Ovarian inseminated sperm impacts spawning success in zebrafish, Danio rerio (Hamilton, 1822) even in the absence of a male stimulus.

Reproductive obstacles have led scientists to develop novel techniques/technologies for artificial reproduction. We aimed to investigate the possibility of propagating zebrafish females using sperm ovarian lavage with and without presence of male stimulus. This experiment consisted of several treatments: traditional spawning approaches with females and wild-type males (AB♀ × AB♂); no males present with non-manipulated females (AB♀); no males present with females inseminated with NaCl into ovarian lobes [AB♀(inj.NaCl)]; no males present with females inseminated with sperm from transgenic males into ovarian lobes [AB♀(inj.Tg♂)]; non-manipulated females kept separately from wild-type males (AB♀|AB♂); females kept separately from wild-type males and inseminated with NaCl into ovarian lobes [AB♀(inj.NaCl)|AB♂]; and females kept separately from wild-type males and inseminated with sperm from transgenic males into ovarian lobes [AB♀(inj.Tg♂)|AB♂]. There were no released eggs in both negative control treatments (AB♀ and AB♀|AB♂). Egg production increased (ranged from 0 to 28.5 eggs/female) when females were injected in the presence [AB♀ (inj.NaCl) |AB♂] or absence of male stimulus [AB♀ (inj.NaCl) and (AB♀(inj.Tg♂)]. A further increase in egg production [relative to AB♀, AB♀ (inj.NaCl), and AB♀|AB♂] was detected when females were inseminated with pooled sperm from transgenic males in the presence of male stimulus [AB♀(inj.Tg♂)|AB♂; ranged from 2.5 to 55 eggs/female] or when using traditional spawning approaches (AB♀ × AB♂; ranged from 25 to 131 eggs/female). Females inseminated with sperm produced embryos, although no differences were detected when females were inseminated with pooled sperm from transgenic males in presence (11.8 ± 16.3%) or absence (average = 12.6 ± 9.2%) of male stimulus. Traditional spawning approaches produced the most eggs (81.2 ± 42.3 per female) and highest fertilization rate (81.3 ± 10.4).

Reproductive aspects of freshwater unionid mussel sperm: Seasonal dynamics, male-to male variability, and cell quantification.

Freshwater unionid mussel diversity is decreasing because of species extirpation or extinction. While little can be done to recover lost species, there is an opportunity to develop techniques to save other species. This can be facilitated through gene banking and assisted reproduction. Unfortunately, limited information is available on mussel reproduction, especially relating to sperm quality. Objectives, therefore, were to quantify seasonal changes in sperm concentration and morphology for two unionid mussels, Ligumia subrostrata and Lampsilisstraminea, measure intraspecific heterogeneity for sperm morphometry, and develop an efficient method to quantify sperm concentration using a microspectrophotometer. There were no differences in sperm concentration when cells were extracted from the center or at a half centimeter on either side of the visceral mass, during the spawning season. There was a seasonal change in sperm concentration, such that concentration for L. subrostrata ranged from 1.1 × 109 to 19.60 × 109 cells/mL with there being the largest counts between 26 September to 7 November. L. straminea sperm concentration was greatest (20.0 × 109 cells/mL) on 13 September and subsequently decreased. Sperm were uniflagellated and SEM results for L. subrostrata and L. straminea showed mean head length and width (mid-spawning) were 3.38 ± 0.04 µm and 1.61 ± 0.01 µm and 3.37 ± 0.04 µm and 1.61 ± 0.01 µm, respectively. There were close (R2 ≥ 0.85) quadratic associations between hemocytometer counts and absorbance (300, 600, 700 nm). These results provide baseline information to further investigate sperm quality, fertilizing capacity, and cryopreservation for freshwater mussels.

Growth, survivorship, and predator avoidance capability of larval shortnose sturgeon (Acipenser brevirostrum) in response to delayed feeding.

