Cryopreservation of marine early-life stages E. Paredes Doctoral Thesis Cryopreservation of marine invertebrate early-life stages: applications in marine water quality assessment and aquaculture. Author: Estefania Paredes Rosendo Supervisors: Dr. Juan Bellas and Dr. Ricardo Beiras UniversidadedeVigoǦFacultadedeCienciasdoMar i assessmentandaquaculture. applicationsinmarinewaterquality invertebrateearlyǦlifestages: UniversidadedeVigoǦFacultadedeCienciasdoMar EstefaniaParedesRosendo Cryopreservationofmarine DoctoralThesis iii Picturesprovidedby À Ǧ ǣ Ǥ UniversidadedeVigoǦFacultadedeCienciasdoMar ǡͳͲʹͲͳͶ ÓǤ × Ǥ v vii Ǥǣ ÀǦ UniversidadedeVigoǦFacultadedeCienciasdoMar Ǥǣ À ǡ ǡͳͲʹͲͳͶǤ ×ǡ Ǥ × × ǡ dz ÓǤ Ǧ ǣ Dz ǡÓ À ÀǦǡ À ǣ UniversidadedeVigoǦFacultadedeCienciasdoMar ǡͳͻʹǤ Ǥ ToJose Tomyfamily ix Ǥ AKNOWLEDGEMENTS xi UniversidadedeVigoǦFacultadedeCienciasdoMar Ǥ ǡ ǡ × ǡ ǡ ǣ Ǥ Ǥ ǡ Ǣ Ǥ Ȍǡ Ȍǡ ǦǦ ȋ ǡ ȋǡǡ ǣ Ǥ ǤǤǤ ǡ ǡ Ǥ Ǥ ǡ Ǥ ǡ ƲǤ ǡ ǡ Ǥ ǣǤ ǯǤ Ǥ Ǧ Ǥ Ǧ ǡ ǨǨ Ǣ ǯ ǡ Ǥ Ǥ Ǥ UniversidadedeVigoǦFacultadedeCienciasdoMar xiii ͳǤʹǤͳ ͳǤʹǤʹ ͵ͳ ʹǤͶ ͺ ʹǤ͵ ȋParacentrotuslividusȌ ʹ ǣ Ǥ ʹǤʹ Ǧ ǣ Ǥ Ͷ ʹǤͳ ȋParacentrotuslividusȌ Ǥ 29 ʹͶ ʹ͵ ʹʹ ʹͳ ͳͻ ͳͻ ͳͺ ͳ ͳͷ ͳͶ ͳ͵ ͳͲ ʹ Pages CHAPTER2.DEVELOPMENTANDSTANDARDIZATIONOFTHESEAURCHIN CRYOPRESERVATIONPROTOCOL Crassostreagigas ͳǤͶǤͶ ͳǤͷ Mytilusgalloprovinciallis ͳǤͶǤ͵ Pernacanaliculus ͳǤͶǤʹ Paracentrotuslividus ͳǤͶǤͳ ͳǤͶ ͳǤ͵Ǥͳ ͳǤ͵ ͳǤʹ ͳǤͳǤͳ ͳǤͳ CHAPTER1.GENERALINTRODUCTION Contents xv ͳͳͲ ͻͻ ANNEXES ͳͷ͵ ͳͶͺ 200 183 175 159 UniversidadedeVigoǦFacultadedeCienciasdoMar 3.Publications 2.Resumen 1.Listoffiguresandtables BIBLIOGRAPHY ͳͶ ͷǤʹ ͷǤͳ 148 CHAPTER5.GENERALDISCUSSIONANDCONCLUSIONS ͶǤ͵ ͶǤʹ ǣ ȋCrassostreagigasȌȋMytilus galloprovincialisȌǤ ͳ͵͵ ͶǤͳ ̻ȋPernacanaliculusȌ Ǥ ͳͳ CHAPTER4.DEVELOPMENTOFBIVALVECRYOPRESERVATIONPROTOCOLS ANDAPPLICATIONS 115 ͵ǤͶ ͵Ǥʹ ȋParacentrotuslividusȌȂ Ǥ Ǥ ͵Ǥͳ ȋParacentrotus lividusȌ ͺͷ Ǥ CHAPTER3.SEAURCHINCRYOPRESERVEDEMBRYOSAPPLICATIONS 83 UniversidadedeVigoǦFacultadedeCienciasdoMar GeneralIntroduction౹Chapter1౹ xvi UniversidadedeVigoǦFacultadedeCienciasdoMar ͳǤͷ ͳǤͶǤͶ Crassostreagigas galloprovincialis ͳǤͶǤ͵Mytilus ͳǤͶǤʹ Pernacanaliculus ͳǤͶǤͳ Paracentrotuslividus ͳǤͶ ͳǤ͵Ǥͳ ͳǤ͵ ͳǤʹǤʹ ͳǤʹǤͳ ͳǤʹ ͳǤͳǤͳ ͳǤͳ CHAPTER1.GENERALINTRODUCTION CryopreservationofmarineinvertebrateearlyǦlifestages 1 FUNDAMENTALSOFCRYOBIOLOGY 2 UniversidadedeVigoǦFacultadedeCienciasdoMar ͳͻͻͳȌǡ ȋ ͳͻͻͶȌǡ ȋǯ ʹͲͲͲȌǡ ȋ Ǥ ʹͲͲͲǡ ʹͲͲʹǡetal.ͳͻͻʹͲͲǡ Ȍǡ ȋǤǤ ǡ ǡ et al. ǡ ʹͲͲͶǡ ʹͲͲǡ et al. ʹͲͲͲȌǤ ǡ ȋ Ǥ ǡ ǣ ȋʹͲͲͶǡʹͲͲȌǤͳͻʹ ǣ ǡ dz ǣ ͳͻͷ Dz ȋʹȌǤǡ ͳͻͷͲ Ǥ ͳͻͷͲ Ǥ ȋ Ȍ et al. ͳͻͶͻ ʹͲͲȌǤ ȋ ʹͲͲͶǡ Ǧ Ǥ ȋ ʹͲͲͶȌǤ Ǧ ǡ ȋȌ Dz dzǤ ǡ ǡ 1.1 1.GENERALINTRODUCTION GeneralIntroduction౹Chapter1౹ 3 Ǥ Ȍ Crioprotectingagents ʹͲͳͳȌȋͳǤͳȌǤ UniversidadedeVigoǦFacultadedeCienciasdoMar et al. ͳͻͻǡ ʹͲͲʹǡ et al. ʹͲͲͻǡ et al. ʹͲͲͻǡ ʹͲͳͳǡ et al. ȋȌȋȌȋǤǤ ͳͻ͵ǡ ȋʹȌǡ ȋ Ȍǡ ȋ Ȍǡ ȋȌǡ ȋ et al. ʹͲͲͲȌǤ ǣ ǡ ǡ ǡ Ǧ Ǧǡ Ǧ Ȃ Ǧ ǡ ȋͳͻͲǡͳͻǡͳͻͺͲǡʹͲͲͶǡetal.ʹͲͲͶȌǤ ǡ ǡ ǡ ȋetal.ʹͲͲͶȌǤǡ ǡ ǡ Factorstobeconsideredinmarineinvertebratecryopreservation: ȋetal.ʹͲͲͶȌǤ ȋ Ȍǡ ǡ ǡ ǡ ǡ Ȁ ȋʹͲͲͺǡʹͲͲʹȌǤ ȋǯʹͲͲͶȌȋetal.ʹͲͳͳȌǡ ȋÓ ͳͻͻͷȌǡ ȋ et al. ʹͲͳ͵Ȍǡ CryopreservationofmarineinvertebrateearlyǦlifestages 4 GeneralIntroduction౹Chapter1౹ ͳǤͳ ǣ ȋʹȌǡ ȋ Ȍǡ ȋ Ȍǡ ȋȌǡ ȋȌǡȋȌǤ ͵ͺʹ ʹʹ ʹ ʹ ȋǦͳȌ ͺǤͳ͵ ȋȀ ͵ȌʹͶ ͑ ͳǤͳ ʹǤͲ ͳǤͳͳ ǤͲͻ ͳǤͲͶ Ǧ ȋͻȌ ͳʹʹʹͳͳ ͳʹʹʹͳͳ ʹͶʹǤ͵ ͳǤͷͺ ʹͶʹǤ͵ ͳǤͷͺ ʹͷͲͲǦʹͷͲͲͲͲͲͲͲ ͳǤʹ UniversidadedeVigoǦFacultadedeCienciasdoMar 5 ǡ DzǦdz b) CPAadditionmethodology UniversidadedeVigoǦFacultadedeCienciasdoMar ǡ ǣ ǡ ǡǤ ǡ ȋ ʹͲͲȌǤ ǢǡǤ ǡ ǡ ȋʹͲͲͶȌǤ ȌǤ ȋ ȌȋʹͲͲͶȌǤ ȋ ȋetal.ʹͲͲͷȌǤ Ǥǡ ǡ Ǥ ǡ Ǥ ǡ DzǦdz ǡ ȋʹͲͲȌǤ ǡ Ǥ ǡ ȋ ʹͲͲȌǤ ǡ CryopreservationofmarineinvertebrateearlyǦlifestages 6 c) Coolingrate UniversidadedeVigoǦFacultadedeCienciasdoMar Ǥ ͳǤͳǤ Ǧͳͻ͑Ǥ Mazuretal.(1980)Ǥ ȋͳǤͳȌǤ ǡ Ǥ ȋͳͻͺͲǡʹͲͲͶȌǡ Ǥ Ǥ ǡ ȋ ͳͻͲǡ ͳͻǡ ͳͻͺͳǡ ʹͲͲȌǤ ǡ ǡ ǡǡǡ GeneralIntroduction౹Chapter1౹ 7 Pegg(2007). UniversidadedeVigoǦFacultadedeCienciasdoMar ͳǤʹǤ Ǥ ʹͲͲȌǤ ȋ ͳͻͲǡ ͳͻͳǡ et al. ͳͻͶǡ ʹͲͲͶǡ Ȍǡ ȋͳǤʹȌǤ ȋ Ǥǡ ǡ ȋȌ ȋʹͲͲͶȌǤ ȌǤ ȋ Ǥ ǡ ȋetal.ʹͲͲͲǡʹͲͲͶȌǤ Ǥ CryopreservationofmarineinvertebrateearlyǦlifestages 8 UniversidadedeVigoǦFacultadedeCienciasdoMar Ǥ Ǥǣ Ǥǣ ͳǤ͵Ǥ̺ Ǥǣ ǡ etal.ʹͲͲͲǡʹͲͲͲȌǤ Ǥȋ Ǥ ȋ ͳǤ͵Ȍǡ ȋʹͲͲͲȌǡ Ȍǡ ȋ ǡ ȋet al. ʹͲͲͲȌǤ ǡ ǡ ǡ d) Seeding GeneralIntroduction౹Chapter1౹ 9 UniversidadedeVigoǦFacultadedeCienciasdoMar ǡ Ǥ ȋ ʹͲͲȌ Ǥ ǡ ǡ Ǧ g) CPAremoval Ǥ Ȍǡ Ȍ ȋ Ǥ ǣ Ȍ ȋȌ ǡ ȌǤ ȋǦ ǡ ǡ Ȍ Ǧ ȋʹͲͲͶȌǣ Ǧ Ǥ ȋ Ȍǡ ǡ Ǥ f) Thawing ȋ etal.ͳͻͺͶȌǤ Ǧ ʹͲͲͲ Ǥ ʹͲͲʹȌǤ ȋ ͳͻͻͳǡ ͳͻͺͲǡ ȋǦͳͻ ͑Ȍ e) Liquidnitrogenconservation CryopreservationofmarineinvertebrateearlyǦlifestages 10 MARINEINVERTEBRATECRYOPRESERVATION UniversidadedeVigoǦFacultadedeCienciasdoMar Ǥ ȋ ʹͲͲͶǡ ʹͲͲȌǤ ǡ ǡ ǡ Paracentrotuslividus Ǥ Ǥǡ Perna canaliculus ȋ ʹͲͲͻȌǡ Mytilus galloprovincialis Crassostreagigas ͳͻͲǯǡ ǡ Ǥ ǡ ͳǤ͵ ǡ ǡȋǦetal.ʹͲͲͲȌǤͳǤʹ ǡ 1.1.1 ȋʹͲͲͲȌǤ ǡ ǡ ǡ Ǧ ǡ Ǥ GeneralIntroduction౹Chapter1౹ 11 etal.ȋʹͲͲȌ etal.ȋʹͲͳͳȌ etal.ȋʹͲͳ͵Ȍ ȋʹͲͲͻȌ etalǤȋͳͻͺͻȌ etal.ȋʹͲͳʹȌ etal.ȋʹͲͲͻȌ etal.ȋʹͲͳͳȌ etal.ȋͳͻͻͶȌǡȋʹͲͲͲȌ ȋͳͻͻͶȌǡȋʹͲͲͲȌǡȋʹͲͳͳȌǡ etal.ȋʹͲͲʹȌ etalǤȋͳͻͻȌǡetal.ȋͳͻͻȌ etal.ȋͳͻͻͶȌǡetal.ȋͳͻͻȌ etal.ȋͳͻͻ͵Ȍ ȋʹͲͳͳȌ etal.ȋʹͲͳͳȌ ȋʹͲͲͺȌ Ǧ etal.ȋͳͻͻͺǡʹͲͲͳȌ Ǧ etal.ȋͳͻͻͺǡʹͲͲͳȌ ȋͳͻͻʹȌǡetal.ȋͳͻͻȌǡȋͳͻͻȌ etalǤȋͳͻȌǡȋͳͻͻͳȌǡetal.ȋͳͻͻ͵Ȍǡ ȋͳͻͻͷȌǡȋͳͻͻȌǡetalǤȋͳͻͻͻȌǡetalǤȋʹͲͳ͵Ȍ etal.ȋͳͻͺͻȌǡetal.(ʹͲͲͷȌǡetal.(ʹͲͳͳȌ ȋͳͻͳȌǡȋͳͻȌǡ ȋͳͻͻͷȌǡetal. ȋʹͲͲͺȌǡȋʹͲͳͳȌ Reference UniversidadedeVigoǦFacultadedeCienciasdoMar Mytilustrossulus Mytilus galloprovincialis Mytilusedulis Pernacanaliculus Haliotis diversicolor Meretrixlusoria Ostreaedulis Saccrostrea glomerata Crassostrea virginica stage Developmental Crassostreagigas Molluscspecies Table1.2. Ǥ CryopreservationofmarineinvertebrateearlyǦlifestages .ȋʹͲͳͳȌ ȋʹͲͲͻȌ etal.ȋʹͲͲȌ etal.ȋʹͲͲͻȌ etal.ȋʹͲͲͶȌ ȋͳͻ͵Ȍ etal.ȋͳͻͻǡͳͻͻǡͳͻͻȌ etal.ȋͳͻͻǡͳͻͻǡͳͻͻȌ etal.ȋͳͻͻǡͳͻͻǡͳͻͻȌ etal.ȋͳͻͻǡͳͻͻǡͳͻͻȌ etal.ȋͳͻͻǡͳͻͻǡͳͻͻȌ etal.ȋͳͻͻǡͳͻͻǡͳͻͻȌ ȋͳͻͺͻȌ etal.ȋͳͻͻͲȌ ȋͳͻͺȌǡ etal.ȋͳͻͺͺȌǡ etal.ȋͳͻͻͳǡͳͻͻͺȌ ȋͳͻͺȌǡetal.ȋͳͻͻͳǡ ͳͻͻͺȌ ȋͳͻͻȌ ȋͳͻͺȌ ȋͳͻͺȌ Reference UniversidadedeVigoǦFacultadedeCienciasdoMar Paracentrotuslividus EvechinusChloroticus Strongulocentrotus drobachiensis Tetrapigusniger Loxechinusalbus Pseudocentrotus pulcherrimus Anthocidariscrassispina Strongylocentotus intermedius Strongylocentrotusnudus stage Developmental Hemicentrotuspulcherrimus Echinoidspecies Table1.3. Ǥ GeneralIntroduction౹Chapter1౹ 12 FUNDAMENTALSOFECOTOXICOLOGY 13 ȋ UniversidadedeVigoǦFacultadedeCienciasdoMar ǡ Ȍ (2002). ͳǤͶǤ Ǥ fromWright&Welbourn ʹͲͲʹȌȋͳǤͶȌǤ Ȍ ȋ ͳͻͻͷǡ ȋ Ȍǡ ǡ ȋ ʹͲͲʹȌǤ ȋ ͳͻͻͷǡ ǡ dzǤ ǡ ǡ ǡȋ Ȍǡ ǡ ͳͻͻ ̶ 1.2 CryopreservationofmarineinvertebrateearlyǦlifestages 14 SEAURCHINBIOASSAYS ͳͻͷͳȌǤ UniversidadedeVigoǦFacultadedeCienciasdoMar ͳͻͷͲǯ ȋ ȋetalǤͳͻͺͶȌǤ ǡ ǡ ͳͻͻͷǡ ʹͲͲʹȌǤ Ȍȋetal. Ȁ ȋ ʹͲͲͺǡʹͲͳͲȌǤ ȋǤǤetal.ͳͻͻͻǡͳͻͻͷǡetal.ʹͲͲ͵ǡ ǡ Ǧ ȋ ʹͲͳ͵Ȍ ǡ 1.2.1 ͳͻͻͷǡʹͲͲʹȌǤ ȋȌ ȋ ͳͻͻ͵ǡ ȋȌ ȋȌ ȋȌǡ Ǥǡ ǣ ǡ ͷͲΨ ȋͷͲȌǡ ʹͲͲʹȌǤ ǣ ȋ Ǥ Ǧ ǡ ȋ ͳͻͻ͵ǡ ʹͲͲʹȌǤ GeneralIntroduction౹Chapter1౹ 15 ECOTOXICOLOGYANDCRYOPRESERVATION UniversidadedeVigoǦFacultadedeCienciasdoMar ͳͻͻͶȌǤ ȋ ͳͻͻʹǡ Ǧ 1.2.2 ȋetal.ͳͻͻͻȌǤ ǡ Ǧ ǣ ǡ ȋetal.ʹͲͲͲȌǤ Ȍ ʹͲͲͲȌǤ ȋetal. Ȍ Ǧ Ǧ ǤȋʹͲͲʹȌǤ Ǥ ȋ et al. ʹͲͲͷȌǤ Ȍ ǡ al.ʹͲͲͷȌǤ Ȍ ȋ et al.ʹͲͲͷȌǤ ȋet ȋetal.ʹͲͲͲȌǡ ǡ Ȍ CryopreservationofmarineinvertebrateearlyǦlifestages 16 AQUACULTUREOFMARINEINVERTEBRATES UniversidadedeVigoǦFacultadedeCienciasdoMar ͳʹʹȋʹͲͳ͵ǡͳǤȌǤ ǡ Ǥ Ǧ ǡ ǡ ȋ ʹͲͲ͵ǡʹͲͲȌǤ Ǧ ǡ ȋ ʹͲͳ͵Ȍǡ ȋ ͳǤͷȌǤ Ǧ ͳͻͻͲǯ ȋ ʹͲͲ͵ȌǤ ǡ ǡ Ǥ ͳͻͲǯǡ ǡ ǡ ǡ ȋ ʹͲͲǡ ʹͲͲ͵ȌǤ 1.3 Ǥ ǡ ǡ Ǥ ǡ Ǧ GeneralIntroduction౹Chapter1౹ 17 (YROXWLRQRIWKHHFRQRPLFYDOXHRIPDULQHLQYHUWHEUDWHV SURGXFHGE\DTXDFXOWXUH86' UniversidadedeVigoǦFacultadedeCienciasdoMar ȂʹͲͳ͵Ǥ ʹͲǤ ͳǤǤ ʹͲǤ ȂʹͲͳ͵Ǥ ͳǤͷǤ CryopreservationofmarineinvertebrateearlyǦlifestages ǡ Ǥʹ 18 AQUACULTUREANDCRYOPRESERVATION UniversidadedeVigoǦFacultadedeCienciasdoMar ȋetal.ʹͲͳʹȌǤ ǡ Ǥ Ǧ al. ʹͲͲͶȌǤ ǡ ȋ ͳͻͻͷǡ Ǧ et al. ͳͻͻͺǡ ʹͲͲͻǡ et ʹͲͲͺȌǤ ǡ ȋ ʹͲͳͳȌǤǡ Ǧ ȋet al. ȋʹͲͲͶǡetal.ʹͲͳͳȌǤ ǡ Ȍ ȋ ǡ ǡ Ǧ Ǥ ȋ et al. ʹͲͳͳȌ ǡ Ǧ ȋ ǡͳͻͻ͵ȌǤ Ȃ Ǧ Ǥ 1.3.1 ȋ ȌǤ ǡ ʹͲͲȌǡ ǡ ȋ ǡ ȋʹͲͳ͵Ȍǡ GeneralIntroduction౹Chapter1౹ 19 THESEAURCHINParacentrotuslividus 1.4.1 UniversidadedeVigoǦFacultadedeCienciasdoMar ȋ ʹͲͲȌǤ ȋ ͳǤͺȌ Ǥ ǡ ǡ ʹͲͲǡ ǡ ʹͲͲȌǤ ȋͶͲͲǦͷͲͲρȌ ͶͺǦȋ ʹͲ͑ǡ ʹͶǡͶǦ ȋ ͳʹͺ ͳͲͲρȌͺ ȋ ͻͲ ρȌǡ ȋ ͳǤȌǤ ǡi.e. ǡ ǡ Ǥ Paracentrotus lividus ǡ GENERALBIOLOGYOFTHETESTSPECIES 1.4 ʹͲͲͶȌǤ Ǧȋ ʹͲͳͳǡ et al. ͳͻͻͳǡ et al. ͳͻͻȌǤ ȋ ͳͻͻʹǡetalǤͳͻͺͻǡetal. ȋetal.ʹͲͳͳǡetal.ͳͻͻ͵ǡetal.