Flow Cytometric Evaluation of Mitochondrial Activity and Membrane Integrity in Fresh and Cryopreserved Rainbow Trout (Oncorhynchus mykiss) Spermatozoa

Baulny, B. Ogier de; Vern, Yves Le; Kerbœuf, Dominique; Maisse, Gérard

doi:10.1006/cryo.1996.1992

Cited by 119 publications

(56 citation statements)

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“…After double staining with Rh123 and PI, we found 74.8% of post-thaw sperm showed membrane integrity and mitochondrial function. In rainbow trout (Ogier de Baulny et al, 1997), the plasma membrane and mitochondrial function were better protected with 10% DMSO.…”

Section: Discussionmentioning

confidence: 95%

“…Samples were diluted and counterstained with 5 mg/mL of propidium iodide (PI, Sigma Chemical Co.). After 10 min, sperm samples were analyzed with flow cytometry (FACSvantage SE flow cytometer; Becton Dickinson, Mountain View, CA, USA) as previously described for trout sperm (Ogier de Baulny, et al, 1997). Sperm populations were identified according to their relative red and green fluorescence (staining with PI and Rh123, respectively).…”

Section: Rhodamine 123 Propidium Iodide and Flow Cytometrymentioning

confidence: 99%

See 1 more Smart Citation

Marine Fish Sperm Cryopreservation and Quality Evaluation in Sperm Structure and Function

Liu¹,

Xiao²,

Shao³

et al. 2012

Current Frontiers in Cryopreservation

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Section: Discussionmentioning

confidence: 95%

Section: Rhodamine 123 Propidium Iodide and Flow Cytometrymentioning

confidence: 99%

Marine Fish Sperm Cryopreservation and Quality Evaluation in Sperm Structure and Function

Liu¹,

Xiao²,

Shao³

et al. 2012

Current Frontiers in Cryopreservation

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“…Similarly, straight line and curvilinear velocities observed in fresh semen were higher compared to cryopreserved semen because spermatozoa were damaged by cryopreservation and thawing. Ogier de Baulny et al (1997) reported losses in seminal properties during cryopreservation, with only a small amount of viable sperm cells, thus reducing the fertilizing capacity of cryopreserved semen. Martinez and Pardo (2010) stated that, even when effectiveness is known for a given cryoprotectant in a fish species, the variation in its concentration could have toxic effects that can affect velocity.…”

Section: Discussionmentioning

confidence: 99%

Insemination of bocachico fish (Prochilodus magdalenae) with fresh or cryopreserved semen: effect of spermatozoa/oocyte ratio

García¹,

Espinosa²,

Martínez

et al. 2015

Rev Colomb Cienc Pecu

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Insemination of bocachico fish (Prochilodus magdalenae) with fresh or cryopreserved semen: effect of spermatozoa/oocyte ratio ¤ Inseminacion de bocachico (Prochilodus magdalenae) con semen fresco o crioconservado: efecto de la proporción espermatozoide/oocito Inseminação do bocachico (Prochilodus magdalenae) usando sêmen fresco o criopreservado: efeito da razão espermatozoide/oócitoVíctor J Atencio García 1* , MSc; José A Espinosa 1 , MSc; José G Martínez 2 , MSc; Sandra C Pardo Carrasco 3 , PhD. (Received: September 4, 2014; accepted: January 11, 2015) doi: 10.17533/udea.rccp.v28n4a07 Summary Background: cryopreservation is an important biotechnological tool in the conservation of biodiversity, particularly for endangered species. Objective: to evaluate six different spermatozoa/oocyte ratios using fresh and cryopreserved semen in bocachico fish (Prochilodus magdalenae). Methods: fresh semen was collected and its quality determined to verify cryopreservation feasibility. The semen was put in 5 mL straws and mixed with a solution (5.5% glucose, 12% egg yolk, and 10% dimethyl sulfoxide -DMSO-) in a 1:4 dilution (semen:solution). The semen was frozen in nitrogen vapor dry shipper for 30 min, rapidly transferred to storage thermos, and submerged directly into liquid nitrogen (LN;. Straws were thawed at 60 ºC for 45 seconds. Motility, velocity, and sperm progressivity of fresh and cryopreserved semen were assessed using Sperm Class Analyzer (SCA ® ) software. Each proportion of spermatozoa/oocyte was assessed with 2 g of eggs (1,630 ± 87 eggs/g) to evaluate fertility (F), hatching (H), and larval survival (LS) rates. Results: the best reproductive performance for fresh semen was obtained inseminating with 160,000 spermatozoa/oocyte (F = 75.0%, H = 67.7%, LS = 32.7%). Similarly, the best reproductive performance for cryopreserved semen was achieved with 320,000 spermatozoa/oocyte (F = 70.0%, H = 48.6%, LS = 19.5%). Conclusion: it is possible to achieve adequate reproductive performance in bocachico fish using cryopreserved sperm (10% DMSO, 5.5% glucose, and 12% egg yolk) at twice the spermatozoa/oocyte ratio used with fresh semen.Keywords: artificial reproduction, cryobiology, fertility, Prochilontidae. Atencio VJ et al. Fresh and cryopreserved semen in bocachico fishResumen Antecedentes: la crioconservación es una herramienta biotecnológica importante para la conservación de la biodiversidad, particularmente de especies en peligro. Objetivos: evaluar seis proporciones diferentes entre espermatozoides/ovocito en la fertilización de bocachico (Prochilodus magdalenae), usando semen fresco o crioconservado. Métodos: se colectó semen y se determinó su calidad para verificar su viabilidad de crioconservación. El semen fue colocado en pajillas de 5 mL y mezclado con una solución crioconservante (5,5% glucosa, 12% yema de huevo y 10% dimethyl sulfoxido -DMSO-) en una dilución 1:4 (semen:solución). El semen fue congelado en un termo de vapores de nitrógeno por 30 min y rápidamente se transfirió a termos de almacenami...

