Gonadal Histology Research Articles

Female-biased sex ratios and delayed puberty in two fish species with different Ecologies in an Anthropogenically affected urban lake

In aquatic ecosystems, endocrine-disrupting compounds (EDCs) pose a growing concern for their potential adverse effects on fish reproduction and development. In lake Pyhäjärvi, located in the urban boreal region of Tampere, Finland, a significant number of sexually immature pikeperch (Sander lucioperca) individuals have been identified in size and age categories that are expected to be sexually mature. To explore if this phenomenon is attributed to estrogenic endocrine disruption, we conducted a comprehensive study comparing fish from lake Pyhäjärvi with those from a nearby reference lake, lake Näsijärvi. Roach (Rutilus rutilus), known for its susceptibility to EDCs, was also included for comparison. We examined various parameters in both pikeperch and roach, including size, condition factor, age, reproductive indicators, biometric indices and gonadal histology. We also assessed liver vitellogenin mRNA levels and genetic sex in roach, and measured estrogen levels in lake waters and wastewater treatment plant effluents. Results revealed that approximately one-third of fish in both species exhibited sexual immaturity in lake Pyhäjärvi, with a female-biased sex ratio. Surprisingly, we found no signs of estrogenic endocrine disruption, indicated by the absence of intersex fish in both species. Furthermore, vitellogenin levels in roach closely resembled those in the reference lake. Estrogens were undetectable in the lake waters, suggesting that factors other than estrogenic EDCs, including other potential endocrine disruptors such as PCBs or heavy metals, may be influencing delayed sexual maturity and skewed sex ratios. Further inquiry is needed to pinpoint these underlying causes. Our study provides essential baseline information on fish sexual development in lake Pyhäjärvi, emphasizing the need for ongoing monitoring and research to understand delayed sexual maturity and biased sex ratios. This is vital given the increasing concern about EDC impacts on aquatic ecosystems and the necessity for effective management strategies to protect these ecosystems' health and integrity.

The regulatory effects of water probiotic supplementation on the blood physiology, reproductive performance, and its related genes in Red Tilapia (Oreochromis niloticus X O. mossambicus)

Probiotics are becoming increasingly popular as eco-friendly alternatives in aquaculture. However, there is limited research on their impacts on the reproductive efficiency of Red Tilapia (Oreochromis niloticus x O. mossambicus) broodstock. Therefore, this experiment aimed to explore the combined effects of selective probiotics Bacillus subtilis and B. licheniformis (BSL; 1:1) added to water on blood hematology, serum metabolites, gonadal histology, reproductive performance, and reproductive associated genes in Red Tilapia broodstock. Tilapia broodfish weighing 140–160 g were stocked in four treatment groups: control (T0), and the other three groups were added different levels of BSL to the water as follows: T1 (0.01 g/m3), T2 (0.02 g/m3), and T3 (0.03 g/m3), respectively. Results indicate that BSL administration significantly improved RBCs, hemoglobin, hematocrit, MCH, and MCHC, with the highest improvement seen in the T3 group (P < 0.05). BSL added to the fish water significantly enhanced serum protein fractions (total protein, albumin, and globulins), while AST, ALT, ALP, creatinine, uric acid, and glucose were significantly diminished in a dose-dependent way (P < 0.05). Adding 0.02–0.03 g/ m3 of BSL resulted in higher antioxidant status (superoxide dismutase and catalase) compared to other groups (P < 0.05). Testosterone levels were higher in T3 than in other groups (P < 0.05). All female hormones (LH, FSH, estradiol, and progesterone) were substantially augmented by the addition of BSL. Additionally, the BSL groups exhibited higher GSI, HSI, VSI (male only), egg diameter (mm), mean number of fry/fish, and mean fry weight (g) compared to the control group (P < 0.05). Expression of reproductive-associated genes (vasa, nanos1a, nanos2, dnd1, pum1, AMH, and vtg) were significantly up-regulated in the gonads of fish in the 0.03 g/m3 treatment. The histological gonadal structure exhibited that BSL improved gonad maturation in both genders of Tilapia fish. Overall, adding a mixture of B. subtilis and B. licheniformis (0.03 g/m3 water) can accelerate reproductive performance in Red Tilapia through up-regulation of reproductive genes and enhance the health profile.

