Identification of ultrastructural and functional damages in sperm from six-banded armadillos (Euphractus sexcinctus) due to cryopreservation

Abstract

RESUMO.-[Identificação de danos ultraestruturais e funcionais em espermatozoides de tatus-peba (Euphractus sexcinctus) oriundos da criopreservação.] O objetivo deste estudo foi criopreservar o sêmen de tatus-peba (Euphractus sexcinctus) em diluente Tris-gema e glicerol, e determinar os danos causados pelo processo de congelação-descongelação, utilizando marcadores fluorescentes e análise ultraestrutural. As amostras de sêmen (n=11) coletadas de 4 tatus-peba adultos por eletroejaculação foram criopreservadas em diluente Tris acrescido de 20% de gema de ovo e 3% de glicerol, em curva rápida de congelação. A análise clássica das amostras foi realizada após a diluição, refrigeração e descongelação, seguida por análise de fluorescência, utilizando uma combinação de sondas flu- The aim of the study was to cryopreserve the semen of six-banded armadillos (Euphractus sexcinctus) in Tris-yolk and glycerol diluent, and to determine the damage caused by the freezing-thawing process, using fluorescent markers and ultrastructural analysis. Semen samples (n=11) collected from 4 adult six-banded armadillos by electroejaculation were cryopreserved in Tris diluent plus 20% egg yolk and 3% glycerol, in a fast freezing curve. Classical analysis of samples was performed after dilution, refrigeration and thawing, followed by fluorescence analysis, using a combination of fluorescent probes to assess membrane integrity (propidium iodide -PI and Hoechst -H342), and mitochondrial activity (CMXRos -Mito Tracker Red ® ). We also used the ultrastructural analysis to verify possible morphological alterations caused by cryoinjuries. When compared with fresh samples, we verified a significant decline in all the armadillos' semen parameters after thawing, in which only 6.1% motile sperm were found. However, the percentage of sperm which remained with viable (13%) and functional (24.7%) membranes after thawing suggests that some cells could be live but immotile. Analysis using fluorescent markers revealed that the mitochondria of armadillos' sperm is highly sensible to the freezing protocol and the findings through ultrastructure analysis proved this statement. Additionally, the images obtained by transmission electron microscopy revealed that frozen-thawed sperm presented damaged plasma membrane, nuclear modifications as changes in chromatin and acrossomal changes relative to sperm capacitation. In conclusion, this study is the first attempt to cryopreserve the semen of an armadillo species, and to help us to identify critical points on the freezing-thawing procedure in order to improve the protocol. Além disso, as imagens obtidas por microscopia eletrônica de transmissão revelaram que espermatozoides congelados-descongelados apresentaram membranas plasmáticas danificadas, modificações nucleares como alterações na cromatina, e alterações acrossomais relativas à capacitação espermática. Em conclusão, este estudo é a primeira tentativa de criopreservação de sêmen em uma espécie de tatu, e nos auxiliou a identificar pontos críticos no processo de ...

Cryopreservation Effects on Ram Sperm Ultrastructure

“…However, sperm cryodamage involves cellular damage, including changes in the membranes, mitochondria, acrosomes, and axonemes. [1][2][3][4][5] Such cellular cryodamage adversely affects spermatozoa fertility by decreasing the viability of the cells.…”

Comparison of different extenders on the recovery and longevity of epididymal sperm from Spix's yellow-toothed cavies (Galea spixii Wagler, 1831)

“…In rodents, the use of fluorescent probes such as the PI (Varisli et al , 2013), CMXRos (Li et al , 2015) and Hoechst 342 stain to isolate samples (Yamashita et al , 2007) has been described in the literature. However, this time is the first in which the combined use of fluorophores has been reported for cavies, following a methodology initially described for bovines (Celeghini et al , 2010) and adapted for armadillos (Sousa et al , 2016) by our team. Such combination allows simultaneous evaluation of cavies’ sperm membrane integrity and mitochondrial activity, which declined with time in the samples using both extenders in a similar way.…”

Evaluación de la adición de quercetina y vitamina E al medio de criopreservación de semen ovino sobre la fertilidad in vivo

“…La membrana juega un papel importante en la viabilidad del espermatozoide y del éxito en la fertilización del ovocito, por lo que es importante que se encuentre intacta al momento del descongelamiento (Martínez-Pastor et al, 2004). El daño de la cromatina por lo general comienza en la región peri-acrosomal, las regiones basales del espermatozoide y posteriormente se expande a otras regiones del núcleo durante la criopreservación (Sousa et al, 2016), afecta considerablemente el estado del acrosoma o incluso la vitalidad del espermatozoide, lo que conlleva a una incapacidad para fertilizar. La baja fertilidad reportada en inseminación artificial cuando se utiliza semen criopreservado podría deberse a los daños ocasionados en los espermatozoides durante el proceso de criopreservación-descongelación (Sousa et al, 2016).…”

“…El daño de la cromatina por lo general comienza en la región peri-acrosomal, las regiones basales del espermatozoide y posteriormente se expande a otras regiones del núcleo durante la criopreservación (Sousa et al, 2016), afecta considerablemente el estado del acrosoma o incluso la vitalidad del espermatozoide, lo que conlleva a una incapacidad para fertilizar. La baja fertilidad reportada en inseminación artificial cuando se utiliza semen criopreservado podría deberse a los daños ocasionados en los espermatozoides durante el proceso de criopreservación-descongelación (Sousa et al, 2016). Por lo anterior, se ha propuesto la adición de antioxidantes en el semen para prevenir los daños producidos en los espermatozoides y mejorar la fertilidad del semen en la inseminación artificial.…”