7th International Workshop on the Biology of Fish Gametes 3-6 Sept. 2019, Rennes, France Book of abstracts Printed in Rennes, France, August 2019
Editors: Alexandra Depincé, Audrey Laurent
Cover design: Eric Beaumont, Alexandra Depincé, Audrey Laurent Crédit photo : INRA-Didier Marie (œufs)
Edition: 1st
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7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 4 WELCOME ADDRESS
Dear participants to the 7th International Workshop on the Biology of Fish Gametes
We are happy to welcome you this year, for the 7th edition of this biennial workshop initiated in 2007 by Otomar Linhart and his colleagues in Vodňany. Before these workshops, which have now become a tradition, the oldest among us may remember the single Workshop on Gamete and Embryo Storage and Cryopreservation in Aquatic Organisms held in 1992 in Marly le Roy, near Paris. The organizer was Professor Roland Billard, who had imagined with Jacky Cosson, Christian Fauvel, Gérard Maisse and Maurice Loir that the aquatic research community should meet and share knowledge and future directions. This is where we could for the first time put faces on the names of E. Kopeika, O. Linhart, A. Ciereszko, E. Lubzens, F. Lahnsteiner, M. Suquet, T. Zhang, O. Chereguini or N.H. Chao. We could not have imagined that so many years later, this community would have expanded to where we are today, and would have become this closely linked community, held by shared research interests and true friendship. Roland passed away last March. This meeting will be an opportunity for the oldest to remember, and for the youngest to get to know the exceptional researcher Roland Billard was. He paved the way for most of our research on the biology of fish gametes. Jacky Cosson agreed to do an obituary for Roland, and we are grateful to him for that.
You came from 16 countries to give and share together the presentation of 34 oral and 66 poster communications. You made the program thanks to your proposals, which the local scientific committee organized in sessions dedicated to I. Spermatogenesis and Sperm Quality, II. Germinal Stem Cells and Biotechnologies, III. Refrigeration and Cryopreservation, IV. Oogenesis and Egg Quality, and V. Sperm Motility and Fertilization. It is noteworthy that our research interests are gradually shifting from gametes to germline stem cells. As a whole, our community is developing research that spans from sperm cryopreservation and germline stem cell transplantation to crispr/cas9 genome editing and gamete proteomics, together with studies on gamete interactions and motility mechanisms. We are pleased that more than 30 students are participating to the student prize competition that IMV Technologies is sponsoring this year. We are also pleased that IMV initiated a partnership with the Workshop on Biology of Fish Gametes, with the intention of sponsoring the workshop for at least two more editions. This will be a great help to our research community.
We hope you will enjoy your stay in Rennes, and that you will enjoy the whole meeting on the beautiful AgroCampus site. A very enthusiastic local group from INRA LPGP took care of thousands of small details to make everything work as well as possible, we hope. And if you are here, it is because you have passed a very demanding test: registration on our website. So you all succeeded, congratulations, everything that can happen next will seem easy and smooth to you.
Good workshop, good science.
Catherine Labbé
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 5 INTERNATIONAL SCIENTIFIC COMMITTEE
Juan Asturiano Universitat Politècnica de València, Spain Igor Babiak Faculty of Biosciences and Aquaculture, Norway Julien Bobe INRA, France Elsa Cabrita CCMAR, Portugal Oliana Carnevali Università Politecnica delle Marche, Italy Andrzej Ciereszko Polish Academy of science, Poland Ákos Horváth Szent István University, Hungary Catherine Labbé INRA, France Luz Pérez Universitat Politècnica de València, Spain Martin Pšenička University of South Bohemia, Czech Republic Vanesa Robles IEO, Spain Ina Wagenaar University of Johannesburg, South Africa Anna Wargelius IMR, Norway Daniel Żarski Polish Academy of science, Poland
LOCAL SCIENTIFIC COMMITTEE
Julien Bobe Catherine Labbé Jean-Jacques Lareyre Audrey Laurent Pierre-Yves Le Bail Violette Thermes
LOCAL ORGANIZING COMMITTEE
Eric Beaumont Stéphane Crespel (Agrocampus Ouest) Alexandra Depincé Stéphanie Gay Agnès Girard Anne-Sophie Goupil Nathalie Huet Thao-Vi Nguyen Laurine Piquemal Guylène Roudaut Charlène Rouillon Elisabeth Sambroni
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 6 TABLE OF CONTENTS
WELCOME ADDRESS ...... 5 SCIENTIFIC PROGRAM ...... 9 POSTERS LIST ...... 17 PLENARY LECTURES ...... 25 ORAL SESSIONS ...... 31 SEESSION I. Spermatogenesis and sperm quality ...... 32 SESSION II. Germinal stem cells and biotechnologies ...... 44 SESSION III. Refrigeration and cryopreservation ...... 47 SESSION IV. Oogenesis and egg quality ...... 59 SESSION V. Sperm mobility and fertilization ...... 73 POSTER SESSIONS ...... 81 SESSION I. Spermatogenesis and sperm quality ...... 82 SESSION II. Germinal stem cells and biotechnologies ...... 109 SESSION III. Refrigeration and cryopreservation ...... 122 SESSION IV. Oogenesis and egg quality ...... 148 SESSION V. Sperm motility and fertilization ...... 165 PARTICIPANTS LIST ...... 171
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SCIENTIFIC PROGRAM
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 9 MONDAY, SEPTEMBER 2ND, 2019
4.30pm - 6.30pm Registration at Agrocampus Ouest
TUESDAY, SEPTEMBER 3RD, 2019
8.00am - 9.00am Registration at Agrocampus Ouest
9.00am - 9.15am Welcome speech by Catherine Labbé and Julien Bobe
9.15am Plenary lecture - Fish spermatology: contribution to the legacy of the novelties PL1 during the 40 past years Jacky Cosson (chairman: Hilal Güralp) University of South Bohemia in České Budějovice, Faculty of Fisheries and Protection of Waters, Vodňany, Czech Republic
SESSION I. SPERMATOGENESIS AND SPERM QUALITY Chairmen: Elsa Cabrita and Olga Bondarenko
10.00am Osmoregulation in fish spermatozoa: involvement in motility activation and O1 impact on short-term storage outcomes Fabio Herrera University of South Bohemia in České Budějovice, Faculty of Fisheries and Protection of Waters, Vodňany, Czech Republic
10.20am Transcriptomic profile during teleost haploid germ cell maturation and O2 differentiation to spermatozoa François Chauvigné IRTA-Institute of Biotechnology and Biomedicine (IBB), Universitat Autònoma de Barcelona, Bellaterra (Barcelona), Spain
10.40am Aneuploid sperm of Danio rerio female x D. nigrofasciatus male hybrid O3 Mitsuru Endoh Hokkaido University, Hakodate, Japan
11.00am - 11.40am Coffee break
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 10 11.40am Effect of age on the sensitivity of zebrafish (Danio rerio) sperm O4 Tímea Kollár Szent István University, Department of Aquaculture, Gödöllö, Hungary
12.00am Metabolic stress decreases sperm quality in goldfish associated with O5 changes in hormonal functions of hypothalamus-pituitary-testis axis Hadi Alavi University of Tehran, Iran 12.20am Proteomic analysis of semen maturation in sex-reversed females of rainbow O6 trout Andrzej Ciereszko Department of Gametes and Embryo Biology, Institute of Animal Reproduction and Food Research of Polish Academy of Sciences, Olsztyn, Poland
12.40am - 2.30pm Lunch
2.30pm First steps on the application of techniques for the control of reproduction O7 of elasmobranch species in captivity Pablo García Salinas Instituto de Ciencia y Tecnología Animal, Universitat Politècnica de València, València, Spain
2.50pm Short-term storage-induced changes in proteome of carp spermatozoa O8 Mariola Dietrich Department of Gametes and Embryo Biology, Institute of Animal Reproduction and Food Research of Polish Academy of Sciences, Olsztyn, Poland
3.10pm Developmental transcriptomics reveals novel mechanisms of teleost O9 spermiogenesis Júlia Castro-Arnau IRTA-Institute of Biotechnology and Biomedicine (IBB), Universitat Autònoma de Barcelona, Bellaterra, Spain
3.30pm – 4.30pm Coffee break
4.30pm – 6.00pm POSTER SESSION A
Session I. Spermatogenesis and sperm quality Session IV. Oogenesis and egg quality
6.00pm Happy Hour
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 11 WEDNESDAY, SEPTEMBER 4TH, 2019
9.15am Plenary lecture - Non model fish transcriptome and genome de novo PL2 assemblies Christophe Klopp (chairman: Anna Wargelius) MIAT INRA Toulouse, Castanet-Tolosan, France PL2 SESSION II. GERMINAL STEM CELLS AND BIOTECHNOLOGIES Chairmen: Catarina Oliveira and Martin Pšenička
10.00am The unexpected diversity of two genetically similar hybrid females in their O10 reproductive pathways Joëlle Lafond Université de Montréal, Montréal, Canada
10.20am Intraperitoneally grafted blastomeres can differentiate into functional O11 gametes in zebrafish Roman Franěk University of South Bohemia in České Budějovice, Faculty of Fisheries and Protection of Waters, Vodnany, Czech Republic
10.40am Transcriptomic analysis of Dead end knockout salmon testis O12 Anna Wargelius Institute of Marine Research, Bergen, Norway
11.00am - 11.40am Coffee break
11.40am Sterilization in sturgeons for surrogate production O13 Abdul Rasheed Baloch University of South Bohemia in České Budějovice, Faculty of Fisheries and Protection of Waters, Vodnany, Czech Republic
12.00am Is the hybrid tiger trout a good recipient for the transplantation of early- O14 stage germ cells? Ákos Horváth Szent István University, Godollo, Hungary
12.20am Virgin Salmon - a sustainable solution for coexistence of farmed and wild O15 salmon strains without mixing Hilal Güralp Institute of Marine Research, Bergen, Norway
12.40am Evolution of germline gene expression profiles during rainbow trout O16 ontogenesis and puberty onset Jean-Jacques Lareyre Department of Fish Physiology and Genomics, INRA, Rennes, France
1.00pm - 2.30pm Lunch
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 12 SESSION III. REFRIGERATION AND CRYOPRESERVATION Chairmen: Marina Morini and Daniel Żarski
2.30pm The effect of glucose, methanol concentration and time of equilibration on O17 post-thaw sperm motility of rainbow trout semen Sylwia Judycka Institute of Animal Reproduction and Food Research PAS, Olsztyn, Poland
2.50pm Cryopreservation of pufferfish sperm: effect on motility, fertilization rates O18 and hatching success Victor Gallego Universitat Politècnica de València, Valencia, Spain
3.10pm Preservation of ovarian tissue in European eel (Anguilla anguilla) O19 Nevena Kitanović Szent Istvan University, Godollo, Hungary
3.30pm Technology transfer of cryopreservation to the industry: example of Atlantic O20 salmon Lucie Gavin-Plagne R&D department, IMV Technologies, L’Aigle, France
3.50pm - 4.40pm Coffee break
4.40pm Visit of the breeding facility at the LPGP : registration during the meeting
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 13 THURSDAY, SEPTEMBER 5TH, 2019
9.15am Plenary lecture - Cellular models available for cryopreservation as a tool for PL3 managing biodiversityON PL2 Bertrand Pain (chairman: Audrey Laurent) INSERM U846 - USC 1361 INRA, Institut Cellule Souche et Cerveau, Bron, France
SESSION IV. OOGENESIS AND EGG QUALITY Chairmen: Oliana Carnevali and Juan Asturiano
10.00am Functional and proteomic characterisation of salmonid coelomic fluid O21 Aurélie Guého Department of Fish Physiology and Genomics, INRA, Rennes, France
10.20am The effects of pharmaceuticals on gamete development in Oreochromis O22 mossambicus (Peters, 1852) after a chronic exposure Ina Wagenaar University of Johannesburg, Johannesburg, South Africa
10.40am Difficulties to compare egg quality: insights from a transcriptomic analysis O23 Tainá Rocha de Almeida Université de Lorraine, Vandœuvre-Lès-Nancy, France
11.00am - 11.40am Coffee break
11.40am Daily rhythms of in vitro fertilization and transcriptome of fish oocytes O24 Juan Fernando Paredes Salas University of Murcia, Spain
12.00am Transgenerational effects of BPA on zebrafish female reproduction and O25 embryo development Oliana Carnevali Department of life and environmental sciences, Università Politecnica delle Marche, Ancona, Italy
12.20a m Very high genetic correlation for egg production traits between two O26 successive spawning in rainbow trout Anastasia Bestin SYSAAF, Rennes, France
12.40am VisEgg: an automatic phenotyping tool to assess rainbow trout unfertilized O27 egg viability Emilie Cardona Department of Fish Physiology and Genomics, INRA, Rennes, France
1.00pm - 2.20pm Lunch
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 14 2.20pm Transcriptomic profiling reveals novel biomarkers of egg quality in O28 domesticated pikeperch, Sander lucioperca Daniel Żarski Department of Gametes and Embryo Biology, Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, Poland
2.40pm What makes a good egg: new insights from crispr/cas9 genome editing in O29 fish model species Julien Bobe Department of Fish Physiology and Genomics, INRA, Rennes, France
3.00pm - 4.30pm POSTER SESSION B
Session II. Germinal stem cells and biotechnologies Session III. Refrigeration and cryopreservation Session V. Sperm motility and fertilization
4.30pm - 5.00pm Coffee break
5.00pm - 5.30pm Obituary to Roland Billard Jacky Cosson University of South Bohemia in České Budějovice, Faculty of Fisheries and Protection of Waters, Vodňany, Czech Republic
6.30pm - 0.00am Gala Dinner Departure from Agrocampus Ouest (1 bus, 2 roundtrips)
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 15 FRIDAY, SEPTEMBER 6TH, 2019
9.15am Plenary lecture - Don't judge a book by its cover, a Trojan horse could be PL4 hidden in the sperm Paz Herraez (chairman: Andrzej Ciereszko) Universidad de León, Spain p.29 SESSION V. SPERM MOTILITY AND FERTILIZATION Chairmen: Mariola Dietrich and Ina Wagenaar
10.00am Guidance and selection during fertilization in fresh water fish: theory and O30 practice Sergii Boryshpolets University of South Bohemia in České Budějovice, Faculty of Fisheries and Protection of Waters, Vodňany, Czech Republic
10.20am Fish sperm competition in hatcheries and between wild and hatchery origin O31 fish in nature José Beirão Nord University, Bodø, Norway
10.40am Egg-sperm interaction in sturgeon: role of ovarian fluid O32 Vitaliy Kholodny University of South Bohemia in České Budějovice, Faculty of Fisheries and Protection of Waters, Vodňany, Czech Republic
11.00am - 11.40am Coffee break
11.40am Social status impacts on sperm competition in fish O33 Elvira Fatsini Fernández Centre of Marine Sciences-CCMAR, University of Algarve, Faro, Portugal
12.00am Steps towards understanding motility of morphologically complex sperm in O34 cartilaginous fishes Borys Dzyuba University of South Bohemia in České Budějovice, Faculty of Fisheries and Protection of Waters, Vodňany, Czech Republic
12.20am Award distribution
SOCIAL EVENT
1.20pm Departure to Dinan, pique-nique on the way on
7.30pm Farewell Crêpes party
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POSTERS LIST
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SESSION I. SPERMATOGENESIS AND SPERM QUALITY
P1 ANALYSİS OF THE SPERM QUALITY PARAMETERS OF KOI FISH IN THE AQUAPONICS SYSTEM Merve TINKIR
P2 THE INFLUENCE OF WATER TEMPERATURE INCREASE ON THE DURATION OF SPERMATOGENESIS IN A NEOTROPICAL FISH, Astyanax altiparanae (CHARACIDAE, CHARACIFORMES) Rosicleire VERÍSSIMO-SILVEIRA
P3 SPERMATOGENESIS IN MATURE MALE SCALLOPED HAMMERHEADS (Sphyrna lewini) FROM KWA-ZULU NATAL, SOUTH AFRICA: A FIRST PRELIMINARY DESCRIPTION Ina WAGENAAR
P4 CHARACTERIZATION OF MALE STERILITY IN HYBRIDS OF cobitis GENUS Tomáš TICHOPÁD
P5 BIOLOGICAL CHARACTERISTICS AND HISTOLOGICAL ANALYSIS OF THE TESTIS IN THE SICHEL POPULATION (Pelecus cultratus, LINNAEUS, 1758) AT LAKE BALATON Levente VÁRKONYI
P6 STRESS REACTION AND SPERM QUALITY IN HORMONALLY TREATED STERLET (Acipenser ruthenus) FOR SPERMIATION Sayyed Mohammad Hadi ALAVI
P7 A SYSTEMATIC REVIEW OF CROSSTALK BETWEEN THYROID HORMONES AND FERTILITY IN MALE FISH Sayyed Mohammad Hadi ALAVI
P8 SPERM HANDLING IN AQUATIC ANIMALS FOR ARTIFICIAL REPRODUCTION José BEIRÃO
P9 DIETARY AMINO ACIDS IMPACT SPERM PERFORMANCE TRAITS FOR A CATADROMOUS FISH, Anguilla anguilla REARED IN CAPTIVITY Juan F. ASTURIANO
P10 COLD SEAWATER INDUCES SPERMATOGONIA PROLIFERATION IN THE EUROPEAN EEL Juan F. ASTURIANO
P11 EFFECT OF PROBIOTICS ON MALE TELEOST REPRODUCTION AND BEHAVIOUR Vanesa ROBLES
P12 DETERMINATION OF ANNUAL REPRODUCTIVE CYCLE IN STERLET, ACIPENSER RUTHENUS USING HISTOLOGY AND ULTRASOUND IMAGING Martin PŠENIČKA
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P13 EFFECT OF BREEDING WATER TEMPERATURE ON SPERM DNA METHYLATION Audrey LAURENT
P14 DAILY RHYTHMS OF MELATONIN RECEPTORS IN HIGH AND LOW QUALITY SPERM SAMPLES IN SENEGALESE SOLE, Solea senegalensis Elsa CABRITA
P15 ADVANCES ON REPRODUCTIVE BIOLOGY OF KILLIFISH (Aphanius iberus AND Valencia hispanica): SPERM MOTILITY, SPERMATOZOA MORPHOLOGY AND GAMETE PRESERVATION Víctor GALLEGO
P16 EFFECTS OF ANESTHETIC TRICAINE ON PLASMA CORTISOL LEVELS AND SPERM MOTILITY OF SOUTH AMERICAN SILVER CATFISH Danilo P. STREIT
P17 TESTICULAR ASPECTS AND GONADO-SOMATIC INDEX (IGS) IN MALES OF PIRACANJUBA Brycon orbignyanus (CHARACIFORMES, BRICONIDAE) OF DIFFERENT AGES Cristiane Fernanda BENEVENTE
P18 TRANSFERRIN IDENTIFICATION IN STERLET, Acipenser ruthenus REPRODUCTIVE SYSTEM Miaomiao XIN
P19 CODING AND NON-CODING RNAS IN TELEOST LOW SPERM QUALITY SAMPLES: EFFECT ON PROGENY Vanesa ROBLES
P20 DETERMINATION OF DAY/NIGHT MELATONIN LEVELS IN SEMINAL PLASMA IN RELATION TO SPERM QUALITY IN SENEGALESE SOLE Catarina OLIVEIRA
SESSION II. SPERMATOGENESIS AND SPERM QUALITY
P21 PRODUCTION AND USE OF TRIPLOID ZEBRAFISH FOR SURROGATE REPRODUCTION Roman FRANĚK
P22 GERM CELL MANIPULATION AS A TOOL FOR COMMON CARP ISOGENIC LINES PRODUCTION AND MANAGEMENT Roman FRANĚK
P23 ONLY MALE INHERITANCE INDUCTION IN STURGEONS AND HATCHING OUT PADDLEFISH FROM STERLET EGGS USING COLD SHOCK ANDROGENESIS Roman FRANĚK
P24 CHARACTERIZATION OF A NEW EMBRYONIC CELLULAR MODEL FOR NUCLEAR TRANSFER IN FISH: THE EMBRYOID BODY Charlène ROUILLON
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P25 FLUORESCENCE-ACTIVATED CELL SORTING (FACS) OF STERLET (Acipenser ruthenus) SPERMATOGONIA BASED ON LIGHT SCATTER PROPERTIES Xuan XIE
P26 NON-INVASIVE SEX DETERMINATION IN LUMPFISH (Cyclopterus lumpus l.) USING ULTRASOUND TECHNOLOGY Maren MOMMENS
P27 LABELLING OF PRIMORDIAL GERM CELLS IN STURGEON USING IRON OXIDE NANOPARTICLES Michaela FUČÍKOVÁ
P28 THE DEVELOPMENT OF A NOVEL TECHNIQUE FOR PARTIAL CLEAVAGE INHIBITION ON STURGEON EMBRYO MODEL Mujahid Ali SHAH
P29 TOWARDS IMPROVING THE PRACTICABILITY OF GERM STEM CELL GRAFTING IN COMMERCIAL SALMONIDS FARMS Sarah TREMPONT, Anne-Sophie GOUPIL
P30 GERM CELL TRANSPLANTATION IN THE WHITE STURGEON, Acipenser transmontanus Amie L. T. ROMNEY
SESSION III. REFRIGERATION AND CRYOPRESERVATION
P31 CHILLING SENSITIVITY OF Astyanax altiparanae EMBRYOS AND THE PERFORMANCE OF FATTY ACIDS AND CRYOPROTECTANTS IN THE MAINTENANCE OF LARVAL VIABILITY Alexandre NINHAUS-SILVEIRA
P32 METHOD FOR LARGE-SCALE PRODUCTION OF STURGEON EMBRYOS USING CRYOPRESERVED SPERM Konstantin KOVALEV
P33 IS IT POSSIBLE TO STORE SPOTTED WOLFFISH (Anarhichas minor) SPERM BY REFRIGERATION? José BEIRÃO
P34 SPOTTED WOLFFISH (Anarhichas minor) SPERM CRYOPRESERVATION IN 5 ML CRYOVIALS José BEIRÃO
P35 IN VITRO SPERM MATURATION IN ACIPENSERIFORMES: FAMILY SPECIFIC FEATURE AND APPLICATION IN CRYOBANKING Viktoriya DZYUBA
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P36 THE GROWTH AND SURVIVAL RATE IN HATCHERY REARED NORTHERN PIKE (Esox lucius) LARVAE OBTAINED FROM 3 DIFFERENT LARGE-SCALE SPERM CRYOPRESERVATION METHODS Gergely BERNÁTH
P37 THE EFFECT OF ARACHIDONIC ACID ON RAINBOW TROUT SPERMATOZOA ACTIVATION AND SHORT-TERM STORAGE Olga BONDARENKO
P38 INHERITANCE OF SPERM CRYORESISTANCE IN ZEBRAFISH (Danio rerio) Bernadett PATAKI
P39 CHANGES IN SPERM CRYOPRESERVATION PROCEDURE FOR INDUSTRIAL NEEDS Catherine LABBÉ
P40 TRIALS FOR THE IMPROVEMENT OF PUFFERFISH (Takifugu alboplumbeus) SPERM EXTENDERS Juan F. ASTURIANO
P41 LONG-TERM ANTIBIOTIC-FREE PRESERVATION OF RAINBOW TROUT MILT (Onchorhynchus mykiss) Lucie GAVIN-PLAGNE, Na LI
P42 EFFECT OF CRYOPRESERVATION ON EPIGENETIC PROFILE IN OVARIAN FOLLICLES OF ZEBRAFISH (Danio rerio) Fernanda de MELLO
P43 COMPARATIVE ANALYSIS OF GLOBAL DNA METHYLATION IN SPERMATOZOA FROM FISH, MOLLUSCS, BIRD AND MAMMALS: EFFECT OF CRYOPRESERVATION Alexandra DEPINCÉ
P44 THE RESPONSE OF Crassostrea angulata (PORTUGUESE OYSTER) LARVAE TO CRYOPROTECTANTS EXPOSURE AND CRYOPRESERVATION Catarina ANJOS
P45 OXIDATIVE STRESS IN CRYOPRESERVED SEMEN OF NORMAL MALES AND SEX- REVERSED FEMALES RAINBOW TROUT IN RELATION TO GLUCOSE CONCENTRATION IN THE EXTENDER Sylwia JUDYCKA
P46 LABORATORY EGG INCUBATION TECHNIQUE IN STERLET (Acipenser ruthenus) Yu CHENG
P47 QUANTITY DOSING OF ENDOCELLULAR CRYOPROTECTANTS DURING CRYOPRESERVATION OF FISH SPERM Aleksandra A. KRASILNIKOVA
P48 CRYOPROTECTANTS USED IN ZEBRAFISH SPERM CRYOPRESERVATION MODULATE OFFSPRING SKELETAL DEVELOPMENT Patricia DIOGO
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P49 CRYOPRESERVATION PROTOCOLS OF ZEBRAFISH GAMETES — A SYSTEMATIC REVIEW Danilo P. STREIT JR.
SESSION IV. OOGENESIS AND EGG QUALITY
P50 CHANGES IN mRNA ABUNDANCE OF SELECTED TRANSCRIPTS DURING OOCYTE AGEING IN ZEBRAFISH Danio rerio Swapnil Gorakh WAGHMARE
P51 THE ROLE OF OXIDATIVE STRESS ON THE PROGRESS OF OOCYTE AGEING IN COMMON CARP Cyprinus carpio Azin MOHAGHEGHI SAMARIN
P52 LIPID AND PROTEIN OXIDATION CHANGES DURING OOCYTE AGEING IN AFRICAN CATFISH Clarias gariepinus Azin MOHAGHEGHI SAMARIN
P53 FROM OOGENESIS TO EMBRYONIC DEVELOPMENT: HOW CLIMATE CHANGE MAY AFFECT REPRODUCTIVE PERFOMANCE OF ATLANTIC COD Maud ALIX
P54 IDENTIFICATION AND FUNCTIONAL CHARACTERIZATION OF OVARIAN AND MATERNALLY-INHERITED miRNAs IN MEDAKA Stéphanie GAY
P55 A DE NOVO TRANSCRIPTOME ASSEMBLY SHEDS NEW LIGHT IN MOLECULAR DYNAMICS OF OVARIAN MATURATION IN THE MEDITERRANEAN SWORDFISH (Xiphias gladius) Giorgia GIOACCHINI
P56 CHARACTERIZATION OF MICROPYLE FORMATION IN MEDAKA (Oryzias latipes) miR-202 -/- MUTANTS Mariana ROZA DE ABREU
P57 INFLUENCE OF THERMAL MANIPULATIONS DURING GAMETOGENESIS ON REPRODUCTIVE MATURATION OF MEAGRE (Argyrosomus regius), SPAWNING SUCCESS, AND GAMETE AND EGG QUALITY Paulo H. de MELLO
P58 A NOVEL METHOD FOR RAPID ELIMINATION OF EGG STICKINESS USING SODIUM HYPOCHLORITE IN THREE FRESHWATER FISH SPECIES Martin PŠENIČKA
P59 UNUSUAL EGG QUALITY PHENOTYPE IN THE ASPE VALLEY: A CASE STUDY IN RAINBOW TROUT Emilien SEGRET
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P60 topaz1, A CRUCIAL GENE FOR MEDAKA (Oryzias latipes) REPRODUCTION Thaovi NGUYEN
P61 EFFECT OF DIFFERENT EQUILIBRIUM TIMES AND CRYOPROTECTOR SOLUTIONS IN THE VITRIFICATION OF ZEBRAFISH OVARIAN TISSUE: PRELIMINARY DATA Danilo P STREIT Jr
P62 FACTORS AFFECTING SECONDARY SEX CHARACTERISTICS IN THE YELLOWTAIL TETRA Astyanax altiparanae Diógenes H. SIQUEIRA-SILVA
SESSION V. SPERM MOTILITY AND FERTILIZATION
P63 INHIBITORY ANALYSIS OF ENERGY SUPPLYING PATHWAYS OF MOTILE AND IMMOTILE SPERMATOZOA IN SIBERIAN STURGEON (Acipenser baerii) Deepali RAHI
P64 HUNDREDS OF SPERMATOZOA ARE SUFFICENT FOR FERTILIZATION OF STERLET (Acipenser ruthenus) EGGS Miaomiao XIN
P65 EFFECT OF PH AND IONS ON SPERM MOTILITY IN PUFFERFISH (Takifugu alboplumbeus) Luz PÉREZ
P66 SPERM MORPHOLOGY AND MOTILITY SIGNALING IN ATLANTIC COD, Gadus morhua Sayyed Mohammad Hadi ALAVI
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PLENARY LECTURES
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 25 PLENARY LECTURE PL1
FISH SPERMATOLOGY: CONTRIBUTION TO THE LEGACY OF THE NOVELTIES DURING THE 40 PAST YEARS.
Jacky COSSON1*
1 University of South Bohemia in České Budějovice, Faculty of Fisheries and Protection of Waters, Research Institute of Fish Culture and Hydrobiology, Zátiší 728/II, 389 25, Vodnany, Czech Republic
When, in the 1980s, I became interested in the spermatology of fish under the light microscope, active spermatozoa were only visible thanks to their head presenting a sort of "tremor". This situation was quite frustrating given the lack of possible information regarding the motor part called flagellum, the structure of the latter being otherwise well described by EM. By analogy with studies developed over the past two decades by sperm specialists from other species such as sea urchin, we decided to apply simple technologies, including photography. Due to the high speed of the moving fish flagellum, the microscope illumination used a pulsed light strobe combined with a dark field microscope to record the flagellum image despite its small diameter (<0.5 μm). From simple photographs, several parameters could already be quantified after analysis of these images: beat frequency, cell velocity, trajectory linearity, waveform, rectitude, symmetry, amplitude, wavelength etc. This approach was rather tedious because of its "manual" aspect and the low number of followed sperm flagella, the results obtained were difficult to consolidate statistically. Soon, a similar approach used high-speed cinematographic microscopy (up to 200 fps), as well as video cameras to record sperm flagella with correct resolution. At the end of the 1990s, an automatic moving object video tracking system began to be commercialized, more commonly known as CASA when applied to spermatozoa. Its main advantages include: a) a large number of cells tracked, which greatly improves statistics, b) computer assistance allowing an automatic analysis that provides many motility parameters, including those mentioned above. Nevertheless, CASA systems were and are still unable to provide information about fish sperm flagella that move fast. During the 1990s, the translation of the microscope stage associated with the stroboscopic lighting of the glass slide containing the drop of sperm represented an important advance. At that time, analog video camera technologies allowed acquisition of flagellum images with high enough resolution for detailed analysis. The next step has been reached more recently, since the 2000s, with the use of high-speed video cameras, allowing the acquisition of images at a much higher resolution and at a much higher frequency, up to 10,000 frames per second. Since it became possible to visualize the flagella in motion, the latter acquired a noble function, which is added to that of a propeller: a flagellum is also a rudder allowing the spermatozoon to respond to certain specific signals, some leading to the control by the egg of its guidance. Several examples of new knowledge derived from observations and results on fish spermatozoa will be briefly illustrated, among which energetic aspects will be presented.
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 26 PLENARY LECTURE PL1
Where do we go, now? The great interest of CASA has been and remains its ability to obtain a statistical analysis of a large number of moving sperm heads. The equivalent for automatic flagella analysis took longer to become functional. A pioneering software was originally developed by Schöevaert et al. in (1990) for the analysis of flagellate algae, then applied to carp sperm. C. Brokaw (1984) developed a semi-automatic system at CalTech-USA and another was developed by Baba et al. in Japan, but both remained of limited application. It is only very recently that a MatLab plug-in has been developed by Gallagher et al. (2019) from Birmingham Univ. (UK) and appears to be among the most promising for an automated flagella analysis of fish spermatozoa.
Keywords: flagellum, sperm guidance, CASA, signaling.
Acknowledgements: Part of this work was supported during the period before my retirement, by the CNRS in France and after, by several grants from Czech Republic: CENAKVA LM2018099, CZ.02.1.01./0.0/0.0/16_025/0007370 and from European Union’s Horizon 2020 research and innovation program (No 652831 AQUAEXCEL2020). The author is highly thankful to all co-workers who friendly contributed to a better knowledge of the movement of the fish sperm flagellum.
References: Baba S. A. and Mogami, Y. (1985) An approach to digital image analysis of bending shapes of eukaryotic flagella and cilia. Cell Mot. 5: 475-489. Brokaw, C. J. (1984) Automated methods for estimation of sperm flagellar bending parameters. Cell Mot. 4: 417-430. Gallagher, M. T. et al. (2019) Rapid sperm capture: high-throughput flagellar waveform analysis. Hum. Reprod. 34: 1173–1185. https://doi.org/10.1093/humrep/dez056 Schöevaërt, D. et al. (1990) A new automated method of image analysis: Comparison of ciliary and flagellar beats. Biorheology 27:567-579.
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 27 PLENARY LECTURE PL2
NON MODEL FISH TRANSCRIPTOME AND GENOME DE NOVO ASSEMBLIES
Christophe Klopp1*
1 MIAT INRA Toulouse, Castanet-Tolosan, France
In the last ten years, the sequencing landscape witnessed two major changes. First, the second generation sequencer per base production costs decreased which enabled to produce transcriptomic data for large numbers of conditions and species in a single project. The Phylofish database is a good example of how to provide a community with valuable material for gene evolution studies. Second, the maturity of third generation sequencers enabled to produce chromosome level high quality reference genomes for non model species. Genofish, SexN’Perch, STURGEONOMICs are several projects using long, linked and Hi-C reads to build de novo references. Software packages and processing strategies have evolved with the data.
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 28 PLENARY LECTURE PL3
CELLULAR MODELS AVAILABLE FOR CRYOPRESERVATION AS A TOOL FOR MANAGING BIODIVERSITY
Bertrand Pain1*
1 Univ Lyon, Université Lyon 1, INSERM, INRA, Stem Cell and Brain Research Institute, U1208, USC1361, Bron, France
Cryopreservation of cells is a critical issue for many approaches in reproductive biotechnology. Many protocols have been developed to obtain the best conditions for freezing and thawing semen and embryos according to the species. In addition to these semen-targeted approaches, the development of new cell types remains an interesting way to maintain, study and manage the bio-diversity of species. Pluripotent stem cells (PSCs) and embryonic stem cells (ESCs) in particular have the unique characteristics of self-renewal and differentiation in vitro and in vivo in all embryonic lineages. For ESCs alone, there is colonization of the germ line with the property of producing descendants with the same genotype as that of the cells when they are injected into a recipient embryo. If PSCs have been isolated in several species, this strict definition of germinal colonization and thus identity of ESCs is currently restricted to rodents only. In the other species, PSCs have been isolated, amplified and established in lines with self-renewal and differentiation properties. PSCs have been indeed described in many species, among which, without being exhaustive, humans, non-human primates, rent animals such as ruminants, sheep, goats, horses, rabbits, etc... pets, including dogs and cats, birds such as chicken and duck, fishes such as medaka, zebrafish, cod, turbot ... etc. Most of these cells have been characterized by their proliferation potential, in vitro differentiation and by the presence of certain markers such as surface antigens including SSEA1, SSEA3 and SSEA4, antigens initially identified in mice, but whose reactivity crossed with other species proved to be very important in identifying these cells. However, among all the cells and species, very few have been demonstrated to contribute to embryonic morphogenesis with the noticable exceptions of chicken and zebrafish PSCs. Germ cells are another particularly interesting cell type in some species such as birds and fishes, because isolated and maintained in in vitro culture, they retain their ability to colonize the gonads. In particular, the primordial germinal cells (PGCs) of chicken, isolated from an embryo, can be established in long term in vitro culture and still maintain their property of colonization of the gonads. They are therefore a powerful tool for isolating and keeping a genotype of interest in avian species. Finally, somatic reprogramming is one of the most promising discoveries in the PSCs over the last decades. In 2006, Pr. S.Yamanaka demonstrated for the first time that the introduction of a gene cocktail into a differentiated cell (a fibroblast) changed the cell's fate by giving it the same properties as PSCs. This reprogramming process was achieved by the combination of four transcription factors, the Oct4, Sox2, Klf4 and c-MYC gene products (OSKM), also called Yamanaka combination. This promising approach is currently used in several mammalian and non-mammalian species with varying degrees of success. We will illustrate and discuss the advantages and disadvantages of these different cell types of interest in different species for the managment of the biodiversity.
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 29 PLENARY LECTURE PL4
DON'T JUDGE A BOOK BY ITS COVER, A TROJAN HORSE COULD BE HIDDEN IN THE SPERM
María Paz HERRAEZ1*
1 Dpt. Molecular Biology, Universidad de León, Spain
The zygote, the first cell of a new individual, contains an equal amount of paternal and maternal genome, but an astonishing content of cytoplasmic components directly inherited from the oocyte. Up to midblastula stage, the oocyte provides the embryo with the proteins and energy resources required for the first steps of development. From this moment onwards, once the embryo starts to generate their own proteins, the cytoplasmic factors are also involved in the control of embryo gene expression, demonstrating an outstanding maternal control of development. But this is just a part of the story…. The sperm, far from being a single carrier of the haploid genome, which is required to generate a diploid embryo, has a crucial role in the control of gene expression from the earlier stages of development. The integrity of the sperm chromatin, the presence of specific RNAs in their cytoplasm or subtle modifications in the epigenetic marks, can define the fate of the embryo: survival or death, proper or impaired development, stable or variable population. Recent advances in the field of the paternal contribution to development in fish will be presented, focusing on the effects that DNA damage and epigenetic changes have on the control of embryonic gene expression and the subsequent developmental consequences.
Keywords: DNA damage, DNA repair, epigenetics, paternal inheritance, embryo development, gene expression control.
Acknowledgements: This work was funded by the Spanish Ministry of Economy and Competitiveness (Projects AGL2011-27787 and AGL2014-53167-C3-3-R), and represents part of the PhDs from Cristina Fernández-Díez and Marta Lombó.
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 30
ORAL SESSIONS
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 31 SESSION I. SPERMATOGENESIS AND SPERM QUALITY O1
OSMOREGULATION IN FISH SPERMATOZOA: INVOLVEMENT IN MOTILITY ACTIVATION AND IMPACT ON SHORT-TERM STORAGE OUTCOMES
Fabio HERRERA1*, Olga BONDARENKO1, Borys DZYUBA1, Vitaliy KHOLODNYY1, Sergeii BORYSHPOLETS1
1 Faculty of Fisheries and Protection of Waters, University of South Bohemia in České Budějovice, Vodňany, Czech Republic
In marine and freshwater oviparous fishes spermatozoa must become motile to reach the egg for fertilization. Motility is activated after spermatozoa release into the water and is triggered by changes in sperm extracellular osmolality pressure or ion composition. These abrupt changes experienced by the cell evoke an osmotic shock resulting in the flux of water across the cell membrane. Generally, water transport mechanisms and regulation of cell volume are important features which allow the cell to maintain its structure and function integrity. The goal of this study is to analyse basic mechanisms of water transport in the connection with its role in fish spermatozoa physiology and focusing on its osmoresistance, volume regulation, motility and survivability during short-term storage. The ability of the plasmatic membrane of fish spermatozoa to resist dramatic changes of environmental ion composition, osmolality and pH is an essential prerequisite for motility and successful fertilization. In the light of this, the involvement of specific water channels (aquaporins) in regulation of cell volume and motility is highly probable. Aquaporins provide water flux across the membrane, allowing to balance the osmotic pressure on it. On the other hand, the fluidity of membrane plays quintessential role in spermatozoa viability under osmotic stress. Current theories of membrane fusion suggest that fluidity and flexibility of cell membranes are mainly dependent on their lipid composition. Spermatozoa exposed to short-term storage are susceptible to damage due low temperatures, availability of oxygen, dilution and storage duration, which can have a direct negative impact on membrane properties and as result on motility and fertilizing capacity. In this study we summarize experimental and theoretical data about osmoregulation in freshwater fish spermatozoa with particular attention to cell ability to resist osmotic shock during activation and short-term storage. Among others the role of aquaporins and membrane lipid composition/fluidity appear the most probable key players of osmosregulation in freshwater fish sperm and require further study.
Keywords: osmotic pressure, aquaporins, water transport, volume regulation.
Acknowledgements: The study was financially supported by the Ministry of Education, Youth and Sports of the Czech Republic - projects “CENAKVA” (LM2018099), by project Biodiversity (CZ.02.1.01. /0.0/0.0/16_025/0007370) and by the Grant Agency of the University of South Bohemia in Ceske Budejovice (125/2016/Z) and by the Czech Science Foundation (18- 12465Y).
SEESSION I. Spermatogenesis and sperm quality
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 32 SESSION I. SPERMATOGENESIS AND SPERM QUALITY O2
TRANSCRIPTOMIC PROFILE DURING TELEOST HAPLOID GERM CELL MATURATION AND DIFFERENTIATION TO SPERMATOZOA
François CHAUVIGNÉ1*, Jessica GÓMEZ-GARRIDO2, Anna ESTEVE-CODINA2, Tyler ALIOTO2, 3, Joan CERDÀ1
1 IRTA-Institute of Biotechnology and Biomedicine (IBB), Universitat Autònoma de Barcelona, Bellaterra (Barcelona), Spain 2 CNAG-CRG, Centre for Genomic regulation (CRG), Barcelona Institute of Science and Technology (BIST), Barcelona, Spain 3 Universitat Pompeu Fabra (UPF), Barcelona, Spain
The molecular pathways regulating the developmental transformation of post-meiotic haploid germ cells into spermatozoa have scarcely been investigated in teleosts. An excellent piscine model for studying this process is the marine flatfish Senegalese sole (Solea senegalensis), which exhibits a semi-cystic type of spermatogenesis where mature spermatids released into the lumen of the seminiferous tubules, which express the luteinizing/choriogonadotropin receptor receptor (Lhcgrba), differentiate to spermatozoa in response to Lh. To investigate the molecular regulation of spermatid maturation and release, we performed RNA-seq analysis on both immature and mature spermatids, which were isolated from the cortical and medullar regions of the testis, respectively, by fluorescence- activated cell sorting (FACS). The transcriptome was de novo assembled and annotated using Trinity v2.8.4 and Blast2go. Homology with other pleuronectiformes was also assigned. Among the 38,297 protein-coding genes found, only 758 were differentially expressed between immature and mature spermatids. Gene ontology (GO) and KEGG analyses revealed the downregulation of transcripts related to protein folding, transport and modification, DNA repair, and proteolysis, during spermatid maturation. In contrast, transcripts potentially involved in the regulation of mRNA processing, and signal transduction, as well as germ cell development and meiosis, were upregulated. Interestingly, amongst the upregulated mRNAs in mature spermatids we found a duplicated paralog of the Lhcgr (Lhcgrbb) which was not identified in previous studies. Using FACS-isolated mature spermatids we further investigated the changes in the transcriptome during Lh-induced spermatozoa differentiation in vitro. In this case, we identified 129 upregulated and 128 downregulated genes. Various transcripts potentially involved in the inositol phosphate signaling pathways, protein biosynthesis, and components of the sperm flagellar machinery, were upregulated, while transcripts related to endocytosis, DNA replication or alternative mRNA splicing, were downregulated. Altogether, these results provide the first insight into the molecular pathways involved in the Lh regulation of spermatozoa differentiation in teleosts.
