Porcine sperm vitrification I: cryoloops method

Abstract

The aims of this study were to evaluate porcine sperm vitrification in cryoloops, with and without two different cryoprotectants and assess two warming procedures. Extended (n = 3; r = 4) and raw (n = 5; r = 2) semen was diluted in media without and with cryoprotectants (4% dimethylformamide and 4% glycerol) to a final concentration of 20 × 10 spermatozoa ml and vitrified using the cryoloops method. Two warming procedures were evaluated: rapid method (30 s at 37°C) and an ultra-rapid method (7 s at 75°C, followed by 30 s at 37°C). Total motility (phase contrast), sperm viability (6-carboxifluorescein diacetate and propidium iodide stain), membrane function (hypo-osmotic swelling test), acrosome integrity (phase contrast), chromatin condensation (toluidine blue stain) and chromatin susceptibility to acid denaturation (acridine orange stain) were evaluated before and after vitrification and analysed using Friedman's test. In all media, the only seminal parameters that were maintained after vitrification were chromatin condensation and integrity. Vitrification of porcine spermatozoon using cryoloops, both in the presence or absence of cryoprotectants and independent of the warming procedure used, permits conservation of sperm chromatin condensation and integrity. It would be interesting to further verify this by producing porcine embryos using vitrified spermatozoon with intracytoplasmic sperm injection.

Evaluación de la calidad del ADN espermático en camélidos sudamericanos y otras especies domésticas

“…Esto permite, utilizar al DTT o al ME como controles positivos de la tinción con AT. Esta tinción se ha utilizado en varias especies para detectar defectos en la condensación de la cromatina (humano: Barrera et al, 1993;Erenpreiss et al, 2001;Erenpreisa et al, 2003;Tsarev et al, 2009;toro: Beletti y Mello, 1996;Beletti et al, 2005;conejo: Beletti y Mello, 2004;padrillo equino: Naves et al, 2004;Sardoy et al, 2008;Carretero et al, 2012a) y actualmente se ha empleado para evaluar la cromatina en caninos, felinos y porcinos (Monachesi y Carretero, 2014;Allera et al, 2016;Arraztoa et al, 2016).…”

“…La concentración del colorante utilizado en la tinción varia entre las especies, sin embargo, en la llama, alpaca, guanaco, equino, felino, canino y porcino se observaron los mismos patrones de coloración a pesar de la diferente concentración de AT: coloración celeste (negativos, sin alteración en la condensación normal de la cromatina), violeta claro (intermedios, algún grado de descondensación) y violeta-azul oscuro (positivos, alto grado de descondensación) (Sardoy et al, 2008;Carretero et al, 2009;2010a, 2010b2012a;Monachesi y Carretero, 2014;Allera et al, 2016;Arraztoa et al, 2016). Los patrones AT positivos y AT intermedios son considerados espermatozoides con alteración en la condensación de la cromatina.…”

“…Por otra parte, distintos autores han utilizado diferentes soluciones para fijar los extendidos: metanol (Barrera et al, 1993), etanol-ácido acético (3:1) más etanol 70% (Beletti y Mello, 2004;Beletti et al, 2005), etanolacetona (1:1) (Gagnon 1999;Erenpreiss et al, 2001;Erenpreisa et al, 2003;Tsarev et al, 2009) pero muchas de estas sustancias pueden ser altamente tóxicas si son inhaladas durante su manipulación (Macedo y Nava Muñoz, 2000). En CSA, equino, felino, canino y porcino, la fijación simple con etanol 96° durante 2 minutos fue efectiva (Sardoy et al, 2008;Carretero et al, 2009;2010a, 2010b2012a;Monachesi y Carretero, 2014;Allera et al, 2016;Arraztoa et al, 2016), dado que se encontraron patrones de coloración similares a los observados en otras especies (Barrera et al, 1993;Gagnon 1999;Erenpreiss et al, 2001;Erenpreisa et al, 2003;Beletti y Mello, 2004;Beletti et al, 2005;Tsarev et al, 2009). Todas las características mencionadas hacen que el AT sea una técnica sencilla de realizar, económica y rápida para determinar el grado de condensación de la cromatina de los espermatozoides.…”

Dog sperm DNA: Raw semen evaluation with Toluidine blue stain

“…Many of these techniques require complex instruments that often are not available in animal reproduction centres. TB is simple and inexpensive and has been used to evaluate the degree of chromatin condensation in various species (humans: Barrera, Mazolli, Pelling, & Stockert, ; Erenpreiss, Bars, Lipatnikova, Erenpreisa, & Zalkalns, ; Erenpreiss et al, ; Erenpreisa et al, ; Tsarev et al, ; bulls: Beletti, Fontoura Costa, & Mendes Guardieiro, ; Beletti & Mello, ; Vieytes, Cisale, & Ferrari, ; Dogan et al, ; rabbits: Beletti & Mello, ; stallions: Carretero, Arraztoa, et al, ; Naves et al, ; Sardoy, Carretero, & Neild, ; South American Camelids: Carretero, Arraztoa, et al, ; Carretero, Giuliano, et al, ; Carretero, Giuliano, Casaretto, Gambarotta, & Neild, , pigs: Arraztoa et al, ; González et al, , and cats: Allera, Comercio, Gonzales Vera, Miragaya, & Carretero, ). To increase the sensitivity of the technique, many of the above‐mentioned studies used acid hydrolysis prior to staining with TB (Beletti et al, ; Beletti & Mello, ; Erenpreisa et al, ; Erenpreiss et al, , ; Tsarev et al, ); however, this increases the length and the cost of the technique.…”

“…To increase the sensitivity of the technique, many of the above‐mentioned studies used acid hydrolysis prior to staining with TB (Beletti et al, ; Beletti & Mello, ; Erenpreisa et al, ; Erenpreiss et al, , ; Tsarev et al, ); however, this increases the length and the cost of the technique. In dogs, there is only one report of using hydrolysis prior to staining (Flores et al, ), and no study has reported TB staining canine sperm without prior hydrolysis as has been described in other species (Allera et al, ; Arraztoa et al, ; Carretero, Arraztoa, et al, ,; Carretero, Giuliano, et al, ; Carretero et al, ; Sardoy et al, ). For this reason, the objectives of this study were as follows: (a) simplify the TB technique for evaluating chromatin in canine raw semen, (b) verify the staining patterns for this species using this simplified technique and (c) establish a protocol for using dithiothreitol (DTT) as a positive control for TB staining in dogs.…”