Mammalian embryos that rapidly reach the two-cell stage in culture have a higher probability of becoming viable blastocysts. Our goal was to separate two-cell bovine embryos based on their zygotic cleavage timing, and to assess their global mRNA levels. Following in vitro fertilization, all embryos that cleaved by 29.5 hpi (early) were cultured separately from those that divided at 46 hpi (late). The blastocyst rates were 46.1 ± 3.7% and 6.1 ± 3.4% for early- and late-cleavers, respectively (P < 0.01). Seven replicates of selected two-cell embryos were collected at each time point for microarray characterization (n = 4) and quantitative reverse-transcriptase PCR (n = 3); the rest were left in culture for blastocyst evaluation. A total of 774 and 594 probes were preferentially present in early- and late-cleaving embryos, respectively (fold change ± 1.5, P < 0.05), with important contrasts related to cell cycle, gene expression, RNA processing, and protein degradation functions. A total of 12 transcripts were assessed by quantitative PCR, of which ATM, ATR, CTNNB1, MSH6, MRE11A, PCNA, APC, CENPE, and GRB2 were in agreement with the hybridization results. Since most of these molecules are directly or indirectly associated with cell-cycle regulation, DNA damage response, and transcription control, our results strongly suggest key roles for those biological functions in mammalian preimplantation development.

2020) Effect of Moringa oleifera seed extract on antioxidant activity and sperm characteristics in cryopreserved ram semen, ABSTRACT Semen cryopreservation damages sperm due to oxidative stress. This study evaluated the antioxidant capacity of Moringa oleifera seed extract in cryopreserved ram semen and the impact of the extract on sperm characteristics. Semen from eight hair rams (four rams per sampling) was allocated into four groups, according to their treatment prior to cryopreservation: Control (no extract), 0.5 (M0.5), 5.0 (M5.0), and 10.0 (M10) mg/mL of M. oleifera extract. The antioxidant activity (ferric reducing antioxidant power, FRAP) and the spermatic characteristics (sperm viability; progressive motility; fast motility; slow motility; acrosome damage; membrane damage; and mitochondrial activity) were assessed post-thawing. Variables were evaluated with analysis of variance followed by Tukey test. While no significant differences were detected in acrosomal damage, mitochondrial activity, fast-, or slow motility, the antioxidant activity was higher (P < 0.05) in M0.5 and M5 treatments. Viability and progressive motility increased in the M0.5 group (P < 0.05), whereas sperm membrane damage was lower (P < 0.05) in the same treatment. In conclusion, supplementation of ram semen with M. oleifera seed extract enhances antioxidant activity, sperm membrane integrity, viability, and progressive motility after thawing. This suggests that M. oleifera extract could be used as an antioxidant to improve the outcome of semen cryopreservation. Highlights. It is widely known that semen cryopreservation induces sublethal damage to sperm, deteriorating spermatic characteristics, which is largely attributed to oxidative stress. . There are scarce studies regarding the use of plant extracts as a replacement for conventional antioxidants to conserve sperm viability in cryopreserved ram semen. . Addition of Moringa oleifera seed extract to a concentration of 0.5 and 5.0 mg/mL prior to ram semen freezing increased antioxidant activity after cryopreservation. . M. oleifera seed extract at 0.5 mg/mL decreased post-thawing damage to the sperm membrane, increasing both viability and progressive motility. . M. oleifera seed extract could be potentially used as a replacement for conventional antioxidants added to maintain sperm viability in cryopreserved ram semen. ARTICLE HISTORY

The effect of elicitation in butterhead lettuce on carotenoid and polyphenol metabolism was evaluated. Different concentrations of arachidonic acid (AA), salicylic acid (SA), methyl jasmonate (MJ) (15, 45, and 90 μM) and Harpin protein (HP) (30, 60, and 120 mg/L) were applied on red and green butterhead lettuces. Total phenolic and flavonoid content were incremented by MJ (90 μM) in green and red lettuce. Carotenoids were increased in red lettuce (AA; 45 μM). Green lettuce modifies their phenolic acid profile after elicitation with AA and MJ; meanwhile, red lettuce incremented mainly in hydroxycinnamic acids and flavonols, MJ being the elicitor with the highest effect. There was an impact on secondary metabolite enzyme gene transcript concentration. Phenylalanine ammonia-lyase (PAL) and lycopene beta cyclase (LBC) increased in both varieties after elicitation. A relationship between phytochemical increase and the activation of the metabolic pathways after elicitation in butterhead lettuce was observed.

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The sequence of a 3' untranslated region (3'UTR) of mRNA governs the timing of its polyadenylation and translation in mammalian oocytes and early embryos. The objective of this study was to assess the influence of cis-elements in the 3'UTR of the developmentally important ATF1 and ATF2 transcripts on their timely translation during first cleavages in bovine embryos. Eight different reporter mRNAs (coding sequence of green fluorescent protein [GFP] fused to the 3'UTR of short or long isoforms of cattle ATF1 or -2, with or without polyadenylation) or a control GFP mRNA were microinjected separately into presumptive bovine zygotes at 18 hr post-insemination (hpi), followed by epifluorescence assessment for GFP translation between 24 and 80 hpi (expressed as percentage of GFP-positive embryos calculated from the total number of individuals). The presence of either polyadenine or 3'UTR sequence in deadenylated constructs is required for GFP translation (implying the need for polyadenylation), and all exogenous mRNAs that met either criteria were translated as soon as 24 hpi-except for long-deadenylated ATF2-UTR, whose translation began at 36 hpi. Overall, GFP was more visibly translated in competent (cleaving) embryos, particularly in long ATF1/2 constructs. The current data shows a timely GFP translation in bovine embryos depending on sequences in the 3'UTR of ATF1/2, and indicates a difference between short and long isoforms. In addition, cleaving embryos displayed increased translational capacity of the tested constructs. Functional confirmation of the identification cis-sequences in the 3'UTR of ATF1/2 will contribute to the understanding of maternal mRNA translation regulation during early cattle development.