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Reduced oocyte quality has been associated with poor fertility of high-performance dairy cows during peak lactation, due to negative energy balance. We examined the role of nonesterified fatty acids (NEFAs), known to accumulate within follicular fluid during under- and overnutrition scenarios, in causing endoplasmic reticulum (ER) stress of in vitro maturated cattle cumulus-oocyte complexes (COCs). NEFA concentrations were: palmitic acid (150 μM), oleic acid (200 μM), and steric acid (75 μM). Abattoir-derived COCs were randomly matured for 24 h in the presence of NEFAs and/or an ER stress inhibitor, salubrinal. Total and hatched blastocyst yields were negatively impacted by NEFA treatment compared with controls, but this was reversed by salubrinal. ER stress markers, activating transcription factor 4 (Atf4) and heat shock protein 5 (Hspa5), but not Atf6, were significantly up-regulated by NEFA treatment within whole COCs but reversed by coincubation with salubrinal. Likewise, glucose uptake and lactate production, measured in spent medium samples, showed a similar pattern, suggesting that cumulus cell metabolism is sensitive to NEFAs via an ER stress-mediated process. In contrast, while mitochondrial DNA copy number was recovered in NEFA-treated oocytes, oocyte autofluorescence of the respiratory chain cofactor, FAD, was lower following NEFA treatment of COCs, and this was not reversed by salubrinal, suggesting the negative impact was via reduced mitochondrial function. These results reveal the significance of NEFA-induced ER stress on bovine COC developmental competence, revealing a potential therapeutic target for improving oocyte quality during peak lactation.

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Accepted (peer-reviewed) VersionSelf-archiving of the accepted version is subject to an embargo period of 12-24 months. The embargo period is 12 months for scientific, technical, and medical (STM) journals and 24 months for social science and humanities (SSH) journals following publication of the final article.The accepted version may be placed on:• the author's personal website • the author's company/institutional repository or archive • certain not for profit subject-based repositories such as PubMed Central as listed below Articles may be deposited into repositories on acceptance, but access to the article is subject to the embargo period. stimulates FAD and NAD(P)H auto-fluorescence within oocytes, without changing the REDOX 57 ratio. Hence, the aim of this study was to investigate if BMP15-induced increased NAD(P)H 58 was due to NADPH production. Cattle COCs were cultured with FSH and/or recombinant 59 human BMP15 (BMP15). Following culture with BMP15, there was a significant decrease in 60 glucose 6-phosphate dehydrogenase activity (P<0.05). Treatment with an inhibitor of isocitrate 61 dehydrogenase (IDH) decreased NAD(P)H intensity 3-fold in BMP15 treated oocytes, 62 suggesting BMP15 stimulates IDH and NADPH production via the TCA cycle. As NADPH is a 63 reducing agent, reduced glutathione (GSH), H2O2 and mitochondrial activity were measured. 64 FSH alone decreased GSH levels within the oocyte, with the combination of BMP15 and FSH 65 recovering levels. Expression of genes encoding glutathione-reducing enzymes were also 66 lower in oocytes cultured in the presence of FSH. However, BMP15 supplementation 67 promoted mitochondrial localisation patterns consistent with enhanced developmental 68 competence. Metabolomics revealed there was significant consumption of glutamine and 69 production of alanine by COCs +FSH +BMP15 compared to the control (P<0.05). Hence, this 70 study demonstrates that BMP15 supplementation differentially modulates reductive metabolism 71 and mitochondrial localisation within the oocyte. In comparison, FSH-stimulation alone 72 decreases the oocyte's ability to regulate cellular stress and therefore utilizes other 73 mechanisms to improve developmental competence. 74