ͳͻͻȌǤ ǡ CryopreservationofmarineinvertebrateearlyǦlifestages 20 Ǧ ͶǦ UniversidadedeVigoǦFacultadedeCienciasdoMar ͳǤǤ Paracentrotuslividus. GeneralIntroduction౹Chapter1౹ 21 THEGREENSHELLTMMUSSELPernacanaliculus UniversidadedeVigoǦFacultadedeCienciasdoMar ͳǤͳͲǤ Ǥ ȋ ʹͲͲ͵ȌǤ ǡ ȌʹͶǦͶͺȋͳͷͲǦʹͷͲρȌǤ ȋ ͺͲǦͳͲͲ ρȌ Ǧ ȋǦ Ͳ ρǤ ͳǦʹͲ ȋʹͲ ͑Ȍ P.canaliculus Ǥ ͳǤͻȌǤ Ǥ ʹͶͲ ȋ Ǥ Ǧ ǡ Perna canaliculus 1.4.2 Ǥ Ǥ ȋ Ȁ Ȍ ȋ Ȍ ͳǤͺǤ ȋȌ ȋȌ CryopreservationofmarineinvertebrateearlyǦlifestages 22 THEMEDITERRANEANMUSSELMytilusgalloprovincialis ͳǤͳͲȌǤ UniversidadedeVigoǦFacultadedeCienciasdoMar ʹͲ ͑ Ͷͺ Ǧ ȋͳͲͲǦͳʹͲ ρȌ ȋ Ǥ ͺͲǦͻͲρǡ ʹͲǦʹͺ edulisǤ ǡMytilus ǡǦȋʹͲͳ͵Ȍ.Mytilusgalloprovincialis ǡ ǡ ǡ ǡ ǡ 1.4.3 Ǧ Ǥ ͳǤͻǤ Ǧ Ǥ Ǥ GeneralIntroduction౹Chapter1౹ 23 THEPACIFICOYSTERCrassostreagigas UniversidadedeVigoǦFacultadedeCienciasdoMar ʹͲͲ͵ȌȋͳǤͳͲȌǤ ʹʹ͑Ǥ ǦʹͶǦͶͺȋʹͲͳ͵ǡ ρ ȋ ǤͷͲρȌ ͳͶ Ǥ ʹͲǦͶͲ Ǥ C. gigas ǣ ͷͲǦʹͲͲ ǡ ǡ ǡ ǡ ǡ ǡ 1.4.4 ͳͷͲȋ ǦʹͲͲȌǤ Ǥ ȋ ʹͲͲ͵ȌǤǦ Ǧ Ǥ ͶͲ Ǧ ǦǦ CryopreservationofmarineinvertebrateearlyǦlifestages D E E D Ǧ E D F Ǧ F 24 UniversidadedeVigoǦFacultadedeCienciasdoMar ͳǤͳͲǤ ǤȋȌPernacanaliculusǢȋȌMytilusgalloprovincialisǢȋ Ȍ CrassostreagigasǤ GeneralIntroduction౹Chapter1౹ OBJECTIVES 25 UniversidadedeVigoǦFacultadedeCienciasdoMar Objective5ǣ Ǧ ȋǡ Ȍȋ ͶȌǤ Objective 4: ǡ ȋ ͶȌǤ Objective3ǣ ȋ ͵ȌǤ Objective 2ǣ Ǧ ȋ ͵ȌǤ ȋ ʹȌǤ ǡ ǣ ǡ ǡ ǡ ǡ Ȁ ǣ Objective 1ǣ Ǧ ǡ . ǣ Perna canaliculusǡ lividusǡCrassostreagigas Mytilusgalloprovincialis Ǧ ǣ Paracentrotus 1.5 CryopreservationofmarineinvertebrateearlyǦlifestages 26 UniversidadedeVigoǦFacultadedeCienciasdoMar GeneralIntroduction౹Chapter1౹ UniversidadedeVigoǦFacultadedeCienciasdoMar 27 ʹǤ͵ ȋParacentotuslividusȌ ǣ Ǥ ʹǤͶ ʹǤʹ ǣ Ǥ ʹǤͳ ȋParacentrotuslividusȌ Ǥ CHAPTER2.DEVELOPMENTAND STANDARDIZATIONOFTHESEAURCHIN CRYOPRESERVATIONPROTOCOL CryopreservationofmarineinvertebrateearlyǦlifestages 28 UniversidadedeVigoǦFacultadedeCienciasdoMar Developmentandstandardization౹Chapter2౹ 29 UniversidadedeVigoǦFacultadedeCienciasdoMar ȋ ͵ȌǤ ǡ Ǥ ǡ ȋ ʹ͵ȌǤ ǡ ȋ ʹȌǤ ǡǦ ǡ Ǥǡ ǡ ǡ ȋ ͳȌǤ ǡ Ǥ Ǧ Ǥǯ ǡ ȋ ǡ ǡ ȋ Ȍǡ ȋ Ȍǡ ȋȌ ȋȌȌǡ ȋ ȋʹȌǡ Ǥ ǡ Ǥ Ǥ CHAPTER2:DEVELOPMENTANDSTANDARDIZATIONOFTHESEAURCHIN CRYOPRESERVATIONPROTOCOL SUMMARY CryopreservationofmarineinvertebrateearlyǦlifestages Developmentandstandardization౹Chapter2౹ ǣ 30 UniversidadedeVigoǦFacultadedeCienciasdoMar ǤʹͲͳͶǤ lividusȌ ǣ Article 3Ǧ Ǥ ǡ Ǥ ǡ (Paracentrotus ͳͶǦͳͺͲǤ ǣ Ǥ ʹ ȋʹͲͳͳȌ Article 2.Ǧ Ǥ ǡ Ǥ ǡ lividusȌ ǤͷͻȋʹͲͲͻȌ͵ͶͶǦ͵ͷͲǤ Article1ǤǦǤǡǤǡ ȋParacentrotus 31 UniversidadedeVigoǦFacultadedeCienciasdoMar ǡ ǡ ǡ Ǧ ȋetal.ʹͲͲ͵ǡetal.ʹͲͲͺȌǤ Ǥ ǡ 1.Introduction ǡ Ǥ ͶʹǤͻΨͲǤͷΨǡ ͑ ǦͳǤ ͶͳǤͷΨ ͺǤͷΨǡ ʹǡ ǦʹͲ͑ͲǤͷ͑Ǧͳǡ Ǧ͵ͷ͑ͳ ͶǦͳʹ͑ͳ͑Ǧͳǡ Ǥ Ǥ ǡ P.lividus Paracentrotus lividusǤ ǡ ǡ ȋȌ ǡ ȋǡ Ǣ ǡ Ȍ ȋ ǡ ʹǢ ǡ Ǣ ǡ Ȍ Ǧ Ǥ ǡ Ǥ Ǥ Ǧ Abstract 2.1.CRYOPRESERVATIONOFSEAURCHINEMBRYOS(Paracentrotuslividus) APPLIEDTOMARINEECOTOXICOLOGICALSTUDIES. CryopreservationofmarineinvertebrateearlyǦlifestages Developmentandstandardization౹Chapter2౹ 32 UniversidadedeVigoǦFacultadedeCienciasdoMar Ǥ Ǣ ȋȌ Ƥ ȋ Ȍ ȋȌǡ ǡ ǣ ȋȌ Ǧ Ǥ ͳͻʹǡetal.ͳͻͻͻǡetal.ͳͻͻͳǡͳͻͻͳȌ ǡ ȋǤǤ Ȍ ȋ ͳͻȌǡ ȋ ͳͻͺǦͳͻͻǡetal.ͳͻͻǡͳͻͻǡetal.ͳͻͻȌǤǡ ȋetal.ʹͲͲǡ al.ʹͲͲͳǡetal.ʹͲͲͷȌǤ ǡ ͳͻͻͷǡetalǤʹͲͲͶǡǦ ʹͲͲͳǡͳͻͻͳǡet ȋǤǤ et al. ͳͻͻǡ Ǥ ǡ ǡ ǡ ȋ ǦʹͲͲȌǤ ǡǦ ǡ ȋ et al. ͳͻͻͻȌǤ Ǧ ǡ ǡ Ǧ Ǥ ǡ Ƥ ȋǤǤetal.ͳͻͻͻǡͳͻͻͷȌǤ ǡ 33 UniversidadedeVigoǦFacultadedeCienciasdoMar ȋǤǤ et al. ʹͲͲǡ Ǥ ǤʹǤͳ ȋȌǤ Ȃ ȋ Ȍǡ Ǧ ȋȌ ȋʹȌǡ ȋ Ȍǡ Ƥ Ǥ Experimentalsolutions Ͷ͑Ǥ Ǥ ͳͲͲ Ǥ Ǧ ȋʹͲͲȌǤ Ulva lactucaǤ ͳǤ À ȋ ǡ Ȍ Ǥ ,P.lividusǡ Biologicalmaterial 2.Materialsandmethods ʹͲͲͳȌǤ P.lividusȋ Ƥ ȋetal.ʹͲͲͷȌǤǡ ǡ ͳͻͻͲȌǤ ȋ ʹͲͲͳǡ ǡ ͳͺͳȌǡ Paracentrotus lividus ȋ CryopreservationofmarineinvertebrateearlyǦlifestages Developmentandstandardization౹Chapter2౹ 34 UniversidadedeVigoǦFacultadedeCienciasdoMar Ǥ ȋͻͲ ρȌǤ Ƥ ͵ͷ ȋ Ȍǡ ͳͲͲȌǡȋα͵ͷȌ ǤƤ ͶͲΨǡ ͶǦȋα Ǥ ʹͲǡ ͶͺͶǦ ͷͲǦρǡ ͲǤͲ͵ͲǤͳ͵Ǥǡ ͑ͳʹƤͳ ͳͷǤ ͵ͲͶ ǡ ȌͲǤͳ͵ȋƤ ʹȌǤ ǤͲǤͲ͵ȋƤ ͲǤͷ al.ʹͲͲͺȌǤͳͷƤͳǡƤʹ ȋǤǤetal.ʹͲͲǡet ȋ et al. ͳͻͶȌǤ ǡ ǡ ǡ Ǥ ʹ ͳƤ ȋȌǤ ǡ ǡ Ǥ ͷͲͲ ǡ ǡ ȋÚ etal.ͳͻͺͷǡetal.ͳͻͺͲȌǤ Ǥ Toxicitytests ʹͲͲͷȌǤ ͳͻǦͳͻͺǦͳͻͻǡ et al. ͳͻͻͶǡ ͳͻͻͷǡ et al. ͳͻͻͻǡ et al. 35 ȋȌ ͲǤͳǡͲǤͷǡ1ǡ1.5ǡʹ 1ǡ1.5ǡ2 0.68ǡ1.36ǡ2.04ǡʹǤʹǡ͵ǤͶ ͲǤͲͳǡͲǤͲʹǡ0.03ǡ0.04ǡͲǤͲͷ 0.085ǡ0.425ǡ0.75 ʹ Ϊ0.085ǡ0.425ǡ0.75 Ϊ0.03ǡ0.04 Ϊ0.085ǡ0.425ǡ0.75 Ϊ0.03ǡ0.04 Ϊ0.085ǡ0.425ǡ0.75 Ϊ0.03ǡ0.04 ȋȌ UniversidadedeVigoǦFacultadedeCienciasdoMar ǡǡ Ǥ Ǥͳ͑ͳǤ ǦͺͺͲͲȋ ǤȌ Ǥ ʹ ͳƤ ȋȌǤ Ǥ ͷͲͲ ǡ Cryopreservationtrials Ǥ ʹǤͳǤ Ǥ ͲǤͺǦͳʹǡͺǤͲͳάͲǤͳͶȋάȌǤ ͵ͶǤͺ ά ͲǤͶͷ ǡ Ǥʹ͵ ά Ǥ ͻͲΨǤ CryopreservationofmarineinvertebrateearlyǦlifestages Developmentandstandardization౹Chapter2౹ 36 UniversidadedeVigoǦFacultadedeCienciasdoMar ǡͳͷƤͳǤ ǡƤ ͲǤͷȋδͲǤͲͳȌǤ ǡ ͲǤͳʹǡ ͳͷƤǤ Ƥ ͳͷƤ Ǥ ʹ ʹǤͳ P.lividus Individualcryoprotectants 3.Results ͳͻͻͷͳͻͻͷǤ Ƥ Ǧ ȋ ͳͻͻͳȌǤ ȋȌǤǡ ǯ ȋȌ Ǧ ȋȌ Ǥ ͳͷǤͲ Statisticalanalyses 37 ǡ Ǧ Ǥ UniversidadedeVigoǦFacultadedeCienciasdoMar ͳͳǤͷ ǡͳǤͷʹǤ ʹͲǤͷͳ ǡ ȋʹǤ͵ȌǤ ǡ P. lividus Ƥ ǡ ͶǦ ȋ ʹǤʹ ʹǤ͵ȌǤ ǡ ǡ ǡ Ǥ ǡ ʹͳͷƤǤǤ ͳͷ Ƥ Ǥ ȋȌ ͵Ͳ ͵Ͳ ʹ ʹǤͳǤ P.lividusǤȋȌ CryopreservationofmarineinvertebrateearlyǦlifestages Developmentandstandardization౹Chapter2౹ 38 ͲǤͷ ͳ ͲǤͺ ͲǤͲͷ ͲǤͷ ͳ Ǧ ͳ ʹ ʹǤͲͶ ʹǤͲͶ ʹ ʹΪ ʹΪ Ϊ Ϊ Ϊ Ϊ ʹǤͲͶ ʹǤͲͶ ͳ ʹ ͳ Ǧ ͲǤͷ ͲǤͲͷ ͲǤͺ ͳ ͳ ȋȌ ʹǤͲͶ ʹǤͲͶ ʹ ʹ ͳ Ǧ ͲǤͷ ͲǤͲͷ ͳǤ͵ ͳ ͳǤͷ Ǧ Ǧ ͳǤͷ Ǧ ͳǤͷ ͳ Ǧ Ǧ ͳǤ͵ ͳǤͷ ͳ Ǧ Ǧ ͳǤͷ Ǧ ͳǤͷ ͳ Ǧ Ǧ ͳǤ͵ ͳǤͷ ͳǤͷ ȋȌ Ǧ Ǧ Ǧ Ǧ ͳǤͷ ͳ Ǧ Ǧ ʹǤͲͶ ͳǤͷ ʹ UniversidadedeVigoǦFacultadedeCienciasdoMar ʹȋͳȌΪ ȋʹǤʹȌǤ Ƥ ʹǤʹȌǤǡʹǡ ǡ ʹ ȋʹǤʹ Ǥǡ ʹ ʹ ʹǤ ʹ ʹ Ǧ ʹǡ ǡ Ǥ Cryoprotectantscombination ʹǤʹǤ Ǥ ȋʹǤʹʹǤ͵ȌǤ ͳǤ͵ ʹǤͲͶ ȋ ʹǤʹȌǤ Ǧ ͲǤͺͳǤ͵ ǡ ͳ ͳǤͷ Ǥ ǡ 39 CryopreservationofmarineinvertebrateearlyǦlifestages ǤʹǤʹǤ P.lividus͵Ͳ ǡ ǡ ʹ ǤȋȌʹͳΪǢȋȌ2ͳǤͷΪǢȋȌʹͳΪǢȋȌʹͳǤͷΪǢȋȌʹʹΪǤǤ UniversidadedeVigoǦFacultadedeCienciasdoMar 40 ౹Chapter2౹ ǤʹǤ͵Ǥ P.lividus͵Ͳ ǡ ǡ ǤȋȌ ͳΪǢȋȌ ͳǤͷΪǢȋȌ ʹΪǢȋȌ ͳΪǢȋȌ ͳǤͷΪǢȋȌ ʹΪǤǤ UniversidadedeVigoǦFacultadedeCienciasdoMar 41 ȌǤ UniversidadedeVigoǦFacultadedeCienciasdoMar ͶͲǤͷΨ ȋʹΨ ͶǦ Ȍ ͺǤͷΨ ȋʹͷǤͻΨ ͶǦ ͵ͲǤ͵Ψȋ ȌͲǤͷΨȋ Ȍǡ ͶǦ ȌǤ ǡ ͶͳǤͷΨȋͳ͵ΨͶǦȌͶͲǤͷΨȋʹΨ ͶʹǤͻΨȋ Ȍ ǡ ͵ͲǤͳΨȋ Ȍ ǡ ǦʹͲ͑ͲǤͷ͑Ǧͳ Ǧ͵ͷ͑ͳ͑ǦͳǤ Ǧ͵ͷ͑ͳ͑ǦͳǡȋȌ ͶǦͳʹ͑ͳ͑Ǧͳǡʹ ǣȋȌ ͶǦͳʹ͑ͳ͑Ǧͳǡʹǡ ȋetal.ʹͲͲǡ ͳͻͻͷǡetal.ͳͻͻͻȌǡ ȋʹȌΪȋͲǤͷȌǤ ǡ ǡ Cryopreservationtrials Ǥ ȋ ʹǤͶ ʹǤʹȌǤ ǡ Ϊ ǡ ǡ Ƥ ȋʹǤ͵ʹǤʹȌǤ Ϊ ǡ ǡ Ǥ ǡƤ Ϊ ͳǤͷ ǡǤ ǤǡƤ Ϊ ͳ Ǥ ͲǤͷ ǡ Ƥ ͳ ʹǡ ǡ ʹ Ϊ ǡ Ǥ ǡ CryopreservationofmarineinvertebrateearlyǦlifestages 42 ౹Chapter2౹ ǤʹǤͶǤ P.lividus͵Ͳ ǡ ǤȋȌ ͲǤͺΪǢȋȌ ͳǤ͵ΪǢȋȌ ʹǤͲͶΪǢȋȌ ͲǤͺΪǢȋȌ ͳǤ͵ΪǢȋȌ ʹǤͲͶΪǤ Ǥ UniversidadedeVigoǦFacultadedeCienciasdoMar 43 ͳ ͳǤͷ ʹ ͲǤͺ ͳǤ͵ ʹǤͲͶ ʹǤʹ ͵ǤͶ ͲǤͲͳ ͲǤͲʹ ͲǤͲ͵ ͲǤͲͶ ͲǤͲͷ ͲǤͲͺͷ ͲǤͶʹͷ ͲǤͷ ͻͻǤͺάͶǤ ͻͻǤͲάǤͲ ͳͲͲά͵ǤͲ ͻͻǤͷάͶǤͺ ͳͲͲάǤͲ ͳͲͲάͶǤ ͳͲͲάͺǤ͵ ͳͲͲά͵Ǥͷ ͻǤͷάǤͲ ͺͻǤͻάͷǤͲ ͻǤ͵άͶǤʹ ͷͻǤʹάǤͳ ͵ͻǤͺάǤͻ ͻͻǤʹάʹǤ͵ ͺͻǤͲάͳǤͷ ǤͶάͶǤ ͻǤͻάͲǤͻ ͻǤͺάͶǤͷ ͻͶǤͳάͷǤͶ ͺͻǤάǤʹ ͵ǤͲάͳͶǤ ͻǤά͵Ǥʹ ͻǤͻάʹǤ ͻͷǤʹάǤ ͻ͵Ǥά͵Ǥͷ ͻͻǤͷάͳǤ͵ ͻͺǤάʹǤͻ ͻǤάͲǤͳ ͻͺǤͳάͳǤͺ ͳͲͲάͲǤͳ ͻͶǤͷάʹǤ ͺͲǤά͵ǤͲ ͷǤά͵ǤͲ ͶǤͻάͲǤͳ ͻǤάͳǤʹ ͻͶǤͷάʹǤͳ ͻͲǤͳάǤ͵ ͻǤͻά͵Ǥ͵ ͻǤάʹǤͶ ͻǤʹάʹǤ ͻǤͻάͳǤ ͷǤͳά͵Ǥ ͻͺǤͷάʹǤ ͻͻǤʹάͳǤͶ ͻͺǤͻάʹǤͲ ͻǤͻάͲǤͺ ͻͻǤͷάͳǤͲ ͻͺǤͻάͳǤʹ ͻǤ͵άͳǤͷ ͻͻǤͷάͲǤ UniversidadedeVigoǦFacultadedeCienciasdoMar ͲǤͳ ͲǤͷ ͳ ͳǤͷ ʹ ͻͻǤͻάʹǤͳ ͳͲͲάʹǤ͵ ͳͲͲάͳǤ ͲǤͲͺͷ ͲǤͶʹͷ ͲǤͷ ʹ ͳͲͲάͳǤʹ ͳͲͲάʹǤ ͳͲͲάʹǤͻ ͻͻǤͺάͶǤ͵ ͳͲͲάͷǤͲ ͲǤͲͳ ͲǤͲʹ ͲǤͲ͵ ͲǤͲͶ ͲǤͲͷ ͻͲǤ͵άͳǤʹ ͶǤͻάͺǤͻ ͷǤάͳ͵Ǥͳ ͶǤʹάͳͺǤͲ ʹͻǤͶάͷǤͳ ͺͷǤͲάͺǤ ͵ͲǤͺάʹǤͲ ͶͺǤͷάǤʹ ͷ͵ǤͺάͷǤͷ ͳͷǤͷάǤʹ ͻͺǤάͳǤʹ ͵͵ǤʹάͷǤͳ Ͳ ͻͷǤʹάͶǤͷ ͶǤͺάʹͳǤͶ ͷǤͺάͳǤͻ ͲǤͺ ͳǤ͵ ʹǤͲͶ ʹǤʹ ͵ǤͶ ͻͺǤͳάͳǤ ͳͲͲάͳǤͶ ͺͳǤͶά͵Ǥͷ ͵ͶǤͲάͳͲǤͻ ʹǤͶάͳ͵Ǥͻ ͺǤͳάͶǤͲ ͻǤͷάǤͷ ͺͶǤάǤ͵ ͵ͶǤͷάͳͲǤͻ ʹǤάͳǤͶ ͵Ǥͻά͵Ǥͻ ͻͶǤͶάǤͳ ͻǤͲάͷǤͳ ͻǤͶάͳǤͺ ͻͲǤάͺǤ ͻǤ͵άʹǤͺ ͻǤͻάͺǤͻ ǤͻάͳͷǤ ͻͻǤͺάͲǤͶ ͲǤͲάͳǤͺ ʹͳǤͲάͷǤʹ ͺǤͻάͳǤͳ ͶǤͳά͵ǤͲ Ͳ ͻͻǤͻάʹǤͲ ͻͺǤͶάͳǤͷ ʹͻǤͺάʹǤͺ ǤͶάͳͲǤͷ Ͳ ΨͶǦ ͺǤͳάͶͻǤͳ ͳͲͲάͳǤͲ ͳͲͲά͵ǤͲͺ ͳ ͳǤͷ ʹ ͲǤͲͺͷ ͲǤͶʹͷ ͲǤͷ ͳͲͲάͷǤͻ ͳͲͲάʹǤͳ ͻǤʹάͳͲǤͺ ͺͻǤάͳͲǤͺ ͻͶǤͺάͳǤͳ ͲǤͲͳ ͲǤͲʹ ͲǤͲ͵ ͲǤͲͶ ͲǤͲͷ ͳͲͲάͳʹǤͶ ͷʹǤͺάͳͷǤͳ ͳǤͷͳάͳͶǤ ͲǤͳ ͲǤͷ ͳ ͳǤͷ ʹ ͲǤͺ ͳǤ͵ ʹǤͲͶ ͺͷǤͻά͵Ǥ ͶǤάͷǤͻ ͳǤάʹǤͶ ͳͲͲάͳͶǤ ͻͳǤͻάͲǤͶ ʹͶǤͺά͵Ǥͳ ͲǤͳά͵Ǥͻ Ͳ Ψ ʹ ͳ ͳǤͷ ʹ ͲǤͳ ͲǤͷ ͳ ͳǤͷ ʹ ʹ ȋȌ ǡǡȋ ȌǤ ǡ ʹǡ ǡ ʹǤ͵Ǥ P.lividu͵Ͳ CryopreservationofmarineinvertebrateearlyǦlifestages Developmentandstandardization౹Chapter2౹ 44 UniversidadedeVigoǦFacultadedeCienciasdoMar ʹ ʹȋetal.ʹͲͲȌǤ Ǥ ǡ ǡ Hemicentrotus pulcherrimus Evechinus chloroticusǡ ʹ ͶǦ ǡȋͳͻͺȌetal.ȋʹͲͲȌ Ǥ ǡ ͳǡ ǡ ǡ P.lividus Ǥ ǡ ǡǦ ͵Ͳ P. lividusǤ ǡ ʹ ʹǡ ǡ ǡǦǡ Ǥ ǡ Ǥ ǡ ǡ ǡ ǡǡ ȋʹͲͲͷǡͳͻͻͷȌǤ ʹͲͲʹȌǡ ȋ ͳͻͻͷǡ et al. ʹͲͲǡ et al. ǡ ʹ Ȍ ǡ Ǥ ǡ ȋ ǡ ǤǤ Ƥ ȋ et al. ʹͲͲͺǡ et al. ʹͲͲͺȌǤ ǡ 4.Discussion 45 UniversidadedeVigoǦFacultadedeCienciasdoMar Ǥ Ǥ lividus Ǥ ǡ ʹ ͲǤͷ ǡ P. ǡ ͳͻͻǡetal.ͳͻͻǡetal.ͳͻͻǡetal.ͳͻͻͳȌǡ ʹ ȋ et al. ʹͲͲǡ ʹ Ǥ ǡ ʹ Ǥǡ P.lividus Ǥ Ǥ ǡ ǡ ǡ ǡ P.lividusǡ Ƥ ǡ ȋetal.ͳͻͻͶȌǤ Ǥǡ Ǧ Ƥǡ Ƥ ǡͳͷƤͳͷ ȋetal.ʹͲͲǡetal.ʹͲͲͺȌǤ Ƥ ǡ Ǥ ȋ ͳͻͻͷǡetal.ʹͲͲͺȌǢ CrassostreagigasHaliotismidaeǡ ʹ etal.ʹͲͲͷȌǤǡ ȋetal.ͳͻͻͶǡ etal.ʹͲͲͷǡ ǡǡ ǡ ʹǡ ǡ CryopreservationofmarineinvertebrateearlyǦlifestages Developmentandstandardization౹Chapter2౹ 46 ǡ UniversidadedeVigoǦFacultadedeCienciasdoMar Ǥ ǡ P.lividusǤ ǡ ǡ Ǥ ǡ ʹ Ƥ Ǥ ǡ ǡ Ƥ P.lividusǤ ǡ Ǥ ǡ ͶʹǤͻΨ ͲǤͷΨǡ ͶͳǤͷΨ ͺǤͷΨǡ ǦǤ ǡ ͳʹ ͑ Ǥ ʹ ͲǤͷ Ǧ ǡ Ǥ 47 UniversidadedeVigoǦFacultadedeCienciasdoMar ǡ ȋǤǤ et al. ʹͲͲͺǡ ͳͻͻʹǡ ǡ ǡ ǮǮ ǯǯǡ Ȃ ǡ ȋͳͻͷͳȌǡ Ƥ 1.Introduction Ǥ ͻ Ǥ Ƥ Ǥ Ȃ Ȍ ʹȂ ǡ ȋͳȂͻͺΨ Ǥ Ǧͳ ͑Ǧͳ ǦͺͲ ͑Ǥ Ƥ ʹ ʹǡ Ͷ ͑ ʹ ǡ Ǧͳ ͑ Ǧͳ Ǧͳʹ ͑Ǥ ͳǤͷͲǤͲͶǤ ǡ Ǥ ǡ ǡ ƪ ǡ Ȃ Ǥ Ȃ Ǥ ǡ Abstract APPLICATIONINMARINEQUALITYASSESSMENT. 2.2. ADVANCES IN THE CRYOPRESERVATION OF SEA URCHIN EMBRYOS: POTENTIAL CryopreservationofmarineinvertebrateearlyǦlifestages Developmentandstandardization౹Chapter2౹ 48 UniversidadedeVigoǦFacultadedeCienciasdoMar ƪ ǡ ȋʹͲͲͻȌǤ ȋParacentrotus lividusȌ ȋȌ ǡ ͳͻͻͳǡͳͻͻͳȌǤ ȋʹͲͲͺǡͳͻͻͻǡͳͻͻͷǡetalǤ ǡǡ Ƥ ǡ ȋ ͳͻͻʹǡ ͳͻͻͶȌǤ Ǧ ͳͻͻǡ et al. ͳͻͻȌǤ ǡ ȋ et al. ʹͲͲǡ ͳͻǡ ͳͻͺǡ ͳͻͻǡ et al. CrassostreaȌǡ Ƥǡǡȋ ͳͻͻͻǡǦ ʹͲͲͳǡetal.ʹͲͲͷȌǤ ǡȋǤǤʹͲͲͳǡ etal.ʹͲͲͻǡetal. Ǥ ǡ ǡ ǡ Ǥ ǡ ʹͲͲͻȌǤ ǡ ȋ ǡ Ǥǡ ǡ ͳͻͳǡ ͳͻͺͳǡ ͳͻͻ͵ȌǤ 49 UniversidadedeVigoǦFacultadedeCienciasdoMar Ͷ͑ Ǥ Ƥ ȋǡʹǤͶȌ Ǥ Ǥ ͳͲͲ Ulva lactucaǤ ͳǤ À ȋ ǡ Ȍ Ǥ ǡP.lividusǡ Biologicalmaterial 2.Materialsandmethods ʹͲͲͳȌǤ Ƥ ȋ ȋʹͲͲͳȌǤ ǡ P.lividus ȋetalǤʹͲͲͷȌǤǡ ǡƤ ʹͲȋʹͲͲͳǡͳͻͻͲȌǤ ǦǡǦ ǡ ǡ Ǥ ǡ ǡ P.lividusȋ ͳͺͳȌǡ P.lividusǤ Ƥ ǤƤǡ ǡ CryopreservationofmarineinvertebrateearlyǦlifestages Developmentandstandardization౹Chapter2౹ 50 ͵ ʹʹ ʹͶ ʹʹʹ ʹͶͳͲʹ ͲǤʹͲͲ ʹͶǤͷͲ ͳͲǤͺ ͶǤͲͲ ͳǤͶ ͲǤͲͲ ͲǤͳͲͲ ͲǤͲͶͷ ͲǤͲʹͶ ͲǤͲͲ͵ ȋǦͳȌ UniversidadedeVigoǦFacultadedeCienciasdoMar Ǥ ǡ ͳǣͳ etal.