Keywords: Germ cell development, flatfish, RNA-seq, Gonadotropin
Acknowledgements: This work was supported by the Spanish Ministry of Economy, Industry and Competition (MINECO) (Grant nº AGL2017-84013-R to F.C., and Ramón y Cajal Program nº RYC-2015-17103 to F.C.)
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 33 SESSION I. SPERMATOGENESIS AND SPERM QUALITY O3
ANEUPLOID SPERM OF Danio rerio FEMALE x D. nigrofasciatus MALE HYBRID
Mitsuru ENDOH1,2*, Fumika SHIMA1, Rei ASANUMA1, Miloš HAVELKA1, Etsuro YAMAHA3, Takafumi FUJIMOTO1, Katsutoshi ARAI1
1 Hokkaido University, Laboratory of Aquaculture Genetics and Genomics, Division of Marine Life Science, Faculty and Graduate School of Fisheries Sciences, 3-1-1, Minato, Hakodate, Hokkaido, 041-8611 Japan; [email protected] 2 Research Fellow of Japan Society for the Promotion of Science 3 Hokkaido University, Nanae Fresh-Water Station, Field Science Center for Northern Biosphere, 2-9-1 Sakura, Nanae, Kameda, Hokkaido, 041-1105 Japan.
In Danio species, interspecific hybridization has been conducted in several combinations of related species. Among them, only the hybrid between zebrafish D. rerio female and spotted danio D. nigrofasciatus male was reported to have fertility. However, gametes of the hybrid have not been investigated genetically as well as reproductive biologically. Here, we induced the hybrid by interspecific cross fertilization so as to study its developmental capacity, reproductive performance and gamete characteristics. There was no difference during embryonic stages between the hybrid and zebrafish control group, and viable progeny occurred. Hybrid nature was genetically verified by PCR-RFLP analysis of rhodopsin gene. All the hybrid samples possessed PCR-RFLP fragments derived from both parental species. External appearance of gonads (testis and ovary) of the hybrid was morphologically normal and similar to that of control zebrafish. However, hybrid males produced sperm with various head sizes as confirmed by light microscopy. Flow cytometry showed that sperm of the hybrid males were aneuploidies with various ploidy range up to diploidy. In backcross between zebrafish female and hybrid male, fertilization rates were lower than the control and most resultant progeny showed abnormal appearances as well as various aneuploidies approximately ranging from haploidy to triploidy. Most aneuploid progeny were inviable due to abnormalities, but only one progeny with 2.5N hyperdiploidy showed normal appearance at 3 days post-fertilization. Though developing oocytes were observed in a hybrid female, ovulated eggs could not be obtained in the hybrid female so far in the present study. In conclusion, the hybrid D. rerio x D. nigrofasciatus produced aneuploid sperm with low fertility. Backcross progenies showed various ploidy levels depending on sperm which contributed to fertilization. There was possibility that aneuploid progeny from hybrid sperm could survive to adulthood.
Keywords: Aneuploidy, backcross, fertility, interspecific hybridization, spotted danio, zebrafish
Acknowledgements: This work was financially supported in part by the Japan Society for the Promotion of Science (JSPS), KAKENHI Grant Number 17J02645 and 15H02457.
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 34 SESSION I. SPERMATOGENESIS AND SPERM QUALITY O4
EFFECT OF AGE ON THE SENSITIVITY OF ZEBRAFISH (Danio Rerio) SPERM
Tímea KOLLÁR1*, Bernadett PATAKI1, Gyöngyi GAZSI1, Béla URBÁNYI1, Ákos HORVÁTH1
1 Szent István University, Department of Aquaculture, Gödöllő, Hungary
Effect of age on the sensibility of zebrafish sperm against Hg-exposure was investigated in the present study. In the last years, in vitro test systems are extensively used in ecotoxicological studies particularly because of the aspects of animal protection. Based on the favourable properties (non-invasive collection, easily measurable parameters, dose- and time-response etc.), fish sperm can also be used as an alternative model. Although results of the use of sperm from mature individuals for toxicity tests have been published, there is no information about the exact age of the fish in some cases, which can affect the results. For this reason, our goal was to compare the response of sperm collected from different-aged zebrafish to Hg exposure. Pooled sperm (5 µL) was collected by stripping from same aged zebrafish into 25 µL cyprinid immobilizing solution (N=3). The pool was divided into 5 sub-groups (5 µL sample in each) which were further-diluted in 1:1 (v/v) ratio with immobilizing solution containing different concentrations of Hg to reach the final testing concentrations (0, 0.5, 1, 2.5 and 5 mg/L Hg). The experiment was carried out on the sperm of zebrafish from 3 different groups of age: 7, 12 and 18 month-old males were used. The same males were stripped again at least after one week. The motility parameters of sperm (progressive motility (%), curvilinear velocity (VCL)) were measured by Computer-assisted Sperm Analysis system, at 30th, 120th and 240th minutes of exposure. For activation, system water was used (in which zebrafish are held) supplemented with BSA. During the exposure, the samples were stored on melting ice. It was found that there were significant differences in PMOT as well in VCL in many cases among the three different aged groups. In PMOT, differences were observed at 0.5, 1 and 2.5 mg/L Hg-concentrations at 30 min, at 0.5 and 1 mg/L at 120 min as well as at 0.5 mg/L at 240 min. In VCL, significant differences were found at each tested concentration at 30 min as well as at 0.5 and 2.5 mg/L at 240 min. At 120 min, there was no significant difference in VCL. We found that the age of zebrafish can influence the sensitivity of its sperm. This concerns not only toxicology tests, but many techniques in fish breeding in which sperm is treated before use (cryopreservation, pressure shock etc.). Furthermore, it can be problematic in other species, as well (e.g. human aspect).
Keywords: mercury, progressive motility, PMOT, curvilinear velocity, VCL, CASA
Acknowledgements: The work was supported by the EFOP-3.6.3-VEKOP-16-2017-00008 project co-financed by the European Union and the European Social Fund
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 35 SESSION I. SPERMATOGENESIS AND SPERM QUALITY O5
METABOLIC STRESS DECREASES SPERM QUALITY IN GOLDFISH ASSOCIATED WITH CHANGES IN HORMONAL FUNCTIONS OF HYPOTHALAMUS-PITUITARY-TESTIS AXIS
Sayyed Mohammad Hadi ALAVI1*, Mahdi GOLSHAN2, Azadeh HATEF3,4, Magdelena SOCHA5, Sylvain MILLA6, Mirosława SOKOŁOWSKA-MIKOŁAJCZYK5, Suraj UNNIAPPAN4, Otomar LINHART7
1School of Biology, College of Science, University of Tehran, P. O. Box: 14155-6455, Tehran, Iran. 2Iranian Fisheries Science Research Institute, Agricultural Research, Education and Extension Organization, P. O. Box: 1331-5745, Tehran, Iran. 3Toxicology Center, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5B3, Canada 4Department of Veterinary Biomedical Sciences, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5B4 Canada 5Department of Ichthyobiology and Fisheries, University of Agriculture, Kraków, Poland 6AFAP Research Unit, University of Lorraine, Nancy, France 7South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses, Faculty of Fisheries and Protection of Waters, University of South Bohemia in České Budějovice, Vodňany 38925, Czech Republic
* [email protected], [email protected]
Similar to vertebrates, hormones of the hypothalamus-pituitary-testis axis (HPT-axis) regulate spermatogenesis and sperm maturation in fish. Metabolic hormones and neuropeptides act on parts of the hypothalamus that control expression and release of gonadotropin-releasing hormone (GnRH). Thus, hormonal functions of HPT-axis depend on an adequate supply of energy. Studies have shown roles of energetics in gonadal development, and reproductive disorders during energy deficiency. However, physiological relationships between metabolic and nutritional status with reproduction is largely unknown. In the present study, we measured sperm quality in the male goldfish following a 7-day fasting, and investigated changes in reproductive and metabolic hormones or peptides including mRNA levels of gnrh3, ghrelin, and nucb-2 (expressing nesfatin-1), and circulatory levels of luteinizing hormone (LH), 11-ketotestosterone (11-KT), and 17β-estradiol (E2). Both spermatozoa motility and velocity were decreased in fasted goldfish (P<0.05), however sperm mass showed a trend toward decrease. Frequency of spermatozoa with velocity >150 µm/s was lower in fasted males. These were associated with a decrease in 11-KT levels in fasted goldfish, but E2 levels remained unchanged. The brain gnrh3 and ghrelin mRNA levels, and circulatory LH were increased in fasted goldfish, but the brain nucb-2 mRNA levels were decreased (P<0.05). However, testicular mRNA levels of ghrelin and nucb-2 remained unchanged. Overall, fasting decreases sperm quality in goldfish due to metabolic-induced disruption of hormonal functions of HPT-axis. Our results provide novel data illustrating a crosstalk between metabolism and reproduction.
Keywords: Metabolic peptides; Neuroendocrine hormones; Sex steroids; Sperm production.
Acknowledgements: Ministry of Education, Youth and Sports of the Czech Republic: CENAKVA (LM2018099), CENAKVA (CZ.1.05/2.1.00/01.0024), CENAKVA II (LO1205 under the
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 36 SESSION I. SPERMATOGENESIS AND SPERM QUALITY O5
NPU I program); Biodiversity (CZ.02.1.01./0.0/0.0/16_025/0007370); University of Tehran; Discovery Grant from the Natural Sciences and Engineering Research Council of Canada.
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 37 SESSION I. SPERMATOGENESIS AND SPERM QUALITY O6
PROTEOMIC ANALYSIS OF SEMEN MATURATION IN SEX-REVERSED FEMALES OF RAINBOW TROUT
Joanna NYNCA1, Mariola SŁOWIŃSKA1, Sylwia JUDYCKA1, Ewa Liszewska1, Stefan DOBOSZ2, Andrzej CIERESZKO1*
1 Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, Poland 2 Inland Fisheries Institute, Department of Salmonid Research, Rutki, Żukowo, Poland
The production of masculinized females, named sex-reversed females (srf) is much more economically desirable and offers many advantages in salmonid aquaculture in comparison to mixed-sex populations. However, masculinization of females leads to incomplete testicular development and therefore spermatozoa must be collected directly from the testes. Consequently, the testicular srf semen is immature with no or low potential for motility, so such potential must be acquired through the incubation of sperm suspensions in buffered saline solutions with a high pH that mimic seminal plasma (ASP - artificial seminal plasma). It is unknown at present if proteins are involved in srf sperm maturation and acquiring the sperm motility. The aim of the study was to investigate the effect of 2 h incubation of srf sperm in ASP on the sperm motility parameters and changes in proteins abundance by 2D difference gel electrophoresis (2D-DIGE). The 2 h incubation of sperm in ASP increased percentage of sperm motility (from 26 to 66%), curvilinear velocity (from 112 to 131 µm s-1), straight-line velocity (from 14 to 25 µm s-1), linearity (from 14 to 19 %), the amplitude of lateral head displacement (from 11 to 16 µm) and had no effect on average path velocity. Incubation did not affect sperm viability, but significantly decreased the proportion of ROS-positive cells, from 12.9% and 9.1%. of fresh sperm and 2 h incubated in ASP led to the detection of 116 differentially protein spots in srf sperm (1.3-fold change, p < 0.05), out of which, the 113 spots were identified by MALDI-TOF/TOF. STRING analysis revealed that the majority of proteins are involved in cilium movement organization, small molecule metabolic process, protein folding and carboxylic acid metabolic process. Analysis with the use of g:Profiler indicated that cillium and flagellum movement and organization were top biological process, whereas small molecule, nucleotide and unfolded protein binding were assigned to top molecular functions. These results indicated that maturation process of srf spermatozoa involves the changes in the structural and metabolic proteins implicated in cell movement. The mechanisms of described protein changes likely involve post-translational modifications of their structure. The identified proteins could be biomarkers of mature spermatozoa and could be strictly evaluated in subsequent studies to verify their specific functions in spermatozoa, as well as their role in reproduction. Such information provides new contributions to understanding the maturation process of the fish male gametes involved in the development of male fertility.
Keywords: fish, spermatozoa, motility, proteins, 2- DIGE
Acknowledgements: supported by Project 2015/17/B/NZ9/01542 from the National Science Centre and funds appropriated to the Institute of Animal Reproduction and Food Research, Polish Academy of Sciences
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 38 SESSION I. SPERMATOGENESIS AND SPERM QUALITY O7
FIRST STEPS ON THE APPLICATION OF TECHNIQUES FOR THE CONTROL OF REPRODUCTION OF ELASMOBRANCH SPECIES IN CAPTIVITY
Pablo GARCíA-SALINAS1*, Víctor GALLEGO1, Marina MORINI1, Miriam GARCÍA-COLL1, Luz PÉREZ1, Daniel GARCíA-PÁRRAGA2, Juan F. ASTURIANO1
1 Universitat Politècnica de València. Instituto de Ciencia y Tecnología Animal. Grupo de Acuicultura y Biodiversidad. Camino de Vera s/n 46022 Valencia, Spain 2 Fundación Oceanogràfic de la Comunitat Valenciana, Gran Vía Marqués del Turia 19, 46005 Valencia, Spain
Sharks and rays are among the most threatened vertebrates on the planet, especially due to overfishing and habitat destruction (IUCN, 2019). Despite this level of threat, techniques to control their reproduction has been scarcely used as an approach to protect these animals. The main aim of this study is to apply techniques commonly used in other fish species to achieve control of the reproduction of elasmobranchs. Four model species are the target of the study (3 sharks: Scyliorhinus canicula; S. stellaris and Galeus melastomus, and one ray: Raja montagui) with which to develop techniques that can be extrapolated to other bigger and/or threatened ones. Target species were obtained from fishing by-catch and from animals kept in public aquaria. To date, urogenital and reproductive system has been anatomically described, and hepatosomatic and gonadosomatic indexes has been calculated for by-catched species. Oocyte diameter from mature females was measured every month to establish their reproductive status through the year. Also, both in vivo and post-mortem extraction of sperm has been achieved, by massage and gentle pressure near the urogenital pores or by catheterization with a veterinary nasogastric canula. Sperm cells are motile even when extracted from the seminal vesicle of dead fish stored at 7 °C for 24 h. The sperm obtained from the cephalic portion of the epididymis is immotile in the 4 species, while it can be activated with a Ringer solution adapted to elasmobranchs when obtained from the caudal portion of the epididymis. Sperm volume and spermatozoa characteristics, such as size of head, midpiece and flagellum, complexity and type of sperm aggregates (established by Tanaka et al., 1995) and number of helical gyres of the sperm head, has been determined. Despite the sperm activity, cells size (total length: 116-198 µm) made impossible to evaluate their motility by CASA protocols. The techniques developed up to date with our target species were also used in others such as nurse shark (Gynglymosotoma cirratum) and lesser devil ray (Mobula hypostoma).
Keywords: Sperm; Assisted Reproduction; Shark; Ray.
Acknowledgements: Co-funded by the Fundación Biodiversidad (PRCV00683) and the UPV. PGS and MGC have PhD grants from the European Union through the Operational Program of the European Social Fund (ESF) of the Comunitat Valenciana 2014-2020 ADIF 2018 (ACIF/2018/147 and /136, respectively). VG and MM have postdoc grants from the MICIU (Programa Juan de la Cierva-Incorporación; IJCI-2017-34200) and UPV (PAID-10-18),
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 39 SESSION I. SPERMATOGENESIS AND SPERM QUALITY O7 respectively.
Tanaka, S., Kurokawa, H. & Hara, M. (1995). Comparative morphology of the sperm in chondrichthyan fishes. Mémoire du Muséum National d’Histoire Naturel 166, 313–320
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 40 SESSION I. SPERMATOGENESIS AND SPERM QUALITY O8
SHORT-TERM STORAGE-INDUCED CHANGES IN PROTEOME OF CARP SPERMATOZOA
Mariola DIETRICH 1*, Mariola SŁOWIŃSKA1, Sylwia JUDYCKA1, Halina KAROL1, Andrzej CIERESZKO1
1 Department of Gametes and Embryo Biology, Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, Olsztyn, Poland
Most fish spermatozoa are quiescent in the seminal plasma; therefore, no energy is consumed for motility. This characteristic makes them most suitable for short-term storage. The semen storage for several days at low temperatures is mostly applied in transport of semen, to synchronize male and female gamete availability and in avoiding the aging of sperm. However, there is a significantly decrease on sperm functions during storage. To our knowledge information regarding proteome changes in fish semen upon short-term storage is lacking. The aim of the present work was to evaluate the changes in protein composition of the extracellular medium (EM; diluted seminal plasma) during storage of carp semen to monitor proteins released from damaged spermatozoa. The carp semen (n=6) was diluted 10-fold in the immobilizing buffer (94 mM NaCl, 27 mM KCl, 50mM glycine, 15mM Tris–HCl, pH 7.5) and stored for 120 h at 4ᴼC. Every 24 hours semen quality was monitored using CASA analysis (sperm motility) and flow cytometry (viability, reactive oxygen species (ROS) positive cells). For proteomic analysis 48h and 120h of storage were selected. Two-dimensional difference gel electrophoresis was performed to compare extracellular fluid from the fresh semen and semen stored for 48h and 120h. The differentially abundant proteins (fold change 1.2; p<0.05) were identified using MALDI- TOF/TOF mass spectrometry and functionally analyzed using Ingenuity Pathway Analysis. A steady increase in protein concentration in EM was observed during storage, a significant increase (2-fold) occurred after 48h. After 120h of semen storage the percentage of sperm motility and viability decreased (from 80±10.6% to 62±5.2% and from 96±3.2% to 91±4.3%, respectively) while number of ROS-positive cells increased (from 1.48±0.3% to 6.8±2.4%). Short-term storage resulted in increasing abundance of 75 protein spots (corresponding to 53 proteins) in EM. Proteins involved in metabolism were found to be highly enriched in EM, including enzymes involved in glycolysis, gluconeogenesis, the citric acid cycle, creatine metabolism, purine metabolism and methylation. Moreover, proteins associated with cellular stress response, cilium assembly, signal transduction and proteolysis were identified. The top enriched molecular network contained proteins involved in organismal injury and abnormalities, respiratory disease (score 61; number of molecules 24). The identification of a high number of proteins released from spermatozoa provides new insights into the mechanism of sperm damage to the particular sperm structure and to specific metabolic pathways affected by short term storage. The availability of a catalogue of carp sperm proteins altered by semen storage provides a crucial tool for the development of novel potential biomarkers of sperm injuries and for the improvement of a short-term sperm preservation procedure.
Keywords: fish, spermatozoa, proteins, electrophoresis, 2D-DIGE, proteomics
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 41 SESSION I. SPERMATOGENESIS AND SPERM QUALITY O8
Acknowledgements: This study was supported by grant from the National Science Centre, Poland 2016/21/B/NZ9/03620 and founds from IAR&FR PAS in Olsztyn.
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 42 SESSION I. SPERMATOGENESIS AND SPERM QUALITY O9
DEVELOPMENTAL TRANSCRIPTOMICS REVEALS NOVEL MECHANISMS OF TELEOST SPERMIOGENESIS
Júlia CASTRO-ARNAU1*, François CHAUVIGNÉ1, Jessica GÓMEZ-GARRIDO2, Anna ESTEVE- CODINA2, Tyler ALIOTO2,3, Joan CERDÀ1
1 IRTA-Institute of Biotechnology and Biomedicine (IBB), Universitat Autònoma de Barcelona, Bellaterra (Cerdanyola del Vallès), Spain 2 CNAG-CRG, Centre for Genomic Regulation (CRG), Barcelona Institute of Science and Technology (BIST), Barcelona, Spain 3 Universitat Pompeu Fabra (UPF), Barcelona, Spain
In teleost fishes, the molecular mechanisms involved in the transformation of haploid germ cells (HGCs) into spermatozoa (spermiogenesis) are largely unknown. Here, we investigated this process in the gilthead seabream (Sparus aurata) through the examination of the changes in the transcriptome between the HGCs and ejaculated sperm (SPZ). RNA-seq analysis revealed a total of 7202 differentially expressed genes (DEGs) (p-value < 0.01) between both cell types, of which approximately half were upregulated in SPZ compared to HCGs. In addition, 296 genes de novo annotated in SPZ were mostly related to immunity, metabolism, transcription and translation, and ion and protein transport. Intriguingly, we also found ~3600 long non-coding RNAs upregulated in SPZ. Among the DEGs identified, 435 upregulated mRNAs in SPZ are related to transcription, translation and chromatin organization, 140 encode for components of the cytoskeleton, and 139 encode for water and ion channels, exchangers and transporters. The SPZ also differentially accumulated 263 receptor-coding mRNAs, of which over a half are enzyme-linked receptors, 89 are G protein- coupled receptors, 13 are nuclear receptors, and 9 are channel-linked receptors. Pathway analysis performed using the Panther version 14.1 software identified a total of 986 transcripts belonging to 120 different signaling pathways. Further analysis showed that these mRNAs are enriched in important pathways related to the metabolism of carbohydrates, lipids, nucleotides and nucleosides, and to receptor-activated transduction mechanisms. Among the latter, we identified 24 receptor pathways upregulated in SPZ of which, surprisingly, the most enriched correspond to the gonadotropin-releasing hormone receptor and platelet derived growth factor signaling pathways. These data therefore reveal the possible involvement of novel endocrine mechanisms during the differentiation and/or maturation of spermatozoa, and pave the way for future studies to investigate the molecular regulation of spermiogenesis in teleosts.
Keywords: Gilthead seabream, RNA-seq, spermiogenesis, transcriptomics
Acknowledgements: This work was supported by the Spanish Ministry of Economy, Industry and Competition (MINECO) (Grant no. AGL2016-76802-R to J.C.). Participation of F.C. and J. C-A. was funded, respectively, by a “Ramon y Cajal” contract (RYC-2015-17103) and a predoctoral grant (BES-2017-080778) from Spanish MINECO.
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 43 SESSION II. GERMINAL STEM CELLS AND BIOTECHNOLOGIES O10
THE UNEXPECTED DIVERSITY OF TWO GENETICALLY SIMILAR HYBRID FEMALES IN THEIR REPRODUCTIVE PATHWAYS
Joëlle LAFOND1*, Philippe HÉNAULT1, Christelle LEUNG1 and Bernard ANGERS1
1 Department of Biological Science, Université de Montréal, Succ. Centre-Ville Montreal, Quebec, Canada.
Triploid vertebrates from unisexual complexes often perpetuate themselves asexually. In the fish Chrosomus eos×eos-neogaeus, triploids are continuously produced by diploid hybrids by a failed gynogenesis. Cybrids C. eos are their only known progeny, which is surprising since they are rare in some lakes where the triploids are abundant. This study thus aims to investigate the oogenesis of these triploid hybrids through experimental crosses to complete the knowledge of the oogenesis of these fishes. A total of 337 larvae from triploid females were fertilized with C. eos sperm and genetically characterized. The detection of C. eos as progeny of triploid hybrids confirmed the occurrence of a pathway similar to meiotic hybridogenesis, but only for half of the triploids. The presence of tetraploid offspring for all these females revealed the formation of unreduced triploid eggs as a probable failure of meiotic hybridogenesis. The remaining female produced diploid and triploid hybrids, in a pathway similar to gynogenesis, but a little bit modified. Triploids excluded the haplome from their grandfather and produced eggs with the diploid hybrid genome through an ameiotic hybridogenesis (modified gynogenesis). Both types of hybridogenesis occurred in a mutually exclusive manner. This leads us to consider two hypothetical scenarios: first, any female triploids can perform either type of hybridogenesis. Secondly, ameiotic hybridogenesis occurs in triploids of the first generation (from diploid mothers), while meiotic hybridogenesis occurs in triploids of the second generation (from triploid mothers). Finally, the male’s spermatozoid could have an impact on the progeny’s ploidy, possibly because of sexual genes, that are still unknown in this species. The population dynamics of the Chrosomus eos-neogaeus complex thus appears a step more complicated than previously expected.
Keywords: Experimental crosses; hybridogenesis; gynogenesis; fish oogenesis; polyploidy
Acknowledgements: This research was performed under institutional animal care guidelines (permit #18–019 delivered by the Université de Montréal), and conforms to the mandatory guidelines of the Canadian Council on Animal Care. Sampling permits were provided by the Quebec Ministry of Natural Resources and Wildlife (MRNF). This research was supported by a research grants from the Natural Sciences and Engineering Research Council of Canada (NSERC) to B.A. (#238600). The work of P.H. was supported by a NSERC – Undergraduate Student Research Undergraduate Student Research Awards.
References: Lafond, J., Hénault, P., Leung, C., and Angers, B. (2018). Unexpected Oogenic Pathways for the Triploid Fish Chrosomus eos-neogaeus. Journal of Heredity. SESSION II. Germinal stem cells and biotechnologies
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 44 SESSION II. GERMINAL STEM CELLS AND BIOTECHNOLOGIES O11
INTRAPERITONEALLY GRAFTED BLASTOMERES CAN DIFFERENTIATE INTO FUNCTIONAL GAMETES IN ZEBRAFISH
Roman FRANĚK1*, Taiju SAITO1,2, Tomáš Tichopád1, Martin PŠENICKA1
1 University of South Bohemia in České Budějovice, Faculty of Fisheries and Protection of Waters, South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses, Zátiší 728/II, 389 25 Vodňany, Czech Republic. 2 Nishiura Station, South Ehime Fisheries Research Center, Ehime University, Uchidomari, Ainan, Ehime, 798-4206, Japan.
Intraperitoneally grafted blastomeres from vas:EGFP strain at 1-4k cell stage were demonstrated to have capability to survive and give rise primordial germ cells (PGCs) from the transplanted cell mass. Donor-derived PGCs colonized host’s genital ridge and later proceeded gametogenesis and gave rise to viable donor-derived gametes. This technique combines advantages of blastula transplantation which can be conducted very early in development and poorly viable embryos can be rescued as well take advantage of intraperitoneal transplantation when recipient larvae are tolerant enough to transplantation procedure resulting in good survival post-transplantation. To optimize the technique, blastomeres were grafted into different recipients - AB zebrafish line, AB zebrafish line with depleted PGCs and AB zebrafish x pearl danio hybrid. Similar germ line chimera induction rate was observed in nonsterile and sterile zebrafish, however, transplantation into the hybrids was unsuccessful. After sexual maturation, germline chimeras produced either donor-derived eggs or sperm exclusively in case of transplantation into PGCs depleted host, or a mixture of donor-derived and host-derived gametes in case of non-sterile recipients. Donor-derived origin of gametes produced from transplanted cells was confirmed by GFP signal detection and by PCR analysis of embryos after in vitro fertilization of germline chimera males with control zebrafish females from AB line. Presented study is the first report of surrogate egg production in zebrafish.
Keywords: germ cell transplantation, surrogacy, zebrafish
Acknowledgements: The study was financially supported by the Ministry of Education, Youth and Sports of the Czech Republic, projects CENAKVA (LM2018099) and Biodiversity (CZ.02.1.01/0.0/0.0/16_025/0007370), by the Grant Agency of the University of South Bohemia in České Budějovice GAJU 097/2019/Z and 034/2017/Z. The Czech Science Foundation (project No. 17-19714Y) and NAZV QK1910248
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 45 SESSION II. GERMINAL STEM CELLS AND BIOTECHNOLOGIES O12
TRANSCIPTOMIC ANALYSIS OF DEAD END KNOCKOUT SALMON TESTIS
Lene KLEPPE1, Rolf BRUDVIK EDVARDSEN, Tomasz FURMANEK1, Eva ANDERSSON1, Kai Ove SKAFTNESMO1, Frida THYRI SEGAFREDO, Anna WARGELIUS1*
1 Institute of Marine Research, P.O. Box 1870, Nordnes, NO-5817, Bergen, Norway 2 Cflow Fish Handling AS, Holsneset 25, N-6030 Langevåg, Norway
A significant amount of research is being performed to find possible solutions to sustainability challenges for the salmon farming industry. As such, use of sterile salmon can solve problems with precocious puberty and genetic introgression from farmed escapees to wild populations. Recently, sterile salmon was produced by knocking out the germ cell- specific dead end (dnd) gene[1]. Several approaches could potentially be applied to inhibit the function of Dnd[2]. Since it is challenging to develop a successful treatment against a gene product already existing in the body, alternative targets are also being explored. Germ cells are surrounded by, and dependent on, gonadal somatic cells. Targeting genes that are essential for the survival of gonadal somatic cells could potentially be good alternative targets for future sterility treatments. The aim of this study was to identify and characterize novel germ cell and gonadal somatic factors in Atlantic salmon. We have for the first time analysed RNA-sequencing data from germ cell-free (GCF)/dnd knockout and wild type (WT) salmon testis and searched for genes preferentially expressed in either germ cells or gonadal somatic cells. To exclude genes with expression in extra-gonadal tissues, our dataset was merged with available multi-tissue transcriptome data. We identified 389 gonad specific genes, of which 194 were preferentially expressed within germ cells, and 11 were confined to the somatic part of the gonad. Interestingly, 5 of the 11 gonadal somatic transcripts represented genes encoding secreted TGF-β factors; gsdf, inha, nodal and two bmp6-like genes, all representative vaccine targets. Of these genes, gsdf and inha had the highest transcript levels. The expression of gsdf and inha was further confirmed to be gonad specific, and their spatial expression was restricted to Granulosa and Sertoli cells of the ovary and testis, respectively. Finally, we show that inha expression increases with puberty in both ovary and testis tissue, while gsdf expression does not change or decreases during puberty in ovary and testis tissue, respectively. This study contributes with novel transcriptome data on Atlantic salmon testis tissue with and without germ cells. In this context we provide a list of novel and known germ cell- and gonad somatic specific transcripts in salmon.
Keywords: 4-6 items. Sterility, transcriptome, salmon, somatic gonad
Acknowledgements: Norwegian research council projects STERWELL and SALMOSTERILE
References: 1. Wargelius A, Leininger S, Skaftnesmo KO, Kleppe L, Andersson E, Taranger GL, Schulz RW, Edvardsen RB: Dnd knockout ablates germ cells and demonstrates germ cell independent sex differentiation in Atlantic salmon. Scientific reports 2016, 6:21284. 2. Wong TT, Zohar Y: Production of reproductively sterile fish: A mini-review of germ cell elimination technologies. General and comparative endocrinology 2015, 221:3-8.
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 46 SESSION II. GERMINAL STEM CELLS AND BIOTECHNOLOGIES O13
STERILIZATION IN STURGEONS FOR SURROGATE PRODUCTION
Abdul Rasheed BALOCH1* and Martin PŠENIČKA1
1 University of South Bohemia in České Budějovice, Faculty of Fisheries and Protection of Waters, South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses, Zátiší 728/II, 389 25 Vodňany, Czech Republic
Sturgeons are also known as living fossils that have existed for a minimum of 200 million years. These ancient giants became target of intensive illegal fisheries due to high caviar value that in-turn led to collapse in their populations. Some sturgeon species have life span over 100 years and attain sexual maturity between 20 and 25 years; however, amongst, sterlet (Acipenser ruthenus) has fastest reproductive cycle that matures from 3 to 7 years. Thus, this species can be used as host for sturgeon surrogate production. However, sterility of host or germ cell free host is pre-requisite for sturgeon surrogacy. Primordial Germ Cells (PGCs) are origin of all germ cells, and the dead end (dnd1) is essential for their migration. In order to produce germ cell free host for aforementioned purpose, we have established various methods in our lab. i) We did knockdown of dnd1 by using antisense morpholino oligonucleotide (MO) approach. PGCs as marker of dnd1-knockdown were observed under fluorescent stereomicroscope at 4 and 21 dpf (days post fertilization). No PGCs were seen in MO-injected embryos as compared to control group injected with FITC-dextran only to label PGCs. Further, data was confirmed by histology and in situ hybridization that showed gonads lacking the germ cells. ii) In second attempt, germ cells in sterlet were removed by means of ultraviolet (UV) irradiation at the VP (vegetal pole) of embryos. Reduction in PGCs number was proportional to dose of UV irradiation; however, some PGCs were observed after UV treatment, they significantly reduced when embryos grew. iii) CRISPR/Cas9 is a cutting-edge genome editing technology, thus, we opted to use it to knockout sterlet dnd1. Crispants lacked PGCs; however, interestingly, some of the crispants showed malformations. Malformations were presumed to be because of off-target effects and/or double injections at AP (animal pole) and VP for dnd1 knockout and PGCs labelling, respectively. Furthermore, we also compared all above-mentioned methods to reach at conclusions in terms of survival and hatching rates of embryos. Higher embryo survival and hatching rates were observed in CRISPR/Cas9, UV and MO, respectively. Apart from these established methods for sterility in sturgeons, iv) the sturgeon hybrids having odd ploidy level have also been suggested to be sterile (females) and limited sterility in males as well. v) Preliminary data from our very recent study, where we injected polystyrene nanoparticles into VP of sterlet embryos, they presumably caused the death/removal of PGCs. This study, however, will be continued in our forthcoming experiments to get more insights of using nanoparticles in sturgeon surrogate production. SESSION III. Refrigeration and cryopreservation Keywords: Antisense morpholino oligonucleotide; CRISPR/Cas9; Hybridization; Sterlet; polystyrene nanoparticles; UV-irradiation.
Acknowledgements: The study was financially supported by the Ministry of Education, Youth and Sports of the Czech Republic -project CENAKVA (LM2018099) and Biodiversity
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 47 SESSION II. GERMINAL STEM CELLS AND BIOTECHNOLOGIES O13
(CZ.02.1.01/0.0/0.0/16_025/0007370) and the Czech Science Foundation (grant number 17- 19714Y), by the European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement No. 642893 (IMPRESS).
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 48 SESSION II. GERMINAL STEM CELLS AND BIOTECHNOLOGIES O14
IS THE HYBRID TIGER TROUT A GOOD RECIPIENT FOR THE TRANSPLANTATION OF EARLY- STAGE GERM CELLS?
Ákos HORVÁTH1*, Zoran MARINOVIĆ1, György HOITSY2, Márton HOITSY3, Boglárka HOITSY1,2, Béla URBÁNYI1, Jelena LUJIĆ1
1 Department of Aquaculture, Szent István University, H-2100 Gödöllő, Páter Károly u. 1., Hungary 2 Hoitsy & Rieger Kft., H-3517 Miskolc, Erzsébet stny. 55., Hungary 3 University of Kaposvár, H-7400 Kaposvár, Guba Sándor u. 40., Hungary
The hybrid tiger trout (Salmo trutta m. fario × Salvelinus fontinalis) was used as a recipient of early-stage germ cells (spermatogonia and oogonia) of the rainbow trout (Ocorhynchus mykiss) in order to produce donor-derived gametes. When selecting potential recipients for surrogate production, several factors have to be taken into account: the recipient needs to be related to the donor, must be relatively easy to spawn and should preferable be sterile. The tiger trout is a hybrid of the brown trout and brook trout that is known to have an impaired gametogenesis which makes it an ideal recipient. Spermatogonia and oogonia were isolated from the gonads of one-year old rainbow trout and transplanted by microinjection into 371 alevins of the tiger trout 5 days post hatch in February and March, 2017. Following transplantation, the recipient fry were grown at a commercial trout farm. Of these, 55 juveniles were alive by May, 2017 and 17 individuals reached the age of 22 months by December, 2018. At a routine check-up, one male was found spermiating and sperm as well as gonad samples were collected from it. Part of the sperm was used for fertilization of rainbow trout eggs. Of the 2490 eggs fertilized, 2328 (93%) reached eyed stage and hatched. Hatched fry were grown at the same fish farm. Currently, 300 of them are still grown there and phenotypically they all appear to be rainbow trout. Genetic analysis of the progeny will be carried out in order to confirm their donor-derived origin. This study has shown that the hybrid tiger trout can be used as a recipient in surrogate production of salmonid fish, however, high mortalities during juvenile growth need to be taken into account.
Keywords: spermatogonia, oogonia, salmonids, surrogate production, transplantation.
Acknowledgements: This study was supported by the National Research, Development and Innovation Office of Hungary (grant SNN 116912), EFOP-3.6.3-VEKOP-16-2017-00008 project co-financed by the EU and the European Social Fund, the Stipendium Hungaricum Scholarship Programme in Hungary, as well as the Higher Education Institutional Excellence Program (1783-3/2018/FEKUTSRAT) awarded by the Ministry of Human Capacities within the framework of water related research of Szent István University.
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 49 SESSION II. GERMINAL STEM CELLS AND BIOTECHNOLOGIES O15
VIRGIN SALMON- A SUSTAINABLE SOLUTION FOR COEXISTENCE OF FARMED AND WILD SALMON STRAINS WITHOUT MIXING
Hilal GÜRALP1*, Kai O. SKAFTNESMO1, Anne Hege STRAUME1, Erik KJÆRNER-SEMB1, Lene KLEPPE1, Rolf B. EDVARDSEN1, Anna WARGELIUS1
1 Institute of Marine Research, Bergen, Norway
Salmon farming is a major industry in Norway, with an annual value of over forty billion NOK. A bottleneck in the salmon farming industry is the crossing of escaped farmed salmon with wild populations. To solve this problem, the industry can use sterile salmon in production. Sterile salmon is currently produced through triploidization of embryos. However, triploid salmon are generally more sensitive to suboptimal production environment, which may lead to several welfare problems. Therefore, the food authority has been critical to this production method. As previously shown by our research group, we can produce sterile immature salmon by preventing the formation of germ cells [1, 2]. In this project, we are exploring a new method where we create broodstock fish with germ cells, which in turn can produce 100% sterile offspring for production. This approach solves the problems of genetic introgression upon escape and premature maturation in farmed salmon and ensures stable production of 100% sterile fish, and thus represents a significant commercial potential. Since this type of salmon is produced through targeted mutations, they are with the current legislation considered as genetically modified organisms (GMO). In some countries, such as Argentina and Australia the legislation has been revised and fish subjected to this the type of genetic editing will not be considered GMOs. It is expected that similar regulatory changes will happen in other countries in the near future, including in Norway, where currently a more modern GMO legislation suggested is soon to be evaluated. The commercial potential of developing a new method to produce sterile salmon is significant since it will solve some of the key challenges in salmon farming including genetic introgression of farmed salmon into wild strains and precocious maturity.
Keywords: crispr-cas9, fish escapes, genome editing, infertility, micromanipulation
Acknowledgements: Institute of Marine Research, Bergen, Norway
References: 1. Kleppe L, Andersson E, Skaftnesmo KO, Edvardsen RB, Fjelldal PG, Norberg B, Bogerd J, Schulz RW, Wargelius A: Sex steroid production associated with puberty is absent in germ cell-free salmon. Sci Rep 2017, 7(1):12584. 2. Wargelius A, Leininger S, Skaftnesmo KO, Kleppe L, Andersson E, Taranger GL, Schulz RW, Edvardsen RB: Dnd knockout ablates germ cells and demonstrates germ cell independent sex differentiation in Atlantic salmon. Scientific Reports 2016, 6.
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 50 SESSION II. GERMINAL STEM CELLS AND BIOTECHNOLOGIES O16
EVOLUTION OF GERMLINE GENE EXPRESSION PROFILES DURING RAINBOW TROUT ONTOGENESIS AND PUBERTY ONSET
Ahmed MAOUCHE1, Anne-Sophie GOUPIL1, Edouard CURRAN1, Elisabeth SAMBRONI1, Jean- Jacques LAREYRE1*
1 INRA, Fish Physiology and Genomics, 35042 Rennes Cedex
In mammals, germ stem cells of the male embryos undergo distinct processes of differentiation, first while they reach the gonad and then after birth but prior puberty. The male germ stem cells are successively named primordial germ cells (PGC), gonocytes or prospermatogonia (ProSpg), and spermatogonial stem cells (SSC). From spermatogenesis onset and throughout lifespan, SSC differentiate to form progenitor cells that are capable to reconstitute the stock of germ stem cells or to continue their spermatogenic differentiation. Although they remain misunderstood, the different stages of the germ stem cell differentiation are accompanied by changes in gene expression profiles. The present study aimed to investigate whether the molecular signature of the undifferentiated spermatogonia evolves during ontogenesis and prior to puberty onset in a fish species. Homologs of the mammalian nanos2, dnd, vasa, gfra1, gdnf, kitr, and kitlg genes were identified in Gnathostomes and reliable orthologous relationships were established using phylogenetic reconstructions and analyses of syntenic chromosomal fragments. Gene duplications and losses occurred specifically in teleost fish and members of the Salmoninae family. The cellular expression profile of the candidate genes was determined in rainbow trout using germ cell and somatic cell fractions. Undifferentiated type A spermatogonia expressed higher levels of kitb and kita1 making them possible target cells for the gonadal kitlgb and somatic kitlga before the onset of spermatogenesis. Interestingly, the germ cell specific nanos2, dnd, gdnfa and gdnfb genes were poorly expressed before the onset of spermatogenesis. Expression of nanos2 and dnd genes increased concomitantly with the emergence of progenitor cells forming doublets or chains. The expression level of gdnfb was correlated with that of the vasa gene suggesting that the late increased abundance of gdnfb during spermatogenesis onset was due to the increased number of A and B spermatogonia. gfra1a2 was expressed in undifferentiated type A spermatogonia whereas gfra1a1 was mainly detected in somatic cells. These observations indicate that the germinal Gdnfb ligand could exert autocrine and paracrine functions on spermatogonia and on testicular somatic cells, respectively. Members of the Kitlg/Kit regulatory pathway were not hormono-dependent. In contrast, Fsh and androgens inhibited gfra1a2 and gdnfb whereas gfra1a1 was stimulated by Fsh, androgens, and 17α,20β progesterone. Altogether, our data provide evidences that the molecular signature of the male germ stem cells changes during ontogenesis and prior to spermatogenesis onset as observed in mammals.
Keywords: Germ stem cell, spermatogenesis, paracrine regulation
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 51 SESSION II. GERMINAL STEM CELLS AND BIOTECHNOLOGIES O16
Acknowledgements: supported by the EU Horizon 2020 research infrastructure project under grant agreement n°652831 (AQUAEXCEL2020 project) and by the French National Research Agency under grand agreement n°11-INBS-0003 (CRB anim project).
