Embryo development after ICSI, using spermatozoa from bovine testicular tissue treated with three membrane-destabilizing agents

ABSTRACT Objective: To determine differences in embryo development of bovine oocytes fertilized by frozen/thawed spermatozoa (F/T), or by intracytoplasmic sperm injection (ICSI) using F/T or spermatozoa from fresh (FTT) or cryopreserved testicular tissue (CTT) using three spermatozoa membrane destabilizers. Methods. Treatment (TRT) 1- In vitro fertilization (FIV) with F/T, TRT-2 ICSI with F/T, TRT-3 ICSI with FTT, TRT-4 ICSI with CTT. The spermatozoa membranes were destabilized using Triton X-100 (TX), Lysolecithin (LL) or Heparin--Glutathione (Hep-GSH). Embryo cleavage at 48 h and grade 1 and 2 blastocyst on day 8 post fertilization were recorded. The comparison among main effect means were analyzed based on the least significant difference of Fisher. Results. At D8 there was no difference in percentage of blastocyst formation among ICSI TRTs (F/T 13 ± 3, FTT 6 ± 3 and CTT 6 ± 3 p>0.05), but they were lower than control (FIV 23 ± 5). With Hep-GSH destabilizer, there was a lower cleavage at 48 h than the LL and TX (35± 5, vs 50± 5 and 56± 5 p<0.05). Cleavage at 48 h was better for the ICSI with F/T and LL, while for D8, the best percentage to blastocyst was for TX. Conclusion. It is possible to produce blastocysts using ICSI with spermatozoa obtained from fresh or cryopreserved testicular tissue. Sperm cells treated with TX or LL produced more BL than those treated with Hep-GSH. More experiments using spermatozoa obtained from different sources are necessary to improve embryo development after ICSI. Keywords:  ICSI, Vitrification, Testicular tissue, Oocytes, Bovine, Fertilization.R Objective. To determine the differences in the embryo development of bovine oocytes fertilized with frozen/thawing (F/T) spermatozoa or with the intracytoplasmic sperm injection (ICSI) of F/T, spermatozoa from fresh testicular tissue (FTT), and cryopreserved testicular tissue (CTT), using three spermatozoa membrane-destabilizing agents. Methodology. Four treatments were used. Treatment (TRT-1): In vitro fertilization (IVF) with F/T. TRT-2: ICSI with F/T. TRT-3: ICSI with FTT. TRT-4: ICSI with CTT. The spermatozoa membranes were destabilized with Triton X-100 (TX), Lysolecithin (LL), and Heparin-Glutathione (Hep-GSH). Embryonic division was recorded at 48 h and grade 1 and 2 blastocysts (BL) were recorded 8 days (D8) after the fertilization. The means were compared using Fisher’s least significant difference method. Results. At D8, the blastocysts formation between ICSI treatments (F/T 13 ± 3, FTT 6 ± 3, and CTT 6 ± 3, p>0.05) were lower than control (IVF 23 ± 5). There was a lower cleavage at 48 h using Hep-GSH than when LL and TX were used (35 ± 5 vs 50± 5 and 56± 5, p<0.05). Embryo division at 48 h obtained better results with the ICSI + F/T and LL treatment, while the highest blastocyst percentage at D8 was obtained using TX. Conclusions. Blastocysts can be produced through ICSI, using spermatozoa from fresh or cryopreserved testicular tissue. The spermatozoa treated with TX and LL produced a higher percentage of BL than the spermatozoa treated with Hep-GSH. Further experiments should be carried out using spermatozoa obtained from different sources, in order to improve embryo development after the ICSI