Elevated temperature impairs gonadal development by suppressing the expression of the genes for kisspeptin, GnRH1 and GTH subunits in Nile tilapia Oreochromis niloticus

Temperature is a preeminent factor in the regulation of fish reproduction and hinders gonadal development beyond a specific threshold. To comprehend the molecular mechanism responsible for reproductive suppression at different temperature, expression of the genes encoding kisspeptin (kiss2), gonadotropin-releasing hormone (gnrh1) and their receptors (gpr54, gnrh1r) in the brain, and the gonadotropin (GTH) subunits (fshb and lhb) in the pituitary were studied in juvenile Nile tilapia (Oreochromis niloticus) along with gonadal histology. Fish were acclimatized to three distinct temperatures, including 31 °C, 34 °C and 37 °C for 14 days. The mRNA levels of kiss2, gpr54, gnrh1, and gnrh1r were significantly decreased at 37 °C compared to 31 °C and 34 °C in the both sexes. In parallel, the expression level of fshb in the both sexes and lhb in the female were significantly lower at 37 °C in the pituitary. Histologically, the gonads of both sexes had normal growth of gametes at control temperature (31 °C), whereas the spermatogenesis and oocyte maturation were slowed down and atretic oocytes were found in the ovary at 37 °C acclimation temperature. Taken together, the results imply that elevated temperature beyond the specific threshold may have a negative impact on reproduction by suppressing the gene expressions of kisspeptin/GnRH1/GTH system and eventually restrains normal growth and maturation of gametes in the both sexes of Nile tilapia.

Histologic analysis of gonadal tissue in patients with Turner syndrome and Y chromosome material

This study investigates gonadal histology in individuals with Turner syndrome assigned female at birth and Y-chromosome material (TS+Y) who underwent prophylactic gonadectomy. Despite case reports suggesting spontaneous menarche and pregnancies in TS+Y, this research reveals the absence of germ cells, indicating low fertility likelihood. Germ cell neoplasia in-situ was present in some patients, emphasizing a non-negligible risk of cancer precursor. As no malignancies were found even in older individuals, the study challenges the immediate need for prophylactic gonadectomy upon TS+Y diagnosis. Limited fertility benefits are suggested, emphasizing the need for further research on optimal timing and criteria for the procedure.

Assessing the temporal isolation barrier between two closely related species of the genus Fissurella off the southwestern coast of South America

ABSTRACT Knowledge of the reproductive isolation mechanisms that prevent gene flow between species is essential for understanding the process of speciation. In this study, we investigate the spawning periods in Fissurella cumingi and F. latimarginata, two closely related sympatric species of keyhole limpets with external fertilization, as a potential prezygotic barrier between them. The reproductive cycle was assessed in two distinct locations in northern Chile using the gonadosomatic index (GSI) and gonadal histology. Species, locality, and sex were designated as predictive variables, while average GSI values, as well as those of partial and total spawning, were considered dependent variables. Both species exhibit a continuous gametogenic cycle throughout the year, characterized by multiple spawning events. A generalized linear model (GLM) factorial ANOVA design revealed that neither species, locality nor sex were significant factors with respect to the GSI and partial spawning scores. However, sex was found to be the only significant factor related to total spawning. No significant interactions were observed among predictive factors. These findings suggest that reproductive periods may not serve as effective barriers to gamete encounters between species. Our study prompts questions about how closely-related externally-fertilizing species maintain reproductive isolation.