ʹͲͲͷȌǡǡ ȋȌ et al. ʹͲͲͻǡ et al. ͳͻͻǡ et al. ͳͻͻǡ et al. ͳͻͻǡ et al. ͳͻͻͻǡ Ƥ ǡ ȋ ȋʹͲͲͻȌǤǡ ȋʹȌ ȋȌ ȋ Ȍ ȋ Ȍ ǡ ʹʹ ʹǤͶǤƤ Ǥ ʹ ͳǤ Ȍ ȋ ǦȌ Ǥ ͷͲͲ ȋͳǦ Cryopreservationexperiments 51 UniversidadedeVigoǦFacultadedeCienciasdoMar Ǥ Ǥ ȋͻͲρȌǤ ȋ Ȍǡ ƤƤ͵ͷ ȋ Ǧ et al. ʹͲͳͲȌǤ ǡ ǡ Ǧ ǡ Ǥ ȋ α ͵ͷȌ Ǥ ͶͲΨǤ ͶǦȋαͳͲͲȌ Ǥ ʹͲǡ ͶͺͶǦ ͷͲρǡ ͳʹ Ƥ Ǥ ǡ ͳ ͑ ͳ ʹͻǤ ͑ Ͷͺǡ ǤǦ͵ͷǦͺͲ ǡ Ǥǡ Ǥ ͺͲ͑ǡƤ ǡͲǤͷ͑ͷ͑Ǧͳǡ Ǥǡ Ǧ͵ͷǦ Ǥ Ͳǡʹͷ ͳ͑ǦͳǦͳʹ͑ǡ ͺͺͲͲȋ ǤȌǤͶ͑ʹ ǡ Ƥ ʹ Ǥ ȋʹͲͲͻȌǤ ͳͷ Ƥ ǡ ͲǤͲͶǤ ǡͳǤͷʹǡͳǤͷ ǡʹ ǡͲǤͷ CryopreservationofmarineinvertebrateearlyǦlifestages Developmentandstandardization౹Chapter2౹ 52 UniversidadedeVigoǦFacultadedeCienciasdoMar ͳǦͺͲ͑ȋʹǤͷȌǤ ǦͳǡǦͲǤͷ͑ǦͳǦʹͲ͑ͳ͑Ǧ ͺͲ͑ȋ͵ͳΨ Ȍ ͳ͑ ͳʹǤͷ͑ǦͳǤʹΪǦ ǡ Ϊ ʹΪ Ϊͳ͑ǦͳǦǤ ȋʹǤȌǤ Ǥ ͑ǦͳǦʹͲ͑ͳ͑ǦͳǦ͵ͷ͑ǡʹ Ǧ ͳǡʹǤͷ͑ǦͳȋǦ͵ͷ͑ȌǦͲǤͷ PostǦholdingcoolingrates ǡ Ǥ ǡͷͷΨͶͺΨ Ǥͷ Ϊ ǡ ͷͷΨ ͷΨ ǡ ʹ Ϊ Ϊ ʹ ǦͳǦ͵ͷ͑ʹƤǤ ʹͷ ͳʹ͑Ǥǡ ͳ͑ Ͷ͑ʹǡ ͳ͑ǦͳͲǡ ȋǦ͵ͷ͑Ȍ Ǥ ʹǤͷ Durationoftheseedinghold 3.Results ȋȌ Ǥ Ǥ Ǧ ͳͷǤͲ Statisticalanalyses ODUYDOJURZWK ODUYDOJURZWK E D PLQ E D (*393 D 3*393 D E D D PLQ D %ODVWXODH )HUWLOLVHGHJJV E E 6HHGLQJ VWHS GXUDWLRQ DW& PLQ E E E D D 0H6275( D D D E E &PLQ D D (*393 E 3*393 E 0H6275( D D D E %ODVWXODH )HUWLOLVHGHJJV D D ʹǤǤ P. lividus Ǥ Ƥ Ǧ ͳǡʹǤͷ͑Ǧͳ ǦͲǤͷ͑ǦͳʹͲ͑ ͳ ͑ Ǧͳ Ǧ͵ͷ ͑Ǥ ǣ Ͷ͑ʹǢ Ǧͳʹ͑ͳ ͑ǦͳǢʹǢ Ǧ͵ͷ͑ ͳ ʹǤͷ ͑ Ǧͳ ǡ ǦʹͲ ͑ ͲǤͷ ͑ ǦͳǦ͵ͷ͑ͳ͑ǦͳǢƤʹ Ǥ ͵ͻͺ ρ ȋ Ǥ ǢδͲǤͲͷȌǤ 53 && PLQ 3RVWKROGLQJFRROLQJ UDWHV &PLQ D D D UniversidadedeVigoǦFacultadedeCienciasdoMar CryopreservationofmarineinvertebrateearlyǦlifestages ʹǤͷǤ P. lividus Ǥ Ͳǡ ʹ ͷ Ǧͳʹ ͑Ǥ ǣ Ͷ ͑ ʹ Ǣ ͳʹ ͑ ͳ ͑ ǦͳǢ Ͳǡ ʹ ͷǢ Ǧ͵ͷ͑ͳ͑ǦͳǢƤ ʹǤ ͵ͳ ρ ȋ Ǥ Ǣ δ ͲǤͲͷȌǤ ODUYDOJURZWK ODUYDOJURZWK ODUYDOJURZWK ODUYDOJURZWK Developmentandstandardization౹Chapter2౹ 54 ͷ͑Ǧͳ ͳ͑Ǧͳ ͵ͲǤάͳͲǤͻʹ ͳͷǤͶάͺǤͲ͵ Ψ Ψ ͳͳǤ͵άǤͷͺ ʹǤͶάǤͲ Ͳ ͲǤͻάʹǤʹͷ ͲǤͷ͑Ȁͳ͑Ǧͳ UniversidadedeVigoǦFacultadedeCienciasdoMar ȋ Ǧ͵ͷ ǦͺͲ ͑Ȍ Plungingintoliquidnitrogen ͵ͻͶρǤǡǢδͲǤͲͷǤ ǡ ǦʹͲ͑ͲǤͷ͑Ǧͳ ǦͺͲ͑ͳ͑ǦͳǢƤʹǤ Ͷ͑ʹǢ Ǧͳʹ͑ͳ͑ǦͳǢʹǢ ǦͺͲ͑ͳ͑Ǧͳͷ͑Ǧͳ ǦͺͲ͑ȋάȌǤ ǣ ʹΪ ǦͺͲ͑ ʹǤͷǤ Paracentrotus lividus ǦͺͲ͑Ǥ ͵ͳΨ Ǥ ǡ Ȍǡ ʹ Ϊ ǦͺͲ ͑ ʹ Ϊ Ǧ͵ͷ ͑ ȋͷΨ ͳΨ ȋ ʹǤȌǤ ǦͺͲ͑ǡ ͳ ͑ Ǧͳ Ǧ͵ͷ ͑ Finalfreezingtemperature 55 D D D (*393 3*393 0H6275( E %ODVWXODH )HUWLOLVHGHJJV E E & E E E )LQDOIUHH]LQJWHPSHUDWXUH & D D D UniversidadedeVigoǦFacultadedeCienciasdoMar ͳʹ͑ͳ͑ǦͳǢʹǢ Ǧ͵ͷǦͺͲ͑ͳ͑ǦͳǢƤʹǤ ǡǦ͵ͷǦͺͲ͑Ǥ ǣͶ͑ʹǢ Ǧ Ǥ Ƥ ʹǤǤ P.lividus Ϊ ΪǤ ʹ Ϊ Ƥ Ǥ ǡ ǦͺͲ͑ǡƤ ǤǡʹΪǡ ͳΨ Ȍ ͶͲΨ Ȍ ǡ ʹΨ ȋͳͲΨ ͶǦ Ǧ͵ͷ͑ͺʹΨȋͳΨͶǦ ͵ͷ͑Ǥ ǡʹΪ ȋʹǤͺȌǤ Ǧ CryopreservationofmarineinvertebrateearlyǦlifestages ODUYDOJURZWK ODUYDOJURZWK ODUYDOJURZWK Developmentandstandardization౹Chapter2౹ 56 UniversidadedeVigoǦFacultadedeCienciasdoMar ǤǢδͲǤͲͷȌǤ Ǥ ͵ͻͲρȋ ͑ǦͳǢʹǢ Ǧ͵ͷ͑ǦͺͲ͑ͳ͑ǦͳǢƤʹǢ Ǧ͵ͷǦͺͲǤ ǣͶ͑ʹǢ Ǧͳʹ͑ͳ Ǥ ʹǤͺǤ P.lividus ǢδͲǤͲͷȌǤ ͶͲͳ ρ ȋ Ǥ CryopreservationofmarineinvertebrateearlyǦlifestages 57 E K D 'XUDWLRQ RI WKH ODUYDO UHDULQJ & K D & D 0H62 75( K D D UniversidadedeVigoǦFacultadedeCienciasdoMar ǤǢδͲǤͲͷȌǤ ͶͺǡʹͻǤ ͵ρȋ ͵ͷǦͺͲ͑ͳ͑ǦͳǢƤʹǢǢ ǣͶ͑ʹǢ Ǧͳʹ͑ͳ͑ǦͳǢʹǢ Ǧ Ͷͺǡ ʹ ͻ Ǥ Ǧ͵ͷ͑ǦͺͲ͑Ǥ ʹǤͻǤ Ǥ ʹΪǤ ͻͺΨǤ ʹǤͻ͵Ψǡ ͻǤ ͵ʹΨ ǡ ǡǦͺͲ͑ǡ ͶͺǤ Ǥ Ǥ Ǧ͵ͷ ͑ ͶǦ Ǥ ʹǤͻ Ͷͺǡ ʹ ͻ ǡ Ƥ ǡ Ǧ͵ͷ ǦͺͲ ͑ ǡǤ Durationofthelarvalrearing ODUYDOJURZWK Developmentandstandardization౹Chapter2౹ 58 UniversidadedeVigoǦFacultadedeCienciasdoMar ʹ Ǥʹ etal.ȋʹͲͲͺȌǤ ǡ Ǥ Ǧͳʹ͑Ǥǡ Evechinuschloroticus ǡ et al. ʹͲͲ Ƥ Ǧ ʹͲͲͺǡetal.ʹͲͲͷȌǤ ʹͲͲͻȌ ȋ et al. ͳͻͻǡ ͳʹ͑Ǧȋ lividus Ǧ ȋ ʹͲͲȌǤ P. ǡ Ǥ ǡ ǡ Ǥ Ȃ Ǥ ʹͲͳͲǡ et al. ʹͲͲͷȌǤ ǡ ȋͳͻͻͷǡetal.ʹͲͲͺǡetal.ʹͲͲǡ ǡ 4.Discussion 59 UniversidadedeVigoǦFacultadedeCienciasdoMar ȋetal.ͳͻͻȌǤ ʹ ͑ Ǧͳ ʹ Ͷ ͑ Ǧͳ Ǥ ǡǡ Ǥ ǡ ǦͳȌ ǡ ȋͳ͑ etal.ʹͲͲǡͳͻǡͳͻͺǡͳͻͻǡetal.ͳͻͻǡͳͻͻȌǤ ȋʹǤͷͳͲ͑ǦͳȌȋ ͲǤ͵ͷͲ͑Ǧͳǡ ȋͲǤ͵ ʹ ͑ ǦͳȌǤ ǡ ͲǤͳ ʹͲ ͑ Ǧͳǡ ʹͲͲͳǡ Ǧ et al. ʹͲͲͺǡ et al. ʹͲͲͷȌǤ et al. ͳͻͻǡ ͳͻͻͻǡ et al. ʹͲͲͷǡ Ǧ ȋetal.ʹͲͲǡetal.ʹͲͲͻǡ ʹͲͲȌǤ ǡ ȋͳͻͻͳǡ ǡ ǡǡ ȋet al. ʹͲͲ͵ȌǤ ǡ ȋetal.ͳͻͻͻȌǤ ǡ ǡ ʹͲͲͳȌǤ ȋǦ ʹͲͲͻǡ et al. ͳͻͻǡ et al. ͳͻͻͻǡ ʹͲͲͺǡ Ǧ et al. ʹͲͲͺȌǡ ǡͷͳͷȋetal. Ϊ Ǥ Ϊ Ϊǡͷ CryopreservationofmarineinvertebrateearlyǦlifestages Developmentandstandardization౹Chapter2౹ 60 Ƥ UniversidadedeVigoǦFacultadedeCienciasdoMar ȋ͵ͶΨȌ Ǥǡ ǦͺͲ͑ǡ ȋ ͺʹΨ ʹΨȌ Ǧ͵ͷ ͑Ǥ ǡ Ǥǡ ǡ gigas Ǥ ǡ etalǤȋͳͻͻͻȌͺΨ C. etal.ͳͻͻͳͲȂͳͷΨ C.gigas ͳͻͻͷ͵ͺΨ ȋC.gigasȌ ǡ Ǥ Ǧͳͻ ͑Ǥ ȋ ͳͻͻͳǡ ͳͻͺͲȌǤ ǡ Ǧͳ͵Ͳ ͑ ǡ ǡǦͺͲ͑Ǥ Ǥ ǡ ǦͺͲ ͑ǡ ͵ͲΨ ǦͺͲ͑Ǥ ǡ ʹΪ Ǧ͵ͷ͑ ǡ Ǧ͵ͲǦͶͲ͑Ǥ ǦͷͲ ͑Ǥ ǡ ȋͳͻͻͷȌ Crassostrea gigas ȋHemicentrotus pulcherrimus Strongylocentrotus intermediusȌ Ǥ ǡȋͳͻǡͳͻͺǡͳͻͻȌ ǡ ǡ ͳͻͺǡͳͻͻǡ ͳͻͻͷǡǦetal.ʹͲͲͺȌǤ Ƥ ȋ et al. ʹͲͲǡ et al. ʹͲͲͻǡ Ǥǡ 61 Ǥ UniversidadedeVigoǦFacultadedeCienciasdoMar Ȃ Ǥ Ƥ ͻ Ǥǡ ǡ ǡ ǡͶͺ Ǥ ͳ͑ǦͳǦͺͲ͑ǤƤʹǦͺͲ͑ ʹǡ ͳ ͑ Ǧͳ Ǧͳʹ ͑Ǥ Ͷ ͑ ʹ ǡ ʹͳǤͷΪͲǤͲͶǤ ǡ ǡ Ǥ ǡ Ǥ ǡ Ƥ ȋͻͺΨ Ȍ ͻ͵Ψ ʹ Ǥ ǡ ͻ Ǧ ǡ ͶǦǤ ͶͺǡʹͻǤǡ ǡ ǦͺͲ ͑ ǡ Ǥ ǡǡ ǡ ǦͺͲ͑ͶǦǦ Ǥ ǡ CryopreservationofmarineinvertebrateearlyǦlifestages Developmentandstandardization౹Chapter2౹ 62 UniversidadedeVigoǦFacultadedeCienciasdoMar ȋʹͲͲȌǡ ǡ Ǥ Ǧ ȋǤʹͲͲʹȌǤ ǡ ǡ ǡ 1.Introduction Ǥ À Ǥ ǤǤǡ ȋǤǤȌǡ ǤǤ ǡ ǡ Ǥ ǡ Ǥ ȋParacentrotus lividusȌ Ǥ Ǧ Ǥ Abstract REPRODUCTIVESEASONALITY. CRYOPRESERVED EMBRYOS: EFFECTS OF CRYOPRESERVATION PARAMETERS AND 2.3. SURVIVAL AND GROWTH OF SEA URCHIN (Paracentrotus lividus) 63 UniversidadedeVigoǦFacultadedeCienciasdoMar ȋ ʹͲͲͻǡ ʹͲͳͳȌǤ ǡ ȋʹͲͲͳȌǤ ʹͲͲͷȌǤ ǡƤ ȋetal. ʹͲͲͳǡ ͳͻͻͲȌǤ ǡ ȋ ȋ ͳͺͳȌǡ Paracentrotus lividus etal.ʹͲͳͳȌǤ ȋ ȌȋʹͲͲͶǡetal.ʹͲͳͳȌǡ ǡ ȋ ǡ ȋetal.ʹͲͳͳȌǤ Ǧ ȋ ǡ ͳͻͻ͵ȌǤ ǡ ȂǦ ǡ ȋʹͲͳͳǡ etalǤʹͲͳ͵ȌǤ ȋ ʹͲͲͶȌǡ ȋ et alǤ ʹͲͳʹȌǡ ȋetal.ʹͲͲʹǡetal.ʹͲͲ͵ǡetal.ʹͲͲͺȌǡ ʹͲͲʹǡ ʹͲͲͺǡ et al. ʹͲͳ͵Ȍǡ ȋ et alǤ ʹͲͳ͵Ȍ ǣ ȋ ǡ ǡ Ǥ ȋ et al. ʹͲͲͶȌǤ et alǤ ʹͲͳͳȌǡ ȋÚ CryopreservationofmarineinvertebrateearlyǦlifestages Developmentandstandardization౹Chapter2౹ 64 UniversidadedeVigoǦFacultadedeCienciasdoMar ǤǤ Ǥ Ǥǡ Ǥ Ǥ Ǥ ȋȌǡǡʹͲ͑ͺǡ Ǣ εͻͲΨȌǤ ȋ Ǥ ȋͳͺιǡ͵ͷǤͷΩȌǤ ǡ Ǥ ͷ ǡ ȋ ǡȌ ǡ ǡParacentrotuslividusǡ À Biologicalmaterial 2.MaterialsandMethods Ȍ Ǥ ǡ ǤǤǦǣ ǡ ǣ ȋ Ǧ ȋʹ ͲǤʹͷ ȌǤ ȋȌǡ ǡ ʹͲͳͳȌǤ ǡ ȋ ʹͲͲͻǡ ǡ ǣ Ȁ ǡ ǡ ȋȌǡ ǡ 65 UniversidadedeVigoǦFacultadedeCienciasdoMar ȋǦȌȋͳǤͷΩȌǤ ͳǤͷʹΪͲǤͲͶ ȋȌ ȋ ͵ͷΩȌǡ CPAsaqueousmedia Methodologicalparameters ǢΨαȋȟȀȟ ȌȗͳͲͲǤ ȋȟ Ȍǡ Ȍǡ ȋͲȌǡȟαǦͲǤ ͵ͷ ȋȌȋα͵ ǤȋȟȌ ͶͺǡʹͻǤʹͶͲΨ ʹͲͳͳȌǡ ͶͺǡȀȋ P. lividus ͶǦ Ǥ ȋʹͲ͑ǡȌͶǦ ͳͻάͳ͑ǤʹͲ ȌǤ ͳʹ ͳ ά ͳ ͑ ȋά ʹ ǦͺͲ͑Ǥ ʹ ͳ ͑ Ǧͳ ǦͺͲ ͑Ǥ Ƥ ʹ ǡ ͳ͑ǦͳǦͳʹ͑Ǥ ȋ ȌͶ͑ ʹ ȋͳǣͳȌǡͳͻάͳ͑Ǥ ʹ ͳ ȋ Ȍ ǡ ͳͷ ͳǤͷȋʹȌΪͲǤͲͶȋȌǡͳ ȋ ʹͲͳͳȌǤ Cryopreservationmethodology CryopreservationofmarineinvertebrateearlyǦlifestages Developmentandstandardization౹Chapter2౹ 66 Ǥ UniversidadedeVigoǦFacultadedeCienciasdoMar Ǥ ͵͵ ǡ ͶǦ ȋʹ ͲǤʹͷ Ȍǡ ǡ Ȍǡ ȋ ǡ Ǥ ǡ ǡ Ǥ ȋ Ȍ Cryopreservationcontainers ͶǦǦ Ǥ ʹͷǦͳȌʹͲ ǡͷͲͲȋ ͷǡͲͲͲǢʹǡͷͲͲǢͳǡʹͷͲͷͲͲȀǡʹǤ ȋ ȌǡǣͳͲǡͲͲͲǢ ǡ Embryodensity ͻ Ǥ ǡ ǡ ͶǦ Ǥ ǦǦʹͲ Ǥ Ͷ Ǥ ͳ ȋͳǣͳ ȌǤ ͳ 67 UniversidadedeVigoǦFacultadedeCienciasdoMar ͷρ Ǥ ǡ Ǥ ͳͻͷȌȋȌ ǡͻͻΨǡ ȋͳǣʹǣ͵ǣ͵ǣͳ ǯ ǡ ǡ Histologicalanalysis ȋ ǦͳȌǤ ȋʹͶǤȌǤ ǡ Ͳ͑ ȋʹͶȌ ͲǤͷ Ǥ ǡ ʹͲ Ɋ Ǥ Ǥ Oocytesweight Ǥ ǤǤ ǡ Ͳ͑ ȋͶͺǤȌ Ǥ etalǤͳͻͻͺǡetalǤʹͲͲǡ ʹͲͲȌǤ ȋͳͻͻͲǡetalǤͳͻͻͷǡ ǤǤ ǡ ȀȌέͳͲͲǤ ȋǤǤȌ ͳͻͻͲǣǤǤαȋ ConditionIndex Biologicalparameters CryopreservationofmarineinvertebrateearlyǦlifestages Developmentandstandardization౹Chapter2౹ 68 UniversidadedeVigoǦFacultadedeCienciasdoMar ȋ ʹǤͳͲȌǡ ǡ ͺΨ ά ʹ͵ ͻ CPAsaqueousmedia Methodologicalparameters 3.Results Ǥ ȋȌ Ǥ Ǥ Ǧ ̺ ͳͷǤͲ Statisticalanalises Ǥ Ǥ Ȃ Ǧǣ Ǥ ǤͷȂǣ ǤͶȂ ǣ ǡ ǣ ǤʹǦ ǣ Ǥ͵Ȃ Ǧ ǣ ͳ Ǧ ǣ ȋͳͻͻͲȌǤ ǡ Ȍ ȋ et al. ͳͻͻͷǡ et alǤ ͳͻͻͷȌǤ ǦÓetal.ʹͲͲͶȌǡȋ Ȍ ȋ ͳͻͻͲǡ ͳͻͻͲǡ et alǤ ͳͻͻͺǡ ǡ ȋ Ǥ Ǥ ǯǤǡ 69 UniversidadedeVigoǦFacultadedeCienciasdoMar ǣʹͲǤʹͷǤ ȋ ȌǤȋȌ Ǥȋ Ȍ ͻ ǤȋȌǣȋȌ ʹǤͳͲ Ȁ ͶǦ ǤͷͲΨ Ǥ ȋ ȌǤ ʹǤͳͲǡ Embryodensity Ǥ ǡ CryopreservationofmarineinvertebrateearlyǦlifestages Developmentandstandardization౹Chapter2౹ 70 Ǥ UniversidadedeVigoǦFacultadedeCienciasdoMar ʹǤͳͳǤ ȋǤǤȌ ÀȋǤȌ ǡ ȋαͲǤͶǡαͲǤͲͳͳǡαͳ͵ȌǤ ȋǦȌǡ ǤǤ ȋ ʹǤͳͳȌǤ ǤǤ ǡ ǡ ǤǤ ǡ Ǥ ParacentrotuslividusÀ ConditionIndex Biologicalparameters ǡǡ ȌǤ ʹȋʹǤͳͲ ȌǤʹΨάͳʹȋͷͺǤͻǤͺΨ ͲǤʹͷ Cryopreservationcontainers 71 ȋʹȌǤ UniversidadedeVigoǦFacultadedeCienciasdoMar ȋͳȌ Ǥ ǡ ǡ Ǧ ȋȌ Ǧ Ǥ ȋͷȌ Ǧǡ ȋͶȌ Ǧǡ ȋͳͻͻͲȌǤ ǡ ȋ͵Ȍ ȋ͵ǡ Ͷǡ ͷ Ȍ ʹͲͳʹǡ ʹǤͳ͵ Histologicalanalysis ʹǤͳʹǤ ǤǤ À ȋ ȌǤ αͲǤͶǡαͲǤͲͳͻǡαͳ͵ȌǤ ǤǤ ȋ ʹǤͳʹǢ ͷǤͲέͳͲǦ ȋ Ȍ ͷǤͻέͳͲǦͳͲ ȋ Ȍǡ ȋͷͲ ͲǤ ȌǤ Oocytesweight CryopreservationofmarineinvertebrateearlyǦlifestages 72 Developmentandstandardization౹Chapter2౹ ʹǤͳ͵Ǥ ȋͳͻͻͲȌǤ͵Ȃ ǦǤͶȂ ǤͷȂ ǤȂǦǤ UniversidadedeVigoǦFacultadedeCienciasdoMar CryopreservationofmarineinvertebrateearlyǦlifestages 73 UniversidadedeVigoǦFacultadedeCienciasdoMar ȋParacentrotus lividusȌ Ǥ ǡ 4.Discussion ͶͺǡʹͻʹͲ͑Ǥ Ǥ ʹǤͳͶǤ ͶǦ Ȁ Ǥ ǤǤǡ ͻ Ǥ ǡ ȋ Ǧ ȌǤ ǡ ǡ Ͷͺǡ ʹ ͻ ǡǡͳͻǤͶΨάǤͺǡ͵ͲǤͺΨάͳͶǤͶǡͷͲΨάͳͲǤͺ ȋ ʹǤͳͶȌǤ ͶǦ Cryopreservationofseaurchinembryos Seaurchincryopreservedembryosapplication౹Chapter3౹ 74 UniversidadedeVigoǦFacultadedeCienciasdoMar ǡ ǡ Ǥ et al. ʹͲͳͳǡ ͳͻͻͷȌǤ ǡ ȋȌȋetal.ʹͲͳͳǡ ǡ Ǥ Ͷ Ȍ ǡ ȋetal.ʹͲͳͳȌǡΨǦȋʹ ͵ ǡ ͲǤʹͷ ǡ ʹ Ͷ Pernacanaliculus Ǥ ǡ ȋ ʹͲͲͲǡ ʹͲͲʹȌǤ Ȍ ʹ ǡ ȋ Ǥ Ȁ Crassostrea gigas ȋ et al. ʹͲͲͷȌ ʹέͳͲ Ȁ ǡ Mytilus galloprovincialis ȋ et al. ʹͲͳͳȌ ͵έͳͲͳͷ ȋȌǤ ǡ ͷͲǡͲͲͲ Ȁǡ ǡ Ǥ ǡ ʹͷͲ ͷͲͲͲ ȀǤ Ǥ CryopreservationofmarineinvertebrateearlyǦlifestages 75 UniversidadedeVigoǦFacultadedeCienciasdoMar ǤǤǡ Ǥ ǡ ȋ Ȍ À ʹͲͲ͵ǡ at el. ʹͲͲǡ Ú ʹͲͲȌǡ ȋ et al. ʹͲͲͲǡ Ǧ ÀǦ Ǥ ǡ ǡ ǡ ǡ ȌǢ ǡ ȋ ȋ ʹͲͲͳȌǤ Ǧ Ǧ ǡ ǤǤ al.ʹͲͲͲȌǤ ȋet ǡ ǡ Ǥ ȋ Ȍ ȋ ʹͲͲͷȌǡ Ǧ Ǥ ȋ etal.ʹͲͲͲǡetal.ʹͲͲʹȌ Ǣ ǡ Ǥ Ǥ ͻ ǡ ǡ Seaurchincryopreservedembryosapplication౹Chapter3౹ 76 UniversidadedeVigoǦFacultadedeCienciasdoMar Ȍ ͶǦ ͻ Ǥ ǡ ȋ Ǥ ǡ etal.ʹͲͲͲǡetal.ʹͲͲʹȌǡ Ͷͺ ʹͲͲͷȌ ȋetal.ʹͲͲ͵ǡ ǡ ȋ Ͷͺ ǡǡʹͲΨ Ǥ ǡ ǡ Ͷͺ ʹͲ ͑ǡ ͶͲͲǦͷͲͲ ρǤ Ǥ ͶǦ ÀǦÀʹͲͲ͵ǡetal.