioning. Only 45 out of l21 triploids (37%) were induced to spawn by thermal shock, with only one spawning as a male. By comparison, 75% of diploid clams spawned with a 1:1 ratio of males to females. Mean fecundity of triploids was significantly lower than that for diploids, at 0.497 compared with 1.54 million eggs per female. Compared with eggs from diploid females, eggs from triploids were larger and significantly fewer of them developed into D-larvae when fertilized by sperm from diploid males. Triploid clams were heavier and had a higher condition index and carbohydrate content than diploids of the same age, but lipid levels were similar. Potential advantages of producing and cultivating 100% triploid batches of Manila clam seed are discussed.     

Growth and gonadal development in diploid and triploid turbot (Scophthalmus maximus)

This study determined the effect of triploidy on the survival, growth and gonadal development of turbot from 6 to 48 months of age. From 6 to 24 months of age (first sexual maturity), survival was similar in both ploidies (P > 0.05). From 24 to 48 months of age, after the first sexual maturity, survival was 91.9% in diploids and 100% in triploids, which did not exhibit the post-spawning-associated mortality. Growth was similar for both ploidies during the first year of life. After that, triploids grew significantly (P < 0.05) more that diploids, with more marked differences after each spawning season. From 24 to 48 months, the average weight difference between both ploidies was 11.4 ± 1.9%, ranging from 4.3 to 23.0%. At 47 months of age, the biomass of triploids was 10.3% greater in total weight and 14.3% greater in eviscerated weight. Gonads of triploid males were similar to that of diploids, whereas in triploid females, they were significantly smaller and rudimentary. A histological analysis carried out at 47 months of age showed complete sterility of triploids in both sexes. Sex ratio was 1 male (M):0.6 female (F), for diploids, significantly (P < 0.05) different from 1:1, and 1 M:3.3 F for triploids, significantly (P < 0.05) different from 1:1 and from the diploids. Since females grow more than males, culture of triploids benefited from the high female ratio, which helped to reduce size dispersion. In addition, their sterility allowed better performance by avoiding the reduction in growth that takes place during the spawning periods. Together, these observations indicate that triploidy induction can be an interesting option for turbot aquaculture, especially for the production of large-size fish of more than 2 years of age.     

Hormonal profile of growing male and female diploids and triploids of the blue tilapia,Oreochromis aureus,reared in intensive culture

Triploidy as a result of thermal shock exposure of fertilized eggs decreases the growth rate ofOreochromis aureus as compared to their diploid controls, but this is due to the higher female ratio present in triploids (86%) and the lower growth rate of females. When females and males are considered separately, the growth rate is not significantly different in diploids and triploids. Since triploidy results in a malfunctioning steroidogenesis in females (mainly testosterone (T) and 17β-estradiol (E₂)), but does not affect the growth rate, it is concluded that female gonadal steroids do not influence growth unless in pharmacological concentrations. These low levels of gonadal steroids are generally accompanied by higher levels of gonadotropin (GtH), but the difference is not always significant. Despite their lower growth rate diploid females have higher plasma concentrations of growth hormone (GH) during several months compared to the triploid females and diploid males. 3,5,3′-triiodo-L-thyronine (T₃) levels, however, are comparable between diploid and triploid females (except for 1 month), but higher in diploid males in 4 of the 5 months studied. 11-ketotestosterone (11kT) is always higher in males. These results indicate that the higher growth rate of males may be related to the high circulating levels of T₃ and 11kT.     

Studies on sperm of diploid and triploid tench, Tinca tinca (L.)

