Fish Sperm Research Articles

Influence of Dietary Fatty Acids on Fish Sperm Tolerance to Cryopreservation

ABSTRACTTo meet growing fish demand, aquaculture must develop sustainable, cost‐effective, and high‐performance fish feed formulations for industry advancement. Plant‐based lipids are a viable alternative to forage fish ingredients in finfish diets. However, most plant‐derived lipids lack long‐chain polyunsaturated fatty acids (LC‐PUFA) like EPA and DHA, which are nutritionally essential for fish. This review summarizes recent findings on the nutritional aspects of fish brood stock and sperm quality, emphasizing the effects of fatty acids on reproductive outcomes. Studies indicate that substituting fish oil with plant oils alters the fatty acid profiles in fish tissues and gametes, potentially compromising sperm quality, cryopreservation success, and progeny viability. Sperm membrane lipids, including EPA, DHA, and ARA, are vital for motility and fertilization rates post‐thaw. Additionally, cryopreservation has been linked to molecular and epigenetic alterations in sperm, which may negatively affect offspring quality. The role of mRNA and noncoding RNA in regulating sperm function and embryonic development is well known, and thus any damage to these molecules as a consequence of cryopreservation might induce long‐term effects on offspring. Proteomic analyses reveal that cryopreservation can lead to significant protein loss in sperm, diminishing their functional and fertilization capabilities. Optimizing cryoprotectant protocols and freezing techniques is crucial to reduce damage, while dietary fatty acids play a key role in preserving sperm quality during cryopreservation stress. Further research with omic technologies is crucial to fully understand sperm tolerance to cryopreservation, which will improve reproductive outcomes and enhance sustainability and quality in aquaculture.

Multivalent Weak Protections Ultimately Enable Customizable DNA Grafting on Pristine Ag Colloids for Nanoplasmonics

Silver nanoparticles (AgNPs) have superior plasmonic properties surpassing other metals. However, a long‐standing difficulty in valence/density‐tunable DNA grafting of AgNPs disfavors their use in DNA‐directed nanoplasmonics. Herein a close‐to‐ideal surface protection of pristine AgNPs against various notorious stability issues of Ag is achieved based on multidentate weak nucleobase bindings of non‐programming FSDNA (fish sperm DNA). This further allows grafting of thiolated DNA with tunable valence/density on AgNPs. The end‐on format of the thiolated DNA grafts and the very thin FSDNA layer benefit DNA hybridization and plasmon coupling, respectively. Significantly promoted optical coupling and Raman enhancing are achieved. The compatibility of FSDNA‐capped AgNPs with Au enables DNA‐bonded symmetry‐broken Au‐Ag heterodimers with strong near‐field coupling and an easily seen Fano‐induced feature. Our work provides a treasured freedom of using AgNPs in DNA‐programmed, better‐behaving plasmonic devices.

Aquaporin-3a Dysfunction Impairs Osmoadaptation in Post-Activated Marine Fish Spermatozoa.