Larval shortnose sturgeon, reared at 17°C, were subjected to delayed feeding treatments of 0, 5, 10, 15, 18, and 23 days post-yolk absorption to examine effects of food deprivation on growth, survival, swimming activity, and escape capabilities. Starvation affected growth and survival but despite degree of starvation, larvae were able to resume growth and experience high survivorship following feeding. Specific growth rate based on larval dry weight for the period directly following first feeding was highest for the day 15 and 18 delayed feeding treatments. There were no differences in survival between the 0 and 5 day treatments, however survival was reduced to 71.2%, 45.4%, and 28.8% for 10, 15, and 18 day delayed feeding treatments, respectively. Shortnose sturgeon had a point-of-no-return (PNR; 55.7% initiated feeding) at ~19 days (or 42 days post-fertilization) following the full absorption of yolk. Mean percent swimming activity and swimming speeds showed an interaction between delayed feeding treatment and larval age, such that no differences were detected at 1 and 6 days post-yolk absorption, while these swimming behaviors generally increased or spiked as feeding was delayed for 10, 15, and 18 days post-yolk absorption. At 23 days post-yolk absorption, only swimming speed increased for larvae that were denied food for 18 days. While there was an interaction between delayed feeding treatments and age for proportion of larvae exhibiting an escape response, generally, larvae from all feeding treatments exhibited a positive escape response. There were also interactions between delayed feeding treatments and age post-yolk absorption for mean and maximum escape speeds, such that less aggressive escape responses were typically detected the longer larvae were denied food. Our research suggests that larval shortnose sturgeon increase physical activity during periods of starvation to find a food patch while remaining vigilant but maybe not as capable to defend against a predatory attack as fed individuals.

Spatiotemporal expression of activin receptor-like kinase-5 and bone morphogenetic protein receptor type II in the ovary of shortfinned eel, Anguilla australis.

In the eel ovary, the expression of growth differentiation factor-9 (Gdf9) appears to be largely confined to the germ cell in early stages of oogenesis. However, both the target tissue and the function of Gdf9 in fish remain unknown. This study aimed to describe the abundance and localization of activin receptor-like kinase-5 (Alk5) and bone morphogenetic protein receptor type II (Bmpr2), which together mediate the Gdf9 signal, in the ovary of a basal teleost, the shortfinned eel, Anguilla australis, during early folliculogenesis. The cDNA encoding eel alk5 and bmpr2 genes were cloned, characterized and the transcript abundances of these receptors quantified by quantitative real-time PCR. Ovarian transcript abundance for both receptors, along with that of gdf9 and of its paralogue bmp15, increased from the previtellogenic to early vitellogenic stage. Localization of receptor mRNAs by in situ hybridization revealed that these receptors are located in the somatic cells surrounding the oocyte. Furthermore, tissue distribution analysis showed that the expression of alk5 and bmpr2 were highest in ovary and thyroid, respectively. Unexpectedly, however, bmpr2 mRNA levels were lower in the ovary than in any of the other 17 tissues examined, and indeed, lower than ovarian gdf9 transcript abundance. These findings, together with the ovarian expression pattern of Gdf9, suggest that Gdf9, and conceivably, Bmp15, from the oocyte can signal through receptors that are located on the somatic cells surrounding the oocyte; this, in turn, facilitates elucidation of the function of these growth factors during oogenesis in teleost fish.

Outcomes of in vitro fertilization with frozen-thawed sperm: An analysis of post-thaw recovery of sperm, embryogenesis, offspring morphology, and skeletogenesis for a cyprinid fish.

BACKGROUND: Gamete cryopreservation causes cellular damage and death. This study develops cryopreservation techniques for Levantine scraper, and deciphers how early offspring development is affected when eggs are sired with fresh and frozen-thawed sperm. RESULTS: Cryopreserved sperm did not affect embryogenesis at two- and four-cell stages, but impaired embryonic development at eight-cell stage. Embryonic viability decreased at organogenesis, where only 34-49% of embryos showed viability with frozen-thawed sperm. Hatching success and percentage of normal hatched embryos declined when fertilized with frozen-thawed sperm. Considering only frozen-thawed cells the dimethyl sulfoxide (DMSO)-5%, methanol (METH)-5%, and METH-10% treatments yielded highest hatch, while METH-5% and propylene glycol-5% yielded the most normal hatched embryos. Larval spinal torsion was higher for fresh than frozen-thawed sperm, where larvae with spinal torsion showed vertebral fusion and shape alterations during exogenous feeding. Both fresh and cryopreserved treatments showed abnormalities in caudal skeleton, while rates of defective yolk-sacs were higher for cryopreserved sperm, where larvae with defective yolks showed oversized yolk extension. Percentage of larvae with defective heads/eyes were also higher for cryopreserved sperm. CONCLUSIONS: Results show how frozen-thawed sperm impairs embryonic/larvae development and identifies frequency and position of abnormalities. Future studies should investigate how sperm DNA damage may have caused these alterations.