The tench Tinca tinca is an interesting fish from the viewpoint of polyploidy and related atypical reproduction aspects. Triploid tench were produced artificially. Studies of spermiation as well as of sperm motility and structure were performed on several triploid and diploid males simultaneously with individual experimental crosses with diploid females to define their reproductive capacities. The testes of triploids visually looked less developed in the most of cases with lower sperm production (0.05 cm³ sperm per male), GSI and weight of testes compared to diploids (0.58 cm³ sperm per male). Analysis of variance showed significant influence of ploidy level on the percentage of motile spermatozoa. Triploidy did not change percentage of live spermatozoa and velocity of spermatozoa at the first time of sperm movement. The study of sperm structure by scanning electron microscopy revealed that most sperm cells were of normal structure with some anomalies. Sperm heads of triploid and diploid males were mostly round-shaped, 1.86±0.2 and 1.6±0.18 μm in diameter. The midpiece of triploid spermatozoa was slightly narrower than that of diploid ones with typical cylindrical shape. Flow cytometry revealed sperm cells of triploids to be largely aneuploid (1.47 n) with high mosaic DNA, oscillating from haploid DNA content (1.0 n) to diploid DNA content (1.9 n). Experimental crosses between triploid males and diploid females revealed that these males were capable to stimulate effective development with relatively high level of fertilization and hatching rates from 0 to 70%. In conclusion, triploidization does not seem to guarantee sterility of tench.     

The growth and reproductive endocrinology of adult triploid Pacific salmonids

This paper describes the effect of triploidy on growth and reproductive endocrinology in the months leading up to and including spawning in rainbow trout,Salmo gairdneri, and pink salmon,Oncorhynchus gorbuscha. Growth rates were the same for diploid and triploid rainbow trout, but triploid female pink salmon were smaller than maturing diploid females and diploid and triploid males of the same age. Triploid males of both species developed typical secondary sexual characteristics and had normal endocrine profiles, although their cycle appeared to be delayed by about one month. Triploid females remained silvery in appearance and showed no endocrine signs of maturation, even at the level of the pituitary. Thus, although triploids of both sexes are genetically sterile, only the females do not undergo physiological maturation.Reported in part at the Third International Symposium on Reproductive Physiology of Fish, St. John's, Newfoundland, August 2–7, 1987.     

Reproductive ability of second generation ornamental (koi) carp (Cyprinus carpio L.) × goldfish (Carassius auratus L.) hybrids and characteristics of their offspring

The purpose of this study was to investigate the reproductive ability of second generation (F₂) koi (Cyprinus carpio L.) × goldfish (Carassius auratus L.) hybrids. Only diploid F₂ males and females were fertile and used in crosses. A significant increase was recorded in male fertility in F₂ versus F₁. In contrast with an earlier study in which only one fertile F₁ male was found, about 20% of F₂ males produced sperm. The observed reproductive ability of F₂ hybrids was similar to that demonstrated by the only fertile F₁ male and F₁ females. F₂ males produced diploid spermatozoa and generated triploids when crossed with koi females. All triploid fish in these progenies were males indicating that F₂ males had a sex chromosome constitution of XY. F₂ females produced diploid eggs and generated mostly triploids when crossed with koi males. In progenies obtained by crosses of F₂ males with F₁ and F₂ females, most of the surviving juveniles (63%–100%) were diploid; a minority of juveniles were aneuploid (ploidy ranged from 2.1n to 3.6n). Diploid fish in these progenies were presumably the result of spontaneous androgenesis and gynogenesis, by the same mechanisms observed earlier in progenies obtained by crossing the F₁ male with F₁ females.     

Reproductive ability of triploid ornamental (koi) carp (Cyprinus carpio L.) × goldfish (Carassius auratus L.) hybrids and characteristics of their offspring

The purpose of this study was to investigate reproductive ability of backcross triploid koi (Cyprinus carpio L.) × goldfish (Carassius auratus L.) hybrids. These triploids have been obtained by crossing of F₁ hybrid females producing diploid eggs with males of parental species. Triploid hybrid females, when crossed with goldfish or koi males, produced mostly aneuploid fish with ploidy range from approximately 2.2n–3.2n with a mean value 2.5n; some fish in crosses of triploid females with koi males were tetraploid (4.0n). Since analysed fish had in their genomes one haploid set from parental males, the data indicate that triploid hybrid females mostly produced aneuploid eggs with ploidy range from approximately 1.2n–2.2n and a mean ploidy around 1.5n while some eggs were triploid. Triploid hybrid males were completely sterile and have not released any sperm after hormonal injection. Despite their low viability, some aneuploid fish obtained from triploid hybrid females were raised in indoor recirculating systems until the age of 2 years and their reproductive ability has been evaluated. One aneuploid female with ploidy 2.1n produced larvae with ploidy range from 2.9n to 3.4n with a mean ploidy of 3.1n when crossed with a koi male; about 60% of obtained larvae had ploidy from 3.0n to 3.2n. These data indicate that this female produced mostly eggs with unreduced ploidy level.     

