Effects of Nobiletin supplementation on the freezing diluent on porcine sperm cryo-survival and subsequent in vitro embryo development

Nobiletin (NOB) is a bioflavonoid compound isolated from citrus fruit peels. The present study aimed to elucidate whether NOB facilitates the porcine sperm cryosurvival and embryo development after in vitro fertilization (IVF). To this end, spermatozoa were diluted and cryopreserved in a freezing extender supplemented with 0 (control), 50, 100, 150, and 200 μM Nobiletin. The kinematic patterns of frozen-thawed (FT) sperm were assessed after 30 and 90 min incubation using a Sperm Class Analyzer (SCA). Viability, acrosome integrity, and mitochondrial membrane potential (MMP) were measured by fluorescence microscopy 30 min after thawing using SYBR-14/PI, PSA/FITC, and R123/PI, respectively. Lipid peroxidation was determined using MDA assay after incubation for 90 min. The addition of 100 μM and 150 μM NOB to the extender significantly improved sperm progressive motility, and acrosome integrity compared to the control group (P < 0.05). The proportion of viable spermatozoa was significantly higher in the 150 μM NOB group. MDA levels were less in 50 μM and 150 μM NOB treated groups compared to the control. In addition, IVF with FT sperm was used to assess the embryo developmental competence. Treatment with 150 μM NOB before cryopreservation increased the cleavage and blastocyst formation rates compared to the control group. Furthermore, the relative expression of POU5F1 and AMPK, genes related to pluripotency and cell differentiation were significantly upregulated in embryos resulting from NOB-treated sperm compared to the control group. These results suggest that Nobiletin is a functionally novel phytochemical to mitigate oxidative stress during the freezing-thawing of porcine spermatozoa as reflected by improved FT sperm quality and IVF outcome.