Study question Are there effects of high non-esterified fatty acids exclusively during bovine in vitro fertilization on cell lineage allocation of blastocysts? Summary answer Under the conditions of the present study, high exposure to NEFA during bovine IVF significantly decreases embryo production and alters cell allocation of resultant blastocysts. What is known already Cattle models have shown that a high exposure to non-esterified fatty acids (NEFA) such as steric acid (SA), palmitic acid (PA]) and oleic acid (OA) during in vitro oocyte maturation and embryo development can disrupt both embryo formation and quality. However, the fertilization process per se have been less studied, which is needed to identify developmental stages were potential therapies could be develop to ameliorate NEFA toxicity during the periconceptional period Study design, size, duration Day–8 blastocysts were immunostained for CDX2, a transcription factor involved in trophectoderm differentiation, to examine cell allocation of blastocysts derived from oocytes fertilised under high NEFA levels. VF (19 h) was carried with different NEFA levels(4 replicates) representing physiological (Control–1[C1], 28μM SA, 23μM PA, 21μM OA) and pathophysiological (NEFA, 280μM SA, 230μM PA, 210μM OA) relevant concentrations. A second control (C2) group contained solvent. Blastocysts (C1; n = 14, C2; n = 12, NEFA; n = 8) Participants/materials, setting, methods All blastocysts were examined by confocal microscopy and cell counting was done with the Imaris software. Data were analysed by ANOVA (mean±SEM) with percentage data arcsine transformed before analysis. Main results and the role of chance Blastocyst formation was decreased by high NEFA levels (C1=25.6±2.7%, C2=26.0±2.3%, NEFA=9.4±0.4%, P < 0.001) which was associated with a decreased cleavage rate (C1=70.1±6.5%, C2=71.5±3.1%, NEFA=42.5±4.1%, P = 0.006) and an increase in embryo degeneration (C1=47.6±3.5%, C2=47.7±5.8%, NEFA=63.0±4.9%, P = 0.05). A lower total cell (TC) the number was observed in high NEFA-derived blastocyst (C1=125.2±6.6, C2=132.3±8.4, NEFA=67.3±5.6, P < 0.001) associated with a low cell number in both the trophectoderm (CDX2 positive cells, C1=90.2±5.9, C2=96.7±6.4, NEFA=41.3±4.1, P < 0.001) and the inner cell mass (ICM, C1=35.0±2.4, C2=35.7±3.5, NEFA=26.0±2.2, P < 0.001). Furthermore, high NEFA-derived blastocyst showed an increased allocation of cells towards the ICM (ICM/TC proportion, C1=28.2±1.7%, C2=26.9±1.8%, NEFA=39.1±2.2%, P < 0.001) Limitations, reasons for caution It will be better if the number of blastocysts reached increases. Wider implications of the findings: such research can be widely applied to the human model due to the similarities between both specie. Trial registration number Royal Embassy of Saudi Arabia Cultural Bureau

Study question Do high concentrations of non-esterified fatty acids (NEFA) affect sperm quality? Summary answer In vitro exposure to high NEFA concentrations induced detrimental effects on acrosome integrity, mitochondrial membrane potential, plasma membrane integrity, and DNA damage in bovine spermatozoa. What is known already High NEFA levels present in obese individuals and in cows experiencing negative energy balance has been linked with impaired reproductive success. In vitro bovine models have showed that a high NEFA microenvironment (i.e. steric acid [SA], palmitic acid [PA], oleic acid [OA]) during oocyte maturation (Reproduction 2013, 145:33-44), fertilisation (ESHRE meeting 2021, P-176), and embryo culture (BMC Genomics 2016, 17:1004) is detrimental for blastocyst formation. However, the effect of high NEFA levels on sperm quality has been less explored. Study design, size, duration Bovine sperm from two bulls was prepared to a maximum concentration of 20x106/mL then incubated in in vitro fertilisation (IVF) medium for 4 h under different NEFA levels representing physiological (Control-1[C1], 28 μM SA, 23 μM PA, 21 μM OA) and pathophysiological (High-NEFA, 280μM SA, 230μM PA, 210μM OA), serum levels found in cows experiencing negative energy balance) concentrations. A second control (C2) group contained solvent. Participants/materials, setting, methods Fluorescence assays (Toxicity 2018, 393:52-50) were used to evaluate simultaneously acrosome integrity (FITC-PSA), mitochondrial membrane potential (JC-1) and plasma membrane integrity (DRAQ7, Hoechst 33342). Spermatozoa (n = 800 per group, four replicates) were assessed with epifluorescence microscopy and Ziess ZEN software. Sperm DNA damage was also assessed with the Halosperm G2® kit (C1; n = 812, C2; n = 879, High-NEFA; n = 930, six replicates). Data were arcsine transformed before analysis (ANOVA, followed by LSD post hoc test). Main results and the role of chance There was no significant difference between the control groups or between bulls in any of the variables analysed. The percentage (mean ± SEM) of spermatozoa with intact acrosome was higher in the control groups compared with high-NEFA (C1=64.50 ± 6.34%, C2=69.13 ± 4.08%, High-NEFA=49.75 ± 6.14%), but it reached statistical significance only when compared to C2 (C2 vs High-NEFA P = 0.037, C1 vs High-NEFA P = 0.096). Similarly, the percentage of spermatozoa with low mitochondrial membrane potential was increased following exposure to high concentrations of NEFA (C1=50.88 ± 5.69%, C2=45.00 ± 4.03%, High-NEFA=61.62 ± 3.38%), but a significant difference was only observed when compared to C2 (C2 vs High-NEFA P = 0.027, C1 vs High-NEFA P = 0.124). The high-NEFA group showed a higher plasma membrane damage (59.50 ± 1.06%) compared to controls (C1=47.50 ± 4.07% P = 0.01, C2=46.75 ± 1.79% P = 0.008). DNA damage was also increased in the High-NEFA group (20.00 ± 0.64%) compared to controls (C1=8.90 ± 0.71% P < 0.001, C2=6.82 ± 0.78% P < 0.001). Limitations, reasons for caution In vitro animal models do not reflect accurately in vivo conditions and cannot be directly extrapolated to humans, but they are useful for the development of conceptual models that could eventually be tested in the target species (e.g. obese individuals). Wider implications of the findings Our results indicate that high NEFA concentrations can impair sperm quality, and given that sperm with DNA damage can achieve fertilization (Human Reproduction 2010, 25:1594-1608), our data partially explain the decreased blastocyst formation previously observed in this model of high NEFA exposure (ESHRE meeting 2021, P-176). Trial registration number 000