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 52 SESSION III. REFRIGERATION AND CRYOPRESERVATION O17
THE EFFECT OF GLUCOSE, METHANOL CONCENTRATION AND TIME OF EQUILIBRATION ON POST-THAW SPERM MOTILITY OF RAINBOW TROUT SEMEN
Sylwia JUDYCKA1*, Mariola SŁOWIŃSKA1, Joanna NYNCA1, Ewa LISZEWSKA1, Andrzej CIERESZKO1
1 Department of Gametes and Embryo Biology, Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, Olsztyn, Poland
The standardization of the freezing and thawing process is required to develop commercial applications of sperm cryopreservation for fish. Recently, a successful attempt has been made to standardize the cryopreservation protocol for many salmonid species, however these studies focused on standardization of glucose and final sperm concentration in straw. To date, standardized approach has not been applied to the testing the effect of methanol concentration as well as the effect of time of equilibration of semen in extender on post- thaw sperm motility. Filling of the mentioned gaps is important for further improving of standardized cryopreservation procedure. The aim of this study was to test the effect of methanol concentration and time of equilibration of semen in extender on post-thaw sperm motility of rainbow trout. Additionally, we tested the effect of final glucose concentration on post-thaw sperm motility, because such data are not available for rainbow trout. Sperm motility measured by CASA was the end point for the evaluation of cryopreservation success. The final glucose concentrations in the straw were tested within the range from 0.11 to 0.23 M in 7.5% methanol combined with a constant final sperm concentration in the straw (1 × 109 spermatozoa ml-1. Final methanol concentrations were evaluated within the range from 0 to 20% in 0.15 M glucose at sperm concentration of 1 × 109 spermatozoa ml-1. The effect of time of equilibration (5, 15, 30, 60, 120, 180 min) was tested on extended semen samples (1.0 × 109 spermatozoa ml-1 and final glucose concentration at 0.15 M in 7.5% methanol) after 5, 15, 30, 60, 120 and 180 min of post-thaw storage. Diluted semen was loaded into 0.5 ml plastic straws and frozen in liquid nitrogen vapour for 5 min. The straws were thawed by immersion in a water bath at 40°C for 10 s. The highest post-thaw sperm motility (63-75%) was recorded from 0.13 to 0.17 M of glucose and from 5.0 to 10.0% of methanol, with the highest peak at 7.5% (70% of post-thaw sperm motility). The equilibration of semen in extender up to 60 min did not affect sperm motility, however a decrease was recorded after 120 min. However, after cryopreservation the post- thaw sperm motility did not differ among almost all equilibration times, except 180 min where decrease was noticed. Our results clearly indicate that post-thaw sperm motility of rainbow trout is highly influenced by glucose and methanol concentration. Those parameters should be taken into consideration during development of sperm cryopreservation protocols for different fish species. The extended time of equilibration (up to 60 min) without decrease of sperm motility allows for prolong handling time which should facilitate cryopreservation of semen for large scale production.
Keywords: fish, spermatozoa, cryopreservation, standardization
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 53 SESSION III. REFRIGERATION AND CRYOPRESERVATION O17
Acknowledgements: This work was supported by funds of the National Science Centre granted on research project 2018/29/N/NZ9/00761 and funds appropriated to the Institute of Animal Reproduction and Food Research, Polish Academy of Sciences.
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 54 SESSION III. REFRIGERATION AND CRYOPRESERVATION O18
CRYOPRESERVATION OF PUFFERFISH SPERM: EFFECT ON MOTILITY, FERTILIZATION RATES AND HATCHING SUCCESS
Víctor GALLEGO1*, Luz PÉREZ1, Manabu YOSHIDA2, Juan F. ASTURIANO1
1 Grupo de Acuicultura y Biodiversidad. Instituto de Ciencia y Tecnología Animal. Universitat Politècnica de València. Camino de Vera s/n 46022 Valencia, Spain. 2 Misaki Marine Biological Station. Graduate School of Science. University of Tokyo. 1024 Misaki-Koajiro. 238-0225 Miura, Kanagawa, Japan.
Cryopreservation of fish gametes has many potential applications both for ecological, scientific and aquaculture purposes. To date, sperm of more than 200 fish species have been successfully cryopreserved and techniques of thawed sperm management have been established for freshwater and marine fish. Nevertheless, there is no study for the long-term conservation of pufferfish (Takifugu alboplumbeus, before T. niphobles) sperm, thus the objectives were i) to develop a new cryopreservation protocol for pufferfish sperm using different sperm:extender ratios and vials; and ii) to assess the fertilization capacity of cryopreserved sperm through IVF trials. Fish were caught in Arai Beach (Miura, Japan) and maintained in aquaria in Misaki Marine Biological Station (MMBS). All the experiments started with the selection of sperm samples showing >80% of motility by CASA-Mot evaluation. Samples were diluted 1:20 and 1:50 in Seminal-Like-Solution (SLS) and then cryoprotectant (methanol, 10% v/v) was added to the diluted sperm. Different vials (straws of 0.5 mL and cryotubes of 2mL and 5 mL) were used for cryopreserving sperm. Sperm motility (MOT) was immediately tested after thawing process and also in the next 1, 2, 3, 5 and 7 days. Cryopreserved sperm samples showed excellent motility results when they were frozen in straws, reaching post-thawed motilities of >60% in both dilutions (1:20 and 1:50). Cryotubes also showed good motility results (around 50% of motile cells after thawing) but slightly lower than those obtained in the straws. In relation to short-term storage after thawing process, sperm samples from straws (dilute at 1:50) were able to keep acceptable motility values (of around 40%) after 7 days of chilled storage. Finally, and due to excellent results obtained in the cryopreservation trials, in vitro fertilization tests were carried using different sperm:egg ratios (1:103, 1:104, 1:105 and 1:106) both in fresh and cryopreserved sperm. High (>90%) fertilization (FR) and hatching (HR) rates were reached using 1:104 and 1:105 ratios (fresh and cryopreserved sperm, respectively). These results mean that cryopreservation process affects negatively the spermatozoa kinetic parameters and then, fertilization and hatching success. In this sense, the sperm:egg ratio necessary to achieve high FR and HR in pufferfish is 10-times higher for cryopreserved than for fresh sperm. This study has laid the bases for the establishment of cryopreservation protocols in pufferfish, that will be helpful for further reproduction in captivity programs and genetic cryobanking.
Keywords: fugu, larvae, sperm quality, spermatozoa
Acknowledgements: Generalitat Valenciana funded the stay in Japan of JFA, LPI and VG
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 55 SESSION III. REFRIGERATION AND CRYOPRESERVATION O18
(BEST 2018/093, BEST 2018/112 and BEST 2018/124, respectively). VG has a postdoc grant from the MICIU (Programa Juan de la Cierva-Incorporación; IJCI-2017-34200).
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 56 SESSION III. REFRIGERATION AND CRYOPRESERVATION O19
PRESERVATION OF OVARIAN TISSUE IN EUROPEAN EEL (ANGUILLA ANGUILLA)
Ilija ŠĆEKIĆ1, Nevena KITANOVIĆ1*, Zoran MARINOVIĆ1, Tamás MÜLLER1, Béla URBÁNYI1, Jelena LUJIĆ1, Ákos HORVÁTH1
1 Department of Aquaculture, Szent István University, Páter Károly u. 1, H-2100 Gödöllö, Hungary
European eel (Anguilla anguilla) is considered to be an economically important fish species for the global markets. In last few decades, a decrease in the numbers of this species with no indication of possible natural recovery has led to listing this species in the IUCN red list as critically endangered. Anthropogenic factors such as oversfishing negatively influence natural recovery of this species. Additionally, long and expensive hormonal treatments, problems related to gamete synchronization and absence of larvae feeding protocol hinder reproduction of this fish in captivity. Therefore, novel biotechnology and conservation tools are needed for developing strategies for preservation and conservation this fish. The aim of this study was to develop cryorpeservation protocols for European eel ovarian tissue. Development of a freezing protocol was preformed through 5 sequential experiments where in each experiment the best outcome was used with a newly changed parameter for further testing. In the first trial, the highest viability was observed using Me2SO (79.7±3.2%). Subsequently, similar results were observed when utilizing 1.5 M Me2SO. Varying sugar and protein supplementation did not provide any significant differences, therefore 1.5 M Me2SO supplemented with 0.1 M glucose and 1.5% BSA was used for further experiments. When comparing the effects of different tissue sizes and equilibration times, equilibration of 50 mg tissue pieces for 30 min on ice resulted in the highest viability (93.8±5.8%). No significant differences were observed between the tested cooling rates and thawing in a 10 ℃ water bath, while reciprocal thawing rates yielded slightly lower viability rates. During the needle- immersed vitrification (NIV) procedure, only vitrification solutions displayed statistically significant influence on cell viability. The utilization of ES3 (containing 1.5 M PG + 1.5 M Me2SO) and VS3 (containing 3 M PG + 3 M Me2SO) yielded the highest viability rates (84.3±11.5%) indicating the beneficial effects of using lower and equal amounts of two cryoprotectants in vitrification solution by mitigating the toxic effects exerted by high amounts of each individual cryoprotectant. Methods developed in this study can aid in developing alternative conservation strategies for the European eel and present the onset of applying germline stem cell biotechnology tools for the conservation of this species.
Keywords: cryopreservation, freezing, vitrification, conservation
Acknowledgements: This study was supported by the National Research, Development and Innovation Office of Hungary (grant FK 124585), EFOP-3.6.3-VEKOP-16-2017-00008 project co-financed by the EU and the European Social Fund, the Stipendium Hungaricum Scholarship Programme in Hungary, as well as the Higher Education Institutional Excellence Program (1783-3/2018/FEKUTSRAT) awarded by the Ministry of Human Capacities within the framework of water related research of Szent István University
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 57 SESSION III. REFRIGERATION AND CRYOPRESERVATION O20
TECHNOLOGY TRANSFER OF CRYOPRESERVATION TO THE INDUSTRY: EXAMPLE OF ATLANTIC SALMON
Lucie GAVIN-PLAGNE1*, Na LI1, Eric SCHMITT1, Richard LE BOUCHER1
1 R&D department, IMV Technologies, L’Aigle, France
For years, cryopreservation in salmon production has been for genetic use only. It started in the nineties as a tool for genetic resource storage. The use of cryopreservation is the origin of the implementation of many breeding programs, especially with recent development in genomics and disease resistance selection. However, it had not crossed the line of salmon production farms until 2016, when IMV Technologies transferred production-based laboratories to the salmon hatcheries. Nowadays, different organization schemes exist around the world for preserving genetic resources in salmon production. Farms are offered off-site cryopreservation services by public institutes (USA, Denmark), cooperatives (France) or private companies (Chile, Norway). IMV Technologies offers a different type of service by installing laboratories within the farm. Farmers can directly collect sperm, freeze samples and store straws on-site, allowing more flexibility. In order to implement this scheme, and to transfer the technology from research to the salmon industry, the need of “innovators” and “early adopters” (customers willing to try the technique) is essential. Indeed, the product range is developed according to the Technology Readiness Levels (TRL) combined with Marketing Readiness Level (MRL). During this development, the projects are time-consuming and require technical and financial support, especially on the field. This now represents 50 farms visits in 23 countries. This is why customers play a key role in this development, particularly for the prototype validation in a large-scale environment. Since the success of the first installation in 2016, more and more examples of salmon production farms have given satisfactory results. Nowadays, in the salmon production, the fertility rate with cryopreserved sperm reaches the level of the fresh sperm (around 70%) on an industrial scale (repeatable data, 600K eggs). Depending on the size of the farm, freezing sperm two days per week is feasible with the storage of 24K medium French straws per day, corresponding to 1.5L of raw milt. Moreover, with the use of ProSorter (accurate sorting system for salmon eggs), egg quality can be correlated with the performance of sperm cryopreservation. The effect of cryopreservation can be consequently assessed on the fertility rate but also the egg quality. To conclude, cryopreservation is validated in large scale in salmon production. On-site cryopreservation allows high quality sperm at optimum maturation and the large diffusion of genetic resources.
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 58 SESSION IV. OOGENESIS AND EGG QUALITY O21
FUNCTIONAL AND PROTEOMIC CHARACTERISATION OF SALMONID COELOMIC FLUID
Aurélie GUEHO1*, Hélène RIME1, Emmanuelle COM2, Régis LAVIGNE2, Blandine GUEVEL2, Jérôme MONTFORT1, Charles PINEAU2, Julien BOBE1
1 INRA Fish Physiology and Genomics 2 Protim-Irset Inserm U1085
While in most fish species egg viability rapidly decreases after ovulation, Salmonids can hold their eggs in the body cavity for a week without any significant loss of egg developmental competence. In the body cavity, eggs are bathed in a biological fluid called ovarian fluid (OF) or coelomic fluid (CF). This fluid appears to be directly responsible for the good conservation of eggs. Indeed, eggs remain viable after several days of storage in this fluid, while their viability rapidly decreases when stored in artificial medium mimicking the mineral composition of CF. Understanding the biological properties of coelomic fluid and identifying the actors involved in this process would be of great interest for aquaculture and for further understanding the biological mechanisms preserving egg viability. It would particularly help improving egg storage conditions. We have used a proteomic approach to characterize rainbow trout (Oncorhynchus mykiss) coelomic fluid proteome. Proteins involved in immunity, part of the extracellular matrix, or involved in lipids metabolism and transport are the most highly present classes in this fluid. Comparison of this proteome with transcriptomic data of the PhyloFish database suggests the presence in the rainbow trout coelomic fluid of proteins specifically expressed in salmonids. To further characterize CF, rainbow trout fluid has been fractionated by HPLC on a gel filtration column. After 4 days of incubation in those fractions, trout eggs were fertilized. In some fractions, eggs exhibited a good developmental rate, even though lower than in non- fractionated coelomic fluid. Proteins present in those fractions are currently being identified. In parallel, proteomic comparison of salmonid coelomic fluids with non-salmonids ovarian fluids will allow us to identify proteins specifically present in salmonids coelomic fluid. Together, those experiments will help us identify potential protein candidates participating in preserving egg viability and unraveling pathways conferring this unique biological activity to salmonid coelomic fluid.
Keywords: Fish reproduction, Gamete quality, mass spectrometry, aquaculture
Acknowledgements: This work was funded by ANR under grant # 16-CE20-001 EggPreserve to JB
SESSION IV. Oogenesis and egg quality
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 59 SESSION IV. OOGENESIS AND EGG QUALITY O22
THE EFFECTS OF PHARMACEUTICALS ON GAMETE DEVELOPMENT IN Oreochromis mossambicus (PETERS, 1852) AFTER A CHRONIC EXPOSURE
Clémentine NIBAMUREKE1; Irene BARNHOORN2 and Ina WAGENAAR1*
1 Department of Zoology, University of Johannesburg, South Africa, 2006 2 Department of Zoology, University of Venda, South Africa, 0950
The antiretroviral (ARV) drug nevirapine (NVP) is one of the main ARVs used as first-line treatment for the prevention of the Human Immunodeficiency Virus (HIV) in Africa. Together with NVP, the antibiotics sulfamethoxazole (SMX) and trimethoprim (TMP) are part of the HIV therapy to treat opportunistic infections. Studies on rats treated with ARVs reported histological alterations in the reproductive organs (Oyeyipo et al. 2018). All three pharmaceuticals, NVP, SMX and TMP have been frequently detected in surface waters across Africa. Information on their potential effects on fish reproduction is limited. This study investigated the chronic effects of environmentally relevant concentrations of NVP (1.48 and 3.74 µg/L) as a single toxicant and in a mixture with SMX (3.68 µg/L) and TMP (0.87 µg/L) on the reproductive organs of male and female O. mossambicus, which occurs widely in African surface waters. The exposed female O. mossambicus recorded higher gonad indices compared to males. Statistical analysis revealed a significant difference in female gonad indices across the groups (Chi-square = 11.020, p = 0.026, df = 4, n = 27) while no significant difference in male fish gonad indices was found (Chi-square = 3.021, p = 0.554, df = 4, n = 24). The main histological changes detected in the ovaries of female fish were oocyte atresia and vacuolation of oogonial nests occurring mainly in the fish exposed to NVP (3.74 µg/L) and to the mixture. In general, regressive changes susceptible of causing changes in the organ function were the most prominent. Somehow, the presence of NVP and the antibiotics (SMX and TMP) in water triggered massive atresia of late vitellogenic and mature oocytes in exposed fish. The observed abnormal high rate of atretic oocytes in the fish from this study could result in impaired fish reproduction. In conclusion, the results from this study showed that NVP in African surface waters may have long-term negative effects on fish reproduction and those effects may be increased by the presence of antibiotics such as SMX and TMP.
Key words: antiretrovirals, antibiotics, fish reproduction, histopathology, surface water.
Acknowledgements: This research is supported in part by the National Research Foundation of South Africa (Unique Grant Number 119318) for rated researchers and the Global Excellence and Stature Scholarship to UMC Nibamureke.
Reference: Oyeyipo, I.P., Skosana, B.T.; Everson, F.P., Strijdom, H. & du Plessis, S.S. (2018). Highly Active Antiretroviral Therapy alters sperm parameters and testicular antioxidant status in diet-Induced obese rats. Toxicological Research, 34(1): 41-48. doi:10.5487/TR.2018.34.1.041
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 60 SESSION IV. OOGENESIS AND EGG QUALITY O23
DIFFICULTIES TO COMPARE EGG QUALITY: INSIGHTS FROM A TRANSCRIPTOMIC ANALYSIS
Tainá ROCHA DE ALMEIDA1*, Julien BOBE2, Aurélie LE CAM2, Christophe KLOPP3, Jérôme MONTFORT2, Pascal FONTAINE1, Dominique CHARDARD1 and Bérénice SCHAERLINGER1
1 University of Lorraine, INRA, UR AFPA, F-54000 Nancy 2 INRA, UR1037 Fish Physiology and Genomics, Campus de Beaulieu, Rennes, France. 3INRA, Sigenae, MIAT, CS 52627, Castanet-Tolosan, Toulouse, France.
The literature devoted to fish eggs quality is abundant. However, variable scientific context render compilation and data analyses more complex. Thus, comparing data from different studies may lead to misinterpretation because the context may not be properly considered. On the other hand, the quality of eggs is determined by their intrinsic properties, including their molecular content, and an incorporation and/or synthesis defect could lead to developmental impairments. Meanwhile, many molecular mechanisms are shared by vertebrates allowing theoretically to make reliable comparisons between studies. To investigate how molecular data may be affected when the scientific context varies, we studied the impact of the developmental criterion and the thresholds chosen to limit two quality groups (low and high) on the transcriptomic profile of unfertilized eggs in Eurasian perch (Perca fluviatilis). A microarray analysis was performed to describe the transcriptomic profiles of eggs presenting different developmental potential. For each criterion used (survival, hatching or deformities), various thresholds (i. e. rates) delimitated each group of quality. For example, when the criterion was survival at 48hpf, the spawn presenting rates >90%(a) and >80%(b) represented the high quality groups, while the spawn presenting survival <30%(c) and <45%(d) represented the low quality groups. Then, the comparisons a vs. c, a vs. d, b vs. c and b vs. d were performed. As for different criteria we sometimes had the same threshold, we could test the effect of both parameters (criterion and threshold) in the analysis. In total, 28 spawn were analyzed and 10 independent comparisons between high vs. low quality groups were performed. We checked that each group of quality contained at least five biological replicates (spawns) for the analyses to be statistically robust. Results show a stronger relationship between early survival rates and the mRNA profiles in eggs than for hatching and deformities rates that did not highlight any differentially expressed genes (DEG). It suggests that these defects may have other causes beyond maternal inherited mRNA, most likely after the zygotic genome activation. Comparisons using early survival rates (24 or 48hpf) and different thresholds (6 comparisons) led to different data showing that parameters choices drastically influence molecular results. Only one common DEG was identified and was significantly up-regulated in all low quality groups in every comparison. In three of the comparisons, several gene ontology terms related with translation and immune system were overrepresented. Taken together, our results demonstrate the importance of the choice of criteria and thresholds used to assess eggs quality in the objective to draw reliable comparisons between studies. In the future, a particular attention should be paid to these parameters and it would be helpful to find standards not only for a given species but also between species. This will also increase meta-analyses feasibility and reliability.
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 61 SESSION IV. OOGENESIS AND EGG QUALITY O23
Keywords: microarray, egg content, gene ontology, mRNA, Perca fluviatilis.
Acknowledgements: We thank the French National Research Agency (Maternal Legacy, ANR- 13-BSV7-0015), the INRA PHASE and the Brazilian National Council for Scientific and Technological Development (CNPq-233389/2014-8).
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 62 SESSION IV. OOGENESIS AND EGG QUALITY O24
DAILY RHYTHMS OF IN VITRO FERTILIZATION AND TRANSCRIPTOME OF FISH OOCYTES
Juan Fernando PAREDES1*, José Fernando LOPEZ OLMEDA1, Gonzalo DE ALBA1, José Antonio MUÑOZ-CUETO1, Prabhugouda SIRIYAPPAGOUDER1, Jorge M. FERNANDES1 and Francisco Javier SANCHEZ-VAZQUEZ1
1 Department of Physiology, Faculty of Biology, Regional Campus of International Excellence, Campus Mare Nostrum, University of Murcia, 30100 Murcia-Spain.
Predictable environmental changes such as the alternation of day/night have pressured organisms to develop a biological clock to keep track of daytime. Such a timing mechanism has fostered the development of a wide range of rhythmic adaptive strategies such as reproduction to occur at specific times of the day to increase the survival of the offspring. In fish, the timely secretion of gonadotropins and sexual steroids are crucial to synchronize spawning, and preliminary research revealed that oocytes, but not sperm, drive daily rhythms in in vitro fertilization (IVF). The aim of this research was to investigate the existence of daily rhythms in the transcriptomic oocyte profile that may explain IVF rhythms during the day. To this end, we kept zebrafish broodstock under 14L:10D light-dark cycle and we performed IVF trials at different Zeitgeber Times: ZT0, ZT1, ZT2, ZT3, ZT4, ZT7, ZT15, ZT21, ZT23, with lights on at ZT0 and lights off at ZT14. IVF success was highest at ZT23-ZT1- ZT2 (75.6-97.1-71.6%), and decreased sharply outside this fertilization window. Samples for transcriptomic analysis by RNA sequencing (RNA-Seq) in the oocytes were collected at ZT1, ZT7, ZT15 and ZT21. The cosinor analysis (p < 0.05) revealed the existence of a statistically significant daily rhythm for in vitro fertilization. In addition, one-way ANOVA also unveiled statistically significant differences between fertilization time points (p < 0.05). Transcriptomic analysis revealed the differentially expressed genes (DEGs) (adjusted p-value < 0.05, fold change ≥ 1.5) in all groups when compared to the control group (ZT1). Additionally, specific reproductive functions (such as regulation of fertilization, sperm-egg recognition, binding of sperm to zona pellucida) were found in each group explaining the variations of the IVF success rates during the day (GO analysis, GeneRatio/BgRatio, adjusted p-value < 0.05). These results suggest that the success for IVF strongly depends on the time of day most likely due to oocyte transcript variations. These data should be considered when establishing protocols for in vitro fertilization in broodstock fish farming.
Keywords: oocyte transcript, daily rhythms, in vitro fertilization
Acknowledgements: This research was funded by the project “SOLEMBRYO” (AGL2013- 49027-C3-1-R) and “BLUESOLE” (AGL2017-82582-C3-3-R) granted by the Spanish Ministry of Economic Affairs and Competitiveness (MINECO) co-funded with FEDER, and “CHRONOHEALTH” granted by Fundación Seneca (19899/GERM/15) to FJSV. J.F.LO was funded through a research fellowship granted by MINECO (Ramón y Cajal Program).
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 63 SESSION IV. OOGENESIS AND EGG QUALITY O25
TRANSGENERATIONAL EFFECTS OF BPA ON ZEBRAFISH FEMALE REPRODUCTION AND EMBRYO DEVELOPMENT
Stefania SANTANGELI1, Claudia CONSALES2, Francesca PACCHIEROTTI2, Hamid R HABIBI3, Francesca MARADONNA 1, Oliana CARNEVALI 1,4*
1 Dipartimento Scienze della Vita e dell’Ambiente, Università Politecnica delle Marche, Via Brecce Bianche, 60131 Ancona, Italy. [email protected]; 2 Laboratory of Biosafety and Risk Assessment, Division of Health Protection Technologies, ENEA Casaccia Research Center, Via Anguillarese 301, 00123 Rome, Italy. [email protected]; [email protected]; 3 Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada. [email protected]. 4 INBB Consorzio Interuniversitario di Biosistemi e Biostrutture, 00136 Roma, Italy.
Bisphenol A (BPA) is an ubiquitous environmental contaminant and the concern for its transgenerational effects is increasing. To induce transgenerational effect, an epigenetic mark should be mitotically and meiotically stable and not reprogrammable in primordial germ cells and embryos. To test the transgenerational effect of BPA, zebrafish females were exposed via mother to environmental dose (20 µg/L) of BPA and then crossed with untreated males till to reach F3 generation. The BPA affected adult female fertility up to F2 generation. In F0, F1 and F2 ovaries, the transcription of esr, star, lhcgr and fshr gene was affected. Focusing on genes involved in gonadal differentiation in 24hpf embryos, amh transcript level was reduced in 24 hpf embryos, up to the F3 generation. Changes of dnmt1 were evident along the generations. To investigate the possible epigenetic mechanisms of the gene expression modulation found in our study, we studied the promoter DNA methylation in three different genes dnmt1, amh and fshr genes. We investigated the percent of 5-methylcytosine (5-mC)/total cytosine. The analysis of 11 CpG sites across the promoter of dnmt1 identified the decrease of their methylation in the ovary of F0 BPA females, despite an increase in F1 BPA females. No changes in the percentage of DNA methylation was observed in 24 hpf F1, F2 and F3 embryos, and in the ovary of F2 females in the BPA group. Evaluating the percentage of 5mC of three CpG of the amh promoter, we observed an increase in the BPA groups in all the generations analyzed (both 24 hpf embryos and adult females) compared to controls, except for the ovary of F0 females. In the ovary of F0-F2 generations, the analysis of three CpG sites relative to the promoter of fshr demonstrated a decrease of DNA methylation only in F0 BPA treated females while no changes were observed in the following generations. To investigate the possibility that gene expression alterations in embryos of BPA group could be caused by inherited epigenetic instability, H3K4me3 and H3K27me3 enrichment were assessed in the transcription start site (TSS) and in its two flanking regions by evaluating dnmt1 and amh in F1 and F2 24hpf embryos. The H3K4me3 enrichment was reduced in the TSS of dnmt1 and amh in BPA treated 24hpf F1 embryos compared to control while increased H3K27me3 enrichment was observed only in the TSS of dnmt1 in the BPA treated 24hpf F1 embryos. No changes were detected in the other regions of F1 and F2 embryos.
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 64 SESSION IV. OOGENESIS AND EGG QUALITY O25
These findings provide evidence for transgenerational effects of BPA in zebrafish and demonstrate that amh is susceptible to stable epigenetic alterations.
Keywords: Epigenetic effects, amh, ovary, steroidogenic enzymes,
Acknowledgements: This work was supported by Grant PRIN 2010–2011 to OC and NSERC grants to HRH
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 65 SESSION IV. OOGENESIS AND EGG QUALITY O26
VERY HIGH GENETIC CORRELATION FOR EGG PRODUCTION TRAITS BETWEEN TWO SUCCESSIVE SPAWNING IN RAINBOW TROUT
Anastasia BESTIN1*, Ana ACIN PEREZ2, Frédéric CACHELOU2, Daniel GUEMENE3, Pierrick HAFFRAY1
1 SYSAAF, Campus de Beaulieu, Bâtiment 16A, Allée Henri Fabre, 35042 Rennes cedex, France, 2 Viviers de Sarrance, Pisciculture Labedan, 64490 Sarrance, France 3 SYSAAF, Centre INRA Val de Loire UMR BOA, 37380 Nouzilly, France
Since the mid-90s, French commercial hatcheries of rainbow trout initiated selective breeding to improve growth performances, focusing on carcass traits thereafter. Now more than 800 million eggs derived from these programs are sold worldwide. It is common practice to reproduce the same female up to 3 times (using photoperiodic regimes). Yet there is no result documenting the genetic parameters of successive spawning of the same individual. This is what this work focused on through the rearing of a commercial batch from Viviers de Sarrance originating from 8 successive generations of selective breeding. 30 sires and 38 dams were crossed in a partly full factorial mating design which created 254 full and half sib families being reared in common environment (spring water, 14°C during pre- growing phase, 9°C afterwards). A sub-batch of 835 pit-tagged and DNA-sampled females was kept under natural photoperiod and phenotyped at their 1st spawning (2-year-old, 2kg mean BW): individual length (L), body weight after spawning (BW), drained spawn weight (SW) and size of the eggs (ES). The same females were kept under natural photoperiod until their 2nd spawn at 3-year-old and the same measurements were recorded on 526 individuals at 3.6kg mean BW. The gonado-somatic index (GSI) was calculated. Traits followed by ‘_1’ and ‘_2’ were recorded at 1st and 2nd spawn. Heritabilities and genetic correlations (rG) were estimated using VCE software. Accuracy of breeding values was estimated as the square root of the coefficient of determination (CD). All traits are heritable: e.g. from 0.32 ± 0.12 for GSI_1 to 0.55 ± 0.10 for SW_1. Heritabilities tended to decrease at nd st nd 2 spawn: e.g. 0.24 ± 0.10 for ES_2 or 0.38 ± 0.10 for SW_2. Either at 1 or 2 spawn, the rG between ES and SW were positive (between 0.53 ± 0.14 and 0.58 ± 0.18). rG between BW or BL and GSI were negative. The SW is tightly correlated with GSI (0.95 ± 0.02 and 0.97 ± 0.02). All traits at 1st spawn were highly correlated with the same traits at 2nd spawn (0.81 for ES, 0.84 for SW, 0.89 for GSI). Accuracies were rather high in this design: e.g. up to 0.72 for ES_1 and 0.63 for GSI_1. They decreased but remain high at 2nd spawn, e.g. 0.56 for ES_2 or 0.67 for SW_2. Spawning traits are heritable at 1st and 2nd spawn. In this population, ES is not correlated with BW and BL, and is positively correlated with other spawning traits. However, selecting for large size trout at spawning would deteriorate their GSI. The accuracies of st st nd breeding values are higher at 1 spawn, and rG between traits at 1 and 2 spawn are high and positive. This means that since the 1st spawn it is possible to rank correctly the best females which will outcompete their relatives even for the next spawning events. Choosing to reproduce these females evaluated on their 1st spawn performances would limit the generation interval, and thus optimize the genetic gain on these traits especially if males are evaluated on their sib breeding values.
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 66 SESSION IV. OOGENESIS AND EGG QUALITY O26
Acknowledgements: The study is part of BestOv project supported by the European Maritime and Fisheries Fund (EMFF) and by the French Government through the FranceAgriMer national body (n°: 2016-0152).
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 67 SESSION IV. OOGENESIS AND EGG QUALITY O27
VisEgg: AN AUTOMATIC PHENOTYPING TOOL TO ASSESS RAINBOW TROUT UNFERTILIZED EGG VIABILITY
Emilie Cardona1,2*, Jérôme Bugeon1,2, Emilien Segret1, Julien Bobe1
1 INRA, UR1037 Physiologie et génomique des poissons, 35000 Rennes, France 2 equal contribution
In aquaculture, assessing female fish reproductive performances requires a thorough evaluation of egg production, including egg number, egg size, egg viability as well as egg developmental potential through the monitoring of embryo survival after fertilization. While embryonic success relies, at least in part, on paternal contribution, some parameters are strictly related to egg characteristics, the main one being the viability of the egg when immerged into water during spawning. Our aim was i) to develop a simple and rapid system to capture images of the eggs combined with computerized processing of these images to perform a fully automatic individual characterization of trout eggs features (number, size and color) ii) to estimate unfertilized egg viability through the monitoring of the percentage of eggs that will not survive to water exposure. For this, unfertilized eggs (approximatively 400 eggs) were hydrated with water. After 24h, a picture of the egg sample was obtained using a dedicated shooting system consisting of light tablet and digital SLR camera (canon EOS 1000D, resolution: 10.1 M pixels) equipment. An image processing algorithm was then developed and coded as a VBA macro with Visilog 7.3 software (Thermo Scientific). This software allows the automatic detection and separation of the eggs in order to make fully automatic measurements for each egg (number, size, white egg percentage). The presence of white egg was interpreted as an indirect measure of egg integrity, the “whitening” being the result of water entry into the egg and more specifically through the vitelline membrane (Gray, 1932). These white eggs were therefore considered as non-viable, as a result of their lack of physical integrity. Fertilization assays were performed in parallel using a sample of the same egg batches. After incubation, hatching rate was calculated. A simple linear regression between white egg percentage after hydration and hatching rate measured individually on 105 egg batched shows a significant correlation between both parameters (Pearson coefficient =-0.557, p<0.001) in consistency with the fact that non-viable egg will not allow successful embryonic development. VisEgg is a convenient and reliable tool to obtain individuals measures on trout eggs. It can be used to assess not only egg size but also unfertilized egg viability before fertilization. This method is complementary of a full evaluation of developmental success that is performed on fertilized eggs to separate the origin of egg quality defects (non-viable eggs vs. viable eggs that do not allow developmental success).
Keywords: VisEGG- Image analysis – Phenotyping method – Rainbow trout- Egg viability
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 68 SESSION IV. OOGENESIS AND EGG QUALITY O28
TRANSCRIPTOMIC PROFILING REVEALS NOVEL BIOMARKERS OF EGG QUALITY IN DOMESTICATED PIKEPERCH, Sander lucioperca
Daniel Żarski1*, Joanna Nynca1, Aurelie Le-Cam2, Christophe Klopp3, Sławomir Ciesielski4 Jerome Montfort2, Pascal Fontaine5, Andrzej Ciereszko1, Julien Bobe2
1 Department of Gametes and Embryo Biology, Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, Poland 2 INRA, UR1037 Fish Physiology and Genomics, F-35000 Rennes, France 3 Sigenae, UR875, INRA, 31326 Castanet-Tolosan, France 4 Department of Environmental Biotechnology, University of Warmia and Mazury, Olsztyn, Poland 5 University of Lorraine, INRA, UR AFPA, F-54000 Nancy, France
Intensive production technology of pikeperch, Sander lucioperca, requires to grow broodstock indoor under artificial environmental conditions. This potentially alters the oogenesis process, when compared to wild-origin fish, and affects egg quality. Besides, low spawning time predictability requires the use of hormonal stimulation of ovulation which can have negative effect on egg quality. Ovulated egg maternally inherited RNA investigation is a powerful tool allowing identification and understanding of processes involved in creation of the egg developmental competences. However, this requires first to understand the causes of lowered egg quality. This study aimed at comparative transcriptomic analysis of high quality (HQ) eggs and the ones with fragmented lipid droplets (FLD) being already known as an indicator of increased incidence of deformed larvae and reduced hatching. We have sequenced 8 different tissues and designed a pikeperch-specific microarray probe set (8x60k, Agilent). The eggs for the study were obtained following controlled reproduction with the use of hormonal treatment to induce spawning. Following hand-stripping egg subsamples were snap-frozen in liquid nitrogen for RNA analysis. Other subsamples were evaluated under the binocular for oil droplet morphology and later were fertilized in vitro. For each egg batch embryonic survival rate (SR), hatching rate (HR) and deformity rate of hatched larvae (DR) were determined. From among 85 egg batches for further analysis n=8 characterized by HQ and n=8 represented FLD were taken. Eggs from HQ group were characterized by higher HR, SR and lower DR, when comparing to FLD (P<0.01). Transcriptomic analysis revealed 179 differentially expressed genes (DEGs) p<0.01, with at least fold change = 2) between HQ and FLD. All DEGs were downregulated in FLD group. Among DEGs, cathepsin B and ubiquitination process involved genes, have already been reported to be linked with egg quality. More than 100 DEGs are potential novel Teleosts egg quality biomarkers. STRING functional analysis revealed that DEGs were mostly related with cell cycle, cellular response to stress or regulation of apoptotic processes. GO enrichment analysis suggests that positive regulation of metabolic processes are the key processes conditioning embryonic developmental success. The data obtained in this study provides the first insight into the transcriptomic portrait of high quality eggs in pikeperch and reveals the repertoire of genes being potential species specific egg quality molecular markers.
Keywords: percids, gamete quality, fragmented lipid droplet, egg quality indicators
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 69 SESSION IV. OOGENESIS AND EGG QUALITY O28
Acknowledgements: Study was funded by project 2016/22/M/NZ9/00590 from National Science Centre, Poland.
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 70 SESSION IV. OOGENESIS AND EGG QUALITY O29
WHAT MAKES A GOOD EGG: NEW INSIGHTS FROM CRISPR/CAS9 GENOME EDITING IN FISH MODEL SPECIES
Caroline T. CHEUNG1, Thaovi. NGUYEN1, Stéphanie. GAY1, Amélie PATINOTE1, Emilie 1 1 1 1 CARDONA , Mariana ROZA DE ABREU , Violette THERMES , Julien BOBE *
1 INRA, UR1037 Fish Physiology and Genomics, F-35000 Rennes, France
Our current understanding of the mechanisms behind egg quality defects, including lack of fertilization and early embryonic failure, remains incomplete. In order to better understand the molecular processes that control egg quality, we have initiated a series of experiments to document the importance of various molecular factors (messenger RNAs and non-coding RNAs) for the production of a fully fertilizable and viable egg using a genome editing approach in model fish species. Experiments were fully approved by INRA institutional and CREEA ethics committees and conducted using zebrafish (Danio rerio) or medaka (Oryzias latipes), two model species with short generation times and daily spawning. Candidate genes for genome editing (i.e. knock- out) were selected due to their predominant expression in the ovary in fish and, in some cases, in vertebrates. This strategy was used for the following genes: nucleoplasmin 2a (npm2a), nucleoplasmin 2b (npm2b), micro RNA 202 (miR-202), forkhead box R1(foxr1, formerly known as foxn5), OTU deubiquitinase with linear linkage specificity a (otulina), and solute carrier family 29, member 1a (slc29a1a). Mutations (nucleotide insertions/deletions) in target genes were generated using CRISPR/Cas9. When possible, homozygote -/- females were generated and used for reproductive phenotyping. Whenever it was not possible to transmit the mutation to the next generation, because of heavily impaired reproductive success, phenotypes were assessed in eggs produced by F0 females after checking that corresponding target genes were sufficiently knocked out. In the case of otulina and slc29a1a, eggs originating from mutant zebrafish females exhibiting a reduced expression of target genes could not be fertilized. Similarly, mutant npm2a females produced eggs that were not cellularized, a phenotype resembling previously described unfertilized eggs (Cheung et al., 2018a). A similar phenotype was observed in miR-202 -/- mutant medaka females that also exhibited a dramatically reduced number of eggs per spawn and were therefore infertile or subfertile (Gay et al., 2018). In the case of foxr1, mutant female zebrafish produced eggs that could be fertilized but did not develop or developed abnormally and systematically died within 24h post fertilization (Cheung et al., 2018b). Finally, npm2b mutant zebrafish females produced eggs that could be fertilized but developed abnormally and systematically died within 24h post fertilization (Cheung et al., 2018a), a phenotype consistent with our prior observations showing a developmental arrest at mid-blastula transition when maternally-inherited npm2b mRNA translation was blocked using morpholino injection in the eggs. Here we reveal the importance of several genes (otulina, slc29a1a, npm2a, and miR-202) that are required for the production of fertilizable eggs. We also confirm the critical role of maternally-inherited npm2b for early embryonic development in consistency with the differential abundance of npm2b mRNA in eggs of varying quality in rainbow trout and report the key role played by foxr1 in determining the ability of the eggs to support early
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 71 SESSION IV. OOGENESIS AND EGG QUALITY O29 development, once fertilized. Further investigations are in progress to understand the mechanisms in which the above studied genes are involved.
References : Cheung, C.T., Pasquier, J., Bouleau, A., Nguyen, T., Chesnel, F., Guiguen, Y., Bobe, J., 2018a. Double maternal-effect: duplicated nucleoplasmin 2 genes, npm2a and npm2b, with essential but distinct functions are shared by fish and tetrapods. BMC Evol. Biol. 18, 167. https://doi.org/10.1186/s12862-018-1281-3 Cheung, C.T., Patinote, A., Guiguen, Y., Bobe, J., 2018b. foxr1 is a novel maternal-effect gene in fish that is required for early embryonic success. PeerJ 6, e5534. https://doi.org/10.7717/peerj.5534 Gay, S., Bugeon, J., Bouchareb, A., Henry, L., Delahaye, C., Legeai, F., Montfort, J., Le Cam, A., Siegel, A., Bobe, J., Thermes, V., 2018. MiR-202 controls female fecundity by regulating medaka oogenesis. PLoS Genet. 14, e1007593. https://doi.org/10.1371/journal.pgen.1007593
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 72 SESSION V. SPERM MOBILITY AND FERTILIZATION O30
GUIDANCE AND SELECTION DURING FERTILIZATION IN FRESH WATER FISH: THEORY AND PRACTICE
Sergii BORYSHPOLETS1*, Vitaliy KHOLODNYY1, Hermes GADELHA1,2 , Jacky COSSON1
1 University of South Bohemia in České Budějovice, Faculty of Fisheries and Protection of Waters, South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses, Zátiší 728/II, 389 25 Vodňany, Czech Republic 2 Department of Engineering Mathematics, University of Bristol, Bristol, UK
During the last decades the common belief that fertilization occurs randomly, via simple diffusion of a large number of spermatozoa, was replaced by the guidance hypothesis. This hypothesis is successful in explaining different aspects of reproduction such as the directed approach of spermatozoa towards egg and specific mechanisms for spermatozoa selection. It finds a confirmation in several species from invertebrates to mammals, in situations where sperm cells use various sensing mechanisms, including chemotaxis, rheotaxis, and thermotaxis, to gather physical or chemical cues to spot the egg. These mechanisms are highly sensitive to changes in the environment, thus driving a high degree of selectiveness among the total sperm population. To date, however, no clear-cut demonstration of guidance exists for fresh water fish species. Our recent experiments shed light to different guidance strategies that may be operating for different fresh water fish species. Externally fertilizing species, especially freshwater fish, reproduce in an environment that is very harsh for gametes. They are under pressure of various external factors (temperature, water flow, pH, osmolality, ion composition, viscosity, presence of ovarian fluid etc.), thus limiting lifetime of gametes to only several minutes or less. The gametes have variable responses to these parameters, ranging from the activation of egg or initiation of sperm motility, to changes in speed, duration and direction of motion, contributing to appearance of the wide spectrum of reproduction strategies. Thus, the success of fertilization in these species is directly related to the ability of their spermatozoa to reach the egg in an open environment but during a very short period of time. So the existence of specific mechanisms for guiding sperm to find the egg would be critical under these conditions. Here we show that factors, such as part of ovarian fluid or substances released directly by the egg, significantly affect the behavior of male gametes by adjusting their motility. We hypothesize that such coordination may influence the fertilization success in open water. However, the specific mechanisms supporting the selection by externally fertilizing in freshwater females are still unclear. Our observations suggest that guidance during fertilization is a critical phenomenon during fresh water fish reproduction. SESSION V. Sperm mobility and fertilization Keywords: Chemotaxis, sperm motility, fertilization, ovarian flued.
Acknowledgements: The study was financially supported by the Ministry of Education, Youth and Sports of the Czech Republic - projects „CENAKVA“ (LM2018099), by project Biodiversity (CZ.02.1.01./0.0/0.0/16_025/0007370) and by the Grant Agency of the University of South Bohemia in Ceske Budejovice (125/2016/Z) and by the Czech Science Foundation (18- 12465Y).
