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.

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.

Paternal identity impacts embryonic development for two species of freshwater fish.

Paternal, compared to maternal, contributions were believed to have only a limited influence on embryonic development and larval fitness traits in fishes. Therefore, the perspective of male influence on early life history traits has come under scrutiny. This study was conducted to determine parental effects on the rate of eyed embryos of Ide Leuciscus idus and Northern pike Esox lucius. Five sires and five dams from each species were crossed using a quantitative genetic breeding design and the resulting 25 sib groups of each species were reared to the embryonic eyed stage. We then partition variation in embryonic phenotypic performance to maternal, paternal, and parental interactions using the Restricted Maximum Likelihood (REML) model. Results showed that paternal, maternal, and the paternal×maternal interaction terms were highly significant for both species; clearly demonstrating that certain family combinations were more compatible than others. Paternal effects explained 20.24% of the total variance, which was 2-fold higher than the maternal effects (10.73%) in Ide, while paternal effects explained 18.9% of the total variance, which was 15-fold higher than the maternal effects (1.3%) in Northern pike. Together, these results indicate that male effects are of major importance during embryonic development for these species. Furthermore, this study demonstrates that genetic compatibility between sires and dams plays an important role and needs to be taken into consideration for reproduction of these and likely other economically important fish species.

First report on facultative parthenogenetic activation of eggs in sterlet sturgeon, Acipenser ruthenus.

This study reported facultative parthenogenetic cleavage development of sterlet sturgeon Acipenser ruthenus eggs and quantified the percentage of parthenogenetically developed eggs in relation to the fertilization ability of different females. When eggs were activated in freshwater, 5.1-13.7% of eggs developed parthenogenetically, while among those activated eggs 3.6-9.4% developed to 2 cells, 0.4-4.5% developed to 4 cells, and 0-0.8% developed to 8 cells. The mean percentage of fertilized and parthenogenetically activated eggs among the females was negatively correlated (R(2)=0.71, p=0.036), which indicates that parthenogenetic activation rate of sterlet eggs depends on the quality of eggs in terms of fertilization rate.

Effects of preincubation of eggs and activation medium on the percentage of eyed embryos in ide (Leuciscus idus), an externally fertilizing fish.

Standardization of fertilization protocols is crucial for improving reproductive techniques for externally fertilizing fish in captive breeding. Therefore, the objectives of this study were to determine the effects of preincubation of eggs and activation medium on the percentage of eyed embryos for ide (Leuciscus idus). Pooled eggs from five females were preincubated in three different activating media for 0, 30, 60, 90, and 120 seconds and then fertilized by pooled sperm from five males. At the eyed-egg stage, the percentage of viable embryos was later calculated. Results showed that preincubation time was significant for the freshwater activation medium (P < 0.001), such that the percentage of eyed embryos declined across the preincubation time gradient. Additionally, there was an effect on the percentage of eyed embryos when eggs were incubated with Woynarovich solution (P < 0.001), such that a decline was detected at 90 seconds, whereas no effect was detected for the saline water medium. Activating medium had a significant effect on the percentage of eyed embryos for each preincubation time (P < 0.05). More precisely, freshwater produced the lowest percentage of eyed embryos at all preincubation times (ranged from 1.9% at 120 seconds to 43.6% at 0 seconds), whereas saline water and Woynarovich solution produced the highest percentage of eyed embryos at 0 seconds and 30 seconds before incubation. Woynarovich solution produced the highest percentage of eyed embryos at 60 seconds (65.26%), whereas saline water produced the highest percentage at 90 seconds (68.37%). No difference was detected between saline water and Woynarovich solution at 120 seconds. Examination of sperm traits showed no impact of activating medium on computer assisted sperm analysis parameters. Together, these results suggest that saline water or Woynarovich solution improve fertilization rate in ide during IVF; thus, these media are useful for standardizing fertilization protocols and controlled reproduction for this species.

Effects of salinity and sea salt type on egg activation, fertilization, buoyancy and early embryology of European eel, Anguilla anguilla.

Improper activation and swelling of in vitro produced eggs of European eel, Anguilla anguilla, has been shown to negatively affect embryonic development and hatching. We investigated this phenomenon by examining the effects of salinity and sea salt type on egg dimensions, cell cleavage patterns and egg buoyancy. Egg diameter after activation, using natural seawater adjusted to different salinities, varied among female eels, but no consistent pattern emerged. Activation salinities between 30-40 practical salinity unit (psu) produced higher quality eggs and generally larger egg diameters. Chorion diameters reached maximal values of 1642 ± 8 µm at 35 psu. A positive relationship was found between egg neutral buoyancy and activation salinity. Nine salt types were investigated as activation and incubation media. Five of these types induced a substantial perivitelline space (PVS), leading to large egg sizes, while the remaining four salt types resulted in smaller eggs. All salt types except NaCl treatments led to high fertilization rates and had no effect on fertilization success as well as egg neutral buoyancies at 7 h post-fertilization. The study points to the importance of considering ionic composition of the media when rearing fish eggs and further studies are encouraged.

