Beauvericin (BEA) is a mycotoxin that disturbs nuclear and cytoplasmic in vitro maturation (IVM) of prepubertal sheep oocytes [1]. The aim of this study was to examine long-term carry-over effects of BEA exposure during IVM on embryo cleavage and blastocyst quality. Cumulus-oocyte complexes recovered from the ovaries of slaughtered prepubertal sheep (<6 months) were cultured for IVM in presence of 0 (Control), 0.5, 1 and 3 M BEA [1,2]. After IVM, oocytes were subjected to in vitro fertilization and in vitro embryo culture [2]. Embryo developmental stage was assessed at day 8. Blastocysts were classified according to expansion and hatching status and their diameter was evaluated. After that, embryos were stained with Hoechst 33258 to evaluate number of nuclei and chromatin integrity, and with MitoTracker Orange CMTM Ros and 2′,7′-dichlorodihydrofluorescein diacetate to asses mitochondrial activity and ROS levels (expressed as the percentage of the signal of the control sample). Data were analyzed by the Chi-square test except for mitochondrial activity and ROS levels data compared by one-way ANOVA (significance at P<0.05). Data are presented for 0.5, 1, 3 μM BEA vs Control, respectively. A total of 523 oocytes were used (n.5 replicates). The result of percentages of embryos at the 16-31 cell after oocyte exposure to BEA were 16/134, 12%, 6/134, 4.5%, 6/128, 4.7% vs 15/127, 12% (P<0.05), and for morula- stage 4/134, 3%, 2/134, 1.5%, 2/128, 1.6% vs 9/127, 7% (P<0.05). From these results it is possible to underline that the exposure to 1 and 3 μM BEA decreased these rates. After exposure to BEA, the 2–3 cell stages were 8/134, 6%, 15/134, 11.2%, 15/128, 12% vs 3/127, 2.4% (P<0.01) but in this case 1 and 3 μM BEA increased the rate. No significant effects were found on blastocyst formation rates (3/134, 2%, 6/134, 4.5%, 4/128, 3% vs 4/127, 3%). Blastocysts derived from oocytes exposed to 1 and 3 μM BEA had not hatched after 8 days, in contrast, a few embryos obtained after exposure to 0 and 0.5 μM BEA did hatch (1/3, 0/6, 0/4, vs 1/4). Blastocyst diameter (131±34, 125±16, 125±13 vs 133±24 μm) and numbers of nuclei (79±15, 87±16, 87±9 vs 82±9) did not vary. Interestingly, increased percentages of embryos with more than 20% affected blastomeres with lobulated nuclei and/or micronuclei were observed after BEA exposure (1/3, 4/6, 3/4 vs 0/4). No effects on mitochondrial pattern and activity were detected (91±30, 103±36, 95±30 vs 100±21) whereas oocyte exposure to any BEA concentration reduced ROS generation ability (64±16, 87±23, 78±20 vs 100±23; P<0.01), possibly indicating viability loss. In conclusion: exposure to BEA during maturation of sheep oocytes compromised embryo development and blastocyst quality, possibly reducing fertility. [1] Mastrorocco et al., Proc. LXXII SISVet Congress, Torino 20-22 Giugno 2018; p203; [2] Martino et al. Reprod Toxicol 2016;65:204-211
THE MYCOTOXIN BEAUVERICIN IMPAIRS EMBRYO DEVELOPMENT AND BLASTOCYST QUALITY IN THE JUVENILE SHEEP
Antonella Mastrorocco;
2019-01-01
Abstract
Beauvericin (BEA) is a mycotoxin that disturbs nuclear and cytoplasmic in vitro maturation (IVM) of prepubertal sheep oocytes [1]. The aim of this study was to examine long-term carry-over effects of BEA exposure during IVM on embryo cleavage and blastocyst quality. Cumulus-oocyte complexes recovered from the ovaries of slaughtered prepubertal sheep (<6 months) were cultured for IVM in presence of 0 (Control), 0.5, 1 and 3 M BEA [1,2]. After IVM, oocytes were subjected to in vitro fertilization and in vitro embryo culture [2]. Embryo developmental stage was assessed at day 8. Blastocysts were classified according to expansion and hatching status and their diameter was evaluated. After that, embryos were stained with Hoechst 33258 to evaluate number of nuclei and chromatin integrity, and with MitoTracker Orange CMTM Ros and 2′,7′-dichlorodihydrofluorescein diacetate to asses mitochondrial activity and ROS levels (expressed as the percentage of the signal of the control sample). Data were analyzed by the Chi-square test except for mitochondrial activity and ROS levels data compared by one-way ANOVA (significance at P<0.05). Data are presented for 0.5, 1, 3 μM BEA vs Control, respectively. A total of 523 oocytes were used (n.5 replicates). The result of percentages of embryos at the 16-31 cell after oocyte exposure to BEA were 16/134, 12%, 6/134, 4.5%, 6/128, 4.7% vs 15/127, 12% (P<0.05), and for morula- stage 4/134, 3%, 2/134, 1.5%, 2/128, 1.6% vs 9/127, 7% (P<0.05). From these results it is possible to underline that the exposure to 1 and 3 μM BEA decreased these rates. After exposure to BEA, the 2–3 cell stages were 8/134, 6%, 15/134, 11.2%, 15/128, 12% vs 3/127, 2.4% (P<0.01) but in this case 1 and 3 μM BEA increased the rate. No significant effects were found on blastocyst formation rates (3/134, 2%, 6/134, 4.5%, 4/128, 3% vs 4/127, 3%). Blastocysts derived from oocytes exposed to 1 and 3 μM BEA had not hatched after 8 days, in contrast, a few embryos obtained after exposure to 0 and 0.5 μM BEA did hatch (1/3, 0/6, 0/4, vs 1/4). Blastocyst diameter (131±34, 125±16, 125±13 vs 133±24 μm) and numbers of nuclei (79±15, 87±16, 87±9 vs 82±9) did not vary. Interestingly, increased percentages of embryos with more than 20% affected blastomeres with lobulated nuclei and/or micronuclei were observed after BEA exposure (1/3, 4/6, 3/4 vs 0/4). No effects on mitochondrial pattern and activity were detected (91±30, 103±36, 95±30 vs 100±21) whereas oocyte exposure to any BEA concentration reduced ROS generation ability (64±16, 87±23, 78±20 vs 100±23; P<0.01), possibly indicating viability loss. In conclusion: exposure to BEA during maturation of sheep oocytes compromised embryo development and blastocyst quality, possibly reducing fertility. [1] Mastrorocco et al., Proc. LXXII SISVet Congress, Torino 20-22 Giugno 2018; p203; [2] Martino et al. Reprod Toxicol 2016;65:204-211I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