Reproductive biology and nuptial coloration of Trachinus draco (Trachinidae) from Atlantic Spanish waters (SW Europe)

Studies focusing on reproductive biology play a key role in deciphering fish population dynamics. We studied the main features of the reproductive biology of the greater weever Trachinus draco based on gonad histology and digital image analysis techniques and the main hypothesis of the study is that males of T. draco exhibit a nuptial colouration. The biological samples of 311 individuals along the Atlantic coast of Spain, covering the twelve months of the year (from 2020 to 2022), in combination with the gonadosomatic index trend revealed a spawning season from May to August, consistent with similar studies in different geographical areas. Length-weight relationship presented differences by sex (females reach higher weight at the same length). The maturity ogive for females indicated that 50 % were mature at a length of 182 mm. Histological analysis confirmed asynchronous development of secondary growth oocytes, pointing to a batch spawning strategy. Digital image analysis of the nuptial coloration showed that the presence of a black spot above the pectoral fin in males was significantly correlated with the spawning capable phase, while blueish coloration was influenced by sex, size and maturity phase, with larger spawning males having the largest areas of blueish coloration. The results of this study represent a significant improvement of our understanding of the reproductive biology of T. draco.

Macroscopic and histologic characteristics of sexual maturation in the Burmeister’s porpoise Phocoena spinipinnis from Peru

The morphology and maturation process of gonads of 70 Burmeister’s porpoises Phocoena spinipinnis, with body lengths ranging 135 - 183 cm (n = 34 females) and 64.5 - 182 cm (n = 36 males) are described. Samples were collected in six ports of central and northern Peru from 1987 to 1999. In the field, sexual maturity was determined through macroscopic examination of gonads. Ovarian corpora indicated mature females (resting, lactating, pregnant). Semen in epididymides was considered evidence for mature males. The ovaries of Burmeister’s porpoises are ovoid or bean-shaped and flattened, with corpora modifying surface appearance. In the laboratory, ovaries were examined macro- and microscopically, measured, weighed and sliced. The number of corpora ovarica (lutea, albicantia, atretica) and their morphology were documented. The follicles, oocytes, and nuclei were measured. Follicular development was predominantly left-sided, but occurred in both ovaries in 16.3% of females, mainly in those with numerous corpora. Macroscopically, several corpora atretica with luteinization had characteristics similar to those of corpora albicantia, making microscopic determination essential. Inconclusive evidence of recent ovulation was reported for January. Two of three immature females showed good follicular development in March and April. The testes of Burmeister’s porpoise are elongated and cylindrical. Of 36 males examined macroscopically, seven were immature, five pubescent and 24 mature. The histological analysis allowed to confidently determine sexual maturity status. Field evaluation of maturity based on the presence of sperm in the epididymides is a useful but in 8.3% of cases not an exact method. No histological evidence of male reproductive seasonality was found. Spermatogenesis was perceptible year-round and tubule diameters showed stochastic monthly variations. The body length at 50% sexual maturity was estimated at 157 cm and 152.7 cm, for males and females, respectively. There was no evidence of reproductive senescence. This study is the first evaluation of the sexual maturation process in the Burmeister’s porpoise, using both macroscopic observations and elaborate gonads histology. It provides important new information on the natural history of the species, including parameters (e.g., mean body length at maturation, reproductive seasonality) highly relevant to its conservation.

Larviculture of Nile tilapia (Oreochromis niloticus) in biofloc and clear water systems: masculinization with 17 α-methyltestosterone

We evaluated the use of the hormone 17α-methyltestosterone during the larviculture of Nile tilapia in biofloc and clear water systems. A completely randomized experimental design was adopted with four treatments and four replications: biofloc system without dietary hormone incorporation (BFT-D); biofloc system with dietary hormone incorporation (BFT-H); clear water without hormone incorporation in the diet (CLW-D); and clear water with hormone incorporation in the diet (CLW-H). The 28-day larviculture of Nile tilapia was carried out using 16 tanks with capacity of 15 L. The evaluations included water parameters, growth, survival, and gonad histology. The biofloc system displayed higher values for alkalinity, total suspended solids, settling solids, and turbidity compared to the clear water system (p &lt; 0.05). Nevertheless, no difference (p &gt; 0.05) was observed for in final weight, weight gain, feed conversion factor, survival, and growth rate between treatments. Then, tilapia post-larvae can be successfully reared in both biofloc and clear water systems without any negative impact on their zootechnical performance. Nonetheless, such results showed that tilapia can be reared in alternative systems, which can increase the production of this species.

