High-throughput sperm cryopreservation of eastern oyster Crassostrea virginica

Yang, Huanming; Hu, Eric; Cuevas‐Uribe, Rafael; Supan, John; Tiersch, Terrence R.

doi:10.1016/j.aquaculture.2012.03.018

Cited by 28 publications

(32 citation statements)

References 15 publications

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“…Among marine invertebrates, the edible oysters are most widely studied [36], but 95% of the work has focused on broadcasting species [18]. The post-thaw sperm motility achieved in spermcasting species is low [21,39] compared to broadcasting species [13,41] probably because of the difference in their spermatological characteristics. The spermcasting species release clusters of spermatozeugma [19] whereas broadcasting species release individual sperm.…”

Section: Introductionmentioning

confidence: 99%

Improvement of post-thaw sperm survivals using liquid nitrogen vapor in a spermcasting oyster Ostrea angasi

Hassan

Qin

2017

Cryobiology

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Low survival of cryopreserved sperm impedes the application of cryopreservation technique in spermcasting oyster species. This study developed a simple method of liquid nitrogen vapor freezing to improve post-thaw sperm survival in the spermcasting oyster Ostrea angasi. The results indicate that the permeable cryoprotectants, dimethyl sulfoxide (DMSO), ethylene glycol (EG) and propylene glycol (PG) were non-toxic to sperm up to 20% concentration and 90 min exposure whereas methanol at 10% or higher was toxic to sperm for any exposure over 30 min. Among the treatments with permeable cryoprotectants, 15% EG produced the highest post-thaw sperm motility. Sperm motility was further improved by the addition of non-permeable cryoprotectants (trehalose and glucose), with 15% EG + 0.2 M trehalose resulting in the highest post-thaw sperm motility among all the combinations evaluated. The durations of 20, 30 and 60 min equilibrations produced a higher post-thaw sperm motility and plasma membrane integrity (PMI) than 10 min. Higher post-thaw motility and PMI were achieved by freezing sperm at the 8 cm height from the liquid nitrogen surface than at the 2, 4, 6, 10 or 12 cm height. Holding sperm for 10 min in liquid nitrogen vapor produced higher post-thaw motility and PMI than for 2, 5 or 20 min. The cryopreservation protocol developed in this study improved both post-thaw motility and PMI of O. angasi sperm at least 15% higher than those cryopreserved using programmable freezing method. Liquid nitrogen vapor freezing might have greater applicability in improving post-thaw sperm quality of spermcasting oyster species.

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Section: Introductionmentioning

confidence: 99%

Improvement of post-thaw sperm survivals using liquid nitrogen vapor in a spermcasting oyster Ostrea angasi

Hassan

Qin

2017

Cryobiology

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“…For flat oysters O. edulis (SUQUET et al, 2010) and O. chilensis (ALIPIA et al, 2014), complete anaesthesia was obtained after 3 h using 50-72 g L -1 and 30 g L -1 MgCl2, respectively. No visible effect of relaxation was shown in the pearl oyster P. albina (NORTON et al, 1996) after 1 h of induction using 30 g L -1 MgCl2, or in the American oyster Crassostrea virginica (YANG et al, 2013) after 36 h of treatment with 5% Dead Sea salt (containing 33.3% MgCl2).…”