Ovarian fluid and its impacts on spermatozoa performance in fish: A review.

Factors such as gamete quality can profoundly affect fertility, but the spawning micro-environment that surrounds the spermatozoa and eggs during gamete contact has largely been neglected. In fishes, understanding these gametic interactions is crucial because each female creates a unique spawning environment by simultaneously expelling her distinct ovarian fluid (OF) along with an egg batch. In turn, OF has been shown to influence spermatozoa performance traits by modifying spermatozoa behaviors and fertilization outcomes. Here, we shed light on these gametic interactions by overviewing literature on OF and how it impacts spermatozoa performance traits. Fish OF is clear or has slight coloration and can constitute ≤10-30% of egg mass. Viscosity of the OF is ∼2- to 3-fold higher than water and its pH ranges 6.2 to 8.8. Osmolality of the OF is lower in freshwater (190-322 mOsmol/kg) than marine species (289-514 mOsmol/kg). Na+ (98.3-213.7 mmol/L) and Cl- (89.8-172.7 mmol/L) are predominant ions in OF, while K+ (1.7-19.3 mmol/L), Mg2+ (0.4-8.1 mmol/L), and Ca2+(0.5-9.7 mmol/L) ions are detected at lower concentrations. Protein levels can be high in OF and exhibit intra- and inter-species variation (54-826 mg/100 mL). Fish OF also contains a series of organic components and substances that enhance and/or attract sperm towards the vicinity of an egg. OF can also differentially impact sperm based on genetic relatedness of mates, male phenotype (i.e. alternative reproductive tactics), or geographic origin. To conclude, when testing further reproductive paradigms, we suggest a shift from classic spermatozoa activation medium (water only) to more natural spawning media, which encompass OF-spermatozoa interactions.

Embryogenesis and early larval development in wild-caught Levantine scraper, Capoeta damascina (Valenciennes, 1842).

Levantine scraper, Capoeta damascina is a candidate species for future stock assessments, conservation studies, and hatchery efforts. Herein, we documented embryonic and early larval development, from egg activation to the exogenous feeding period, using morphological and histological landmarks. Embryos were obtained by in vitro fertilization from hormonally induced wild-caught broodstock, and subsequent development was monitored at temperatures coinciding with native conditions. Embryonic development from fertilization to hatch lasted ~105-110 hr. Larvae emerged with unpigmented eyes and body morphology, as well as an undifferentiated digestive tract. The mouth was closed at hatch by the oropharyngeal membrane and opened by the early endogenous feeding period. Trabeculae cartilage, quadrate bone, and Meckel's cartilage of the endoskeleton were present during the endogenous feeding period. During this period, the larvae underwent considerable changes in craniofacial morphology, locomotion, and organogenesis of the digestive tract. The cartilaginous floor of the neurocranium developed and the first four ceratobranchials appeared simultaneously at the end of endogenous feeding period. The digestive tract was differentiated into buccopharynx, esophagus, and small intestine during the endogenous feeding period. The intestinal valve and numerous longitudinal folds at the posterior region of the intestine formed together by the endo-exogenous feeding period. Major developmental events in retinogenesis occurred during the endogenous feeding period. When larvae entered exogenous feeding the mouth was fully-functional. Additionally, liver size and eye diameter increased. Our analysis of embryonic and early larval development in Levantine scraper aligned with other freshwater fishes.

Spawning performance, serum sex steroids, and ovarian histology in wild-caught Levantine scraper, Capoeta damascina (Valenciennes, 1842) treated with various doses of sGnRHa + domperidone.

