Birgitta Norberg

Control of puberty in farmed fish

Puberty comprises the transition from an immature juvenile to a mature adult state of the reproductive system, i.e. the individual becomes capable of reproducing sexually for the first time, which implies functional competence of the brain-pituitary-gonad (BPG) axis. Early puberty is a major problem in many farmed fish species due to negative effects on growth performance, flesh composition, external appearance, behaviour, health, welfare and survival, as well as possible genetic impact on wild populations. Late puberty can also be a problem for broodstock management in some species, while some species completely fail to enter puberty under farming conditions. Age and size at puberty varies between and within species and strains, and are modulated by genetic and environmental factors. Puberty onset is controlled by activation of the BPG axis, and a range of internal and external factors are hypothesised to stimulate and/or modulate this activation such as growth, adiposity, feed intake, photoperiod, temperature and social factors. For example, there is a positive correlation between rapid growth and early puberty in fish. Age at puberty can be controlled by selective breeding or control of photoperiod, feeding or temperature. Monosex stocks can exploit sex dimorphic growth patterns and sterility can be achieved by triploidisation. However, all these techniques have limitations under commercial farming conditions. Further knowledge is needed on both basic and applied aspects of puberty control to refine existing methods and to develop new methods that are efficient in terms of production and acceptable in terms of fish welfare and sustainability.

Gonadal development and associated changes in liver size and sexual steroids during the reproductive cycle of captive male and female Atlantic cod (Gadus morhua L.)

Gametogenesis in female and male Atlantic cod (Gadus morhua L.) was investigated by sampling blood plasma and gonadal tissue from 19 to 33-month-old fish. The reproductive cycles of both female and male Atlantic cod are characterized by distinct annual variations in gonadal size and developmental stage and these are associated with changes in sex steroids and liver size. I(H) did not change during early gonadal development, but both spent females and males had lower I(H) than late maturing females and spermiating males, respectively. In females I(G) was correlated to plasma E2 levels and they were highest in spawning females. The lowest levels during the reproductive cycle were observed in spent females. Plasma T levels were low throughout ovarian development, and were at a minimum in spent females. 11-ketotestosterone in plasma of males increased rapidly during spermiation, while T increased at earlier testicular stages and reached maximum during spermiation. High plasma levels of steroids in male and female cod during spawning serve to promote further development and growth of less advanced stages of germ cells.

Changes in plasma vitellogenin, sex steroids, calcitonin, and thyroid hormones related to sexual maturation in female brown trout (Salmo trutta).

Female brown trout (Salmo trutta) from a wild strain (Baltic sea trout) and a cultured strain were sampled individually for blood plasma at regular intervals during the period around final sexual maturation. The plasma samples were analyzed for vitellogenin (VTG), estradiol-17 beta, testosterone, 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one (17,20 beta-P), calcitonin, tri-iodothyronine (T3), thyroxine (T4), and total and free plasma calcium. In the wild fish, VTG, estradiol-17 beta, and testosterone peaked 30 days before ovulation, while 17,20 beta-P had a sharp peak at ovulation. Both T3 and T4 declined at the beginning of the sampling period, reached minimal levels 30 days before ovulation, and rose sharply at the time of ovulation. Calcitonin levels were elevated during final maturation. Total plasma calcium correlated with plasma VTG levels. In the cultured strain, sampling was started 2 weeks before ovulation. The levels of VTG, estradiol-17 beta, and testosterone decreased throughout the sampling period. 17,20 beta-P and calcitonin concentrations were high during the period close to ovulation. Plasma thyroxine remained at basal levels in cultured trout. The discrepancies observed between wild and cultured females may be due to differences in stress susceptibility, environmental conditions, life cycles, or to genetic divergence between the strains.

Photoperiod regulates the timing of sexual maturation, spawning, sex steroid and thyroid hormone profiles in the Atlantic cod (Gadus morhua)

Abstract Atlantic cod of both sexes were held under four different photoperiod regimes from age 1 year until second sexual maturation at 3 years. Annual photoperiod cycles were compressed into 6 or 9 months, held at 12 months, or extended to 18 months, in each case followed by one 12-month cycle (termed 6+12, 9+12, 12+12, and 18+12, respectively). Photoperiod alterations caused shifts in the cyclic patterns of plasma calcium, sex steroid, and thyroid hormones, and also produced correlative changes in the timing of spawning. Initial spawning was advanced in the compressed (6+12) photoperiod group, followed by further advancement in the timing of the second spawning. Conversely, spawning was delayed in the 18+12 group. Patterns in circulating hormones observed under these experimental conditions were consistent with the coupling of photoperiod to spawning by way of established endocrine transducers. These results demonstrate that manipulation of the annual photoperiod cycle is a powerful hatchery technique in the maintenance of reproductive stocks of Atlantic cod that spawn at various times of the year.

