Cryopreservation of the whole testes of Asian sea bass (Lates calcarifer) and its effects on apoptosis, germ cell-specific gene expression, germ cell transplantability, and DNA methylation.

Cryopreservation of spermatogonia could be a useful tool to preserve the genetic resources of fish, which could be further restored via germ cell transplantation. In this study, the protocol for the cryopreservation of the spermatogonia of Asian sea bass (Lates calcarifer), an economically important fishery resource in the Indo-West Pacific, was optimised. The impact of the cryopreservation technique on cell viability and apoptosis, expression of several genes related to immature germ cell markers, transplantability in allogeneic recipients, and global DNA methylation was evaluated. The slow-freezing method was performed for the cryopreservation of immature testis tissue, which contains a high proportion of spermatogonia. The optimal condition that yielded the highest recovery rate of post-thawed spermatogonia included a cryomedium containing Leibovitz's (L-15) medium and 10 % dimethyl sulfoxide, ice equilibration for 60 min before freezing, and subsequent thawing at 4 °C for 8 min. Moreover, a higher number of early and late apoptotic cells was detected in the cryopreserved than in the fresh testes, suggesting that apoptosis could result in reduced viability. The expression levels of dazl decreased in the cryopreserved testes; however, there were no significant differences in the expression levels of nanos2 or nanos3 between the fresh and cryopreserved testes. Although qRT-PCR showed lower vasa expression in cryopreserved testicular cells, in situ hybridisation showed expressed vasa in the cryopreserved testicular cells. Post-thawed spermatogonia could be incorporated into the genital ridge of allogeneic recipients, suggesting that cryopreserved spermatogonia exhibit transplantability characteristics. Compared with fresh testes, significant changes in the proportion of DNA methylation (decreased 5-mC and 5-caC) were observed in cryomedium-free testicular cells, whereas those of the cryopreserved cells were not significantly different. Therefore, the method we developed for the cryopreservation of the spermatogonia of Asian sea bass enabled post-thaw cells to retain several stemness characteristics and maintain their epigenetic stability.

Effects of dietary supplementation of estradiol-17ß during fry stage on growth, physiological and immune parameters and gonadal gene expression in adult snakeskin gourami.

In snakeskin gourami (Trichopodus pectoralis), females are generally larger than males, and estradiol-17ß (E2)-sex reversal to produce female monosex has gained interest in this species. In this study, we aimed to investigate the effects of E2-induced sex reversal on growth, physiological and immune parameters, and gonadal gene expression in adult snakeskin gourami. Fry (7 days posthatching) were divided into different experimental groups based on the dose of E2: control (no E2 (0 mg kg-1) supplementation), E2-100 (100 mg kg-1), E2-200 (200 mg kg-1), and E2-300 (300 mg kg-1), fed with the E2 doses for 90 d and cultured for 11 months (adult stage). The findings revealed that E2 supplementation produced 88.89-100% of female population. After 11 months of culture, the effects of sexual dimorphism on the growth performance of the E2-100 group were not significant compared to that on the growth performance of the control male and female groups; however, it improved significantly in the E2-200 and E2-300 groups (P < 0.05). E2 elevated the CP and fat contents in body in E2-200 and E2-300 groups (P < 0.05) compared to that in the control group. No sex differences in blood metabolites, haematological values, or immune parameters were identified. Nevertheless, E2-200 and E2-300 groups showed increased blood glucose, triglyceride, haemoglobin, and total immunoglobulin (P < 0.05) compared to control male fish. In addition, all concentrations of E2 increased alternative complement 50 (P < 0.05). Several genes, including bHLH, cyp19a1, daz, deadend, esrb, esrrg, gnrhr, gpa, gsg1l, hsd17ß, mospd1, nanos2, p53, piwi2, rerg, rps6ka, tgfb, and vgr, showed differential expression between testis and ovary in control female and E2-treated groups. The expression patterns of the genes were similar in the ovary of the control female and E2-200-treated fish. In conclusion, the findings demonstrate that a feminisation duration of 7-97 days and two doses of E2 at 200 or 300 mg kg-1 successfully produced all-female stocks in snakeskin gourami. Furthermore, the findings showed that E2-treated females were maintained throughout adulthood and exhibited several superior characteristics to male fish. Together with the information generated on differentially expressed sex-related genes, these findings could enable the culturing of faster-growing sex to increase productivity and contribute to the development of intensive snakeskin gourami farming.

