Non-invasive assessment of porcine oocyte quality by supravital staining of cumulus–oocyte complexes with lissamine green B

Abstract

SummaryWe evaluated the usefulness of lissamine green B (LB) staining of cumulus-oocyte complexes (COC) as a non-invasive method of predicting maturational and developmental competence of slaughterhousederived porcine oocytes cultured in vitro. Cumulus cells of freshly aspirated COCs were evaluated either morphologically on the basis of thickness of cumulus cell layers, or stained with LB, which penetrates only non-viable cells. The extent of cumulus cell staining was taken as an inverse indicator of membrane integrity. The two methods of COC grading were then examined as predictors of nuclear maturation and development after parthenogenetic activation. In both cases LB staining proved a more reliable indicator than morphological assessment (P < 0.05). The relationship between LB staining and cumulus cell apoptosis was also examined. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay for DNA fragmentation revealed that oocytes within COCs graded as low quality by either LB staining or visual morphology showed significantly greater DNA fragmentation (P < 0.05) than higher grades, and that LB and visual grading were of similar predictive value. Expression of the stress response gene TP53 showed significantly higher expression in COCs graded as low quality by LB staining. However expression of the apoptosis-associated genes BAK and CASP3 was not significantly different between high or low grade COCs, suggesting that mRNA expression of BAK and CASP3 is not a reliable method of detecting apoptosis in porcine COCs. Evaluation of cumulus cell membrane integrity by lissamine green B staining thus provides a useful new tool to gain information about the maturational and developmental competence of porcine oocytes.

Characterization of porcine oocytes stained with Lissamine Green B and their developmental potential in vitro

“…In Dutta et al (2016) successful maturation rate was reached 79.9% in A grade group, which is very similar to our control group (74.03%). With porcine A grade oocytes (high quality oocytes after LB staining), the successful maturation rate reached 84.70%, which is almost identical to our LB-oocytes (82.24%).…”

“…Their study also observed a higher competence of C grade group (lower quality after LB staining) to reach MII (62.26%), which is significantly different as our result in LB+ group (22.80%). In Dutta et al (2016), very similar differences were observed after parthenogenetic activation too. Based on the results, we can say that the detection of membrane damage leads to a significant reduction in oocyte developmental potential, which is more important than the detection of damaged cumulus cells.…”

“…In comparison with the control group, we can conclude that the LB-group achieved a higher maturation rate (82.24%) than both the control group (74.03%) and LB+ group (22.80%). Our results can be compared with Dutta et al (2016), who focused on the evaluation of cumulus cells by Lissamine Green B.…”

“…Unstained (LB-) COCs with 0% stained cumulus cells were classified as high-quality COCs and stained (LB+) COCs with green ooplasm were designated as COCs with poor quality. In the LB+ group percentage of stained cumulus cells was not considered as an important marker ( Figure 1) (Dutta et al, 2016). Classification of porcine oocytes after Lissamine Green B staining.…”

“…Due this fact, Lissamine Green B (LB) could be very elegant solution of these negatives. Lissamine Green B is a non-toxic and synthetic stain composed of an organic acid dye with two aminophenyl groups (Kim et al, 2009) and was already used for the selection of porcine oocytes (Dutta et al, 2016). This vital staining can detect cell damage, which means that LB diffuses through the damaged membrane.…”