Reproduction biology of mullet fish (Mugil dussumieri) found in Bandar Khalipah Waters Serdang Berdagai Regency, North Sumatera Province

The Mullet fish (Mugil dussumieri) is one of the types of marine fish that is caught in waters Bandar Khalifah. This limited information reproductive aspects of Mugil dussumieri, such as sex ratio, gonad maturity level, gonad maturity index, fecundity, egg diameter, and gonad histology, that can be used as a guideline for the management of mullet resources to ensure sustainable utilization. This study was conducted from January to March 2023, and method used in this research was random purposive sampling. Fish samples were taken in 3 station is estuary, Pandang River and the coastal waters of Bandar Khalifah District, Serdang Berdagai Regency, North Sumatera Province, which was divided into 3 stations using a census sampling method. The total catch was sampled once a month for 3 months. The fishing gear used was a Gillnet with a mesh size of 1.5 cm. The data analysis included measurements of the samples, gonad maturity level, fecundity, and gonad histology. The reproductive results showed an unbalanced sex ratio of 1:3. The gonad maturity level of the mullet fish ranged from gonad maturity level I to gonad maturity level IV. The egg fecundity ranged from 963 to 129,311 eggs, and based on the egg diameter, the Mullet fish is classified as a partial spawner. These studies are crucial for obtaining information that can be used as a guideline for the management of Mugil dussumieri resources to ensure sustainable utilization.

Effect of various local natural ingredients fortified in fish feed on the gonad histology and water quality of tilapia (Oreochromis niloticus)

The addition of natural ingredients fortified in fish feed was developed to enhance good formulation engineering. Currently, there is a need to improve follow-up research strategies for designed feed formulations using natural ingredients in tilapia farming. It is hoped that a sustainable and eco-friendly fish farming system will be improved. This study aimed to evaluate the effect of local natural ingredients fortified in fish feed on the gonad histology and water quality of tilapia (O. niloticus). Following these results, the histology test of tilapia showed that more than 50% of the gonad tended to be male in all treatments. This showed that the use of natural ingredients in the appropriate dosage in feed would affect the masculinization of tilapia. Furthermore, the specific growth rate (SGR) with the addition of natural ingredients to the feed showed results comparable to that of synthetic hormones. It can be concluded that the use of natural ingredients such as honey, cow testes, and Tribulus fortified in fish feed revealed a positive effect on the masculinization of tilapia and water quality levels in the fish farming system.

Triclosan, an antimicrobial drug, induced reproductive impairment in the freshwater fish, Anabas testudineus (Bloch, 1792).

Triclosan (TCS), an antimicrobial drug, is known to occupy different compartments in aquatic ecosystems. The present study focused to evaluate the reproductive toxicity of triclosan, at environmentally relevant (0.009 and 9μg L-1) and sublethal (176.7μg L-1) concentrations for 90days in the pre-spawning phase of the fish, Anabas testudineus. The reproductive biomarkers, namely, gonadal steroidogenic enzymes, expression of aromatic genes, levels of serum gonadotropins, sex hormones, and histology of gonads were analyzed. The weight of the animal, brain weights along with gonadosomatic index decreased while mucus deposition increased significantly at all concentrations of triclosan as the primary defensive mechanism to prevent the entry of toxicants. Triclosan disrupted gonadal steroidogenesis as evidenced by a reduction in the activities of gonadal steroidogenic enzymes. The expressions of cyp19a1a and cyp19a1b genes were up-regulated in the brain of both sexes and testis, while down-regulated in the ovary indicating estrogenic effects of the compound. The endocrine-disrupting effects of triclosan were confirmed. The current results suggest that chronic exposure to triclosan altered reproductive endpoints thereby impairing normal reproductive functions in fish.