Effects of pre-incubation of eggs in fresh water and varying sperm concentration on fertilization rate in sterlet sturgeon, Acipenser ruthenus.

Standardization of fertilization protocols for sterlet Acipenser ruthenus is crucial for improving reproductive techniques and for conservation purposes. Our objectives were to determine the number of sperm (tested 430,000:1, 43,000:1, 4300:1, 430:1 sperm to egg) required to fertilize eggs and explore how pre-incubation of eggs in freshwater for 0min, 0.5min, 1min, and 10min interacts with different sperm ratios. Fertilization success ranged from 29.7% at 430:1 to 84.2% at 430,000:1. Pre-incubation time had no effect on fertilization success at 430,000:1 and 43,000:1 sperm to egg ratios, while it was significant at the 4300:1 and 430:1 ratios. The use of adequate experimental suboptimal sperm to egg ratio revealed a positive effect of pre-incubation time, such that at the 430:1 ratio, 0.5min pre-incubation increased the fertilization rate than 10min. At 0min pre-incubation the proportion of fertilized eggs increased at the 430,000:1 ratio, while at 1min fertilization increased at the 4300:1 ratio. At the 10min pre-incubation time, fertilization increased at the 43,000:1 ratio. Moreover, at the 0.5min pre-incubation time, the 43,000:1 ratio increased the fertilization rate than the 430:1 ratio. Generally, for 430:1 ratio, the fertilization rate is lower than in control. Transmission electron microscopy showed that pre-incubation of eggs in water for <10min does not trigger a cortical reaction or the formation of a perivitelline space. Results suggest that with a low sperm to egg ratio 0.5 to 1min pre-incubation of eggs in freshwater prior to fertilization can enhance fertilization rate of sterlet.

Impact of dietary fatty acids on muscle composition, liver lipids, milt composition and sperm performance in European eel.

In order for European eel aquaculture to be sustainable, the life cycle should be completed in captivity. Development of broodstock diets may improve the species' reproductive success in captivity, through the production of high-quality gametes. Here, our aim was to evaluate the influence of dietary regime on muscle composition, and liver lipids prior to induced maturation, and the resulting sperm composition and performance. To accomplish this fish were reared on three "enhanced" diets and one commercial diet, each with different levels of fatty acids, arachidonic acid (ARA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA). Neutral lipids from the muscle and liver incorporated the majority of the fatty acid profile, while phospholipids incorporated only certain fatty acids. Diet had an effect on the majority of sperm fatty acids, on the total volume of extractable milt, and on the percentage of motile sperm. Here, our results suggest that the total volume of extractable milt is a DHA-dependent process, as we found the diets with the highest DHA levels induced the most milt while the diet with the lowest DHA level induced the least amount of milt. The diet with the highest level of ARA induced medium milt volumes but had the highest sperm motility. EPA also seems important for sperm quality parameters since diets with higher EPA percentages had a higher volume of milt and higher sperm motility. In conclusion, dietary fatty acids had an influence on fatty acids in the tissues of male eel and this impacted sperm performance.

Seminal plasma biochemistry and spermatozoa characteristics of Atlantic cod (Gadus morhua L.) of wild and cultivated origin.

Our objectives were to compare spermatozoa activity, morphology, and seminal plasma (SP) biochemistry between wild and cultivated Atlantic cod (Gadus morhua). Swimming velocities of wild cod spermatozoa were significantly faster than those of cultivated males. Wild males had a significantly larger spermatozoa head area, perimeter, and length, while cultivated males had more circular heads. Total monounsaturated fatty acids and the ratio of n-3/n-6 were significantly higher in sperm from wild males, while total n-6 from cultivated males was significantly higher than the wild males. Significantly higher concentrations of the fatty acids C14:0, C16:1n-7, C18:4n-3, C20:1n-11, C20:1n-9, C20:4n-3, C22:1n-11, and C22:6n-3 were observed in wild males, while significantly higher concentrations of C18:2n-6, C20:2n-6, and C22:5n-3 occurred in cultivated males. Osmolality, protein concentration, lactate dehydrogenase and superoxide dismutase activity of SP of wild males were significantly higher than the cultivated males. Antioxidant capacity of SP was significantly higher in cultivated males, while pH and anti-trypsin did not differ between fish origins. Four bands of anti-trypsin activity and nine protein bands were detected in SP. Performing a discriminant function analysis, on morphology and fatty acid data showed significant discrimination between wild and cultivated fish. Results are relevant to breeding programs and aquaculture development.