Alpha-linolenic acid is an important polyunsaturated fatty acid that exhibits anticancer, anti-inflammatory, and antioxidative effects. In this study, we investigated the protective effects of alpha-linolenic acid on the cell proliferation and differentiation of C2C12 cells under essential amino acid-deficient conditions. Different concentrations of alpha-linolenic acid and essential amino acids were added to the growth and differentiation media. The concentrations of 10 μM of alphalinolenic acid and 2% essential amino acid were chosen for subsequent experiments. Supplementation with alpha-linolenic acid and essential amino acids improved the proliferation and differentiation of C2C12 cells and significantly increased the mRNA levels of catalase, superoxide dismutase, B-cell lymphoma-2, and beclin-1 as well as the protein levels of PPARγ coactivator-1α compared to those in the controls. Moreover, supplementation with alpha-linolenic acid and essential amino acids reduced the levels of phosphorylated H2A.X variant histone, Bcl-2-associated X, p53, and light chain 3 during C2C12 cell proliferation, and increased the expression levels of myogenic factors 4 (myogenin) and 5 during C2C12 cell differentiation. Overall, we determined that alpha-linolenic acid and essential amino acids maintained the cell proliferation and differentiation of C2C12 cells via their anti-oxidative, anti-apoptotic, and anti-autophagic effects.

The aim of present study was to investigate regulatory mechanism of alpha-linolenic acid (ALA) during in vitro maturation (IVM) on nuclear and cytoplasmic maturation of porcine oocytes. Basically, immature cumulus-oocyte complexes (COCs) were incubated for 22 h in IVM-I to which hormone was added, and then further incubated for 22 h in IVM-II without hormone. As a result, relative cumulus expansion was increased at 22 h after IVM and it was enhanced by treatment of ALA compared with control group (p < 0.05). During IVM process within 22 h, cAMP level in oocytes was decreased at 6 h (p < 0.05) and it was recovered at 12 h in ALA-treated group, while oocytes in control group recovered cAMP level at 22 h. In cumulus cells, it was reduced in all time point (p < 0.05) and ALA did not affect. Treatment of ALA enhanced metaphase-I (MI) and MII population of oocytes compared with oocytes in control group at 22 and 44 h, respectively (p < 0.05). Intracellular GSH levels in ALA group was increased at 22 and 44 h after IVM (p < 0.05), whereas it was increased in control group at 44 h after IVM (p < 0.05). In particular, the GSH in ALA-treated oocytes during 22 h of IVM was higher than control group at 22 h (p < 0.05). Lipid amount in oocytes from ALA group was higher than control group (p < 0.05). Treatment of ALA did not influence to absorption of glucose from medium. Cleavage and blastocyst formation of ALA-treated oocytes were enhanced compared with control group (p < 0.05). These findings suggest that supplementation of ALA could improve oocyte maturation and development competence through increasing GSH synthesis, lipid storage, and regulation of cAMP accumulation during early 22 h of IVM, and these might be mediated by cumulus expansion.

This present study was conducted to investigate protective effect of discontinuous Percoll gradient containing alpha-linolenic acid (ALA) before freezing process on viability, acrosome damage, mitochondrial activity, and oxidative stress of frozen-thawed boar spermatozoa. The separation of spermatozoa by discontinuous Percoll gradient was performed by different concentration of Percoll solution (45/90%) containing ALA combined with bovine serum albumin (BSA), and collected sperm in each Percoll layer was cryopreserved. To evaluate viability, acrosome damage, mitochondrial activity, and reactive oxygen species (ROS) level of frozen-thawed sperm, flow cytometry was used. Morphological abnormalities were observed under light microscope. In results, viability of sperm from 90% Percoll layer was higher than control and 45% Percoll group (p < 0.05). Separated sperm in 90% Percoll layer had lower acrosome damage and morphological abnormalities than control as well as viability, whereas 45% Percoll group was higher (p < 0.05). Similar with acrosome damage and abnormalities, mitochondrial activity was slightly enhanced and the population of live sperm with high ROS level was decreased by 90% Percoll separation, however, there was no significant difference. Supplementation of 3 ng/mL ALA into Percoll solution increased sperm viability and decreased population of live sperm with high ROS compared to control (p < 0.05). In conclusion, discontinuous Percoll gradient before freezing process could improve efficiency of cryopreservation of boar sperm through selection of sperm with high freezing resistance, and supplement of ALA during Percoll gradient might contribute suppression of ROS generation via stabilizing of plasma membrane during cryopreservation.

Alpha-linolenic acid (ALA; n-3 18:3), a one of omega-3 fatty acid, is mainly contained in chloroplast of plant and ALA is an essential fatty acid, not synthesized in mammalian body, it must be supplied from foods. Polyspermy is especially high on in vitro fertilization (IVF) in pigs, which is a major obstacle to in vitro embryo production systems. In our previous study, when ALA was supplemented during in vitro maturation (IVM), the methaphase-II rate and gluthathione level was increased. The objective of this study was to evaluate the effects of alpha-linolenic acid (ALA) supplementation during IVM and subsequent of IVF in pigs. The cumulus-oocyte complexes (COCs) were submitted to IVM medium containing 0, 25, 50, and 100 μM ALA for 44 h. After 44 h of IVM, denuded oocytes were co-cultured with spermatozoa during 18 h. After 18 h of in vitro fertilization, oocyte were using aceto-orcein method, to evaluated penetration rate, monospermy (number of monospermy oocytes/total oocytes), and the IVF efficiency (number of monospermy/total penetrated oocytes). In results, 25 and 50 μM ALA groups were significantly increased on penetration rate compared with 100 μM ALA group (p<0.05). Similarly, monospermy rate were significantly increased 25 and 50 μM ALA groups than control group (p<0.05). IVF efficiency was no significant difference between control and ALA treatment groups. Our findings suggested that treatment of ALA supplementation during in vitro maturation (IVM) and subsequent of in vitro fertilization in pigs, ALA can increase IVF efficiency by effectively blocking polyspermy and increasing monospermy some mechanism in porcine oocytes. However, the study of mechanism by which ALA blocks polyspermy are needed, and this study suggests that ALA has a positive effect on in vitro production of porcine oocytes by decreasing polyspermy. This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (Ministry of Education) (2016R1D1A1B03931746).