As global aquaculture continues to expand, increasing efforts are focusing on assisted reproductive technologies. This study sought to test whether salmon GnRH [D-Arg6, Pro9NEt]) analogue (sGnRHa) + domperidone injection at 0.25 mL/body weight (BW; 5-µg sGnRHa + 2.5-mg domperidone), 0.5 mL/kg BW (10-µg sGnRHa + 5-mg domperidone), or 1.0 mL/kg BW (20-µg sGnRHa + 10-mg domperidone) affects spawning performance and gamete quality in wild-caught Levantine scraper, Capoeta damascina. The ability of these treatments to elicit a response was further examined by in vivo stimulation of estradiol (E2) and 17α,20ß-dihydroxy-4-pregnen-3-one (DHP) and by its in vivo potency to induce oocyte maturation (OM). Females that received saline injection (control) did not spawn, whereas sGnRHa + domperidone induced ovulation and spawning across the hormonal gradient. Spawning success was highest with the 0.5 mL/kg dosage (80%) and female latency period decreased with increasing dosage. Females treated with 0.5 mL/kg had a significantly higher fecundity than those injected with 0.25 or 1.0 mL/kg. Mean oocyte diameter significantly increased in females treated with 0.5 or 1.0 mL/kg. Fertilization success, hatching rate, larvae morphology, and survival were not affected by hormonal treatment. At 12 h postinjection, E2 levels significantly declined in females treated with 0.5 or 1.0 mL/kg, whereas DHP levels significantly increased across the hormonal gradient. This steroidogenic shift is supported by histological analyses, where OM was accelerated by administration of sGnRHa + domperidone in a dose-dependent manner. In conclusion, the 0.5 mL/kg dosage of sGnRHa + domperidone is recommended for assisted reproduction of Levantine scraper.

Effect of hCG and Ovaprim™ on reproductive characteristics of male Levantine scraper, Capoeta damascina (Valenciennes, 1842).

Species richness and abundance within the genus Capoeta has been depleted. As such, there is great need for developing assisted reproductive technologies for controlling reproduction in captivity. Here, we conducted in vivo studies with single administrations of human chorionic gonadotropin (hCG) and Ovaprim™ [(D-Arg6, Pro9NEt)-sGnRH + domperidone] in wild-caught Levantine scraper, Capoeta damascina and then evaluated milt characteristics, fertilization success, serum sex steroids, and spermatogenesis via histological testicular development. Spermiation responses were significantly stronger for Ovaprim injected fish than those injected with hCG or saline. hCG had a negative effect on milt quality by reducing the percentage of motile sperm and fertilization success at 12-48 h post injection (hpi), which was not observed after treatment with Ovaprim or the saline injection. Hormonal therapy resulted in higher sperm densities and spermatocrit, although sperm longevity was not impacted. Sex steroids were not impacted by hCG or saline injection, but Ovaprim effectively induced androgen and progestin release, as evident by higher serum levels of testosterone, and 17α,20ß-dihydroxy-4-pregnen-3-one. Consequently, their levels peaked at 12 hpi, which coincided with maximal milt production. Histological analysis of the testes and quantification of germ cell types revealed that Ovaprim significantly stimulated spermiogenesis, as a higher number of accumulated spermatozoa were observed at 12 h and 24 hpi. Testes from saline and hCG-injected fish remained unchanged through the experiment, and contained all stages of germ cells, predominantly spermatocytes with few spermatozoa. In conclusion, Ovaprim treatment successfully induced steroidogenesis and maturation of spermatogenic germ cells, leading to spermiation and milt production without having any negative impacts on sperm quality and fertility in wild-caught C. damascina.

Hormonal induction of ovulation using Ovaprim™ [(D-Arg6, Pro9NEt)-sGnRH+domperidone] and its impact on embryonic development of wild-caught Longspine scraper, Capoeta trutta (Heckel, 1843).