ʹͲͲͷȌǤ ȋ ͳͻͻͲǡ ͳͻͻͷǡ et al. ͳͻͻͺǡ Ǧ ǡ ǡ ȋǤǤ ǡ ǡ ȌǤ ǡ ǡ À Ǥ ǯ Ȍǡ ǤǤȋͶ Ǥ ȋͷͲ ͲǤ Ȍǡ ǡ Ǥ ǡ ǤǤ ȋʹͲͲͳȌ CryopreservationofmarineinvertebrateearlyǦlifestages 77 UniversidadedeVigoǦFacultadedeCienciasdoMar Ǥ Ǧ Ǥ Ǥǡ ȋ et al. ʹͲͲ͵ȌǤ ǡ ȋȌ ǡ Ȍǡ ȋǤǤ ǡ ǡ ǡ Ǥ ǡ Ǧ Ǥ ʹͲͲͲǡ ʹͲͲͳǡ et al. ʹͲͲͳȌǤ ȋ Ǥ Ǥ ǡ ͶǦ ͻ ǡ Ȍ Ǥ ȋǤǤǡ ǡǤ etal.ʹͲͳ͵Ȍǡ galloprovincialisȋetalǤʹͲͳͳǡetalǤʹͲͳ͵ȌCrassostreagigasȋ Perna canaliculus ȋ et al. ʹͲͳͳȌǡ Mytilus Seaurchincryopreservedembryosapplication౹Chapter3౹ 78 UniversidadedeVigoǦFacultadedeCienciasdoMar ͺͲ͑ Ǥ ͳ͑ǦͳǦͺͲ͑ǤƤʹǦ ʹǡ Ͷ͑ʹǡ ͳ͑Ǧͳͳʹ͑Ǥ 7. P.lividusǣ Ǥ ʹΪǦͺͲ͑ ǡ ǡ 6. Ǥ P.lividusǤ ͳ͑Ǧͳ ǦǤ 5. ͳʹ ͑ Ǥ Ǥ 4. ȋǦ Ȍ ǡ Ƥ Ǥ 3. ͳͷƤͳͷƤ Ǥ 2. Ǥ ǡ Ǧ ȋ ǡ Ȍ͵Ͳǡ 1. ʹ 2.4CONCLUSIONS 79 UniversidadedeVigoǦFacultadedeCienciasdoMar Ǥ ȋǤǤǡ Ȍ 11. ͶͺǤ Ȁǡ ͶǦͻ 10. ǡ Ǥ 9. Ǥ ʹͷͲ ͷͲǡͲͲͲ Ȁǡ 8. CryopreservationofmarineinvertebrateearlyǦlifestages 80 UniversidadedeVigoǦFacultadedeCienciasdoMar Seaurchincryopreservedembryosapplication౹Chapter3౹ UniversidadedeVigoǦFacultadedeCienciasdoMar ͵Ǥ͵Ǥ Ǥ ͵ǤʹǤ ȋParacentrotuslividusȌȂ Ǥ ͵Ǥͳ ȋParacentrotuslividusȌǤ CHAPTER3.SEAURCHINCRYOPRESERVED EMBRYOSAPPLICATIONS CryopreservationofmarineinvertebrateearlyǦlifestages 81 82 UniversidadedeVigoǦFacultadedeCienciasdoMar Seaurchincryopreservedembryosapplication౹Chapter3౹ CryopreservationofmarineinvertebrateearlyǦlifestages 83 UniversidadedeVigoǦFacultadedeCienciasdoMar ȋ ͷȌǤ ǡ ǡ Ǥǡ Ǧ Ǥ Ǥ ǡ Ǥ ǡ Ǧ ȋP.lividusȌ ȋ ͶȌ ǡǡ ǡ Ǥ Ǥ SUMMARY CHAPTER3:SEAURCHINCRYOPRESERVEDEMBRYOSAPPLICATIONS ǣ Seaurchincryopreservedembryosapplication౹Chapter3౹ 84 UniversidadedeVigoǦFacultadedeCienciasdoMar Ǥǡ Ǥ Article 5ǤǦ Ǥ ǡ Ǥ ǡ ȋParacentrotus lividusȌ Ȃ ȋParacentrotuslividusȌǤǡǤ Article 4ǤǦ Ǥ ǡ Ǥ ǡ CryopreservationofmarineinvertebrateearlyǦlifestages UniversidadedeVigoǦFacultadedeCienciasdoMar Ǥ ȋ ʹͲͲͺȀͷȀȌǤ ǡ ǡ ȋȌ ǡ ȋ ʹͲͲͲȀͲȀȌǤ ǡ ǡ ǡ ǡ ȋȌ 1.Introduction Ǥ Ǥ Ǧ ͵ͲͲǤρǦͳͶǦǦ ȋͶǦȌǤ ͷ͵ǤρǦͳ ǡͺͳǤͲρǦͳǡ͵ͲͲǤρǦͳǦ͵ȋǦ͵Ȍ Ǥ ͷͲ ǡ ȋ ǡȌǤ Ȍ Ͷ Ǧ ǡ ȋ ȋParacentotuslividusȌ Abstract 85 3.1.THEUSEOFCRYOPRESERVEDSEAURCHINEMBRYOS(Paracentrotuslividus)IN MARINEQUALITYASSESSMENT. Seaurchincryopreservedembryosapplication౹Chapter3౹ 86 UniversidadedeVigoǦFacultadedeCienciasdoMar Ǧ Ǥ ȋ etal.ʹͲͳʹȌǤ ȋSparus aurata) SelenastrumcapricornutumǤ ȋ ǡ Ȍ etal.ȋͳͻͻȌǡ ȋͳͻͻͷǡ etal.ͳͻͻͻǡʹͲͲͺȌǤ ǡ ǡ ǡ ȋ ͳͻͻʹǡ ͳͻͻͶȌǤ Ǧ Ǥ ǡ Ǥ Ǧ ȋetal.ͳͻͻͻȌǤ ǣ ǡ Ǧ Ǥ ǡ ȋ ͳͻͻͷǡ et al. ͳͻͻͻȌǡ ʹͲͳͲȌ ȋetalǤͳͻͻͻǡͳͻͻͷǡetal.ʹͲͲ͵ǡʹͲͲͺǡ ǡǦ ǡǤ CryopreservationofmarineinvertebrateearlyǦlifestages 87 UniversidadedeVigoǦFacultadedeCienciasdoMar Ǧ ͺͲ ͑ Ǥ ǡ ͳ͑ǦͳȂͺͲ͑ǤƤʹ ͳ͑ǦͳǦͳʹ͑Ǥǡʹ Ȍǡͳͻάͳ͑Ǥ Ͷ͑ʹǡ ͳǡͳͷȋͳǣͳ ʹͳǤͷΪͲǤͲͶǤ ʹͲ ͑Ȍ ȋʹͲͳͳȌǤ ȋͺ Cryopreservationprotocol ͳǤ ȋ ͵ǤͳȌǡ ǡ Ͷ ͲǤͳΨȋȀȌǤ Ǥ ʹ ͶǦͷ͑Ǥ ȋʹȌ ȌǦ͵ȋǦ͵Ȍ ȋǡǡȌǤ ȋȌ ȋ ʹͲͲʹȌǤ ͶǦǦ ȋͶǦ ͳ Ǧͳ Ǥ ȋ ǡȌǡ ǡȋȌǡ Samplecollectionandexperimentalsolutions 2.MaterialsandMethods ȋ ǡȌǤ ǡͶ ǣ ͶǦǦ Ǧ͵Ȍ ǣȋ Ȍǡ ȋ Seaurchincryopreservedembryosapplication౹Chapter3౹ 88 UniversidadedeVigoǦFacultadedeCienciasdoMar ȋ et al. ʹͲͳʹȌǤ ȋȌ ͵Ͳ Ͳ ǡ ͳǣͶ Elutriationprocedure Ǥ Ǥ ͶǦ Ǥ ȋǣͺͲǦͳȌ ʹͲ͑ͻ ȌǤ ȋ Cryopreservedembryobioassay ʹͲ͑Ȍ Ǥ ȋͺ Ǥ Ǥ Ǧetal.ȋʹͲͳͲȌǤ ȋα͵ͷ Ȍǡ ͶͲΨ Ȍ ͵ͷ ȌʹͲ͑ȋ Ͷͺ ȋ ͶͲ Ǧͳ Ǥ Ȍinvitroǡ Ǧ ȋ Standardandfreshblastulabioassays. Bioassays ͳͻάͳ͑ Ǥ ͳʹ ͳ ά ͳ ͑ Ǥ CryopreservationofmarineinvertebrateearlyǦlifestages 89 ʹͲͳͲȌǤ ȋǡ ǡ UniversidadedeVigoǦFacultadedeCienciasdoMar ͵ͶǤͳ ρ Ǧͳǡ ͷͲ ͵ǤʹǤ ͷͲ 3.Results ȋαͶȌ Ǥ Ǧ Ǥ Ǥ ͷͲ ǤͲ ǡ Ǧ Ǧ Ǧ ȋ et al. ʹͲͲʹȌ ǦͻͷΨ ͷͲǦ ͷͲΨ Statisticalanalyses ʹʹ ƬȋʹͲͳʹȌǤ ȋͲͷǡȌ͵ȋʹȌͲǤͳͷΨ ȋȌ ǡǡ Chemicalanalysis Ǥ Ǥ ǡ Ͷ ʹͲ͑Ͷͺͻ ͺͲǦͳǡ ͶͲ Ǧͳǡ ʹͷ ͳͲΨ Ǥ ǡͳͲͲǡͷǡͷͲǡ ǡȋ͵ǤͳȌǤ ǡ ǡ Ȍ al. ͳͲ ʹȋ Ǧet Ǧ ȋȌ Seaurchincryopreservedembryosapplication౹Chapter3౹ 90 UniversidadedeVigoǦFacultadedeCienciasdoMar Ǥ ȋα͵ͷȌǤ ǡ ͵ǤͳǤ Ǥ ȋͺͳǤͲρǦͳȌȋ͵ǤͳȌǤ ǦͳȌǡ ͳȌǡ ȋͷʹρ ȋ͵ǤͳȌǤͷͲȋ͵ǤʹȌȋͶʹρǦ δͲǤͲͷ ȋͷ͵Ǥ ρ ǦͳȌ ȋ͵ʹǤʹρǦͳȌǡͷͲ CryopreservationofmarineinvertebrateearlyǦlifestages 91 ͺιǯͷǯǯ Longitude(W) Ͳ O2(mgLǦ1) S2Ǧ(mgLǦ1) δͲǤͲʹ Cd2+(μgLǦ1) ͳͲͲ ͳͲͲ 25% 10% ͳͲͲ ͳͲͲ 25% 10% ʹͳǤ͵ Ͳ Ǥͻͺ ͵ͷǤͳ ʹͳǤͶ δͲǤͲʹ ʹʹά͵ ͲǤάͲǤͲͳ ͲǤͶͳάͲǤͲͳ ͳͲͲ ͳͲͲ ͳͲͲ ͳͲͲ ͳͲͲ ͳͲͲ ͳͲͲ ͳͲͲ ͳͲͲ ͳͲͲ ͳͲͲ ͳͲͲ %LarvalGrowthcryopreservedblastulabioassay ͳͲͲ ͳͲͲ ͳͲͲ ͳͲͲ %LarvalGrowthStandardbioassay δͲǤͲʹ ͵ͺά͵ ͲǤͷάͲǤͲ ͳǤͷάͲǤͶ Metalconcentrationmeasuredintheelutriates Ͳ ͺǤͲͺ ͵ͷ ͺιͶǯ͵ǯǯ Ͷʹιʹʹǯʹǯǯ ʹͲǤͲǤͳ͵ Vi2 Elutriateparameters ͺιͶǯͻͺǯǯ Ͷʹιʹʹǯͺͺǯǯ ʹͲǤͲǤͳ͵ Vi1 SedimentSamplingLocation UniversidadedeVigoǦFacultadedeCienciasdoMar ͳͲͲ 50% ͷǤ 75% 100%elutriate ͳͲͲ 50% ͺǤͶ 75% 100%elutriate ʹͻάͷ Zn2+(μgLǦ1) ͲǤ͵άͲǤͲͷ Cu2+(μgLǦ1) Pb2+(μgLǦ1) ͲǤάͲǤʹ Ǥͳ pH ͵ͷ ͺǤͳͳ Salinity(‰) Tª(ºC) ʹͳǤͶ Ͷʹιʹǯǯǯ Latitude(N) ʹͲǤͲǤͳ͵ Vi0 Date Ǥ δͲǤͲʹ άͳ ͳͲͲ ͳͲͲ ͳͲͲ ͳͲͲ ͳͲͲ ͳͲͲ ͳͲͲ ͳͲͲ ͲǤͶͻάͲǤͲ ͲǤͶάͲǤͲͳ Ͳ Ǥ ͺǤʹ ͵ͷǤͳͷ ʹͳǤͶ ͺιʹǯʹǯǯ ͶʹιʹͶǯͳͺǯǯ ʹͲǤͲǤͳ͵ Vi4 ǤȋΨȌ Ǧ ǡ ͵ǤͳǤ ȋ Ȍǡ Seaurchincryopreservedembryosapplication౹Chapter3౹ 92 Ͷʹͷȋʹ͵ǤͺǦͷͻͲǤͳȌ ͶͲͶͺǤȋͳͻͷͲǤȂʹͳͺǤȌ ͵ͺͻǤʹȋʹͷͶǤͺȂͷʹ͵ǤȌ Pb BPǦ3 4ǦMBC ͶͶͺǤͷȋʹͶ͵ǤͲȂͷͶǤͲȌ ͲͲͲȋǤ ǤȌ ͷʹȋͺͺǤǦͺͳǤʹȌ ͵ʹǤʹȋʹͻǤͳǦ͵ͷǤ͵Ȍ ͵ͲͲǤȋͳͶͳǤʹȂͶͲǤͲȌ ͳͷͶͳȋͳʹͷǤͷȂͳͺʹͶǤͷȌ ͺͳȋͻǤͳǦͺ͵ǤͲȌ ͷ͵ǤȋͷͳǤͻǦͷͷǤͷȌ ͷͲǦ͵ȋ͵ǤʹȌͶͲͶͻρǦ ͵ͶǤͳȋ͵ͳǤͻǦ͵ǤͶȌ Cu UniversidadedeVigoǦFacultadedeCienciasdoMar δͲǤͲʹρǦͳǤ ǡ Ǥ ͷͲ ͵Ǥͳǡ ͲǤͻȋ͵Ǥ͵ȌǤ ʹαͲǤ ʹαͲǤͻͻͳǤ ǡ ͷͲ Ǥ ȋ͵ǤʹȌǤ ǡ ǡ ͷͲ ǣ ͵ͺͻǤʹ ρ Ǧͳǡ ͶͶͺǤͷ ρ Ǧͳǡ ͵ͲͲǤ ρ Ǧͳǡ ͶǦǤ ȋͳͷͶͳ ρ ǦͳȌ ȋ δͲǤͲͷȌ ȋ ͵ǤʹȌǤ ȋͲͲͲ ρ ǦͳȌǡ ͷͲ ͳǡ ͷͲ EmbryoǦlarvalbioassaysEC50(μgLǦ1) ǯ ǦǤ ǣDz dzǤ ǡ Ǧ͵ ͶǦǡ Ǯǯǡ Ǯ ǯ Ǯ ͵ǤʹǤͷͲȋρǦͳȌͻͷΨ ȋ Ȍ ǡǦ CryopreservationofmarineinvertebrateearlyǦlifestages 93 ͳͲͲΨ ͳǡ ʹ Ͷǡ UniversidadedeVigoǦFacultadedeCienciasdoMar ͷǤΨ ǡ ǡ Ǥ ǤͲ Ǥ ȋα͵ͷȌǤ ǡ ͵ǤʹǤǦ Ǧ͵ ͶǦǤ Seaurchincryopreservedembryosapplication౹Chapter3౹ 94 UniversidadedeVigoǦFacultadedeCienciasdoMar ͳͻͻͷȌǡ Ǧ ȋ Ǥ ȋ ͳͻʹǡ ͳͻͺͶǡ et al. ͳͻͻͻȌ 4.Discussion ȋȌǤǤ ȋȌǡ ȋȌ ȋȌ ȋȌǡ ȋȌ ͵Ǥ͵ǤͷͲ Ǥ Ǥ ͺǤͶΨ ǡ CryopreservationofmarineinvertebrateearlyǦlifestages 95 UniversidadedeVigoǦFacultadedeCienciasdoMar Ǧͳǡetal.ʹͲͳ͵ȌǤ ͷͲ Paracentrotuslividusȋͺͷ͵ǤͶρ ͶͶͺǤͷρǦͳǤ ǣ ͵ͲͲǤ ρ Ǧͳ ǡ ͵ͺͻǤʹ ρ Ǧͳ Ǥ ͷͲ ͶǦ ǡ ȋ͵ʹͺͲ ρ Ǧͳǡ et al. ʹͲͳ͵Ȍǡ ρ Ǧͳǡ ͶͲͶͺ ρ Ǧͳǡ ͲͲͲ ρ Ǧͳǡ ȌǤ ͷͲ δαǡȋͳͷͶͳ ǡ ͷͲ Ǧ͵ ǣ Ǥ ǡ ȋʹͲͲͳȌǡ ͳȌǤͷͲ ParacentrotuslividusͷͲͻρǦͳ δδȋͺͳρǦͳǡͶʹͷρǦͳǡͷʹρǦ ͷͲ ǣ Ǥ ǡ ͷͲ ȋͷ͵Ǥ ρ ǦͳȌ et alǤ ʹͲͲʹǡ et alǤ ͳͻͺͳ et alǤ ͳͻͻǡ ȌǤ ͵ͷǤǡ ͶͳǤͳǡ ͷͲǦͳͲͲ ͶǤ Ǧ ͵Ǥ ρ Ǧͳ et alǤ ͳͻͺǡ et al. ʹͲͲǡ ρǦͳȌǡParacentrotuslividusǡȋ͵ͳǤͺǡ ȋ͵ʹǤʹ ǤͷͲ ȋ͵ͶǤͳρǦͳȌ ǡ ȋ Ȍǡ ͶǦ Ǧ͵ǡ Ǧ ǡ ǡ Ǧ Ǥ ǡ Ǥ ǡ 96 ǡ Seaurchincryopreservedembryosapplication౹Chapter3౹ UniversidadedeVigoǦFacultadedeCienciasdoMar ʹͲͲͳǡ et al. ʹͲͲͺȌǤ ȋȌ ȋ± ȋͺǤͶΨȌǤ ǡ ȋͷǤΨȌ Ͳǡ Ǥ Ǥ Ǥ ǡ ǡǡ ȋȌǤ ȋ ͳͻͻͳȌǡ ǡ ʹ etal.ͳͻͻȌǤǡ P.lividusȋetalǤͳͻͺͳǡ ͷͲ Ǥ ǡ ǡ ǡ ǡ ǡ ǤͷͲ ȋʹͲͲ͵ǡʹͲͲʹȌǡ ǡ et al. ȋͳͻͻȌǡ ǡ Ǥ ǡǤǤ ǡ ͲǤͻǡ ǡ ȋͺ Ǥ Ȍ Ǥ ǡ ͳǡ ͷͲ CryopreservationofmarineinvertebrateearlyǦlifestages 97 Ȁ UniversidadedeVigoǦFacultadedeCienciasdoMar Ǧ ǡ ͶͺǤ ͻǤǡ ͷͲ Ǥ ͶͺǤ Ǥ Ǥ ȋͺǤǦȌ Ǧ ǡ Ǥ ͳͻͺͶǡ et al. ͳͻͻͻȌ ǡ ȋ ͳͻʹǡ ǤǦ ͳͻͻͷǡ et al. ͳͻͻͻǡ ʹͲͲͺȌǡ ǡ ȋ ȋSparusaurata)Ǥ ȋͳͻͻȌǡ ǡ et al. ȋʹͲͳʹȌǡ et alǤ Ǥ ǡ ȋǦ Ȍ Ǧ ʹͲ ǡ ȋͳͻͻʹȌ ȋͳͻͻͶȌ ǡǤ Ͳ ͳǡʹͶǡ δ ͲǤͷ ǡ ǡ Ǥ ǤͳǡͷͲΨ ͷͲ α ȏȐȀͷͲǡ Seaurchincryopreservedembryosapplication౹Chapter3౹ 98 UniversidadedeVigoǦFacultadedeCienciasdoMar Ǥ ǡ ǡ ǡǡ Ǥ ȋParacentrotuslividusȌ ȋʹͲͳͳȌǤ ͻǤ ʹͲ͑ǡ ͷͲΨ ͶǦ CryopreservationofmarineinvertebrateearlyǦlifestages 99 UniversidadedeVigoǦFacultadedeCienciasdoMar ȋ et al. ʹͲͲͷȌǤ ǡƤ ȋʹͲͲͳǡͳͻͻͲȌǤ ǡ Paracentrotuslividusȋ ͳͺͳȌ 1.Introduction ʹͷΨ Ǥ Ǥ Ψǡ ͺǦ ǡ ǡ ͶǦͺǡǦͳͲȌǤͳ͵ ͳͲȋ ͶǦ ͻ Ǥ ǡ ʹͲȌǡ ͳΨ ǡ Ǧ Ǥ ͷͲΨ͵ʹͻΨȋ ǡ Ǥ ǡ Ǥ Paracentrotus lividus Abstract CRYOPRESERVEDEMBRYOS. 3.2. SEA URCHIN (Paracentrotus lividus) LARVAL REARING – CULTURE FROM Seaurchincryopreservedembryosapplication౹Chapter3౹ 100 UniversidadedeVigoǦFacultadedeCienciasdoMar Paracentrotuslividusǡ ȋ ʹͲͳͳȌ ȋetal.ͳͻͻͳȌǤ ǡ etal.ȋͳͻͻǡͳͻͻȌǡorEvechinusChloroticusetal.ȋʹͲͲͶǡʹͲͲȌǡ ȋͳͻͺǡͳͻͻȌ ȋͳͻͻͳǡͳͻͻͺȌǡTetrapigusniger Strongylocentrotus nudus species Hemicentrotus pulcherrimus, ȋǡ ȌǤ al. ʹͲͳͳǡ ʹͲͳͳǡ Ǧ et al. ʹͲͳͳȌǡ ȋʹͲͳͳȌǡǡȋet ȋetal.ʹͲͳͳȌǡ ȋetal.ʹͲͳͳǡʹͲͲͶȌǤ ȋ et al. ʹͲͳͳȌ Ǧ ǡǤǤ ȋʹͲͲͶȌǡ ȋ Ǧetal.ʹͲͲͻȌǤ ʹͲͲͳǡ ʹͲͲͲǡ et al. ʹͲͲͷȌǡ ȋ ʹͲͲȌǡ ȋ et alǤ ȋ etal.ʹͲͲͳǡ etal.ʹͲͲͶȌǡ ȋͳͻͻȌǡ ǣ Ǧ ȋ ʹͲͲȌǡ ȋetal.ʹͲͲʹȌ Ǧ Ǥ ȋ et alǤ ʹͲͲʹȌǤ ǡ ǡ ǡ ȋetalǤʹͲͲʹǡʹͲͳͲǡetal.ʹͲͳͲȌǤ P. lividus ǡ ǡ ȋ ʹͲͲͳȌǤ CryopreservationofmarineinvertebrateearlyǦlifestages 101 UniversidadedeVigoǦFacultadedeCienciasdoMar ȋʹȌΪͲǤͲͶȋȌǤͳ ȋͺ ʹͲ ͑Ȍǡ ͳǤͷ ȋʹͲͳͳȌǤ Cryopreservationprotocol ȋȌǤ Ȍ Cylindrotheca closterium ȋ Ȍ Chaetoceros gracilis ȋ Ȍǡ Phaeodactilum tricornutum ȋ Tetraselmis suecica ȋͻͲͶȌǡ Isocrysis aff. galbana clon TǦISO ȋͳ͵ʹͶȌǡ Ǥ Ǥ ʹͲ͑ ͺ ȋǡetal.