Spermatozoon volume regulation is an essential determinant of male fertility competence in mammals and oviparous fishes. In mammals, aquaporin water channels (AQP3, -7 and -8) have been suggested to play a role in spermatozoon cell volume regulatory responses in the hypotonic female oviduct. In contrast, the ejaculated spermatozoa of marine teleosts, such as the gilthead seabream (Sparus aurata), experience a high hypertonic shock in seawater, initially resulting in an Aqp1aa-mediated water efflux, cell shrinkage and the activation of motility. Further regulatory recovery of cell volume in post-activated spermatozoa is mediated by Aqp4a in cooperation with the Trpv4 Ca2+ channel and other ion channels and transporters. Using a paralog-specific antibody, here, we show that seabream spermatozoa also express the aquaglyceroporin AQP3 ortholog Aqp3a, which is highly accumulated in the mid posterior region of the spermatozoon flagella, in a similar pattern to that described in mouse and human sperm. To investigate the role of Aqp3a in seabream sperm motility, we used a recently developed AQP3 antagonist (DFP00173), as well as the seabream Aqp3a-specific antibody (α-SaAqp3a), both of which specifically inhibit Aqp3a-mediated water conductance when the channel was heterologously expressed in Xenopus laevis oocytes. Inhibition with either DFP00173 or α-SaAqp3a did not affect sperm motility activation but did impair the spermatozoon motion kinetics at 30 s post activation in a dose-dependent manner. Interestingly, in close resemblance to the phenotypes of AQP3-deficient murine sperm, electron microscopy image analysis revealed that both Aqp3a inhibitors induce abnormal sperm tail morphologies, including swelling and angulation of the tail, with complete coiling of the flagella in some cases. These findings suggest a conserved role of Aqp3a as an osmosensor that regulates cell volume in fish spermatozoa under a high hypertonic stress, thereby controlling the efflux of water and/or solutes in the post-activated spermatozoon.

Formation and characterization of artificial gynogenetic northern snakehead (Channa argus) induced by inactivated sperm of mandarin fish (Siniperca chuatsi)

Artificial gynogenesis is a pivotal technique in the realm of aquaculture breeding. Northern snakehead (Channa argus) is confronting a critical issue of germplasm degradation. To solve this problem, we produced viable northern snakehead offspring via gynogenesis using UV-irradiated sperm from mandarin fish (Siniperca chuatsi). As expected, all gynogenetic northern snakeheads were female, as verified by sex-specific markers. To investigate the characterization of the gynogenetic northern snakehead, including whether the mandarin fish DNA fragments integrate into gynogenetic northern snakehead, we compared their morphological features, growth characteristics, DNA contents, chromosomal numbers, karyotypes, mitochondrial DNA sequences, and microsatellite DNA sequences. The gynogenetic northern snakehead exhibited dominant maternal patterns regarding morphological features, DNA contents, chromosomal numbers, karyotypes, and mitochondrial DNA traits. However, microsatellite assessments confirmed the successful integration of mandarin fish DNA fragments into the gynogenetic northern snakehead genome. Notably, the gynogenetic northern snakehead displayed superior growth compared to the maternal. We induced spawning in the 12-month-old gynogenetic northern snakehead and successfully obtained offspring. This study holds significant commercial potential for northern snakeheads and offers valuable insights for breeding.

Synthesis and characterization of polysaccharide-cryogel and its application to the electrochemical detection of DNA

The main goal of our study is to demonstrate the applicability of the PPy-cryogel-modified electrodes for electrochemical detection of DNA. First, a polysaccharide-based cryogel was synthesized. This cryogel was then used as a template for chemical polypyrrole synthesis. This prepared polysaccharide-based conductive cryogel was used for electrochemical biosensing on DNA. Carrageenan (CG) and sodium alginate (SA) polysaccharides, which stand out as biocompatible materials, were used in cryogel synthesis. Electron transfer was accelerated by polypyrrole (PPy) synthesized in cryogel networks. A 2B pencil graphite electrode with a diameter of 2.00 mm was used as a working electrode. The prepared polysaccharide solution was dropped onto a working electrode as a support material to improve the immobilization capacity of biomolecules and frozen to complete the cryogelation step. PPy synthesis was performed on the electrodes whose cryogelation process was completed. In addition, the structures of cryogels synthesized on the electrode surface were characterized by thermogravimetric analysis (TGA), Fourier transform infrared spectroscopy (FTIR), and scanning electron microscopy (SEM). Surface characterization of the modified electrodes was performed by energy-dispersive X-ray spectroscopy (EDX) analysis. Electrochemical determination of fish sperm DNA (fsDNA) was performed using a PPy-cryogel-modified electrode. The use of a porous 3D cryogel intermediate material enhanced the signal by providing a large surface area for the synthesis of PPy and increasing the biomolecule immobilization capacity. The detection limit was 0.98 µg mL−1 in the fsDNA concentration range 2.5–20 µg mL−1. The sensitivity of the DNA biosensor was estimated to 14.8 µA mM−1 cm−2. The stability of the biosensor under certain storage conditions was examined and observed to remain 66.95% up to 45 days.Graphical