Glucose injection into the yolk influences intermediary metabolism in adult Nile tilapia fed with high levels of carbohydrates.

Nutritional programming is a concept proposed to be applied in the field of fish nutrition to improve the use of new diets in aquaculture. This study aimed to investigate for the first time the effects of a glucose injection into the yolk at the alevin stage on intermediary metabolism and growth in adult Nile tilapia (Oreochromis niloticus) at 32-37 weeks later in the life. The early stimulus was performed through direct microinjection of 2 M glucose into yolk sacs of Nile tilapia alevin. Subsequently, in adult tilapia, the long-term effects of glucose stimulus on growth performance, blood metabolites, chemical composition in the liver and muscle, expression of genes involved in glucose transport and metabolism (glycolysis and gluconeogenesis) and related pathways (amino acid catabolism and lipogenesis) were investigated. Our results showed that, even though early glucose injection had no effect on growth performance in adult fish, very few significant effects on glucose metabolism were observed. Furthermore, to evaluate the potential metabolic programming after a dietary challenge, a 2 × 2 factorial design with two early stimuli (0.85% NaCl or 2 M glucose) and two different dietary carbohydrate intakes (medium-carbohydrate diet, CHO-M; high-carbohydrate diet, CHO-H) was performed between weeks 33 and 37. As expected, compared with the CHO-M diet, the CHO-H diet led to decreased growth performance, higher glyceamia and triglyceridemia, higher glycogen and lipid levels in the liver as well as down-regulation of gluconeogenesis and amino acid catabolism gene expressions. More interestingly, although early glucose injection had no significant effect on growth performance, it enhanced the capacities for lipogenesis, glycolysis and gluconeogenesis, particularly in fish that were fed the CHO-H diet. Thus, the nutritional programming of tilapia linked to glucose injection into the yolk of alevins is always visible at the adult stage albeit less intense than what we previously observed in juvenile.

Characterization of proopiomelanocortin in the snakeskin gourami (Trichopodus pectoralis) and its expression in relation to food intake.

Proopiomelanocortin (POMC) is the precursor of several hormones involved in physiological systems including feed intake. The snakeskin gourami (Trichopodus pectoralis) POMC complementary DNA (TpPOMC) was cloned and characterized. Phylogenetic analysis showed that TpPOMC was clustered in a major POMC lineage in fish. Analysis of the Ka to Ks ratios for the entire POMC sequence and for each hormonal segment suggested that different POMC-derived peptide segments were subject to different evolutionary pressures. High expression level of TpPOMC was observed in all brain regions, with the highest levels in the diencephalon and pituitary gland. In situ hybridization also revealed that TpPOMC-expressing cells were distributed in discrete brain regions. The transcription level of TpPOMC was also found at moderate levels in several peripheral tissues, including gills, liver, head kidney, trunk kidney, stomach, intestine, spleen, ovary and testis, and at a low level in muscle. The expression level of TpPOMC was evaluated in each brain region (telencephalon, mesencephalon, metencephalon, and diencephalon together with the pituitary gland) at 1 h before the first and the last meals of the day and compared with expression levels at a time interval between the first and the last meals of the day. Low expression levels of TpPOMC were found at 1 h before the last meal of the day (P < 0.05). These finding suggest that decreased POMC expression level may lead to reduced melanocyte-stimulating hormones, which may in part be responsible for stimulating food intake. The effect of short-term fasting (24 h) on TpPOMC expression level in each brain region was also investigated. In telencephalon and diencephalon together with the pituitary gland, TpPOMC messenger RNA reached a nadir at 12 h of fasting, whereas TpPOMC transcript showed a nadir at 6 h of fasting in metencephalon and mesencephalon. A peak of TpPOMC level was observed at 18 h of fasting in metencephalon and diencephalon together with the pituitary gland. These findings suggest that TpPOMC expression is affected by nutritional status.