Nauplii Production from Wild,Cultivated, and Mixed Populations of Blue Shrimp,Penaeus stylirostris

ABSTRACT

Due to the low nauplii production of cultivated broodstock, and to reduce the dependence on the wild stock, an experiment was carried out with 400 adult blue shrimp, Penaeus stylirostris, from wild and cultivated (F6) populations. Four treatments, each in duplicate, were applied: (1) wild females and males (W-W); (2) wild females and cultivated males (W-C); (3) cultivated females and wild males (C-W); and (4) cultivated females and males (C-C). More than 300 individual spawns were monitored to evaluate the egg and nauplii production per female. Mixed model ANOVA for factorial arrangements (4 × 3 × 2³ and 3 × 2³) were conducted. The factors considered besides the treatments were: rematuration (number of successive spawnings for a female), ovarian maturity, integrity of the spermatophore attached (complete spermatophore, “wings,” or “remnant”) and condition of spawning (partial or complete). The introduction of both wild females and males was a successful measure to improve the overall egg and nauplii production. Both mixed populations outperformed the cultivated broodstock, but were inferior to the wild stock (average production of eggs and nauplii: W-W-l 12,713 and 34,682, respectively; W-C-l 13,215 and 22,038, respectively; C-W-82,702 and 11,715, respectively; C-C-66,948 and 7,653, respectively). Populations with wild females produce a larger number of eggs, and wild males contribute to higher hatching rates. Other observations indicate the need to select for spawning only those females showing an advanced degree of ovarian maturity, having a complete spermatophore attached to the thelycum, and spawning completely.     

Growth and gonadal development in diploid and triploid Atlantic cod (Gadus morhua)

Induction of triploidy has been suggested as an effective tool to prevent spawning of farmed fish. This experiment examined the growth potential of triploid cod when reared communally with diploid ones after the juvenile stage. Pressure treatment was used to induce triploidy in a batch of cod eggs in April 2009. The resulting offspring were reared separately from their diploid counterparts until they reached the proper size for PIT tagging. At the age of 8 months, an equal number of 115 diploids (135.5 ± 3.95 g) and triploids (93.6 ± 2.63 g) were communally reared in a circular flow-through tank until the age of 22 months. By the end of this rearing period, diploids (1,002.4 ± 39.9 g) were significantly heavier than triploids (654.6 ± 27.7 g), but the specific growth rate did not differ significantly during the growth trial. Gonadal development at the age of 22 months was also lower among triploids than diploids, especially for females (5.3 and 91.9 %) but also for males (32.5 and 72.7 %). Sterility among female triploids was evident by the reduced size and dysfunctional gonads, but gonadal development in male triploids was less suppressed. Prevalence of body deformities was, however, significantly higher among triploids (62.6 %) than diploids (33.9 %). Higher prevalence of deformities in triploid cod underlines the need for further fine-tuning of the triploidization procedure or finding other methods of sterilization. At present, triploid cod are still far from being established as an alternative for commercial production.     

Induction of triploidy in mud loach (Misgurnus mizolepis) and its effect on gonad development and growth

Triploidy was induced in mud loach (Misgurnus mizolepis) by cold shocking fertilized eggs 5 min post-fertilization at 2°C for 15 to 60 min. Best results were obtained when eggs were shocked for 60 min; 98% of fish examined in that treatment were triploids. Triploidy was confirmed by erythrocyte measurements and chromosome counts. Diploids had 48 chromosomes, while triploids had 72. Histological analysis of 9-month-old triploid ovaries showed an appreciable number of oocytes at the chromatin nucleolus stage with considerable interstitial tissue. However, diploids had well developed oocytes. Diploid testes from diploid males exhibited normal spermatids and spermatozoa, while a few were seen in triploid males. Growth rate was evaluated over a 9-month growth trial. Although male and female triploids were slightly heavier than their diploid counterparts from the third to the ninth month, their growth rates were not significantly different compared to their diploid controls.     

