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Journal of Assisted Reproduction and Genetics

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01-12-2014 | Fertility Preservation

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

Authors: A. Langbeen, E.P.A. Jorssen, N. Granata, E. Fransen, J.L.M.R. Leroy, P.E.J. Bols

Published in: Journal of Assisted Reproduction and Genetics | Issue 12/2014

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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Title: Effects of neutral red assisted viability assessment on the cryotolerance of isolated bovine preantral follicles
Authors: A. Langbeen
E.P.A. Jorssen
N. Granata
E. Fransen
J.L.M.R. Leroy
P.E.J. Bols
Publication date: 01-12-2014
Publisher: Springer US
Published in: Journal of Assisted Reproduction and Genetics / Issue 12/2014
Print ISSN: 1058-0468
Electronic ISSN: 1573-7330
DOI: https://doi.org/10.1007/s10815-014-0340-y

At a glance: The ONWARDS insulin icodec trials

Springer Medicine

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

Abstract

Purpose

Methods

Results

Conclusions

At a glance: The ONWARDS insulin icodec trials

Springer Medicine

Abstract

Purpose

Methods

Results

Conclusions

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