Aqp4a and Trpv4 mediate regulatory cell volume increase for swimming maintenance of marine fish spermatozoa

Volume regulation is essential for cell homeostasis and physiological function. Amongst the sensory molecules that have been associated with volume regulation is the transient receptor potential vanilloid 4 (TRPV4), which is a non-selective cation channel that in conjunction with aquaporins, typically controls regulatory volume decrease (RVD). Here we show that the interaction between orthologous AQP4 (Aqp4a) and TRPV4 (Trpv4) is important for regulatory volume increase (RVI) in post-activated marine fish spermatozoa under high osmotic stress. Based upon electrophysiological, volumetric, and in vivo and ex vivo functional experiments using the pharmacological and immunological inhibition of Aqp4a and Trpv4 our model suggests that upon ejaculation and exposure to the hypertonic seawater, spermatozoon shrinkage is initially mediated by water efflux through Aqp1aa in the flagellar tail. The shrinkage results in an increase in intracellular Ca2+ concentration, and the activation of sperm motility and a Na+/K+/2Cl− (NKCC1) cotransporter. The activity of NKCC1 is required for the initiation of cell swelling, which secondarily activates the Aqp4a-Trpv4 complex to facilitate the influx of water via Aqp4a-M43 and Ca2+ via Trpv4 and L-type channels for the mediation of RVI. The inhibitory experiments show that blocking of each of these events prevents either shrinkage or RVI. Our data thus reveal that post-activated marine fish spermatozoa are capable of initiating RVI under a high hypertonic stress, which is essential for the maintenance of sperm motility.

Protamine and Heparin Interactions: A Narrative Review.

Protamine, first isolated from salmon fish sperm and now produced through recombinant biotechnology, is an antidote that neutralizes the anticoagulant properties of heparin. Protamine function is based on the capacity to dissociate the heparin-antithrombin III (AT III) complex (an important link that promotes blood fluidification by inhibiting coagulation), forming the inactive heparin-protamine complex. Protamine has itself dose-dependent anticoagulant properties: It interferes with coagulation factors and platelet function; it stimulates fibrinolysis; it can lead to thrombocytopenia and reduction in thrombin-related platelet aggregation; it decreases platelet response to thrombin receptor agonist in a dose-dependent manner. In this review, we will focus on protamine and its interaction with heparin. Notably, protamine is able to antagonize not only unfractionated heparin (UFH) but also low molecular weight heparins to various degrees. Protamine-allergic and anaphylactoid systemic reactions may affect up to 1 in 10 people and should be prevented and treated early.

Donepezil-Squaric Acid Hybrid: Synthesis, Characterization and Investigation of Anticholinesterase Inhibitory, DNA Binding and Antioxidant Properties

A novel donepezil-squaric acid (DS) hybrid compound was designed, synthesized and biologically evaluated as multi-target-directed ligands against neurodegenerative disease by fusing a fragment of donepezil (D) and squaric acid (S). This study focuses on investigating the binding mechanism of double-stranded fish sperm DNA (Fsds-DNA) with DS and S. The interaction between DS and S with Fsds-DNA was explored using spectrophotometric and viscometric methods. Besides, DNA binding constants (Kb) were determined. The results reveal that DS binds to Fsds-DNA via the minor groove binding mode. The hybrid molecule demonstrates potent inhibitory activity against acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). Furthermore, it exhibits significant antioxidant activity, surpassing the scavenging ability of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals compared to squaric acid alone. In conclusion, these results suggest that the hybrid molecule may serve as a potential multifunctional agent for the treatment of Alzheimer’s disease.

