title: Morphological characterization and conservation of bovine spermatogenic cells by refrigeration at 4°C and freezing using different cryoprotective molecules.
abstract: The objectives of this study were study a practical method to characterize bovine spermatogenic cells and test the efficiency cells conservation by refrigeration at 4°C and cryopreservation in different solutions using two cooling curves. Cellular identification was performing by analysis of shape, size and morphology, associated with nucleus positioning and nuclear-cytoplasm ratio (NCR). Cellular samples were kept at 4°C for a period of 96 h in refrigeration solution and every 24h plasma membrane and DNA integrity were evaluated. Cryopreservation of cells was carried out using solutions containing 10% Dimethyl sulfoxide, 5% Dimethylformamide, 7% Glycerol and 7% Ethylene glycol, using a controlled and non-controlled cooling curve. Results of cellular characterization demonstrated that spermatocytes II presented a cylindrical shape, NCR of 1:1.5 and diameter ranging from 14.5 to 17.5 μm. Round spermatids presented diameter ranging from 7.6 to 13.4 μm, acrosomal cap and NCR of 1:2. Elongation and elongated spermatids showed to marked divergence in shape. There was a daily significant loss of viability of cooled cells until third day of storage, however they presented 72.77±5.16% viability after 4 days of storage at 4°C. There was no difference among the cryoprotectant solutions and cooling curves. In conclusion we demonstrated that association of microscopes and staining was a practical method to identify bovine spermatogenic cells. Furthermore, refrigeration at 4°C is an important strategy to preserve over 70% of viable cells after 4 days and cryopreservation, regardless of cryoprotectant solution or cooling curve used, can maintain over 50% of cells viable.
authors.affiliation: Laboratory of Reproduction, Center for Technology Transference of Zebu Dairy Cows-CTZL, Embrapa Cerrados, DF 180, Km 18, Brasília, Federal District, Brazil. Electronic address: carlos.martins@embrapa.br.
authors.forename: C F
authors.initials: CF
authors.lastname: Martins
authors.affiliation: Embrapa Genetic Resources and Biotechnology, Brasília, Federal District, Brazil. Electronic address: emidiofd@yahoo.com.
authors.forename: A E D Feliciano
authors.initials: AE
authors.lastname: Silva
authors.affiliation: Embrapa Genetic Resources and Biotechnology, Brasília, Federal District, Brazil. Electronic address: margot.dode@embrapa.br.
authors.forename: M N
authors.initials: MN
authors.lastname: Dode
authors.affiliation: Embrapa Genetic Resources and Biotechnology, Brasília, Federal District, Brazil. Electronic address: rodolfo.rumpf@embrapa.br.
authors.forename: R
authors.initials: R
authors.lastname: Rumpf
authors.affiliation: Laboratory of Reproduction, Center for Technology Transference of Zebu Dairy Cows-CTZL, Embrapa Cerrados, DF 180, Km 18, Brasília, Federal District, Brazil. Electronic address: heidi.cumpa@embrapa.br.
authors.forename: H C B
authors.initials: HC
authors.lastname: Cumpa
authors.affiliation: Laboratory of Reproduction, Center for Technology Transference of Zebu Dairy Cows-CTZL, Embrapa Cerrados, DF 180, Km 18, Brasília, Federal District, Brazil; University of Brasília, Brasília, Federal District, Brazil. Electronic address: carolgonzaless@gmail.com.
authors.forename: C G
authors.initials: CG
authors.lastname: Silva
authors.affiliation: University of Brasília, Brasília, Federal District, Brazil. Electronic address: pivato@unb.br.
authors.forename: I
authors.initials: I
authors.lastname: Pivato
mesh_terms: D000177:Acrosome / Q000502:physiology; D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000502:physiology; D048430:Cell Shape / Q000187:drug effects; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents / Q000494:pharmacology; D004247:DNA / Q000235:genetics; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D004126:Dimethylformamide / Q000494:pharmacology; D019855:Ethylene Glycol / Q000494:pharmacology; D005615:Freezing; D005990:Glycerol / Q000494:pharmacology; D008297:Male; D008853:Microscopy / Q000295:instrumentation / Q000379:methods; D012034:Refrigeration; D013091:Spermatogenesis; D013094:Spermatozoa / Q000166:cytology* / Q000187:drug effects; D013997:Time Factors
chemical_list: D003451:Cryoprotective Agents; D004126:Dimethylformamide; D004247:DNA; D019855:Ethylene Glycol; D005990:Glycerol; D004121:Dimethyl Sulfoxide
keywords: Assisted reproduction; Cryopreservation; ICSI; Male germplasm; Spermatids