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 73 SESSION V. SPERM MOBILITY AND FERTILIZATION O31
FISH SPERM COMPETITION IN HATCHERIES AND BETWEEN WILD AND HATCHERY ORIGIN FISH IN NATURE
José BEIRÃO1*, Torvald B. Egeland1, Craig F. Purchase2, Jarle T. Nordeide1
1 Faculty of Biosciences and Aquaculture, Nord University, NO – 8049 Bodø, Norway 2 Biology Department, Memorial University of Newfoundland, St. John's, NL, A1B 3X9, Canada
Males compete pre- and post-mating to fertilize the maximum number of eggs. In polyandry, sperm competition occurs when sperm from two or more males compete to fertilize eggs from a female. In this work we review how sperm competition from hatchery origin fish can cause loss of genetic variability in fish populations kept in captivity and in wild populations. In fish hatchery practices, sperm competition occurs in mass spawners that release gametes in tanks, and in artificial fertilizations when pooled semen is used. In mass spawnings sperm competition is difficult to tease apart from pre-mating competition and other post-mating selective mechanisms, whereas, studies focused on the use of pooled semen in different fish species have shown a clear relationship between sperm motility parameters and precedence in fertilization. In both situations, sperm competition will result in a loss of genetic variability that accumulates over generations, but hatchery protocols can be adjusted to mitigate it. Another source of concern regarding sperm competition for hatchery produced fish is the spatial and temporal overlap in spawning with wild individuals, either via aquaculture escapees or purposeful stocking programs. This may result in sperm competition between hatchery origin and wild males and impact natural populations. Our review suggests that in order to give every adult selected as broodstock an equal opportunity to produce offspring in captivity, mass spawning and the use of pooled semen should be limited.
Keywords: polyandry, aquaculture, artificial fertilization, stock enhancement
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 74 SESSION V. SPERM MOBILITY AND FERTILIZATION O32
EGG-SPERM INTERACTION IN STURGEON: ROLE OF OVARIAN FLUID
Vitaliy KHOLODNYY1*, Borys DZYUBA1; Hermes GADÊLHA1,2, Jacky COSSON1, Sergii BORYSHPOLETS1
1 University of South Bohemia in České Budějovice, Faculty of Fisheries and Protection of Waters, South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses, Zátiší 728/II, 389 25 Vodňany, Czech Republic; e-mail: [email protected] 2 Department of Engineering Mathematics, University of Bristol, Bristol, BS8 1UB, UK
Fertilization of fresh water fish occurs in an environment which affects negatively the lifespan of the gametes, therefore the male gametes should reach their counterpart, the female gamete, as soon as possible because spermatozoa motility becomes affected within minutes or less due to osmotic shock. Existence of mechanisms controlling the encounter of gametes would be highly expedient in these conditions. By analogy with other groups of species where guidance was demonstrated it is likely that that this control may be performed by ovarian fluid or substances released by egg. The research was done in sturgeons (sterlet) which exhibit a specific sperm characteristics comparing to other externally fertilizing fresh water fish (e. g. presence of acrosome and several micropyles in the egg), i. e. the strategy of reproduction and gametes interaction may differ from other freshwater fish species. The aim was to study the effect of ovarian fluid and egg-released substances on sterlet spermatozoa behavior. The latter was assessed in various conditions (presence of ovarian fluid, various activation media, in particular “egg water”, i. e. the media where eggs were incubated during certain time, etc.), as well as fertilization test was performed. It was found that presence of particular concentration of ovarian fluid has inhibiting effect on spermatozoa motility initiation. For the activated spermatozoa no effect of ovarian fluid presence in the medium on the spermatozoa trajectories was found. The same absence of effect was found in case of using different osmolarity of activating medium, or egg water. Test of chemotactic response (using a microcapillary injection of fluids into suspension of motile spermatozoa) also showed no effect of ovarian fluid on spermatozoa behaviour, at the same time the effect of egg water was evident. Moreover, the latter depended on the presence of Ca2+ ions in the activating medium. The results of fertilization test showed that presence of ovarian fluid prevented the eggs from losing the fertilizing ability due to the contact with water, as well as promoted the spermatozoa to fertilize the eggs during longer period of time. We conclude that ovarian fluid may serve as a protector for the eggs and spermatozoa against effect of fresh water during fertilization, but do not exhibit chemotactic effect on the male gametes in our experimental conditions. At the same time, the attraction of spermatozoa may be provided by some substances released from the eggs during their contact with fresh water. Thus, the combined physico-chemical action of “female factors” may provide the guidance of sterlet male gametes during fertilization.
Keywords: sperm motility, fertilization, ovarian fluid, egg water, chemotaxis
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 75 SESSION V. SPERM MOBILITY AND FERTILIZATION O32
Acknowledgements: The study was financially supported by the Ministry of Education, Youth and Sports of the Czech Republic - projects „CENAKVA“ (LM2018099), by project Biodiversity (CZ.02.1.01./0.0/0.0/16_025/0007370) and by the Grant Agency of the University of South Bohemia in Ceske Budejovice (125/2016/Z) and by the Czech Science Foundation (18- 12465Y)
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 76 SESSION V. SPERM MOBILITY AND FERTILIZATION O33
SOCIAL STATUS IMPACTS ON SPERM COMPETITION IN FISH
Elvira FATSINI1*, Patrícia DIOGO1, Gil MARTINS1, Catarina ANJOS1, Paulo J. GAVAIA1,2 and Elsa CABRITA1
1 Centre of Marine Sciences (CCMAR), University of Algarve, Campus de Gambelas, 8005-139 Faro (Portugal) 2 Department of Biomedical Sciences and Medicine, University of Algarve, Campus de Gambelas, 8005-139 Faro (Portugal)
Sperm function and quality are essential elements of male reproductive role and consequently fitness. Dominance, which is a critical point to determine social status, and physical competition affect sperm traits in a wide range of taxa. Dominant males may avoid subordinate mating with females and monopolise spawning sites preferred by females. Moreover, reproductive strategy is very important to define gamete quality features, particularly in those species where courtship behaviour is implicated in spawning. This may be the case of Senegalese sole (S. senegalensis) and greater amberjack (S. dumerili), two important commercial species in the South of Europe. Hence, there are males that prefer to invest more energy in being dominant and others that select to spend energy in other physiological characteristics to obtain an opportunity to share their genetic material. Usually, the cost of this rivalry may promote diverse physiological conditions in males that may affect fertilization success. In this view, the effects of dyadic zebrafish D. rerio (used as model fish species) male competition on sperm quality was assessed using CASA to determine motility and flow cytometry to evaluate cell viability. For this purpose, 21 pairs of males were randomly established in high standard competition for 5 consecutive days, where behaviours were recorded every morning during 5 min, evaluating territorial behaviour. On day 6, one mature female was introduced in the tank to stimulate direct competition in relation to reproductive stimulus. After one-day cohabiting, the two males and the female were moved to a spawning cage to allow them to reproduce naturally. The next day, sperm was collected from all males to evaluate its quality. A hierarchical cluster based on the representative behavioural variables categorised by PCA was run to classify the animals in subordinates and dominants. Total motile cells, TM (%), and curvilinear velocity, VCL (m/s) were the two most descriptive variables of the sperm quality variables. Hence, TM was significantly higher in subordinate (55.9 ± 19.8 %) fish than dominant (47.4 ± 16.7 %) at 10 sec post-activation on those groups that did not spawn. However, no differences were observed in terms of sperm velocity and viability between subordinate and dominant males. No significant differences were observed in TM, VCL and viability in sperm from males that reproduced. However, these findings suggest that being dominant may have positive effects in terms of obtaining better surrounding conditions, such as territory, feeding places, and possible reproductive success, but the high cost of keeping this social status may implicate a general adverse cost in sperm traits.
Keywords: Gamete quality; dominance behaviour; sperm fitness; commercial fish species
Acknowledgements: This study received Portuguese national funds from FCT through project
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 77 SESSION V. SPERM MOBILITY AND FERTILIZATION O33
UID/Multi/04326/2019. E. Fatsini was supported by a postdoctoral Fellowship by MAR2020- 16-02-01-FMP-0059/MAR-02.01.01-FEAMP-0064. P. Diogo was supported by FCT through the doctoral grant SFRH/BD/97466/2019.
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 78 SESSION V. SPERM MOBILITY AND FERTILIZATION O34
STEPS TOWARDS UNDERSTANDING MOTILITY OF MORPHOLOGICALLY COMPLEX SPERM IN CARTILAGINOUS FISHES
Borys DZYUBA1*, Viktoriya DZYUBA1, Alexandre NINHAUS-SILVEIRA2, Martin KAHANEC1, Rosicleire VERISSIMO-SILVEIRA2, Vitaliy KHOLODNYY1, Marek RODINA1, Sergii BORYSHPOLETS1
1 University of South Bohemia in České Budějovice, Faculty of Fisheries and Protection of Waters, South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses, Vodňany, Czech Republic 2 São Paulo State University, Faculty of Engineering, Department of Biology and Zootechny, Ilha Solteira, SP, Brazil
Cartilaginous fishes diverged from other vertebrate classes more than 400 million years ago. Interestingly, that extant cartilaginous fishes keep very specific structural features of their spermatozoa, which are absent in actinopterygian species, but can be found in representatives of other, later diverged classes of vertebrates. These features are: quite big size (several hundred µm of total length), very elongated (compared to the length of flagellum) screw-shape head, and flagellum possessing longitudinal columns. All these features could be considered as plesiomorphic for vertebrates with internal fertilization. Considering that restricted data related to motility description of these morphologically complex spermatozoa is currently available, we focused our study on description of basic characteristics of motile spermatozoa of ocellate river stingray Potamotrygon motoro – the representative of freshwater cartilaginous fishes. We combined the data obtained from scanning and transmission electron microscopy and light video microscopy of low frame rate (25 fps) to assess spermatozoa kinetic parameters by CASA and high speed one (2000 fps) for the description of sperm flagella movement. To characterize spermatozoa motility, we used swimming media of natural origin – seminal fluid (SF) and uterine fluid (UF) and artificial fluids mimicking ionic composition of SF and UF. Electron microscopy allowed to conclude that head, flagellum and flagellar internal cytoskeletal structure (9+2 axoneme together with two longitudinal columns) have spiral configuration. Video microscopy analysis showed that motility of stingray spermatozoa is characterized by low velocity (around 30 µm/s for VCL; 20 µm/s for VAP and 15 µm/s for VSL) associated with whole cell rotation along longitudinal axis (range 14–20 Hz) and helical shape of flagellum – screwing inside of the medium. Progressive motility was observed in case of SF or UF, while in artificial media most of cells rotated without effective straightforward propagation and active flagellum beating was associated with increased head rotation rate (25–35 Hz). Analysis of the data indicates that despite taxonomical distance specific features of spermatozoon morphology and motility mode of propagation are the closest to ones in different vertebrate groups, other than fish.
Keywords: Potamotrygon motoro, CASA, flagellum beating, spermatozoon structure
Acknowledgements: The study was supported by the Czech Science Foundation (No. 16- 03754S), by the Ministry of Education, Youth and Sports of the Czech Republic (“CENAKVA”,
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 79 SESSION V. SPERM MOBILITY AND FERTILIZATION O34
LM2018099), by project Biodiversity (CZ.02.1.01./0.0/0.0/16_025/0007370) and by the Grant Agency of the University of South Bohemia in Ceske Budejovice (125/2016/Z)
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 80
POSTER SESSIONS
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 81 SESSION I. SPERMATOGENESIS AND SPERM QUALITY P1
ANALYSİS OF THE SPERM QUALITY PARAMETERS OF KOI FISH IN THE AQUAPONICS SYSTEM
Devrim MEMİŞ1, Merve TINKIR1*, Momin MOMIN1, Gökhan TUNÇELLİ1
1 Istanbul University, Faculty of Aquatic Sciences, Ordu Str. No:8, Laleli, Istanbul, Turkey
In this study, Koi fish (Cyprinus carpio sp.) was reared in the aquaponics system with Cress (Lepidium sativum). The sperm quality parameters of mature koi fish were analyzed in the Sapanca Inland Water Fish Production, Research and Applied Station of Istanbul University, Sakarya district of Turkey. Total 43 fish were kept in the aquaponics system where only 6 males were found matured for the first time. Sperm was taken from 6 male koi fish (1+ years) with 17.86±0.53 cm mean length and 95.03±12.53 g mean weight, fish were kept for 135 days at the mean temperature of 18.56±2.38 °C, dissolved oxygen of 6.24±0.77 mg/l and 7.13±0.09 of pH in this aquaponics system. And also, PO4, NO2, NO3, Total phosphate and Fe of the system’s water was measured as 2.3±0.88 (mg/l), 0.15±0.11 (mg/l), 0.97±0.68 (mg/l) and 0.25±0.08 (mg/l), respectively. When white sperm was coming out by gentle massage then Carp pituitary hormone at a rate of 4 mg per kg was applied on the dorso-lateral side of the male individual 12 h prior the stripping. Total motility, progressive motility, VCL, VSL, VAP, STR and LIN were analyzed and the sperm activation was done by the aquaponics system’s water. Sperm quality parameters were analyzed using the CASA between 13th to 14th February in 2019. As a result of the study, the mean total motility and progressive motility were 71% and 25%, respectively. Among kinematic parameters, VAP, VCL, VSL, STR and LIN were 50.23 µm/sec, 39.58 µm/sec, 55.63 µm/sec, 78.68% and 72.36%, respectively. However, sperm quality parameters were analyzed to check if the sperm of the koi fish reared in the aquaponics system is similar with the koi fish reared with the normal culture system.
Keywords: Lepidium sativum, sperm motility, kinematic parameters, Cyprinus carpio.
SESSION I. Spermatogenesis and sperm quality
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 82 SESSION I. SPERMATOGENESIS AND SPERM QUALITY P2
THE INFLUENCE OF WATER TEMPERATURE INCREASE ON THE DURATION OF SPERMATOGENESIS IN A NEOTROPICAL FISH, astyanax altiparanae (CHARACIDAE, CHARACIFORMES)
Rosicleire VERÍSSIMO-SILVEIRA1*, Patricia POSTINGEL-QUIRINO12, Maira DA SILVA RODRIGUES3, Elis Marina DA SILVA CABRAL1, Diógenes Henrique DE SIQUEIRA-SILVA4, Ricardo HIDEO MORI1, Alexandre NINHAUS-SILVEIRA1
1 Neotropical Ichthyology Laboratory (LINEO), Department of Biology and Zootechny, São Paulo State University (UNESP) – Brasil Avenue, 56, 15085-000, Ilha Solteira, São Paulo, Brazil 2 Institute of Biosciences of Botucatu (IBB), São Paulo State University (UNESP) 3 Reproductive and Molecular Biology Group, Department of Morphology, IBB, UNESP 4 Group of Studies on the Reproduction of Amazon fish (GERPA/LANEC), PPG in Biodiversity and Biotechnology (BIONORTE), University of South and Southern of Pará (Unifesspa), Marabá, Pará, Brazil.
In view of the established climate change scenery and the consequently changes in global temperature, it is essential to study its effects on animal spermatogenesis. Therefore, the objective of this study was to characterize, morphologically and stereologically, the cell types found in spermatogenesis of yellow-tetra Astyanax altiparanae, in addition to determining the testicular phases and verifying the spermatogenesis duration in different temperatures. For this purpose, ninety-six male and adult specimens of A. altiparanae were kept in a closed circulation system under water temperature stabilized at 27ºC (comfort temperature) and 32ºC. Subsequently, the specimens received pulses of BrdU (bromodeoxyuridine) at a concentration of 100 mg/kg/day for 2 consecutive days, and the samples were collected daily for a period of 15 days. Their testes were removed, fixed, processed in historesin and sectioned in 3 μm, submitted to Hematoxylin/Eosin and to BrdU immunodetection. Partial results of the optimum temperature allowed to classify A. altiparanae in spermatogonia Aind*, Aind, Adiff and type B spermatogonia, spermatocytes, spermatids and spermatozoa. The spermatogenesis duration was determined in approximately six days for animals under the temperature of 27 °C and one day for animals at 32 °C. The elevated temperature was also responsible for increasing cell proliferation in spermatids, spermatozoa, and cell death (cells pyknotic).
Keywords: Yellow-Tetra, Reproduction, Characiformes
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 83 SESSION I. SPERMATOGENESIS AND SPERM QUALITY P3
SPERMATOGENESIS IN MATURE MALE SCALLOPED HAMMERHEADS (Sphyrna lewini) FROM KWA-ZULU NATAL, SOUTH AFRICA: A FIRST PRELIMINARY DESCRIPTION
Helene COETZEE 1 and Ina WAGENAAR1*
1Department of Zoology, University of Johannesburg, South Africa, 2006
Sharks are abundant, widely spread and play role a vital in marine ecosystems, but not extensively studied in terms of the biology and reproduction. Three of the nine species of the family Sphyrnidae (hammerheads and bonnet heads) are found in South African oceans. The aim of this study was to describe the germ cell development in the testes of mature male Scalloped Hammerheads (Sphyrna lewini). Three mature male S. lewini were caught at Zinkwazi, Kwa-Zulu Natal, South Africa. Total length, total weight, and testes measurements were taken. A standard necropsy was performed on each shark and the testes were collected for histological processing. The histological assessment revealed that the testes of this species of sharks consist of seminiferous tubules which form part of a larger lobular structure with germ cells in different stages of development. Germ cell development takes place in the seminiferous tubules, with initial development from the basal membrane on the periphery of the seminiferous tubule. The spermatogonia originate from the basal membrane of the seminiferous tubule through the process of mitosis. Spermatogonia then undergo meiosis to form primary and secondary spermatocytes. Secondary spermatocytes then undergo haploid division to form spermatids. Immature spermatozoa develop from the spermatids which then move to the periphery of the basal membrane. Mature spermatozoa clump together to from a distinctive spiral structure in the eosinophilic matrix to mature near the basement membrane of the seminiferous tubule. In conclusion the histological methods proved successful to further describe spermatogenesis and reproduction of mature male Scalloped Hammerheads. The various stages of development identified are similar to a recent study done on the Black-spotted Smooth Hound shark (GraIan and Lackovi, 2016).
Keywords: Histology, Testes, Sharks, Reproduction
Acknowledgements : This research is supported in part by the National Research Foundation of South Africa (Unique Grant Number 119318) for rated researchers and the Global Excellence and Stature 4IR Scholarship to H. Coetzee. Ms Kristina Naidoo and Dr G Cliff from the Kwa-Zulu Natal Sharks Board, South Africa are acknowledged for their support and assistance with the sampling.
References : GraIan R and Lackovi G. 2016. Histological and Morphological Aspects of Reproduction in Male Blackspotted Smooth-Hound Mustelus punctulatus in the Adriatic Sea (Eastern Mediterranean Sea). Journal of Marine Biology. 2016: 1-6. http://dx.doi.org/10.1155/2016/3438678
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 84 SESSION I. SPERMATOGENESIS AND SPERM QUALITY P4
CHARACTERIZATION OF MALE STERILITY IN HYBRIDS OF cobitis GENUS
Tomáš TICHOPÁD1*, Jan ROSLEIN2,3, Oldřich BARTOŠ2,4, Roman FRANĚK1, Alena ZIKMUNDOVÁ2,3, Martin PŠENIČKA1, Karel JANKO2,3
1 Laboratory of Germ Cells, Faculty of Fisheries and Protection of Waters, University of South Bohemia in České Budějovice, Vodňany, Czech Republic 2 Laboratory of Fish Genetics, Institute of Animal Physiology and Genetics, The Czech Academy of Sciences, Liběchov, Czech Republic 3 Department of Biology and Ecology, Faculty of Science, University of Ostrava, Ostrava, Czech Republic, 4Department of Zoology, Faculty of Science, Charles University, Praha, Czech Republic
Polyploidization and hybridization played a crucial role in the formation of new species but drastic changes, such as whole genome duplication or disruption of allelic crosstalk, are often linked with reduced fertility of allodiploids and allopolyploids. Asexual reproduction appears as another correlate of genome merging and our recent data demonstrated that tendency of hybrids to reproduce asexuality evolves hand in hand with hybrid sterility as a side-effect of differentiation of parental species. This is a case of fish from genus Cobitis, where hybridization between two sexually reproducing yet substantially diverged species gives rise to fertile gynogenetically reproducing females and sterile males with meiotic arrest and absence of functional spermatozoa. Interestingly, recent RNA-seq analysis demonstrated that distortion of female hybrid reproductive mode towards asexuality is not associated with any massive alterations of gene expression. In this work, we present gene expression profiles of male gonadal as well as somatic tissues. We compared diploid and triploid hybrids with both parental species and showed that, in contrast to females, hybridization leads to massive dysgenesis in males in gonadal tissue during the reproductive season, with almost 1/3 of all analyzed genes being differentially expressed in hybrids compared to their parents. Our data demonstrate that hybrid sterility from the gene expression perspective, albeit evolving simultaneously with asexuality, is associated with massive gene deregulation. In contrast, no major deregulations, neither as a cause nor as consequence, are connected with the switch from sexuality to asexuality.
Keywords: polyploidy, asexuality, hybridization, spermatogenesis, transcriptomics
Acknowledgements: The study was financially supported by the Ministry of Education, Youth and Sports of the Czech Republic - projects „CENAKVA“ (No. CZ.1.05/2.1.00/01.0024), “CENAKVA II“ (No. LO1205 under the NPU I program), project Biodiversity (CZ.02.1.01/0.0/0.0/16_025/0007370), by the Czech Science Foundation (project No. 17- 09807S) and the Grant Agency of the University of South Bohemia in Ceske Budejovice (projects No. 125/2016/Z).
References: 1) Characterization of male sterility in hybrids of Cobitis genus 2) Bartoš, O., Röslein, J., Kotusz, J., Paces, J., Pekárik, L., Petrtýl, M., Halačka, K., Štefková Kašparová, E., Mendel, J., Boroń, A., et al. (2019). The legacy of sexual ancestors in
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 85 SESSION I. SPERMATOGENESIS AND SPERM QUALITY P4 phenotypic variability, gene expression and homoeolog regulation of asexual hybrids and polyploids. Mol. Biol. Evol.
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 86 SESSION I. SPERMATOGENESIS AND SPERM QUALITY P5
BIOLOGICAL CHARACTERISTICS AND HISTOLOGICAL ANALYSIS OF THE TESTIS IN THE SICHEL POPULATION (Pelecus cultratus, LINNAEUS, 1758) AT LAKE BALATON
Levente VÁRKONYI1*, Zoltán Bokor1, Árpád FERINCZ1, Ádám STASZNY1, Jozsef MOLNÁR1, Zita BIRKÓ-SULYOK1, Vera JUHÁSZ1, Flóra KEREKES1, Borbála NAGY1, Ferenc FODOR2, Zsolt SZÁRI2, Béla URBÁNYI1, Gergely BERNÁTH1
1Department of Aquaculture, Szent István University, 1. u. Páter Károly, H-2100 Gödöllő, Hungary/Tópart u. 5309/8 Hrsz, H-2484 Agárd (Gárdony), Hungary 2Balaton Fish Management Non-Profit Ltd, Horgony u. 1., H-8600 Siófok, Hungary
Sichel (Pelecus cultratus) is an endemic cyprinid fish at Lake Balaton. In the last decades, its catch decreased notably (decreasing of natural food, increasing of the number if competitor species and angling activity). The propagation of the species has not been developed (stress sensitive fish species) yet. The aim of our research was to measure the ichtyometric parameters of the population (standard length, average body weight, sex of the individuals) located in the Lake Balaton. Our study focused also on the determination of the sperm production period using histological analysis in sichel males. Samples were collected monthly using a benthic gill net (36x1,5m / 12 panels / EN 14757) from April to September. Basic ichtyometric parameters were measured in both males and females. Samples were taken from the male gonads where hematoxylin and eosin painting was used. The amounts of four different cell types (spermatogonia, spermatocyte, spermatid, and spermatozoa) were counted in each testis section. In our experiment 127 individuals (male: N=61, female: N=66) were caught. In general, a notable difference in average body weight was recorded in males (146±38 g) and females (168±53 g) where standard length was similar in both sexes (males: 25±2 cm and females: 26±3 cm). In April, a low amount of spermatogonia (40±42), spermatid (53± 50) and an elevated number of spermatocyte (105±68) were recorded. During the expected spawning season (May-June) a high amount of spermatozoa (in May: 92±97, in June: 107±68) was found. Contrarily, a reduced number of spermatocyte (in May 16±27, in June: 2±3) and spermatid (in May: 70±41, in June: 5±8) were observed in all samples. In August, a small number of spermatozoa (7±12) and a large amount of spermatogonia (194±50) were recorded. Similarly, an elevated number of spermatogonia (226±37) and a reduced amount of spermatocyte (4±7) were observed in September. As a conclusion, a significant correlation was observed between the average body weight and the sex of the individuals. In contrast, the standard length did not show a similar tendency. According to the histological analysis, the spermiation of sichel starts already in May and prolonged until the end of summer. The results obtained in September (elevated amount of spermatogonia) indicate that individuals have begun the preparation for the next spawning season.
Keywords: sichel, ichtyometric parameters, spawning season, spermatogenesis, Lake Balaton
Acknowledgements: The work was supported by the project GINOP 2.3.2–15–2016–00004: "Establishing the sustainable angling-aimed management of Lake Balaton“. The publication is supported by the EFOP-3.6.3-VEKOP-16-2017-00008 project. The project is co-financed by
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 87 SESSION I. SPERMATOGENESIS AND SPERM QUALITY P5 the European Union and the European Social Fund and by the ÚNKP-18-3 New National Excellence Program of the Ministry of Human Capacities Fellowships.
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 88 SESSION I. SPERMATOGENESIS AND SPERM QUALITY P6
STRESS REACTION AND SPERM QUALITY IN HORMONALLY TREATED STERLET (Acipenser ruthenus) FOR SPERMIATION
Forough CHELENGARI1, Sayyed Mohammad Hadi ALAVI1*, Azadeh HATEF2, Josef VELÍŠEK3, Marek RODINA3, Otomar LINHART3
1 School of Biology, College of Science, University of Tehran, P. O. Box: 14155-6455, Tehran, Iran. 2 Toxicology Center, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5B3, Canada 3 South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses, Faculty of Fisheries and Protection of Waters, University of South Bohemia in České Budějovice, Vodňany 389 25, Czech Republic.
Fish species that do not reproduce in captivity need hormonal treatment to induce gamete maturation. However, cortisol level rise after hormonal treatment indicating a stress reflection. In males, decrease in sperm quality, sperm production and number of spermiating males have been reported under stress condition (Bayunova et al., 2002; Castranova et al., 2005). Thus, it might be possible that induction of spermiation also affect sperm quality. In the present study, sperm quality was investigated in sterlet treated with 4 mg/kg b.w. carp pituitary extract (CPE) or 3 pellets/kg b.w. ovopel. Ovopel contains metoclopramide which is a dopamine receptor antagonist. Each group composed of 8 mature males, and one group was injected with physiological saline (PS). No male was spermiated in PS treated fish, but all fish treated with CPE or ovopel were spermiated. Cortisol levels were increased in PS and CPE treated fish, however remained unchanged in ovopel treated fish. Sperm mass, sperm density, and the percentage of motile spermatozoa after activation were not differed between CPE and ovopel treated fish, however spermatozoa velocity was higher in ovopel treated fish than CPE treated fish. In CPE treated fish, there were negative correlations between cortisol levels and sperm mass, sperm density or spermatozoa motility (P<0.05). However, in ovopel treated fish, only positive correlation was observed between cortisol and spermatozoa velocity (P<0.05), and there were no correlations between cortisol levels and other parameters (P>0.05). These suggest that application of ovopel is better than CPE in sturgeon aquaculture, probably due to inhibition of dopamine that interfere with hormonal regulation of spermiation. When data of CPE and ovopel treated fish were pooled, no correlations were observed between cortisol and sperm quality parameters. Overall, stress reflection and sperm quality differ by the type of hormones used for spermiation induction.
Keywords: Carp pituitary; Cortisol; Ovopel; Sperm motility; Sperm production.
Acknowledgements: CENAKVA: LM2018099, CZ.1.05/2.1.00/01.0024, LO1205 under the NPU I program; Biodiversity CZ.02.1.01./0.0/0.0/16_025/0007370; University of Tehran
References: Bayunova, L., Barannikova, I., et al., 2002 Sturgeon stress reactions in aquaculture. J. Appl. Ichthyol., 18:397–404
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 89 SESSION I. SPERMATOGENESIS AND SPERM QUALITY P6
Castranova, D.A., King W.V., et al., 2005. The effects of stress on androgen production, spermiation response and sperm quality in high and low cortisol responsive domesticated male striped bass. Aquaculture 246:413–422.
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 90 SESSION I. SPERMATOGENESIS AND SPERM QUALITY P7
A SYSTEMATIC REVIEW OF CROSSTALK BETWEEN THYROID HORMONES AND FERTILITY IN MALE FISH
Sara ALIJANPOUR1, Sayyed Mohammad Hadi ALAVI1*
1 School of Biology, College of Science, University of Tehran, P. O. Box: 14155-6455, Tehran, Iran.
In all vertebrates, thyroid hormones (THs) regulate metabolism, and are essential for development and metabolic-dependent biological systems including reproduction. It has been reported that THs influence hormonal functions of hypothalamus-pituitary-gonad axis (HPG) via THs receptors. However, physiological interactions between THs and reproductive hormones shows variations among vertebrates. Moreover, in fishes, THs effects on HPG vary among species, and thus it is unclear how THs regulate reproduction. Endocrine regulation of THs synthesis differs between fish and mammals. In fish, thyrotropin-releasing hormone is absent, and corticotropin-releasing hormone released from hypothalamus stimulates pituitary to synthesis and release thyroid-stimulating hormone that trigger thyroxine (T4) synthesis in the thyroid gland. Conversion of T4 into triiodothyronine (T3) is catalyzed by type 1 and type 2 deiodinases. In fish, conversion of T4 to T3 occurs mainly in peripheral tissue (including liver, brain, kidney, gill) rather than in the thyroid itself. However, similar to other vertebrates, availability of free iodide and T4 are limiting factors to produce T4 and T3, respectively. Moreover, the thyroid follicles are not encapsulated in an organ in fish, and are dispersed among the afferent branchial arterioles of the ventral region of pharynx. It has been reported that inhibition of T3 synthesis and deficiency of deiodinases influence sex differentiation, puberty, and spermatogenesis. The effects are highly dependent on developmental stage in which the fish are treated with THs or chemicals that cause hypo- or hyperthyroidism (Matta et al., 2002; Sharma and Patiño, 2013; Houbrechts et al., 2019). Available information demonstrates that these effects are associated with changes in syntheses of gonadotropin-releasing hormone, gonadotropins and sex steroids. A recent study reports that fertilization is reduced when male and female zebrafish have deficiency of deiodinases (Houbrechts et al., 2019). Similarly, in human, hypothyroidism in men results in decrease in sperm density and motility, and cause abnormalities to sperm morphology (Nikoobakht et al., 2012). Thus, it is possible that dysfunction of hormonal functions of thyroid gland results in infertility via modulation of HPG. The present study is a systematic review of crosstalk between THs and endocrine regulation of reproduction in male fish with emphasize of spermatogenesis and fertility, and notes future perspectives that await elucidation.
References Nikoobakht, M.R., et al., 2012. The Role of Hypothyroidism in Male Infertility and Erectile Dysfunction. Urology J., 9:405-409. Houbrechts, A.M., Van Houcke, J., Darras, V.M., 2019.Disruption of deiodinase type 2 in zebrafish disturbs male and female reproduction. J. Endocrinol., 241;111-123.
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 91 SESSION I. SPERMATOGENESIS AND SPERM QUALITY P7
Sharma, P., Patiño R., 2013. Regulation of gonadal sex ratios and pubertal development by the thyroid endocrine system in zebrafish (Danio rerio). Gen. Comp. Endocrinol., 184:111- 119.
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 92 SESSION I. SPERMATOGENESIS AND SPERM QUALITY P8
SPERM HANDLING IN AQUATIC ANIMALS FOR ARTIFICIAL REPRODUCTION
José BEIRÃO1*, Myrina BOULAIS2, Victor GALLEGO3, Justine K. O’BRIEN4, Silvio PEIXOTO5, Todd R. ROBECK6, Elsa CABRITA7
1 Faculty of Biosciences and Aquaculture, Nord University, NO – 8049 Bodø, Norway 2 University of Brest, CNRS, IRD, Ifremer, LEMAR, rue Dumont d’Urville, F-29280, Plouzané, France 3 Grupo de Acuicultura y Biodiversidad, Instituto de Ciencia y Tecnología Animal, Universitat Politècnica de València, Valencia, Spain 4 Taronga Institute of Science and Learning, Taronga Conservation Society, Bradleys Head Rd, Mosman NSW, 2088, Australia 5 Departamento de Pesca e Aquicultura, Universidade Federal Rural de Pernambuco (UFRPE), Recife, Brazil 6 SeaWorld Species Preservation Lab, SeaWorld Parks and Entertainment, 2595 Ingraham Road, San Diego, CA, 92019, USA 7 CCMAR, University of Algarve, Campus of Gambelas, 8005-139 Faro, Portugal
Artificial reproduction involves collection and handling of gametes in a way that secures their quality and maximizes the fertilization outcome. In addition to initial sperm quality, numerous steps can affect the final result of fertilization, from the sperm collection process until gamete mixing (or co-incubation) when the spermatozoon enters or fuses with the oocyte. In this review, we summarize the whole process of sperm handling, from collection until fertilization for fish, penaeid shrimp, bivalve mollusks and marine mammals. To obtain sperm from captive animals, techniques vary widely across taxa, and include stripping by abdominal massage or testis surgical removal in fish, spermatophore collection in penaeid shrimps, gonadal scarification or temperature shock in bivalve mollusks, and voluntary collection via positive reinforcement in mammals. In most cases, special care is needed to avoid contamination by mucus, seawater, urine, or feces that can either activate sperm motility and/or decrease its quality. In this work, we also review techniques and extender solutions used for refrigerated storage of sperm across the aforementioned taxa. Finally, we give an overview of the different protocols for in vivo and in vitro fertilization including activation of sperm motility and methods for gamete co-incubation. The present study provides valuable information regarding breeder management either for animal production or species conservation.
Keywords: sperm extraction, aquaculture, spermatozoa:oocyte ratio, in vitro fertilization
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 93 SESSION I. SPERMATOGENESIS AND SPERM QUALITY P9
DIETARY AMINO ACIDS IMPACT SPERM PERFORMANCE TRAITS FOR A CATADROMOUS FISH, Anguilla anguilla REARED IN CAPTIVITY
Ian A.E. BUTTS1, Guðrún S. HILMARSDÓTTIR2,3, Vahid ZADMAJID4, Victor GALLEGO5, Josianne G. STØTTRUP3, Charlotte JACOBSEN2, Maria KRÜGER-JOHNSEN3, Sebastian POLITIS3, Juan F. ASTURIANO5*, Jonna TOMKIEWICZ3
1 School of Fisheries, Aquaculture, and Aquatic Sciences, Auburn University, Auburn, AL, USA 2 National Food Institute, Technical University of Denmark, Lyngby, Denmark 3 National Institute of Aquatic Resources, Technical University of Denmark, Lyngby, Denmark 4 Department of Fisheries Science, University of Kurdistan, Sanandaj, Iran 5 Grupo de Acuicultura y Biodiversidad. Instituto de Ciencia y Tecnología Animal. Universitat Politècnica de València, Valencia, Spain.
Little is known about the role of dietary amino acids on male reproductive performance and gamete quality in fishes. Thus, the objective of this study was to investigate how “enhanced” feeds (EH-4, EH-5, EH-6), with modified fatty acid and amino acid composition, impact body composition, milt biochemistry, and sperm performance compared to the standard on- growing diet (DAN-EX), in male European eel. Fatty acid composition of EH-4, EH-5 and EH-6 was similar but differed to that in DAN-EX, while amino acid composition varied between all four diets. Diet did not influence organ-somatic indices (e.g. HSI, GSI), while males fed EH-4 were heavier than other groups. Arginine, alanine, and lysine were the most abundant amino acids in milt, followed by glycine, aspartic acid, valine, glutamic acid, and leucine. Diet impacted milt arginine, serine, proline, methionine, and histidine levels. Specifically, DAN-EX, EH-4, and EH-5 had the highest percentages of arginine, while EH-4 to EH-6 had higher percentages of serine. Proline was most abundant in DAN-EX, EH-5, and EH-6. Both methionine and histidine were detected at low percentages, and were impacted by diet, where EH-4 and EH-5 had higher percentages of methionine, and DAN-EX, EH-4, and EH-6 had the highest percentage of histidine. Eels fed EH-4 and EH-6 produced the freest flowing milt and production increased over time, where milt volume reached 3.22 ± 0.42 mL/100 g eel after 9 weeks of injections. Spermatocrit was 43.1 ± 1.8% and did not differ between the diets. Dietary regime had an impact on sperm motility, such that eels fed EH-5 and EH-6 had the greatest percentage of motility, while EH-5, EH-6 and also DAN-EX had the fastest sperm. Spermatogenic maturity index of hormonally treated eels varied within groups but did not differ between dietary treatment groups after 9 weeks of injections. The most interesting amino acids to scrutinize from PCA plots were proline, histidine, and valine as well as lysine and arginine. Here, eels with highly motile sperm had milt with high relative proportions of proline, histidine, and valine, but were low in lysine and arginine. Our findings add evidence that certain amino acids regulate milt biochemistry, and that male ejaculate traits may be promoted by amino acid intake.
Keywords: Aquaculture; Broodstock diet; Assisted Reproduction; Gamete quality; European eel
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 94 SESSION I. SPERMATOGENESIS AND SPERM QUALITY P9
Acknowledgements: Funded by the Innovation Fund Denmark, USDA – NIFA (I.A.E.B), and V.G. has a postdoc grant from the MICIU (Programa Juan de la Cierva-Incorporación; IJCI- 2017-34200).
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 95 SESSION I. SPERMATOGENESIS AND SPERM QUALITY P10
COLD SEAWATER INDUCES SPERMATOGONIA PROLIFERATION IN THE EUROPEAN EEL
Christoffer ROZENFELD, Juan Germán HERRANZ-JUSDADO1, Luz PÉREZ1, Víctor GALLEGO1, Marina MORINI1, David S. PEÑARANDA1, Juan F. ASTURIANO1*
1 Universitat Politècnica de València. Instituto de Ciencia y Tecnología Animal. Grupo de Acuicultura y Biodiversidad. Camino de Vera s/n 46022 Valencia, Spain
The European eel (Anguilla anguilla) is an endangered species (IUCN) and a highly valued and demanded resource. Our hypothesis was that environmental factors, such as temperature, could have a positive effect on eel reproductive performance. In particular, the exposure to cold water, which has been reported to improve sexual maturation in many fish species. Moreover, previous trials demonstrated the effect of low temperature on the maturation of male (Peñaranda et al. 2016) and female eels (Pérez et al. 2011). European eel males were acclimatized to seawater and 20 °C during 3 weeks. Later, fish were exposed to 3 temperature regimes for 2 weeks. The 3 regimes included 2 with constant temperature of 10 °C (T10) or 20 °C, and one with a variable temperature regime: alternating 10 and 20 °C every 12 hours (to mimic the natural daily temperature fluctuation that the eels experience during their oceanic migration). Sampling of brain, pituitary, testis (the BPG axis), and blood was performed after acclimation (20 fish as control group) and once the experiment was finished (3x20 fish). From these samples, the transcriptome profile of the BPG axis was analyzed, in 3 fish per group, using high throughput sequencing technology and differential expression analysis for sequence count data (DEseq). Further, plasma levels of testosterone (T) and 11-ketotestosterone (11KT) in all fish, were analyzed using RIA, and testis histology was done to determine the stage of development and cell-type counting. Males treated with cold seawater (10 °C) for 2 weeks showed an increase in the proliferation of differentiated spermatogonial type A cells, and elevated T and 11KT levels. Transcriptomes from the tissues of the BPG axis of T10 samples revealed a differential gene expression profile compared to the other experimental groups, with clustering in a principal component analysis and in heat maps of all differentially expressed genes. Furthermore, a functional analysis of differentially expressed genes revealed enriched gene ontology terms involved in the regulation of circadian rhythm, histone modification and meiotic nuclear division. These results indicate that a cold water pretreatment may prepare European eel males for artificial maturation, which potentially could minimize the percentage of non-responders or increase their reproductive potential, reducing the cost of the hormonal treatments.
Keywords: Anguilla anguilla; RNA-sequencing; Epigenetics; Temperature; Migration
Acknowledgements: Funded by the project REPRO-TEMP (MINECO; AGL2013-41646-R) and the European Union’s Horizon 2020 MSCA-ETN grant agreement No 642893 (IMPRESS). VG and MM have postdoc grants from the MICIU (Programa Juan de la Cierva-Incorporación; IJCI-2017-34200) and UPV (PAID-10-18), respectively.
Peñaranda et al. 2016. Temperature modulates testis steroidogenesis in European eel.
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 96 SESSION I. SPERMATOGENESIS AND SPERM QUALITY P10
Comp. Biochem. Physiol. A. Mol. Integr. Physiol. 197, 58–67 // Pérez et al. 2011. Influence of temperature regime on endocrine parameters and vitellogenesis during experimental maturation of European eel (Anguilla anguilla) females. Gen. Comp. Endocrinol. 174, 51–59.
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 97 SESSION I. SPERMATOGENESIS AND SPERM QUALITY P11
EFFECT OF PROBIOTICS ON MALE TELEOST REPRODUCTION AND BEHAVIOUR
David G. VALCARCE1, Marta F. RIESCO1, Juan Manuel MARTÍNEZ-VÁZQUEZ1 and Vanesa ROBLES1,2*
1 Spanish Institute of Oceanography, Santander (Spain) 2 MODCELL GROUP, Dpt. of Molecular Biology, Universidad de León, León (Spain)
Nutrition has a crucial impact on male reproduction. Developing an efficient oral supplement with direct effects on sperm quality is a promising tool for animal production and human reproduction fields. The present study was performed to evaluate the effect of a short-time probiotic supplementation on teleost sperm quality and male behavior. A two-strain probiotic mixture consisting on: Lactobacillus rhamnosus CECT8361 and Bifidobacterium longum CECT7347 was used to validate our hypothesis. These bacteria present antioxidant and anti-inflammatory properties. We used adult zebrafish as animal model. Each animal within the experiment was tagged with Visible Implant Elastomers (VIE) to easy tracking along the study. Inclusion criteria within the experimental design was an initial spermatic total motility ≤ 60%. 36 males were included in the final experiment. Three homogeneous groups of males (n=12) were created 1) CONTROL: fed with a normal standard diet; 2) MALTO (vehicle control): fed with maltodextrin and 3) PROBIO: fed with a probiotic preparation (109 CFU) based on a mixture (1:1) of both cited strains. The feeding regime was 21 days (a single spermatogenesis in zebrafish). Sperm quality was analyzed at t=0 d and t=21 d after the beginning of the diet ingestion using CASA analysis. Behaviour analysis were performed using a Novel Tank Test (NTT) at t=-1 and t=20. Our results demonstrated that in zebrafish model, probiotic-fed males increased sperm quality. In particular, in terms of sperm counts, 11 of the 12 males within the PROBIO group showed an improvement in concentration. Total motility enhanced with a 1,7 fold change after bacteria ingestion. Concomitantly, progressive motile cells were also improved and probiotic-fed males showed higher percentages of fast spermatozoa after treatment. Tracking analysis revealed a modulation in behaviour after probiotic ingestion. The number of crossings between the upper and lower subareas of the novel tank used in the experiment was statistical significant only in PROBIO group. These data highlight the promising use of this probiotic mixture to improve reproductive performance in teleost and furthermore it seems to promote a lower anxiety-like behaviour pattern.