Quantitative study on a simple electrochemical dsDNA-pregabalin biosensor; multi-spectroscopic, molecular docking and modelling studies

Pregabalin (PGB) is a γ-aminobutyric acid (GABA) alkylated analog prescribed to treat neuropathic pain, fibromyalgia, and postherpetic neuralgia. Using analytical, spectroscopic methods and molecular docking and molecular dynamics (MD) simulations, a detailed experimental and theoretical investigation was conducted into the binding process and interactions between PGB and double-stranded fish sperm deoxyribonucleic acid (dsDNA). It was evident from the collected experimental results that PGB binds with ds-DNA. PGB attaches to dsDNA via minor groove binding, as demonstrated by the results of electrochemical studies, UV–Vis absorption spectroscopy, and replacement study with ethidium bromide and Hoechst-32588. PGB's binding constant (Kb) with dsDNA, as determined by the Benesi-Hildebrand plot, is 2.41×104 ± 0.30 at 298 K. The fluorescence investigation indicates that PGB and dsDNA have a binding stoichiometry (n) of 1.21 ± 0.09. Molecular docking simulations were used in the research to computational determination of the interactions between PGB and dsDNA. The findings demonstrated that minor groove binding was the mechanism by which PGB interacted with dsDNA. Based on the electrochemically responsive PGB-dsDNA biosensor, we developed a technique for low-concentration detection of PGB utilizing differential pulse voltammetry (DPV). The voltammetric analysis of the peak current decrease in the deoxyadenosine oxidation signals resulting from the association between PGB and dsDNA enabled a sensitive estimation of PGB in pH 4.80 acetate buffer. The deoxyguanosine oxidation signals exhibited a linear relationship between 2 and 16 μM PGB. The values for the limit of detection (LOD) and limit of quantitation (LOQ) were 0.57 μM and 1.91 μM, respectively.

Rapid antibody conjugation strategy via instant charge inversion of AuNBPs toward ultrasensitive SERS-LFIA detection of AFP

Alpha-fetoprotein (AFP) is widely utilized as the primary serum marker for diagnosing Hepatocellular carcinoma. Integrating surface-enhanced Raman scattering (SERS) with lateral flow immunoassays (LFIA) displays significant promise for the rapid quantification of AFP. Gold nanobipyramids (AuNBPs) is a kind of SERS probe with high activity showing strong application potential in SERS-LFIA. However, the drastic electrostatic interaction between negative charged antibodies and positive charged AuNBPs will result in uncontrollable aggregation, which poses a challenge for antibody binding and limits its application in SERS-LFIA. Hence, we propose a rapid antibody conjugation strategy with the aid of instant charge inversion of AuNBPs, which enables efficient and expedited antibody coupling onto AuNBPs. The thiol-free fish sperm (FSDNA) was employed to adsorb on the cetyltrimethylammonium bromide (CTAB) coated AuNBPs for immediate charge inversion. The adsorption of FSDNA will cause partial stripping of CTAB from the AuNBPs surface to provide additional active sites for antibodies binding. In this study, approximately average 37 AFP-antibodies were verified bonding on one charge inverted AuNBPs by direct incubation for 30 min. The antibodies modified AuNBPs were further incubated with 4-nitrothiophenol for preparing SERS immune probes, which display excellent colloid stability and specificity. The probes were used to construct SERS-LFIA strips for AFP detection, enabling highly sensitive detection of AFP with the limit of detection (LOD) as low as 0.72 pg·mL−1. The developed SERS-LFIA strip demonstrates excellent stability and accuracy in detecting serum samples, with a recovery rate ranging from 84 % to 114 %. This rapid antibody coupling strategy is applicable to a wide range of positively charged nanoparticles, effectively addressing the challenging issue of utilizing such nanomaterials in LFIA.

