59 Delivery of exogenous sperm microRNAs increases cleavage rates and changes gene expression in embryos, leading to an increment on blastocyst and development rates in low in vitro-production fertility bulls

Bulls may present different in vitro fertility, which represents financial losses and delays in breeding programs. Sperm RNAs play roles in early embryo development by the action of noncoding RNAs, such as microRNAs, which could influence bull fertility. Recently, four microRNAs (A, B, C, and D) were identified exclusively in sperm from high in-vitro-fertility bulls (Hamilton et al. 2021 Reprod. Fertil. Dev. 33). We hypothesised that these microRNAs could improve in vitro embryo production (IVP) of low-fertility bulls. We performed two functional experiments with six Nellore bulls, three with high and three with low IVP fertility, retrospectively selected from commercial IVP manipulations (n = 7,000). In the first experiment (called “rescue”), we performed IVP using the bulls with low fertility; 18 h after fertilisation, zygotes were microinjected with 5–7ρl mimics of the four miRNAs ([mimic group] 100 nM, miRCuryLNA mimic^®, miScript mimic^®, Qiagen). In the second experiment, called “proof of principle,” high IVP fertility bulls were used, and zygotes were microinjected with inhibitors of the same four miRNAs (inhibitor group). For control group, zygotes were microinjected with negative control mimic or inhibitor molecules (scramble groups). After microinjection, zygotes were in vitro cultured. We performed three replicates per bull with 30 oocytes microinjected by experimental group (total of 90 oocytes/bull/group). Cleavage rate (cleaved structures ÷ total oocytes) and gene expression (q-RT-PCR) of miRNA target transcripts (TGB1, CDKN1, HDAC1, PTEN, BCL6, and IRF1) in 2–4 cell embryos (30 embryos/bull/mimic or inhibitor and scramble group) were evaluated. For blastocyst rate and embryo development, three replicates/bull/experimental group were performed with a total of 90 microinjected oocytes/bull/experimental group. SAS System 9.3 was used to evaluate IVP rates by GLM procedure and qPCR data by mixed procedure (Steibel et al. 2009 Genomics 94,146–153). In the rescue experiment, cleavage rate was increased in the mimic group compared to the scramble group (68.30 ± 2.65 vs 54.23 ± 3.69; P < 0.0001). A lower expression of HDAC1 was observed in 2–4 cell embryos in mimic compared to scramble group. Moreover, blastocyst (27.56 ± 4.84 vs 17.18 ± 1.89; P = 0.03) and development (44.21 ± 5.93 vs 32.03 ± 2.64; P = 0.001) rates were increased in mimic group compared to scramble group. No differences were observed in proof of principle experiment. As it was hypothesised, the results showed a positive action of exogenous sperm miRNAs in embryo cleavage when using bulls with low IVP fertility. This is probably because of the increase in global gene expression and consequence of a lower HDAC1 expression (enzyme responsible for histone deacetylation), leading to an increment in blastocyst and development rates.

This research was supported by FAPESP (2018/03871-6).