Combined effects of binary mixtures of 17β-estradiol and testosterone in western mosquitofish (Gambusia affinis) after full life-cycle exposure

Estrogens and androgens are typical steroid hormones and often occur together in contaminated aquatic environments, but their mixed effects in aquatic organisms have been less well reported. In this study, the endocrine disrupting effects of binary mixtures of 17β-estradiol (E2) and testosterone (T) in western mosquitofish (Gambusia affinis) were assessed by analyzing the sex ratio, secondary sex characteristics, gonadal histology, and transcriptional expression of target genes related to the hypothalamic-pituitary-gonadal (HPG) axis in G. affinis (from embryos) continuously exposed to E2 (50 ng/L), T (T1: 50 ng/L; T2: 200 ng/L), and mixtures of both (E2 + T1: 50 + 50 ng/L; E2 + T2: 50 + 200 ng/L) for 119 d. The results showed that exposure to E2 + T1 and E2 + T2 reduced the length ratio of ray 4/6 ratio in male G. affinis, suggesting feminized phenomenon in male G. affinis. Furthermore, 16.7–38.5 % of female G. affinis showed masculinized anal fins and hemal spines when exposed to T alone and in combination with E2. Importantly, the transcriptional levels of certain target genes related to the HPG axis were significantly altered in G. affinis following exposure to E2 and T alone and in combinations. Moreover, exposure to E2 and T in combinations can lead to combined effects (such as synergistic and antagonistic effects) on the transcriptional levels of some genes. These results collectively suggest that exposure to environmentally relevant concentrations of E2 and T alone and in mixtures can impact the endocrine system of G. affinis, and may pose potential risks in aquatic systems.

Interaction of 17α-ethinylestradiol and methyltestosterone in western mosquitofish (Gambusia affinis) across two generations

The interactions between estrogen and androgen in aquatic animals remain largely unknown. In this study, two generations (F0 and F1) of western mosquitofish (Gambusia affinis) were continuously exposed to 17α-ethinylestradiol (EE2, 10 ng/L), methyltestosterone (MT, 10 ng/L (MTL); 50 ng/L (MTH)), and mixtures (EE2+MTL and EE2+MTH). Various endpoints, including sex ratio (phenotypic and genetic), secondary sex characteristics, gonadal histology, and transcriptional profile of genes, were examined. The results showed that G. affinis exposed to MTH and EE2+MTH had a > 89.7 % of phenotypic males in F1 generation, with 34.5 and 50.0 % of these males originated from genetic females, respectively. Moreover, females from F0 and F1 generations exposed to MTH and EE2+MTH exhibited masculinized anal fins and skeletons. The combined effect of MT and EE2 on most endpoints was dependent on MT. Furthermore, significant transcriptional alterations in certain target genes were observed in both the F0 and F1 generations by EE2 and MT alone and by mixtures, showing some degree of interactions. These findings that the effects of EE2+MTH were primarily on the phenotypic sex of G. affinis in offspring generation suggest that G. affinis under chronic exposure to the binary mixture contaminated water could have sex-biased populations.

Growth performance, reproductive status, and chromosomal instability in triploid Nile tilapias.