DNA and bovine serum albumin protein (BSA) interaction of antitumor supramolecular nickel(II) complex: Inference for drug design

To explore the therapeutic potential of supramolecular nickel(II) Schiff base self-assemble complexes, the Schiff base ligand (N, N′-bis(salicylidene)-1,3-diamino-2-propanol) and its Ni(II) complex [Ni2(H2L)2(NCS)2(CH3OH)2](CH3OH)2 were synthesized. Characterization by X-ray crystallography (for the complex), FT-IR, UV–Vis spectroscopy, 1HNMR and elemental analysis (CHN) were performed. Single-crystal X-ray structure of the complex exhibited a distorted octahedral geometry in which each nickel(II) center, in the asymmetric unit, was six coordinated with three N atoms and three O atoms (from Schiff base ligand, methanol and NCS). In vitro Fish Sperm DNA (FS-DNA) and Bovine Serum Albumin (BSA) binding studies of the complex employed UV–Vis and fluorescence spectroscopy, cyclic voltammetry, viscosity measurement and molecular docking. The complex indicated a good binding propensity to BSA and was also bound to FS-DNA, preferably via intercalation. The outputs of the molecular docking study confirmed the experimental results. The complex was found to exert high anticancer activity against the gastric and prostate cancerous cells with putative selective anticancer properties. The death-induced mechanism underlying the complex is mainly related to the intercalator interactions, which may result in apoptosis-programmed cell death.

Induction of diploid gynogenesis in Micropterus salmoides using irradiated heterogeneous sperm from Siniperca chuatsi

The artificial induction of gynogenesis in fish breeding can be used to produce monosex populations. Micropterus salmoides (largemouth bass) is an important freshwater aquaculture fish, and the females grow faster than the males. In this study, we aimed to determine the best way to artificially induce largemouth bass spawning and identify suitable heterologous sperm donors to induce gynogenetic largemouth bass by suppressing extrusion of the second polar body in fertilized eggs. With respect to the artificial induction of spawning in LB, four groups were tested for hormone types. The results showed that a single intramuscular injection of the hormone combination HCG (2000 IU/kg) + LHRH-A2 (20 μg/kg) + DOM (5 mg/kg) into female largemouth bass could yield desirable spawning results, and the mean number of eggs per female in kg was 28,780 ± 4574. With respect to suitable sperm donors, largemouth bass eggs were fertilized with sperm from five fish species, and it was found that mandarin fish (Siniperca Chuatsi) sperm can activate the development of largemouth bass eggs, but most of the embryos died during development, which indicated that mandarin fish are the potential of suitable heterologous sperm donors for gynogenetic experiments with largemouth bass. The suitable optimal induction conditions were detected for chromosome doubling. The results indicated that the most effective method for promoting gynogenesis in largemouth bass was to expose inseminated eggs to a temperature of 3 °C for 15 min, beginning 2 min after fertilization. In addition, the DNA content, karyotype and morphological characteristics of the gynogenetic offspring were consistent with those of the largemouth bass. Examination of sex-specific markers and artificial propagation of the gynogenetic largemouth bass revealed that all the tested fishes were female, indicating female homogamety (XX) in the largemouth bass. In summary, in this study, we established an improved artificial spawning technique for largemouth bass and successfully induced diploid gynogenetic largemouth bass using irradiated heterogeneous sperm from mandarin fish. Our study lays a foundation for the mono-sex breeding of largemouth bass.

Effects of Long-Term Cryopreservation on the Transcriptomes of Giant Grouper Sperm.