ʹͲͲʹȌǡǡ εͻͲΨǤ Ǥ Ǥ Ǧ ȋʹͲͲȌǤ Ulvalactuca. ȋͳͺάʹ ιǡ ͵ͷΩȌ À ȋ ǡ Ȍ ǡ ǡParacentrotuslividusǡ Biologicalmaterial 2.MaterialsandMethods Ǥ Ǥ Seaurchincryopreservedembryosapplication౹Chapter3౹ 102 UniversidadedeVigoǦFacultadedeCienciasdoMar ͳͷȋͲǤʹʹρΪȌȀʹͷ ǡ ͳͲ ͵ͷǡ ȌǤ ǣ ȋαͳͲͲȌǡȋ ǦetalǤȋʹͲͲ͵ȌǤ ͳͶͺͲʹͲȌ etalǤȋʹͲͳʹȌ Isochrysis galbanaǡ ͲǦ ͶͲ ǡ Ͳ iso, Chaetoceros gracilis Phaeodactilum tricornutumǡ ͶͲͺͲȋetraselmissuecica,Isocrysisaff.galvanaclonTǦ ͳͻάͳ͑ʹͶ ȋǤͳρǦʹǦͳȌǡ ͶͲ ρ ȌǤ ͳͲͷ ȋͲǤͷ ρ Ϊ Ȍǡ ȋ ͳͲͲ ȋȌ ʹͲǡ ǡ Ǥ Ǥ Ǥ ǡ ͺ ǡ ǡ ǡ ǣ Ǥ Larvaldevelopmentandsettlement ͳͻάͳ͑ Ǥ ͳʹ ͳ ά ͳ ͑ Ǥ ͺͲ ͑ Ǥ ǡ ͳ͑ǦͳȂͺͲ͑ǤƤʹ Ǧ ͳ͑ǦͳǦͳʹ͑Ǥʹ Ȍǡͳͻάͳ͑Ǥ Ͷ͑ʹǡ ǡͳͷȋͳǣͳ CryopreservationofmarineinvertebrateearlyǦlifestages 103 UniversidadedeVigoǦFacultadedeCienciasdoMar ȋα͵ͷǡδͲǤͲͷȌ ͳͷ ȋ͵ǤͷȌ Ǥ ͵ͺͺǤͷ ρǡ ͲΨ ͶǤͺ ρ ͵Ǧ ȋ ͵ǤͷȌ ȋ ͵ǤȌǤ Ͷ Ǥ ǡ ͶͲǤͷΨ ʹͻΨ ͳͲͲΨ ȋ ͵ǤͶȌǤ ͷͲΨ ͵ 3.Results ̺ͳͷǤͲ Ǥ Ǧ ȋȌ Statistics ͺǦͳͲǤ ǦͳȌͳͺ͑ǡͷͲ ǡ ȋͲǤͷρǦʹ Ǥ ʹͲ Ͳ Ǥ ͳͺ͑ͷͶǤʹρǦʹǦͳ Cylindrotheca closterium Seaurchincryopreservedembryosapplication౹Chapter3౹ ǡ ͳͷǡ 104 UniversidadedeVigoǦFacultadedeCienciasdoMar ͳʹͲǡ Ǥ ͳͶǡǡ ȋ͵ǤȌǡǦͳͳǡͺǦ ͶǦ Ͷͺ ǡ Ǧ ȋα͵ͷǡδͲǤͲͷȌǤ ͳ ͵ǤͷȌǤ Ǥ ȋ Ǥ Ǥ ͵ǤͶǤ ȋαͳͲͲȌǡ 105 ǡ CryopreservationofmarineinvertebrateearlyǦlifestages ͳͲ ǡ ͺǦ UniversidadedeVigoǦFacultadedeCienciasdoMar ȌǤ ʹͺǤʹͳΨ ǤͲͻΨ ȋʹͷǤͳͶΨ ȌǤ ͳͺ ʹǤΨȋͳͻǤΨ ͳͶǤͲͺΨǡ ȋα͵ͷȌǤ Ǥ ͵ǤͷǤǤ ǣ Ǣǣ ȋ͵ǤȌǤ Ȍ ȋ ͺǦ ͺǡǦͳͲȌǤͳ͵ ͺǦ ͳͲ ȋͶǦ ͶǦͻǡ 106 Seaurchincryopreservedembryosapplication౹Chapter3౹ 'D\ 'D\ 'D\ 'D\ 'D\ 'D\ 'D\ 'D\ 'D\ 'D\ 'D\ 'D\ 'D\ 'D\ ͵ǤǤǦʹͲǤ ǣ ǣ Ǥ UniversidadedeVigoǦFacultadedeCienciasdoMar 107 Ǥ CryopreservationofmarineinvertebrateearlyǦlifestages 4.Discussion UniversidadedeVigoǦFacultadedeCienciasdoMar ͻ ǦǤ ǦǤ ǡ ȌǤ ǡ ȋ Ǥ ȋParacentrotus lividusȌ ǡ δͲǤͲͷǤ ͳͲȋαͲȀȌǤǡ Ǥ ͵ǤǤǦ ǡ b Ǥ ͳͲ ͳͺǡ Seaurchincryopreservedembryosapplications౹Chapter3౹ 108 UniversidadedeVigoǦFacultadedeCienciasdoMar ʹǤΨͳͲǤͲͻΨͳͺǤ ȋͲΨȌP.lividusȋetalǤʹͲͳʹȌǤǡ ʹͺǤʹͳΨ ͳͺ Ǥ ͳͲ ͳͶǤͲͺΨ ǡ ǡ ǡ Ǥ ȋͺǦȌǤ ǡ ǡ ͳͲǡ ͳ ȌǤ ǡ ȋ ͲΨ ǡ Ǥ ͻ ǡ ǡ ʹͻΨǤ ȋʹͲͳʹȌ ȋͷ͵ǦͷΨȌǡ et al. ȋʹͲͲͻȌ ȋͶǦΨȌǡ P. lividus et alǤ ȋʹͲͲͻȌ ȋͷǦͺͷΨȌǡ et al. ͶͲǤͷΨǡ ȋetal.ʹͲͲȌǤʹͲ ȋ ʹͲͳͳȌǡ for Evechinus chloroticus ǡ P.lividus P. lividus et al. ȋʹͲͲͻȌ et al. ȋʹͲͲͻȌǤ ȋͶͶǦͺͷǤͷΨ Ȍ Ǥ Ǧ ǡ ǡ Ǥ ȋ Ȍǡ ǡ ͷͲΨ Ǥ ͶǦǡ 109 ȋStrongylocentrotus intermediusȌ CryopreservationofmarineinvertebrateearlyǦlifestages UniversidadedeVigoǦFacultadedeCienciasdoMar ǡǤ ǡ ǡ Ǥ Paracentrotus lividus Ǥ ǡ ǡ ǡ Ǥ ȋ Ȍǡ Ǥ ȌǡǤǡ ȋͳΨ ʹͷ Ψ Ȍǡ P.lividusȋ ǡǤ ͳ ͳǤͷ ǡ ǡ ǡ ǡ ǡʹ ͶǦEvechinuscholoticus.ǡ Ψ ͲǤʹΨ Ǥ ǡ et al. ȋʹͲͲȌ ȋ et al. ͳͻͻͳȌǡ ͲǤͳǦ 110 UniversidadedeVigoǦFacultadedeCienciasdoMar Ǧ ͳͲǤ 7. ͶǦͺǡ Ǥ Ǥ 6. ͻ ͲΨ ʹͻΨǤ 5. ʹͲ Ǥ ǡ 4. Ǥ Ǥ ǡ ͲǤͻǤ ͳǡ 3. ȋ Ȍ ͶǤ Ǧ͵ αα Ǥ δ α ǡ δ δ ǣ α δ 2. Ǥ ͷͲ ǡ 1. Seaurchincryopreservedembryosapplications౹Chapter3౹ 3.4CONCLUSIONS 111 UniversidadedeVigoǦFacultadedeCienciasdoMar ͳͺǤ 8. ʹǤΨ ͳͲ ǤͲͻΨ ȌǤ ȋ ǣͳ͵ ͺǦ CryopreservationofmarineinvertebrateearlyǦlifestages UniversidadedeVigoǦFacultadedeCienciasdoMar Seaurchincryopreservedembryosapplications౹Chapter3౹ 112 Ǥ ȋPernacanaliculusȌ UniversidadedeVigoǦFacultadedeCienciasdoMar ͶǤ͵ gigasȌȋMytilusgalloprovincialisȌǤ ǣ ȋCrassostrea ͶǤʹ CHAPTER4.DEVELOPMENTOFBIVALVE CRYOPRESERVATIONPROTOCOLS ANDAPPLICATIONS. ͶǤͳ CryopreservationofmarineinvertebrateearlyǦlifestages 113 UniversidadedeVigoǦFacultadedeCienciasdoMar Developmentofbivalvecryopreservationprotocols౹Chapter4౹ 114 CryopreservationofmarineinvertebrateearlyǦlifestages 115 ̻ ȋPerna canaliculusȌǡ UniversidadedeVigoǦFacultadedeCienciasdoMar ȋ ȌǤ Ǥ ͳȋǦͳȌǡ ǡ ǡ ȋǡȌCrassostreagigas Ǥ Mytilus galoprivinciallis Ǥ ǡ ȋ ȌǤ Ǧ ͲǤͷ ͳ ι ǦͳǤ Ȁ ǡ Ǥ P.canaliculus Ǥ ǡ Ǥ ǡ ǡ Ǥ Ǣ ǡ SUMMARY APPLICATIONS CHAPTER4:DEVELOPMENTOFBIVALVECRYOPRESERVATIONPROTOCOLSAND ǣ Developmentofbivalvecryopreservationprotocols౹Chapter4౹ 116 UniversidadedeVigoǦFacultadedeCienciasdoMar ȋMytilusgalloprovincialisȌǤȋʹͲͳ͵ȌǦʹͶǦʹͻǤ ǣ ȋCrassostrea gigas) Article 7ǤǦ Ǥ ǡ Ǥ ǡ ǤǤ ǡ ǤͷȋʹͲͳʹȌʹͷǦʹʹǤ ǡ Ǥ ǡ ȋPerna canaliculusȌ Article 6ǤǦ Ǥ ǡ ǤǤ ǡ ǤǤ ǡ ǤǤ ǡ ǤǤ ǡ ǤǤ ǡ ǤǤ CryopreservationofmarineinvertebrateearlyǦlifestages 117 ̻ȋPernacanaliculusȌ UniversidadedeVigoǦFacultadedeCienciasdoMar ȋǤǤ Ǥ ǡ Ǥ ǡ Ǧ ȋ ʹͲͲ͵ȌǤ Ǧ ǦǤǡ ȋʹͲͳͲǡȌǤ ȋȌ Ǥ͵͵ͲͲͲȋ̈́ͳͷʹȌ 1.Introduction Ǥ ʹǤͺΨ Ǥ Ǥ ͳͺ ͷǡ ͲǤͷ͑ǦͳǦ͵ͷ͑ͷ ǣͲ͑ͷǡ ͳ͑ǦͳǦͳͲ͑ǡ Ǧ Ǥ ǡ ͶͲȂͲΨ Ǥ ȌǤ ͳͲȂͳͷΨ ȋ Ȍ ȋͳȂʹͲ Ǥ ǡ Ǥ ̻ ȋPerna canaliculusȌ Abstract 4.1. CRYOPRESERVATION OF GREENSHELLTM MUSSEL (Perna canaliculus) TROCHOPHORELARVAE Developmentofbivalvecryopreservationprotocols౹Chapter4౹ 118 Ǥ UniversidadedeVigoǦFacultadedeCienciasdoMar Ǥ Ǧ ǡ ǡ Ǥ Ǥ galloprovincialis Ǧ ͳΨ Ǥ et al. ʹͲͳͳ ǡ Mytilus ͳͻͺͻ ǡ Mytilus edulisǡ ȋ ͳͻͻͷǡ et al. ʹͲͲͷȌǤ et al. ǡ ȋȌ Crassostrea rhizophoraeȌ Ǥ ȋCrassostrea gigas ǡ ̻ ʹͲͲǡ et al. ʹͲͳʹȌǤ Ǧȋetal.ʹͲͳͳǡetal. ʹΨ Ǥ ȋ͵ͲȂͶͺǦǤȌǤ Ǧǡ et al. ʹͲͲͻȌ Ƥ Ǥ ̻ ȋetal.ʹͲͲͻǡ ʹͲͲͳǡʹͲͲͶȌǤ ȋetal.ʹͲͳͳǡʹͲͳͳǡ Ƥ ȋetal.ʹͲͲͶǡͳͻͻͳȌ Ǥ ǡ ǡ ǡ ȌǤ ȋǤǤ ǡ ǡ Ȍ CryopreservationofmarineinvertebrateearlyǦlifestages 119 ǡ ȋ Ȍ UniversidadedeVigoǦFacultadedeCienciasdoMar Ͷ ̱ʹͶǤʹͲ͑Ǧ Ǥ ǡͶͲǦͳǡ ͳͷ Ƥ ǡ Ǥ ƤǤ ͳǣͳ Ȁǡ ƤȌ ȋͲǡ ͲǤʹ ͲǤͶ ǡ ƤȌǤ Ƥ ȋͲǡͳͲǡͳͷǡʹͲʹͷΨ ǡ ǡ ǡ ǡ ȌǤ Ǥ ȋȌ ȋ ȋȌ Ȃ Ǧ Ǥ Ǥ ͷͲ Ǥ ǡ ρ Ǥ ͳͷ ʹͲ ͑ ͳͷ έ ͳͲ ȀǤ ͳȂʹͲ Ǥǡ ρƤȋȌͳǦͳ ȋȌ ͳͷȂʹͲ ̱ͳͷͲͳ ǦͳǤ ͷͲͲ ͳͲͲͲ ʹͲ ρ Ǥ Ǥ ȋ et al. ʹͲͳͳǡ et alǤ ʹͲͲͻȌǤ Ǥ ǡǡ Gametecollectionandhandling ̻ ȋP. canaliculusȌ 2.Materialsandmethods Developmentofbivalvecryopreservationprotocols౹Chapter4౹ 120 UniversidadedeVigoǦFacultadedeCienciasdoMar Ǧ Ǥ Ǧǡ Ƥ ͳͲΨ Ψ Ǧ Ǥ Ͷͷ ρ ͳͷͳǦͳʹͶǤʹͲ͑ ͲǤͳΨȋȀȌǤ ͳͷ ͳ Ǥ ʹͺ ͑ Ǥ ǡ Ǥ ǦͳͲ͑Ǥ ǣȋͳȌͲǤͷ͑ǦͳǢȋʹȌͳ͑ǦͳǦ͵ͷ͑ǡͷ ͑ ͷ ͳ ͑ Ǧͳ ǦͳͲ ͑ ͷ ͲǤʹȋƤȌǤ ǣͲ ͳͲΨ Experiment1:effectofcoolingrate Ƥǡ ͳͷǤ Ƥ ͳ ͳ ǡ ȌǤ ǡ ȋ Ǥǡ ǡ Cryopreservationexperiments Ȍ Ǥ ǡ Ǧ ȋȌǯǯ Ǥ ȋ ǮǮ ȋȌǯǯ ǮǮ Ǧ Ǥ Ƥ ͶͲͲ ρ ͳͲΨ Ǥ Ͷ 121 UniversidadedeVigo ǦFacultadedeCienciasdoMar ͳͷͲ ͳ Ǧͳ ʹͲ ͑Ǥ Ǧ Ǧ Ǥ ͑ͷ ͲǤͷ͑ǦͳǦ͵ͷ͑ͷ Ͳ͑ͷ ͳ͑ǦͳǦͳͲ Ǥ Ǥ ͲǤʹͷ Ǥ ǡ ͳͲΨ Ϊ ͲǤͶ ȋƤȌ Ǥ Ƥ Ǧ Experiment4:effectofcryopreservationondevelopmentbeyondDǦlarvalstage Ǥ ͳǤ Ǥ ǣͲǤʹͷͲǤͷ ʹͶ ȋͳͲΨ ΪͲǤͶͳͷΨ ΪͲǤʹȌǤ Experiment3:effectoffreezingcontainertypeandvolume ǤǤ ͲǤͷ͑ǦͳǤ Ǧ Ǥ ͳͲȂͳͷΨ ͲǤʹȂͲǤͶ Ǧ ǡ Experiment2:effectofvaryingEGandTREconcentrations Ȍ Ǥ ȋ ͳͲͲΨ Ǧ Ǥ CryopreservationofmarineinvertebrateearlyǦlifestages 122 UniversidadedeVigo ǦFacultadedeCienciasdoMar ͳͷΨ Ǥ ʹͲΨ ǤǡͳͲΨ ͳǦȋδͲǤͲͷȌǤͳͷΨ ʹͲΨǤ ʹͲ ʹͷΨ ǡ ͷ Ǧ ǡͳǦȀƤͳǤƤ ͶǤͳ Toxicitytests 3. Results ȌǤ Ǧ ȋ Ǯǯ Ǥ ʹǤͳʹǤʹ ǯ Ǥ Ǧ ȋȌ Ǥ ͷǤͲ Statisticalanalysis Ǥ Ǧ ͳͲͲͲ ʹȂ͵Ǥ ǡ etal.ʹͲͳͲǤ ȋȌǦȋͶͶͲͲͲȂͳͷͳͲͲͲȌ Ǧ ǡ Ǧ ǦǤ ǡͶͷρ Developmentofbivalvecryopreservationprotocols౹Chapter4౹ VWHSDGGLWLRQ (*75(0&RQFHQWUDWLRQV 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 VWHSDGGLWLRQ CryopreservationofmarineinvertebrateearlyǦlifestages 123 UniversidadedeVigo ǦFacultadedeCienciasdoMar Ǥ Ǥ Ȍ ȋ ȋ ͶǤʹȌǤ ǡ Ƥ ȋδͲǤͲͷȌǦ ͲǤͷ͑Ǧͳ Ǥǡ ͳͲΨ ΪͲǤʹǡ ȋͲǤͷͳ͑ǦͳȌ Experiment1:effectofcoolingrate Cryopreservationexperiments ǤάǤ ͳ ȌǤ ͲȀͲ Ǥ ȋͳͷ Ǥ ͶǤͳǤ Ǧ ̻ 1RUPDO'ODUYDH &PLQ &PLQ 1R%VDWKDZLQJ %VDWKDZLQJ Developmentofbivalvecryopreservationprotocols౹Chapter4౹ 124 Ǥ UniversidadedeVigo ǦFacultadedeCienciasdoMar δ ͲǤͲͷ ȋ ͶǤͳȌǤ Ǧ ͳǢ̱ʹͲΨ ͵Ǥ ͳͷΨ Ϊ ͲǤʹ Ǥ ǡ Ǧ ȋ δ ͲǤͲͷǢ ͶǤ͵ȌǤ ͳͲΨ Ϊ ͲǤͶ Ƥ Ǧ Ǧ ͲǤͷ ͑ Ǧͳ Ǥ Ȁ ǡ Experiment2:effectofvaryingEGandTREconcentrations Ǥ Ǥ ͲǤͷ ͑ Ǧͳ ǡ δ ͲǤͲͷȌ ȋ ͶǤͳȌǤ ȋȌ ǡ ȋʹͲΨ Ƥ Ǧ Ǥ δͲǤͲͷǤάǤ ǦǤ ͳͲΨΪͲǤʹǤ ͶǤʹǤ Ȁ 1RUPDO'ODUYDH 125 ȗ ͳ͑Ǧͳ ȗ ȗ ȗ ȗ ȗ ͳͲΨ ΪͲǤʹ ͳͲΨ ΪͲǤͶ ͳͷΨ ͳͷΨ ΪͲǤʹ ͳͷΨ ΪͲǤͶ ͺǤͲ ȗ ȗ ȗ ȗ ȗ ȗ ȗ ȗ ͳͲΨ ΪͲǤͶ ͳͷΨ ΪͲǤʹ ͳͲΨ ΪͲǤͶ ͳͷΨ ΪͲǤʹ ͳͲΨ ΪͲǤͶ ͳͷΨ ΪͲǤʹ ͳͲΨ ΪͲǤͶ ͳͷΨ ΪͲǤʹ ȗ Ǧ Ǧ ͶǤ͵ ͳǤͶ ʹǤͻ ͲǤͻ ʹǤͶ ͶǤͳ ͲǤͺ ͷǤͲ ͷǤͶ ʹǤͻ ͳͲǤʹ ͺǤʹ Ǥʹ ͳǤʹ ʹǤ ͳǤͻ ͳǤͲ ͷǤͺ ʹǤʹ ͲǤͶ UniversidadedeVigo ǦFacultadedeCienciasdoMar Ͷ Ǧ Ǧ Ǧ ͻǤͺ ͺǤ͵ ͷǤͶ ͺ͵Ǥͺ ͻͳǤ͵ ͳͲͷǤͻ ͺͳǤͶ ͷǤ ͺʹǤ Ǥ ͺ͵Ǥ ͺǤ͵ ȗ ͳͲΨ ͻǤͺ ͳͲǤ ȗ ͳ͑Ǧͳ ͺ͵Ǥ ͳͲʹǤ͵ ȗ ͲǤͷ͑Ǧͳ ͺǤͷ ȗ ͲǤͷ͑Ǧͳ ͺʹǤ͵ ͳͲ͵ǤͲ ȌǡάǤ Ͷ ʹ ͲǤͷ ͲǤʹͷ ͵ ʹ ͳ ȋρȌ Ǧ Ǧ ͶǤ ͺͲǤ ͲǤ͵ ͺǤͶ ͺͷǤͶ ͺͳǤͶ ǤͲ ͷ͵Ǥ ͺǤ͵ ͵Ǥ ͻǤ͵ ͺͳǤ ͺǤͳ ͺͳǤ ͻǤͻ ͺͶǤͲ ȋΨȌ ͶǤͳǤ Ͷͺ ȋ ǡ ͵ͷ CryopreservationofmarineinvertebrateearlyǦlifestages Generaldiscussionandconclussions౹Chapter5౹ 126 Ǥ 0 D 0 D E 0 0 EF (*75(0&RQFHQWUDWLRQV 0 0 D F UniversidadedeVigo ǦFacultadedeCienciasdoMar ȋͶǤͳȌǤ Ǥ ȋ δ ͲǤͲͷȌǡ ǡ ǡ ̱ʹͲΨ ά Ǥ Ǥ δͲǤͲͷǤ ͶǤ͵Ǥ Ǧ ͲǤͷ͑Ǧͳ ͳͲͳͷΨ ͵ ȋͶȌǤ ȋδͲǤͲͷȌ Ǧ ͲǤͷ ʹ Ͷ Ǥ Ƥ ǣͲǤʹͷ Experiment3:effectoffreezingcontainertypeandvolume 1RUPDO'ODUYDH 127 UniversidadedeVigo ǦFacultadedeCienciasdoMar Ǥ δͲǤͲͷǤά ǦǦǤ ͶǤͶǤ ȋͲǤʹͷͲǤͷǢʹͶȌ ʹǤΨά͵ǤάȌǤ ǡ ʹǤͺάͳǤͲΨͳͺȋ Ͷ ͺǤ ǦȋǤǤǦȌǤƤ Ͳ Ƥ Ǥ ͶǤͷ ǤǦȋͳͺȌ ǡ ͳͲΨ Ϊ ͲǤͶ ǡ Ƥ ǡ ͲǤʹͷ Experiment4:effectofcryopreservationondevelopmentbeyondDǦlarvalstage CryopreservationofmarineinvertebrateearlyǦlifestages 'D\V &RQWURO Generaldiscussionandconclussions౹Chapter5౹ &U\RSUHVHUYHG 128 UniversidadedeVigo ǦFacultadedeCienciasdoMar Ǥ ǡ Ǧ Ͷ ǡ Ǥ ǡ Ƥ ͳͺ Ǧ Ǥ ʹͲΨ ȋ ͶǤͳȌǤ ͶǤ ǡ Ͷͺ Ǥ Ǧ ȋǤǤͲα ǦȌǤά ǤǦ ͶǤͷǤ ͳͻ 6XUYLYDO &RQWUROV )UR]HQWURFKV CryopreservationofmarineinvertebrateearlyǦlifestages 129 UniversidadedeVigo ǦFacultadedeCienciasdoMar ǡ ʹȋetal.ͳͻͻǡ ʹͲͲͻȌǤ ǡ Ǥ Ǧȋetal. ʹͲͲͻǡ ̻ ǡ Ǥ ͳͷΨ ȋȌ ȋ ʹͲͲͻȌǤ ǡ ǡ ȋʹͲͳͲȌǤ ȋ et al. ʹͲͳͳȌǤ et al. ͳͻͺͻȌǡ ȋǡʹͲͳͲǡ Ȁ ̻ Ǥ ǡ Ǧ ǡ 4. Discussion Ǥά ͶǤǤͳͻ '/DUYDHVL]HPLFURPV 130 UniversidadedeVigo ǦFacultadedeCienciasdoMar ȋ et alǤ ʹͲͲͺǡ et al. ʹͲͲȌǡ Ǥ ǡ ȋ et al. ʹͲͲ͵ȌǤ ȋȌǤ Ǥ ͲǤͷ͑Ǧͳ Ǧ ͲǤͳΨ ǡ ǡ ͲǤͷ ͳ͑ Ǧͳ Ȍ Ǥ ȋ ȋͷȌ ǡ ͳͲ ͳͷΨǤ ǡ ʹͲΨ ǡ ͷ ȋǦetal.ʹͲͲͺȌǤ Ȍ Ȍǡ ȋ ȋ ͳͷȋʹͲͲͻȌǤ ȋ et al. ʹͲͲȌ ǡ ǡ ȋetal.ʹͲͲͻǡetal.ͳͻͻͶȌͳͲ Ǥǡ ȋǦ ʹͲͲͳȌǤ ̻ ǡǦ Ǥ et alǤ ͳͻͻͶǡ et al. ʹͲͲͳǡ et al. ʹͲͲͷȌ ̻ ǡ Generaldiscussionandconclussions౹Chapter5౹ CryopreservationofmarineinvertebrateearlyǦlifestages 131 UniversidadedeVigoǦFacultadedeCienciasdoMar Ǥǡ ǡ ǡ Ǥǣ ǡ ʹͲΨ ȋͶǤͳȌǤ ǡ Ǧ Ǥ Ǥ ǡ ȋ ȌǤ Ǥǡǡ Ǥ ̻ Ǧ ȋetal.ʹͲͳͳȌǤǡ ǡ Ǥ ΪͲǤʹ Ǥ Ǥ ͳͲΨ ΪͲǤͶͳͷΨ Ǧ ͲǤͷ ͑ Ǧͳ ǡ Ǥ Ǧͳ Ǥǡ ͲǤͷ͑ ǡ Ǥǡ Generaldiscussionandconclussions౹Chapter5౹ 132 Ǥ UniversidadedeVigoǦFacultadedeCienciasdoMar Ǧ Ǧ Ǥ ǤǦ ǦȌǤ ̻ ȋ ȌǤ ǡ ǡ ȋ Ǧ ȌǤ ȋ ǡǡ ǡ Ǧ ȋ Ͷ Ȍ Ǥ ǣ Ǧ Ȍǡ ǡ ȋ ͻΨ ǤǡͳΨǦ ͷΨʹ ǦǡMytilusgalloprovincialis Ǥetal.ȋʹͲͳͳȌ Ǥ Ǥ ǡ ͻ ǡ ͵ΨǤ Ͷ ͲΨ ͺǤ ȋȌǤ ǡ ͳͺ Ǥ Ǥ CryopreservationofmarineinvertebrateearlyǦlifestages 133 UniversidadedeVigoǦFacultadedeCienciasdoMar ʹͲͳͲȌ ǡ ʹͲͳͲǤ ȋͶͷͷǡ ʹͲͲͷ ͵͵ͲͲ ȋ̈́ʹͲ Ȍ ǡ ʹ͵Ͳ ʹͲͳͲ ȋ ʹͲͳͲȌǤ ǡ Crassostrea gigas, ʹͶǤͷ 1.Introduction ǦȋͶͺǤͻάǤΨǦȌǤ ǡ Ǧ ͑ Ǧͳ ͳͲΨ ͲǤͷ ͑ ǦͳǤ ǡ ͲǤͷ͑Ǧͳͳ ȋȌǡ ͳͲΨ Ǥ ǦͳǦͳͲ͑ͷ ͲǤͷ͑Ǧͷ͑ǡͷ ǤΨǦȌͲ͑ͷ ͳ͑ ȋͲǤͲά Ǧ Ǥ ȋͳͶǤʹͲǤȌ Ǥ Abstract MUSSEL(Mytilusgalloprovincialis) SPECIES OF BIVALVES: PACIFIC OYSTER (Crassostreagigas) AND MEDITERRANEAN 4.2.COMPARATIVECRYOPRESERVATIONSTUDYOFTROCHOPHORELARVAEFROMTWO 134 UniversidadedeVigoǦFacultadedeCienciasdoMar ȋ et al. ͳͻͺͻȌǤ ǡ Ǧ edulis ǡ ǡ C.gigasC.rhizophoraeǡM. ǡ Ǥ etal.