Reproductive control is one of the biggest challenges in tilapia production and triploidy was developed as an alternative to sterilization. In general, polyploids present chromosomal instability but for triploid Nile tilapia it has yet to be reported. This study evaluated the chromosomal instability from juveniles to adulthood, growth performance and gonadal status of tilapia hatched from eggs submitted or not to heat shock for triploid induction. Nile tilapia oocytes were fertilized (1,476 oocytes), half of the eggs were subjected to a four-minute shock in 41 °C water four minutes after fertilization and the other half were not (Control group). The eggs were incubated (at 27°C) and 160 larvae from the treated group hatched and survived after yolk sac absorption. The determination of ploidy was performed by flow cytometry at 85th (juveniles) and 301st (adults) days of age post yolk sac absorption. At the time of the first cytometry analysis there were 73 surviving juveniles from the treated group, and only 14 were confirmed triploid. However, at the analysis of adult ploidy, one out of 8 surviving adult tilapias from the 14 confirmed triploid juveniles remained triploid. Gonadal histology showed that the non-remaining triploids continued to produce gametes. The growth performance of triploid tilapia was initially superior to that of diploid tilapia during the juvenile phase, but similar in adults. Once the chromosome sets are lost and the tilapias become diploid again, at least in tissues with a high proliferation rate, such as the hematopoietic tissue that was analyzed (and possibly in gonads), all possible advantages of triploids are probably lost. Thus, our results suggest that, due to genomic instabilities, the triploid generation of tilapia has low efficiency.

Alteration in the histology of gonads and hormone levels of fish Channa punctatus on exposure to 17-α ethynylestradiol

It has been demonstrated that effluents discharged from sewage treatment facilities allow synthetic estrogens, widely employed in contraceptives and other pharmaceutical applications, to reach the aquatic environment. One of the most biologically active xenoestrogens in the aquatic environment is the synthetic hormone 17-α ethynylestradiol(EE2), raisingreproductive issues in the fish population. The present study chose Channa punctatus fish to study the effects of EE2 on gonad histology and the calculation of estrogen and testosterone levels. The experimental setting was separated into four groups, with Group I as control with 0 ng/L of EE2. The other three groups viz., Group II to IV, had 5 ng/L, 10 ng/L, and 20 ng/L concentrations of EE2, respectively, and samples were obtained after the durations of 7, 14, 21, and 28 days. The findings showed that as 17-α ethynylestradiol concentrations increased, testosterone levels decreased from 10.88±0.24 ng/dL to 4.91±0.24 ng/dL,while estrogen levels increased from 42.7±2.22 pg/mL to 120.18±4.54 pg/mL. The number percentage of primary growth oocytes, previtellogenic oocytes and vitellogenic oocytes in the histology of ovary decreased from 28.45±1.42 to 10.43±0.47; 16.22±0.81 to 3.15±0.16 and 14.81±0.89 to 2.14±0.11 respectively while in testis, the number percentage of spermatogonia, spermatocytes decreased from 21.4±0.86 to 15.1±0.68 and 97.1±3.88 to 54.2±3.25. In contrast, the percentage of mature spermatids increased from 20.8±0.94 to 40.2±2.41. Since synthetic estrogens adversely affect aquatic animals, especially fish, they should be treated properly before releasing them into the aquatic environment.

Otolith microchemistry reveals diverse habitat uses and migratory patterns of two Coilia species (Engraulidae) in the Min River Estuary, southern China

A recent study based on gonad histology revealed that the existence of the spawning grounds for Gray's grenadier anchovy (Coilia grayii) and Osbeck's grenadier anchovy (C. mystus) in the Min River Estuary, the largest in Fujian Province, southern China. Further confirming their natal sources and migratory patterns is essential to understand their life histories. We used otolith microchemistry to assess the origins and habitat uses of 23 C. grayii and 22 C. mystus, collected the Min River Estuary and the adjacent waters. The results showed that C. grayii spawned in both freshwater (n‍ = 14) and brackish water (n = 9), and C. mystus spawned mainly in brackish water (n‍ = 20) with minor in freshwater (n‍ = 1) and marine water (n‍ = 1). The migratory patterns of C. grayii (four types) and C. mystus (five types) were diverse, mainly exhibiting anadromous and semi-anadromous behaviors. The first migratory behavior of C. grayii and C. mystus occurred within the age of the first year. The findings have significant implications for fishery stock management of the Min River Estuary and its adjacent waters.