Effects of photoperiod on sexual maturation and somatic growth in male Atlantic halibut (Hippoglossus hippoglossus L.).

A major obstacle in modern, intensive aquaculture is the precocious maturation of male fish, leading to decreased somatic growth and reduced filet quality. Effects of photoperiod on sexual maturation and growth in male Atlantic halibut were therefore examined. In June 1996, 1300 1+ fish of both sexes were distributed in two indoor tanks supplied with continuous light (LL) or a simulated natural photoperiod (SNP). In December 1996 and June 1997, 200 individuals were exchanged between the tanks creating six experimental groups that were followed until June 1998. LL stimulated growth and accelerated timing of first maturation by approximately 3 months. LL also appeared to interrupt circannual rhythmicity in sexual maturation. Sexual maturation led to reduced growth from 3 months pre-spawning and throughout the spawning season. Males that did not mature during the experiment attained the highest final body weight. All males reared on LL from June 1997 reached sexual maturity the following season. In contrast, only 26% of the males matured in the group transferred from LL to SNP in June 1997, and this group also had the highest final body weight. The results indicate a possible route for reducing the problem of precocious maturation in male halibut.

Induction, isolation and a characterization of the lipid content of plasma vitellogenin from two Salmo species: Rainbow trout (Salmo gairdneri) and sea trout (Salmo trutta)

Vitellogenin synthesis is induced in juvenile rainbow trout (Salmo gairdneri) and juvenile sea trout (Salmo trutta) by estradiol-17 beta. A purification procedure for vitellogenin from trout plasma by precipitation with MgCl2-EDTA and subsequent anion exchange chromatography on DEAE-Sephacel is described. The total lipid contents of purified rainbow trout and sea trout vitellogenins are 18 and 19%, respectively. Approximately 2/3 of the lipids are phospholipids, while the remainder consists of triglycerides and cholesterol. Phosphorus determinations on delipidated vitellogenin yield a phosphorus content of 0.63% in rainbow trout and 0.58% in sea trout vitellogenin. Native (dimeric) vitellogenins from rainbow trout and sea trout both have an apparent molecular weight of 440,000, when estimated by gel filtration on Sepharose 6B.

Spermatogenesis and related plasma androgen levels in Atlantic halibut (Hippoglossus hippoglossus L.)

Spermatogenesis in male Atlantic halibut (Hippoglossus hippoglossus L.) was investigated by sampling blood plasma and testicular tissue from 15-39-month-old fish. The experiment covered a period in which all fish reached puberty and completed sexual maturation at least once. The germinal compartment in Atlantic halibut testis appears to be organized in branching lobules of the unrestricted spermatogonial type, because spermatocysts with spermatogonia were found throughout the testis. Spermatogenesis was characterized histologically, and staged according to the most advanced type of germ cell present: spermatogonia (Stage I), spermatogonia and spermatocytes (Stage II), spermatogonia, spermatocytes and spermatids (Stage III), spermatogonia, spermatocytes, spermatids and spermatozoa (Stage IV), and regressing testis (Stage V). Three phases could be distinguished: first, an initial phase with low levels of circulating testosterone (T; quantified by RIA) and 11-ketotestosterone (11-KT; quantified by ELISA), spermatogonial proliferation, and subsequently the initiation of meiosis marked by the formation of spermatocytes (Stage I and II). Secondly, a phase with increasing T and 11-KT levels and with haploid germ cells including spermatozoa present in the testis (Stage III and IV). Thirdly, a phase with low T and 11-KT levels and a regressing testis with Sertoli cells displaying signs of phagocytotic activity (Stage V). Circulating levels of 11-KT were at least four-fold higher than those of T during all stages of spermatogenesis. Increasing plasma levels of T and 11-KT were associated with increasing testicular mass throughout the reproductive cycle. The absolute level of, or the relation between, testis growth and circulating androgens were not significantly different in first time spawners compared to fish that underwent their second spawning season. These results provide reference levels for Atlantic halibut spermatogenesis.

Fertility and motility of sperm from Atlantic halibut (Hippoglossus hippoglossus) in relation to dose and timing of gonadotrophin-releasing hormone agonist implant

