Isolate^® and Optiprep^® minigradients as alternatives for sperm selection in bovine in vitro embryo production

Abstract

. 2014. Isolate † and Optiprep † minigradients as alternatives for sperm selection in bovine in vitro embryo production. Can. J. Anim. Sci. 94: 35Á42. The objective of this study was to evaluate alternatives in small volumes to conventional gradient of Percoll † on semen quality, in vitro embryo production, sex ratio and embryo survival after vitrification. Thawed semen was randomly allocated to one of four density gradient selection methods: (1) conventional Percoll † (P), (2) MiniPercoll (MP), (3) MiniIsolate (MI), and (4) MiniOptiprep (MO). Sperm kinetics and quality were evaluated. Use of P, MP and MI gradients did not affect sperm motility (P!0.05). However, there was a decrease in total and progressive sperm motility in MO (70.8 and 51.3% vs. 87.3 and 69.5% for P; 87.3 and 73% for MP; 92.3 and 78.8% for MI; PB0.05). The MO had lower membrane integrity compared with P, MP and MI (39.7 vs. 70.5, 72.3, 63.8%, respectively, PB0.05). The percentage of blastocysts produced was higher in MI than in MP and MO (21.1 vs. 16.1 and 16.9%, PB0.05) and similar to P (18.4%; P!0.05). Sex ratio and embryo survival after vitrification were similar among groups (P!0.05). Semen selected by Isolate and Optiprep gradient, at the concentrations and small volumes used, demonstrated similar characteristics and in vitro embryo production to conventional Percoll † gradient. † , en petits volumes, sur la qualite´de la semence, la production in vitro d'embryons, le rapport des sexes et la survie de l'embryon apre`s vitrification. Des semences de´congele´es ont e´te´attribue´es ale´atoirement a`une de quatre me´thodes de se´lection par gradient de densite´: (1) Percoll † traditionnel (P), (2) MiniPercoll (MP), (3) MiniIsolate (MI), et (4) MiniOptiprep (MO). La cine´tique et la qualite´des spermatozoı¨des ont e´te´e´value´es. L'utilisation des gradients P, MP et MI n'ont eu aucun effet sur la motilite´des spermatozoı¨des (P!0,05). Il y avait, cependant, une diminution des motilite´s totales et progressives dans le gradient MO (70,8 et 51,3 % c. 87,3 et 69,5 % pour P; 87,3 et 73 % pour MP; 92,3 et 78,8 % pour MI; PB0,05). Le gradient MO avait une inte´grite´de membrane infe´rieure aux gradients P, MP et MI (39,7 c. 70,5; 72,3; 63,8 % respectivement, PB0,05). Le pourcentage de blastocystes produits e´tait plus e´leve´apre`s gradient MI qu'apre`s les gradients MP et MO (21,1 c. 16,1 et 16,9 %, P B0,05) et similaire apre`s gradient P (18,4 %; P!0,05). Le rapport des sexes et la survie de l'embryon apre`s vitrification e´taient similaires parmi tous les groupes (P !0,05). Les semences choisies par gradients Isolate et Optiprep, aux concentrations et aux petits volumes utilise´s, de´montrent des caracte´ristiques et une production in vitro d'embryons similaires a`celles choisies par gradients Percoll † traditionnels.

Gradient sperm selection for reproductive techniques in cattle: Is Isolate a suitable replacement for Percoll?

“…Our results agree with those of Vianna et al. (), who investigated the effect of Percoll ® and Isolate ® gradients and observed similar motility parameters between both methods. The principle of separation of both gradients relies on the different buoyant densities of spermatozoa.…”

“…The principle of separation of both gradients relies on the different buoyant densities of spermatozoa. However, Isolate ® gradient is used more often in human reproductive techniques, owing to its lower cell and embryo toxicity (Mousset‐Siméon, Rives, Masse, Chevallier, & Mace, ), and has been reported to influence on sex ratio (Vianna et al., ). The densities used here were similar to both separation system (discontinuous gradient of 45% upon 90%); therefore, we hoped to get similar sperm qualities post‐selection, because the immotile and/or damaged spermatozoa, without sufficient motility cannot be transferred to the final pellet where the spermatozoa with greatest motility are collected (Guimarães et al., ).…”

“…On the other hand, Isolate ® is a colloidal suspension of silica particles stabilised with covalently bound hydrophilic silane in HEPES‐buffered HTF medium, which has been filtered through a membrane and processed under aseptic conditions. A small number of studies have used Isolate ® as sperm selection method for IVF in cattle (Rodríguez‐Villamil et al., ; Vianna et al., ), and therefore, the effect of this compound on spermatozoa has not been extensively evaluated. Due to the major role of the sperm selection method in ARTs and the need to develop alternative methods with lower embryo toxicity in ARTs in cattle, this study set out to compare the effect of Isolate ® and Percoll ® sperm selection methods on different sperm quality and functional parameters including the assessment on the in vitro embryo development.…”

Bovine epididymal spermatozoa treatment for in vitro fertilization: Heparin accelerates fertilization and enables a reduction in coincubation time

“…The same results were not observed for the EJ that were selected using P and for EP that was only subjected to washing, since in both groups no difference from the expected 1:1 ratio was noted. These findings do not corroborate with that from other studies conducted by Martins et al [3] and Vianna et al [16] using EJ sperm, in which different discontinuous gradients have not affected the embryo sex ratio. One possibility for the contradictory results could be the effect of the sires, since effect of the bull on sex ratio has been demonstrated in several studies [22, 30, 31].…”

“…In fact, studies in pigs have shown that methods of processing spermatozoa can affect the in vitro fertilization parameters [13]. The various steps involved in the IVF, such as the sperm selection process and its influence on quality and embryo sex [16, 17, 18], the presence and concentration of heparin in fertilization media [11, 15, 17] and the time necessary for sperm-oocyte coincubation [19], are well established for EJ sperm. However, in regard to the use of EP, little is known regarding the changes that the sperm experience after being exposed to the different treatments and in vitro conditions.…”