ʹͲͲͶǡetal.ʹͲͲͻȌǤ ǡ Ǧ ȋǤǤ ȋetal.ͳͻͺͻǡetal.ʹͲͲͻǡetal.ʹͲͳͳȌǤ ʹͲͲͷǡetalǤʹͲͲͷǡetalǤʹͲͲͺȌǡ ȋetal.ͳͻͻǡetal.ͳͻͻͻǡetal. Ǥ Ǥ ǡ Ǥ ǡ Ǥ ǡ ǡ alǤͳͻͻͻǡetalǤʹͲͲͶȌǤ Ǧ ȋ et ǡ Ǧ ǡ Ǥ ǡ ǡ ǡ Ǧ ͳȌǡ Ǧ Ǧ ǤͳȋǦ ǡ ǡ Ǥǡ C.gigas ʹͲͳͲȋǡʹͲͳͲȌǤ Mytilus galloprovincialisǡ ͺʹΨ ǯ Generaldiscussionandconclussions౹Chapter5౹ 135 UniversidadedeVigo ǦFacultadedeCienciasdoMar ʹͲ͑ͳέͳͲ ȀǤ ρȋȌͳǦͳ ȋȌǡ ͳͷʹͲ ͵Ͳͳ Ǥ ǡ et al. ȋʹͲͲͷȌǤ × À Ó ȋȌǤ ȋ ǡȌ ȋMytilusgalloprovincialisȌ ͳͷέͳͲ ȀǤ ͳǦͳ ȋȌǡʹʹι ͳͷʹͲǡ ̱ͳͷͲ ȋͷͲͲ Ȁ Ȍ ǡͶιǤ Ǥ ͳɊȋȌǤ Ͷι ͳ Ǥ dzǤ Ǥ Dz ǡ ǡ ȋCrassostrea gigasȌ Gametecollectionandhandling 2. MaterialsandMethods ǦǤ Ǥ ȋ et al. ʹͲͳʹȌ CryopreservationofmarineinvertebrateearlyǦlifestages 136 ͳͶǤʹͲǤǡ Generaldiscussionandconclussions౹Chapter5౹ UniversidadedeVigoǦFacultadedeCienciasdoMar Ǥ Ǥ ȋȌǤ Ǧ ȋȌ Ǧ ȌǤ Ǧ ǤȋͳΨȀ ͶͲǦͳǡʹͶǤʹͲǦʹʹ͑ Ǥ ͳͷ ͳǣͳ ȋȌǤ Ǥ ǡ Toxicitytests Ǧ ȌǤ Ǥ ȋȌ ȋ ȌǤǡȋȌǦ Ǧ ȋǡǡȌǡȋǦ ȋ ȌȋȌǡ Experimentalreagents ƤǦǦǤ et alǤ ȋͳͻͻȌǤ ǡ ͶͺǦǦ Ǧ Ǥ Ǧ Ǥ Ǥ ǡ Ǥ ǡ ͷͲ Ǥ ͳͷρ Ǧ CryopreservationofmarineinvertebrateearlyǦlifestages 137 ʹͺ ͑ Ǥ UniversidadedeVigoǦFacultadedeCienciasdoMar Ǥ Ǧ Ͷͺǡʹͻ ΪͲǤʹ Ǥ ͳͲΨ Effectsofcryopreservationonlarvaldevelopment Ǥ ȋͳΨȀ ȌΨǦ ǤǦ ͶͷɊ ʹͶ Ǥ ȋ ̱͵ͲͲǡͲͲͲȀ Ȍ ʹͲǦʹʹ ͑ǡ Ǧ ͳͷʹͲ ͳǣͳ ͲǤͳΨȋȀȌ Ǥ ǦͳͲ͑ ͳǦ͵ͷ͑ǡͷȋȌǤ ͳ͑ǦͳǦͳͲ͑ͷ ͲǤͷ͑Ǧͳͳ͑Ǧ ͲǤͷ͑Ǧͳͳ͑Ǧͳ ǣͲ͑ͷ ȋ ǡ Ȍ Ǥ Ǥ ͲǤʹͷ ͳ ǡͳͷ ȋ Ǧ Ȍ ͳ ȋ ȌǤ ǡ Cryopreservationexperiments ǡ Ǥ ȋͳͻͶͺȌǣαȋǦ ȀͳͲͲǦ ȌέͳͲͲǡ ǯ Generaldiscussionandconclussions౹Chapter5౹ 138 ͳͷΨ ʹͲΨ Ǥ UniversidadedeVigoǦFacultadedeCienciasdoMar ͻ͵ǤͳͲάʹǤͶ͵ȋȌǤ ȋ ͶǤͺȌǤ ͳͷΨ ͲǤʹͲǤͶ. ǡͳͲΨ ͳǤͶȋǡȌǤ Ǥ ͻͷǤʹͷ ά ǡ ͳͲΨ ͳͷΨ ȋ ͶǤȌǤ Ǧ ǡ ʹͲΨ ͳͷΨ ʹͲΨ Ǥ Toxicitytests 3.Results ǡǦͷΨ Ǥ ȋ ȌǤ Ǧ ȋȌǤ ʹǤͳʹǤʹ ǯ ȋȌ ȋȌǡ ǯ Ǧ ͷǤͲ Ǥ Statisticalanalysis 1RUPDO'ODUYDH 0 75( 0 75( 393 0 75( 0 393 75( 393 (* (* (* (* (* (* 0 0 0 393 0 75( 75( 75( 75( 393 393 (* (* (* (* (* (* 0 0 0 0 393 75( 75( 75( 75( 393 393 (* (* (* (* (* (* 0 0 0 393 0 75( 75( 75( 75( 393 393 (* (* (* (* (* (* 0 0 0 0 393 75( 75( 75( 75( 393 393 )6: 139 UniversidadedeVigo ǦFacultadedeCienciasdoMar ǤάȋȌǤ ȋȌȀͳΨǤ Ǥ ͶǤǤ Ǧ 1RUPDO'ODUYDH CryopreservationofmarineinvertebrateearlyǦlifestages 1RUPDO'ODUYDH 1RUPDO'ODUYDH 1RUPDO'ODUYDH (* (* )6: (* 75( (* 75( (* 75( (* 75( (* 75( 75( (* (* (* (* 75( (* 75( (* 75( Generaldiscussionandconclussions౹Chapter5౹ (* 75( (* 75( (* 75( 140 ǡ Ǧ UniversidadedeVigoǦFacultadedeCienciasdoMar ͲǤͷ͑ǦͳͳͷΨ ΪͲǤͶȋͶǤͳͲȌǤ ͲǤͷ͑Ǧͳͳ͑Ǧͳ ͳͲΨ Ǥ Ǧ Ǥ ͲǤͷ͑ǦͳȋͶǤͻȌǤ ȋͲǤͲάǤΨǢ ά Ȍ ͳͲΨ Ǧ Cryopreservationexperiments ȋȌǤ ȋȌǤ Ǥ ά ͶǤͺǤ Ǧ 1RUPDO'ODUYDH 1RUPDO'ODUYDH 1RUPDO'ODUYDH 1RUPDO'ODUYDH 1RUPDO'ODUYDH 1RUPDO'ODUYDH (* (* 75( 75( 393 (* &PLQ (* 75( 75( 393 &PLQ CryopreservationofmarineinvertebrateearlyǦlifestages 141 (* (* 75( (* 75( (* &PLQ (* 75( (* 75( &PLQ Ǧͳ ȋȌǤ UniversidadedeVigoǦFacultadedeCienciasdoMar ͳͷΨ ȋȌǤ Ǥά Ǧͳ ͶǤͳͲǤ Ǧ ͲǤͷ͑ ͳ͑ ͳͲ ȋȌǤ ȋȌͳΨȋȌǤ Ǥά ͲǤͷ͑Ǧͳͳ͑ǦͳͳͲͳͷΨ ȋ Ȍ ͲǤʹ ͶǤͻǤ Ǧ 1RUPDO'ODUYDH 1RUPDO'ODUYDH 142 Ǧ ȋͳǤͻΨ Generaldiscussionandconclussions౹Chapter5౹ 5B 5A &RQWURO (* (*75( 75( 393 (* (*75( 75( 393 $YHUDJH UHGXFWLRQ 6( /HQJKW UHGXFWLRQ FRPSDUHGWR FRQWURO 'ODUYDHIURPWKDZHGWURFKRSKRUHV $YHUDJHODUYDH VL]H ,QFXEDWLRQWLPHKRXUV )UHVKFRQWUROV UniversidadedeVigoǦFacultadedeCienciasdoMar Ȁ Ͷͺ Ǥ ǤάȋȌǤǣǦ ͶǤͳͳǤǣǦ Ȁ Ͷͺǡʹͻ ȋͳͲǤ͵ΨǢͶǤͳͳȌǤ ȋ ͶǤͳͳȌǤ Ǧ ǡ Ȍ Ǥ 'ODUYDHOHQJWKPLFURPV 143 ǦͳǤ UniversidadedeVigo ǦFacultadedeCienciasdoMar ͲάΨǦ ͲǤͷ͑ ͳͲΨ ʹͲΨ Ǥ ǡ Ȁ ȋetal.ʹͲͳʹȌ ǡ ǡ Ǥ ʹͲͲͳǡetalǤʹͲͲͷȌǤǡ ȋ etalǤʹͲͲͷǡetal.ͳͻͻǡ ͳͻͻͷǡ Ȁ ǡ al.ʹͲͳʹȌǤǡ ȋet ȀǤ Ǥ ǡ ͳͷΨ ʹͲΨ Ǥ ǡ ̻ȋPernacanaliculusǢetalǤʹͲͳʹȌǤ ͳͻͻͷǡ ʹͲͲͷȌǤ ȋ et al. ͳͻͻǡ 4. Discussion CryopreservationofmarineinvertebrateearlyǦlifestages 144 ǦȋͺͲΨȌ Generaldiscussionandconclussions౹Chapter5౹ Ǧ UniversidadedeVigoǦFacultadedeCienciasdoMar ͲǤͲάǤΨͶͺǤͻάǤΨǤ ͲǤͷ͑ǦͳǤ ǤǡͳͷΨ ǡ ͳ ͑ Ǧͳ ͳͲΨ ǡ ǡͲǤͷ͑ Ǧͳ Ǥ ǡ Ȁ Ȍ ǡ ǡ ȋ Ǥ ȋ ͶȀ Ȍǡ ǡǡ Ǥ Ǧ et alǤ ȋʹͲͳͳȌ Mytilus galloprovincialis Ǧ Ǥ etal.ȋʹͲͳʹȌǡ Ͷ ǡ ȋͳǤͻΨ Ȍ ǡ ͷͲΨǤ ǡ ͶͺǤͺάͳǤͷΨǡ ǡ Ǥ ȋͲǤͷ͑ǦͳȌǡ ǦͶͺǤͻάǤΨǡͶǤͷάͳͳǤʹΨ ͳͲΨ ΪͲǤʹͳͲΨ ΪͲǤͶȋͳ͑ǦͳȌǡͳͷΨ ΪͲǤͶ ǡ Ǥ ǡͳͲΨ ΪͲǤʹǡ ͲǤͷ͑ǦͳǤ 145 ǡ CryopreservationofmarineinvertebrateearlyǦlifestages UniversidadedeVigoǦFacultadedeCienciasdoMar Ǥ ͲǤʹͷ ǡ ͳͲͲͲͲͲǦ ǡ Ǧ ǡ Ǧ ǡ Ǥ ȋM.galloprovincialisC.gigasȌ Generaldiscussionandconclussions౹Chapter5౹ 146 UniversidadedeVigoǦFacultadedeCienciasdoMar ȌǤ ȋͳͲǤ͵Ψ ͳǤͻΨ 9ǤǦ Ǧ ͲǤͷ͑ǦͳͳͷΨ ΪͲǤͶ̱ͷͲΨǦǤ ͲǤͷ͑Ǧͳͳ͑Ǧͳ ͳͲΨ Ǥ 8ǤǦ (Mytilus galloprovinciallisȌǡ Ǧ ͳͲΨ ͲǤͷ͑ǦͳǤ 7ǤǦ Ǧ ͲΨ ͳͷΨ ʹͲΨ Ǥ 6ǤǦ ʹǤͺΨǤ 5ǤǦ ͳͻ Ǥ 4ǤǦ ̻ Ǧ PernacanaliculusȌǤ ȋȌ ǡ ȋʹͲΨ 3ǤǦ Ƥ Ǧ Ƥ ȋδͲǤͲͷȌǦǤ 2ǤǦ ͲǤͷ͑Ǧͳ ʹͲΨ Ǥ ʹͲΨPernacanaliculusǤͳͷΨ 1ǤǦ Ƥ 4.3.CONCLUSIONS UniversidadedeVigo ǦFacultadedeCienciasdoMar CHAPTER5.GENERALDISCUSSIONAND CONCLUSIONS CryopreservationofmarineinvertebrateearlyǦlifestages 147 GENERALDISCUSSION 5.1 148 UniversidadedeVigo ǦFacultadedeCienciasdoMar ǡͲǤͳǦͲǤʹΨǤ Ǥ ȋ et alǤ ͳͻͺͺǡ ͳͻͻͳǡ ͳͻͻͺȌ ͳͻǡͳͻͺȌǤ ͵ ȋ ͳͻͳǡ nudus Ǧ ͻͲΨ H. pulchehimus, S. intermcdius S. ǡ ǡ Ǥ ǡ Ǥ ǡ ǡʹ Ǥ ǡ ǡ ǣ ǡ Ǥ ǡ ǡ ǡ Ǥ ǡ ǡǡ ǡ GENERALDISCUSSIONANDCONCLUSIONS 5. Generaldiscussionandconclussions౹Chapter5౹ 149 UniversidadedeVigo ǦFacultadedeCienciasdoMar ȋ ǤǤͳͲǦʹͲΨȌǤ ǡ ͶͲǦͲΨ Ǧ Ǥ ǡ ǦȋͶͺȌǡ MytilusgalloprovincialisCrassostreagigas Ǥ Perna Canaliculus, Ǧ Ǥ ǡ ʹͲͳʹǡetal.ʹͲͳ͵ǡʹͲͳʹǡetalǤʹͲͳͶǡetalǤʹͲͳͳȌǤ ȋetal. ʹͲͲǡ etalǤ ʹͲͳͳǡ et alǤ ȋetalǤʹͲͲͻǡetal.ʹͲͲͲȌǤǡͷ Ǧǡ Ǧǡ Survival,Growthanddevelopment Ǥ canaliculus and Mytilus galloprovincialis Crassostrea gigas ǣ Perna Mytilus galloprovincialis Ǥ ǡ ǡ et al. ȋͳͻͺͻȌ ǡ ǦǤ et alǤ ʹͲͳͳȌǡ ȋetalǤʹͲͳͳǡetal.ʹͲͲͻǡ ǡ Ǥ ǡ Ǧ ǡ Paracentrotus lividus CryopreservationofmarineinvertebrateearlyǦlifestages 150 Mytilusgalloprovincialis Generaldiscussionandconclussions౹Chapter5౹ UniversidadedeVigoǦFacultadedeCienciasdoMar ȋǤͳͻͻǡ ͳͻͻͷǡʹͲͲͳǡetal. ȋ et alǤ ʹͲͲͷȌǤ Ǥ ʹͲͳͲǡ et al. ͳͻͺͻȌ ǡ ȋ et alǤ ʹͲͲͻǡ ǣ Cryopreservationprotocols Ǥ ǡ ʹͻΨ ʹͲ Ψ Ǥ ǡ Ǥ Ǧ ǡ ʹͲΨ ǩ Ͷͺ ǡ Ǥ ͷͲΨ ǡ ͻǡ ǡ ǡ Ǧǡ Ǧ Ǥ Ǧ ʹǤͺΨͳͺǤ ͺ Ǥǡ Ȍǡ ͲΨͶȋ ȋͳͺ Ȍ ǦͻǤPernacanaliculus CryopreservationofmarineinvertebrateearlyǦlifestages 151 UniversidadedeVigoǦFacultadedeCienciasdoMar ǡ ʹǤͺΨǡ Ǥ Applicationsofcryopreservationprotocols Ǥ ȋ ʹͲͲͷȌǡ ȋ et al. ʹͲͲ͵Ȍ ȀȀǤ ǡ ǡ ǡ Ǥ Ǥ ȋǦͳͻ͑Ȍ ǡ Ǥ ǡ͵ǦͶ Ǥ Ǥ ǡ ǡ Ǥ ǡ ǡ Ǥ ǡ ǡ ʹͲͲͷȌǡ ǡ ǡ Generaldiscussionandconclussions౹Chapter5౹ 152 UniversidadedeVigoǦFacultadedeCienciasdoMar Ǥ ǡ ǡ Ǥ ǡ ǡ Ǥ ǡ ǡ Ǥ ǡ Ǥ ǡ Ǥ Ǥ ǡ Futureviews ǦǤ Ǥ ͷͲ Ǥ ǡǤ ǡʹΨΨ ǡ ǡ Ǥ CryopreservationofmarineinvertebrateearlyǦlifestages 153 CONCLUSSIONS UniversidadedeVigoǦFacultadedeCienciasdoMar ͻȋʹͲ͑ȌǤ ͶǦ ͷͲΨ ͳʹǤ Ȁ Ǥ ͳ ͑ ͳ͑ǦͳǦͺͲ͑ǤƤʹǦͺͲ͑ ʹǡ Ͷ͑ʹǡ ͳ͑ǦͳǦͳʹ͑Ǥ ͳͷͳǤ ȋͷǤͳȌ ȋͳǤͷʹΪͲǤͲͶ Ȍ ǣ ȋͺǡʹͲ͑Ȍ Ǥ Objective1 Ǧ 5.2 Ǥ ǡǡ ǡ Ǥ Ǥ Ǥ Generaldiscussionandconclussions౹Chapter5౹ 154 UniversidadedeVigoǦFacultadedeCienciasdoMar ͷͲǡǦǤ Ǥ ǡ ȋͷǤʹȌ Ǥ Objective2 Ǧ ǡ Ǥ ͷǤͳ (P. lividus) Ǥ ͻͲ CryopreservationofmarineinvertebrateearlyǦlifestages 155 UniversidadedeVigoǦFacultadedeCienciasdoMar Ǥ ͳͺ Ǧ ȋ ͷǤ͵ȌǤ ͷͲΨͻǡʹΨʹͲΨ Ǥ Objective3ǤǦ Ǥ ͷǤʹǤ Generaldiscussionandconclussions౹Chapter5౹ 156 UniversidadedeVigoǦFacultadedeCienciasdoMar ͲΨǦͶͺȋͷǤͶȌǤ ʹͺ͑ Ǥ ͑ǡ ͷ Ǥ ͳ͑ǦͳǦͳͲ͑ͷǡ ͲǤͷ͑Ǧ͵ͷ ǡ Ͳ͑ͷ ͳͲΨ ȋƤȌǤȋͲǤʹͷȌ ȋͳͶǤʹʹ͑Ȍ Crassostrea gigas ǣ ǡͲΨͶʹǤͺΨͳͺǤ ͷͲΨ Ǧ Ͷͺ Ǥ Ǥʹͺ͑ Ǥ ͲǤͷ͑ǦͳǦ͵ͷ͑ǡͷ ǡ ͳ͑ǦͳǦͳͲ͑ͷǡ Ȍ ǡ Ͳ͑ͷ ͳͲΨ Ϊ ͲǤͶ ȋƤȌǤ ȋͲǤʹͷ ȋͳǦʹͲǤʹͲ͑Ȍ Perna canaliculus ǣ Ǥ Objective4 Ǥ ͷǤ͵Ǥ Ǥ 157 Mytilusgalloprovincialis ǣ CryopreservationofmarineinvertebrateearlyǦlifestages UniversidadedeVigoǦFacultadedeCienciasdoMar Ǥ ͷǤͶǤCrassostreagigasǦ ǦǡͶͺǤͻΨͶͺ Ǥ galloprovincialis Ǧ ͲΨ Ǧ Ͷͺ ȋ ͷǤͶȌǤ Mytilus ʹǤͺΨ ͳͺǤ Crassostrea gigasǡ ǡǦͲΨͶ Perna canaliculus ͷͲΨ Ǧ Ͷͺ Ǥ Ǥ Objective5 Ͷͺ Ǥ ǤͷͲΨ ǦǦ ʹͺ͑ Ǥ ͳͲΨ ͲǤͷ ͑ Ǧͳ ͳͷΨ Ǥ ǣ ͲǤͷ͑Ǧͳͳ͑Ǧͳ Ǥ ȋʹͲ Ǥ ʹͲ ͑Ȍ UniversidadedeVigoǦFacultadedeCienciasdoMar ౹Bibliography౹ 158 UniversidadedeVigoǦFacultadedeCienciasdoMar BIBLIOGRAPHY CryopreservationofmarineinvertebrateearlyǦlifestages 159 UniversidadedeVigoǦFacultadedeCienciasdoMar ౹Bibliography౹ 160 CryopreservationofmarineinvertebrateearlyǦlifestages 161 UniversidadedeVigoǦFacultadedeCienciasdoMar ǤǡǤǡ Ǥͳͷ ȋͳͻͺȌǡǣͳʹʹǦͳʹǤ ǤǤǡǤǤǡǤǦǡǤǤǡǤǤ ǡǤǤǡǤǡǤǡǤǤǡ ǤǤ Ǥ Ǥ ǣ ǡ ʹ EditionǤǤǤ ǤǤ ǡȋǤȌ ȋʹͲͳͳȌǡǡǣ ͳǦʹ͵Ǥ ǤǤ ǡ ǤǤ ǡ ǤǤ ǡ Ǥ Ǥ ǡ ǤǤ ǡ ǤǤ ǡ Ǥ Ǥ ǡ ǤǤ ǡ ǤǤ Ǥ ǣȋCrassostreagigasǡȌǡȋPernacanaliculusȌ ȋHaliotis irisȌǤ ǣ ǡ ʹ Ǥ Ǥ Ǥ Ǥ Ǥ ȋǤȌ ȋʹͲͳͳȌǡǡǣͷʹǦͷ͵Ǥ ǤǤǡǤǤǡǤǤǡǤǤǡǤǤǡǤǤǡ ǡ ǡ ǦǦ Ǥ ͳȋʹͲͲʹȌǡǣͶȂͶǤ Ǥ ǡ Ǥ ǡ Ǥ ǡ Ǥ ȋʹͲͲͳȌǡǣ͵ͳǦ͵͵Ǥ ǤǤǡǤǡǤǡǤ ǤǡǤǤǡǤǤǤǡǤǤǡǤǡ Ǥ ǡ ǤǤ ǡ Ǥ ǡ ǤǤ ǡ Ǥ ǡ Ǥ ǡ ǤǤ ǡ ǤǤ ǡ ǤǤ Ǧ ÓǡǤǡǤǤǡǤǤǡǤǤǡǤǡǤǤǤǡǤǤ ǡǤǤ ǡ ǤǤ ǡ ǤǤ ǡ Ǥ ǡ Ǥ Ǥ ǣ ǡ Ǥ ͶͲǤ ǡ Ǥǡ ǡ ǤǤǡ ǡ Ǥǡ ǡ ǤǤǤȋȌǤ ȋʹͲͲʹȌǡǣ͵Ͷ͵ǦͶʹǤ Ǥ ǡ Ǥ ǡ Ǥ ͳͶȋͳͻȌǡǣͲ͵Ǥ ǤǤ ǡ ǤǤ ǡ ǤǤ ǡ ǤǤ ǡ ǤǤ ǡ Ǥ Ǧǡ ǤǤ ǡ ǤǤ ǡ ǤǤ ǡǤǤǤ ȋPernacanaliculusȌ Ǥ ͷͺȋʹͲͲͻȌǡǣͻǦͶǤ ǤǤ ǡ ǤǤ ǡ ǤǤ ǡ ǤǤ ǡ Ǥ Ǥ ǡ ǤǤ ǡ ǤǤ ǡ ǡ Crassostrea gigas ȋȌǤ ͵ͻǡͳ͵ȋʹͲͲͺȌǡǣͳͶ͵ͶǦͳͶͶʹǤ ǤǤ ǡ ǤǤ ǡ ǤǤ ǡ ǤǤǡ ǤǤ ǡ ǤǤ ǡ ǤǤǡ ǤǤ ǡ Ǥ Ǥ ǡ ȋCrassostreagigasȌǣ ǡ ʹͶʹȋʹͲͲͶȌǡǣʹͳǦʹͺʹǤ ǤǤǡǤǤǡǤǤǡ ǡEvechinus chloroticusǤͷʹȋʹͲͲȌǡǣͳ͵ͻǦͳͶͷǤ ǤǤ ǡ ǤǤ ǡ ǤǤ ǡ Ǥ ǡ Ǥ Ǧǡ ǤǤ ǡ Ǥ ǡ ǤǤ ǡ ǤǤ ǡȋPernacanaliculusȌ ǤͷͷȋʹͲͲȌǡǣ͵ͶͺǦ ͵ͶͻǤ ǤǤǡǤǤǡǤǤǡ ǡEvechinus chloroticusǤͷʹȋʹͲͲȌǡǣͳ͵ͻǦͳͶͷǤ ǤǤ ǡ ǤǤ ǡ ȋ Ȍ Ǥ ǡ ʹͷǡͶȋʹͲͲͶȌǡǣʹͺǦʹͻͻǤ ǤǤ ǡ ǤǤ ǡ Ǥ ǣ Ǥ ǤǤ ǡ ǤǤǡǤǤǡǤǤȋͳͻͻͷȌǡǣͳͻǦͶͶǤ ౹Bibliography౹ 162 UniversidadedeVigoǦFacultadedeCienciasdoMar Ǥ ǡ Ǥ ǡ ǤǤ ǡ Ǥ ǡ À ȋ ǡ Ȍǣ Ǥ ʹ ȋʹͲͲͺȌǡǣͺʹǦͺ͵ͷǤ Ǥ ǡ Ǥ ǡ Ǥ ǡ ȋParacentrotuslividusȌȋMytilusedulisȌǤͷͲȋʹͲͲͷȌǡǣͳ͵ͺʹȂͳ͵ͺͷǤ Ǥ ǡ ǦǤ ͺͺȋʹͲͲͺȌǡǣ͵ͲͻǦ͵ͳͷǤ ǤǡǤǡǤǡǤ ǦÀǡ ǣParacentrotuslividus Ǥ ͷͳȋʹͲͳʹȌǤ Ǥ ǡ Ǥ ǡ Mytilus galloprovincialis Ǣ Ǥ ʹȋʹͲͲͺȌǡ ǣʹͲͺǦʹͳʹǤ ǤǡǤ Ǧǡ Ǧ ǤǡǡƬǡͳȋʹͲͲȌǡǣͶͷǦͶǤ ǤǡǤǡǤǡǤǡǤ ǦǡǤǡ ǡ ǡ Ȃ ǤͷʹȋʹͲͲ͵ȌǡǣͳʹͲͻȂͳʹʹͶǤ Ǥ ǡ Ǥ ǡ Ǥ ǡ ǤǤ ǡ Ǥ Ǧ Àǡ ȋ ȌǢ ǡ ȋMytilus galloprovincialisȌǦParacentrotuslividusCionaintestinalisǤ ͷȋʹͲͲ͵Ȍǡǣͷ͵ͳȂͷͷ͵Ǥ Ǥ ǡ ǤǤǡ ǤǤǡ ǤǤ ǡ ǤʹͶǡͶȋʹͲͲ͵ȌǡǣʹͳǦʹͺǤ Ǥ ǡ Ǥ ǡ ǤǤ ǡ ʹǤ͵ͷȋͳͻͻȌǡǣ͵ͶͳǤ Ǥ ǡ Ǥ ǡ Ǥ ǡ ǤǤ ǡ Ǥ Ǥ ǡ Ǥ ǣ Ǥ Ǥ ǡǤȋǤȌǤǡȋͳͻͻȌǡǤʹͳͷǦʹʹͲǤ Ǥ ǡ Ǥ ǡ ǤǤ ǡ Ǥ Ǥ ǡ ȋTetrapigus nigerȌ ȋLoxechinusalbusȌͳǤͲʹǤ͵͵ȋͳͻͻȌǡǣͶǤ ǤǡǤǤǡǤǤǡ Ǥǣ ȋȌǤ Ǥ ǡ Ǥ ȋǤȌǤ ȋȌ ǤȋʹͲͲ͵ȌǡǡǡǤͶʹͻǦͶ͵ͶǤ Ǥ Ǣͳͷ͵Ȃ ͻͷǢ ǡ ͳͻͻͷǡ ͳͳǤͲͶǢ ǡ Ǥ Ǥ ǡ Ǥ ǡ Ǥ ǡʹͳȋͳͻͻȌǡǣͶʹ͵ǦͶ͵ͲǤ 163 UniversidadedeVigo ǦFacultadedeCienciasdoMar ǤǤ Ǥ ǡǤǤ ǡ ̵ ȋ Ȍǡ ǡ ǡ ǡ ǤǤ ǡ Ǥ ǡ Ǥ Ǥ Ǥ ǣ Ǥ Ȁ ȋȌǤ ͶǡȀȋͳͻͺͶȌǡǣͷ͵ǦͺͻǤ ǤǤǡ Ǥ͵ȋͳͻʹȌǡǣ ͳͻͲǦͳͻʹǤ ǤǤ ǤǤ ȋ Ȍ Ǥ ʹʹȋʹͲͲȌǡ ǣͳ͵ǦͳǤ ǦǤ ǡ ǦǤ ǡ ǦǤ ǡ ǦǤ ǡ Ǥ ͳȋͳͻͺͻȌǡǣͳͻǦʹͲͳǤ ǦǤ ǡ ǦǤ ǡ Ǥ Ǥ ǣ ǡ ʹ Ǥ ǤǤ ǤǤ ǡ ȋǤȌ ȋʹͲͳͳȌ ǡ ǡǣͲͶǦͳͷǤ ǦǤǡǤǤǡǤ ͳͻȋʹͲͲͳȌǡ ǣͳͳǦͳͻͺǤ ǦǤǡǦǤǡǦǤǡǦǤǡǦǤǡ Ǥ ͳͷͷȋͳͻͻȌǡǣ͵ͳǦͶͶǤ ǦǤ ǡ ǦǤ ǡ ȋ Ȍ Ȃ Ǥ͵ͳȋͳͻͻͶȌǡǣͷ ǦǤ ǡ ǤǤ ǡ ǤǤ ǡ ǤǤ ǡ ǤǤ ǡ Ǥ ȋͳͻͻͶȌǡǣͻͻǦͳͲͶǤ ǤǡǤǡǤǡǤǡǤǡǤǡ ǡ ͳͺͳǡ Ǥ ʹͲͳͳǡ Ǥ ǤǤǡǤǤ ǡǤǡ Ǥ ǤǤǤ ǤͷͳȋʹͲͲȌǡǣͷ͵ǦͷͻǤ ǤǤǡǤǤǡǦǦ ǤǤ ǤȋͳͻͻʹȌǡǣͳͻǦ͵ͲǤ ǤÓǡǤǤǤ Ǥ ǡͳ͵ȋͳͻͻͷȌǡǣʹǦʹͻͲǤ Ǥ Ǧǡ Ǥ ǡ ×Ǥ ǣ À × ȋ Ȍ ǤǤȋǤȌ ȋʹͲͲȌǡǣͷͻǦ ͻǤ ǤǤǡǤǡ ParacentrotuslividusǤǣ ǣ Ǥ ǤǤ ȋǤȌǤǡȋȋʹͲͲͳͳǡȌǡʹͲͲʹȌǡǣʹͶǦʹͺ ǤǡǤǤǡǤǤǡǣ Ǥ Ǥ ǤǤͳǡ͵ȋͳͻͻȌǡǣͷͲͷǦͷͲͺǤ ǤǡǤǡ Ǧ ǣ Ǥʹǡ͵ȋʹͲͳͳȌǡǣͳͶǦͳͺͲǤ CryopreservationofmarineinvertebrateearlyǦlifestages 164 UniversidadedeVigo ǦFacultadedeCienciasdoMar ǡ ǡ ǣȀȀǤǤȀ ȀͲͲ͵Ȁ͵͵ʹȀ͵͵ʹͲͲǤ ǡǤǤʹͲͳʹǡʹͲͳ͵Ǥ ǤǤǡǤǤǤǡǤǡ ǤǦ Ǥ ǡͳʹǡʹȋʹͲͲͲȌǡǣ͵ͳȂ͵Ǥ Ǥ ǡ Ǥ ǡ ǡ Ǧ Ǥ ͳͲȋʹͲͲͳȌǡǣ ʹ͵ȂʹͳǤ Ǥ ǡ ǤǤ ǡ ȋ ǣ Ȍ Ǥ ʹͲͶȋʹͲͲͲȌǡǣͳ͵ͳǦͳͶͳ Ǥ ǡ ȋ ǣ Ȍ ȋ Ȍ Ǧ ǤǤͳͳͺǡͶȋͳͻͻȌǡǣͳ͵Ǧͳ͵ͺͶǤ ǤǤǡ ǤͲȋʹͲͳͲȌǡǣͶͷǦͷ͵Ǥ ǤǤǡ Dz dz Ǥ ͳͺ ȋͳͻͺͳȌǡǣͶ͵ǦͶͺʹǤ Ǥ ǡ Ǥ ǯǡ Ǥ ǡ ǤǤ ǡ Ǥ ǡ Ǥǡ ǡ Ǥ ǡ Ǥ ǡ Ǥ ǡ ǣ Ǥ ͺͶȋʹͲͳʹȌǡǣʹͻ͵ǦʹͻͺǤ ǤǡǤǡ ǡ ȋ ǡȌǣǤͺͷǡͶȋʹͲͳͳȌǡǣͳͺͺͺǦͳͺͻǤ Ǥ ǡ Ǥ ǡ Ǧ Ǥ ʹͻȋʹͲͳͲȌǡǣͳͳͻʹǦͳͳͻͺǤ ǤǤǡǤ ǡ Ǥͷͳȋͳͻ͵Ȍǡǣ ǦͻǤ ʹͲͲͲȀͲȀ ǣȀȀ ǤǤȀȀȀǦ Ȁ ʹͲͲͺȀͷȀ Ǥ ǣȀȀ ǤǤȀȀȀ̴Ǥ ǡǡǤǤǡǤǡ ǤǡȋMytilus galloprovincialisȌǤͷͺǡͳȋʹͲͲͻȌǡǣͳͶͷǦͳͷͲǤ ǤǤ ǡ Ǥ ǡ ǤǤ ǡ Ǥ ǣ ǤǤǤ ǡǤǤȋǤȌǤ ȋʹͲͲͲȌǡǣͷͻǦͶ ǤǡǤǤ ǡǤǡȋ Ȍ ʹͲͲǦʹͲͲͻǤ ǡʹͲͲͻǤ Ǥ Ǥ ǡ ǤǤ ǡ Ǥ ǣ ǤǤǤȋǤȌǡ ǡȋʹͲͲͷȌǡǣͳͷǦͳͺǤ ̵ȋ ȌǤͶͳǡͷȋͳͻͻͶȌǡǣͳͲͳͳǦ ʹͳǤ ౹Bibliography౹ 165 UniversidadedeVigo ǦFacultadedeCienciasdoMar Ó ×Ǥ ǤͳʹȀͳʹȀʹͲͳʹ ǤǤǡǡ ǤʹͻȋʹͲͲͺȌǡǣͶ͵ǦͷʹǤ ǤǤǡǤǤǡ ǤǤǤǡǤǡǤ ǤȋͳͻͻͷȌǡ ǣͳǦͳʹǤ Ǥ ǡ Ǥ ǡ ǤǤǤ ǡ Ǧ ǡǣ ͵ǤǤǤǡǤǤǡǤǤȋǤȌǤ ǡ ȋͳͻͻͻȌǡǤͳǦͳͺǤ ǤǤ ǡ ǤǤ ǡ Ǧ ǡ Ǥ ʹǡ ǡǡͳͻͻͲǤ ǤǤ Ǥǡ Ǥ ǣ Ǥ ͳǤ ǤǤǤǤǡǤǤǤȋǤȌǤ ǡȋͳͻͻͳȌǡǣ͵ͻǦͳͲͷǤ ǤǤ ǡ Ǥ ǣ ǡ ǡ ȋǤȌǤ Ǧ ǤͶǦͷǡǡǡǤ ȋͳͻͻ͵ȌǡǣͳǦͳͻȌǤ ǤǡǤǡǤǤǡ ǤǡͳȋʹͲͳ͵ȌǡǣͳǦʹ͵ ǦǤ ǡǤǤǡǤǤǡ ȋ ȌǤ ǡͷͺǡͺȋʹͲͲʹȌǡǣͳͷ͵ǦͺǤ ǦǤ ǡ ȋCrassostrea gigasȌ Ǥ Ͷ͵ ȋͳͻͻͷȌǡ ǣ ͳͳ͵Ǧ ͳͳͶǤ Ǥ ǡ Ǥ ǡ Ǥ ǣ ȋ ǡ ȌǤͳͳͺȋͳͻͻͺȌǡ ǣ͵ͳǦͶ͵Ǥ Ǥ ǡ Ǥǡ Ǥ ʹͲͲ͵Ǥ ǤǤ ǡǤ ǤǡǤǤǡ ͺǦ ǦͳͻǤ ͲȋͳͻͺͶȌǡǣʹʹͻǦʹ͵ͶǤ ǤǤ ǡ ǤǤ ǡ ǤǤ ǡ Ǥ ʹͶʹȋʹͲͲͶȌǡǣʹͳǦʹʹͺ ǤǤ ǡǤǤ ǡǤǤ ǡǤǡǤǡ Ǥ ͳͻͻȋʹͲͲͳȌǡǣ͵ͷ͵Ǧ͵ͻǤ Ǥ ͶȋʹͲͲʹȌǡǣͶͷǦͷ͵Ǥ ǤǤ ÀǡǤǡ ǤǤ ǡ ǤǤ ǡ ǤǤ ǡ ǤǤ ǡ ǤǤ ǡ ǤǤ ǡ ǤǤ ǡ Perna canaliculus Ǧ Ǥ ͷͻȋʹͲͲͻȌǡǣͶͳͶǤ ǤǤ ǡ ǤǤ ǡ ǤǤ ǡ ǤǤ ǡ ǤǤ ǡ ǤǤ ǡ ǤǤ ǡ ǤͳͻȋͳͻͺͺȌǡǣͳͷǦͳͺͲǤ CryopreservationofmarineinvertebrateearlyǦlifestages ౹Bibliography౹ 166 UniversidadedeVigoǦFacultadedeCienciasdoMar ǤǤ ǡ Ǥ ǡ ǤǤ ǡ ǤǤǤͺͻȋͳͻͶȌǡǣͻǦͺͺǤ Ǥ Ǥ ǡ Ǥ Ǥ ǡ Ǥ Ǥ ǡ ȋ ȌǤ ͷ͵ǡͶȋʹͲͲȌǡǣʹͻͻǦ͵Ͳ͵ ǤǡǤǡǤǡ Ǥ͵ȋͳͻͺͻȌǡ ǣʹͳͷǦʹͳͻǤ Ǥ ǡ Ǥ ǣ ǡʹǤ ǡǤǤǡ ǡǤǤȋȌǤ ʹͲͳͳͶͲͻǦͶʹͲǤ ǤǤǡǦ Crassostreagigasǡ Ǥ ͺȋͳͻͳȌǡǣͷͻͻǦͲͳǤ ǤǤǡǤǡǤǤ ǡǤǤ ǡ Ǥ ͺǡʹȋͳͻͻȌǡǣʹǦʹͳ ǤǤ ǡǣ Ǣ ǤȋǤȌȋʹͲͲȌǡǣͳǦͻ ǤǤ Ǥ ǡ Ǧ Ǥ Ǥͺǡ ͷ ȋͳͻͳȌǡ ǣǣͶͶȂͶͷʹǤ Ǥ ǡ Ǥ ǣ ǡǤͻǤǤǤȋǤȌǤ ǤǡȋͳͻͻͷȌǡǣͷ͵ͻȂͲͻǤ Ǥǡ ǡ ǤǤǤǤǤʹȋͳͻͺͳȌǡǣͳʹ͵Ǧͳ͵͵Ǥ Ǥǡ ǡ ǤǤǤǤǤͳͺȋͳͻͳȌǡǣ͵ͻǦͶͲǤ Ǥ Ú ǡ Ǥ ǡ Ǥ òǡ Ǥ Ǥ ǡ ǡ¡͵ͻȋͳͻͺͷȌǡ ǣͳǦͳͻǤ ǤǤ ǡ Ǥ ǡ Ǧ Ǥ Ǥ ǤͳͲͺǦͳȋʹͲͳͲȌǡǣʹ͵ǦʹͶǤ Ǥǡ Ǥ ʹͲͲ͵ǦͲͷǡ Ͳ͵ͳͲͳȋʹͲͲ͵Ȍ ǤǤǡ ǦǦ Ǥ ʹͷ͵ȋʹͲͲȌǡǣʹͻͲǦ͵ͲͲǤ ǤǤ ǡ ǤǤ ǡ Dama damaǤ ǣ ǤǤ ǡ ǤǤ ȋ ǤȌ ǣ Ǥ ǡ ǡǡȋͳͻͻͳȌǣͶͺͷȂͶͻͳǤ Ǥ ǡ Ǥ ǡ Ǥ ǡ Ǥ ǡ ǡ ȋCrassostreagigasȌǡͶͻ ȋʹͲͲͶȌǡǣʹͷͲǦʹͷǤ 167 UniversidadedeVigo ǦFacultadedeCienciasdoMar Ǥǡ Ǥ ͳͲȋͳͻͺͲȌǡǣͳ͵ǦͳͷͻǤ Ǥǡ ǤͳͶ ȋͳͻȌǡǣʹͷͳǦʹʹǤ Ǥǡǣ Ǥ ͳͺȋͳͻͲȌǡǣͻ͵ͻǦͻͶͻǤ ǤǡǤǤǡǤ Ǥǣ ǤǤǤǡǤǡǤǤȋǤȌǤȋʹͲͲͶȌǡǣ͵ͳǦ͵ͻʹǤ Ǥ Ǥ ǡ Ǥ ǡ Ǥ ǡ ͳͻͻͳǡ ȋȌ Ǥ ͳͻȋͳͻͻͳȌǡǣʹͶͳǦʹͶͺǤ Ǥ ǡ Ǥ ǡ Ǥ ǡ Ǧ Paracentrotus lividusǣ Ǥ ͳͺȋʹͲͲͷȌǡǣͶͷǦͷͶǤ ǤǤ ǡ Ǥ ǡ Ǥ ǡ Ǥ ǡʹͷǡǤͳȋʹͲͲȌǡ ǣ͵ȂͶͶǤ ǤǤǡǤǡǤǡ Ǥ ͷͺȋʹͲͲʹȌǡǣʹǦͶͳ Ǥǡǡ ȋSaccostreaglomerataȌǤǤ ǤͷȋʹͲͲͺȌǡǣͳͳʹͷǦͳͳʹͺǤ ǦǤ ǡ ǦǤ ǡ Ǥ ǣ ǡʹǤ ǡǤǤǡ ǡǤǤȋȌǤ ȋʹͲͳͳȌǡǣͲͶǦͳͷǤ ǦǤ ǡ ǦǤ ǡ Ǥ ǣ ǤǤǤ ǡǤǤȋǤȌ ȋʹͲͲͲȌǡǣʹͶͲǦʹͷͲǤ ǤǦǤ ǡ ǤǦǤ ǡ ǤǦǤ ǡ ȋCrassostrea gigasȌ Ǥ͵ͻȋͳͻͻͻȌǡǣͳͻʹǦͳͻǤ ǦǤǡǦǤǡǦǤǡ Ǥ͵ͳȋͳͻͻͶȌǡǣ ͷǤ ǦǤ ǡ ǦǤ ǡ ǦǤ ǡ Ǥ͵Ͳȋͳͻͻ͵ȌǡǣͳͶǤ ǤǤǤ ǡ Ǥ ǣ ǣ ǤǤǤǤǡǤ ǤǤȋǤȌǤǡǡȋͳͻͻͳȌǡǤʹ͵ͳǦ ʹͶͶǤ ǤǤ ǡ Ǥ ǡ Ǧ Ǥ ͷȋʹͲͲʹȌǡǣ͵Ͳ͵Ǧ͵ʹǤ ǤǤ ǡ Ǥ Ǥ ǣ Ǥ ǤǤ ǡ ǤǤȋǤȌǤ ȋʹͲͲͲȌǡǣͷǦͺ͵Ǥ CryopreservationofmarineinvertebrateearlyǦlifestages ౹Bibliography౹ 168 UniversidadedeVigoǦFacultadedeCienciasdoMar ǤǤǡ Ǥ ǡ ǡ ǡǡͳͻͻͷǤ ǤǤ ǡǤǤǡǤǤǡ ǤǡǤǤǡǤ ǡ ǡ ͷͳȋʹͲͲͷȌǡǣͳͳ͵ǦͳͳǤ ǤǤ ǡ ǤǤ ǡ Ǧ ǤʹͶȋͳͻͻͺȌǡǣʹͲ͵ǦʹͲǤ ǤǤǡǤǤ ǡǤǤǡǤǤǡ Ǥǣ ǤǤǡǤǡǤǡǤǡ ǤȋǤȌǤǤȋͳͻͻͳȌǡǣʹͳȂʹͻǤ ǤǤǡǤǤ ǡǤǤǤǡǤǤ ǡ ǣ Ǥ ǣǤǤǤǡǤǡǤǤȋǤȌȋʹͲͲͶȌǡǣǦͳͲͺǤ ǤǤǡǤǤǡǤǤǡǤ ǡǤǤǡǤǤ ǡ ̻ȋPernacanaliculusȌ Ǥ Ͷ ǡͳͻǦʹ͵ȋʹͲͲͻȌǡǡʹͳǤ ǤǦǡǤǡǤǡǤǡǤ ǡ Ǥǣ ǤßǡǤ Ǥ ǡǤǤȋǤȌǤ ȋʹͲͲ͵Ȍǡǣʹͷ͵Ǧ͵ͳͺǤ ǤǤǡǤǤ ǡǤǡǦǣǡ ǡ ͵ͳȋʹͲͲʹȌǡǣ Ͷ͵ͻȂͶͷͷǤ ǤǤ ǡ ǡ ͳǤͳͺȋͳͻȌǡǣ͵ͷǦ͵ͺʹǤ Ǥ ǡ Ǥ ǡ ǣ Ǥ ǣ Ǥ ǤǤǡǡǤ ȋǤȌǡǡȋʹͲͲȌǡǣ ʹͷǦʹͺʹǤ ǤǤ ǡ Ǥ Ǧ ǤǤ ǤǡǤǤ ȋǤȌǤ ǡȋͳͻͻͷȌǡǣͳͶͷǦͳͶͻǤ ǤǤ ǡ Ǥǡ ǤǤ ǯǡ ǤǤ ǡ ǣ Ǥ ǡʹǡʹȋʹͲͲȌǡǣʹͺͳǦʹͻͶǤ ǤǤǡǤǤǡǤǤǡǤǡǤǡǤ ǡ Ǥ ǣ ǡ ʹ Ǥ ǡ ǤǤǡ ǡ ǤǤ ȋȌǤ ȋʹͲͳͳȌ͵Ǧ͵ͺͲǤ ǤǤǤ ǡǤǤǡ Ǧ ǤǤ ǤǤǤͳͲ͵ȋͳͻͻͶȌǡǣͳͲ͵ǦͳͲͻǤ Ǥ ǡ ǣ Ǥ ǤǤ ǡ Ǥ ǡ ǤǤ ȋǤȌ ǤȋʹͲͲͶȌǡǣ͵ǦͷǤ ǤǤ ǡ Ǥ Ǥ ǡ Ǥ ǣ ǣ Ǥ ǤǤ ȋǤȌȋʹͲͲȌǡǣͳǦͺͷǤ ǤǤǤ ǡ ǤǤ ǤǤǤͻͳȋͳͻͻʹȌǡǣʹͳͷǦʹʹͲǤ 169 UniversidadedeVigo ǦFacultadedeCienciasdoMar Ǥ ǡ ǤǤ ǡ ǤǤ ǡ ǤȋͳͻͶͻȌǣͳͶǦͳǤ ǤǤ ǡ Ǥ ǣ Ǧ Ǥ Ǥ Ǥǡ ǤǤ ȋȌǤ ͵ͺȋʹͲͲȌǡǢ͵ͻǦͷ Ǥ ǡ Ǥ ǡ Ǥ ǡ ǤǤ ǡ Ǥ ǡ ǡ ǡ ǡǤȋʹͲͳ͵ȌǡǣͳͲǤͳͲͳȀǤ ǤʹͲͳ͵ǤͳͲǤͲͷ͵Ǥ Ǥ ǡ Ǥ ǡ ǤǤ ǡ ǣ ȋ Ȍ ȋ ȌǤ ǡ͵ȋʹͲͳ͵ȌǡǣʹͶǦʹͻǤ Ǥ ǡ ǤǤ ǡ ǤǤ ǡ ǤǤ ǡ ǤǤ ǡ ǤǤ ǡ ǤǤ ǡ Ǥ ǡ ȋPernacanaliculusȌ Ǥͷǡ͵ȋʹͲͳͳȌǡ ǣʹͷǦʹʹǤ Ǥ ǡ Ǥ ǡ ȋ Ȍ ǤͷͻȋʹͲͲͻȌǡǣ͵ͶͶǦ͵ͷͲǤ Ǥ ǤǦǡǤǤ ǡǤǤǡǤǤ ǡ Ǥǣ ǡʹǤ ǡǤǤǡ ǡǤǤȋȌǤ ȋʹͲͳͳȌǡǣͷͻͷǦͲ͵Ǥ Ǥ ǤǦǡǤǤ ǡ ǡͶ͵ȋʹͲͲͳȌǡǣʹͳͳǦʹʹ͵Ǥ Ǥ Ǥ Ǧǡ ǤǤ ǡ ǤǤ ǤǤ ǡ Ǥǣ ǤǤǤ ǡǤǤȋȌȋʹͲͲͲȌǡǦʹ͵ͲǦʹ͵ͻǤ Ǥ ǤǦǡǤǤ ǡǤǤ ǡ Ǥ ͳȋͳͻͻͺȌǡǣʹ͵ͶǦʹ͵Ǥ Ǥ Ǥ Ǧǡ ǤǤ ǡ ǤǤ ǡ ǤǤ ǡ ǤͳͻȋͳͻͻͺȌǡǣʹͺ͵ǦʹͻʹǤ ǤǤ ǡ Ǥ ǡ ǤǤ ǡ ǡ ǡͶͲȋʹͲͲȌǡǣʹͶǦʹͺǤ ʹͲͳ͵ ǣȀȀǤǤȀ ȀȀ ȀͲͲͷͺͻȀͲͲͷͺͻ̴ ΨʹͲ ΨʹͲ ΨʹͲ ΨʹͲ Ǥ ǤǡǤǦÓǡǤǡǤǤǤ ǣ Ǥ ǡȋʹͲͲͻȌǡǣʹͺʹǦʹͺ͵ ǤǤ ǯǡ ǤǤ ǡ Ǧ ȋDicerorhinus sumatrensisȌ (Diceros bicornisȌǤǤǤǤͳͳͺȋʹͲͲͲȌǡǣʹ͵ǦʹͳǤ ǤǤ ǡ Ǥ ǡ Ǥ ǡ ǤǤ ǡ ǤǤ Óǡ Ǥ ǡ ǣ ×ǡ ×Ǥ ÀʹͲͳͲ CryopreservationofmarineinvertebrateearlyǦlifestages ౹Bibliography౹ 170 UniversidadedeVigoǦFacultadedeCienciasdoMar ǤǡǤǤ ǡǤǤǡǤǡǤǤǡǤǤǡ ǣ Ǥ ǡǡͳ͵ȋʹͲͲͷȌǡǣͳͳǦͳͺǤ Ǥ Ǧǡ ǤǤ ǡ ǤǤ ǡ ǡ Crassostrea gigasǡ ǤͷȋʹͲͲͺȌǡǣͶ͵ǦͷʹǤ Ǥ Ǧǡ ǤǤ ǡ ǤǤ ǡ ǤǤ ǡ ǤǤ ǡ ǡ Crassosstrea gigasǡ ǤͷȋʹͲͲͺȌǡǣʹͺǦ͵ͷǤ Ǥ ǦǡǤǡǤǤǡǤǡ Ǧ ȋȌ Ǥ ͵ȋʹͲͳͲȌǡǣͶͻͳǦͶͻͻǤ Ǥ Ǥ ǤȋʹͲͳʹȌ Ǥ ǡ Ǥ ǡ Ǥ Ǧǡ ȋHaliotismidaeȌǤͷȋʹͲͲͺȌǡǣ ͵ͲͺǦ͵ͳͳǤ Ǥ ǡ Ǥ ǡ ǣ Crassostrea gigas Pectenmaximus Ǥ ͳʹǡʹȋͳͻͻͻȌǡǣͳʹͳǦͳ͵ͲǤ Ǥ ǡǤǡǤǤǡǤǡǤǤǤ ͷͺʹȋʹͲͲͺȌǡǣͳͺ͵ȂͳͺǤ ǤǤ ǡ ǤǤ ǡ ǤǤ ǡ Ǥ ǡ ǤǤ ǡ ǤǤ ̵ǡ ȋ ȌǤȋʹͲͳͳȌǣʹ ǤǤ ǡǤǤǡǤǡǤǡǤǤǯǡǤǡǤ ǡǤǤ ǡǤǡ ǤǤǡ Ȃ ȋ ȌǤ ͳʹͻȋʹͲͲͶȌǡǣͷͷͻǦͶ ǤǤǡǦ ǡǡǡ Isognomoncalifornicumǡ ͳͶͻȋͳͻͻͳȌǡǣͷͷǦǤ Ǥǡ Crassostreagigasǣ ǡ ͻʹȋͳͻͻͳȌǡǣͶ͵ǦͷǤ ǤǤ ǡ ǤǤ ǡ Ǥ ǡ Ǥ ǡ ǤǤ òǡ Ǥ ǡ Sillago japonicaǡ ͷ ȋʹͲͲͺȌǡ ǣ ͳͲǦͳͶǤ ǤǤ ǤǤǤ͵ȋʹͲͲȌǣʹͳͷǦʹʹͷǤ ǤǤ Ǥǡ Ǥ ǡ Ǥ ǡ ǤǤ ǡ Chaetoceros calcitrans ȋȌ,PernacanaliculusǤ ͵ͲȋʹͲͳͲȌǡ ǣʹͲǦʹͺͲǤ 171 UniversidadedeVigo ǦFacultadedeCienciasdoMar Ǥǡ Ǥ ǤȋͳͻͻͷȌǤ ǤǤ ǡ ǤǤ ǡ ǤǤ ǡ ǤǤ ǡ ǤǤ ǡ ǤǤ ǡ ǤǤ ǡ ȋCrassostreagigasȌ ǤͷͳȋʹͲͲͷȌǡǣͳͶʹǦͳͷͳǤ Ǥ ǡ Ǥ ±ǡ Ǥ ǡ Ǥ ǡ Ǥ ǡ Ǥ ǡ Ǥ ǡ Ǥ ǡ Ǥ ǡ ǡ Ǥ ǡ ǡ ȋȌǤͻǡͶȋʹͲͳͶȌǡǣͳͲǤͳ͵ͳȀǤǤͲͲͻ͵ͶͺǤ ǤǡǤǡǤǡǤǡǤǤǤ ǡ͵ͳȋʹͲͲͲȌǡǣʹ͵ͳǦʹͶ͵Ǥ ǤǤǤ ǡ Ǥ ǡ Ǥ ǡ ǤǤ ǡ Ǥ ǡ Ǥ ǡ ǤǡǦǤ±ǤǤǤǤ.ͳͻȋͳͻͺͲȌǡǣ͵ʹʹȂ ͵͵ʹǤ ǡǤǢ ǡ ǤǤǡǡ ǤǤǡ ǡ ǤǤǡǡ ǤǤǡǡ ǤǤǡǡ Ǥǡǡ ǤǤǡǡ Ǥǡ Ǧǡ ǤǤǤǡǡ ǤǤ Ǧ Ǥ ͳʹǡ ͶǡʹͲͳʹǡǣͶͷǦͶͷǤ ǤǤǡǤǤǡǤ ǡǡ ȋͳͻͻͷȌǤ ǤǤǡǤǤǡǤǤ ǡǤǤ ǡǤǤǡǤǤǡ ȋPerna canaliculusȌ Ǥ Ǥ Ǥ ͵͵ͶǦ͵͵ ȋʹͲͳʹȌǡ ǣ ͳͻͻǦʹͲͶǤ ǤǤ ǡ ǤǤ ǡ ǤǤ ǡ ǤǤ ǡ ǤǤ ǡ Ǥ Ǥ Ǥ,ͳͲǡʹͲͲǡ ȋ ͵Ǧ͵ʹȌ,ǣͳͳͷǤ ǤǤ ǡ ǤǤ ǡ ǤǤ ǡ ǤǤ ǡ ǤǤ ǡ ǤǤ ǡ ǤǤ ǡ ǡǡ ͳȋʹͲͲͳȌǡ ǣ͵ͳǦ͵ͶǤ ǤǤ Ǥ ǡ ǡ ʹ ȋͳͻͷͲȌǡ ǣ ͻͳͲȂͻͳͳ ǤǡǤǤ ǡǤ ǡǤǡǤǦǡ Ǥ ʹͶͷȋʹͲͲͷȌǣͳͲͳǦͳͲͻǤ Ǥ ǡǤǣ ǤǡǤȋǤȌ ȋͳͻͻͳȌǡǣͺͻ͵ǦͻͲͺǤ ǣȀȀǤǤ Ǥ ǤǤǡǤǦǡǤǡǤǦǡ ǡ ơ ǡ Ǥ͵ͳȋʹͲͲʹȌǡǣͳͺ͵ǦͳͺͺǤ CryopreservationofmarineinvertebrateearlyǦlifestages ౹Bibliography౹ 172 UniversidadedeVigoǦFacultadedeCienciasdoMar Ǥǡ ǤǣǤǤǤǡ ǤǡǤǤȋǤȌȋʹͲͲͶȌǡǣͶͳͷǦͳ͵Ǥ ǤǤ ǡ Ǥ ȋȌǡ Ǥ ʹͲͲʹǤ ǤǤ ǡ ǣ Ǥ ͷȋͳͻͺͶȌǡǣͳǦʹͳǤ Ǧ ǤǡǤǡǤǡ Ǥ ǤͷȋͳͻͻͲȌǡǣͶͻǦͷͶǤ ǤǡǤǡǤǦǡǤǡǤ ǡǤǤ ǡ C.virginicaǤ ͵ͶͶǦ͵ͶͻȋʹͲͳʹȌǡǣʹʹ͵Ǧʹ͵ͲǤ ǤǤǡ ǤǤǤǤ ǤǤ͵ͲȋͳͻͷͳȌǡǣͳǦʹͲǤ ǤǤǡ Ǥǣ Ǥǡ ǤǤǡǡǤȋǤȌǤ ȋͳͻͺͳȌǡǣͳͺͻǦʹͳͺǤ Ǥ ǡ Ǥǡ ǡ Ǥǡ ǡ Ǥǡ ǡ Ǥǡ ǡ Ǥǡ ǡ Ǥǡ ǡ Ǥ ͳͷȋͳͻͻȌǡǣͳͻ͵ͳǦͳͻ͵Ǥ Ǥ ǡ Ǥ ǡ Ǥ ǡ Ǥ ǡ Ǥ ǡ Ǥ ǡ ǡ Mytilus galloprovincialis Ǥ Ͷʹ ȋʹͲͳͳȌǡ ǣ ͳͺͳǦͳͺʹ͵Ǥ Ǥ ǡǤǡǤǡǤǤ ǡ Paracentrotus lividus ȋȌǤ ǣ ǤʹǤ ǤǤ ȋǤȌǡ ȋʹͲͲͷȌǡǤͳͶǦͳͺǤ ǤǤǤǡǤǡǤǤǡ Ǥǡ ȋǤǡͳͷͺȌǤ͵ǡʹȋʹͲͳͳȌǡǣͳͳͺǦʹͶǤ ǤǤ ǡ ǤǤ ǡ ǣ Ǥ ǤǤǤʹǡͳ͵Ͷȋͳͻͻ͵Ȍǡǣͳʹ͵ͷǦͳʹͶͷǤ Ǥ ǡ ǤǤ ǡ Ǥ ǡ Ǥǣ ǣ ǡ ǤǤǤǡǤȋǤȌǤ ǡȋʹͲͲͷȌǡǣͶ͵ǦͺǤ ǤǤ ǡ Ǥ ǡ Ǥ Ǥ Ǥ ȋͳͻͺͻȌǡǣͳͳǤ Ǥ ǡ Ǥ ǡ Ǥ ǡ ǡ CrassostreagigasǤ ͺǡͶȋʹͲͲͷȌǡ ǣͷǦʹǤ ǤǡǤǤ ǡǡ ǦǤǤȋʹͲͲȌǡǣ͵ͺ͵Ǧ͵ͻͲǤ UniversidadedeVigo ǦFacultadedeCienciasdoMar LISTOFFIGURESANDTABLES CryopreservationofmarineinvertebrateearlyǦlifestages 173 UniversidadedeVigoǦFacultadedeCienciasdoMar ౹FiguresandTables౹ 174 UniversidadedeVigoǦFacultadedeCienciasdoMar Figure 1.6 ʹͲ Ǥ ȂʹͲͳ͵Ǥ Figure1.7ǤǦ Paracentrotuslividus. Figure1.8.Ǧ ȋȌ ȋȌ Ǥ ȋ Ȁ Ȍ ȋ Ȍ Ǥ ȂʹͲͳ͵Ǥ ʹͲ Ǥ Figure 1.5. Ǧ fromƬȋʹͲͲʹȌǤ Figure 1.4 .Ǧ Ǥ Table1.3.Ǧ Ǥ Table1.2.Ǧ Ǥ Ǥ Ǥ ǣ Ǥǣ Figure 1.3Ǥ Ǧ ̺ Ǥ ǣ ǡ (2007). Figure1.2.