Keywords: sperm quality, zebrafish, motility, CASA, conduct
Acknowledgements: Project AGL2015-68330-C2-1-R (MINECO-FEDER), RED AQUA CIBUS CYTED 318RT0549, PTA 201611987-I, Stolt Sea Farm and staff from Planta de Cultivos El Bocal.
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 98 SESSION I. SPERMATOGENESIS AND SPERM QUALITY P12
DETERMINATION OF ANNUAL REPRODUCTIVE CYCLE IN STERLET, ACIPENSER RUTHENUS USING HISTOLOGY AND ULTRASOUND IMAGING
Amin GOLPOUR1, Caroline BROQUARD2, Sylvain MILLA2, Hadiseh DADRAS1, Abdul Rasheed BALOCH1, Taiju SAITO1, Martin PŠENIČKA1*
1 Faculty of Fisheries and Protection of Waters, South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses, Research Institute of Fish Culture and Hydrobiology, University of South Bohemia in České Budějovice, Vodňany, Czech Republic 2 Unité de Recherche Animal et Fonctionnalités des Produits Animaux, Université de Lorraine, Vandoeuvre-lès-Nancy, France
During annual sexual cycle of male sterlet, stages of gonad maturity examined using histology and ultrasonography approaches. The histology identified males at different stages of maturity among fish sampled monthly. According to the seasonal changes in the testes, reproductive cycle was divided into four stages including resting, pre-spawning, spawning and post-spawning. The histology examination revealed considerable variation in testicular developmental stages. These changes were identified based on persistent spermatogenesis and asynchronous gonad development in testes, showing regulation of annual gonadal cycle is influenced by season. Also, the results obtained using ultrasound suggested that reproductive stages can be identified based on morphology and tissue echogenicity. In addition, gonadosomatic index, water temperature and number of spermatogenic cysts at identified phases of testicular development were changed accordingly. The major significance of the present study is determination of reproductive stage profile and asynchronous testicular development of sterlet. This study presents basic knowledge about reproductive biology in sterlet that contributes to optimal broodstocks management and comparison of reproductive development among sturgeon species.
Key words: Breeding season; Reproductive cycle; Spermatogenesis; Sterlet; Testicular development
Acknowledgements: The study was financially supported by the Ministry of Education, Youth and Sports of the Czech Republic – project CENAKVA (LM2018099) and Biodiversity (CZ.02.1.01/0.0/0.0/16_025/0007370) and the Czech Science Foundation (grant number 17- 19714Y), by the European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement No. 642893 (IMPRESS).
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 99 SESSION I. SPERMATOGENESIS AND SPERM QUALITY P13
EFFECT OF BREEDING WATER TEMPERATURE ON SPERM DNA METHYLATION
Audrey LAURENT1*, Stéphanie KICA1, Catherine LABBE1
1INRA LPGP (Fish Physiology and Genomics), F-35000 Rennes, France
During the post-meiotic phase of spermatogenesis, important chromatin remodelling events not only allow the compaction of paternal DNA, they also contribute to its protection and programming for progeny development. These coordinated functions of chromatin rely on the epigenetic marking of DNA and DNA interacting proteins. In the spermatozoa, while most histones are evicted and replaced by highly condensing protamines, important epigenetic information lies in the DNA modifications. Because epigenetic marking is sensitive to the environment, and fish are particularly exposed, a fortiori in rearing conditions, we aim to evaluate the stability of the paternal epigenome under environmental and biotechnological constraints. Here, we present a starting project in which the effect of breeding water temperature on rainbow trout sperme DNA methylation will be assessed.
Keywords: epigenetics, DNA methylation, spermatogenesis, water temperature
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 100 SESSION I. SPERMATOGENESIS AND SPERM QUALITY P14
DAILY RHYTHMS OF MELATONIN RECEPTORS IN HIGH AND LOW QUALITY SPERM SAMPLES IN SENEGALESE SOLE, Solea senegalensis
Marina MORINI1, Elvira FATSINI2, Leonor FERRÃO2, Victor GALLEGO1, Paulo FRIAS2, Catarina OLIVEIRA2, Elsa CABRITA2*
1 Grupo de Acuicultura y Biodiversidad, Instituto de Ciencia y Tecnología Animal, Universitat Politècnica de València, Camino de Vera s/n, 46022 Valencia,Spain 2 Center for Marine Sciences-CCMAR, University of Algarve, 8005-139 Faro, Portugal
The Senegalese sole (Solea senegalensis) is a flatfish with a high commercial interest, being considered as an emerging species in European aquaculture, particularly in Spain and Portugal. However, different aspects of its culture still need to be solved and optimized before its production on a large scale. In fish reared under intensive culture conditions (F1 generation), females very often fail to spawn and males produce low sperm volume with poor fertilization capacity. Studies characterizing sperm quality traits in relation to other male reproductive parameters are very important to reach a conclusive explanation about this main reproductive issue. Melatonin is a hormone linked to many biological functions such as reproduction, growth, feeding and behavior, playing a crucial role in the entrainment of daily, lunar and annual physiological rhythms. As Senegalese sole F1 individuals present an altered rhythmicity, we decided to evaluate and correlate the daily rhythms on melatonin receptors in spermatozoa with sperm quality traits. Sperm samples were collected from Senegalese sole males (6 animal/tank, 6 tanks) every two weeks, alternating between Mid- Light (ML, 1.30pm) and Mid-Dark (MD, 1.30am). Data as sperm density, sperm volume and spermatozoa kinetic parameters were first obtained to evaluate the sperm quality for each sample. RNA extraction and RT-qPCR analyses were performed to evaluate the expression of the three melatonin receptors, MT1, MT2, Mel1C. The day–night expression profile of the three melatonin receptors was examined in sperm samples of both high and low quality. Sperm quality in Senegalese sole breeders seemed to follow a daily rhythm. Our qPCR results showed day–night variations in expression patterns of melatonin receptors, which may be related with sperm quality variations. This study has a potential application on the improvement of sperm quality in Senegalese sole, which is of special importance for aquaculture, helping to produce gametes and larvae of better quality.
Keywords: sperm motility, MT1, MT2, Mel1C, gene expression
Acknowledgements: This research was funded by ReproF1 Project (Programa Operacional Mar2020, MAR-16-02-01-FMP-0059), Portuguese national funds from FCT - Foundation for Science and Technology through project UID/Multi/04326/2019, AquaExcel project (AE130010).
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 101 SESSION I. SPERMATOGENESIS AND SPERM QUALITY P15
ADVANCES ON REPRODUCTIVE BIOLOGY OF KILLIFISH (Aphanius iberus AND Valencia hispanica): SPERM MOTILITY, SPERMATOZOA MORPHOLOGY AND GAMETE PRESERVATION
Marta BLANES1, Miriam GARCÍA-COLL1, Marina MORINI1, Pablo GARCÍA-SALINAS1, Pilar RISUEÑO2, Luz PÉREZ1, Juan F. ASTURIANO1, Víctor GALLEGO1*
1 Grupo de Acuicultura y Biodiversidad. Instituto de Ciencia y Tecnología Animal. Universitat Politècnica de València. Camino de Vera s/n, 46022 Valencia, Spain. 2 Centro de Conservación de Especies Dulceacuícolas de la Comunitat Valenciana (CCEDCV). Plaza 04, El Palmar, Valencia, Spain.
Spanish toothcarp (A. iberus) and Valencia toothcarp (V. hispanica) are cataloged among the most threatened species on the UICN. One of the strategies for conserving threatened species is the gamete preservation, which would allow to own a genetic resources bank in the case that they become extinct. Due to the lack of information on the reproduction of these species, the goals of this study were i) to increase our knowledge on different aspects of their reproductive process and ii) to develop gamete preservation protocols for long-term storage. Spanish toothcarps were caught from the CCEDCV facilities, while Valencia toothcarps caught from some wetlands of the Albufera Natural Park and moved temporally to the CCEDCV. Sperm samples (1-5 µL) were obtained by stripping and then diluted 1:20 and 1:50 (A. iberus and V. hipanica, respectively) in PBS. Sperm kinetic parameters were assessed by a CASA-Mot system and sperm morphology was analyzed by ASMA software. Samples showing high motility (>60%) were selected for cryopreservation trials. Egg preservation was carried out inducing the diapause by drying the embryos; and reactivation were done 15 and 30 days later. Sperm kinetic patterns were similar for Spanish and Valencia toothcarp. Both species showed high motility (>70%) at 10, 20 and 30s post-activation, with a marked decline just after (50% at 60s). Both species showed a high percentage (>70%) of fast spermatozoa during the first 20s, with a continue decrease until getting 10% at 60s. Sperm swimming period was significant different, with some Spanish toothcarp samples able to move until 30 min, while maximum was 10 min for Valencia toothcarp. Regarding sperm morphology, ASMA analysis revealed a circular head with approx. 2.3 x 2.5 µm, similarly to other killifishes. On the other hand, cryopreservation trials lead first results for killifish species, with post- thawed sperm samples showing acceptable motility values of around 20 and 25% (A. iberus and V. hipanica, respectively). Considering these results, we recommend using a sperm:extender ratio of 1:50 plus the addition of methanol (10% v/v) as cryoprotectant. Regarding the egg preservation by inducing the diapause, experiments will be carried out at the end of the spawning season so results will be presented further on. Summing up, this study has improved the knowledge on the reproductive biology of killifish and has laid the bases for the establishment of cryopreservation protocol for Spanish and Valencia toothcarps sperm, that will be helpful for further reproduction in captivity programs.
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 102 SESSION I. SPERMATOGENESIS AND SPERM QUALITY P15
Keywords: Spanish toothcarp, Valencia toothcarp, cryopreservation, diapause
Acknowledgements: VG and MM have a postdoc grant from the MICIU (IJCI-2017-34200) and the UPV (PAID-10-18), respectively. PGS and MGC have PhD grants from the European Union through the Operational Program of the European Social Fund (ESF) of the Comunitat Valenciana 2014-2020 ADIF 2018 (ACIF/2018/147 and /136, respectively).
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 103 SESSION I. SPERMATOGENESIS AND SPERM QUALITY P16
EFFECTS OF ANESTHETIC TRICAINE ON PLASMA CORTISOL LEVELS AND SPERM MOTILITY OF SOUTH AMERICAN SILVER CATFISH
Nathalia S. TEIXEIRA1, Lis S. MARQUES1, Rômulo B. RODRIGUES, Darlan GUSSO2, Ana A. FOSSATI², Danilo P. STREIT1*
¹ Animal Science Research Program of Federal University of Rio Grande do Sul, Porto Alegre, RS, Brazil 2 Programa de Pós-Graduação em Biologia Celular e Molecular, Laboratório de Neuroquímica e Psicofarmacologia, Escola de Ciências, Pontifícia Universidade Católica do Rio Grande do Sul, Porto Alegre, RS, Brazil
In both research and animal production, it is important to understand the effects of the anesthetic on the animal and the tissues of interest, in order to guarantee the validity of the data and to improve animal welfare. The aim of this study was to evaluate the effects of different concentrations (0, 100, 200 and 300 mg / L) of the tricaine methanesulfonate anesthetic (MS-222) on cortisol levels and their influence on the sperm motility of Rhamdia quelen. After hormonal induction, 28 sexually mature males (363.00 ± 71.24 g) were randomly distributed among treatments, followed by collection of semen and blood. The semen was diluted and activated (58 mM NaCl solution in a ratio of 2:20 mL). Under optical microscopy the sperm motility rate was estimated from 0 to 100%. Plasma cortisol concentration was measured by enzyme-linked immunosorbent assay (ELISA) using blood plasma samples. Motility data were analyzed using ANOVA followed by Tukey's test and cortisol data were analyzed by Kruskal-Wallis analysis. In the motility data, a linear regression model was applied. The sperm motility rate was significantly higher in the control (90.00 ± 4.47%) when compared to the 300 mg/L treatment (66.25 ± 5.65%), but did not differ from the treatments 100 and 200 mg/L. In the plasma cortisol levels, there was no difference among the control (96.86 ± 7.08 ng/mL) and the treatments 100 (143.43 ± 24.97 ng/mL), 200 (138.29 ± 23.20 ng/mL) and 300 mg/L (182.50 ± 42.03 ng/mL). The MS-222 concentrations studied were not able to prevent the increase of plasma cortisol levels. It is possible that stress-related hormones cause blood plasma dilution and alteration in seminal plasma osmolarity, causing pre-activation of spermatozoa and, consequently, decreased sperm motility.
Keywords: fish sperm, osmolarity, MS-222, seminal plasma
Acknowledgements: This research was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 104 SESSION I. SPERMATOGENESIS AND SPERM QUALITY P17
TESTICULAR ASPECTS AND GONADO-SOMATIC INDEX (IGS) IN MALES OF PIRACANJUBA Brycon orbignyanus (CHARACIFORMES, BRICONIDAE) OF DIFFERENT AGES
Cristiane Fernanda BENEVENTE 1,2*, Luciane GOMES-SILVA 1,2*, Patrícia POSTINGEL- QUIRINO1,2*, Maria Luiza DELGADO1, Guilherme Antonio de FREITAS3, Alexandre NINHAUS- SILVEIRA1, Rosicleire VERÍSSIMO-SILVEIRA1 a Neotropical Ichthyology Laboratory (LINEO), Departament of Biologia e Zootecnia, São Paulo State University (UNESP) – Ilha Solteira Campus, Ilha Solteira, São Paulo, Brazil b Institute of Biosciences of Botucatu (IBB), São Paulo State University (UNESP) - Post- Graduation Program in Biological Sciences (Zoology) c BIG FISH Ltda, Zootecnista, Mestre em Aquicultura, São Paulo, Brazil
Brycon orbignyanus is a species of neotropical fish with a commercial interest. However, as a result of anthropic actions, with habitat fragmentation, hydroelectric construction, and overfishing, it has led to their suppression in the natural environment and is now considered a threatened species, almost extinct in nature. In view of the importance of this, it was sought to understand the testicular development in specimens of 3 different ages, for a better understanding of the reproduction of the species, by means of histological analyzes together with the gonadosomatic Index (IGS). Thus, B. orbignyanus specimens aged 1, 2 and 3 years were euthanized in benzocaine solution. Their testes were removed, fixed, processed in historesin and sectioned in 3 μm, submitted to Hematoxylin/Eosin. Histological analysis allowed the identification of three stages of testicular development: Immature, Initial Maturation for 1-year-old animals and Final Maturation for 2 and 3-year-old animals. In all the morphometric parameters analyzed, there was a statistical difference between the three ages. The IGS of the one-year-old animals was 0.02 ± 0.01, whereas the sexually mature two and three-year-old animals showed statistical difference in relation to the IGS in the testicular phase capable of reproduction, and the mean 1.37 ± 0.24 and 1.93 ± 0.36, respectively. There was no difference in the amount of spermatozoa in the lumen of the anastomosed tubules in animals of 2 and 3 years. Thus, the IGS values for B. orbignyanus males during the reproductive period vary with the age of the animal.
Keywords: reproduction, testis, morphology, age
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 105 SESSION I. SPERMATOGENESIS AND SPERM QUALITY P18
TRANSFERRIN IDENTIFICATION IN STERLET, Acipenser ruthenus REPRODUCTIVE SYSTEM
Miaomiao XIN1,2*, Pavlina VECHTOVA3,4, Anna SHALIUTINA-KOLESOVA1, Dmitry LOGINOV3, Yana LOGINOVA3, Zoltan FUSSY3, Borys DZYUBA1, Serhii BORYSHPOLETS1, Marek RODINA1, Otomar LINHART1, Jan STERBA3,4
1 University of South Bohemia in Ceske Budejovice, Faculty of Fisheries and Protection of Waters, South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses, Research Institute of Fish Culture and Hydrobiology, Zatisi 728/II, 389 25 Vodnany, Czech Republic 2 Sino-Czech Joint Laboratory of Fish Conservation and Biotechnology: Key Laboratory of Freshwater Biodiversity Conservation, Ministry of Agriculture of China, Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Wuhan, China 3 University of South Bohemia in Ceske Budejovice, Faculty of Science, Institute of Chemistry, Branisovska 1760, 37005 Ceske Budejovice, Czech Republic 4 Biology Centre of Academy of Sciences of the Czech Republic, Institute of Parasitology, Branisovska 31, 37005 Ceske Budejovice, Czech Republic
Transferrins are a superfamily of iron-binding proteins and are recognized as multifunctional proteins. Its function in male reproductive system is related to the spermatogenesis and sperm quality. The present study focused in sterlet Acipenser ruthenus on (1) the confirmation of transferrin expression and identification of the various transferrin isoforms in the organs of reproductive tract – the testes, kidney, and Wolffian duct; (2) the comparison of expression levels of transferrin genes in reproductive organs between spermiating and out-of-spawning males; (3) the identification of the transferrin proteins in seminal plasma and spermatozoa by mass spectrometry. The results showed that 9 transferrin transcripts were identified in the transcriptome of sterlet, and these transcripts were qualified as two different transferrin genes, serotransferrin (7 isoform) and melanotransferrin (2 isoform), with several isoforms present for both of them. The relative abundance of serotransferrin isoforms was higher in all three studied organs in the spermiating males compared to out-of-spawning males. In addition, transferrin was detected in sterlet seminal plasma, but not in sterlet spermatozoa extract using anti-transferrin antibody. These results were also confirmed by mass spectrometry identification of transferrin in seminal plasma but not in spermatozoa. Achieved results in this study indicate that transferrin may play a critical role in sterlet spermatogenesis regulation and sperm maturation.
Keywords: kidney, sperm, testes, transcriptome, Wolffian duct
Acknowledgements: The study was supported by the Czech Science Foundation (No. 16- 02407Y), by the Ministry of Education, Youth and Sports of the Czech Republic (“CENAKVA”, LM2018099), by project Biodiversity (CZ.02.1.01./0.0/0.0/16_025/0007370).
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 106 SESSION I. SPERMATOGENESIS AND SPERM QUALITY P19
CODING AND NON-CODING RNAs IN TELEOST LOW SPERM QUALITY SAMPLES: EFFECT ON PROGENY
Marta F. RIESCO1, David G. VALCARCE1, Juan Manuel MARTÍNEZ-VÁZQUEZ1 and Vanesa ROBLES1,2*
1 IEO, Spanish Institute of Oceanography, Planta de Cultivos El Bocal, 39012, Monte, Santander, Spain. 2 MODCELL GROUP, Department of Molecular Biology, Universidad de León, 24071, León, Spain.
During recent years, several studies have demonstrated that sperm cell is not a simple vehicle for male DNA delivery to the embryo during fertilization. The male contribution, far from being confined to DNA, has been extended to a wide variety of molecules including coding and non-coding RNAs. Evidence of their importance has been provided in several studies that demonstrated that sperm cells can be a vehicle for certain phenotype transmission through some molecules like miRNAs. Taking into account this fact, the possibility of select sperm samples under a molecular point of view could be ensure the fertilization success and avoid the transmission of undesired phenotypes. For this reason, the first objective of this work was to demonstrate that miRNAs could represent important molecular biomarkers to select good breeders. The second objective was to evaluate the effect of sperm altered miRNA pattern on early development using zebrafish as a model. Different analyses were performed in sperm samples to correlate sperm quality parameters with miR expression (miR-200a-5p, miR-122-5p and miR-141-3p, previously described as biomarkers in human sperm). Once breeders were characterized according to sperm quality parameters, fertilization trials were done and fertility rates were recorded together with malformation rates and survival during development. At molecular level, the relative expression of the studied miRNAs was analyzed in different developmental stages in the progeny (blastula and 24 hpf stages). In addition, some mRNA targets of such miRNAs were predicted and their relative expression were measured in the embryos. In this work we found that low quality sperm samples have an altered sperm miRNA pattern. Moreover, progenies derived from low quality sperm samples showed higher malformation rates and aberrant expression of miR-141-3p and 200a-5p and their targets: kita and dmrt1. The importance of fertilizing with high sperm quality samples get relevance from a new perspective: to avoiding molecular alterations in the progeny that could remain masked and could have different unexpected consequences in it.
Keywords: miRNAS, mRNAs, zebrafish model and early development.
Acknowledgements: We acknowledge Planta de Cultivos el Bocal (IEO) staff. This work was supported by AGL2015 68330-C2-1-R project (MINECO/FEDER), PTA2016-11987-I contract (MINECO/FEDER) and AQUA-CIBUS international net 318RT0549 funded by CYTED (Programa Iberoamericano de Ciencia y Tecnologia para el Desarrollo).
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 107 SESSION I. SPERMATOGENESIS AND SPERM QUALITY P20
DETERMINATION OF DAY/NIGHT MELATONIN LEVELS IN SEMINAL PLASMA IN RELATION TO SPERM QUALITY IN SENEGALESE SOLE
Victor GALLEGO1, Leonor FERRÃO2, Elvira FATSINI2, Marina MORINI1, Paulo FRIAS2, Elsa CABRITA2, Catarina OLIVEIRA2*
1 Grupo de Acuicultura y Biodiversidad, Instituto de Ciencia y Tecnología Animal, Universitat Politècnica de València, Camino de Vera s/n, 46022 Valencia, Spain. 2 Center for Marine Sciences-CCMAR / University of Algarve, 8005-139 Faro, Portugal.
Melatonin is a major key-player in the synchronization of biological rhythms in vertebrates, including reproduction, providing the cells with information about daytime and season. Bloodstream melatonin is produced in the pineal organ during night-time, being inhibited during day-time. In addition, gonads and other organs also produce melatonin, which is not released into the bloodstream and is likely to have an antioxidative function locally. In this sense, recent studies in mammals have detected melatonin in seminal fluid, showing its beneficial effects on sperm cells. On the other hand, Senegalese sole (Solea senegalensis) is a promising species for European aquaculture diversification but still presents bottlenecks to overcome for intensive production. Therefore, the objective of this research was to determine melatonin concentrations in seminal plasma in Senegalese sole breeders both during day- and night-times, and further relate it with bloodstream values and sperm quality parameters. Sperm samples from S. senegalensis breeders were collected both at Mid-Light (ML) and at Mid-Dark (MD) during the natural spawning seasoning (natural photoperiod and temperature). Sperm kinetic parameters were immediately assessed by a CASA-Mot system at 15, 30, 45 and 60s and afterwards seminal plasma was separated by centrifugation and stored at -80ºC for further melatonin determination by ELISA. Sperm quality seemed to follow a daily rhythm, and sperm motion parameters from MD breeders were slightly higher than ML breeders. Regarding total motility, sperm from ML and MD breeders was similar at 15 and 30s, but sperm from MD group showed high motility values at 45 and 60s. However, in the rest of sperm motion parameters analysed (progressive motility, VSL and VCL), MD breeders showed higher values than ML breeders at all post-activation times. In this sense, swimming period (sperm longevity) was longest in MD breeders, increasing the spermatozoa capacity for fertilization success. Correlations between melatonin seminal plasma levels and sperm quality will be further discussed. Understanding melatonin action at gonadal level to design strategies to ameliorate gamete quality and production will be fundamental to improve techniques for controlling the artificial reproduction.
Keywords: sperm motility, kinetic parameters, biorhythm, circadian rhythms
Acknowledgements: This research has been funded by ReproF1 Project (Programa Operacional Mar2020, MAR-16-02-01-FMP-0059), Portuguese national funds (FCT-Foundation for Science and Technology) through project UID/Multi/04326/2019, AquaExcel project (AE130009 & AE130010). VG and MM have a postdoc grant from the MICIU (IJCI-2017-34200) and the UPV (PAID-10-18), respectively.
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 108 SESSION II. GERMINAL STEM CELLS AND BIOTECHNOLOGIES P21
PRODUCTION AND USE OF TRIPLOID ZEBRAFISH FOR SURROGATE REPRODUCTION
Roman FRANĚK1*, Tomáš TICHOPÁD1, Michaela FUČÍKOVÁ1, Christoph STEINBACH1, Martin PŠENIČKA1
1 University of South Bohemia in České Budějovice, Faculty of Fisheries and Protection of Waters, Zátiší 728/II, 389 25 Vodňany, Czech Republic
Triploid fish are potential candidates as surrogates for germ cells transplantation. However, triploidy does not always imply functional sterility when sperm obtained from triploid in some species can be competitive to normal haploid sperm from a diploid individual thus validation of triploid surrogates needs to be performed in species of interest. We report for the first-time comparison of two approaches for zebrafish triploid production using cold shock and heat shock treatment. Subsequently, produced triploid zebrafish were used as recipients for intraperitoneal transplantation of ovarian and testicular cells originating from vas:EGFP strain in order to verify their suitability for surrogate reproduction. Heat shock treatment was far more effective evaluated as success rate of triploid production and viability in comparison to cold shock. As expected, all zebrafish triploids were males. Subsequently, germ cell transplantation revealed that triploids are suitable surrogate hosts. Only donor-derived sperm was obtained from triploid surrogates even after oogonia transplantation. Production of donor-derived sperm was achieved in 23% and 16% of triploids transplanted by testicular and ovarian cells, respectively. Success of the transplantation was confirmed by positive GFP signal detected in gonads of dissected fish and stripped sperm. Germline transmission was confirmed by fertilization tests followed by PCR analysis of embryos. Reproductive success of germline chimera triploids evaluated as fertilization rate and progeny development was comparable to control males. To the best of our knowledge, this study provided the first report of the suitability of zebrafish triploid swim-up larvae as a recipient for intraspecific transfer of germ cells and production of donor- derived gametes. Zebrafish triploid surrogates might be utilized further to improve sperm production in lines exhibiting lower reproductive performance or for sex determination studies in case of donor-derived sperm from oogonia.
Keywords: chromosome manipulation, sterility, germ cell transplantation
Acknowledgements: The study was financially supported by the Ministry of Education, Youth and Sports of the Czech Republic, projects CENAKVA (LM2018099) and Biodiversity (CZ.02.1.01/0.0/0.0/16_025/0007370), by the Grant Agency of the University of South Bohemia in České Budějovice GAJU 097/2019/Z and 034/2017/Z. The Czech Science Foundation (project No. 17-19714Y) and NAZV QK1910248.
SESSION II. Germinal stem cells and biotechnologies
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 109 SESSION II. GERMINAL STEM CELLS AND BIOTECHNOLOGIES P22
GERM CELL MANIPULATION AS A TOOL FOR COMMON CARP ISOGENIC LINES PRODUCTION AND MANAGEMENT
Roman FRANĚK1*, Taiju SAITO1,2, Tomáš TICHOPÁD1, Michaela FUČÍKOVÁ1, Zoran MARINOVIĆ3, Jelena LUJIĆ3, Ákos HORVÁTH3 Vojtěch KAŠPAR 1, Martin PŠENIČKA1
1University of South Bohemia in Ceske Budejovice, Faculty of Fisheries and Protection of Waters, Zátiší 728/II, 389 25 Vodňany, Czech Republic. 2Nishimura Station, South Ehime Fisheries Research Center, Ehime University, Uchidomari, Ainan, Ehime 798-4206, Japan. 3Department of Aquaculture, Szent István University, Páter Károly u. 1, H-2100, Gödöllő, Hungary.
Isogenic lines are recognized to be a convenient approach how to control genetic background of the study across used animals. In fish, uniparental inheritance induction such as mitotic gynogenesis and androgenesis is used to produce doubled haploid (DH). Then DH specimens are reproduced again using uniparental inheritance and isogenic line (IL) is obtained. Although DH induction procedures have been developed in many fish species, only a few of them were successfully propagated to obtain ILs and further maintained to facilitate their use in experiments. Main problems of ILs are yield of doubled haploid and reproductive issues. Also, further interferences are needed to produce the next isogenic generations. We attempted to overcome disadvantages connected with the conventional approach for IL generation using germ cell manipulation. Cryopreservation protocols for testicular and ovarian tissue yielded 40-60% post-thaw viability. Recovery of cryopreserved germ cells (GCs) was confirmed after transplantation into sterile goldfish when cryopreserved GCs retained colonization rate comparable to non-cryopreserved control. GCs from DHs were transplanted into sterilized goldfish surrogates, which we previously confirmed to be capable to accept common carp GCs further differentiating into testis or ovary, giving chance that sperm and eggs can be produced when single DH fish is used as a donor. Goldfish surrogates are believed to provide a more convenient environment for carp DH germ cells increasing the chance that gametes will be produced, and IL will be established. So far, GCs from 14 different DH donors were transplanted within frame of three years, while two groups reached maturity and donor-derived sperm originating from transplanted DH oogonia was obtained.
Keywords: isogenic line, common carp, chromosome manipulation, surrogacy, goldfish
Acknowledgements: The study was financially supported by the Ministry of Education, Youth and Sports of the Czech Republic, projects CENAKVA (LM2018099) and Biodiversity (CZ.02.1.01/0.0/0.0/16_025/0007370), by the Grant Agency of the University of South Bohemia in České Budějovice GAJU 097/2019/Z and 034/2017/Z. The Czech Science Foundation (project No. 17-19714Y) and NAZV QK1910248.
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 110 SESSION II. GERMINAL STEM CELLS AND BIOTECHNOLOGIES P23
ONLY MALE INHERITANCE INDUCTION IN STURGEONS AND HATCHING OUT PADDLEFISH FROM STERLET EGGS USING COLD SHOCK ANDROGENESIS
Roman FRANĚK1*, Miloš HAVELKA2, Marek RODINA1, Katsutoshi ARAI3, Martin PŠENIČKA1
1 University of South Bohemia in České Budějovice, Faculty of Fisheries and Protection of Waters, Zátiší 728/II, 389 25 Vodňany, Czech Republic. 2 Nishiura Station, South Ehime Fisheries Research Center, Ehime University, Uchidomari, Ainan, Ehime, 798-4206, Japan. 3 Faculty and Graduate School of Fisheries Sciences, Hokkaido University, 3-1-1 Minato, Hakodate, Hokkaido 041- 8611, Japan.
Androgenesis is a mode of uniparental inheritance with only paternal genome transmission to offspring. The basic principle of androgenesis is the inactivation of the maternal genome to produce androgenetic haploids. Embryos are subsequently exposed to shocks blocking the first cell division and doubled haploids are produced. Conventional methods for maternal genome inactivation are using UV or Gamma radiation, thus there are certain requirements for equipment, especially for Gamma rays which are difficult to be available. Moreover, eggs of sturgeon cannot be irradiated by UV prior to androgenesis due to thick chorion and heavy pigmentation. A low-cost method for androgenesis induction was introduced using immediate exposition of embryos in cold shock to inactivate maternal genome. This method was successfully applied in loach, Japanese flounder and zebrafish. Here we reported successful application of cold shock androgenesis for haploid induction in sterlet (Acipenser ruthenus). Cold shock treatment was optimized firstly using different temperatures and durations. Then, heat shock treatment for rediploidization of androgenetic embryos was optimized. Only paternal inheritance was confirmed using microsatellite loci covering different linkage groups. Developed protocol for androgenetic haploid induction was also tested on several sturgeon species – Russian sturgeon, Siberian sturgeon and beluga. Surprisingly, we succeeded in viable androgenetic paddlefish (Polyodon spathula) production, when sterlet eggs fertilized with paddlefish sperm were facilitated to cold shock and subsequently heat shock treatment. Developed protocol for androgenesis induction has exceptional potential for application in big and long maturing sturgeon species when eggs from a target species can be substituted with small and fast maturing sterlet.
Keywords: chromosome manipulation, androgenesis, sturgeons
Acknowledgements: The study was financially supported by the Ministry of Education, Youth and Sports of the Czech Republic, projects CENAKVA (LM2018099) and Biodiversity (CZ.02.1.01/0.0/0.0/16_025/0007370), by the Grant Agency of the University of South Bohemia in České Budějovice GAJU 097/2019/Z and 034/2017/Z. The Czech Science Foundation (project No. 17-19714Y) and NAZV QK1910248.
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 111 SESSION II. GERMINAL STEM CELLS AND BIOTECHNOLOGIES P24
CHARACTERIZATION OF A NEW EMBRYONIC CELLULAR MODEL FOR NUCLEAR TRANSFER IN FISH: THE EMBRYOID BODY
Charlène ROUILLON1*, Alexandra DEPINCE1, Pierre-Yves LE-BAIL1, Catherine LABBE1
1 INRA LPGP-Fish Physiology and Genomics, Rennes, France
In fish, one of the means to regenerate breeders is to master nuclear transfer using cells from somatic origin. One difficulty of this biotechnology lies in the epigenetic reprogramming defects of the somatic chromatin in the embryo, which would explain the low clone production rates. Nuclear transfer using embryonic cells should prevent to some extent those reprogramming defects, thanks to the less differentiated nature of embryonic cells. However, the use of such cells for nuclear transfer raises other difficulties. Indeed, before the mid-blastula transition (MBT) stage when embryonic genome is not fully activated, embryonic cells are undifferentiated but they are still interconnected by intercellular bridges, they are too large and they are always engaged in some steps of the cell division process. On the contrary, older embryonic cells after MBT are smaller and have slowed cell division, but they have passed the embryonic genome activation stage are already engaged in differentiation pathways. It was therefore necessary to develop an intermediate model with small but still undifferentiated cells. Goldfish embryonic cells at MBT stage were separated from the yolk, and these cellular mounds cultivated for 24 hours produced a multicellular structure called the embryoid-body. Embryoid bodies appeared less complex and less organized than whole 24h embryos, and the suitability of embryoid bodies for nuclear transfer was studied, both for their cellular characteristics and their differentiation status. Embryoid body cells were significantly reduced in size compared to MBT embryonic cells (embryoid body: 9.6µm ; MBT: 33.8µm), and most were no longer in the mitotic stages (embryoid body: 95.1 % ; MBT 26.1 %). Therefore, embryoid body cells should be better suited for nuclear transfer than MBT cells. However, expression of some marker genes indicated that embryoid body cells had an intermediate degree of differentiation, between MBT cells and 24h old embryos. The effect of the epigenetic reprogramming agent, trichostatin A, was tested during embryoid body formation (embryoid body-TSA). DNA methylation level of the early marker gene nanog was reduced in embryoid body-TSA compared to untreated embryoid bodies (embryoid body-TSA: 1.4% of methylation; embryoid body: 5.0% ; MBT : 1.3%), showing some maintenance of MBT cells characteristics. In parallel, the expression of marker genes involved in late development and tissue differentiation such as sox2 and ck8/49 has been reduced as well in embryoid body- TSAs and in MBT cells, contrarily to untreated embryoid bodies. These findings suggest that embryoid body-TSA cells have maintained a more undifferentiated state, likely more compatible with the chromatin reprogramming necessary for the development of a clone via nuclear transfer.
Keywords: Carassius auratus, reprograming, trichostatin A, DNA methylation, transcription.
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 112 SESSION II. GERMINAL STEM CELLS AND BIOTECHNOLOGIES P24
Acknowledgements: CR is recipient of an INRA-PHASE and Région Bretagne fellowship. This work is funded by the French CRB Anim project «Investissements d’avenir», ANR-11-INBS- 0003.
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 113 SESSION II. GERMINAL STEM CELLS AND BIOTECHNOLOGIES P25
FLUORESCENCE-ACTIVATED CELL SORTING (FACS) OF STERLET (Acipenser ruthenus) SPERMATOGONIA BASED ON LIGHT SCATTER PROPERTIES
Xuan XIE1*, Galina KISLIK2, Pavel ABAFFY3, Radek ŠINDELKA3, Roman FRANĚK1, Martin PŠENIČKA1
1 University of South Bohemia in Ceske Budejovice, Faculty of Fisheries and Protection of Waters, South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses, Vodňany, Czech Republic 2 Imaging Methods Core Facility at BIOCEV, operated by Faculty of Science, Charles University in Prague, Czech Republic 3 Laboratory of Gene Expression, Institute of Biotechnology, Academy of Sciences of the Czech Republic, Průmyslová595, Vestec 252 50, Czech Republic
Sturgeons belong to the order Acipenseriformes, which are among the most ancient of actinopterygian fishes. Nowadays 64% of sturgeon species are listed as critically endangered, due to their habitat alteration caused by damming of rivers, pollution and overharvesting. Moreover, most sturgeon species are large and late maturing, which makes their culturing and conservation very costly and time consuming. Therefore, germ cell culture and xenotransplantation of germ cells could be used as an affordable and a time-efficient method for surrogate production of endangered sturgeons. Germ stem cells are a crucial component in reproduction, with the ability to self-renew to maintain the stem cell population as well as to differentiate into other types of germ cells. In transplantation system, germ cells are capable of undergoing gametogenesis after their incorporation into the genital ridges of recipients. Besides, in germ cell culture, somatic cells and other types of germ cells could compete with germ cells and inhibit their growth. Thus, enrichment of germ cells can be expected to increase the efficiency of culture and transplantation. Besides, the morphology as well as cellular and molecular mechanisms of sturgeon germ cell development have been poorly understood and remain a question of broad interest. Development of new methods for enrichment of germ cell populations in vitro is very essential. In our study, we established a method for identification and isolation of sturgeon germ cells using FACS. Flow cytometry analysis of freshly prepared whole testicular cell suspension showed a few distinguished cell populations formed according to different values of light scatter parameters. FACS of these different cell populations was performed directly on glass slides for further immunocytochemistry to identify germ cells. The same cell populations were also single cell sorted into 384-well plates for q-PCR of vasa gene. Results of q-PCR and immunocytochemistry of post sorted cells, as well as immunohistochemistry and histology observation of whole testis sections showed that the cell population in gate P1 on a flow cytometry plot (with high forward scatter (FSC) and high side scatter (SSC) parameter values) contains the highest amount of germ cells – 81.92 ± 2.71% compared to a non-sorted sample – 11.59 ± 1.86%. We expect that the use of this FACS strategy can improve the production of sturgeons with surrogate broodstock and further study of cellular and molecular mechanisms of sturgeon germ cell development.
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 114 SESSION II. GERMINAL STEM CELLS AND BIOTECHNOLOGIES P25
Keywords: FACS, flow cytometry, germ cells, sturgeon, spermatogonia
Acknowledgements: The study was financially supported by the Ministry of Education, Youth and Sports of the Czech Republic -project CENAKVA (LM2018099) and Biodiversity (CZ.02.1.01/0.0/0.0/16_025/0007370) and the Czech Science Foundation (grant number 17- 19714Y), and the Czech Science Foundation (grant number 16-02407Y).
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 115 SESSION II. GERMINAL STEM CELLS AND BIOTECHNOLOGIES P26
NON-INVASIVE SEX DETERMINATION IN LUMPFISH (Cyclopterus lumpus l.) USING ULTRASOUND TECHNOLOGY
Maren MOMMENS1*, Rikard HAGESKAL1, Ingun NÆVE1, Nina IVERSEN2, Helge TVEITEN3, Velmurugu PUVANENDRAN4
1 AquaGen AS, P.O. 1240, Sluppen, N-7462 Trondheim 2 Namdal Rensefisk AS, Storlavika Industriområde, Lauvsnes, N-7770 Flatanger 3 University of Tromsø, Hansine Hansens veg 18, 9019 Tromsø 4 Nofima, PO Box 6122, N-9291 Tromsø
The lumpfish (Cyclopterus lumpus L.) is used as a cleaner fish in the Atlantic salmon farming industry providing a non-medicinal treatment against sea lice infection. Lumpfish production relies on the capture of wild broodstock which may not be sustainable in the future, limits year-round production, and the possibility to select for desired traits such as robustness and sea lice grazing efficiency. Lumpfish develop secondary sexual characteristic when becoming sexually mature and display a sex-dependent growth dimorphism. Mature males are smaller, are richly colored and possess a relatively larger suction disc compared to mature females. However, small immature fish of either sex cannot be easily distinguished outside breeding season and have similar sized suction discs. Both sexes loose color and separation of the sexes by external characteristics becomes less easy at equivalent weights. Our goal was therefore to test the use of ultrasound (US) as a non-invasive method for sex determination in lumpfish of commercial size and during broodfish production. We used a MyLab Alpha (Esaote, Italy) US machine to determine sex in juvenile, commercial size lumpfish and broodstock recruits. In juvenile, commercial sized lumpfish (48 ± 14 to 72 ± 16g), sex was correctly determined by US examination in 87 to 100% of lumpfish, with highest accuracy in lumpfish of 72 ± 16g. In larger lumpfish (192 ± 53g to 742 ± 310g), sex determination accuracy varied between 69 to 100% for both sexes. In females, an accuracy of 100% was achieved at a smaller size (392 ± 141g) compared to males (742 ± 310g). In all larger lumpfish (up to 1438 ± 872g), sex was correctly determined and was related to an increase in GSI. Male coloration became visible in males at 392 ± 141g and increased from 44% to 96% during the last examination at 1438 ±872g. The sex of immature males lacking coloration was determined with 100% accuracy using ultrasound in ≥ 742 ± 310g males. Ultrasound seems to be well suited as a non-invasive method for sex determination in lumpfish. We will further compare our observations to external morphological measurements of lumpfish, sex hormone profiles and gonad histology.
Keywords: Cleaner fish, gender, broodstock production
Acknowledgements: The project Reproductive biology of lumpfish (Cyclopterus lumpus): a key to successful selective breeding (CycloBreed) is funded by the Norwegian Seafood Research, project number 901418.
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 116 SESSION II. GERMINAL STEM CELLS AND BIOTECHNOLOGIES P27
LABELLING OF PRIMORDIAL GERM CELLS IN STURGEON USING IRON OXIDE NANOPARTICLES
Abdul Rasheed BALOCH1, Michaela FUČÍKOVÁ1*, Marek RODINA1, Brian METSCHER2, Tomáš TICHOPÁD1, Mujahid Ali SHAH1, Roman FRANĚK1, Martin PŠENIČKA1
1 University of South Bohemia in České Budějovice, Faculty of Fisheries and Protection of Waters, Zátiší 728/II, 389 25 Vodňany, Czech Republic. 2 Department of Theoretical Biology, Althanstraße 14, 1090 Vienna, Austria
Nanoparticles (NPs) are finding increasing applications in diagnostics, imaging and therapeutics in medicine. Iron oxide nanoparticles (IONs) have received significant interest of scientific community due to their distinctive properties. For the first time, we have used IONs in labelling of germ cells in any species. Our results showed that Primordial Germ Cells (PGCs) labelled with IONs could be detected until 7 days post fertilization (dpf) under fluorescent microscope and at 22 dpf by micro-CT. Precise labelling of cells with IONs could be helpful for studying of germ cell biology and improvement of germ cell-based bio- technologies as isolation of PGCs using magnetic activated cell sorting or application of hyperthermia for host sterilization purpose. Intriguingly, in our study, we did not find any toxic effects of IONs on survival and hatching rates of sturgeon embryos.
