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Effects of neutral red assisted viability assessment on the cryotolerance of isolated bovine preantral follicles

  • Fertility Preservation
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Journal of Assisted Reproduction and Genetics Aims and scope Submit manuscript

Abstract

Purpose

Fertility preservation strategies warrant non-invasive viability assessment of preantral follicles (PAF) such as staining with Neutral Red (NR) that is incorporated by viable follicles. To optimize the procedure, we firstly determined the lowest concentration and shortest exposure time needed for optimal viability screening of isolated bovine PAF. Secondly, we combined this protocol to a vitrification procedure to assess cryotolerance of the stained follicles.

Methods

Isolated PAF (900, divided over 6 replicates) were cultured in DMEM/Ham’s F12 (Culture Medium - Cm) for 4 days (38.5 °C, 5 % CO2). On D0, D2 and D4, follicles were stained, by adding NR medium (NRm = Cm with different concentrations NR) after which viability was assessed by counting stained/non-stained PAF every 30 min for a period of 2 h.

Results

Following a binary logistic regression analysis with staining as a result (yes/no) versus log-concentration, a probability model could be fitted, indicating that the proportion of stained follicles remained stable after 30 min when 15 μg/ml NR was used, without compromising follicular health and viability. Consequently, using this protocol, no significant effect of staining prior to vitrification, was found on PAF viability immediately after warming or following 4 days of culture.

Conclusions

In conclusion, we propose NR staining as a non-invasive, non-detrimental viability assessment tool for PAF, when applied at 15 μg/ml for 30 min, being perfectly compatible with PAF vitrification.

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Acknowledgments

The authors thank Silke Andries and Els Merckx for their excellent technical assistance and the local slaughterhouses for their cooperation in sample collection.

Conflict of interest

All (co-)authors state that the funding of this research is provided by the independent Operational Costs of the University of Antwerp. E. P. A. Jorssen acknowledges support from a Research Grant from the Belgian Government (Federale Overheidsdienst Volksgezondheid, Veiligheid van de Voedselketen en Leefmilieu, Cel Contractueel Onderzoek) ‘Embryoscreen RF6222’. There is nothing to disclose and there are no conflicts of interest, financially nor personal, for none of the (co-)authors.

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Authors and Affiliations

  1. Faculty of Pharmaceutical, Biomedical and Veterinary Sciences, Laboratory of Veterinary Physiology and Biochemistry, University of Antwerp, Universiteitsplein 1, Gebouw U, B-2610, Wilrijk, Belgium

    A. Langbeen, E.P.A. Jorssen, N. Granata, J.L.M.R. Leroy & P.E.J. Bols

  2. StatUa Center for Statistics, University of Antwerp, Prinsstraat 13, B-2000, Antwerp, Belgium

    E. Fransen

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  1. A. Langbeen
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  2. E.P.A. Jorssen
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  3. N. Granata
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  5. J.L.M.R. Leroy
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  6. P.E.J. Bols
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Correspondence to A. Langbeen.

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Capsule Applied at a concentration of 15 μg/ml for 30 minutes, Neutral Red is proposed as a non-invasive viability assessment tool for isolated bovine preantral follicles, without compromising follicle cryotolerance.

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Langbeen, A., Jorssen, E., Granata, N. et al. Effects of neutral red assisted viability assessment on the cryotolerance of isolated bovine preantral follicles. J Assist Reprod Genet 31, 1727–1736 (2014). https://doi.org/10.1007/s10815-014-0340-y

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