The giant grouper fish (Epinephelus lanceolatus), one of the largest and rarest groupers, is a fast-growing economic fish. Grouper sperm is often used for cross-breeding with other fish and therefore sperm cryopreservation is important. However, freezing damage cannot be avoided. Herein, we performed a transcriptome analysis to compare fresh and frozen sperm of the giant grouper with frozen storage times of 0, 23, 49, and 61 months. In total, 1911 differentially expressed genes (DEGs), including 91 in El-0-vs-El-23 (40 upregulated and 51 downregulated), 251 in El-0-vs-El-49 (152 upregulated and 69 downregulated), and 1569 in El-0-vs-El-61 (984 upregulated and 585 downregulated), were obtained in the giant grouper sperm. DEGs were significantly increased at 61 months of cryopreservation (p < 0.05). GO and KEGG enrichment analyses of the DEGs revealed significant enrichment in the pilus assembly, metabolic process, MAPK signaling pathway, apoptosis, and P53 signaling pathway. Time-series expression profiling of the DEGs showed that consistently upregulated modules were also significantly enriched in signaling pathways associated with apoptosis. Four genes, scarb1, odf3, exoc8, and atp5f1d, were associated with mitochondria and flagella in a weighted correlation network analysis. These genes may play an important role in the response to sperm freezing. The experimental results show that long-term cryopreservation results in freezing damage to the giant grouper sperm. This study provides rich data for studies of the mechanism underlying frozen fish sperm damage as well as a technical reference and evaluation index for the long-term cryopreservation of fish sperm.

Influence of diimine bidentate ligand in the nitrosyl and nitro terpyridine ruthenium complex on the HSA/DNA interaction and antiviral activity

Nitric oxide (NO) acts in different physiological processes, such as blood pressure control, antiparasitic activities, neurotransmission, and antitumor action. Among the exogenous NO donors, ruthenium nitrosyl/nitro complexes are potential candidates for prodrugs, due to their physicochemical properties, such as thermal and physiological pH stability. In this work, we proposed the synthesis and physical characterization of the new nitro terpyridine ruthenium (II) complexes of the type [RuII(L)(NO2)(tpy)]PF6 where tpy = 2,2':6′,2″-terpyridine; L = 3,4-diaminobenzoic acid (bdq) or o-phenylenediamine (bd) and evaluation of influence of diimine bidentate ligand NH.NHq-R (R = H or COOH) in the HSA/DNA interaction as well as antiviral activity. The interactions between HSA and new nitro complexes [RuII(L)(NO2)(tpy)]+ were evaluated. The Ka values for the HSA–[RuII(bdq)(NO2)(tpy)]+ is 10 times bigger than HSA–[RuII(bd)(NO2)(tpy)]+. The sites of interaction between HSA and the complexes via synchronous fluorescence suppression indicate that the [RuII(bdq)(NO2)(tpy)]+ is found close to the Trp-241 residue, while the [RuII(bd)(NO2)(tpy)]+ complex is close to Tyr residues. The interaction with fish sperm fs-DNA using direct spectrophotometric titration (Kb) and ethidium bromide replacement (KSV and Kapp) showed weak interaction in the system fs-DNA-[RuII(bdq)(NO)(tpy)]+. Furthermore, fs-DNA–[RuII(bd)(NO2)(tpy)]+ and fs-DNA–[RuII(bd)(NO)(tpy)]3+ system showed higher intercalation constant. Circular dichroism spectra for fs-DNA–[RuII(bd)(NO2)(tpy)]+ and fs-DNA–[RuII(bd)(NO)(tpy)]3+, suggest semi-intercalative accompanied by major groove binding interaction modes. The [RuII(bd)(NO2)(tpy)]+ and [RuII(bd)(NO)(tpy)]3+ inhibit replication of Zika and Chikungunya viruses based in the nitric oxide release under S-nitrosylation reaction with cysteine viral.