Potent ovarian development as being stimulated by cocktail hormone in the female Scylla olivacea

The mud crab Scylla olivacea is widely cultured for its economic value, but reproduction issues limit its production. Vitellogenesis-inhibiting hormone (VIH), serotonin (5-HT), and gonadotropin-releasing hormone (GnRH) are important neurohormones that control reproduction in crustaceans. Mimicking crab hormone stimulation during reproduction has scarcely been reported. Comparison of the single hormone and multiple hormone approaches to crab hormonal control in S. olivacea is limited. In situ hybridization showed that injection of dsRNA-VIH could abolish its gene expression in neuronal clusters of female S. olivacea eyestalks, potentially reducing its inhibitory effects on ovarian maturation. This was confirmed by assessing the ovarian gonadosomatic index (GSI), hemolymph vitellogenin (Vg), an indicator of vitellogenesis, and gonad histology using dsRNA-VIH and 5-HT/GnRH combinations. Based on our findings, we demonstrated that administration of dsRNA-VIH significantly increased the gonadosomatic index (GSI) on days 14 and 28 post-treatment. The combination cocktail, however, consisting of 5-HT + GnRH + dsRNA-VIH on days 14 and 28, and GnRH + dsRNA-VIH on day 28, was the most efficacious in increasing GSI and enhancing crab ovarian maturation. Upregulation of hemolymph Vg levels was observed solely on the 28th day following treatment with dsRNA-VIH, 5-HT + GnRH + dsRNA-VIH, and GnRH + dsRNA-VIH. Differential gene expression analysis using quantitative RNA-sequencing of the neural tissues (brain and ventral nerve cord), revealed a significant upregulation of certain receptors (5-HTR, GnRHR, LHR, and FSHR), neuropeptides (sNPF, NPF1, NPF2, SIFamide, AKH/Crz, CHH, and RPCH), downstream reproductive-related genes (FAMeT, ESULT, progesterone-like protein), and prostanoid-related genes (phospholipase A and C, COX, Thromboxane A synthase, prostaglandin D, E, and F synthases) following treatment, particularly dsRNA-VIH + GnRH and/or 5-HT-injected individuals. Upregulation of prostaglandin E synthase and estrogen sulfotransferase genes was confirmed by real-time PCR. Since the construction and propagation of dsRNA-VIH is costly, its lower dose application supplemented with synthetic GnRH and/or 5-HT may be an alternative approach to ensure that female S. olivacea attain sufficient reproductive fecundity in aquaculture. Furthermore, we propose that the administration of multiple hormones in crabs may better emulate the physiological conditions of crustaceans in their natural habitat.

The effects of 17α-methyltestosterone on gonadal histology and gene expression along hypothalamic-pituitary-gonadal axis, germ cells, sex determination, and hypothalamus-pituitary-thyroid axis in zebrafish (Danio rerio).

As a synthetic androgen, 17α-methyltestosterone (MT) is widely used in aquaculture to induce sex reversal and may pose a potential risk to aquatic organisms. This ecological risk has attracted the attention of many scholars, but it is not comprehensive enough. Thus, the adverse effects of MT on zebrafish (Danio rerio) were comprehensively evaluated from gonadal histology, as well as the mRNA expression levels of 47 genes related to hypothalamic-pituitary-gonadal (HPG) axis, germ cell differentiation, sex determination, and hypothalamus-pituitary-thyroid (HPT) axis. Adult zebrafish with a female/male ratio of 5:7 were exposed to a solvent control (0.001% dimethyl sulfoxide) and three measured concentrations of MT (5, 51 and 583 ng/L) for 50 days. The results showed that MT had no significant histological effects on the ovaries of females, but the frequency of late-mature oocytes (LMO) showed a downward trend, indicating that MT could induce ovarian suppression to a certain extent. The transcriptional expression of activating transcription factor 4b1 (atf4b1), activating transcription factor 4b2 (atf4b2), calcium/calmodulin-dependent protein kinase II delta 1 (camk2d1), calcium/calmodulin-dependent protein kinase II delta 2 (camk2d2) and calcium/calmodulin-dependent protein kinase II inhibitor 2 (camk2n2) genes in the brain of females increased significantly at all treatment groups of MT, and the mRNA expression of forkhead box L2a (foxl2) and ovarian cytochrome P450 aromatase (cyp19a1a) genes in the ovaries were down-regulated by 5 and 583 ng/L group, which would translate into inhibition of oocyte development. As compared to females, MT had relatively little effects on the reproductive system of males, and only the transcriptional alterations of synaptonemal complex protein 3 (sycp3) and 17-alpha-hydroxylase/17,20-lyase (cyp17) genes were observed in the testes, not enough to affect testicular histology. In addition, MT at all treatments strongly increased corticotropin-releasing hormone (crh) transcript in the brain of females, as well as deiodinase 2 (dio2) transcript in the brain of males. The paired box protein 8 (pax8) gene was significantly decreased at 51 or 583 ng/L of MT in both female and male brains. The above results suggest that MT can pose potential adverse effects on the reproductive and thyroid endocrine system of fish.

