Viability assessment of primary growth oocytes following ovarian tissue vitrification of neotropical teleost pacu (Piaractus mesopotamicus)

Lis S Marques; Ana A Fossati; Martinha S Leal; Rômulo B Rodrigues; Robie A Bombardelli; Danilo P Streit Jr

doi:10.1016/j.cryobiol.2018.03.009

Viability assessment of primary growth oocytes following ovarian tissue vitrification of neotropical teleost pacu (Piaractus mesopotamicus)

Cryobiology. 2018 Jun:82:118-123. doi: 10.1016/j.cryobiol.2018.03.009. Epub 2018 Mar 26.

Authors

Lis S Marques¹, Ana A Fossati², Martinha S Leal², Rômulo B Rodrigues², Robie A Bombardelli³, Danilo P Streit Jr²

Affiliations

¹ Universidade Federal do Rio Grande do Sul (UFRGS), Programa de Pós-Graduação em Zootecnia, 91540-000, Porto Alegre, RS, Brazil. Electronic address: lis.marques@ufrgs.br.
² Universidade Federal do Rio Grande do Sul (UFRGS), Programa de Pós-Graduação em Zootecnia, 91540-000, Porto Alegre, RS, Brazil.
³ Universidade Estadual do Oeste do Paraná (UNIOESTE), Instituto de Pesquisa em Aqüicultura Ambiental (InPAA), 85900-030, Toledo, PR, Brazil.

PMID: 29596843
DOI: 10.1016/j.cryobiol.2018.03.009

Abstract

Vitrification of ovarian tissue containing immature oocytes provides an important tool for protecting the endangered species and genetic diversity in aquatic species. Therefore, the main objective was to assess primary growth (PG) oocytes viability following ovarian tissue vitrification using histological analysis, two staining protocols (trypan blue or fluorescein diacetate combined with propidium iodide) and mitochondrial activity assay (MTT assay). In addition, oocyte histomorphometry was performed to evaluate the morphometric parameters after vitrification and the relationship with the occurrence of damage (nucleus and/or membrane) in PG oocytes. There was no significant difference among the vitrified oocytes using trypan blue dye or FDA + IP staining. Oocyte viability assessed using histological analysis showed that vitrification solution 2.0 M Me₂SO + 2.5 M etilenoglycol +0.5 M sucrose (VS3; 66.43 ± 4.68%) and 1.5 M methanol + 5.5 M Me₂SO + 0.5 M sucrose (VS5; 74.14 ± 3.71%) had the lowest viability rate. Similar results were observed in MTT assay where VS3 (1.63 ± 0.12) and VS5 (1.58 ± 0.09) had the lowest averages when compare with VS1 (2.39 ± 0.14), VS2 (1.78 ± 0.06) and VS4 (2.34 ± 0.19) (P = 0.0002). In membrane damage evaluation by histology, there was no difference among vitrified oocytes and control. However, the highest percentages of nucleus damage were observed in treatments VS3 (26.00 ± 5.55) and VS5 (26.00 ± 5.55). Oocyte diameter did not change after vitrification; however, nucleus diameter was significantly higher in control group (49.03 ± 1.07). Oocyte viability by histological analysis was positive-correlated to the occurrence of nucleus (r² = 0.78) and membrane (r² = 0.45) damage after vitrification/warming. The high viability of PG oocytes obtained after ovarian tissue vitrification of Piaractus mesopotamicus suggests that the protocol applied here might be used successfully in other teleost species for food production.

Keywords: Histomorphometry; Immature oocytes; Ovarian follicle; Ovarian tissue cryopreservation.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Cell Membrane / physiology*
Cell Nucleus / physiology*
Cell Survival
Characiformes / embryology*
Cryopreservation / methods*
Female
Methanol / pharmacology
Mitochondria / metabolism
Oocytes / growth & development*
Ovary / physiology*
Sucrose / pharmacology
Vitrification*

Substances

Sucrose
Methanol