Supplementation of Extender with Melatonin Improves the Motility, Mitochondrial Membrane Potential, and Fertilization Ability of Cryopreserved Brown-Marbled Grouper Sperm.

Sperm cryopreservation is a valuable tool for breeding, conservation, and genetic improvement in aquatic resources, while oxidative damage will cause a decline in sperm quality during this progress. Melatonin (MT), a natural antioxidant hormone, is used as an additive in sperm cryopreservation to reduce cellular damage from oxidative stress. Here, we aimed to investigate the effect of adding MT to the freezing medium in sperm cryopreservation of brown-marbled grouper (Epinephelus fuscoguttatus). Different concentrations of MT (0, 0.1, 0.25, and 0.5 mg/mL) were tested. We evaluated sperm motility, viability, apoptosis, mitochondrial membrane potential (MMP), and fertilization ability to assess the effects of MT supplementation. Our results demonstrated that the addition of MT to the extender improved the post-thaw motility, MMP, and fertilization ability of brown-marbled grouper sperm. The total motility, curvilinear velocity, straight linear velocity, and average path velocity in MT-treated groups (0.1 and 0.25 mg/mL) exhibited significantly higher values than that of the control group. A higher MMP (p < 0.05) was observed in the group treated with 0.25 mg/mL MT, suggesting that supplementation of MT in the extender might be able to protect mitochondrial membrane integrity effectively. Regarding fertilizing ability, 0.25 mg/mL MT yielded a significantly higher hatching rate than the control. An adverse effect was found with the concentration of MT up to 0.5 mg/mL, suggesting the possible toxicity of a high-dose addition. In this study, we optimized the sperm cryopreservation protocol of brown-marbled grouper, which might be valuable for sperm cryopreservation and sample commercialization of groupers and other fish.

Effects of supplementation of cryopreservation media with cysteine on the post-thaw quality and fertility of brown-marbled grouper (Epinephelus fuscoguttatus) spermatozoa.

The cryopreservation process is associated with the generation of excessive reactive oxygen species, which causes a series of cellular damage to spermatozoa. The objective of the current study was to investigate the effect of different concentrations of cysteine on post-thaw sperm quality of brown-marbled grouper sperm. Semen samples were frozen with cysteine supplemented at 0.5, 1, 2, 5, 10 mM and the control group (no additive). After thawing, sperm quality parameters were analyzed. In comparison to the control, cysteine treatment groups yielded relatively higher sperm total motility, progressive motility, and curvilinear velocity. Different concentrations of cysteine had no effect on average path velocity, straight linear velocity and viability (P > 0.05), while an increase in the concentration of cysteine resulted in a significant improvement in the mitochondrial membrane potential, SOD activity, and ATP content (P < 0.05). As for lipid peroxidation, the extent of which in cysteine treated spermatozoa was less than the control, although the differences were not statistically significant (P > 0.05). In terms of fertilizing capacity, a greater hatching rate (91.7 ± 1.2%) was obtained in thawed sperm treated with 2 mM cysteine, compared to the control (84.3 ± 4.2%; P < 0.05). Overall, it is concluded that the addition of cysteine is helpful in maintaining the function of frozen-thawed brown-marbled grouper sperm, which can be recommended as an effective antioxidant to improve the semen cryopreservation efficiency.

Supplementation of the freezing medium with Coenzyme Q10 attenuates oxidative stress and improves function of frozen-thawed giant grouper (Epinephelus lanceolatus) spermatozoa.