Induction of Reproductive Sterility in Coho Salmon (Oncorhynchus kisutch) by an Immersion-Based Gene Silencing Technology

Undesired maturation and reproduction are major challenges in fish aquaculture regarding genetic introgression, precocious maturation, and reproduction-related mortality, which can have profound ecological or economic impacts. Farming reproductively sterile fish can effectively mitigate these challenges. In this paper, we transferred and applied a novel immersion-based, non-transgenic gene silencing technology to sterilize coho salmon for the first time. Unfertilized eggs were bath immersion-treated with csdnd-MO-Vivo in different immersion media. Eyed rates of treated groups ranged from 0.9 to 63.5%. Sterile fish lacking germ cells, and those with arrested germ cells/atretic oocytes, were obtained at 14 and 20 months of age, albeit at a low percentage (2.3 to 10.0% based on females). Gonadal histology and vasa/nanos3 gene expression profile were provided for comparing fertile and sterile gonads, as well as retarded ovaries. Future directions and strategies for optimizing the technology and improving sterility induction were also proposed. The successful production of sterile coho salmon achieved in this study demonstrates the proof of principle for this new sterilization technology. As we continue to expand upon these findings and refine the technology, achieving coho salmon sterile population farming would facilitate the future transfer and application to other commercially important aquaculture fish.

Experimental exploration of estrogenic effects of norethindrone and 17α-ethinylestradiol on zebrafish (Danio rerio) gonads

Synthetic progestins and xenoestrogens found in aquatic habitats are currently gaining attention on global scale. The current study aimed to investigate the time-and dose-dependent effects of synthetic progestin Norethindrone (NET; 100, 500 and 1000 ng/L) and estrogen 17α-ethinylestradiol (EE2; 100 ng/L) individually as well as in binary mixture (1000 ng/L NET + 100 ng/L EE2) on reproductive histology and transcriptional expression profile of genes in adult zebrafish. For this, 20 female (3.15 ± 0.18 cm & 0.33 ± 0.06 g) and 20 male zebrafish in each group (2.93 ± 0.13 cm & 0.29 ± 0.04 g) were exposed to drugs dissolved in water for 30 days in 12 L rectangular tanks. We found that both NET and EE2 exposure reduced the gonadosomatic index in females, while only EE2 exposure caused significant reduction in males (p ≤ 0.05). Interestingly, NET delayed oocyte maturation in females and accelerated spermatogenesis in males, while EE2 consistently suppressed sperm maturation throughout the experiment. Further, qRT-PCR results revealed differential expression pattern of the study genes (er-α, er-β1, er-β2, pgr, vegfaa and p53) in male and female zebrafish. Co-exposure indicated potential inconsistencies in steroidal function in mixtures rather than single exposures. Our findings imply that changes in gonadal histology after NET and EE2 exposure may result from unique regulation of steroid hormone receptors. Additionally, significantly reduced p53 levels (p ≤ 0.05) following co-exposure in both sexes may suggest an elevated risk of neoplastic transformations. Further research with mammalian models will help to explore the mechanisms behind differing effects of alone and co-exposures.