Improved embryo development using high cysteamine concentration during IVM and sperm co - culture with COCs previous to ICSI in bovine
In contrast to other species, intracytoplasmic sperm injection (ICSI) in bovine remains inefficient, resulting in low embryo developmental rates. It is unclear whether such inefficiency is due to the poor response of bovine ooplasms to the injection stimulus, or to the inability of bull sperm to induce oocyte activation. In order to facilitate these events, two strategies were assessed: the use of high concentration of cysteamine [Cys] during IVM; and the selection of sperm attached to cumulus cells after incubation with COCs for ICSI. First, COCs were IVM with increasing [Cys] and subjected to IVF. Zygotes from all groups were cultured under different O2 tensions and development to blastocyst was evaluated. In a second experiment, sperm were co-cultured for 3 h with COCs and acrosome reaction was studied. Afterwards, the best IVM and IVC conditions determined on Experiment 1 were used for ICSI assay. COCs were matured for 21 h with 1 (Cys 1) or 0.1mM Cys (Cys 0.1 groups, standard condition). In addition, COCs were incubated for greater than 3 h with 16 x 10(6) sperm/ml and only sperm attached to cumulus cells were selected for ICSI (ICSI þ Co-cult groups). After chemical activation, embryos were cultured in SOF medium under low O2 tension. Cleavage and blastocyst rates were evaluated at days 2 and 7 of IVC, respectively. Finally, the relative expression of eight genes indicators of embryo quality was compared between ICSI and IVF control blastocysts by qPCR. Cleavage rates were higher for Cys 0.1 ICSI þ Co-cult and Cys 1 ICSI þ Co-cult groups (n ¼ 117, 92% and n ¼ 116, 79%, respectively) compared to their controls (n ¼ 132, 60% for Cys 0.1 ICSI and n ¼ 108, 52% for Cys 1 ICSI) (p smaller than 0.05). Interestingly, the combined treatment (Cys 1 ICSI þ Co-cult) showed higher blastocyst rates than all other ICSI groups (23 vs. 11, 18 and 14% for Cys 0.1 ICSI þ Co-cult, Cys 1 ICSI, and Cys 0.1 ICSI, respectively) (p smaller than 0.05). Moreover, incubation with COCs increased the rates of live acrosome reacted sperm (p smaller than 0.05). The relative abundance of mRNAs coding for INFt, CAT, DNMT1, OCT4, and HDAC3 did not differ between treatments (p smaller than 0.05). SOD2, HADC1 and HADC2 expression was higher for Cys 0.1 ICSI than for IVF embryos (p smaller than 0.05). Group Cys 1 ICSI did not differ from IVF for those three genes, neither did Cys 1 ICSI þ Co-cult, except for HDAC1 (p smaller than 0.05). In conclusion, the use of 1mM Cys during IVM and of sperm incubated with mature COCs might be a good strategy to improve ICSI outcomes in cattle.
| Main Authors: | , , , , , |
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| Format: | Texto biblioteca |
| Language: | eng |
| Subjects: | SPERM INJECTION, CUMULUS CELLS, PRETREATMENT, IN VITRO MATURATION, OXYGEN TENSION, GENE EXPRESSION, |
| Online Access: | http://ceiba.agro.uba.ar/cgi-bin/koha/opac-detail.pl?biblionumber=46115 http://ceiba.agro.uba.ar/cgi-bin/koha/opac-detail.pl?biblionumber= http://ceiba.agro.uba.ar/cgi-bin/koha/opac-detail.pl?biblionumber= http://ceiba.agro.uba.ar/cgi-bin/koha/opac-detail.pl?biblionumber= |
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