The successful production of "sterbel" hybrids using beluga (Huso huso) cryopreserved sperm.

This is the first report of the production of viable "sterbel" hybrids using cryopreserved beluga (Huso huso) sperm. In the present study, beluga cryopreserved sperm were used for fertilization and activation of eggs collected from four females of sterlet (Acipenser ruthenus). Sperm were cryopreserved with the use of a glucose methanol extender with the application of an automatic freezer. The mean sperm concentration of beluga was 1.54 × 109 mL-1. Cryopreservation did not significantly change sperm velocity and trajectory parameters (VCL, VSL, VAP, LIN, STR, ALH, BCF). Cryopreservation affected only the values of percentage of motile sperm (MOT) and progressive motility (PRG). The frozen/thawed sperm were subsequently used for the fertilization of the sterlet ova. The fertilized and activated eggs from each female were incubated in separate experimental incubation cages in the RAS system (at 15 °C). This experiment resulted in the production of inter-generic hybrids that were incubated until hatching. Experimental hybridization was characterized by 20-35% hatching rates and normal development of "sterbel" larvae. Experimental hybrids were verified using molecular and cytogenetic analyses. All produced specimens were characterized by sterlet maternal and beluga paternal genomes and were diploids with 120 chromosomes. This study presents the procedure of hybridization of the sterlet with beluga cryopreserved sperm that can be applied in sturgeon aquaculture or research focused on the biology of sturgeon reproduction.

Constant darkness negatively affects the outcome of hormonally induced reproduction in cultured Eurasian perch females.

This study aimed to assess the effect of constant darkness applied to fish during controlled breeding on reproductive traits in domesticated females of Eurasian perch. Based on the assumption that keeping fish in constant darkness during the reproduction operation may reduce stress, suspected to be responsible for variable spawning effectiveness in this species. Two conditions were assessed (16 h light per day [group 16L] and constant darkness [group 0L], two tank replicates per condition). The reproductive protocol involved a 7-day-long adaptation period for group 0L where photoperiod was reduced by 2.3 h a day down to constant darkness. After the adaptation period, two hormone injections (salmon gonadoliberin analogue) were applied to both groups: priming (10 µg/kg) and resolving (25 µg/kg) with a 7-day interval between them. During the study, morphometric indices were recorded and blood, brain, and pituitary samples were collected to assess stress markers and determine hypothalamic-pituitary-gonadal axis functioning via measuring blood plasma hormones, as well as gonadoliberin and gonadotropins (luteinising hormone [LH] and follicle-stimulating hormone [FSH]) transcript abundance (n = 7 for each group at each sampling point). In addition, kinetics of the final oocyte maturation (FOM) process, ovulation rate, and egg quality of each group was monitored (n = 12 for each group). The results indicated that there were no differences in terms of morphometry, FOM kinetics, and most stress indices between groups throughout the experiment, except haematocrit, which increased immediately following the acclimation period in fish kept in darkness. Constant darkness negatively affected plasma levels of 17α,20ß-dihydroxy-4-pregnen-3-one (DHP) and LH transcript expression at the time of the second hormone injection. This indicated that exposure to constant darkness negatively affected priming of the hormonal dose applied, resulted in the disruption of ovulation, and reduced ovulation rates (50%) for group 0L, as compared to 16L (91%). The findings of this study clearly indicate that constant darkness may have significant deleterious effects on reproductive traits throughout out-of-season induced, hormonally supported, controlled reproduction. Therefore, we advise against the use of constant darkness when managing broodstock reproduction in domesticated Eurasian perch.

Inhibition of ß-N-acetylglucosaminidase by acetamide affects sperm motility and fertilization success of rainbow trout (Oncorhynchus mykiss) and Siberian sturgeon (Acipenser baerii).

ß-N-Acetylglucosaminidase (ß-NAGase) is an enzyme found in the sperm acrosome of numerous animal species including fish. Fish spermatozoa differ in their morphology including acrosome or acrosomeless aquasperm in chondrostean (e.g., sturgeon) and teleostean (e.g., rainbow trout). It has been shown that ß-NAGase exists with high activity in both eggs and sperm of these species. The present study shows the potency of ß-NAGase in fertilization. In rainbow trout, increase in sperm motility parameters (VAP and MOT) were observed in the presence of acetamide, an inhibitor for ß-NAGase. In contrast, sperm motility parameters (VCL, VSL, VAP, MOT, and PRG) were reduced on the Siberian sturgeon in the presence of acetamide. The inhibition of the activity of ß-NAGase in rainbow trout spermatozoa was led to a reduction in the number of fertilized eggs from 79% to 40%, whereas in sturgeon no change was observed in fertilization. Moreover, inhibition of ß-NAGase in both spermatozoa and eggs of trout and sturgeon resulted in significant decrease in fertilization rate from 79% to 1% in rainbow trout and from 84% to 12% in Siberian sturgeon. Our research proves that ß-NAGase can play a significant role in the fertilization process in teleosteans.