Keywords: Acipenser, caviar, hyperthermia, iron oxide nanoparticles, micro-CT, sterilization
Acknowledgements: The study was financially supported by the Ministry of Education, Youth and Sports of the Czech Republic -project CENAKVA (LM2018099) and Biodiversity (CZ.02.1.01/0.0/0.0/16_025/0007370) and the Czech Science Foundation (grant number 17- 19714Y), by the European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement No. 642893 (IMPRESS).
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 117 SESSION II. GERMINAL STEM CELLS AND BIOTECHNOLOGIES P28
THE DEVELOPMENT OF A NOVEL TECHNIQUE FOR PARTIAL CLEAVAGE INHIBITION ON STURGEON EMBRYO MODEL
Mujahid Ali SHAH1* and Martin PSENICKA1
1 Faculty of Fisheries and Protection of Waters, South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses, University of South Bohemia in Ceske Budejovice, Zátiší 728/II, 389 25 Vodnany, Czech Republic
Sturgeons are living fossils that represent ancestral mode of eggs cleavage pattern as in xenopus, so called holoblastic. To study the transition of cleavage pattern (holoblastic to meroblastic) and fate mapping in sturgeons and other basal bony fishes, it is crucial to develop a novel technique for partial inhibition of cleavage. Previous studies have shown that polyunsaturated aldehydes including 2–4 trans decadienal (DD) present an adverse effect on developing embryos of several broadcast spawning species, however, the localized effect of DD on cleavage has not been investigated so far. In this work, we have developed a novel technique for partial inhibition of cleavage in developing embryos of sturgeon, we utilized the minimum concentration of DD (0.001%) and light for irradiation (> 395 nm, ~ 91 W m−2). All embryos were injected at 2 cell stage into one blastomere and gradually n = 3 were irradiated from different stages (4, 8, 16, 32, 64 early blastula and late blastula) under optimized light. In our results, embryos at 4, 8, 16, 32 and 64 cell stage showed arrest of cleavage of treated blastomeres compared to before irradiation / control, whereas, alone DD injection or light irradiation were found to be unable to inhibit the cleavage. Our work is intended as a resource for all workers who wish to employ inhibition of cleavage pattern or fate mapping of single blastomeres.
Key words: 2–4 trans decadienal, light irradiation, arrest of single blastomeres and Acipenser
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 118 SESSION II. GERMINAL STEM CELLS AND BIOTECHNOLOGIES P29
TOWARDS IMPROVING THE PRACTICABILITY OF GERM STEM CELL GRAFTING IN COMMERCIAL SALMONIDS FARMS
Sarah TREMPONT1*, Anne-Sophie GOUPIL1*, Tina TERRENNE2, Francine KRIEG3, Marjorie BIDEAU2, Nicolas DECHAMP3, Laurent LABBE3, Philippe HOCDE4, Lionel GOARDON3, Jean- Jacques LAREYRE1
1 INRA UR 1037 Fish Physiology and Genomics, 35042 Rennes, France. 2 UMR UR 1313 Animal Genetics and Integrative Biology, 78352 Jouy-en-Josas, France. 3 INRA UE 0937 PEIMA, 29450 Sizun, France.
* [email protected] ; [email protected]
The use of the 17-α methyl testosterone (MT) hormone remains a legal and common practice in France for the production of neomales in Rainbow trout (Oncorhynchus mykiss) but MT is an endocrine disruptor released in the effluents. In other species, such as Arctic char (Salvelinus alpinus), attempts to reverse the phenotypic sex of genetic females using MT failed. As a result, there is a need to establish new alternative practices that reduce the environmental impact of endocrine disruptors used to produce monosex populations while maintaining the competitiveness of the fish industry. The study that is part of the BioGerm project aims to improve the practicability of the germ stem grafting in fish farms to produce neomales without the use of exogenous hormone in salmonids including Arctic char and Rainbow trout. Donor germ stem cells were collected from immature or ovulated arctic char females, and injected into the abdominal cavity of recipient embryos hatched for less than 24 hours and previously sterilized by triploidization. We tested different recipients species because they show different age at the first sexual maturity in males (Brook trout: 1 year; Brown trout: mainly 2 years; Rainbow trout: 1 years). The improvement of the embryo handling (control of temperature and oxygenation) allow us to decrease the mortality induced by the injection. The survival rate of the injected embryos was reduced to 10 to 15 % compared to non-injected embryos. We observed that eggs quality in Brook trout drastically influenced the survival rate. No or a low number of surviving embryos was obtained using egg collected from two years old females. In contrast, eggs collected from three years old females supported the development of the injected embryos. Grafted embryos are currently growing and their reproductive performance will be studied in autumn 2020. An additional experiment was carried out previously using the transplantation of purified and non-purified spermatogonia collected from 1 year old neomales of two distinct isogenic trout lines. The success rate of germ stem cell grafting and the reproductive performance of germinal chimera were studied at two years. 80% of the injected females was successfully grafted and produced normal amounts of eggs per body weight. Egg quality was variable depending on the female studied but similar to that observed in the recipient strain. 80% of the injected males was successfully grafted, but only 10% produced at least 1 ml milt with sperm counts reaching 108 spermatozoa/ml or higher. Egg fertilization using the milt led to the production of female monosex populations.
Keywords: Arctic char, Rainbow trout, Germ stem cell, germinal chimera, transplantation
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 119 SESSION II. GERMINAL STEM CELLS AND BIOTECHNOLOGIES P29
Acknowledgements: supported by the EU Horizon 2020 research infrastructure project under grant agreement n°652831 (AQUAEXCEL2020 project) and by the European Maritime and Fisheries Fund (FEAMP 2014-2020) under the BioGerm grant.
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 120 SESSION II. GERMINAL STEM CELLS AND BIOTECHNOLOGIES P30
GERM CELL TRANSPLANTATION IN THE WHITE STURGEON, Acipenser transmontanus
Amie L. T. ROMNEY1*, Tawny N. SCANLAN1, Stuart A. MEYERS1
1 Department of Anatomy, Physiology and Cell Biology, University of California, Davis, School of Veterinary Medicine, Davis, CA
The white sturgeon is the largest freshwater fish in North America. Because of their unique life history characteristics including longevity, late maturation, and long spawning intervals, white sturgeon aquaculture can be a significant investment of resources. Even so, California is the largest producer of white sturgeon meat and caviar in the United States. Germ cell transplantation (GCX) is an innovative technology previously demonstrated in other fish species for the production of a surrogate broodstock. The technique relies upon germ-line stem cell (or primordial germ cell; PGC) isolation, purification, and transplantation. Here, we determine cell types and optimal germ cell recovery from juvenile gonads, as well as the transplantation success and colonization rates into newly hatched larvae. Histological examination revealed that gonads from 1 to 2 year old females and as old as 4 year old males had a high proportion of PGCs compared to other ages. Gonad dissociation using trypsin enzyme yielded high numbers of cells that were subsequently determined as PGCs after Percoll sorting and immunolabeling with the antibody DDX4, or vasa, as a marker specific to germ-line stem cells. Suspensions of PGCs were labeled with a cell membrane dyes and transplanted by microinjection into the peritoneal cavity of newly hatched larvae close to the presumptive genital ridge. Larvae were reared until 1 and 2 months post- transplantation to monitor for colonization and proliferation of PKH26-labeled cells within the recipient gonads. Determining optimal methods for PGC localization and transplantation will support this new reproductive management tool, GCX, to use alternative species for the production of sturgeon, and for hatcheries to utilize natural gamete production without genetic modification. These preliminary findings summarize our efforts in adapting GCX for the improved production of white sturgeon, and the management of other threatened sturgeon species.
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 121 SESSION III. REFRIGERATION AND CRYOPRESERVATION P31
CHILLING SENSITIVITY OF Astyanax altiparanae EMBRYOS AND THE PERFORMANCE OF FATTY ACIDS AND CRYOPROTECTANTS IN THE MAINTENANCE OF LARVAL VIABILITY
Alexandre NINHAUS-SILVEIRA1, Cristiane BASHIYO-SILVA1, Raphael DA SILVA COSTA1, Laís PEDROSO BORGES1, Laícia CARNEIRO LEITE1, Cristiéle DA SILVA RIBEIRO2, Rosicleire VERÍSSIMO-SILVEIRA1
1 Neotropical Ichthyology Laboratory (LINEO) 2 Laboratory of Animal Physiology Studies (LEFISA), Department of Biology and Zootechnics, FEIS / UNESP – São Paulo State University, Brasil Avenue, 56, 15085-000, Ilha Solteira, São Paulo, Brazil.
Studies on embryo cooling have been of great interest in the production and conservation of fish species, since it allows the embryos to be maintained at temperatures close to 0°C for a short period of time, delaying their development without altering their viability. The objective of this study was to evaluate the effect of cooling of embryos of Astyanax altiparanae from parents fed a diet rich in omega 3, at temperatures of 15, 5 and 0ºC for a period of 12 hours, and the action of cryoprotectant propylene glycol, in larval hatching rate. For this, 2400 specimens of A. altiparanae were divided in two treatment groups. One fed with commercial ration (Laguna omnivores alevino®) - GC, and the other fed with same ration plus 5% marine fish oil (Campestre®) - GO. In order to obtain the embryos, the hormonal induction of the reproducers was carried out and the embryos were used in the phase of six somites. To perform the experiments eggs were placed in PVC incubators and inserted into Petri dishes containing: a) 50 mL of tap water and; b) containing 50mL of propylene glycol solution (1M or 2M) and then placed in incubators of type B.O.D. at the proposed temperatures, where they remained for a period of 12 hours, to test the sensitivity to cold and the action of the cryoprotectant in the cooling process. After the cooling period, all incubators with the samples were placed in a container with running water (27°C) until larvae were hatched. The larval hatching rate of GO was significantly (P <0.05) higher than GC in the treatments: Control (27ºC) (GC = 83.96 ± 14.11%, GO = 98.5 ± 2.94%); 15°C - S / Cryoprotectant (GC = 51.0 ± 26.78%, GO = 75.0 ± 18.5%), 1M (GC = 58.3 ± 33.28%, GO = 83.3 ± 27.21% ). At 5°C there was only hatching in the GO, at 1M concentration (GO = 18.39 ± 8.34%) and in GC treatment only (86.67 ± 9.42%) hatching occurred at 15ºC / 2M. We found that GC when cooled in solution of 2M PROP at 15 ° C, presented a significant similarity to the control. However, the embryos exposed to the cooling treatments produced larvae with morphological deformations. The experiments demonstrated that the A. altiparanae embryos exhibit sensitivity to low temperature exposure, that the cryoprotectant increased their resistance to cold and that the embryos from GO showed to be more resistant to cold, but more sensitive to cryoprotective concentration.
Key words: cooling, permeability, omega3, propylene glycol SESSION III. Refrigeration and cryopreservation Acknowledgements: This work had the financial support of the Foundation for Research Support of the State of São Paulo (FAPESP) (Proc.2015/10115-5) and the logistic support of
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 122 SESSION III. REFRIGERATION AND CRYOPRESERVATION P31 the National Center for Research and Conservation of Continental Fishes (CEPTA / ICMBIO), which provided the fish and facilities used in this study.
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 123 SESSION III. REFRIGERATION AND CRYOPRESERVATION P32
METHOD FOR LARGE-SCALE PRODUCTION OF STURGEON EMBRYOS USING CRYOPRESERVED SPERM
Konstantin KOVALEV1*, Natalya PRONINA1, Olga DOKINA1, Vladimir MILENKO1
1Filial of Freshwater Fisheries of Russian Federal Research Institute of Fisheries and Oceanography (Russia)
Cryopreserved sperm can be used to conserve biodiversity and for fish reproduction in case of lack of native sperm. Our cryobank was founded in 1989 and now collection of fish sperm consists of 32 species and 38 breeds including sperm samples from endangered and extinct species and populations. Cryopreserved sturgeon sperm in our collection is of a great value because a number of sturgeon species and population are close to extinction. Methods for cryopreservation of sturgeon sperm are well-developed and successfully used when a small batch of eggs should be fertilized by cryopreserved sperm in the laboratory conditions. There are no reports yet in which cryopreserved sturgeon sperm was applied in massive reproduction using industrial incubation facilities. The aim of our research was to elaborate a method that would allow a large-scale production of sturgeon embryos using cryopreserved sperm. Toward this aim, we carried out a number of experiments with gametes of different sturgeon species, namely Siberian sturgeon (Acipenser baerii), sterlet (A. ruthenus), beluga and kaluga sturgeons (Huso huso and H. dauricus). METHODS. Cryopreserved sperm used in experiments was frozen in basic cryodiluent developed in our laboratory. Basic cryodiluent was composed of (w/w) 0,1% sucrose, 0,08% KCl and 8,0% methanol. Before freezing sperm samples were diluted with cryodiluent at a ratio of 1:1 and poured in 1,5 ml vials. The sperm samples were frozen according to the two- step slow cooling freezing protocol (2-5oC/min from + 5 to - 15oC, then 10-20oC/min from - 15 to - 196oC). Sperm used for fertilization had a post-thaw motility more than 20% and a post-thaw fertilization rate higher than 77% (it was tested previously in laboratory conditions). Cryopreserved sperm of Siberian sturgeon (collected and cryopreserved in 2005), beluga (2009), kaluga (2018), sterlet (2017) and the eggs of Siberian sturgeon, sterlet and beluga were used for fertilization. To estimate eggs quality, we fertilized the same volume of eggs by native sperm when it was possible. The egg: sperm ratio was 100 g of egg and 6 ml of sperm. Sperm thawing was carried out in a water bath at 40°C for 60 s. Thawed sperm was diluted in water at the ratio of 1:40 or 1:70 (depending on sperm quality). Eggs were immediately fertilized by received solution during 3 minutes. Then eggs were washed with hatchery water, de-glued and incubated using techniques specific for a given fish hatchery. RESULTS. 1. Two batches of sterlet eggs (14400 ps or 200 g per batch) were fertilized by cryopreserved or native sterlet sperm. The third batch of sterlet eggs (21600 ps or 300 g) was fertilized by cryopreserved kaluga sturgeon sperm. Fertilization rates were equal to 73 and 87% for sterlet and kaluga cryopreserved sperm comparing to 71% for native sterlet sperm. 2. Fertilization of two batches of beluga sturgeon eggs (3000 ps or 100 g per batch) with cryopreserved or native sperm of beluga resulted in 81 and 97% developing embryos, respectively. 3. Fertilization of 10200 Siberian sturgeon eggs (300 g) with cryopreserved sperm of the same species gave 75% developing embryos.
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 124 SESSION III. REFRIGERATION AND CRYOPRESERVATION P32
Thus, fertilization procedures using cryopreserved sturgeon sperm were successfully optimized to inseminate large volumes of eggs at industrial sturgeon hatcheries. Application of this simple method looks promising in sturgeon propagation.
Keywords: fertilization, endangered species, cryobank, biodiversity.
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 125 SESSION III. REFRIGERATION AND CRYOPRESERVATION P33
IS IT POSSIBLE TO STORE SPOTTED WOLFFISH (Anarhichas minor) SPERM BY REFRIGERATION?
Wendy Ángela GONZÁLEZ-LÓPEZ1, José BEIRÃO2*
1 IRTA Sant Carles de la Rápita, 43540 Sant Carles de la Rápita, Tarragona, España 2 Faculty of Biosciences and Aquaculture, Nord University, NO – 8049 Bodø, Norway
Spotted wolffish (Anarhichas minor) reproduction in captivity is dependent on in vitro reproduction. For this reason, semen storage protocols are an important tool in spotted wolfish hatcheries management. Spotted wolfish sperm is already motile at stripping, and remains motile for up to two days after stripping. Sperm cryopreservation protocols has been the preferred methodology for this species sperm storage. Nonetheless, when sperm only needs to be stored for a few days, sperm refrigeration is a more practical and cheaper option. In this work we test the possibility to store spotted wolfish sperm by refrigeration. During stripping it is difficult to avoid urine contamination, in the first part we try to understand if urine contamination in the samples could be explain the period different individual sperm samples retain motility. Individual sperm samples (n=9) were kept refrigerated (2 ˚C) diluted 1:2 in the solution developed by Smith & Ryan (2010) and undiluted. Sperm motility was measured at different times until 50 h after collection. Urea, glucose and total protein was measured in the seminal plasma from the freshly collected sperm samples. Urea was also measured from pure urine samples and from seminal plasma samples collected from the testis of two sacrificed males. In the second part, individual sperm samples (n=3) were divided into four different sub-samples and stored refrigerated in the following solutions (a sodium based solution, a calcium based solution, a potassium based solution and a glucose based solution). The four solutions had similar osmolality and pH. Sperm motility was checked at different times until 50h after collection. In the third part, individual sperm samples (n=8) were stored refrigerated diluted and undiluted in the Smith & Ryan solution. Samples were analysed for motility, ATP and glucose at 0, 5, 10, 20 and 30 hours after collection. The contamination with urea could not explain the variability in the individual sperm samples storability. No different effect in the sperm storability was observed between the different solutions (sodium, calcium, potassium or glucose). Both ATP and glucose values remained stable during the storage time, thus the two days motility period in spotted wolffish is not limited by the ATP reserves. In all trials the diluted samples retain higher sperm motility and velocity values compared with the undiluted samples.
Keywords: sperm storability, ATP, Glucose, ions
Acknowledgements: Work supported by the WOLFSTORE project (AF0078) funded by MABIT program Norway. Wendy González was supported with a PhD scholarship from CONACYT (Mexico).
References: Smith CC & Ryan, MJ 2010. Evolution of sperm quality but not quantity in the internally fertilized fish Xiphophorus nigrensis. J Evol Biol, 23, 1759-71.
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 126 SESSION III. REFRIGERATION AND CRYOPRESERVATION P34
SPOTTED WOLFFISH (Anarhichas minor) SPERM CRYOPRESERVATION IN 5 ML CRYOVIALS
Stian FLENGSTAD1, Igor BABIAK1, José BEIRÃO1*
1 Faculty of Biosciences and Aquaculture, Nord University, NO – 8049 Bodø (Norway)
Spotted wolffish (Anarhichas minor) is considered a potential candidate for cold water marine aquaculture diversification in the North Atlantic. Under captivity, A. minor fails to spawn naturally and reproduction needs to be conducted in vitro. Due to low sperm volume available and asynchrony in gamete maturation, cryopreservation is used to secure sperm availability when eggs are spawned. On average, each female spawns between one to three L of eggs at the time. Currently, sperm is cryopreserved in 0.5 mL straws, and in order to achieve good fertilization rates, an approximate volume of either 2 mL fresh (Beirão and Ottesen, 2018) or 5 mL of cryopreserved sperm (Santana et al. 2019) is recommended for each L of eggs. This implies thawing a large volume of straws every time a batch of eggs needs to be fertilized with cryopreserved sperm. In order to facilitate A. minor hatcheries procedures, in this study we develop a protocol for sperm cryopreservation in 5 mL cryovials. Sperm was cryopreserved with the extender previously developed (Flengstad et al. 2018). Different freezing rates (1.5, 4.5 and 7.5 cm from the LN2) and thawing rates (5˚C for 10 min, 10˚C for 6 min, 15˚C for 4 min and 20˚C for 3.5 min) were tested. Results were evaluated in terms of % of motile cells and sperm velocity (VCL) with a CASA system, and in terms of sperm plasma membrane integrity (viability) by double staining the cells with propidium iodide and SyBr 14 under a fluorescent microscope. Sperm cryopreserved in the 5 mL cryovials at 1.5 cm from the LN2 and thawed at 10˚C for 6 min presented similar quality in comparison with the sperm cryopreserved with the traditional 0.5 mL straws (45.4% motile cells, 21.91 µm/s for sperm velocity and 75.5% of viable sperm).
Keywords: sperm motility, sperm viability, CASA, 0.5 mL straws
Acknowledgements: Work supported by the WOLFSTORE project (AF0078) funded by MABIT program Norway.
References: Beirão J., Ottesen, O.H. 2018 Optimization of a fertilization protocol for spotted wolffish (Anarhichas minor). Aquaculture 484: 133-138 Flengstad S., Babiak I., Beirão J. 2018 Efficiency of membrane stabilizers and antioxidant in spotted wolffish (Anarhichas minor) sperm cryopreservation. AQUA 2018, Montpellier, France Santana, Eggen B., Beirão J. 2017 Optimization of a sperm cryopreservation protocol for spotted wolffish (Anarhichas minor Olafsen, 1772). AE 2017, Dubrovnick, Croatia
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 127 SESSION III. REFRIGERATION AND CRYOPRESERVATION P35
IN VITRO SPERM MATURATION IN ACIPENSERIFORMES: FAMILY SPECIFIC FEATURE AND APPLICATION IN CRYOBANKING
Viktoriya DZYUBA1*, William L. SHELTON1,2, Jacky COSSON1, Marek RODINA1, Otomar LINHART1, Sergii BORYSHPOLETS1, Borys DZYUBA1
1 University of South Bohemia in České Budějovice, Faculty of Fisheries and Protection of Waters, South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses, Vodňany, Czech Republic 2 University of Oklahoma, Biology Faculty, Norman, USA
Specific anatomy of male excretory system in sturgeons determines natural mixing of testicular spermatozoa (TS) with urine. This mixing is required for TS maturation (ability to initiate motility and fertilize). We aimed our study first, to ascertain if this morphology and physiology are common in both extant families (Acipenseridae and Polyodontidae) of Acipenseriformes and second, to check if in vitro sperm maturation can be applied in practise. Wolffian duct (WD) sperm and TS were collected from sterlet (Acipenser ruthenus) and American paddlefish (Polyodon spathula). The in vitro maturation was performed by incubation of TS in the seminal fluid collected from WD sperm (already naturally mature) in sterlet and American paddlefish, then sperm motility was evaluated in 10 mM Tris, pH 8. Some individuals were euthanized, and dye was injected into the urogenital sinus to visualize the sperm and urinary ducts. We found that the deferent ducts of each species pass through kidneys before entering WDs, and TS is significantly more concentrated than the WD sperm. These two facts confirm a mixing process of sperm with urine that occurs before ejaculation. Motility of sterlet (n = 70) TS was not activated in an activating medium, but this capacity was acquired after in vitro maturation. In paddlefish (n = 4) prior to any in vitro maturation, two samples of TS showed motility activation at 10 – 20 % level, while TS from two other fish remained immotile. Among these same four samples, 100 % motility was observed after 5 min of in vitro maturation in three samples, but motility characteristics of the fourth sample did not improve even after prolonged incubation. We conclude that sterlet and paddlefish have similar morphology of urogenital system which includes natural mixing of sperm and urine. This mixing is a mandatory requirement for sperm maturation in sterlet but might be less important in paddlefish. The knowledge about sperm maturation process in sturgeons was applied during sperm cryobanking when sperm of Russian sturgeon (A. gueldenstaedtii) unable to initiate motility were collected. These sperm samples were incubated with urine that resulted in 50 % motility. Then the samples were frozen and post-thaw sperm motility was about 20 %, high enough for sperm cryobanking.
Keywords: Acipenseridae, Polyodontidae, sperm cryopreservation, sperm motility
Acknowledgements: The study was supported by the Czech Science Foundation (No. 16- 03754S), by the Ministry of Education, Youth and Sports of the Czech Republic (“CENAKVA”,
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LM2018099), by project Biodiversity (CZ.02.1.01./0.0/0.0/16_025/0007370) and by the Grant Agency of the University of South Bohemia in Ceske Budejovice (125/2016/Z).
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 129 SESSION III. REFRIGERATION AND CRYOPRESERVATION P36
THE GROWTH AND SURVIVAL RATE IN HATCHERY REARED NORTHERN PIKE (Esox lucius) LARVAE OBTAINED FROM 3 DIFFERENT LARGE-SCALE SPERM CRYOPRESERVATION METHODS
Gergely BERNÁTH1*, József MOLNÁR1, Levente VÁRKONYI1, Enikő SOLYMOSI 2, Borbála NAGY1, Tibor IZSÁK 1, Levente Zete LÁNG1, Zsolt CSENKI-BAKOS 1, Béla URBÁNYI1, Zoltán BOKOR1
1 Department of Aquaculture, Szent István University, 1 Páter Károly, H-2100 Gödöllő, Hungary 2 Szegedfish Ltd., 2. Nádvágó, H-6728 Szeged, Hungary
Northern pike (Esox lucius) a commercially important predator game fish. The dissection of testis is a traditional method for the collection of high quality and quantity sperm. Sperm cryopreservation can optimize and reduce the costs of the production. In our study, propagation was carried out at hatchery conditions (N= 5 males) using fresh (control) and cryopreserved sperm (5 mL: Polystyrene box and controlled-rate freezer (CRF), 10 mL: CRF). A species specific extender (150 mM glucose, 75 mM NaCl, 30 mM KCl, 1 mM Na2HPO4*12 H2O, 1 mM MgCl2*6H2O, 1 mM CaCl2*2H2O, 20 mM Tris, and 0.5% BSA, pH 8.0±0.2) developed in our former study was applied for freezing pike sperm. The growth (standard length, average body weight) and survival rate was recorded for 10 days at the moment of hatching, the end of the non feeding period and the end of a 5 days feeding period. A significantly lower body weight was recorded in the control group at the hatching and feeding period compare to the P. box (5 mL) and the CRF (10 mL). Similarly, a significantly higher body weight was observed using CRF (5 mL) at the feeding period than in the control. However, body weight in CRF 5 (mL) was significantly lower at hatching period than in the P. box and the CRF (10 mL). Correspondingly, the average standard length showed a similar tendency at all 3 sampling period. Furthermore, no significant difference in survival rate of larvae was observed between fresh and frozen groups at the end of the experiment (N= 1600, Control: 69%, P. box 5 ml: 80%, CRF 5 ml: 74%, CRF 10 mL: 74%). According to our study, large-scale sperm cryopreservation did not affect negatively the survival and growth rate of pike larvae at hatchery condition.
Keywords: Northern pike, large-scale sperm cryopreservation, larvae rearing
Acknowledgements: The work was supported by the GINOP-2.1.1-15-2015-00645 and by the European Fisheries Fund Fisheries Operative Programme III. axis, “European Fisheries Fund for Renewable Fisheries provided by the EU and Hungary”. The research was also funded by the Bolyai János Postdoctoral (BO/00508/18/4, Hungarian Academy of Sciences) and the ÚNKP-18-4 New National Excellence Program of the Ministry of Human Capacities Fellowships of Gergely Bernáth. The publication is supported by the EFOP-3.6.3-VEKOP-16- 2017-00008 project. The project is co-financed by the European Union and the European Social Fund.
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 130 SESSION III. REFRIGERATION AND CRYOPRESERVATION P37
THE EFFECT OF ARACHIDONIC ACID ON RAINBOW TROUT SPERMATOZOA ACTIVATION AND SHORT-TERM STORAGE
Olga BONDARENKO1*, Fabio HERRERA1, Jan MRAZ1, Zdenka MACHOVA1, Sergii BORYSHPOLETS1
University of South Bohemia in České Budějovice, Faculty of Fisheries and Protection of Waters, South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses, Zátiší 728/II, 389 25 Vodňany, Czech Republic
Arachidonic acid (AA) is one of the main participants in different cell types metabolism, including fish spermatozoa, being the substrate to produce prostaglandins, leukotrienes, etc. Moreover, oxidation of AA can lead to membrane damage followed by cell death. Thus, we were interested to investigate the role of AA and its oxidation products in fish spermatozoa viability during short-term storage (up to 48 h). In addition, AA and products of its oxidation are important secondary messengers involved in signaling cascades by inhibition of voltage gated Ca2+ channels. Since it is generally believed that Ca2+ channels are involved in trout sperm activation, the potential effect of AA in sperm motility was also under the study. To investigate the role of AA and its oxidation in rainbow trout spermatozoa storage, sperm from 10 males was incubated 20 min, 24 h and 48 h (4oC) with Arachidonic acid (AA, 10 uM) and inhibitors of AA oxidation such as Eicosatetraynoic acid (ETYA, 10 uM), Octadecynoic Acid (17-ODYA, 10 uM), Clotrimazole (Clo, 1 and 10 uM), and inhibitor of prostaglandin E formation - Aspirin (10 ug/ml). After incubation, sperm motility was activated in 20 mM Tris- 2+ HCl, pH 8.5 with/without 200 uM CaCl2, to distinguish whether increase of extracellular Ca can influence the inhibitory effect of AA. As a control, fresh sperm was activated in presence or absence of AA and inhibitors of its oxidation. To estimate if the amount of AA is changing during storage, the lipid composition analysis was performed according to Mraz and Pickova (2009), with using 22:2n-6 fatty acid as an internal standard. Despite expectations, AA and inhibitors of its oxidation (all but Clo) did not affect motility neither in control conditions nor after 24 h or 48 h of storage. Presence of Clo in both activation and incubation conditions led to significant decrease of spermatozoa motility parameters, that can be related to another Clo property such as inhibition of Ca2+ activated K+ channels. Analysis of lipid composition shows gradual and significant decrease in palmitoleic (C16:1) and α-linolenic acid (18:3n-3) and an increase in palmitic (C16:0) and DPA (C22:5n-3), but not AA, during 24 and 48 h of sperm storage in control. An increase of oleic acid was also detected in sperm stored with Clo 24 h. We suggest that AA is not directly involved neither in mechanisms of rainbow trout sperm motility activation nor in processes of sperm aging during short-term storage. Likely, clotrimazole affect spermatozoa motility rather by inhibition of Ca2+ dependent K+ channels then by inhibition of cytochrome P450 pathway of AA oxidation.
Keywords: Arachidonic acid, oxidation products, spermatozoa motility, lipid composition
Acknowledgements: The study was financially supported by the Ministry of Education, Youth and Sports of the Czech Republic - projects „CENAKVA“ (LM2018099), by project Biodiversity
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(CZ.02.1.01./0.0/0.0/16_025/0007370) and by the Grant Agency of the University of South Bohemia in Ceske Budejovice (125/2016/Z) and by the Czech Science Foundation (18- 12465Y).
References: Mraz J, Pickova J. 2009. Differences between lipid content and composition of different parts of fillets from crossbred farmed carp (Cyprinus carpio). 10.1007/s10695-008-9291-5.
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 132 SESSION III. REFRIGERATION AND CRYOPRESERVATION P38
INHERITANCE OF SPERM CRYORESISTANCE IN ZEBRAFISH (Danio rerio)
Bernadett PATAKI1*, Tímea KOLLÁR1, Zoran MARINOVIĆ1, Jelena LUJIĆ1, Gyöngyi GAZSI1, Roberta Izabella BERTA1, Béla URBÁNYI1, Ákos HORVÁTH1
1Department of Aquaculture, Szent István University, Páter Károly u. 1, Gödöllő H-2100, Hungary
The objective of this study was to investigate the inheritance of sperm cryoresistance in zebrafish (Danio rerio). A previous study has shown that the sperm of rainbow trout males that were produced from cryopreserved sperm had a higher post-thaw fertilizing capacity than that of males produced from fresh sperm. In order to investigate this effect in zebrafish, an F0 generation of 6 males was previously selected from 50 males based on the progressive motility of sperm measured with CASA (Computer-Assisted Sperm Analysis). For fertilization, 6 females were also selected and eggs from each female were separated into two and were fertilized with previously cryopreserved or fresh sperm from one of the F0 males, thus, creating families of full-sib F1 fish. Sperm from F1 fish was collected tubes individually, but was pooled later for fertilization and the sperm concentration measurements. The pooled sperm was cryopreserved using grayling extender (200 mM glucose, 40 mM KCl, 30 mM Tris, pH 8.0) and 8% methanol in a programmable freezer at a cooling rate of 10 C/min. Following thawing, sperm concentration, post-thaw motility and fertilizing capacity of sperm was tested. For fertilization 5000 spermatozoa/egg were used and also fresh sperm was used as a control. No significant difference (p=0.27) was found between the post-thaw progressive motilities of zebrafish sperm from individuals originating from fresh (B) (21±12%) or cryopreserved (A) (25±8%) sperm, neither (p=0.73) between the motilities of fresh sperm in group B (79±11%) and of group A (77±18%). The number of cells after thawing in group A was 5 4 x 105 spermatozoa/ l and 4 3 x 105 spermatozoa/ l in group B and no significant difference (P=0.56) was found between the concentrations. No significant difference (P=0.73) was found between the post-thaw fertilization percentages of group A (1±1%) and group B (1±3%), however, cryopreservation had a significant effect on motility and concentration. Our results show that in the first generation there is no significant difference between the sperm parameters of individuals originating from fresh or cryopreserved sperm.
Keywords: fertilization, motility, cell concentration, generations
Acknowledgements: The work was supported by the NKFIH K129127 as well as the EFOP- 3.6.3-VEKOP-16-2017-00008 project co-financed by the European Union and the European Social Fund.
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CHANGES IN SPERM CRYOPRESERVATION PROCEDURE FOR INDUSTRIAL NEEDS
Catherine LABBE1*, Guy DELHOMME 2, Alexandra DEPINCE 1, Lucie GAVIN-PLAGNE 2, Lionel GOARDON 3, Stéphanie KICA 1, Richard LEBOUCHER 2, Romain MORVEZEN 4, Li NA 2
1 INRA, Fish Physiology and Genomics, Campus de Beaulieu, 35000 Rennes, France. 2 IMV Technologies, Zone Industrielle Numéro 1 EST, 61300 Saint-Ouen sur Iton, France 3 INRA, UE PEIMA, Barrage du Drennec, 29450 Sizun, France 4 SYSAAF, LPGP INRA - Campus de Beaulieu, 35000 Rennes, France
Several efficient methods for rainbow trout sperm cryopreservation have been published and some are routinely used in aquaculture for genome preservation of valuable strains and to some extend for fry production. These methods were selected on the basis of their robustness in field conditions, with transportable and affordable material. In France, cryopreservation of aquatic resources has been centralized in a unique site where trained staffs ensure sperm cryopreservation and storage. As a consequence, the need for field condition robustness has decreased while the need to intensify the quantity of sperm processed in one day of work has increased. The objective of the present work was to change the current sperm cryopreservation method in order to meet this new requirement. The conventional raft cryopreservation tank (200 straws processed / run) was compared to the Digitcool programmable freezer (2000 straws / run). The concentration in the 500 µL straws was increased from 1.5 to 5x109 spermatozoa/mL, and the sperm/cryoprotectant volume ratio was increased while maintaining the final cryoprotectant concentration. Cryoprotectant contained either dimethyl sulfoxide (DMSO) or methanol (MeOH), with sucrose and liposomes (FreezeFish, IMV Technologies, France). Testicular sperm from neomales was used to test these cryopreservation conditions. Motility and fertilization rates assessed at eyed stage were used as estimators of thawed sperm quality. Our results show that cryopreservation with DMSO in the Digitcool programmable freezer yielded the same thawed sperm performance as the raft system (fertilization rate 83 versus 81 % of the fresh control), although the freezing curves were different. The Digitcool programmable freezer yielded less variable and better sperm performances than the raft system when MeOH was used as cryoprotectant (87 versus 56 % fertilization rate). The lowest sperm concentration in 7.5 or 10 % DMSO yielded the same high fertilization rates as the highest sperm concentration (fertilization rates from 84 to 94 % of the fresh control). In all, this set of experiments confirmed that the existing protocols are suitable for medium throughput use, while higher throughput methods with a programmable freezer and higher sperm concentration in the straws yield excellent fertilization rates. Additionally, cryoprotectants with DMSO are more robust in terms of freezing conditions than those with MeOH.
Keywords: Rainbow trout, Oncorhynchus mykiss, neomale, Digitcool, raft, DMSO, MeOH
Acknowledgements: Work funded by the FEAMP BIOGERM (2017, mesure 49) project and by the PIA CRB Anim (ANR-11-INBS-0003). Authors are grateful to Pablo PELISSIER (INRA PEIMA) and Cécile DURET (INRA LPGP) for testicular sperm preparation and embryo care. Jean RUCHE and the Milin Nevez trout farm provided eggs and fresh control sperm.
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 134 SESSION III. REFRIGERATION AND CRYOPRESERVATION P40
TRIALS FOR THE IMPROVEMENT OF PUFFERFISH (Takifugu alboplumbeus) SPERM EXTENDERS
Juan F. ASTURIANO1*, Víctor GALLEGO1, Manabu YOSHIDA2, Luz PÉREZ1
1 Universitat Politècnica de València. Instituto de Ciencia y Tecnología Animal. Grupo de Acuicultura y Biodiversidad. Camino de Vera s/n 46022 Valencia, Spain 2 Misaki Marine Biological Station. Graduate School of Science. University of Tokyo. 1024 Misaki-Koajiro. 238-0225 Miura, Kanagawa, Japan
In 1985, Morisawa described the ionic composition of the pufferfish sperm, which included 0.9 mM of magnesium. In 2003, Krasznai et al. proposed the composition of an extender called SLS (semen-like solution) used from then with this species. However, SLS does not include magnesium at all. Moreover, we found that the pH of the pufferfish sperm is exactly 7.5 (coinciding with the SLS pH proposed by Krasznai et al.), but the seminal plasma pH is 8. On the other hand, experiments carried out by Pérez et al. (this meeting) suggested that 8.5 is an indicated pH for the extenders of pufferfish sperm. Our objective was to optimize the pufferfish sperm diluents to get a longer preservation under refrigeration. We carried 3 experiments to assess the addition of several concentrations of magnesium and NaHCO3 (used in extenders for other fish species), as well as different pHs. Fish were caught in Misaki Bay (Miura, Japan) and maintained in aquaria. All the experiments started with the selection of sperm samples showing >80% of motility after activation with seawater and CASA-Mot evaluation. Samples were diluted 1:50 in the tested extenders and stored in eppendorfs at 4 ºC. Sperm motility was tested after 24, 48 and 72 h, 7, 10, 14 and 21 days, registering the percentage of motile cells (MOT) and some kinetic parameters (p-MOT, VAP, VCL, VSL). Experiment 1. Sperm samples (n=12) were preserved in SLS (as control), in SLS + 1 mM MgCl2 (imitating pufferfish seminal plasma), or in SLS + 20 mM NaHCO3. SLS and SLS+MgCl2 showed similar MOT results and kinetic parameters after 10 days of storage, while SLS+NaHCO3 already showed a reduction of motility. SLS caused the best results (>80% MOT) after 2 weeks. Experiment 2. Sperm samples (n=7) were preserved in SLS or in SLS + 1 or 2 mM MgCl2 with pH 7.5. No important differences were obtained, and all the extenders preserved MOT values >70% and high kinetic parameters after 3 weeks of storage. Experiment 3. Sperm samples (n=7) were preserved in SLS , or in SLS + 1 or 2 mM MgCl2. pH was adjusted at 7.5, 8 or 8.5, generating 9 combinations. Only SLS + 2 mM MgCl2 with pH 7.5 preserved motilities >70% after 14 days. Sperm kinetic parameters showed similar profiles. The use of Mg 2 mM and the pH measured in the sperm of this species (7.5) is recommended.
Keywords: Short-term preservation; fugu; magnesium; spermatozoa; Takifugu niphobles
Acknowledgements: To Sumangala Thiyakeswaran for making last CASA analyses. Generalitat Valenciana funded the stay in Japan of JFA, LPI and VG (BEST 2018/093, /112,
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 135 SESSION III. REFRIGERATION AND CRYOPRESERVATION P40
/124). VG has a postdoc grant from the MICIU (Programa Juan de la Cierva-Incorporación; IJCI-2017-34200).
Krasznai et al. 2003. Gadolinium, a mechano-sensitive channel blocker, inhibits osmosis- initiated motility of sea- and freshwater fish sperm, but does not affect human or ascidian sperm motility. Cell Motility and the Cytoskeleton 5: 232-243. // Morisawa 1985. Initiation mechanism of sperm motility at spawning in teleost. Zoological Science 2: 605-615.
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 136 SESSION III. REFRIGERATION AND CRYOPRESERVATION P41
LONG-TERM ANTIBIOTIC-FREE PRESERVATION OF RAINBOW TROUT MILT (Onchorhynchus mykiss)
Lucie GAVIN-PLAGNE1*, Na LI1*, Lionel GOARDON2, Laurent LABBE2, Eric SCHMITT1, Richard LE BOUCHER1
1 R&D department, IMV Technologies, L’Aigle, France 2 INRA Peima UE0937, Sizun, France
* [email protected] ; [email protected]
In salmonids production, the elimination of antibiotics use in daily farming practices is a strong focus. More and more, it is a farm commitment to obtain sustainability certifications. Tremendous progress has been made in grow-out production stages to develop efficient alternatives to antibiotics. However, in hatcheries, low quantity of antibiotics is used to preserve milt at 4°C. The current trial aimed to evaluate the efficiency of a new antibiotic- free extender (Bsol) on spermatozoa motility. Fresh stripped milt from 26 rainbow trout males (Onchorhynchus mykiss) were analysed after collection (d0) with salmonids settings on IVOS II®, an automatic-stage CASA system (IMV Technologies, L’Aigle, France). Milt was diluted with extender (1:3) and preserved at 4°C in GTBbag® (IMV Technologies, L’Aigle, France), fully filled with pure oxygen (5ml diluted milt filled in 80 ml GTBbag®). In the control batch, Storfish® (IMV Technologies, L’Aigle, France) was used as a preservation extender when in the tested batch Bsol was used. Spermatozoa motility and kinematic parameters were measured on d2, d5, d8, d15 and d22. To standardize the motility evaluation, kinematic parameters were recorded between 6 and 12 seconds after activation. To validate the repeatability of this duration, another trial recorded the motility decrease of spermatozoa during 27 seconds after activation (t0). Statistical analyses were performed on R software using mixed models including time and extender batches as fixed effects and male as a random effect. A significant effect of the time was observed. No significant difference was observed between extender batches except for d2. For total motility, the initial value on the day of collection (d0) was 95.1 ± 3.0 %, indicating a limited individual male variability (CV: 1.5 %). Preserved trout sperm (pooled batches) showed a total motility of 64.7 ± 12.8 % (d2), 56.9 ± 11.1 % (d5), 56.0 ± 7.8 % (d8), 35.3 ± 12.1 % (d15) and 7.6 ± 7.7 % (d22). There was no drastic total motility drop until 17 seconds (89.8 % ± 9.8 %) after activation. The final total motility (27 sec) dropped to 57.0 ± 19.5 % at d0. This study indicates the potential flexibility in egg fertilization industry with the use of GTBbag® (35 % motility at d15). The preservation conditions will be optimized in further experiments for a better milt preservation. Fertility rate and bacterial growth will be studied to complete these results. Trout farming can now move from almost antibiotic-free production to fully antibiotic-free fish production.
