Tauroursodeoxycholic acid enhances the development of porcine embryos derived from in vitro-matured oocytes and evaporatively dried spermatozoa

Abstract

Evaporative drying (ED) is an alternative technique for long-term preservation of mammalian sperm, which does not require liquid nitrogen or freeze-drying equipment, but offers advantages for storage and shipping at ambient temperature and low cost. However, the development of zygotes generated from these sperms was poor. Here, we demonstrated that the supplementation of tauroursodeoxycholic acid (TUDCA), an endogenous bile acid, during embryo culture improved the developmental competency of embryos derived from in vitro matured pig oocytes injected intracytoplasmically with boar ED spermatozoa by reducing the production of reactive oxygen species, the DNA degradation and fragmentation, and the expression of apoptosis-related gene Bax and Bak, and by increasing the transcription of anti-apoptosis gene Bcl-XL and Bcl-2. Furthermore, TUDCA treatment promoted the blastocyst quality manifested by the total cell numbers and the ratio of inner cell mass. Taken together, our data suggest that evaporative drying would be a potentially useful method for the routine preservation of boar sperm in combination with further optimization of subsequently embryo culture conditions.

Figure 1

Effect of TUDCA supplementation during embryo culture on the levels of intracellular reactive oxygen species (ROS) in porcine blastocysts. Representative fluorescence images of blastocysts from in vitro matured oocytes fertilized using ICSI with the conventional freeze-thaw (FT) boar spermatozoa without TUDCA-treatment (a), evaporatively dried boar spermatozoa without TUDCA-treatment (b) or evaporatively dried boar spermatozoa with 200 μM TUDCA-treatment (c). Bar: 100 μm. d. ROS contents were quantified as fluorescent intensity for three independent times. Each time 6–12 blastocysts were measured. Each bar represents the mean ± SD of the fluorescent density. Bars that do not share the same letter are significantly different at p < 0.05.

Figure 2

Relative transcript levels of pro-apoptotic gene of Bax and Bak and anti-apoptotic gene of Bcl-XL and Bcl-2 in blastocysts with or without TUDCA treatment. FT: Blastocysts developed from conventional freeze-thaw boar spermatozoa without TUDCA-treatment; ED: Blastocysts developed from evaporatively dried boar spermatozoa without TUDCA-treatment; ED+ TUDCA: Blastocysts developed from evaporatively dried boar spermatozoa with 200 μM TUDCA-treatment. mRNA levels of Bax and Bak in FT and ED+ TUDCA groups were normalized against those in ED group. GAPDH served as a loading control. Each bar represents the mean ± SD. All experiments were repeated for three times. Bars that do not share the same letter are significantly different at p < 0.05.

Figure 3

Effects of TUDCA addition during embryo culture on the quality of porcine blastocysts. (a) Representative fluorescence images of TUNEL staining. Blue: DNA; Red: apoptotic cell; Bar: 30 μm. The indices of apoptosis (b) and fragmentation (c) are expressed as mean ± SD for four times. Different low case letters above columns indicate statistical differences at p < 0.05 by one way ANOVA followed by LSD test. FT: Blastocysts developed from conventional freeze-thaw boar spermatozoa without TUDCA-treatment; ED: Blastocysts developed from evaporatively dried boar spermatozoa without TUDCA-treatment; ED+ TUDCA: Blastocysts developed from evaporatively dried boar spermatozoa with 200 μM TUDCA-treatment.