Production of interploid triploids by 4n × 2n blunt snout bream (Megalobrama amblycephala. Yih) and their first performance data

Positive and negative interploid triploids of blunt snout bream (Megalobrama amblycephala) were produced by reciprocal crossing autotetraploids with diploids. Fertilization and hatching rate of negative interploid triploid, 2n♀× 4n♂, at different reproductive ages was similar to that of control, but higher than that of positive interploid triploid, 4n♀× 2n♂. Additionally, the development rate of embryos of the positive interploid triploid, 4n♀× 2n♂, was slower than that of control and the negative interploid triploid, 2n♀× 4n♂. Gonad growth of the positive and negative interploid 3n was significantly (P<0.01) affected by parents, and spawning could not be induced both in females and males at the 2+ age. The negative interploid 3n (2n♀× 4n♂) had a significantly higher survival rate than the positive interploid 3n (4n♀× 2n♂), but close to the level of the control (P<0.05). The daily growth rate of the negative interploid 3n was similar to that of the good breed ‘Pujiang No. 1’ blunt snout bream (2n) during the early life stages.     

Production of triploid eastern little tuna,Euthynnus affinis (Cantor, 1849)

Herein, we developed a triploidization method using spawned eggs collected immediately after spawning of eastern little tuna (ELT), Euthynnus affinis. ELT broodstock induced the spawning by hormonal treatment in the tank, with the resulting spawned eggs being used for the triploidy induction. Under optimal conditions, the mean ± SEM triploidization and hatching rates were 97.2 ± 2.8% and 84.5 ± 10.3%, respectively. Although triploid ELT showed growth performance equivalent to that of diploids, the triploids died at a higher rate than the diploids during 2–4 weeks post‐hatching when triploids and diploids were reared in the same tank. Therefore, we propose that it would be necessary in a practical operation to use triploid‐only ELT seedlings to avoid selective cannibalism by the diploids. The ELT triploids exhibited an all‐female phenotype. Because previous studies have reported that female triploids show a greater probability of sterility than male triploids, this characteristic could be a major advantage. Since this triploidization method, using spawned eggs, can be performed without handling the broodstock, it is possible to avoid the physical damage caused by the process of artificial insemination, making it possible to repeatedly produce triploid populations without valuable broodstock loss. Thus, we have developed an efficient method to produce ELT triploids, although further study is essential to evaluate sterility of the triploid ELT.     

Hemoglobin level,metabolic rate,opercular abduction rate and swimming efficiency in female triploid brook trout (Salvelinus fontinalis)

Metabolic (oxygen consumption) rate, opercular abduction rate and tail beat frequency were determined in two strains of diploid and triploid female brook trout (Salvelinus fontinalis) while these fish swam at 0.37±0.02 body lengths per sec in a Blazka respirometer. Total blood hemoglobin level was also measured and opercular condition examined. Total blood hemoglobin levels in diploids and triploids were equal. The opercular abduction rate was the same in diploids and triploids (regardless of whether triploid opercular condition was good or poor) yet triploids had a lower oxygen consumption rate than diploids, indicating that triploids take up less oxygen than diploids per opercular cycle. Tail beat frequency, an indicator of swimming effort, was the same in diploids and triploids, suggesting that triploids require less oxygen than diploids for a similar swimming effort.     

Maturation and Spawning of Blue Shrimp Penaeus stylirostris (Stimpson) under Hypersaline Conditions

Maturation and spawning of blue shrimp Penaeus stylirostris were obtained in hypersaline and turbid water. Results indicate that number of spawnings per female and percentage of viable spawnings are similar to those reported under "standard" conditions by other investigators. However, the number of nauplii produced was 50 to 75% less. A significant correlation was observed between reproductive activity and lunar cycles, showing peak activity at three to four days following new moon. Additionally, working with records of individual females, the number of spawnings per female was found to be extremely variable, with intervals between consecutive spawnings non-uniform. Instead, they tended to group in pairs with less than six days between spawnings, followed by a variable period of time (usually more than 12 days) before the paired spawnings were repeated.     