Keywords: milt activation, trout sperm, CASA system, liquid preservation, motility standardization, sperm velocity
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 137 SESSION III. REFRIGERATION AND CRYOPRESERVATION P42
EFFECT OF CRYOPRESERVATION ON EPIGENETIC PROFILE IN OVARIAN FOLLICLES OF ZEBRAFISH (Danio rerio)
Fernanda de MELLO*, Natalia PVM FARIA2, Leandro C GODOY3, Renata G MOREIRA2
1 University of Sao Paulo, Department of Physiology, Bioscience Institute, Brazil 2 Instituto de Biociências, Departamento de Fisiologia da Universidade de São Paulo, Centro de Biologia Marinha da Universidade de São Paulo (CEBIMar/USP), Brazil 3 Federal University of Rio Grande do Sul, Animal Science Department, Brazil
In fish, epigenetic mechanisms in ovarian follicle remain poorly understood. The gametes are carrying not only genetic but epigenetic information that will be transferred to the progeny. Cryopreservation of ovarian follicles is a promising method to preserve genetic material in fish. However, very little is known on the most with regards to possible epigenetic alterations, as well as the possible impacts of alterations generated by cryogenic temperature. The present study therefore aims to characterize the epigenetic profile and alterations generated by the cryopreservation process of the ovarian follicles on oogenesis in zebrafish. Expression of Dnmt1 (DNA methyltransferase 1), Dnmt3a1 (DNA methyltransferase 3a1), Hat1 (histone acetyl transferases 1) and Hdac1 (histone deacetylation 1) genes were used for characterized the epigenetic profile of fresh and cryopreserved ovarian follicles at stages 1, 2 and 3 of development. Fresh ovarian follicles showed a decrease on expression of the Hat1 (P=0.01), being lower in stage 3. A high expression of Hdac1 was observed in stage 1, already in stage 2, decreases its expression by 3 times and increases the expression in stage 3 by about 2 times. The Dnmt1 and Dnmt3a1 genes present a greater expression in stage 1 (P<0.001) with an abrupt decline in stage 2 and maintenance of the expression in stage 3 (P=0.3). After Vitrification the ovarian follicles showed a general increase in the expression of methylation genes (P<0.001) and histone modification genes (P=0.001), being the ovarian follicles in stage 2 which showed a greater increase, altering the pattern of methylation genes (P<0.0001). Cryopreserved ovarian follicles of stages 1 and 3 differed from fresh ovarian follicles (P<0.001); for Hat1 and Hdac1 genes there was no difference in expression between stage 2 and 3, but differed from the control (P<0.001). Results showed a decrease of methylation throughout the follicular development in zebrafish, evidenced by expression of Dnmt1 and Dnmt3a1 genes. The dynamics of expression between Hat1 and Hdac1 genes in fresh ovarian follicles suggest that chromatin is more compacted in stage 1 decreasing in stages 2 and 3, that is in stages 2 and 3 genes the chromatin is less compacted therefore the DNA is available for transcription. After cryopreservation the epigenetic profile changes significantly with increasing expression of methylation genes in stages 2 and 3, and acetylation / deacetylation in the three stages analyzed. Cryopreservation generated changes in the epigenetic profile by increasing the expression of methylation genes and histone modification.
Keywords: Vitrification; Oogenesis; DNA Methylation; Modification of Histones.
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Acknowledgements: The authors would like to thank the Foundation for Research Support of the State of Sao Paulo (FAPESP).
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 139 SESSION III. REFRIGERATION AND CRYOPRESERVATION P43
COMPARATIVE ANALYSIS OF GLOBAL DNA METHYLATION IN SPERMATOZOA FROM FISH, MOLLUSCS, BIRD AND MAMMALS: EFFECT OF CRYOPRESERVATION
Alexandra DEPINCÉ1*, Hélène JAMMES2, Catherine LABBÉ1
1 INRA, UR 1037 Fish Physiology and Genomics, F-35000 Rennes, France 2 INRA ENVA, UMR 1198 Biology of Reproduction and Development, F-78352 Jouy en Josas, France
Spermatozoa show high differences in morphology and structure between species. Nevertheless, they have one common goal: successful fertilization and offspring development. The highly differentiated mature spermatozoa carry not only the genetic, but also the epigenetic information that is to be transmitted to the embryo. DNA methylation is one epigenetic actor associated with sperm nucleus compaction, gene silencing, and prepatterning of embryonic gene expression. Therefore, the stability of this mark towards reproductive biotechnologies is a major issue in animal production. The aim of this study was (i) to understand how different species with different sperm nucleus organizations may differ in their methylation pattern, ii) to investigate whether spermatozoa show a specific pattern of DNA methylation compared to somatic cells and iii) to explore the stability of DNA methylation in relation to sperm cryopreservation. For this purpose, we performed a comparative analysis of DNA methylation in semen of species from different phyla: 4 mammals (bull, ram, goat, boar), 2 fishes (rainbow trout, zebrafish), 1 bird (chicken) and 2 molluscs (oyster, great scallop). Whole DNA methylation level was estimated using LUMA (LUminometric Methylation Assay) which is based on differential DNA cleavage by methylation-sensitive restriction enzyme HpaII and its methylation-insensitive isoschizomer MspI. After enzymatic digestion, the amount of DNA breaks in CCGG sequences obtained with each enzyme was assessed with a polymerase extension assay by pyrosequencing (Qiagen PyroMark Q96 ID). Despite the high compaction of sperm chromatin in all species, we found significantly different levels of DNA methylation between species. Fish sperm differs with very high levels of global methylation (88.0% for rainbow trout and 78.7% for zebrafish). Molluscs have extremely low levels of global methylation (e.g. 15.7% for oysters); whereas the mammals and birds studied show intermediate levels (e.g. 67.5% for ram and 55.1% for chicken). Similarities in methylation levels between fish, or between mammals, suggest that the global methylation rate of spermatozoa would be conserved within the same phylogenetic group. The global DNA methylation level of the spermatozoon would therefore be related to the species rather than to its highly condensed sperm cell nature. We also point out that spermatozoa and somatic tissues of the same species have similar levels of global DNA methylation. Spermatozoa follow a species-specific pattern rather than displaying a profile that would sign a gamete status. Thanks to this multi-species comparison, we can propose that no sperm-specific tendency (hypermethylation for example) emerges. Finally, we show that global DNA methylation was not affected after cryopreservation except for rainbow trout where cryopreservation induces a slight but significant hypermethylation (paired Wilcoxon test, p value = 0.0039).
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 140 SESSION III. REFRIGERATION AND CRYOPRESERVATION P43
Keywords: epigenetic, biotechnology, gamete.
Acknowledgements: Funding support of “Investissements d’Avenir”ANR-11-INBS-0003 (CRB- Anim 2013-2019); M. Suquet and M. Boulais (IFREMER, Argenton, France); T. Joly (ISARA, Lyon, France); H. Acloque (INRA, Toulouse, France); H. Kiefer (INRA, Jouy en Josas, France); A. Fatet and F. Bordères (INRA, Lusignan, France); E. Blesbois, A. Thélie and I. Grasseau (INRA, Nouzilly, France).
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 141 SESSION III. REFRIGERATION AND CRYOPRESERVATION P44
THE RESPONSE OF Crassostrea angulata (PORTUGUESE OYSTER) LARVAE TO CRYOPROTECTANTS EXPOSURE AND CRYOPRESERVATION
Catarina ANJOS1,2*, Daniel DUARTE1, Patricia DIOGO1, Domitilia MATIAS2, Elsa CABRITA1
1 Centre of Marine Sciences, University of Algarve, 8005-139 Faro, Portugal 2 Portuguese Institute for Sea and Atmosphere, Av 5 de Outubro, 8700-305 Olhão, Portugal
The Portuguese oyster (Crassostrea angulata) was one of the most important shellfish commercial species. However, its natural populations collapsed due to pathologies onset and poor remediation and management measures. Therefore, the conservation of this genetic resource is important not only for aquaculture purpose but also for natural restocking. Larvae cryopreservation is advantageous over gametes since it allows the availability of diploid organisms immediately after thawing. This study aims to develop a larvae cryopreservation protocol for Crassostrea angulata. The toxicity of two different cryoprotectants (CPAs), namely 20% (v/v) dimethyl sulfoxide (DMSO) and 20% (v/v) ethylene glycol (EG), was investigated after CPTs exposure (3 min) and after freezing/thawing. All extenders contained 2% (w/v) polyvinylpyrrolidone 40000 MW (PVP-40) and 400 mM sucrose diluted in milli-Q water. Trochophore and D-larvae in artificial seawater (ASW) (n=5) were added to each extender in a 1:1 (v/v) proportion, in a final concentration of 60000 larvae/ml. Larvae were evaluated according to morphology and motility. Samples were cryopreserved after 3 min of equilibration (at 4°C) in 0.5 ml French straws in a programmable biofreezer, with a cooling rate previously established for Crassostrea gigas (Labbé et al., 2018). Trochophore and D-larvae quality was evaluated 1 h after thawing. There were no morphological alterations in larvae exposed to cryoprotectants. However, prior to cryopreservation, motility was affected, showing alterations in larvae velocity and swimming behavior (exposed velum), suggesting a stress response to the presence of CPAs and cryopreservation. Our study represents the first report on a successful cryopreservation protocol for Portuguese oyster larvae, yielding motile larvae, although with some morphological alterations. Further research should focus on the use of stress markers for post-thaw larvae quality evaluation.
Keywords: Trochophore; D-larvae; Ethylene glycol; Dimethyl sulfoxide.
References: Labbé, C., Haffray, P., Mingant, C., Quittet, B., Diss, B., Tervit, H. R., Adams, S. L., Rimond, F. and Suquet, M. (2018). Cryopreservation of Pacific oyster (Crassostrea gigas) larvae: Revisiting the practical limitations and scaling up the procedure for application to hatchery. Aquaculture 488, 227–234.
Acknowledgements: This research and D. Duarte were supported by 0139_VENUS_5_E (Interreg POCTEP), ASSEMBLE+ JRA2-H2020-INFRAIA-2016-2017 (No 730984), EBB- EAPA_501/2016 (Interreg Atlantic Area) and received national funds through FCT - Foundation for Science and Technology through project CCMAR/Multi/04326/2013. Catarina Anjos and Patricia Diogo were supported by FCT through (SFRH/BD/130910/2017) and (SFRH/BD/97466/2019) respectively.
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 142 SESSION III. REFRIGERATION AND CRYOPRESERVATION P45
OXIDATIVE STRESS IN CRYOPRESERVED SEMEN OF NORMAL MALES AND SEX-REVERSED FEMALES RAINBOW TROUT IN RELATION TO GLUCOSE CONCENTRATION IN THE EXTENDER
Sylwia JUDYCKA1*, Mariola SŁOWIŃSKA1, Joanna NYNCA1, Ewa LISZEWSKA1, Andrzej CIERESZKO1
1 Department of Gametes and Embryo Biology, Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, Olsztyn, Poland
Cryopreservation induces oxidative stress on sperm structure due to significant reactive oxygen species (ROS) generation. Oxidative stress can be induced by osmotic stress, however such effects were not studied for fish semen. The aim of the current study was to determine the sperm quality of normal males and sex-reversed females (SRF) rainbow trout measured as ROS+ production in semen cryopreserved at different osmolalities of extender - optimal (0.15M), low (0.11M) and high (0.19 and 0.21M, for SRF and normal males rainbow trout, respectively) glucose concentrations in 7.5% methanol (GM extender). Semen was diluted in GM extender at three concentrations of glucose (0.11M, 0.15M and 0.19 or 0.21M, for SRF and normal males, respectively) in 7.5% methanol. The final sperm concentrations in the straw were 3.0 and 1.0 × 109 spermatozoa ml-1, for SRF and normal males, respectively. Diluted semen was loaded into 0.5 ml plastic straws, equilibrated for 15 min and frozen in liquid nitrogen vapour for 5 min. The straws were thawed by immersion in a water bath at 40°C for 10 s. The oxidative stress in fresh, equilibrated and frozen/thawed sperm was measured using a portable flow cytometer Muse Cell Analyzer (Millipore, Billerica, MA, USA). The ROS+-positive spermatozoa of fresh semen were 3.3 ± 2.8 % and 3.4 ± 1.2 %, for SRF and normal males, respectively. During equilibration we observed the values of ROS+ within the range 4.3-5.4 % and 4.8-7.1 %, for semen of SRF and normal males, respectively. The cryopreservation of semen caused a significant increase (p<0.05) of ROS+ values in sperm of both tested groups compared to equilibrated semen. No significant differences between glucose concentrations were determined for SRF sperm (8.5-9.8 %). However, for the semen of normal males, the lowest values of ROS+ after cryopreservation were observed at 0.15M of glucose (13.5 ± 4.9 %) compared to 19.0 ± 5.4 % and 17.4 ± 7.0 %, for concentrations of glucose 0.11M and 0.21M, respectively. Moreover, we recorded significantly higher ROS+ production after cryopreservation at 0.11 and 0.19M of glucose in normal males than SRF. Our results clearly indicate, that oxidative stress in SRF and normal males sperm is caused by cryopreservation. Moreover, in the case of normal males rainbow trout sperm, the conditions of cryopreservation (glucose concentration) strongly affect the production of ROS+. It has to be also underlined that spermatozoa of SRF and normal males rainbow trout appeared to differ in response to both cryopreservation and osmotic stress.
Keywords: spermatozoa, cryopreservation, ROS+, flow cytometry.
Acknowledgements: This work was supported by funds of the National Science Centre granted on research project 2018/29/N/NZ9/00761 and funds appropriated to the Institute of Animal Reproduction and Food Research, Polish Academy of Sciences.
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 143 SESSION III. REFRIGERATION AND CRYOPRESERVATION P46
LABORATORY EGG INCUBATION TECHNIQUE IN STERLET (Acipenser ruthenus)
Yu CHENG1,2*, Miaomiao XIN1,2, Marek RODINA1, Vladimíra TUČKOVÁ1, Vojtěch KAŠPAR1, Otomar LINHART1
1 University of South Bohemia in Ceske Budejovice, Faculty of Fisheries and Protection of Waters, South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses, Research Institute of Fish Culture and Hydrobiology, Zatisi 728/II, 389 25 Vodnany, Czech Republic 2 Sino-Czech Joint Laboratory of Fish Conservation and Biotechnology: Key Laboratory of Freshwater Biodiversity Conservation, Ministry of Agriculture of China, Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Wuhan, China
In present study, sterlet Acipenser ruthenus was used as a model fish to 1) compare eggs incubation and larval development at 17 °C in Petri dishes incubated in tank (all 12 dishes in 300 liter running dechlorinated water), in Q-cell incubator box and in the air-conditioned laboratory (in both conditions each dish placed in plastic box with 300 ml dechlorinated water) and 2) develop management technique for easy incubation of eggs up to hatching in Petri dishes in laboratory condition. Until now, there is no publication about the proper technique of fish eggs incubation on Petri dishes during experiments in laboratory condition. The comparison of sturgeon embryos incubation in Petri dishes in small volume of water in plastic box and in large volume water in tank has not yet been reported. Development of simple incubation method in sturgeon is important for performing extensive laboratory experiments in future. Eggs from three individual females (not pooled) were fertilized by pooled sperm from four males with motility higher than 80% in 4 replicates. The results showed that no differences (P > 0.05) in neurulation and cleavage rates were detected between tank and air-conditioned incubation laboratory. Further, no significant differences (P > 0.05) in cleavage, neurulation and hatching were observed between air-conditioned laboratory and Q-cell incubator box. These results illustrated that the use of Petri dish placed each in a small plastic box with 300 ml of dechlorinated water in air-conditioned laboratory is sufficient for successful sterlet eggs incubation. It was very important to follow the principles to: 1) fertilize eggs from female individually, because they cannot be pooled consistently; 2) place the eggs in the Petri dish evenly after fertilization, so that they did not clump together; 3) change water after 24 h during the incubation but not later in neurulation (48 h); 4) remove the non-developing embryos just after neurulation and change the water after neurulation until hatching every day.
Keywords: Artificial reproduction, embryonic development, neurulation, egg incubation, sturgeon
Acknowledgements: The study was supported by the Ministry of Education, Youth and Sports of the Czech Republic (“CENAKVA”, LM2018099), by project Biodiversity (CZ.02.1.01./0.0/0.0/16_025/0007370) and by the Grant Agency of the University of South Bohemia in Ceske Budejovice (125/2016/Z).
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 144 SESSION III. REFRIGERATION AND CRYOPRESERVATION P47
QUANTITY DOSING OF ENDOCELLULAR CRYOPROTECTANTS DURING CRYOPRESERVATION OF FISH SPERM
Aleksandra A. KRASILNIKOVA1*
1 Federal State Budgetary Institution of Science «The Federal research center Southern scientific center of the Russian Academy of Sciences», 41 Chekhov Street, Rostov-on-Don, 344006 Russia
Increasing the viability of the frozen-thawed cells is a special case of solving the problems of cryobiology and is based on the optimization of many interrelated biological and physicochemical parameters. There is a number of issues, the solution of which will allow to develop and standardize the technology of cryopreservation of reproductive cells of male fish. Obviously, an excessive amount of tread, as a toxicant, adversely affects the quality of sperm after a double temperature shock. Consequently, a metered or dosed quantity of the protector in the composition of the cryoprotective media, which is necessary for binding the intracellular fluid in the sex cells, must increase their (cells’) quality after cryopreservation. The goal of the presented research was determining the amount of intracellular water in the fish sperm of different ecological groups and the amount of endocellular protectors, necessary to bind the water in the cell. An excessive number of cryoprotectors, especially of penetrating action, such as DMSO, methanol, as well as toxicants, adversely affect the quality of sperm after double thermal shock. In cryomedia comprising a basic solution, sucrose, mannitol, DMSO and the yolk, on the basis of the received data on the content of intracellular water, the content of DMSO (for the sperm of great sturgeon – 3%, for Russian sturgeon – 4%) was reduced. When the seminal fluid of great sturgeon was frozen in a standard cryomedia containing 10% of dimethyl sulfoxide (DMSO), the mobility was 75%. During cryopreservation with a reduced amount of penetrating protector, an increase in mobility up to 82% is observed. Sperm motility of Russian sturgeon, frozen in a cryoprotective media containing 10% of DMSO was 70%, while with the preserving cryomedia with 4% of DMSO the mobility increased to 78%. The efficiency of reduction of toxic substances in the composition of cryomedia has led to increased viability of frozen-thawed cells. The obtained results allow recommending the adjustment of the concentrations of penetrating protector in cryoprotective solution depending on the amount of intracellular water.
Keywords: Cryopreservation, Fish Sperm, Cryoprotectant, Sturgeon, Survival, Preservation of the Gene Pool
Acknowledgements: Work is performed with the use of the Bioresource collection of rare and endangered species SSC RAS No. 73602 with the financial support of RFBR, research project No. 19-016-00208 (quantity dosing of endocellular cryoprotectants) and grant MK- 68.2019.11 (the freezing rate).
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 145 SESSION III. REFRIGERATION AND CRYOPRESERVATION P48
CRYOPROTECTANTS USED IN ZEBRAFISH SPERM CRYOPRESERVATION MODULATE OFFSPRING SKELETAL DEVELOPMENT
Patricia DIOGO1*, Gil MARTINS1, Rita NOGUEIRA1, Ana MARREIROS2,3, Paulo Jorge GAVAIA1,2, Elsa CABRITA1
1Centre of Marine Sciences, University of Algarve, 8005-139 Faro, Portugal 2Department of Biomedical Sciences and Medicine, University of Algarve, 8005-139 Faro, Portugal 3Algarve Biomedical Center, Campus Gambelas, 8005-139 Faro, Portugal
In the last two decades, thousands of new mutant and transgenic zebrafish lines were developed challenging bioterios space management. Sperm cryopreservation can support this challenge. The synergy obtained by the combination of cryoprotectants is a successful strategy that can be beneficial on the optimization of zebrafish sperm cryopreservation. Therefore, the objective of this study was to select the most adequate cryoprotectant combination that generates high-quality offspring with normal skeletogenesis. Sperm was cryopreserved and stored using an electric ultrafreezer (-150 ºC) with a cooling rate of -66 ºC/min, as previously established by our group (Diogo et al., 2018). Among the several N,N- dimethylformamide (DMF) concentrations studied (5, 7.5, 10, 12.5 and 15%), 12.5% and 15% DMF yielded the highest post-thaw sperm quality and hatching rates. For these two concentrations, the presence of bovine serum albumin (1%), egg yolk (10%), glycine (30 mM) and bicine (50 mM) was evaluated for post-thaw sperm motility, viability, in vitro fertilization success and offspring skeletal quality (30 days post-fertilization). The extender with 15% DMF resulted in offspring with a lower incidence of deformed arches and severe skeletal malformations (lordosis, kyphosis and scoliosis), which suggests a high capacity to protect the cell against cold stress and DNA damage. Bicine and egg yolk were the non-permeating cryoprotectants with the highest post-thaw sperm quality. The use of these compounds resulted in a reduction in vertebral fusions, compressions and severity of skeletal malformations in the offspring. In conclusion, 15% DMF with 50 mM bicine was beneficial for the quality of zebrafish offspring sired by cryopreserved sperm with a -66 ºC/min freezing rate. To the best of our knowledge, this is the first report on skeletal development of the offspring sired by cryopreserved sperm performed with different extender compositions in zebrafish.
Keywords: Cryopreservation, sperm, in vitro fertilization, skeletal malformations.
References: P Diogo, G Martins, R Nogueira, I Quinzico, PJ Gavaia, E Cabrita (2018) Electric ultrafreezer (-150ºC) as an alternative for zebrafish sperm cryopreservation and storage. J Fish Physio Biochem 44 (6) 1443-1455.
Acknowledgments: P. Diogo was supported by Portuguese Foundation for Science and Technology (FCT) through the doctoral grant SFRH/BD/97466/2019. This study was partly funded FCT through UID/Multi/04326/2019 and from the operational programs CRESC Algarve 2020 and COMPETE 2020 through project EMBRC.PT ALG-01-0145-FEDER-022121.
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 146 SESSION III. REFRIGERATION AND CRYOPRESERVATION P49
CRYOPRESERVATION PROTOCOLS OF ZEBRAFISH GAMETES — A SYSTEMATIC REVIEW
Danilo P. STREIT JR.1*, Rômulo B. RODRIGUES1, Paula LASSEN1, Lis S. MARQUES1, Andréa G. GALUPO1, Leandro C. de GODOY1, Tiantian Zhang2
1Animal Science Research Program of Federal University of Rio Grande do Sul (PPGZoot/UFRGS), Porto Alegre - Brazil 2Faculty of Science and Technology, Bournemouth University, UK
Cryopreservation has been used to establish genetic banks of fish, in order to preserve endangered species and optimizing reproductive management in fish farms. Objective: The aim of this systematic review is to perform an updated approach to the main cryopreservation protocols of gametes, embryos and gonadal tissue used in zebrafish. Methods: Electronic searches in the PubMed, Science Direct, Scopus, and Google Scholar databases were performed with the keyword combination: cryopreservation, zebrafish, Danio rerio, vitrification, slow freezing and cryoprotectant. There were no restrictions on publication date, and the language was restricted to Portuguese, English or Spanish. Results: Pubmed resulted in 21 articles; Science Direct in 25 articles; Scopus in 38 articles; and Google Scholar in 43 articles. Forty-four articles were excluded because they were duplicates and 28 were excluded through the title and abstract analysis and 31 were excluded through the text analysis. The remaining 24 articles were used in the systematic review; an article found in the selected references was added in the study, totaling 25 scientific articles. The articles were distributed in six groups: ovarian follicle (22.22%), blastomer (22.22%), embryo (18.51%), semen (14.81%), primordial germ cell (11.12%), and ovarian tissue (11.12%). In the studies two cryopreservation methods were investigated: slow freezing (n=17) and vitrification (n=11). The main cryoprotectants used in cryopreservation protocols were dimethylsulfoxide (n=20), methanol (n=16), ethyleneglycol (n=9), propyleneglycol (n=8) and sucrose (n=6). Conclusion: The cellular viability obtained in the studies was quite variable, probably due to the methodological differences and the particularities of each study, which does not allow a safe evaluation of the efficiency of these protocols. However, among the zebrafish biological systems evaluated, only embryo cryopreservation did not show viability after warming. Scientific evidence presented in this review may contribute to further research on zebrafish cryopreservation.
Keywords: Blastomer, Danio rerio, DMSO, embryo, methanol, semen
Acknowledgements: Research funding by the CAPES and CNPq
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 147 SESSION IV. OOGENESIS AND EGG QUALITY P50
CHANGES IN mRNA ABUNDANCE OF SELECTED TRANSCRIPTS DURING OOCYTE AGEING IN ZEBRAFISH Danio rerio
Swapnil Gorakh WAGHMARE1*, Azin Mohagheghi SAMARIN1, Azadeh Mohagheghi SAMARIN1, Thao Vi NGUYEN2, Roman FRANEK1, Martin PSENICKA1, Tomas POLICAR1, Julien BOBE2
1 University of South Bohemia in Ceske Budejovice, Faculty of Fisheries and Protection of Waters, South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses, Research Institute of Fish Culture and Hydrobiology, Vodňany, Czech Republic 2 INRA, UR1037, Laboratory of Fish Physiology and Genomics, Campus de Beaulieu, 35042 Rennes Cedex, France
Egg quality can be highly affected by the oocyte age after ovulation. So far, little is known about the effect of post-ovulatory oocyte ageing on fertilizing ability and larval development at the molecular level. Oocyte ageing may lead to changes in the mRNA abundance of specific transcripts and thereby result in loss of developmental potential. In the current study the possible changes in the mRNA abundance of selected transcripts were examined during the progress of oocyte ageing in zebrafish Danio rerio. Oocytes were incubated in vitro for 6 hours post-stripping (HPS) at 26 ˚C. The unfertilized oocytes, 2-h embryos and 24- h embryos originating from different aged oocytes were sampled. The hatching rate was 80% for the eggs fertilized immediately after stripping and significantly decreased to 16% after 4 hours of the egg storage. The complete loss of egg viability occurred at 6 HPS. In addition, embryo mortality and larval malformation rates significantly increased with ageing and all larvae originating from 4 HPS oocytes were malformed. The gene related to chromosome segregation, mad2, showed significant decrease in relative mRNA abundance in 3-4 HPS unfertilized oocytes as well as in the 2-h post fertilization embryos originated from 3-4 HPS oocytes. The relative mRNA abundance of gene/s related to oxidative stress (cat), epigenetic modifications (dicer, hat1 and sirt1), spindle and chromosome abnormality (mad2) and apoptosis (caspase3a and wdr6) showed significant decrease in their mRNA abundance in 2-h embryos originating from aged oocytes. The relative mRNA abundance of gene/s related to oxidative stress (cat) and epigenetic modifications (tet3, hdac9b and has3) showed significant decrease in 24-h embryos originating from aged oocyte. The results indicated that oxidative stress, apoptosis and epigenetic modifications are worth to be deeply examined in the future studies for their possible involvements in the progress of fish oocyte ageing.
Keywords: developmental potential, egg quality, embryo mortality, hours post-stripping SESSION IV. Oogenesis and egg quality Acknowledgements: This study was financially supported by the Ministry of Education, Youth and Sports of the Czech Republic - project “CENAKVA” (LM2018099), project Reproductive and Genetic Procedures for Preserving Fish Biodiversity and Aquaculture (CZ.02.1.01/0.0/0.0/ 16_025/0007370) and by the Ministry of Agriculture of the Czech Republic: project NAZV (QK1710310).
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 148 SESSION IV. OOGENESIS AND EGG QUALITY P51
THE ROLE OF OXIDATIVE STRESS ON THE PROGRESS OF OOCYTE AGEING IN COMMON CARP Cyprinus carpio
Azin Mohagheghi SAMARIN1*, Azadeh Mohagheghi SAMARIN1, Tone-Kari Knutsdatter ØSTBYE2, Bente RUYTER2, Sabine SAMPELS3, Viktoriia BURKINA1, Miroslav BLECHA1, David GELA1, Tomas POLICAR1
1 University of South Bohemia in Ceske Budejovice, Faculty of Fisheries and Protection of Waters, South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses, Research Institute of Fish Culture and Hydrobiology, Vodňany, Czech Republic 2 Nofima (Norwegian Institute of Food, Fisheries and Aquaculture Research), Ås, Norway 3 Department of Molecular Sciences, Swedish University of Agricultural Sciences, Uppsala, Sweden
Decreasing egg quality following oocyte ageing is a major restricting factor for the breeding programs. The mechanisms behind this process has not yet been clarified. Oxidative stress has been proposed as the initiator of the oocyte ageing process in higher vertebrates. To examine the possible involvement of oxidative stress on the progress of oocyte ageing, oocytes from 6 females common carp Cyprinus carpio were incubated in vivo for 14 hours post-ovulation (HPO) and in vitro for 10 hours post-stripping (HPS) at 20 °C. Batches of the stored eggs were fertilized separately and the embryo survival rates were examined. The relative mRNA levels of candidate transcripts involved in oxidative injury, mitochondrial function and stress response were quantified during the ageing of the oocytes by real-time PCR. The possible alteration in the oxidation status of the oocytes during ageing was also studied. In addition, the activity of antioxidant enzymes was evaluated. Hatching rates were over 65% up to 4-6 HPO and finally dropped to 1.3% at 12-14 HPO. Hatching rates were more than 70% for the eggs stored in vitro up to 6 HPS and then decreased to 21.3% at 10 HPS. The relative mRNA abundance of the genes related to the oxidative injury and stress response (hsp70, sod and gpx1) were not significantly altered through oocyte ageing (P > 0.05). In addition, the amount of thiobarbituric acid reactive substances (TBARS), an indicator of lipid peroxidation, and the amount of carbonyls, which show the extension of protein oxidation, did not change with elapsing time following ovulation. The activity of antioxidant enzymes, catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPX) and glutathione reductase GR, also remained constant during oocyte ageing. The lack of response of both activities of antioxidant enzymes and oxidation products during oocyte ageing strengthens the conclusion that oxidative stress is unlikely to be a main factor determining the progress of oocyte ageing in common carp.
Keywords: antioxidant enzymes, Cyprinus carpio, mRNA abundance, oocyte ageing, oxidation products
Acknowledgements: The study was financially supported by the Ministry of Education, Youth and Sports of the Czech Republic - project “CENAKVA” (LM2018099), project Reproductive and Genetic Procedures for Preserving Fish Biodiversity and Aquaculture
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 149 SESSION IV. OOGENESIS AND EGG QUALITY P51
(CZ.02.1.01/0.0/0.0/16_025/0007370), the Ministry of Agriculture of the Czech Republic: project NAZV (QK1710310) and by NF-CZ07-MOP-3-184-2015 and NF-CZ07-ICP-3-185-2015.
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 150 SESSION IV. OOGENESIS AND EGG QUALITY P52
LIPID AND PROTEIN OXIDATION CHANGES DURING OOCYTE AGEING IN AFRICAN CATFISH Clarias gariepinus
Azadeh Mohagheghi SAMARIN1, Azin Mohagheghi SAMARIN1*, Sabine SAMPELS2, Cheila Alexandra ALBUQUERQUE DE ALMEIDA1, Tomas POLICAR1
1 University of South Bohemia in Ceske Budejovice, Faculty of Fisheries and Protection of Waters, South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses, Research Institute of Fish Culture and Hydrobiology, Vodňany, Czech Republic 2 Department of Molecular Sciences, Swedish University of Agricultural Sciences, Uppsala, Sweden
Oocyte ageing which refers to the time interval between ovulation and fertilization has been identified as the most important factor affecting egg quality of several fish species after ovulation. The processes and mechanism of oocyte aging have been poorly understood. Increased Reactive Oxygen Species (ROS) and the consequent oxidative damage has been proposed as the initiator of oocyte ageing process in other vertebrates. To identify the role of oxidative stress in the progress of oocyte ageing, the oxidation status of African catfish oocytes was investigated during post-stripping ageing. Stripped ova of 5 females were stored separately at 25 °C and fertilized at 0, 2, 4, 8 and 16 hours post-stripping (HPS). Egg viability percentages and larval malformation rates were examined. TBARS as a marker of lipid oxidation and carbonyls, which show the extent of protein oxidation were measured based on spectrophotometric assays. Additionally, possible changes in the mRNA levels of transcripts involved in oxidative damage and stress response were investigated during oocyte ageing by real-time PCR. A complete loss of egg viability occurred at 16 hours post- stripping (HPS). Embryo mortality and larval malformation rates increased significantly during oocyte ageing and were measured as the highest in the most aged oocytes. With elapsing time following ovulation, the amount of TBARs and carbonyls did not change significantly (P > 0.05). Additionally, our results demonstrated no significant changes in the mRNA levels of hsp70 and cox1 during the progress of oocyte ageing. According to the obtained results in the current study, oxidative stress is not the main initiator of the oocyte ageing process. However, complementary tests and analysis such as microarray analysis, total ROS measurement, mitochondrial dysfunction indicators, ATP content of the eggs, etc., are required to fully evaluate the contribution of oxidative stress to the defect of egg quality during oocyte ageing.
Keywords: Clarias gariepinus, egg viability, oocyte ageing, oxidative stress
Acknowledgements: The study was financially supported by the Ministry of Education, Youth and Sports of the Czech Republic - project “CENAKVA” (LM2018099), project Reproductive and Genetic Procedures for Preserving Fish Biodiversity and Aquaculture (CZ.02.1.01/0.0/0.0/16_025/0007370) and by the Ministry of Agriculture of the Czech Republic: project NAZV (QK1710310).
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 151 SESSION IV. OOGENESIS AND EGG QUALITY P53
FROM OOGENESIS TO EMBRYONIC DEVELOPMENT: HOW CLIMATE CHANGE MAY AFFECT REPRODUCTIVE PERFOMANCE OF ATLANTIC COD
Maud ALIX1*, Lene KLEPPE1, Eva ANDERSSON1, Birgitta NORBERG1, Ørjan KARLSEN1, Jon Egil SKJÆRAASEN1, Olav Sigurd KJESBU1
1 Institute of Marine Research, P.O. Box 1870 Nordnes, 5817 Bergen, Norway
The temperature is one of the key parameters of climate change influencing teleost physiology and population dynamics. In addition, the thermal windows for spawners and early life stages (eggs and larvae) appear to be the narrowest during the life of the fish. The maternal contribution to fish embryogenesis is clearly established as this latter depends mainly upon the molecules incorporated in oocytes during reproductive cycle. As a consequence, temperature fluctuations could have an impact on developmental success, either directly or as a consequence of failures during oogenesis. In Atlantic cod (Gadus morhua), a species of great ecological and commercial interest in the North Atlantic, previous results indicate that the spawning stage seems particularly sensitive to environmental parameters due to this mentioned narrow thermal window. Therefore, to understand how ocean warming could impact cod reproductive performances, we conducted an indoor experiment with a focus on oocyte growth and developmental success. To do so, 200 wild-caught Norwegian coastal cods were tracked over their reproductive cycle and spawning season (from September to April). The experimental setup was composed of 12 tanks (3 replicates per temperature) in recirculated aquaculture system at 3, 6, 9 and 12C. The PIT-tagged fish were sampled every month to follow their growth and condition index as well as oocyte maturation. During the spawning season, egg volumes and embryonic stages were daily recorded. In addition, 20 spawn per temperature were incubated and developmental success was studied from early embryogenesis to hatching. So far, we have found that temperature did not affect the fish ability to spawn, but developmental success is clearly impacted as probably gamete quality. Higher temperature was associated with the worst survival, hatching and deformity rates. To fully understand the mechanisms involved in this variability of egg quality, gene expression will be soon investigated at different time of oogenesis including spawning season. Moreover, an overview of hormonal variations along the gametogenesis could help us to understand the reproductive physiology of Atlantic cod and consider its future using to predictive-type models. Our preliminary results will be presented in the context of global change.
Keywords: reproduction, developmental success, ocean warming, temperature, Gadus morhua
Acknowledgements: Norwegian Research Council for funding this project (no. 268336 ScaleClim), and IMR Matre Biological Station.
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 152 SESSION IV. OOGENESIS AND EGG QUALITY P54
IDENTIFICATION AND FUNCTIONAL CHARACTERIZATION OF OVARIAN AND MATERNALLY- INHERITED miRNAs IN MEDAKA
Stéphanie GAY1*, Jérôme MONTFORT1, Fabrice LEGEAI2, Thaovi NGUYEN1, Thomas DESVIGNES3, Violette THERMES1#, Julien BOBE1#
1 INRA Fish Physiology and Genomics, Rennes, France 2 IGEPP, INRA BP35327, Le Rheu, France 3 Institute of Neuroscience, University of Oregon, Eugene, USA # equal contribution
Maternally-inherited molecules play a crucial role after fertilization to support early development until the onset of the zygotic genome. While most existing studies in vertebrates have focused on protein-coding genes (messenger RNAs and proteins), the role of maternally-inherited non-coding RNAs has received far less attention. In the present study, we aimed at characterizing the maternally-inherited microRNA (miRNA) repertoire in medaka (Oryzias latipes). A total of 24 libraries originating from various organs/tissues (eyes, brain, gills, heart, liver, muscle, liver, kidney, intestine), as well as testis, ovary, ovarian follicles, eggs and embryos at different stages were generated and subjected to small RNA sequencing (50 bp, single-end) with a minimum depth of 7.5 million reads per library. Using the Prost! software (Desvignes et al., 2019) we were able to characterize and annotate 458 mature miRNAs in medaka including 138 maternally-inherited miRNA. We then performed a differential analysis between samples containing germinal cells or maternally-inherited RNA and the remaining samples. This resulted in the identification of 37 ovarian/maternal miRNAs that were kept for further analysis. Of these 37 miRNAs, 15 were also found to be maternally-inherited, while the other ones were mostly present in the ovary. Further investigations, including expression analysis during the reproductive cycle and cellular localization analysis using 3D imaging in the ovary are currently in progress. In addition, CRISPR/Cas9-based mutant have been generated for several candidates, including miR-187-3p to investigated the roles of targeted miRNAs in oogenesis and/or early development in relation with reproductive success.
Keywords: 4-6 items. Non coding RNA, CRISPR/Cas9, Prost, medaka, egg, RNA-seq.
Acknowledgements: supported by ANR Dynamo
References: Desvignes, T., Batzel, P., Sydes, J., Eames, B.F., and Postlethwait, J.H. (2019). miRNA analysis with Prost! reveals evolutionary conservation of organ-enriched expression and post- transcriptional modifications in three-spined stickleback and zebrafish. Sci. Rep. 9, 3913. Gay, S., Bugeon, J., Bouchareb, A., Henry, L., Delahaye, C., Legeai, F., Montfort, J., Le Cam, A., Siegel, A., Bobe, J., et al. (2018). MIR-202 controls female fecundity by regulating medaka oogenesis. PLoS Genet. 14, e1007593.
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 153 SESSION IV. OOGENESIS AND EGG QUALITY P55
A DE NOVO TRANSCRIPTOME ASSEMBLY SHEDS NEW LIGHT IN MOLECULAR DYNAMICS OF OVARIAN MATURATION IN THE MEDITERRANEAN SWORDFISH (Xiphias gladius)
Giorgia GIOACCHINI1*, Luca MARISALDI1, Danilo BASILI1, Michela CANDELMA1, Francesca MARADONNA1, Riccardo AIESE CIGLIANO2, Walter SANSEVERINO2, Gary HARDIMAN3, Paolo PIGNALOSA4, Oliana CARNEVALI1
1 Department of Life and Environmental Sciences (DISVA), Marche Polytechnic University (UNIVPM), 60131 Ancona, Italy 2 Sequentia Biotech, 08193 Bellatera (BCN), Spain 3 School of Biological Sciences & Institute for Global Food Security, Queens University Belfast, BT9 5AG Belfast, UK 4 OCEANIS Srl, Ercolano (NA), Italy
Swordfish (Xiphias gladius) has been recently classified as an overfished species in the Mediterranean Sea and in 2016, the International Commission for the Conservation of the Atlantic Tunas (ICCAT) established a multi-annual management plan to recover this stock. To successfully achieve this goal, knowledge about swordfish reproductive biology is needed. In order to support such conservation effort, RNA-sequencing represents a suitable approach to discover molecular pathways involved in metabolism and reproduction. In this study, by means of Illumina sequencing, ovary and liver mRNA from mature and immature females were analysed in order to gain insights into puberty onset. In addition, by applying a de novo transcriptome assembly approach multidisciplinary approach we characterise the molecular dynamics underlying ovarian maturation. The final swordfish transcriptome assembly was composed by 100.869 sequences, of which 30.398 with a Gene Ontology (GO) annotation and 25.151 unigenes. Moreover, differential expression analysis (DEA) followed by GO and KEGG pathway analyses revealed that most of the genes involved into key biological functions underlying reproductive maturation such as ovarian steroidogenesis, RNA/DNA processing and lysosome formation/maturation, in addition to transport and lipid metabolism, were up-regulated in mature ovaries. In addition, a phylogenetic analysis let us identify a candidate vitellogenin receptor. Finally In the liver we focused our attention on lipid metabolism and vitellogenin and zona radiata proteins synthesis. This is the first swordfish transcriptome assembly and these findings provide in-depth understanding of molecular processes describing ovarian maturation. Moreover, the establishment of a publicly available database containing information on the swordfish transcriptome aims to boost research on this species with the long-term of developing more comprehensive and successful stock management plans.
Keywords: 4-6 items. RNA-seq, puberty, oocyte maturation, vitellogenesis.
Acknowledgements: This research was supported by the Ministry of Agriculture, Food and Forestry Policies, General Directorate of Fisheries and Aquaculture (MiPAAF) - Italy, note 6775, Art.36 Paragraph 1 Reg (UE9 n 508/2014) to OC.
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 154 SESSION IV. OOGENESIS AND EGG QUALITY P56
CHARACTERIZATION OF MICROPYLE FORMATION IN MEDAKA (Oryzias latipes) miR-202 -/- MUTANTS
Mariana ROZA DE ABREU1,2*, Manon LESAGE1, Stéphanie GAY1, Thaovi NGUYEN1, Cécile DURET1, Violette THERMES1#, Julien BOBE1#
1 INRA Laboratoire de Physiologie et Génomique des Poisson, 35042 Rennes. 2 Aquaculture Center of Sao Paulo State University (CAUNESP), SP, Brazil. # equal contribution
*[email protected] miR-202 is a microRNA that is predominantly expressed in gonads in fish and vertebrates. miR-202 is involved in the regulation of follicular recruitment and growth and is necessary for female reproductive success and in particular the regulation of female fecundity and fertility (Gay et al. 2018). Eggs originating from miR-202 -/- knock out (KO) females mated with either wild-type or mutant males cannot be fertilized, while eggs originating from wild- type females mated with a mutant male can be fertilized but yield embryos exhibiting a developmental arrest during the first cleavage stages of embryonic development (Gay et al. 2018). In this context, the aim of our study was to understand why eggs produced by medaka (Oryzias latipes) lacking miR-202 cannot be fertilized. Two working hypotheses have been tested: a) eggs from mutant medaka have a failure in micropyle formation resulting in the impairment of fertilization; b) the micropyle is present but the egg cannot be fertilized even though the sperm penetrates into the oocyte. We first analyzed the presence of micropyle in wild type and miR-202 KO eggs after micropyle staining with a nonspecific protein-staining dye, Coomasie Brilliant Blue G (CB). To analyze the micropylar cell formation during oogenesis we are using immunocytochemistry techniques in ovaries and dissected ovarian follicles of wild type and miR-202 KO. Recent studies have showed that Taz, an effector of Hippo pathway signaling, is required either very early for the differentiation of the micropylar cell or for its specification, as well as for fertilization in zebrafish (Dingare et al. 2018; Yi et al. 2019). Based on these studies we are testing the use of a rabbit anti-Taz as a primary antibody since Taz was showed be specifically enriched in the micropylar cell. Analyses are still in progress, but in our first tests with CB staining, the micropyle was clearly visible and stained in wild type eggs. The micropyle showed a funnel-shape identified on the chorion above the animal pole of these eggs.