Section: Discussionmentioning

confidence: 99%

Anesthesia in oysters of the genus Crassostrea cultured in Brazil

Puchnick‐Legat¹,

Legat²,

Gomes³

et al. 2015

Bol. Inst. Pesca

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This study evaluated: 1) the time required for anaesthesia induction and recovery of oysters Crassostrea rhizophorae, Crassostrea gasar and Crassostrea gigas using magnesium chloride (MgCl2); 2) the survival after anaesthesia and gonad sampling; and 3) the D-larvae generation after anaesthesia. For each species, three groups of 10 animals were kept in 50 g L -1 MgCl2, salinity of 36 and temperature of 22 ºC. One control group was kept in solution without MgCl2. Every 1 h anaesthetised oysters were recorded, sampled to determine sex and placed in clean seawater to assess recovering every 30 min. Gonad samplings were made beside the posterior adductor muscle, using 1 mL syringes and needles (0.60 x 25 mm). Factorial crosses were generated within and between anaesthetised and non-anaesthetised oyster groups to produce D-larvae in C. gigas. The survival after 10 days of anaesthesia was 100% for the three studied species. For C. rhizophorae and C. gigas, 100% of the animals were anaesthetised after 360 min and were recovered after 240 and 150 min, respectively. For C. gasar, 87% were anesthetised after 720 min and recovered after >240 min. There were no significant differences in D-larvae numbers between factorial crosses. The salt MgCl2 served as an efficient relaxant and caused no deleterious effect on the survival of the three studied species, or on the D-larvae generation in C. gigas.Keywords: anaesthetics; magnesium chloride; Crassostrea gasar; Crassostrea rhizophorae; Crassostrea gigas; D-larvae ANESTESIA EM OSTRAS DO GÊNERO Crassostrea CULTIVADAS NO BRASIL RESUMOEste estudo avaliou: 1) o tempo necessário para indução à anestesia e recuperação das ostras Crassostrea rhizophorae, Crassostrea gasar e Crassostrea gigas utilizando cloreto de magnésio (MgCl2); 2) a sobrevivência após anestesia e amostragem gonadal; e 3) a geração de larvas-D após anestesia. Para cada espécie, três grupos de 10 animais foram mantidos em 50 g L -1 de MgCl2, salinidade de 36 e temperatura de 22 ºC. Um grupo controle foi mantido em solução sem MgCl2. A cada 1 h, ostras anestesiadas foram registradas, amostradas para determinar sexo e colocadas em água do mar limpa para avaliar a recuperação a cada 30 min. Amostragens das gônadas foram feitas ao lado do músculo adutor posterior, usando seringas de 1 mL e agulha de 0,60 x 25 mm. Cruzamentos fatoriais foram gerados dentro e entre grupos de ostras anestesiadas e não anestesiadas para produzir larvas-D em C. gigas. A sobrevivência após 10 dias de anestesia foi de 100% para as três espécies. Para C. rhizophorae e C. gigas, 100% dos animais foram anestesiados após 360 min e recuperados após 240 e 150 min, respectivamente. Para C. gasar, 87% foram anestesiados após 720 min e recuperados após >240 min. Não houve diferença significativa no número de larvas-D entre os cruzamentos. O sal MgCl2 serviu como relaxante eficiente e não causou efeitos deletérios sobre a sobrevivência das três espécies estudadas ou na geração de larvas-D de C. gigas.

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“…Sperm cryopreservation was performed based on the protocol developed in 2010 (Yang et al . ). Briefly, sperm samples were mixed with an equal volume of 20% DMSO in Ca‐free HBSS650, and the mixture was packaged into 0.5‐mL French straws (IMV Technologies, Paris, France).…”

Section: Methodsmentioning

confidence: 97%

“…The extenders were artificial seawater or calcium-free Hanks' balanced salt solution (Ca-free HBSS) (Hanks 1975). In 2009-2010, we developed a reliable protocol for sperm cryopreservation of eastern oysters with potential for high-throughput application (for large-scale breeding programs) by systematic evaluation of cryoprotectants, cooling rates and thawing temperatures with post-thaw fertilization of 58% for 0.5-mL French straws and 54% for 0.5-mL CBS straws (Yang, Hu, Cuevas-Uribe, Supan, Guo & Tiersch 2012).…”

Section: Introductionmentioning

confidence: 99%

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Production of inbred larvae through self-fertilization using oocytes and cryopreserved sperm from the same individuals after sex reversal in eastern oysterCrassostrea virginica

Yang

Tiersch

2014

Aquac Res

Self Cite

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The eastern oyster Crassostrea virginica can change sex which makes self‐fertilization possible if sperm can be cryopreserved. In this study, small (~1 year old) and large (~2–3 years old) oysters were biopsied for sperm collection. Survival of the biopsied oysters after 1 year was 50% for small oysters and 17% for large oysters. Oocytes were collected from sex‐reversed females, and self‐fertilized with cryopreserved sperm. Of the 24 cryopreserved samples, 14 individuals had ≤1% fertility when crossed with oocytes from unrelated females, indicating that the cryopreserved sperm had reduced fertility. The other 10 individuals had a fertility of 39 ± 25% when crossed with oocytes from unrelated females (non‐selfing), but showed a significantly lower success of self‐fertilization (12 ± 16%) (P = 0.008), while aliquots of the same oocytes had a fertilization of 83 ± 11% when crossing with fresh sperm. Larvae were produced at day 3 in the self‐fertilized families (12–94% of the fertilized oocytes), and survived to eyed‐larvae stage at days 11–14. Genotyping with 9 microsatellite markers confirmed that the larvae resulted from self‐fertilization in four families. This study demonstrated the feasibility of creating self‐fertilized inbred lines of oysters by use of non‐lethal sperm collection and cryopreservation.

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High-throughput sperm cryopreservation of eastern oyster Crassostrea virginica

Cited by 28 publications

References 15 publications

Improvement of post-thaw sperm survivals using liquid nitrogen vapor in a spermcasting oyster Ostrea angasi

Improvement of post-thaw sperm survivals using liquid nitrogen vapor in a spermcasting oyster Ostrea angasi

Anesthesia in oysters of the genus Crassostrea cultured in Brazil

Production of inbred larvae through self-fertilization using oocytes and cryopreserved sperm from the same individuals after sex reversal in eastern oysterCrassostrea virginica

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