Impact of embryo technologies on secondary sex ratio in rabbit
Introduction
In recent decades, assisted reproductive technologies (ARTs) have become consolidated as a combination of methods intended to overcome fertility difficulties in humans and enhance the genetic advancement in livestock [14,40]. However, we are increasingly aware that the application of these tools on gametes and embryos in the early stages of development introduces modifications in gene expression, which sometimes have phenotypic effects [16,38,39]. Moreover, there is increasing evidence reporting a skewed sex ratio after conventional ART procedures. The first evidence was published in bovine as early as 1991 [2], and this observation has been confirmed repeatedly in several species including mouse, porcine, bovine, rabbit and humans [7,10,19,24,36,[42], [43], [44],47,51]. Today, it is well known that the maternal environment seems to modify offspring sex-ratio (reviewed in Gardner et al. [21]). This fact is of great interest for the reproductive field, where the embryos are mandatorily exposed to non-standardised in vitro condition, such as new media, supplements, incubation conditions, etc. [53]. Interestingly, epidemiologic studies associate some ARTs, such as in vitro fertilisation and blastocyst transfer with higher proportions of the male sex, while other such as intracytoplasmic sperm injection has been linked with a skewed sex-ratio towards the female sex [10,31,36,42].
The underlying hypothesis is that developmental changes caused by embryo technologies are attributed to the unfavourable in vitro conditions that largely fail to reproduce the optimal in vivo conditions [38,39]. These adverse conditions might force embryos to undergone reprogramming to develop adaptive responses (developmental plasticity), but can also be a cause of disturbances in the dynamic epigenetic remodelling that takes place during preimplantation development [16,39]. However, male and female preimplantation embryos show remarkable differences in gene expression and epigenetic processes, exhibiting different proteome and metabolome and, ultimately differences in their cellular phenotype [5,21]. Such molecular and phenotypic differences may logically be involved in the reported differences in growth rates between male and female preimplantation embryos. Consequently, it is not surprising that disturbance of physiological signals during ART procedures will generate different effects in the two sexes, and supporting this, some ART-induced developmental deviations show a sex-dimorphic pattern [11,13,15,30,44].
Nowadays, cryopreservation of embryos has become a routine procedure in both veterinary and human medicine [27]. This technique involves exposing embryos to an environment with toxic chemical agents and shallow non-physiological temperatures, in which they have no intrinsic ability to survive [41]. However, in this field, offspring-derived data are relatively scarce because, although cryopreservation can be lethal to some embryos, for many years, it was considered to be neutral for survivors [1]. Therefore, to date, very few studies have reported the effects of embryo vitrification on offspring sex ratio, and the results are controversial and/or attributed to the embryo grading criteria used, instead of to the cryopreservation procedure per se [7,10,29,51]. In our recent study, we demonstrated that embryo vitrification skews secondary sex ratio in an adequately designed rabbit model [19]. But, it appears that embryo reprogramming may have an epigenetic basis, and may therefore have consequences for the subsequent offspring [8,48]. Nevertheless, the accumulative and long-term transmission of skewed sex-ratio after a MOET program together with the use of vitrification technique (MOVET) have not yet been studied. This study aims to test the hypothesis that a MOVET program could cause a skewed sex ratio and sex-dimorphic effect in the offspring.
Section snippets
Materials and methods
All chemicals, unless otherwise stated, were reagent-grade and purchased from Sigma-Aldrich Química S.A. (Alcobendas, Madrid, Spain).
Skewed SSR following MOVET program in favour of males
The overall SSR was imbalanced towards male sex in MOVET animals compared to those NC (0.59 ± 0.029 vs 0.42 ± 0.023, for the MOVET and NC animals, respectively; p < 0.05). No effect of generation (0.48 ± 0.030 vs 0.54 ± 0.038 vs 0.50 ± 0.028, for F1, F2 and F2c, respectively; p > 0.05) or interaction between experimental group and generation were detected (Table 1). As shown in Table 1, vitrified F1 generation increased SSR by 12% (0.54 ± 0.052 vs 0.42 ± 0.030, for the MOVET and NC animals,
Discussion
Our results indicate that a MOVET program skews the sex ratio in the rabbit, resulting in a higher proportion of males, according to our previous results [19]. This phenomenon was magnified after two consecutive MOVET programs, which can be attributed to a synergic effect of that inherited from the first MOVET program and that added by the second one. Besides, we found a dimorphic impact on the adult body weight after two consecutive MOVET programs. These findings matched those of previous
Conclusion
In summary, this work has demonstrated, for the first time in a rabbit MOVET program, involving MOET together with the use of vitrification technique, skewed offspring sex ratio and induced long-term effects that can be sex-dimorphic. Future studies should be conducted to understand the epigenetic mechanisms holding the reported findings.
Funding
This work was supported by the Ministry of Economy, Industry and Competitiveness (Research project: AGL2017-85162-C2-1-R) is acknowledged. X. Garcia-Dominguez was supported by a research grant from the Ministry of Economy, Industry and Competitiveness (BES-2015-072429). English text version was revised by N. Macowan English Language Service.
Additional information
The authors declare no competing financial interests.
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