title: Sperm pretreatment with heparin and l-glutathione, sex-sorting, and double cryopreservation to improve intracytoplasmic sperm injection in bovine.
abstract: In bovine, intracytoplasmic sperm injection (ICSI) remains inefficient partially due to low levels of sperm decondensation. The aim of this study was to determine whether the injection of normal size sperm pretreated with heparin (Hep) and l-glutathione (GSH), the use of sex-sorted sperm, the double round of sperm freezing/thawing (re frozen), or the combination of these approaches can improve sperm decondensation and embryo development after ICSI in cattle. Cleavage and blastocyst rates were evaluated on days 2 and 7 post ICSI. Quality of ICSI blastocysts was analyzed by the relative expression of four genes by qPCR and the DNA fragmentation index by TUNEL assay. For all assays, semen samples were co-incubated with pCX-EGFP 50 ng/μl before ICSI. GFP expression, which can be detected by fluorescence microscopy, was used as a tool to estimate the success of sperm decondensation after ICSI. The use of normal size sperm pretreated with 80 μM Hep-15 mM GSH for 20 h (Hep-GSH) increased cleavage, blastocyst and EGFP + blastocysts rates (60.8, 19.4 and 61.9%) compared to control ICSI (35, 4.9 and 20%) (p < 0.05). Moreover, HMGN1, GLUT5, AQP3 and POU5F1 transcription levels did not differ between ICSI Hep-GSH and IVF embryos. The use of sex-sorted sperm (X, Y) improved cleavage rates and EGFP expression at day 4 (83 and 30.2% for ICSI Y and 83.2 and 31.7% for ICSI X) compared to non-sorted group (50.9 and 15.1%), not showing differences at the blastocyst stage. Finally, sex sorting (X) was combined with Hep-GSH and/or re frozen treatments. The use of Hep-GSH diminished cleavage rates from ICSI X re frozen group (80.4% vs. 94.2%) and blastocyst development from ICSI X group (3.3% vs. 10%), compared with their controls (p < 0.05). While Hep-GSH pretreatment induced lower transgene expression at day 4, no differences were found at the blastocyst stage between ICSI groups (from 58.3 to 80%). TUNEL assay showed higher DNA fragmentation indexes for all ICSI treatments (p < 0.05), except for ICSI X Hep-GSH, which did not differ from IVF X control. In conclusion, the use of normal size sperm pretreated with Hep-GSH, combined or not with sex-sorting by flow cytometry could improve ICSI outcomes in cattle.
authors.affiliation: Laboratorio de Biotecnología Animal, Facultad de Agronomía, Universidad de Buenos Aires-CONICET, Av. San Martín 4453, P.C. 1417, Buenos Aires, Argentina. Electronic address: ncanel@agro.uba.ar.
authors.forename: Natalia Gabriela
authors.initials: NG
authors.lastname: Canel
authors.affiliation: Laboratorio de Biotecnología Animal, Facultad de Agronomía, Universidad de Buenos Aires-CONICET, Av. San Martín 4453, P.C. 1417, Buenos Aires, Argentina. Electronic address: bevacqua@agro.uba.ar.
authors.forename: Romina Jimena
authors.initials: RJ
authors.lastname: Bevacqua
authors.affiliation: Laboratorio de Biotecnología Animal, Facultad de Agronomía, Universidad de Buenos Aires-CONICET, Av. San Martín 4453, P.C. 1417, Buenos Aires, Argentina. Electronic address: hiriart@iho-argentina.com.ar.
authors.forename: María Inés
authors.initials: MI
authors.lastname: Hiriart
authors.affiliation: Universidade Federal de Juiz de Fora, Embrapa Gado de Leite, Rua Eugenio do Nascimento 610, P.C. 36038-330, Minas Gerais, Brazil. Electronic address: natanarabelo.bio@gmail.com.
authors.forename: Natana Chaves
authors.initials: NC
authors.lastname: Rabelo
authors.affiliation: Universidade Federal de Juiz de Fora, Embrapa Gado de Leite, Rua Eugenio do Nascimento 610, P.C. 36038-330, Minas Gerais, Brazil. Electronic address: luiz.camargo@embrapa.br.
authors.forename: Luiz Sergio
authors.initials: LS
authors.lastname: de Almeida Camargo
authors.affiliation: Instituto de Biología y Medicina Experimental (IBYME)-CONICET, Vuelta de Obligado 2490, P.C. 1428, Buenos Aires, Argentina. Electronic address: marinaromanato@hotmail.com.
authors.forename: Marina
authors.initials: M
authors.lastname: Romanato
authors.affiliation: Instituto de Biología y Medicina Experimental (IBYME)-CONICET, Vuelta de Obligado 2490, P.C. 1428, Buenos Aires, Argentina. Electronic address: lucrepcalvo@gmail.com.
authors.forename: Lucrecia Piñeiro
authors.initials: LP
authors.lastname: de Calvo
authors.affiliation: Laboratorio de Biotecnología Animal, Facultad de Agronomía, Universidad de Buenos Aires-CONICET, Av. San Martín 4453, P.C. 1417, Buenos Aires, Argentina. Electronic address: salamone@agro.uba.ar.
authors.forename: Daniel Felipe
authors.initials: DF
authors.lastname: Salamone
mesh_terms: D000818:Animals; D001755:Blastocyst / Q000502:physiology; D002417:Cattle; D002469:Cell Separation / Q000379:methods / Q000662:veterinary*; D015925:Cryopreservation / Q000662:veterinary; D053938:DNA Fragmentation; D005260:Female; D005434:Flow Cytometry / Q000662:veterinary; D005978:Glutathione / Q000494:pharmacology*; D006493:Heparin / Q000494:pharmacology*; D020287:In Situ Nick-End Labeling; D008297:Male; D012662:Semen Preservation / Q000379:methods / Q000662:veterinary; D012732:Sex Determination Analysis; D020554:Sperm Injections, Intracytoplasmic / Q000662:veterinary*; D013094:Spermatozoa / Q000187:drug effects* / Q000502:physiology
chemical_list: D006493:Heparin; D005978:Glutathione
keywords: Cattle; ICSI; Sex-sorted sperm; Sperm pretreatment; pCX-EGFP