Improved preimplantation development of bovine ICSI embryos generated with spermatozoa pretreated with membrane-destabilizing agents lysolecithin and Triton X-100.
In cattle, intracytoplasmic sperm injection (ICSI) has a low efficiency. The acrosome content may be responsible for this effect because of the large amount of hydrolytic enzymes that are released within the oocyte. With the aim of removing the acrosome and destabilize the membranes, cryopreserved bovine spermatozoa were treated with lysolecithin (LL) and Triton X-100 (TX) at different concentrations. We evaluated the membrane integrity, the acrosome integrity, DNA integrity, and the variation of phospholipase C zeta. The rates of development (cleavage and blastocysts) were also evaluated along with pronuclear formation and the embryo quality. Spermatozoa incubated with LL and TX (0.01%, 0.02%, 0.03%, and 0.04%) decreased (P < 0.0001) sperm viability in a dose-dependent manner. The acrosome reaction was also increased (P < 0.0001) in all tested concentrations of LL and TX achieving 100% at 0.05% concentration in both treatments. Terminal deoxynucleotidyl transferase dUTP nick-end labeling assay reported an increase (P < 0.05) in DNA fragmentation only with the highest concentration of LL (0.06%), whereas all concentrations assessed of TX reported an increased respect to the control. Phospholipase C zeta expression decreased (P < 0.05) in spermatozoa treated with LL and TX at all concentrations tested. A higher cleavage rate was observed in ICSI-TX (66%) and ICSI-LL (65%) groups compared with the untreated control group (51%) and the blastocyst formation rate significantly increased in the ICSI-LL group (29%) compared with the control (21%). No differences were observed in the pronuclear formation and quality of the embryos. In conclusion, the destabilization of the plasma membrane and the release of the acrosomal content with LL and TX before ICSI improve the rate of embryonic development, without affecting the quality of the embryos produced by this technique.
Laboratory of Reproduction, Centre of Reproductive Biotechnology (CEBIOR-BIOREN), Universidad de La Frontera, Temuco, Chile.
Fabiola
F
Zambrano
Laboratory of Reproduction, Centre of Reproductive Biotechnology (CEBIOR-BIOREN), Universidad de La Frontera, Temuco, Chile.
Luis
L
Aguila
Laboratory of Reproduction, Centre of Reproductive Biotechnology (CEBIOR-BIOREN), Universidad de La Frontera, Temuco, Chile.
María E
ME
Arias
Laboratory of Reproduction, Centre of Reproductive Biotechnology (CEBIOR-BIOREN), Universidad de La Frontera, Temuco, Chile.
Raúl
R
Sánchez
Laboratory of Reproduction, Centre of Reproductive Biotechnology (CEBIOR-BIOREN), Universidad de La Frontera, Temuco, Chile; Department of Agricultural Sciences and Natural Resources, Faculty of Agriculture and Forestry Sciences, Universidad de La Frontera, Temuco, Chile. Electronic address: rfelmerd@gmail.com.
Ricardo
R
Felmer
D000177:Acrosome / Q000187:drug effects / Q000502:physiology; D020101:Acrosome Reaction / Q000187:drug effects; D000818:Animals; D002417:Cattle / Q000196:embryology*; D002462:Cell Membrane / Q000187:drug effects; D015925:Cryopreservation / Q000662:veterinary; D053938:DNA Fragmentation / Q000187:drug effects; D047108:Embryonic Development; D005260:Female; D006023:Glycoproteins / Q000494:pharmacology*; D020287:In Situ Nick-End Labeling; D008245:Lysophospholipase / Q000494:pharmacology*; D008297:Male; D017830:Octoxynol / Q000494:pharmacology*; D012662:Semen Preservation / Q000662:veterinary; D020554:Sperm Injections, Intracytoplasmic / Q000662:veterinary*; D013094:Spermatozoa / Q000187:drug effects* / Q000502:physiology / Q000648:ultrastructure; D010738:Type C Phospholipases / Q000032:analysis / Q000378:metabolism
D006023:Glycoproteins; D017830:Octoxynol; D008245:Lysophospholipase; C011711:lysolecithin acylhydrolase; D010738:Type C Phospholipases
Acrosome; Bovine spermatozoa; ICSI; Membrane disruption