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“…Labbe et al (2001) reported a very low increase in the proportion of rainbow trout spermatozoa with altered DNA after cryopreservation compared to the 20 fold increase in the proportion of spermatozoa reported as having damaged membranes and mitochondria (DeBaulny et al 1997). …”

Section: Discussionmentioning

confidence: 97%

“…spermatozoa. Labbe et al (2001) reported a very low increase in the proportion of rainbow trout spermatozoa with altered DNA after cryopreservation compared to the 20 fold increase in the proportion of spermatozoa reported as having damaged membranes and mitochondria (DeBaulny et al 1997). Similarly, a marked decrease in motility and viability was observed in African clawed frog spermatozoa immediately post-thaw (Sargent and Mohun 2005;Mansour et al 2009).…”

Section: Fertilisation Success Following Cryopreservationmentioning

confidence: 97%

Amphibian sperm DNA fragmentation

Pollock¹

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There are extremely limited studies that have investigated DNA fragmentation in amphibians and even less information exists pertaining to DNA fragmentation in the spermatozoa.A DNA fragmentation assay applied and validated for amphibian spermatozoa would not only provide the first evidence of this phenomenon in a new major taxon, but also prove a valuable tool for developing improvements in ART used in the captive propagation of threatened and endangered species. Consequently, the fundamental aim of this project was the development and validation of the sperm chromatin dispersion (SCD) test for amphibian spermatozoa with the purpose of investigating the dynamic behaviour of amphibian sperm DNA in response to cryopreservation.The SCD test was first applied to African clawed frog (Xenopus laevis) as an amphibian sperm model using a species-specific modified lysing solution for protein depletion. Sperm DNA fragmentation (SDF) was assessed immediately following activation of sperm motility (T0) and again after one hour and 24 hours of incubation in order to produce a range of spermatozoa with differing levels of DNA damage. The SCD procedure resulted in the production of three nuclear morphotypes; amphibian sperm morphotype 1 (ASM-1) and ASM-2 showed no evidence of DNA damage, whereas ASM-3 spermatozoa were highly fragmented with large halos of dispersed DNA fragments and a reduced nuclear core. Levels of SDF revealed by the SCD test were highly correlated with results produced using in situ nick hybridisation with DNA-specific molecular probes (ISNT) and the double comet assay (r = 0.9613). The alkaline step of the double-comet assay revealed the extensive presence of structural single-stranded DNA breaks (SSB) and provided grounds for the suggestion that alkali labile sites (ALS) are a constitutive feature of African clawed frog sperm chromatin.SDF is a dynamic process and has been shown to increase with incubation at room temperature as well as following cryopreservation. However, the mechanisms of DNA cryoinjury and the resulting effect on the potential reproductive output of ART remain open to interpretation.Consequently, dynamic changes to the basal level of DNA fragmentation were examined in fresh African clawed frog spermatozoa in comparison with the post-thaw integrity of cryopreserved (-80 °C) and frozen (-20 °C) sperm DNA. SDF was examined initially at T0 and over incubation of up to 60 minutes. The difference in SDF was only marginal for fresh, frozen (-20 °C) and cryopreserved (-80 °C) spermatozoa examined at the onset of the experiment immediately upon thawing, however, a significant increase (p = 0.004) in SDF, albeit of a low magnitude, was observed in activated ii cryopreserved (-80 °C) spermatozoa following incubation. Although African clawed frog spermatozoa appeared to be highly resistant to cryopreservation-induced DNA damage, cryopreserved-thawed spermatozoa yielded a significantly lower (p = 0.0065) fertilisation rate than fresh spermatozoa.In order to provide further insight into cryo...

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Flow Cytometric Evaluation of Mitochondrial Activity and Membrane Integrity in Fresh and Cryopreserved Rainbow Trout (Oncorhynchus mykiss) Spermatozoa

Cited by 119 publications

References 25 publications

Marine Fish Sperm Cryopreservation and Quality Evaluation in Sperm Structure and Function

Marine Fish Sperm Cryopreservation and Quality Evaluation in Sperm Structure and Function

Insemination of bocachico fish (Prochilodus magdalenae) with fresh or cryopreserved semen: effect of spermatozoa/oocyte ratio

Amphibian sperm DNA fragmentation

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