Keywords: Taz, micropylar cells, fertilization, miRNAs, genoma editing.
Acknowledgements: We thank “São Paulo Research Foundation – FAPESP” for research fellowship abroad [grant #2019/05741-5] and ANR Dynamo.
References: Dingare C, et al. (2018) The Hippo pathway effector Taz is required for cell morphogenesis and fertilization in zebrafish. Development 145: dev167023. doi: 10.1242/dev.167023. Gay S, et al. (2018) MiR-202 controls female fecundity by regulating medaka oogenesis. PLoS Genet 14(9): e1007593. https:// doi.org/10.1371/journal.pgen.1007593.
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 155 SESSION IV. OOGENESIS AND EGG QUALITY P56
Yi X, et al. (2019) The effector of Hippo signaling,Taz, is required for formation of the micropyle and fertilization in zebrafish. PLoS Genet 15(1): e1007408. https://doi.org/10.1371/journal. pgen.1007408.
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 156 SESSION IV. OOGENESIS AND EGG QUALITY P57
INFLUENCE OF THERMAL MANIPULATIONS DURING GAMETOGENESIS ON REPRODUCTIVE MATURATION OF MEAGRE (Argyrosomus regius), SPAWNING SUCCESS, AND GAMETE AND EGG QUALITY
Paulo H. de MELLO1,2*, Ioannis FAKRIADIS2, Panagiota TSOUKALI2, Stefano LANCEROTTO2, Luca CALLONI2, Svenja KÖPPER2, Roberta SARRACINO2, Constantinos C. MYLONAS2
1 Aquaculture Center of São Paulo State University, Via Prof. Paulo Donato Castellane, s/n 14884-900, Jaboticabal, São Paulo, Brazil. 2Institute of Marine Biology, Biotechnology and Aquaculture, Hellenic Center for Marine Research, P.O. Box 2214, Iraklion, Crete 71003, Greece.
The meagre (Argyrosomus regius) is a species with a great potential for the diversification of aquaculture production in the Mediterranean region. Its production is increasing rapidly, with large advances and success on spawning induction methods, although reproduction in captivity still remains a problem. Most commercial hatcheries use borehole water for their broodstock for biosecurity reasons (lack of pathogens). The objective of the study was to examine if gametogenesis can take place under constant borehole water temperatures, which would be cost effective if the water does not need to be heated or cooled during the year. The study evaluated the effect of constant and natural cycling temperatures on reproductive maturation, spawning success and egg/sperm quality of meagre. Two broodstock were maintained in separate 15 m3 tanks with natural (Cycling T, 16.4 to 19.6ºC) and constant (Constant T, 19.4±0.6ºC) temperatures. For spawning induction, 4 couples per group were placed in separated 5 m3 tanks after weekly spawning induction with GnRHa, over a period of 4 weeks. Females were induced with GnRHa injections (four administrations) and males with GnRHa implants every 15 days (two administrations). Blood samples were taken at the time of the first spawning induction for sex steroid hormone evaluation. Over the following 4 weeks, we examined spawning success, oocyte and sperm quality (CASA), and embryo and larval survival. Differences in all parameters were examined with two-way Analysis of Variance (ANOVA) or t-test at a level of P˂0.05. The oocyte diameter in the first sampling were significantly higher (P=0.025) in Cycling T group with an average of 598±27 µm and a tendency of higher relative fecundity was observed also so, thermal manipulation during the year affected Constant T group during the reproductive maturation process. Differences in the spawning kinetics between the two groups were observed, with differences in number of spawns after the four injections, there were no significant differences in any of the egg quality and embryo survival. In males, significant difference between groups were observed, and the Constant T individuals did not release any sperm at the beginning of the spawning induction experiment, but in response to the GnRHa implants they produced progressively more sperm. This suggested that males are more affected by the constant temperature than females. Overall, there were no significant differences in sperm quality parameters between the Cycling T and Constant T groups. The study demonstrated that cycling the temperatures are important during the reproductive maturation process in meagre with males being more affected by the constant temperature than females.
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 157 SESSION IV. OOGENESIS AND EGG QUALITY P57
Key words: Temperature, sperm, oocytes, reproduction, GnRHa.
Acknowledgements: NEWTECHAQUA (H2020) and Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP) Processo FAPESP (2019/05290-3).
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 158 SESSION IV. OOGENESIS AND EGG QUALITY P58
A NOVEL METHOD FOR RAPID ELIMINATION OF EGG STICKINESS USING SODIUM HYPOCHLORITE IN THREE FRESHWATER FISH SPECIES
Martin PŠENIČKA1*, Zuzana Linhartová1, Roman Franěk1
1 University of South Bohemia in České Budějovice, Faculty of Fisheries and Protection of Waters, South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses, Research Institute of Fish Culture and Hydrobiology, Zátiší 728/II, 389 25 Vodňany, Czech Republic
Reducing egg stickiness is a crucial step in artificial culture of fish with sticky eggs, with commonly used methods being time consuming or interfering with hatching. Sodium hypochlorite (SH) at varying concentrations and exposure times was tested on eggs of three different fish species (sterlet Acipenser ruthenus, northern pike Esox lucius, and European catfish Silurus glanis), with 0.03% SH for 40 s effectively eliminating stickiness without impairment to eggs, embryo development, or hatching when compared with other de- adhesion methods (clay; NaCl, urea, tannic acid, alcalase enzyme). Field tests showed similar numbers of larvae hatched using SH when compare with other conventional methods. Immunohistochemistry revealed a protein carbonyl group only on the surface of eggs treated with SH, evidence of oxidation as the mode of action and indicating that oxidation did not affect inner egg layers or cytoplasm. Treatment with SH is a rapid, simple, and inexpensive method for de-adhesion of fish eggs. The method was not successful when applied on other fish species as carp Cyprinus carpio or goldfish Carassius auratus probably due to smaller size of eggs.
Key words: egg de-adhesion; sturgeon; artificial reproduction; sodium hypochlorite; protein carbonyl
Acknowledgements: The study was financially supported by the Ministry of Education, Youth and Sports of the Czech Republic – project CENAKVA (LM2018099) and Biodiversity (CZ.02.1.01/0.0/0.0/16_025/0007370) and the Czech Science Foundation (grant number 17- 19714Y and P502/13/26952S).
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 159 SESSION IV. OOGENESIS AND EGG QUALITY P59
UNUSUAL EGG QUALITY PHENOTYPE IN THE ASPE VALLEY: A CASE STUDY IN RAINBOW TROUT
Emilien SEGRET1,2*, Frederic Cachelou1, Emilie Cardona2, Jerome Bugeon2, Sandrine Skiba- Cassy3, Julien Bobe2
1 Viviers de Sarrance, 64490 Sarrance, France 2 INRA, UR1037 Laboratoire de Physiologie et Génomique des Poissons, 35000 Rennes, France 3 INRA, Univ Pau & Pays Adour, E2S UPPA, UMR 1419, Nutrition, Métabolisme, Aquaculture, Saint Pée sur Nivelle, F-64310, France
Optimization of egg production, both in terms of quality and number, is a major issue for aquaculture sustainability. Insufficient egg quality leading to reduced fertilization rates and/or embryonic mortalities can induce major economic losses for hatcheries. The impact of environmental factors and husbandry practices on egg quality has been thoroughly documented in many fish species, including rainbow trout (Oncorhynchus mykiss). In this context, the occurrence of an unusual low egg quality phenotype has been repeatedly observed under production conditions in the Aspe valley. Ovulated eggs collected at ovulation are visually similar to post-ovulatory aged (i.e. over-ripe) eggs and exhibit early reduced egg viability possibly with a lack of fertilization. Surprisingly, this phenotype is observed at optimal temperature (8-9°C) as early as the time of ovulation (+/- 1 week due to weekly detection) and is extremely variable over time during the year ranging from a few % of females affected to 40-50 % of the females in the worst cases. Interestingly, this phenotype is not observed in the neighbor Ossau valley using fish from the same genetic strain and with similar husbandry practices. In the present study we aimed at: (i) thoroughly describing the phenotype, (ii) identifying the mechanisms triggering this unusual phenotype in order to (iii) possibly correct the problem. In order to describe the phenotype, we used the VisEgg methodology (described in this section). Batches of eggs (N=100-200 eggs), freshly collected from broodfish (1 batch per female) from the Aspe valley –good and low quality batch- and from the Ossau valley –good quality batches- were analyzed. Each sample was analyzed after 24 hours of hydration in water. A picture of each egg sample was obtained using a dedicated shooting system (Cardona et al., 2019). Using a VBA macro with Visilog 7.3 software (Thermo Scientific), individual parameters are calculated and compared. The presence of white eggs was interpreted as a measure of low egg integrity thus corresponding to non-viable oocytes. For the Ossau Valley no low quality batches were observed and only 4.19% eggs per batch were non-viable. In the Aspe valley, 22% of the females sampled presented low quality batches; two groups could be identified: “good quality” and “low quality” batches with respectively 6.00% and 51.84% of non-viable eggs (Kruskal-Wallis test- p.value <0.0001). We hypothesize that an unknown phenomenon triggers a loss of integrity of eggs, observable only in the Aspe valley, even though not systematically (in our case for 22% of analyzed females). An experiment is currently ongoing. Females bred at high and low density are followed during late oogenesis. Several physiologic biomarkers are measured in plasma and eggs are
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 160 SESSION IV. OOGENESIS AND EGG QUALITY P59 collected. Oocytes composition and quality will be evaluated. Results will shed new light on the physiological mechanisms triggering this phenomenon.
Keywords: Rainbow trout- Egg viability
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 161 SESSION IV. OOGENESIS AND EGG QUALITY P60 topaz1, A CRUCIAL GENE FOR MEDAKA (Oryzias latipes) REPRODUCTION
Thaovi NGUYEN1*, Amaury HERPIN1, Julien BOBE1
1 INRA Laboratoire de Physiologie et Génomique des Poissons – LPGP. Bâtiment 16A, Campus de Beaulieu, 35042 Rennes
The protein Topaz1 (testis and ovary specific PAZ domain gene 1) contains a domain found in regulatory Piwi-Argonautes-Zwille proteins. This domain is highly conserved in vertebrates. In sheep, Topaz1 is preferentially expressed in the ovary during foetal life, while a predominant expression is found in the testis in adult mice. Topaz1 plays an essential role in germ cell formation in mouse has shown in previous studies. In absence of Topaz1, homozygous male mice are sterile while female fertility is not affected. Fish also have a gene coding for Topaz1. The aim of the present study was to investigate its role in fish reproduction. We studied the expression of topaz1 in medaka, a fish model species, and we analyzed reproductive phenotypes after topaz1 inactivation using the CRISPR/Cas9 system. Expression analysis reveals a high abundance of the transcript in the gonads of wild-type medaka adult, and particularly in the ovary. During embryonic development, topaz1 is highly expressed from the first embryonic stages to the start of gastrulation, after which its abundance dramatically decreases. This pattern suggests a role during embryonic development. The inactivation of topaz1 in medaka leads to sterile animals. Moreover, every XX fish developped a male phenotype (fin shape and gonad). Dissection of the knock-out (KO) animals shows that sex-reversed XX males and XY males have an abnormal testis, with an “empty string” shape. The sex reversal of females and the testis shape suggest a dysfunction in the differenciation of the germ line. To explain these anomalies, we followed the establishment of the germ cells stock, over the very early stages of embryonic development after injecting a mRNA encoding a nanos-gfp fusion protein into 1-cell stage eggs. These results suggest that Topaz- /- embryos have a significantly reduced number of germ cells in comparison to wild-types. This defect is observed very early during the first days (at 12-16 somite-stage) of development. In summary, topaz1 inactivation in medaka demonstrates that this gene is essential for reproduction. Lack of Topaz1 leads to a serious defect of germ cell formation in the embryo resulting in fish sterility in both males and females due to the lack of gamete production. To day, the role of topaz1 in the adult ovary remains to be characterized.
Keywords: CRISPR/Cas9, medaka, sterile, germ cells, sex inversion
References: Baillet A, Le Bouffant R, Volff JN, Luangpraseuth A, Poumerol E, et al. (2011) TOPAZ1, a Novel Germ Cell-Specific Expressed Gene Conserved during Evolution across Vertebrates. PLoS ONE 6(11): e26950. doi:10.1371/journal.pone.0026950 Luangpraseuth-Prosper A, Lesueur E, Jouneau L, Pailhoux E, Cotinot C, Mandon-Pépin B (2015) TOPAZ1, a germ cell specific factor, is essential for male meiotic progression. Developmental Biology 406 (2015) 158-171. doi.org/10.1016/j.ydbio.2015.09.002
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 162 SESSION IV. OOGENESIS AND EGG QUALITY P61
EFFECT OF DIFFERENT EQUILIBRIUM TIMES AND CRYOPROTECTOR SOLUTIONS IN THE VITRIFICATION OF ZEBRAFISH OVARIAN TISSUE: PRELIMINARY DATA
Itamar C GOMES1, Andrea G GALUPPO1, Rômulo B RODRIGUES1, Jhony BENATO1, Thales L FLORES1, Jessica PETRY2, Douglas GAMBA2, Leandro C GODOY1, Danilo P STREIT Jr1*
1Aquaculture Laboratory-AQUAM, Rio Grande do Sul Federal University, RS, Brazil 2Biochemestry Department, Rio Grande do Sul Federal University, RS, Brazil
It is well established that the use of cryoprotectants is essential for the viability success after cryopreservation procedures such as vitrification. However, in addition to the cryoprotective effect these substances are also highly toxic to cells under certain conditions, such as high concentrations and temperatures. Therefore, the objective of this work was to evaluate the effect of different equilibrium times and cryoprotectant solutions on cell viability after vitrification of zebrafish (Danio rerio) ovarian tissue fragments. Zebrafish females were measured (4.1814±0.2993cm), weighed (0.5782±0.1683g), and later the collection of gonads was performed, which were also weighed (0.0611±0.0401g). The ovarian tissue was fragmented (2x2 mm) and distributed among the experimental groups according to the equilibrium times and vitrification solutions. The equilibrium solutions were L-15 90% and 1.5M methanol associated with 2.25M propylene glycol (E1) or 2.75M Me2SO (E2). The vitrification solutions used were L-15, 1.5M methanol and 4.5M propylene glycol associated with 0.5M sucrose (SV1-S) or 2% alginate (SV1-A), and L-15, 1.5M methanol and 5.5M Me2SO associated with 0.5M sucrose (SV2-S) or 2% alginate (SV2-A). The solutions SV1 (S and A) were submitted to E1 and solutions SV2 (S and A) to E2 for 1, 7 or 15 min equilibrium at 4°C. After equilibration the fragments were exposed to respective vitrification solutions (90s) and immediately immersed in N2L. Thawing was performed in a water bath at 38 ° C. The cell viability test used was the MTT assay, and in order to equalize the results due to a possible variation in fragments size the data were analyzed per gram of tissue. The data showed that there was no difference in viability among the times 1, 7 or 15min for the SV1-S solution. SV2-S cell viability was significantly higher when compared 1 versus 15 min, showing that a shorter equilibrium time in this situation is better. When considering SV1-A and SV2-A the best results were obtained with 7 min of equilibrium. However, it is interesting to note that the solutions SV2-S and SV2-A presented similar results between them and with SV1-A. Except for the SV1-S solution, we observed that for all other solutions tested (SV1-A, SV2-S and SV2-A) times under 15 min provided higher viability rates. Our observations lead us to believe that sodium alginate may be used as a substitute for the sucrose, and performing equilibrium under 15 min is an important factor for maintaining cell viability of zebrafish ovarian tissue. These data lead us to rethink the vitrification protocols that use times superior than 7 min in equilibrium. However, our data are preliminary and we hope to obtain clearer results on the best combination of equilibrium time and vitrification solution in order to maximize the survival of oocytes submitted to cryopreservation techniques.
Key words: Cryopreservation, Me2SO, propyleneglycol, sodium alginate, viability test
Acknowledgements: CNPq, CAPES, FAPERGS
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 163 SESSION IV. OOGENESIS AND EGG QUALITY P62
FACTORS AFFECTING SECONDARY SEX CHARACTERISTICS IN THE YELLOWTAIL TETRA Astyanax altiparanae
Diógenes H. SIQUEIRA-SILVA1*, Rafaela M. BERTOLINI2, Nycolas L. PEREIRA3, Nivaldo F. NASCIMENTO3, José A. SENHORINI3, Lucas Henrique PIVA3, José Bento S. FERRAZ4, George S. YASUI3
1 UNIFESSPA – Universidade Federal do Sul e Sudeste do Pará. Instituto de Estudo em Saúde e Biológicas (IESB) Marabá, Pará, Brazil 2 UNESP – Univ. Estadual Paulista, Campus de Botucatu, Programa de Pós-Graduação em Ciências Biológicas (Zoologia) Botucatu, São Paulo, Brazil 3 Centro Nacional de Pesquisa e Conservação da Biota Aquática Continental (CEPTA-ICMBIO), Pirassununga, São Paulo, Brazil 4 USP– University of São Paulo, Faculdade de Zootecnia e Engenharia de Alimentos Departamento de Medicina Veterinária, Pirassununga, São Paulo, Brazil
We aimed to analyze factors affecting secondary sexual characteristics in the yellowtail tetra Astyanax altiparanae. Seventy-five specimens were separated into three different size classes (small, medium and large groups) between summer and winter seasons. In all groups, male fish were consistently bigger in the summer. Females from both seasons presented in media the same length into the size classes. Afterwards, we performed histology of the gonads to first confirm the genus and investigate the phase of maturation of each animal. During the winter, most of the small animals were males (22), most of the large animals, females (23), and the medium size animals followed a tendency of 1:1 ratio (9 male: 16 female). In the summer, male were the majority in both small (20) and medium (20) size. Larger-size animals were female (23). Then, in order to analyze the influence of genus, phase of maturation, season of the year, the number, and length of the animals spinelets, we diaphanized, counted, and measured them in each animal. The spinelets are a sexual secondary characteristic of male genus independently of the size, season and phase of maturation. However, some tendencies were observed. Males bigger than 48 mm always presented spinelets; their size are in media the double in summer in comparison to winter; and summer males presents more rays with spinelets in the summer. Curiously, the larger specimen sampled was a female presenting spinelets in five rays. Lastly, we performed the gonadectomization of the animals and hypothesized that gonad hormones will directly influence this characteristic. The gonadectomization only initially influence on the size and number of spinelets in the anal fin rays, since the thirty-day-gonadectomized animals presented few and smaller spinelets against the control ones. However, the spinelets normalized in ninety-day-gonadectomized specimens. Such a work showed spinelets can be considered a secondary sexual characteristic to distinct male from female and can be used in the management in specimens bigger than 48 mm, but cannot indicate fish sterility.
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 164 SESSION V. SPERM MOTILITY AND FERTILIZATION P63
INHIBITORY ANALYSIS OF ENERGY SUPPLYING PATHWAYS OF MOTILE AND IMMOTILE SPERMATOZOA IN SIBERIAN STURGEON (Acipenser baerii)
Deepali RAHI1*, Borys DZYUBA1 , Miaomiao XIN1 , Yu CHENG1 , Viktoriya DZYUBA1
1 University of South Bohemia in Ceske Budejovice, Faculty of Fisheries and Protection of Waters, South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses, Research Institute of Fish Culture and Hydrobiology, Zátiší 728/II, 389 25 Vodňany, Czech Republic
To date, it is clear that ATP is the only energy source for sperm motility and that can be generated and stored according to different metabolic strategies present in sperm cells. Generally, during fish sperm motility the rate of ATP consumption exceeds its production, which causes the brief motility duration in most of the externally fertilizing freshwater fish species. However, it is not clear if this ‘rule’ is applicable for sturgeon spermatozoa, which are characteristically different from ones of the freshwater teleost fish due to longer motility duration. Consequently, we investigated the role of different bioenergetic pathways in motile and non-motile spermatozoa of Siberian sturgeon, as a representative of Acipenseriformes. Sperm from five mature males were collected following established hatchery protocols. Sperm motility was initiated in activation media with or without inhibitor of oxidative phosphorylation (sodium azide – NaN3) or glycolysis (2-deoxy-D-glucose – DOG) or β- oxidation of fatty acids (sodium fluoride – NaF) or uncoupler of oxidative phosphorylation (carbonyl cyanide m-chlorophenyl hydrazine – CCCP). In addition, sperm was incubated in non-activating medium containing the same inhibitors/uncoupler for 60 min followed by activation in media containing the same reagents. Curvilinear velocity (VCL) of spermatozoa was analyzed through CASA at different post-activation time points – 10 (T1), 60 (T2) and 180 (T3) s. In activation media without inhibitor or uncoupler (control) VCL was non-significantly different at T1 and T2 while significantly decreased at T3. At T1, only NaF significantly reduced VCL while rest of the inhibitors/uncoupler reduced velocity significantly at T2 and T3. Sperm incubated in non-activation media containing inhibitors/uncoupler showed significant reduction in VCL irrespective of post-activation time (T1, T2 and T3) and activation medium composition (with and without inhibitors and uncoupler). Our results suggest that glycolysis, β-oxidation of fatty acids and oxidative phosphorylation are important not only for generation of ATP store during immotile state but also for maintenance of long-lasting motility in sturgeon spermatozoa.
Keywords: Fish, Bioenergetics, Oxidative phosphorylation, Fatty acid oxidation, Glycolysis, Sperm motility SESSION V. Sperm motility and fertilization Acknowledgments: The study was supported by the Czech Science Foundation (No. 16- 03754S), by the Ministry of Education, Youth and Sports of the Czech Republic (“CENAKVA”, LM2018099), by project Biodiversity (CZ.02.1.01./0.0/0.0/16_025/0007370) and by the Grant Agency of the University of South Bohemia in Ceske Budejovice (125/2016/Z).
7th International Workshop on the Biology of Fish Gametes - 2019, Rennes, France 165 SESSION V. SPERM MOTILITY AND FERTILIZATION P64
HUNDREDS OF SPERMATOZOA ARE SUFFICENT FOR FERTILIZATION OF STERLET (Acipenser ruthenus) EGGS
Otomar LINHART1, Yu CHENG1,2, Marek RODINA1, Vladimíra TUCKOVA1, Vojtěch KASPAR1, Miaomiao XIN1,2*
1 University of South Bohemia in Ceske Budejovice, Faculty of Fisheries and Protection of Waters, South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses, Research Institute of Fish Culture and Hydrobiology, Zatisi 728/II, 389 25 Vodnany, Czech Republic 2 Sino-Czech Joint Laboratory of Fish Conservation and Biotechnology: Key Laboratory of Freshwater Biodiversity Conservation, Ministry of Agriculture of China, Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Wuhan, China
Fertilization environment (condition) is usually described as optimum sperm : egg ratio while the volume of activation water or ratio of volume of eggs and activation water is mentioned marginally. Nevertheless, combination of both parameters is important for fertilization and rough calculation of sperm : egg ratio is not good enough to describe fertilization conditions. This study was conducted to better understand relations between neurulation, hatching ability and used sperm quantities together with different volume of activation water (dechlorinated water) used for fertilization in sterlet Acipenser ruthenus. Eggs (50-60) from three individual females (not pooled) were fertilized by pooled sperm from four males with motility higher than 80% in 4 replicates. Firstly, total number of 50000, 250000 and 1250000 spermatozoa in combination with 2, 8 and 32 ml of activation water were applied for fertilization, respectively. Secondly, 10000 and 50000 spermatozoa with 8 and 88 ml of activation water were used. The results showed that even 50000 spermatozoa in 8 ml and 32 ml of activation water (concentration 6250 and 1563 spermatozoa per ml) was sufficient for successful fertilization with 60-90 % neurulation and hatching rate according to quality of eggs from individual female. Surprisingly, no significant effect of the volume of activation medium and quantity of spermatozoa on neurulation and hatching was shown in eggs of two females, which means that 1563 sperm per ml of activation water was sufficient for eggs´ fertilization. In the second experiment, 10000 spermatozoa in 88 ml of activation water (113 spermatozoa per ml) was sufficient for neurulation and hatching reaching 20-30% and also the volume of activation water with sperm quantity had a significant effect on neurulation and hatching. Finally, concentration of 100 - 1500 spermatozoa per ml with around 80% motility in activation water allow that such a good sperm can be divided according to fertility and to express their quality.
Keywords: Acipenser ruthenus, fertilization, activation water, egg, spermatozoa
Acknowledgements: The study was supported by the Ministry of Education, Youth and Sports of the Czech Republic (“CENAKVA”, LM2018099), by project Biodiversity (CZ.02.1.01./0.0/0.0/16_025/0007370) and by the Grant Agency of the University of South Bohemia in Ceske Budejovice (125/2016/Z).
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EFFECT OF PH AND IONS ON SPERM MOTILITY IN PUFFERFISH (Takifugu alboplumbeus)
Luz PÉREZ1*, Victor GALLEGO1, Manabu YOSHIDA2, Juan F. ASTURIANO1
1 Universitat Politècnica de València. Instituto de Ciencia y Tecnología Animal. Grupo de Acuicultura y Biodiversidad. Camino de Vera s/n 46022 Valencia, Spain 2 Misaki Marine Biological Station. Graduate School of Science. University of Tokyo. 1024 Misaki-Koajiro. 238-0225 Miura, Kanagawa, Japan
The process of sperm activation is complex, and known only partially in fish. Sperm is immersed in a seminal plasma that maintains its viability and allows further sperm motility (or capacitation). There are not many studies concerning the seminal plasma characteristics important for sperm capacitation, like ionic composition and pH. In this work, the effect of removal of several ions (Na+, K+, Mg2+) from seminal plasma as well as the effect of seminal plasma and seawater pH on the sperm activation were studied in the pufferfish (Takifugu alboplumbeus, before T. niphobles). Sperm samples were obtained from fish caught in Arai Beach (Miura, Japan) and maintained in aquaria. Samples selected by its high motility, measured by CASA-mot system were used in the experiments. For ion removal, samples were centrifuged (500 g, 5 min, 4 ºC) and resuspended in K-free extender, Na-free extender, K/Na-free extender, and control extender for pufferfish (Krasznai et al. 2003). None of this media reduced the sperm motility in relation to controls, although K-free extender induced a higher percentage of hyperactive spermatozoa, and after 3 days of incubation with the different extenders VCL was reduced in Na-free and Na/K-free extenders. Extender pH was adjusted to several pHs to check its effect on further motility. Extender pH had a strong effect on sperm motility. Samples diluted at pH 6.5 showed a high reduction of sperm motility in comparison with higher pHs (7.5, 8.5 and 9.5.) The inhibitory effect of low pH was reversible, as samples previously maintained at pH 6.5 and resuspended at pH 8.5, total sperm motility was recovered. Regarding the pH of seawater (used as activation medium), there were no significant differences between control (pH 8.2) and pH 6.5, thus indicating that fugu sperm can swim in acidic environments. Finally, nigericin (that equals intracellular pH with extracellular pH) was added, and the highest sperm motility was found at pH 7.0, although the highest percentage of progressive motility was caused by pH 6.8. That indicated that the optimal intracellular pH of pufferfish for sperm motility is 6.8-7.0.
Keywords: Capacitation; Activation; Extender; Takifugu niphobles
Acknowledgements: Generalitat Valenciana funded the stay in Japan of JFA, LPI and VG (BEST 2018/093, /112 and /124, respectively). VG has a postdoctoral grant from the Spanish Ministry of Science, Innovation and Universities (MICIU; Programa Juan de la Cierva- Incorporación; IJCI-2017-34200).
References Krasznai et al. 2003. Gadolinium, a mechano-sensitive channel blocker, inhibits osmosis- initiated motility of sea- and freshwater fish sperm, but does not affect human or ascidian sperm motility. Cell Motility and the Cytoskeleton 5: 232-243.
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SPERM MORPHOLOGY AND MOTILITY SIGNALING IN ATLANTIC COD, Gadus morhua
Sayyed Mohammad Hadi ALAVI1*, Azadeh HATEF2, Ian A. E. BUTTS3, Jacky COSSON4, Igor BABIAK5
1 School of Biology, College of Science, University of Tehran, P. O. Box: 14155-6455, Tehran, Iran. 2 Toxicology Center, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5B3, Canada 3 School of Fisheries, Aquaculture and Aquatic Sciences, Auburn University, Auburn, AL, USA 4 South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses, Faculty of Fisheries and Protection of Waters, University of South Bohemia in České Budějovice, Vodňany, Czech Republic 5 Faculty of Biosciences and Aquaculture, Nord University, Bodø, Norway
Understanding reproductive biology is crucial for sustainable aquaculture. In this context, studying biology of sperm provides valuable information to optimize artificial reproduction. Here, we investigated morphology of sperm using TEM and SEM, and studied spermatozoa motility signaling in the Atlantic cod using computer-assisted sperm analysis. The spermatozoon lacked an acrosome, and consisted of a head, midpiece and flagellum. The head of spermatozoa were round, oval, or elongated in shape, showing high variations in dimensions. There were up to 6 mitochondria that encircled the proximal part of the flagellum. The proximal and distal centrioles were located within the nuclear notch and arranged orthogonal to each other. The axoneme had a 9+2 microtubule structure. In most teleosts, length of the flagellum has reported in range of 30-40 μm (Lahnsteiner and Patzner, 2008), however flagellar length in cod spermatozoa was observed in range 50-90 μm. Spermatozoa were immotile in the seminal plasma. Dilution of sperm into seawater with osmolality of 1100 mOsmol/kg resulted in initiation of motility in 91.0±3.4% spermatozoa with average velocity of 86.2±2.3 μm/s. The duration of spermatozoa motility was >10 min, however the percentage of motile spermatozoa decreased at 60 s post-activation. Decreasing and increasing osmolality of seawater to 575 and 1500 mOsmol/kg, respectively, resulted in decrease in spermatozoa motility (67.8 and 75.7%, respectively) (P<0.05). Spermatozoa motility was not initiated at ≤400 and ≥2500 mOsmol/kg. Using sucrose, spermatozoa motility was initiated at 600 mOsmol/kg, was highest at 800–1100 mOsmol/kg, and was suppressed at 1500 mOsmol/kg. Further experiments using inhibitors for ion channels showed that a hyperosmotic signal triggers spermatozoa motility initiation similar to other marine fishes (Cosson et al., 2008).
Keywords: Flagellum; Ions; Osmolality; Spermatozoa motility; Spermatozoa velocity
Acknowledgements: CENAKVA: CZ.1.05/2.1.00/01.0024; CENAKVA II: LO1205 under the NPU I program; Biodiverzita: CZ.02.1.01/0.0/0.0/16 025/0007370; University of Tehran; USDA.
References Cosson, J., et al., 2008. Marine fish spermatozoa: racing ephemeral swimmers. Reproduction 36:277-294.
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Lahnsteiner, F., Patzner, R.A., 2008. Sperm morphology and ultrastructure in fish. In Alavi, S.M.H., et al., editors. Fish Spermatology, Oxford: Alpha Science Ltd; 2008, p. 1-61.
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PARTICIPANTS LIST
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Alavi Sayyed Mohammad Hadi, S1 O5, S1 P6 P7, S5 Boryshpolets Sergii, S5 O30 P66 University of South Bohemia, Faculty of Fisheries University of Tehran, Iran and Protection of Waters, České Budějovice, Czech [email protected] Republic [email protected] Alix Maud, S4 P53 Institute of Marine Research, Bergen, Norway Cabrita Elsa, S1 P14 [email protected] University of Algarve, Faro, Portugal [email protected] Anjos Catarina, S3 P44 University of Algarve, Faro, Portugal Cardona Emilie, S4 O27 [email protected] INRA LPGP, Rennes, France [email protected] Asturiano Juan F, S1 P6, S1 P9 P10, S3 P40 Instituto de Ciencia y Tecnología Animal, Valencia, Carnevali Oliana, S4 O25 Spain Department of life and environmental sciences, [email protected] Ancona, Italy [email protected] Baloch Abdul Rasheed, S2 O13 University of South Bohemia, Faculty of Fisheries Castro-Arnau Júlia, S1 O9 and Protection of Waters, České Budějovice, Czech IRTA-IBB-UAB, Bellaterra, Spain Republic [email protected] [email protected] Chauvigné François, S1 O2 Barrier-Loiseau Chloé IRTA-IBB-UAB, Bellaterra, Spain Agrocampus Ouest, Rennes, France [email protected]
Beirão José, S1 P8, S3 P33 P34, S5 O31 Chenais Nathalie Nord University, Bodø, Norway INRA LPGP, Rennes, France [email protected] [email protected]
Benevente Cristiane Fernanda, S1 P17 Cheng Yu, S3 P46 UNESP - Universidade Estadual Paulista Júlio de Faculty of Fisheries and Protection of Waters, Mesquita Filho, Ilha Solteira, Brazil Vodňany, Czech Republic [email protected] [email protected]
Bernáth Gergely, S3 P36 Ciereszko Andrzej, S1 O6 Szent István University, Gödöllő, Hungary Institute of Animal Reproduction and Food Research [email protected] PAS, Olsztyn, Poland [email protected] Bestin Anastasia, S4 O26 SYSAAF, Nouzilly, France Cosson Jacky, PL1 [email protected] University of South Bohemia, Faculty of Fisheries and Protection of Waters, Vodňany, Czech Republic Bobe Julien, S4 O29 [email protected] INRA LPGP, Rennes, France [email protected] de Mello Fernanda, S3 P42 Department of Physiology, University of Sao Paulo, Bondarenko Olga, S3 P37 Brazil University of South Bohemia in České Budějovice, [email protected] Faculty of Fisheries and Protection of Waters, České Budějovice, Czech Republic de Mello Paulo, S4 P57 [email protected] Aquaculture Center of São Paulo State University (CAUNESP), Brazil [email protected]
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Depincé Alexandra, S3 P43 Gavin-Plagne Lucie, S3 O20, S3 P41 INRA LPGP, Rennes, France R&D Department, IMV Technologies, L’Aigle, France [email protected] [email protected]
Dietrich Mariola, S1 O8 Gay Stéphanie, S4 P54 Institute of Animal Reproduction and Food Research INRA LPGP, Rennes, France of Polish Academy of Sciences, Olsztyn, Poland [email protected] [email protected] Gioacchini Giorgia, S4 P55 Diogo Patricia, S3 P48 Università Politecnica delle Marche, Ancona, Italy University of Algarve, Faro, Portugal [email protected] [email protected] Girard Agnès Dzyuba Borys, S5 O34 INRA LPGP, Rennes, France University of South Bohemia, Ceské Budejovice, [email protected] Czech Republic [email protected] Goupil Anne-Sophie, S2 P29 INRA LPGP, Rennes, France Dzyuba Viktoriya, S3 P35 [email protected] University of South Bohemia, Faculty of Fisheries and Protection of Waters , Vodnany, Czech Republic Guého Aurélie, S4 O21 [email protected] INRA LPGP, Rennes, France [email protected] Endoh Mitsuru, S1 O3 Hokkaido University, Hakodate, Japan Güralp Hilal, S2 O15 [email protected] Institute of Marine Research, Bergen, Norway [email protected] Fatsini Fernandez Elvira, S5 O33 University of Algarve, Faro, Portugal Haffray Pierrick [email protected] SYSAAF, Rennes, France [email protected] Franěk Roman, S2 O11, S2 P21 P22 P23 University of South Bohemia, Faculty of Fisheries Herraez Paz, PL4 and Protection of Waters (USB FFPW), Vodňany, Universidad de León, Spain Czech Republic [email protected] [email protected] Herrera Rodríguez Francisco Fabio, S1 O1 Fučíková Michaela, S2 P27 University of South Bohemia, Ceske Budejovice, University of South Bohemia, Faculty of Fisheries Czech Republic and Protection of Waters, Vodňany, Czech Republic [email protected] [email protected] Horváth Ákos, S2 O14 Gallego Albiach Víctor, S1 P15, S3 O18 Szent István University, Godollo, Hungary Universitat Politècnica de València, Spain [email protected] [email protected] Huet Nathalie García Coll Miriam INRA LPGP, Rennes, France Universitat Politècnica de València, Spain [email protected] [email protected] Judycka Sylwia, S3 O17, S3 P45 García Salinas Pablo, S1 O7 Institute of Animal Reproduction and Food Research Instituto de Ciencia y Tecnología Animal, Universitat PAS, Olsztyn, Poland Politècnica de València, Spain [email protected] [email protected]
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Kholodnyy Vitaliy, S5 O32 Lesage Manon Faculty of Fisheries and Protection of Waters, INRA LPGP, Rennes, France University of South Bohemia, Vodňany, Czech [email protected] Republic [email protected] Lescat Laury INRA LPGP, Rennes, France Kica Stéphanie [email protected] INRA LPGP, Rennes, France [email protected] Mohagheghi Samarin Azin, S4 P51 P52 Jihoceska univerzita, Ceské Budejovice, Czech Kitanović Nevena, S3 O19 Republic Szent Istvan University, Department of Aquaculture, [email protected] Godollo, Hungary [email protected] Mommens Maren, S2 P26 AquaGen, Trondheim, Norway Klopp Christophe, PL2 [email protected] MIAT INRA Toulouse, Castanet-Tolosan, France [email protected] Morini Marina Instituto de Ciencia y Tecnología Animal, Valencia, Kollár Tímea, S1 O4 Spain Szent István University, Department of Aquaculture, [email protected] Gödöllö, Hungary [email protected] Nguyen Thao Vi, S4 P60 INRA LPGP, Rennes, France Kovalev Konstantin, S3 P32 [email protected] Filial of Freshwater Fisheries of Russian Federal Research Institute of Fisheries and Oceanography, Ninhaus-Silveira Alexandre, S3 P31 Rybnoe (Moscow Region), Russia Neotropical Ichthyology Laboratory (LINEO), São [email protected] Polo, Brazil [email protected] Krasilnikova Aleksandra, S3 P47 SSC RAS, Rostov-On-Don, Russia Oliveira Catarina, S1 P20 [email protected] CCMAR, University of Algarve, Faro, Portugal [email protected] Labbé Catherine, S3 P39 INRA LPGP, Rennes, France Pain Bertrand, PL3 [email protected] Université Lyon 1, INSERM, INRA, Stem Cell and Brain Research Institute, U1208, USC1361, Bron, Lafond Joëlle, S2 O10 France Université de Montréal, Saint-Mathias-Sur-Richelieu, [email protected] Canada [email protected] Paredes Salas Juan Fernando, S4 O24 University of Murcia, Spain Lareyre Jean-Jacques, S2 O16 [email protected] INRA, Rennes, France [email protected] Pataki Bernadett, S3 P38 Szent István University, Department of Aquaculture, Laurent Audrey, S1 P13 Gödöllő, Hungary INRA LPGP, Rennes, France [email protected] [email protected] Pérez Luz, S5 P65 Le Bail Pierre-Yves Universitat Politècnica de Valècia, Instituto de INRA LPGP, Rennes France Ciencia y Tecnología Animal, Valencia, Spain [email protected] [email protected]
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Piquemal Laurine Shah Mujahid Ali, S2 P28 INRA, Le Rheu Cedex, France Faculty of Fisheries and Protection of Waters, [email protected] Vodnany, Czech Republic [email protected] Pšenička Martin, S1 P12, S4 P58 University of South Bohemia, Ceske Budejovice, Siqueira-Silva Diógenes, S4 P62 Czech Republic Unifesspa, Marabá, Brazil [email protected] [email protected]
Rahi Deepali, S5 P63 Streit Jr Danilo, S1 P16, S3 P49, S4 P61 Faculty of Fisheries and Protection of Waters, Federal University of Rio Grande do Sul (UFRGS), Vodnany, Czechia Porto Alegre, Brazil [email protected] [email protected]
Ribeiro Delgado Maria Luiza Thermes Violette Universidade Estadual Paulista Júlio de Mesquita INRA LPGP, Rennes, France Filho - UNESP, Ilha Solteira, Brazil [email protected] [email protected] Tichopád Tomáš, S1 P4 Robles Vanesa, S1 P11, S1 P19 University of South Bohemia, Faculty of Fisheries Instituto Español de Oceanografía, Santander, Spain and Protection of Waters, Vodňany, Czech Republic [email protected] [email protected]
Rocha De Almeida Tainá, S4 O23 Tinkir Merve, S1 P1 Universite de Lorraine, Vandoeuvre-Les-Nancy, Faculty of Aquatic Sciences, Istanbul, Turkey France [email protected] [email protected] Trempont Sarah, S2 P29 Romney Amie, S2 P30 INRA LPGP, Rennes, France University of California, Davis, United States [email protected] [email protected] Várkonyi Levente, S1 P5 Roudaut Guylène Szent István University, Gödöllő, Hungary INRA LPGP, Rennes, France [email protected] [email protected] Veríssimo-Silveira Rosicleire, S1 P2 Rouillon Charlène, S2 P24 São Paulo State University, Ilha Solteira Campus, INRA LPGP, Rennes, France Brazil [email protected] [email protected]
Roza De Abreu Mariana, S4 P56 Wagenaar Ina, S1 P3, S4 O22 Aquaculture Center - UNESP / LPGP - INRA, University of Johannesburg, South Africa Jaboticabal, Brazil [email protected] [email protected] Waghmare Swapnil Gorakh, S4 P50 Sambroni Elisabeth University of South Bohemia, Ceske Budejovice, INRA LPGP, Rennes, France Czech Republic [email protected] [email protected]
Segret Emilien, S4 P59 Wargelius Anna, S2 O12 INRA LPGP, Rennes, France Institute of Marine Research, Bergen, Norway [email protected] [email protected]
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Xie Xuan, S2 P25 Faculty of Fisheries and Protection of Waters, University of South Bohemia, Vodnany, Czech Republic [email protected]
Xin Miaomiao, S1 P18, S5 P64 Faculty of fisheries and protection of waters, Vodnany, Czech Republic [email protected]
Żarski Daniel, S4 O28 Institute of Animal Reproduction and Food Research of Polish Academy of Sciences, Olsztyn, Poland [email protected]
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th 7 International Workshop on the Biology of Fish Gametes 2019 - Rennes