Effects of Sex‐sorted Spermatozoa on the Efficiency of in vitro Fertilization and Ultrastructure of in vitro Produced Bovine Blastocysts

Abstract

Frozen-thawed sexed semen from six bulls (Holstein) was used for studying their efficiency in an in vitro fertilization (IVF)-programme and to compare their ultrastructure with in vitro produced bovine blastocysts produced with non-sorted sperm. Progressive motility of sorted spermatozoa, their IVF rate, development of produced blastocysts and the ultrastructure of the blastocysts were analysed. The cleavage rates of sexed sperm of bulls (groups S1, S2 and S4) were significantly lower than that of unsorted control sperm (P < 0.01). Blastocyst development at day 7 of the sexed semen groups varied between 3.5% and 28.8% versus 33.6% for non-sexed semen. The individual blastocyst yield with sexed semen of group S5 (28.8%) was similar to the mean blastocyst production of the non-sexed control spermatozoa (C, 33.6%; P > 0.05). The remaining five sexed sperm groups resulted in significantly lower developmental rates of blastocysts on day 7 (S1, 4.9%; S2, 0%; S3, 0%, S4, 3.5%; S6, 25.8%, P < 0.01). Group S2 showed microbiological contamination in 50% (four of eight) and S3 in 100% of the experiments (eight of eight). Progressive motility of sexed sperm was significantly lower than that of unsorted sperm (S1, 48 +/- 12.0%; S2, 41 +/- 11.9%; S3, 39.0 +/- 9.9%; S4, 42 +/- 4.6%; P < 0.01; S5, 72 +/- 7.1% and S6, 64 +/- 9.3; P < 0.05 versus C 82 +/- 4.6%). The percentage of progressive motile spermatozoa showed a good correlation with the developmental capacity of blastocysts (r(2): >0.70), the regression parameter was significant (P < 0.01). Furthermore, with a straw containing 10 x 10(6) sexed spermatozoa significantly lower number oocytes was fertilized than with the same concentration of non-sexed sperm (P < 0.01). Our results demonstrate that the suitability of sperm sorting for in vitro fertilization (IVF) is lower than no sexed sperm. Our ultrastructural studies showed that blastocysts produced with flow-cytometrically sex-sorted spermatozoa possessed deviations in the number and structure of organelles like mitochondria, rough endoplasmic reticulum (ER) and nuclear envelope. These morphological alterations may be responsible for compromised development that observed in embryos produced with sex-sorted spermatozoa. Thus, we conclude that sperm sex sorting can markedly affect the efficiency of an IVF-programme.

In vitro and in vivo quality of bovine embryos in vitro produced with sex-sorted sperm

“…It has been reported that IVF of oocytes with sexed sperm led to embryos with slower developmental kinetics, and blastocysts with ultrastructural alterations affecting numbers and structure of mitochondria, rough endoplasmic reticulum, and nuclear membrane [19]. Also, Morton et al [60] describe a reduced expression of developmentally important genes in embryos produced with sexed sperm.…”

“…However, after fertilization with sex-sorted spermatozoa, lower fertility rates have been observed both in vivo [9 -13] (after AI) and in vitro [14 -16]. Thus, in a recent large field trial of AI using sexed spermatozoa from seven Holstein bulls, Frijters et al [17] reported a 13.6% decline in the 56-day no return rate; many works also reported decreases in blastocysts rates after IVF of oocytes with sex-sorted sperm [18,19]. Proposed factors for the reduced fertility rates in vivo include not only the damage to the spermatozoa caused by the sorting procedure [11], but also the lower doses of spermatozoa used [9], and sire effects [20] (for review, see Rath et al [21]).…”

“…Such parameters have been considered as embryo quality indicators. Moreover, ultrastructural studies showed that blastocysts produced with flow-cytometrically sexsorted spermatozoa have a lower quality than their nonsorted counterparts [19]. Puglisi and coworkers [6] described a minor tolerance of the sexed frozen-thawed sperm to the stress, making the manipulation procedures important during IVF.…”

Sex selection of sperm in farm animals: status report and developmental prospects

“…In particular, the mitochondria, rough endoplasmic reticulum and nuclear envelope showed deviations (Palma et al 2008). Additionally, the authors showed a positive correlation of the percentage of progressively moving, sorted spermatozoa, and blastocyst development.…”

Comportamiento del Semen Bovino Sexado Congelado-Descongelado en Fertilización in vitro (FIV) Capacitado Mediante BO en dos Concentraciones versus Percoll

“…El Percoll es un medio muy costoso y a veces difícil de obtener, solo permitido su uso veterinario (Palma et al, 2008). La centrifugación en gradiente de Percoll permite que los espermatozoides se desplacen por el gradiente hasta que alcanzan un punto donde su densidad y la del gradiente son idénticas, por lo cual también se le llama centrifugación isopínica.…”