Knowledge of gamete quality is a prerequisite for developing techniques to fertilize eggs and rear offspring for hatchery production. Our objective was to develop assisted reproductive techniques, via hormonal induction of final oocyte maturation (FOM), for Longspine scraper, Capoeta trutta. Fish were administered injections of salmon gonadotropin-releasing hormone analogue containing anti-dopaminergic drug (Ovaprim™) or saline (control). Effects of Ovaprim on induction of ovulation, gamete quality, embryonic development, and larval survival were later examined with serum steroid hormone levels and ovarian histology. The saline group failed to spawn, whereas Ovaprim accelerated FOM and induced spawning. Fish treated with Ovaprim showed an increase in gonadosomatic index, egg diameter, and wet weight relative to controls. Average absolute fecundity, relative fecundity, fertilization, and hatching rates were 8823 eggs/spawn, 53 eggs/g body weight, 95%, and 91%, respectively. Serum 17α,20ß-dihydroxy-4-pregnen-3-one (DHP) levels were significantly enhanced by ∼4-fold in Ovaprim-treated fish compared to the saline-injected fish, while 17ß-estradiol levels declined upon FOM in hormone treated fish. Embryonic development closely resembled the teleost scheme, despite variations in timing. Larval survival at 6 and 12days post-hatch were 98% and 95%, respectively. Results suggest that Ovaprim is efficient for inducing spawning in C. trutta for stock enhancement or hatchery purposes.

Effects of 11-ketotestosterone and temperature on inhibin subunit mRNA levels in the ovary of the shortfinned eel, Anguilla australis.

Members of the transforming growth factor-b (TGFb) superfamily are important during early oogenesis in mammals. In this study, we tested whether documented effects of 11-ketotestosterone (11KT) on previtellogenic eel ovaries are mediated through affecting the expression of key ovarian TGFb genes. Furthermore, we investigated whether 11KT effects interacted with temperature. Accordingly, three thermal regimes were compared and their interaction with 11KT-mediated actions on expression of TGFb superfamily genes (chiefly inhibin subunits) evaluated in the eel (Anguilla australis). Inhibin subunit mRNA levels were also measured in ovarian explants cultured in vitro with 11KT and in ovaries from eels collected from the wild. In wild eels, inhibin-bA mRNA levels were higher in early than in previtellogenic eels; inhibin-a expression did not differ between stages, whereas that of inhibin-bB first decreased, then recovered with advanced developmental stage. Temperature was ineffective in modulating any of the end points, at least as long as a Q10 adjustment was made to correct for 'metabolic dose'. However, 11KT affected the expression of inhibin-a compared to control fish, while those of inhibin-b subunit genes remained unaffected. In contrast, 11KT dramatically reduced mRNA levels of inhibin-b subunits in vitro, but had inconsistent effects on inhibin-a transcript abundance. We conclude that 11KT affects ovarian inhibin subunit gene expression, but effects are not in keeping with the changes seen during early oogenesis in eels from the wild. We further contend that in vivo temperature experiments are easily biased and that Q10 corrections may be required to identify 'true' temperature effects.

Steroidogenic acute regulatory protein transcript abundance in the eel, Anguilla australis: changes during the induced reproductive cycle and effects of follicle-stimulating hormone during previtellogenesis.

Steroidogenic acute regulatory protein (StAR) mRNA levels in the eel ovary were assayed by quantitative PCR and related to plasma steroid levels throughout oogenesis in order to shed light on the previously considered 'aberrant' prematurational increase in plasma levels of estradiol-17ß (E2). Total ovarian StAR transcript abundance mirrored circulating levels of E2, but not of 11-ketotestosterone (11KT). The study was complemented by evaluation of in vitro effects of follicle-stimulating hormone (FSH) on ovarian StAR transcript abundance and on short-term ('acute') radiolabelled pregnenolone-supported steroid metabolism by ovarian fragments to understand how the production of steroids during previtellogenic oocyte growth is regulated. We observed a significant effect of FSH on StAR mRNA levels within 24h of incubation, but these were no longer evident by 4 days of culture. Unexpectedly, FSH had no effect on substrate-supported steroidogenesis, as comparable yields of steroid products were detected using semi-quantitative HPLC and scintillation counting. We conclude that the eel ovarian follicle can respond to FSH from a very early stage of development (early oil droplet stage) by increasing StAR mRNA levels, but that there is no evidence for acute effects of FSH on bioactive steroid production downstream of cytochrome P450 side-chain cleavage. Furthermore, the prematurational increase in StAR mRNA in vivo is in keeping with general teleost models and is likely to be a 'normal' response to reaching advanced stages of development.