Effect of prolonged photoperiod on growth, gonadal development, sex ratio, and skin coloration of red tilapia (O. mossambicus × O. urolepis hornorum)

Farming of red tilapia is one promising direction in aquaculture. The objective of this study was to investigate the impact of long-day photoperiod on the growth performance, reproductive parameters, maturity, sex ratio, β-carotene content, and skin coloration of red tilapia. Early life larvae were allocated into three treatment groups in triplicates: T1 = control group (12L:12D), T2 = long-day group (18L:6D), and T3 = continuous illumination group (24L:0D) for 90 days. By the end of the experiment, gonadosomatic index (GSI), relative fecundity, steroid hormones, maturity stages, sex ratio, and ovarian and testicular histology were examined; also, growth performance, survival rate, skin color, and total β-carotene content were measured. The results indicated that GSI% and serum testosterone (T) in T3 group significantly increased with greater amount of spermatozoa of male fish. Also, growth parameters and HSI% in female fish displayed significantly increased for T3 group. Females in group T2 showed significant elevation in GSI%, relative fecundity, and estradiol (E2) with higher number of mature stage. Continuous light group (T3) showed shifted male sex ratio, while female percentage was higher in T2 group. The total β-carotene content of T3 group showed significant elevation with less number of black spots. This study demonstrated that continuous light manipulation during red tilapia early life could stimulate the growth and enhance coloration by reducing black spots that significantly affects economic profitability. Long-day application was effective in female reproductive performance and maturity. This study also served as an important reference for red tilapia aquaculture.

Catalase and Uric Acid Prevent Morphological Damage to the Sperm Flagella of Colossoma macropomum During 96 Hours at Low Storage Temperatures.

Oxidative stress is one of the main causes of loss of sperm function during chilled storage. The aim of the current study was to evaluate the effects of a fructose-based extender, which was supplemented with catalase or uric acid, on the motility, viability, morphological integrity, and lipid peroxidation (LPO) of Colossoma macropomum spermatozoa. Sperm was diluted in extenders containing catalase (0; 0.1; 0.8; and 1.5 kU/L) or uric acid (0; 0.25; 0.5; and 1.0 mmol/L) and then stored at 4.3 ± 0.6°C for 96 hours. The chilling storage time had more significant and pronounced effects on practically all the measured sperm quality parameters than the different concentrations of both antioxidants added to the extenders. This was true for sperm motility, motility duration, sperm viability, and the percentage of normal spermatozoa. In fact, for all these parameters, values were higher in the extenders supplemented with catalase or uric acid, than those not supplemented with these antioxidants, especially after 96 hours. The LPO process showed an antioxidant-dependent response. In catalase-supplemented extenders thiobarbituric acid reactive substance (TBARS) levels increased gradually and significantly with time, but remained stable during the 96 hours of chilled storage in all samples in which uric acid was added. Despite this, TBARS levels were lower in the extenders supplemented with both catalase and uric acid than in those not having these antioxidants. Inverse correlations were found between sperm motility and the damage in sperm flagella. Our findings suggest that the supplementation of an extender with catalase or uric acid is beneficial and protects fish sperm membranes from damage caused by oxidative stress during low-temperature storage. The extenders containing 0.1 kU/L of catalase and 0.25 mmol/L of uric acid provided effective antioxidant protection for the spermatozoa of this important Amazonian fish.

Paternal-effect-genes revealed through sperm cryopreservation in Perca fluviatilis

Knowledge about paternal-effect-genes (PEGs) (genes whose expression in the progeny is influenced by paternal factors present in the sperm) in fish is very limited. To explore this issue, we used milt cryopreservation as a specific challenge test for sperm cells, thus enabling selection amidst cryo-sensitivity. We created two groups of Eurasian perch (Perca fluviatilis) as a model – eggs fertilized either with fresh (Fresh group) or cryopreserved (Cryo group) milt from the same male followed by phenotypic-transcriptomic examination of consequences of cryopreservation in obtained progeny (at larval stages). Most of the phenotypical observations were similar in both groups, except the final weight which was higher in the Cryo group. Milt cryopreservation appeared to act as a "positive selection" factor, upregulating most PEGs in the Cryo group. Transcriptomic profile of freshly hatched larvae sourced genes involved in the development of visual perception and we identified them as PEGs. Consequently, larvae from the Cryo group exhibited enhanced eyesight, potentially contributing to more efficient foraging and weight gain compared to the Fresh group. This study unveils, for the first time, the significant influence of the paternal genome on the development of the visual system in fish, highlighting pde6g, opn1lw1, and rbp4l as novel PEGs.

