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. 2023 Aug 14;13(16):2621.
doi: 10.3390/ani13162621.

Brilliant Cresyl Blue Negative Oocytes Show a Reduced Competence for Embryo Development after In Vitro Fertilisation with Sperm Exposed to Oxidative Stress

Lilli Bittner-Schwerda  1 , Carolina Herrera  1 , Sarah Wyck  1 , Eleni Malama  1 , Christine Wrenzycki  2 , Heinrich Bollwein  1
Affiliations

Brilliant Cresyl Blue Negative Oocytes Show a Reduced Competence for Embryo Development after In Vitro Fertilisation with Sperm Exposed to Oxidative Stress

Lilli Bittner-Schwerda et al. Animals (Basel). .

Abstract

The extent of oxidative damage transferred by the damaged sperm to the progeny is likely to be limited by the oocyte's repair and antioxidative capacity. We aimed to assess the association between Brilliant Cresyl Blue (BCB) staining in oocytes and their competence for embryo development after in vitro fertilisation (IVF) with damaged sperm. For this purpose, bovine sperm were incubated without (non-oxidised sperm, NOX S) or with 100 µM H2O2 (oxidised sperm, OX S) and were used to fertilise in-vitro-matured bovine oocytes (BCB-pos./BCB-neg.). Unstained oocytes served as controls (US). Development was assessed at 30, 46, 60 h and on Days (D) 7 and 8 after IVF. Total cell number and apoptotic index were analysed in D7 blastocysts. BCB-neg. oocytes showed lower cleavage rates and blastocyst rates than unstained oocytes after IVF with NOX S (p < 0.05). They showed the highest reduction in D7 blastocyst rate upon fertilisation with OX S and showed a delayed embryo development at 46 and 60 h after IVF compared to embryos produced with NOX S (p < 0.05). Total cell number in blastocysts produced with BCB-neg. oocytes was lower (p < 0.05) in the embryos produced with OX S than in embryos after IVF with NOX S. In conclusion, BCB-neg. oocytes have a lower competence to support embryo development after in vitro fertilisation with oxidised sperm.

Keywords: DNA damage; oocyte quality; sperm quality.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Bovine COCs after staining with Brilliant Cresyl Blue (BCB). (A) BCB-negative (neg.) oocytes were colourless. (B) BCB-positive (pos.) oocytes coloured blue.
Figure 2
Figure 2
(A) Cleavage rates (46 h after IVF) and blastocyst rates on Days (D) 7 and 8 after IVF and (B) Percentages of embryos at different cell stages (2-, 4-, 6/8-, >8-cell stage) at 46 and 60 h after IVF of unstained oocytes (US) and BCB-pos. oocytes “oocyte group +” and BCB-neg. oocytes “oocyte group –“, fertilised in vitro with non-oxidised sperm (NOX S). Values are means + SD. Different letters “a, b” indicate differences (p < 0.05) between oocyte groups fertilised with NOX S.
Figure 3
Figure 3
(A) Cleavage rates (46 h after IVF) and blastocyst rates on Days (D) 7 and 8 after IVF and (B) Percentages of embryos at different cell stages (2-, 4-, 6/8-, >8-cell stage) at 46 and 60 h after IVF of unstained oocytes (US) and BCB-pos. oocytes “oocyte group +” and BCB-neg. oocytes “oocyte group –“, fertilised in vitro with oxidised sperm (OX S). Values are means + SD. Different letters “a, b” indicate differences (p < 0.05) between oocyte groups fertilised with OX S.
Figure 4
Figure 4
(A) Overlay of Figure 2A and Figure 3A with cleavage rates (46 h after IVF) and blastocyst rates on Days (D) 7 and 8 after IVF; (B) Overlay of Figure 2B and Figure 3B with percentages of embryos at different cell stages (2-, 4-, 6/8-, >8-cell stage) at 46 and 60 h after IVF of unstained oocytes (US) and BCB-pos. oocytes “oocyte group +” and BCB-neg. oocytes “oocyte group –“, fertilised in vitro with non-oxidised sperm (NOX S) (white bars) and oxidised sperm (OX S) (black bars). Values are means + SD; the asterisk “*” indicates differences (p < 0.05) between NOX S and OX S within oocyte groups.
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