Larval metamorphosis and morphological characteristic analysis of triploid shrimp Fenneropenaeus chinensis (Osbeck, 1765)

Both MI and MII triploids were successfully produced by heat shock in Chinese shrimp Fenneropenaeus chinensis. The inducing conditions for MI and MII triploids were optimized. The highest inducing rate obtained for MI triploids reached more than 90%, and that for MII triploids reached nearly 100% at the nauplius stage as evaluated using flow cytometry. Comparisons of survival rates at larval stages between triploids and diploids or diploids experiencing treatment and diploids without treatment were performed. At larval stage from nauplii to postlarvae, heat shocks lowered survival at larval stages even if the ploidy was not changed. Ploidy did not affect shrimp larvae survival, and no significant difference was found in the survival of shrimp larvae between MI and MII triploids. Highly significant differences were observed in the morphology of triploids and diploids, and no apparent difference was found in the morphology of MI and MII triploids at the grow‐out stages. Discriminating formulae for triploid and diploid shrimp at grow‐out stage were developed and could be used to distinguish triploids from diploids based on morphological parameters. MI and MII triploids of shrimp have the potential to be used in aquaculture.     

Comparative Growth of Diploid and Triploid Asian Catfish Clarias macrocephalus in Thailand

Wild caught Asian catfish were spawned manually following HCG injection, and a portion of the eggs were subjected to cold-shock at 4 C for 15 min within 2-min post-fertilization. Nuclear diameter measurements of cold-shocked fish revealed that 96% were triploids (3N), while non-shocked fish were all diploids (2N). During larval and fry culture (first 26 d), triploid fish mortality was =50%, while diploid mortality was =25%. Following 8-mo culture in tanks at three stocking densities, triploid fish survival was significantly greater ( P < 0.05), than diploids, with 84.0% and 57.3%, respectively. Triploid live weight was also significantly greater than diploids, with 69.2 and 45.9 g averages, respectively. Ninety-two percent of diploids had welldeveloped gonads after 8 mo; whereas none of the triploids had mature gonads. Gonads were undifferentiated with 31% of the triploids. These sexually undifferentiated fish had greater growth rates than male or female triploids, and greater growth than all diploids. Carcass weight (gutted) of triploids was 95.8% of live weight, compared with 92.5% for diploids. Lastly, triploids had very few deformities compared with diploids, with 1.3% and 17.6%, respectively. Deformities included curved spines, and humped backs just posterior of the head.     

Analysis of growth, weight and relevant indices of diploid and triploid population of tench Tinca tinca (Linnaeus 1758)

This study determined biometric and weight parameters and relevant indices of diploid and triploid tench. Altogether, 137 siblings of tench were studied. The effect of ploidy level appeared in significantly better growth of triploids (P<0.001) as to biometric [total length (TL), standard length (SL), body height (BH), body width (BW)] and weight [fish weight (FW), carcass weight (CW)] parameters of T₃ of both sexes and of T₃₊ females. The effect of ploidy level also appeared as significantly higher dressing percentage (DP; P<0.001) of triploid T₃ females compared with other groups, significantly higher gonad weight (GW) and gonadosomatic index (GSI; P<0.001) of diploid T₃ females, as well as GSI and hepatosomatic index of diploid T₃₊ females. The effect of sex appeared in significantly higher (P<0.001) biometric (TL, SL, BH, BW) and weight (FW, CW) parameters of T₃ females of both ploidy levels, as well as of triploid T₃₊ females. The effect of sex also appeared as significantly higher DP (P<0.001) of males in diploid T₃ fish, as well as of males of both ploidy levels in T₃₊ fish and significantly higher GW and GSI (P<0.001) of females in diploid T₃ fish, as well as of females of both ploidy levels in T₃₊ fish. This study shows evidence for faster somatic growth and bigger final weight of triploid populations of tench compared with diploids in both age categories T₃ and T₃₊.