Ȃ Ǥ Figure1.1ǤǦ Ǧͳͻ͑Ǥ Mazur et al. (1980)Ǥ CryopreservationofmarineinvertebrateearlyǦlifestages Page12 Page13 Page17 Page17 Page20 Page21 Page6 Page7 Page8 Page11 Page4 175 Table 1.1 Ȃ ǣȋʹȌǡ ȋ Ȍǡ ȋ ȌǡȋȌǡ ȋȌǡȋȌǤ Page35 Table 2.1.Ǧ Ǥ Ǥ UniversidadedeVigo ǦFacultadedeCienciasdoMar Table 2.4.Ǧ Ƥ Ǥ Table2.3.Ǧ P.lividus ͵Ͳ ǡ ʹǡ ǡ ǡ ǡ ȋ ȌǤ Figure. 2.4.Ǧ P. lividus ͵Ͳ ǡ ǤȋȌ ͲǤͺΪǢ ȋȌ ͳǤ͵ΪǢȋȌ ʹǤͲͶΪǢȋȌ ͲǤͺΪǢȋȌ ͳǤ͵ Ϊ Ǣ ȋȌ ʹǤͲͶ Ϊ Ǥ Figure. 2.3ǤǦ P. lividus ͵Ͳ ǡ ǡ ǤȋȌ ͳΪǢȋȌ ͳǤͷΪǢȋȌ ʹΪǢȋȌ ͳΪǢȋȌ ͳǤͷΪ ǢȋȌ ʹΪǤǤ Figure. 2.2.Ǧ P. lividus ͵Ͳ ǡ ǡ ʹǤȋȌʹͳΪ ǢȋȌʹͳǤͷΪǢȋȌʹͳΪǢȋȌʹͳǤͷΪ ǢȋȌʹʹΪǤǤ Table 2.2.Ǧ Ǥ Page50 Page43 Page42 Page40 Page39 Page38 Page37 Page24 Figure1.10ǤǦ ǤȋȌPernacanaliculusǢ ȋȌMytilusgalloprovincialisǢȋ ȌCrassostreagigasǤ Figure.2.1.Ǧ Ǥ Ǥ ȋȌ ͵Ͳ ʹ ͳͷƤǤȋȌ ͵Ͳ ʹ ͳͷ Ƥ Ǥ Ǥ Page22 Figure 1.9ǤǦ Ǧ Ǥ ǤǦ Ǥ ౹FiguresandTables౹ 176 UniversidadedeVigo ǦFacultadedeCienciasdoMar Figure. 2.8.Ǧ P. lividus Ǥ Ǧ͵ͷ ǦͺͲ Ǥ ǣͶ͑ʹǢ Ǧͳʹ͑ͳ͑ ǦͳǢʹǢ Ǧ͵ͷ͑ǦͺͲ͑ͳ͑ǦͳǢ ƤʹǢǤ ͵ͻͲρȋ Ǥ ǢδͲǤͲͷȌǤ Figure 2.7.Ǧ P. lividus Ǥ Ƥ ǡ Ǧ͵ͷ ǦͺͲ ͑Ǥ ǣͶʹǢ ͳʹ͑ͳ͑Ǧ ͳǢ ʹ Ǣ Ǧ͵ͷ Ǧ ͺͲ ͑ ͳ ͑ ǦͳǢ Ƥ ʹ Ǥ ͶͲͳ ρ ȋ Ǥ ǢδͲǤͲͷȌǤ Table 2.5.Ǧ Paracentrotuslividus ʹΪ ǦͺͲ͑ Figure. 2.6.Ǧ P. lividus Ǥ Ƥ Ǧ ͳǡʹǤͷ͑ǦͳǦͲǤͷ͑Ǧͳ ʹͲ͑ͳ͑Ǧͳ͵ͷ͑Ǥ ǣͶ ͑ʹǢ ͳʹ͑ͳ͑ǦͳǢʹǢ ͵ͷ͑ͳʹǤͷ͑Ǧͳǡ ʹͲ͑ͲǤͷ͑Ǧͳ͵ͷ͑ ͳ ͑ Ǧͳ Ǣ Ƥ ʹ Ǥ ͵ͻͺ ρ ȋ Ǥ Ǣδ ͲǤͲͷȌǤ Figure. 2.5.Ǧ P. lividus Ǥ Ͳǡ ʹ ͷ ͳʹ ͑Ǥ ǣͶ͑ʹǢ ͳʹ͑ͳ͑ǦͳǢ ͲǡʹͷǢ ͵ͷ͑ͳ͑ǦͳǢƤʹǤ ͵ͳ ρ ȋ Ǥ ǢδͲǤͲͷȌǤ CryopreservationofmarineinvertebrateearlyǦlifestages Page56 Page55 Page54 Page53 Page53 177 UniversidadedeVigoǦFacultadedeCienciasdoMar Figure. 2.9.Ǧ P. lividus ʹ Ϊ Ǥ Ǧ͵ͷ ͑ ǦͺͲ ͑ Ǥ ͶͺǡʹͻǤ ǣͶ͑ʹǢ ͳʹ͑ͳ͑ǦͳǢ ʹǢ Ǧ͵ͷǦͺͲ͑ͳ͑ǦͳǢƤʹ ǢǢ ͶͺǡʹͻǤ ͵ ρ ȋ Ǥ ǢδͲǤͲͷȌǤ Figure 2.10.Ǧ Ȁ ͶǦ ͻ Ǥ ȋȌ ǣ ȋȌ ȋ ȌǤ ȋȌ Ǥ ȋ Ȍ ǣ ʹ ͲǤʹͷ Ǥ Ǥ Figure2.11ǤǦ ȋǤǤȌ ÀȋǤȌ Figure 2.12. Ǧ ǤǤ ÀȋȌǤ Figure 2.13Ǧ ȋͳͻͻͲȌǤ ͵ Ȃ Ǧ Ǥ Ͷ Ȃ Ǥ ͷ Ȃ Ǥ Ȃ ǦǤ Figure 2.14ǤǦ ͶǦ Ȁ Ͷͺǡ ʹ ͻ ʹͲ͑Ǥ Ǥ Figure3.1Ǧ Ǥȋα͵ͷȌǤ ǡ Ǥ Table 3.1.Ǧ ȋ Ȍǡ ǡ Ǥ ȋΨȌ Ǧ Table3.2.ǦͷͲ ȋρǦͳȌͻͷΨ ȋ Ȍ ǡǦǡǦ͵ͶǦǡ Ǯǯǡ Ǯ ǯ Ǯ ǯ Ǧ Ǧ Ǥ ǣDz dzǤ ౹FiguresandTables౹ Page90 Page91 Page92 Page70 Page71 Page72 Page73 Page57 Page69 178 UniversidadedeVigoǦFacultadedeCienciasdoMar Figure3.5.Ǧ ǤǦ Ǥ ǣ Ǣ ǣ ȋα͵ͷȌǤ Ǥ Figure3.6.ǦʹͲ Ǥ ǣ Ǣ ǣ ǤǤ Figure3.7ǤǦ ǡ Ǥ ͳͲ ȋα Ͳ ȀȌǤ ǡ ȋδͲǤͲͷȌǤ Fig. 4.1.Ǧ Ǧ ̻ ȋͳ ͷ ͳȌǤͲȀͲ Ǥ ǤάǤ Fig.4.2ǤǦ Ȁ ǦǤ ͳͲΨ Ϊ Ͳǡʹ Ǥ Ǥ δͲǤͲͷǤάǤ Figure 3.2.Ǧ Ǧ͵ ͶǦǤ ȋα͵ͷȌǤ ǡ Ǥ Figure 3.3Ǧ ͷͲ ǤȋȌ ȋȌǡ ȋȌ ȋȌǡ ȋȌ ȋȌǤ Ǥ Figure 3.4.Ǧ ȋαͳͲͲȌǤ ǣ Ǣǣ Ǥ CryopreservationofmarineinvertebrateearlyǦlifestages Page105 Page106 Page107 Page123 Page124 Page104 Page93 Page94 179 UniversidadedeVigoǦFacultadedeCienciasdoMar Table4.1ǤǦͶͺ ȋ ǡ͵ͷȌǤ άǤ Figure4.3.Ǧ Ǧ ͲǤͷ ͑Ȁ ͳͲ ͳͷΨ ͵ Ǥ Ǥ δͲǤͲͷǤά Figure4.4.Ǧ ȋͲǤʹͷͲǤͷǢʹͶȌ Ǧ Ǧ Ǥ Ǥ δͲǤͲͷǤά Figure4.5ǤǦͳͻ ǤǦ ȋǤǤͲα ǦȌǤά Figure4.6.Ǧͳͻ Ǥά Figure 4.7.Ǧ Ǧ ȋȌ Ȁ ͳΨ Ǥ Ǥ Ǥά ȋȌǤ Figure 4.8.Ǧ Ǧ ȋȌǤ Ǥ άȋȌǤ Figure 4.9.Ǧ Ǧ ͲǤͷ͑Ǧͳͳ͑Ǧͳ ͳͲ ͳͷΨ ȋ Ȍ ͲǤʹ ȋȌ ͳΨ ȋȌǤ ǤάȋȌǤ Figure 4.10Ǧ Ǧ ͲǤͷ͑Ǧͳͳ͑ǦͳͳͲͳͷΨ ȋȌǤ Ǥ ά ȋȌǤ ౹FiguresandTables౹ Page125 Page126 Page127 Page128 Page129 Page139 Page140 Page141 Page141 180 Page155 Page156 Page157 Figure 5.2ǤǦ ǡ Ǥ Figure 5.3ǤǦ Ǥ Figure 5.4ǤǦ Crassostrea gigas Ǧ Ǥ UniversidadedeVigo ǦFacultadedeCienciasdoMar Page154 Page142 Figure 5.1.Ǧ (P. lividus) Ǥ ͻͲ ǡ Ǥ Figure 4.11Ǥ Ǧ ǣ Ǧ Ȁ Ͷͺǡ ʹ ͻ Ǥ ά ȋȌǤ ǣ Ǧ Ȁ Ͷͺ Ǥ CryopreservationofmarineinvertebrateearlyǦlifestages 181 UniversidadedeVigoǦFacultadedeCienciasdoMar ౹Resumen౹ 182 Resumen CryopreservationofmarineinvertebrateearlyǦlifestages Ǥ UniversidadedeVigoǦFacultadedeCienciasdoMar Ǥ ÓǤ ͳǤ͵Ǥ × ̺Ǥ ǣ ǡ ×Ǥ À À ÀǤ À ǡ ±ǡ Ǥ × À ǡ Àǡ ×ǡ À MytilusgaloprovinciallisPernacanaliculus. ParacentrotuslividusǡCrassostreagigas ×ǡ ǣ × Capítulo1 183 184 UniversidadedeVigo ǦFacultadedeCienciasdoMar Ǥ × Ȍ ǣ × ǡ ±Ǥ × × × Ǥ Ǥ ï × ± ± ± ǡ × ± ×ǡ Ȍ ×ǣ ×ǡ Ǥ × ï ×ǡ ± × ǡ À Ȍ À × ǣ × ×Ǥ À ǡ Ȍ ǣ À ï ×ǣ Ǥ × ǡ ± Ǥ × ×ǡ ± À Ó ǡ Fundamentosdecriobiología À Ǥ ×ǡ À À × ౹Resumen౹ UniversidadedeVigo ǦFacultadedeCienciasdoMar ×Ǥ × × × × À À Ǥï± × × ï × Acuiculturadeinvertebradosmarinos: × ǣ × Ǥ × À ǦǤ × × × ×± Óǡ ×ǡ × Ǥ À À × Ǥ À À Fundamentosdecotoxicología ×Ǥ ± Ȍ ǣ × Ǥ Ȍ ×ǣ × Ǥ × × Ȍ × × Àǣ × × À CryopreservationofmarineinvertebrateearlyǦlifestages 185 ౹Resumen౹ 186 ͳ ͲǤͺ ͲǤͲͷ ͲǤͷ ͳ Ǧ ͳ ʹ ʹǤͲͶ ʹǤͲͶ ʹΪ ʹΪ Ϊ Ϊ Ϊ Ϊ ʹǤͲͶ ʹǤͲͶ ͳ ʹ ͳ Ǧ ͲǤͷ ͲǤͲͷ ͲǤͺ ͳ ͳ ʹǤͲͶ ʹǤͲͶ ʹ ʹ ͳ Ǧ ͲǤͷ ͲǤͲͷ ͳǤ͵ ͳ ͳǤͷ Ǧ Ǧ ͳǤͷ Ǧ ͳǤͷ ͳ Ǧ Ǧ ͳǤ͵ ͳǤͷ ͳ Ǧ Ǧ ͳǤͷ Ǧ ͳǤͷ ͳ Ǧ Ǧ ͳǤ͵ ͳǤͷ ͳǤͷ ȋȌ Ǧ Ǧ Ǧ Ǧ ͳǤͷ ͳ Ǧ Ǧ ʹǤͲͶ ͳǤͷ ʹ ± ×ǡ ͲǤͷ ʹ ȋȌ UniversidadedeVigoǦFacultadedeCienciasdoMar ± × Ǥ dz À Dz ǣ ʹǤʹǤ ×Ǥ ×Ǥ dz × À Dz ȋParacentrotus lividusȌ Ǥ × ǡ × Capítulo2 E K D 'XUDWLRQ RI WKH ODUYDO UHDULQJ & K D & D 0H62 75( D K D CryopreservationofmarineinvertebrateearlyǦlifestages × UniversidadedeVigoǦFacultadedeCienciasdoMar Ǥ dz × ȋ Ȍ ǣ Ǥ À Dz ȌǤ± δͲǤͲͷȌǤ ×Ǥ Ó ͵ ρ ȋ × ʹΪǤ Ǧ͵ͷ͑ǦͺͲ͑À ǤʹǤͻǤ P.lividus ODUYDOJURZWK 187 ౹Resumen౹ 188 ʹ × Àʹǣ UniversidadedeVigoǦFacultadedeCienciasdoMar P.lividusǣ ʹͶ͑ǡ × ××ÀǤ ʹΪ ×ǦͺͲ͑ ǡ Ǥ Ǧ ×Ǥ Dzdz ʹ Dzdz Ǧͳʹ ͑ × × × Ǥ ×Dzdz ͳͷ × Ǥ × ×ǡ ǡ ȋ ǡ Ȍ ± ͵Ͳ Ǥ ǡ × Ͷͺǡ ʹǡ ͻ ʹͲ ͑Ǥ × ʹǤͷǤ Ͷ CryopreservationofmarineinvertebrateearlyǦlifestages UniversidadedeVigoǦFacultadedeCienciasdoMar × Ǥ ȋParacentrotuslividusȌdz À Dz ÓǤ × × ǡ × Ǥ À ×À Capítulo3 ×Ǥ ×ǡǡ × Ǥ À × À Ǥ Ǥ À × À × ͻǤ ǡ Ͷ ± ×Ǥ × Ȁǡ Ǥ × × ʹͷͲ ͷͲǡͲͲͲ À Ǥ ±ʹǦͺͲ͑ǡ × ʹǡ ×ͳ͑ǦͳǦͺͲ͑Ǥ ×ͳ͑ǦͳǦͳʹ͑Ǥ Dzdz 189 ౹Resumen౹ 190 UniversidadedeVigoǦFacultadedeCienciasdoMar × Ǥ ǡ ȋParacentrotus lividusȌ Ȃ dz × Ǥ À Dz ± × À × ï ± ǡ Ǥ ǡ × ȋα͵ͷȌǤ ͵ǤͳǤǦ × Ǥ CryopreservationofmarineinvertebrateearlyǦlifestages UniversidadedeVigoǦFacultadedeCienciasdoMar ͲΨ Ó ͻ Ǥ × ʹͻΨǤ Ó Ǥ ǡ × ͻ ȋ ͲǤȌǤ ± Ǥ × ͳǡ ǡ ǡ α α Ǥ Ǥ Ǧ͵ǡ δ α ͶǦ ǡ ǡ δ δ ǡ α δ ͷͲ Ǥ À͵ǣ Ǥ À Ǥ × ͶǤͳǤǦ ȋαͳͲͲȌǡÀ ± 191 ౹Resumen౹ 192 UniversidadedeVigoǦFacultadedeCienciasdoMar × ȋͲȂ ȌǤ × Ǥ ͶǤͷǤǦ ͳͻ × Ǥ × ǡ ±± × ǡ × ï Ǥ ǡ × × × Capítulo4 ÀͳͲʹǤΨͳͺÀǤͲͻΨǤ Ǥ ǡ ͺ À ͳ͵ Ͷ À ͺǡ À ͳͲǡ ± ÀǤ × ȋPerna canaliculusȌǡ CryopreservationofmarineinvertebrateearlyǦlifestages UniversidadedeVigoǦFacultadedeCienciasdoMar Ó À Ǥ × × Ó À ȋ ÓȌǤ × ǡ × ± ͳ ȋǦͳȌ ǡ ȋMytilus galloprovincialisȌdz × × ǣ ȋCrassostrea gigasȌ À Dz άȋȌǤ ȋȌǡ ȋȌǤ Ǥ ×ͲǤͷ͑Ǧͳͳ͑ǦͳͳͲǦͳͷΨ ȋ Ȍ × ͲǤʹ ͶǤ ͵ǤǦ × ± Dz ȋPernacanaliculusȌ dz × × À À ×Ȁ ×Ǥ±× × Ǥ × × 193 (* (* 75( (* 75( (* &PLQ ౹Resumen౹ (* 75( (* 75( &PLQ 194 × ÀͶǣ UniversidadedeVigoǦFacultadedeCienciasdoMar ͳͻʹǤͺΨǤ Ǥ ×ǡ × Perna canaliculus × ʹͲΨ Ó × Ǥ × ×ͲǤͷ͑Ǧͳ × Ǥ ×ǡ ǤͳͷΨʹͲΨ × ʹͲΨ ȋ Ȍ × ȋȌǤ ͶǤͶǤǦ ×ͲǤͷ͑Ǧͳͳ͑ǦͳͳͲǦͳͷΨ 1RUPDO'ODUYDH Crassostrea gigas Mytilus galloprovincialis CryopreservationofmarineinvertebrateearlyǦlifestages UniversidadedeVigoǦFacultadedeCienciasdoMar À Ǥ ͳͻ͑Ȍ × À Ǥ × À ȋǦ × ǡ ͵ǦͶ À Ǥ ǡ À ǡ ǡ Ǥ ǡ Ó ǡ × ǡ ǡ × À ǡ × ×Ǥ ǡ Ǥ × ïǡ Capítulo5 ×ͳͲǤ͵ΨͳǤͻΨ Ǥ Ó × × Ǧͳ ͳͲΨ ͲǤͷ͑Ǧͳ ͳͷΨ ΪͲǤͶǤ × ̱ ͷͲΨ ͲǤͷ ͑ Ǧͳ ͳ ͑ ×ǡ ǡ ͳͲΨ ͲǤͷ͑ǦͳǤ ͳͷʹͲΨǤͲΨ × 195 ౹Resumen౹ 196 Ǥ UniversidadedeVigoǦFacultadedeCienciasdoMar ͷͲ × Ǥ × × Ǥ Àǡ ×± Ǥ Ψǡï × × ǡ ʹΨ ǡ À × × × Ǥ ï × ×ǡ ʹǤͺΨǡ × × Ǥ × × × ±À Ǥ × ×Ȁ ×Ǥ ± ×Ȁ ǡ × × × ǡ± ±ǡ ± À× ÀǤ × ǡ × × × Objetivo 1ǣ × CryopreservationofmarineinvertebrateearlyǦlifestages Objetivo3ǣ UniversidadedeVigoǦFacultadedeCienciasdoMar ΨͳͺǦ Ǥ ͻǡʹΨʹͲÀ × ǡ ͷͲΨ Ǥ ǡ Ǥ × × × × × × ×Ǥ Objetivo2ǣǦ Ǥ ͳ ͑Ǥ ͳʹ ͳ ×ÀǤ × ×Ó ͳ͑ Ǧͳ ǦͺͲ ͑ ʹ × ×ͳ͑ǦͳǦͳʹ͑ȋʹȌǡ × × Ͷ͑ʹǡ± ǡʹͳǤͷΪͲǤͲͶÓͳͷͳǤ × × ×ͺʹͲ͑Ǥ ×Àȋ ÀʹȌǤ ×ǡǡ × ×ǡ × Ǥ × Conclusiones: 197 198 Objetivo 4: × ͵ ǡ ౹Resumen౹ UniversidadedeVigoǦFacultadedeCienciasdoMar ͶͺǤͻΨͶͺǤ Ͷͺ ×Ǥ × ͲΨ ʹǡͺͳͺǤ ×ȋPernacanaliculusȌǡ ͲΨͶǡ × ȋ ÀͶȌ Objetivo 5: ÀǤ ××Ó± ʹͺ͑Ǥ ͑ǡ ͷ × × × ͷͲ͑ × Ǧ͵ͷ ͳͷ × × × MytilusgalloprovinciallisȌǤ × PernacanaliculusʹͲ͑ǡͳͶǤCrassostreagigasʹʹ͑ʹͲǤʹͲ͑ ͷͲǦͲΨǤ × × ×ȋͳǦʹͲǤ ×Ǥ Ǧ × ȋ ÀͶȌ × × × UniversidadedeVigoǦFacultadedeCienciasdoMar PULICATIONSINSCIJOURNALS CryopreservationofmarineinvertebrateearlyǦlifestages 199 UniversidadedeVigoǦFacultadedeCienciasdoMar ౹PublicationsinSCIJournals౹ 200 focussed in field of applications UniversidadedeVigoǦFacultadedeCienciasdoMar ͳͲǤǦǡǤǡǤǡǤʹͲͳͶ ȋParacentrotuslividusȌȂ ǤȋȌ ͻǤǦǤǡǤʹͲͳͶ ȋParacentrotuslividusȌǤȋȌ ͺǤǦ Ǥ ǡ Ǥ ʹͲͳͶ ǣ Ǥ ȋȌ ǤǦ Ǥ ǡ Ǥ ǡ Ǥ ǡ Ǥ ǦÓǡ ǤǤ Ǥ ʹͲͳ͵ × ǣ Nannochloropsis gaditana Rhodomonaslens,Cylindrothecaclosterium,Chaetocerosgracilis,Synechoccocussp.eIsochrysisaff.galbana ǦǤǤ ǤǤ ǤǤǡ ǤǤǡǦ±ǤǡǤȋǤȌǣʹ͵Ǧʹ͵Ǥ ǤǦǤǡǡǤǤǡǤǡʹͲͳ͵ ȋǦ͵ǡǦͶǡͶǦǡȌ Isocrysisgalbana,Mytilusgalloprovincialis,Paracentrotuslividus,andSiriellaarmataǤ ͷǤǦ Ǥ ǡ Ǥ ʹͲͳ͵ ǡ Ǥ ǣ ͳͲǤͳͲͳȀǤ ǤʹͲͳ͵ǤͲͻǤͲͲ͵Ǥ ͶǤǦǤǢǤǢǤǤʹͲͳ͵ ǣ Ƥ ȋCrassostreagigasȌ(MytilusgalloprovinciallisȌǤ ͵ǤǦ ǡ Ǥ Ǣ Ǥ ǤǤǢ ǡ ǤǤǢ ǡǤǤǢ ǤǤ Ǣ ǤǤǡ ǤǤ ǡ Ǥ ʹͲͳʹǤ ȋPernacanaliculusȌ ǤͷʹͷǦʹʹǤ ʹǤǦǤǡǤǤʹͲͳͳ Ǧ ǣ Ǥ ʹȋ͵ȌͳͶǦͳͺͲǤ ͳǤǦ Ǥ ǡ Ǥ Ǥ ʹͲͲͻǤ ȋParacentrotus lividusȌ Ǥ ͷͻȋ͵Ȍ͵ͶͶȂ͵ͷͲǤ assessmentandaquaculture”2014. life stages: Applications in marine quality “Cryopreservation of marine invertebrate earlyǦ its multidisplinary and the UniversityofTennessee(USA), herPhDthesisis research stays in Cawthron Institute (NZ) and counting with seven months accumulated in participated in several research projects and Advance Studies Diploma in 2010. Having Finished her PhD master studies obtaining the specialization on Environment and Pollution. by Universidade de Vigo (Spain) with a cryobiology CryopreservationofmarineinvertebrateearlyǦlifestages Estefania Paredes has a degree in Oceanography 201 UniversidadedeVigoǦFacultadedeCienciasdoMar ǡ Ǥ ౹PublicationsinSCIJournals౹ 202