Abstract In broodstocks of Atlantic halibut, Hippoglossus hippoglossus , male and female gamete production often becomes unsynchronised towards the end of the spawning season—milt becomes very viscous and difficult to express while the females are still producing batches of good quality eggs. Gonadotrophin-releasing hormone agonist (GnRHa) has been shown to stimulate spermiation in a number of fish species. Therefore, we conducted two experiments where male halibut were implanted intramuscularly with pellets containing GnRHa. The effect of the pellets was tested at three periods: before, at the height of and at the end of spermiation. In the middle period, GnRHa was tested at two doses (5 and 25 µg/kg bodyweight). Measurements were made of milt hydration, sperm motility and fertilisation rate. Implanted males began spermiation at least 4 weeks before control males. Both doses of GnRHa increased the fluidity of the milt. This effect lasted for at least 20 days in the low dose group and for 40 days in the high dose group. When applied at the end ofthe season, GnRHa reversed the normal trend for the milt to become more viscous. GnRHa treatments did not affect fertilisation rates obtained with the sperm. However, towards the end of the spawning season, sperm motility was enhanced in males treated with the high dose of GnRHa (25 μg/kg) compared to controls. As described previously, plasma concentrations of the gonadal steroids, 5β-pregnane-3β,17,20β-triol 20-sulphate and 17,20α-dihydroxy-4-pregnen-3-one, were significantly enhanced by GnRHa treatment. Concentrations of testosterone on the other hand decreased when spermiating males were treated with GnRHa. Our data suggest that 17,20β-dihydroxy-4-pregnen-3-one or its metabolites are involved in milt hydration, possibly through affecting ion transport. Crown Copyright

Vitellogenin, steroid plasma levels and spawning performance of cultured female Senegalese sole (Solea senegalensis).

The Senegalese sole (Solea senegalensis) is a high value market flatfish, which aquaculture is compromised by severe reproductive problems; these are mostly found in soles hatched and raised in captivity (F1 generation). To gain knowledge on the reproductive dysfunctions observed in cultured (F1) Senegalese sole, this work aimed at developing a specific vitellogenin (VTG) ELISA, for the measurement of plasma VTG levels in this species. Profiles of VTG were correlated with those of sexual steroids and spawning performance of an F1 broodstock, during three consecutive years. The Senegalese sole VTG (ssVTG) was purified by precipitation with MgCl(2)-EDTA and anion-exchange chromatography and showed a molecular mass of 172 kDa, by SDS-PAGE. Specific antibodies were obtained and used to develop a competitive ELISA, which had a sensitivity of 3.6 ng ml(-1), and inter- and intra-assay coefficients of variation of 9.5% (n=29) and 6.7% (n=12), respectively. Annual profiles of plasma VTG showed a major peak at pre-spawning, and a second minor rise around autumn, which mirrored plasma profiles of both estradiol (E(2)) and testosterone (T) levels. Spontaneous spawning occurred every year in the spring season, but no fertilized eggs were obtained. In conclusion, this study described, for the first time, the purification and development of a sensitive and specific ELISA for Senegalese sole VTG. The endocrine and spawning data suggested that F1 female broodstock showed normal VTG and steroid releasing profiles in captivity with occurrence of spontaneous spawning, but no fertilization of the eggs was recorded.

Molecular characterization and expression of FSHβ, LHβ, and common α-subunit in male Atlantic halibut (Hippoglossus hippoglossus)

To elucidate the role of the gonadotropins in the multiple spawner Atlantic halibut (Hippoglossus hippoglossus) full length cDNAs encoding FSHbeta, LHbeta, and the common alpha-subunit were cloned from pituitary glands by RACE-PCR. The three cDNAs consisted of 614, 595, and 666 nucleotides encoding peptides of 131, 146, and 124 amino acids, respectively. Halibut FSHbeta and LHbeta showed unique structural features among the vertebrate glycoprotein hormones. First, in contrast to all known FSHbeta, which contain either one or two conserved N-glycosylation sites, no potential binding site was found in Atlantic halibut FSHbeta. Second, the conserved glycosylation site in the N-terminus of all vertebrate LHbeta has been substituted with a unique C-terminal binding site in Atlantic halibut LHbeta. Furthermore, a specific cysteine residue of importance for the folding and heterodimerization of mammalian FSH is lacking in the FSHbeta from Atlantic halibut as well as many other teleosts. However, teleost FSHbeta is characterized by an additional N-terminal cysteine, which has likely replaced the missing residue, implicating a modified folding pattern of this subunit. In situ hybridization of mature male pituitaries revealed that FSHbeta and LHbeta mRNA were expressed in distinct cell types throughout the proximal pars distalis of the adenohypophysis, while alpha-subunit mRNA was identified in all parts of the proximal pars distalis, and also along the periphery of pars intermedia. Consistently, Northern blot analysis of pituitary RNA from mature males showed that FSHbeta, LHbeta, and alpha-subunit mRNAs were highly expressed. In juvenile male pituitaries very few cells containing FSHbeta, LHbeta, and alpha-subunit mRNA were identified by in situ hybridization. Low mRNA levels encoding LHbeta and the alpha-subunit were also demonstrated by Northern blot analysis of the juvenile pituitaries, while no FSHbeta mRNA was detected using this less sensitive technique. The results suggest that both FSH and LH play a role during both the very early and the final reproductive stages in Atlantic halibut males.