Incorporation of Coenzyme Q10 (CoQ10) to the freezing medium provides advantageous effect for sperm cryopreservation in a variety of animal species, yet which has not been tested in giant grouper (Epinephelus lanceolatus). This research was designed to elucidate if CoQ10 could be used as a potential additive to improve giant grouper sperm quality after cryopreservation. After the process of freezing and thawing, various sperm quality parameters including motility, viability, apoptosis, mitochondrial membrane potential (MMP), intracellular reactive oxygen species (ROS) generation, DNA fragmentation as well as fertilization rate were evaluated with CoQ10 added at concentrations of 0, 25, 50 and 100 µM. Compared to the control group (0 µm), addition of CoQ10 in the medium yielded significantly higher total motility and curvilinear velocity, whereas the progressive motility, straight-line velocity and average path velocity were not differ from each other. An obvious improvement in viability was observed in spermatozoa cryopreserved with 25 and 50 µM CoQ10, while the apoptosis rate in CoQ10 treated groups (25, 50 and 100 µM) exhibited significantly lower values than that of the control. Besides, the production of ROS was significantly decreased with CoQ10 addition groups when compared with the control. In consistent with the improvement in antioxidant defense, CoQ10 supplementation in the medium also enhanced mitochondrial activity and reduced DNA fragmentation. In addition, freezing medium supplemented with CoQ10 also improved the fertilization success, a significantly higher fertilization rate was recorded at the concentration of 50 µM, but this value was not differ from that of 25 µM. Overall, the antioxidant CoQ10 provided an obvious beneficial effect on post-thaw quality of giant grouper spermatozoa. It was concluded that the optimal concentration of CoQ10 is 50 µM in the freezing medium.

NKB/NK3 system negatively regulates the reproductive axis in sexually immature goldfish (Carassius auratus).

To ascertain the significance of the Neurokinin B/Tachykinin 3 receptor (NKB/NK3) system in goldfish reproduction, two cDNAs encoding tachykinin 3 receptors, namely tacr3a and tacr3b, were cloned. Subsequent studies revealed that the downstream signalling of both Tac3rs can be activated by different NKB peptides, suggesting that the cloned receptors are biologically functional in goldfish. RT-PCR analysis showed that tacr3s are widely expressed in brain regions. During the gonadal development, tacr3a and tacr3b exhibited different expression patterns in the hypothalamus and pituitary. The actions of NKB peptides on reproductive axis was further investigated in vivo. Intraperitoneal injections of NKB peptides significantly reduced the expression of kiss2 and gonadotropin releasing hormone 3 (gnrh3) in the hypothalamus, and the expression of luteinizing hormone beta subunit (lhb) and follicle stimulating hormone beta subunit (fshb) in the pituitary in sexually immature goldfish. Taken together, our findings revealed that NKB/NK3 system plays a negative role in the reproductive axis of immature goldfish.

Molecular cloning and functional characterization of the first non-mammalian 26RFa/QRFP orthologue in Goldfish, Carassius auratus.

In this study, we used data mining approach to predict 26RFa/QRFP precursors from fish, amphibian, reptile and avian species and subsequently cloned a 26RFa/QRFP precursor cDNA from goldfish brain based on the predicted sequences information. The goldfish 26RFa/QRFP precursor cDNA encoded a propeptide of 168 amino acids (aa) with predicted signal peptide of 30 aa at N-terminal and putative mature peptides, including 26RFa (26 aa) and 7RFa (7 aa) located at the C-terminal. Multiple sequence alignment showed almost all of the 26RFa/QRFP mature peptides possessed KGGFXFRF-amide motifs (X=G, S, A or N) at their C-terminus, and the last three residues FRF were fully conserved across vertebrates, indicating that the evolutionary pressure has exerted to conserve several C-terminal amino acid residues among the known and predicted 26RFa/QRFP precursors. Real-time PCR revealed that 26RFa/QRFP gene was expressed abundantly in goldfish hypothalamus, optic tectum-thalamus and testis. The regulation of goldfish hypothalamic 26RFa/QRFP gene expression by negative energy balance and putative role of goldfish 26RFa/QRFP in the control of luteinizing hormone (LH) release were studied. Hypothalamic 26RFa/QRFP gene expression was pronouncedly increased at 4 days after food deprivation. Furthermore, intraperitoneal (IP) injection of synthesized goldfish 26RFa/QRFP at a dose of 1 microg/g bodyweight significantly increased serum LH levels at 1h. However, LH levels were not significantly changed by IP injection of goldfish 26RFa/QRFP at lower dosage or at other time points (3 and 6 h), or by incubation of goldfish primary cell cultures. These results suggested that goldfish 26RFa/QRFP shared some similar features with its mammalian counterparts and partly exerted the regulatory function in energy homeostasis and hypothalamic-pituitary-gonadal (HPG) axis as observed in mammalian species.