Strengths and limitations of reduced representation bisulfite sequencing (RRBS) in the perspective of DNA methylation analysis in fish: a case-study on rainbow trout spermatozoa.

DNA methylation in CpG dinucleotides is an important epigenetic mark in fish spermatozoa since it has been shown that some sperm methylome features are transmitted to the offspring. Reduced representation bisulfite sequencing (RRBS) is one genome-scale methods developed to assess DNA methylation at CpG sites. It allows the sequencing of a reduced fraction of the genome expected to be enriched in CpGs. The aim of this study is to characterize the extent of the CpG sites that can be identified in the RRBS-reduced sequenced fraction of rainbow trout spermatozoa, in order to evaluate the potential of RRBS for sperm DNA methylation studies. We observed that RRBS did provide a reduced amount of genomic data, the sum of the CpGs analyzed on 12 males spanning 9% of the total genomic CpGs. CpGs were only slightly enriched in the RRBS data (×1.7 times the sequenced nucleotides), the possible causes being linked to trout genome structure and sequenced fragments size. All genomic functional features were represented in our CpG dataset, with a noticeable enrichment in exons but, strikingly, not in promoters. The number of CpGs shared between biological replicates was low, but this proportion reached workable values from six biological replicates (46% of the analyzed cytosines) on. The choices that are to be made regarding fragment size selection and the options during bioinformatic data processing are discussed. In all, RRBS is a relevant first-approach method to scan the CpG DNA methylation status of spermatozoa along rainbow trout genome, although in a very reduced pattern among biological replicates.

Effect of Different Natural Extenders in Maintaining the Quality of Sperm Fish (Cyprinus carpio)

High demand on as consume fish nor as decorative fish, causes an increasing production. But, an obstacle occurs on production, due to reproductive characteristic of Common Carp fish. Different time on sperm and eggs production from male and female fish cause difficulties in breeding. Hence, it needed sperm preservation so the breeding could be done in whole year. Natural extenders are the solution to preserve fish sperm without side effect. The purpose of this study was to determine the effect of different types of extenders and different doses on the percentage of sperm motility and viability of carp (Cyprinus carpio) after storage This research was conducted at the Faculty of Fisheries and Marine Sciences, Universitas Brawijaya and the Freshwater Cultivation Installation (IBAT Punten), Batu, East Java. The method in this study was Complete Random Design (CRD) with 6 treatments, K (100ml lactate ringer); A (1ml cider + 99ml lactate ringer); B (1ml coconut + 99ml lactate ringer); C (1ml date juice + 99ml lactate ringer); D (1ml honey + 99ml lactate ringer); E (1ml sugar cane + 99ml lactate ringer). The results showed that the highest motility was obtained in the treatment of using date extract extender with a motility percentage rate of 77.66%; highest viability was obtained in the honey extract with 80.71%; and the highest fertility rate was obtained in the honey extract treatment with 72.67 %.

Novel 3D-Printed Microfluidic Magnetic Platform for Rapid DNA Isolation.

This study presents a novel miniaturized device as a 3D-printed microfluidic magnetic platform specifically designed to manipulate magnetic microparticles in a microfluidic chip for rapid deoxyribonucleic acid (DNA) isolation. The novel design enables the movement of the magnetic particles in the same or opposite directions with the flow or suspends them in continuous flow. A computational model was developed to assess the effectiveness of the magnetic manipulation of the particles. Superparamagnetic monodisperse silica particles synthesized in-house are utilized for the isolation of fish sperm DNA and human placenta DNA. It was demonstrated that the proposed platform can perform DNA isolation within 10 min with an isolation efficiency of 50% at optimum operating conditions.