During the freezing and thawing process, fatty acids in the plasma membrane of sperm are released, which results in a functional damage of sperm. Sperm with functional loss due to cryo-damage result in a decrease in fertility. Previous studies have shown that the addition of one of the fatty acid alpha-linolenic (ALA) with carrier proteins improves the stability of plasma membrane and reduces the damage. In this experiment, we focused on the functional aspects of the plasma membrane of sperm and experimented with motility and morphology. For preparation of ALA-carrier protein complex, 3 ng/ml ALA was mixed with 0.7 μg/ml bovine serum albumin (BSA) or 14 ng/ml methyl-β-cyclodextrin (MBCD) in distilled water. The boar semen was purchased from GUMBO Company. Boar semen was cryo-preserved in 20% egg yolk freezing extender containing ALA, BSA, MBCD, ALA+BSA, ALA+MBCD. The frozen boar sperm was thawed at 37.5 ℃ for 45 sec in water-bath. The sperm motility and morphological abnormalities were evaluated under a phase-contrast microscope at 200 × magnification and randomly counts of 200 sperm each sample. In results, motility of frozen-thawed sperm was increased in all treatment groups. In particular, there has been significant improvement in ALA+BSA and ALA+MBCD treatment groups than control (p<0.05). However, there was no significant difference in ALA, BSA and MBCD treatment groups. Morphological normalities in frozen-thawed sperm was reduced in complex treatment groups (p<0.05). However, there was no significant difference in single treatment groups. In both motility and morphology characteristics, ALA+BSA and ALA+MBCD treatment group was higher than all treatment groups. In conclusion, the addition of ALA with carrier proteins during cryopreservation has a positive effect in its functional aspect. This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (Ministry of Education) (2016R1D1A1B03931746).

Alpha-linolenic acid (ALA) is one of n-3 polyunsaturated fatty acids and found mainly in the chloroplasts. Many studies have been reported that intracellular reactive oxygen species (ROS) in mammalian oocytes were reduced by supplementation of ALA in in vitro maturation (IVM) medium. Based on these reports, we expected that ALA acts as an antioxidant during IVM of porcine oocytes. Therefore, the objective of this study was to investigate the antioxidant effect of ALA supplementation during IVM in porcine oocytes. The cumulus-oocyte complexes (COCs) were incubated in IVM medium containing 200 μM H2O2 or H2O2 with 50 μM ALA for 44 h. Nuclear maturation stage of oocytes was evaluated using aceto-orcein method. For measurement of oxidative stress state, intracellular ROS and glutathione (GSH) levels were measured using carboxy-DCFDA and cell tracker red, respectively. In results, oocytes in metaphase-II (MII) stage development was significantly reduced in H2O2 group compared to non-treated control group (61.84±1.42% and 80.00%, respectively; p<0.05) and it was slightly recovered by treatment of ALA (69.76±1.67%; p<0.05). The intracellular GSH levels was decreased in H2O2 groups compared with control groups, but it was enhanced by ALA treatment (p<0.05). On the contrary, H2O2 treatment increased intracellular ROS level in oocytes and H2O2-induced ROS was decreased by treatment of ALA (p<0.05). Our findings suggested that ALA treatment under oxidative stress condition improve oocyte maturation via elevated GSH and reduced ROS levels in oocytes. Therefore, these results suggest that ALA have an antioxidative ability and it could be used as antioxidant in in vitro production system of porcine embryo.

The aim of this study was to determine the effect of additional alpha-linolenic acid (ALA) supplementation during in vitro maturation (IVM) and culture (IVC) on nucleic maturation and embryo development of pigs. Cumulus-oocyte complexes (COCs) were incubated in IVM medium containing different concentration of ALA (25, 50 and 100 μM) for 44 h. After in vitro maturation, nuclear maturation of oocytes were evaluated by aceto-orcein stain. Mature oocytes with 50 μM ALA were fertilized and cultured in IVC medium with ALA (25, 50 and 100 μM) during early-embryogenesis (48 hours after fertilization). Then, embryos were cultured with 25 μM ALA during early embryogenesis and/or late embryogenesis (120 hours after early-embryogenesis). In results, oocyte maturation were significantly increased by 50 μM ALA treatment groups compared with control groups (p<0.05). Treatment of 25 μM ALA during early-embryogenesis enhanced cleavage rate of embryo compared with other groups (p<0.05), whereas formation and total cell number of blastocyst had no significant difference. Similarly, cleavage rate of embryos were increased by 25 μM ALA supplement during early- or late-embryogenesis than ALA treatment both stage of embryogenesis (p<0.05), but did not influence to blastocyst formation. Interestingly, total cell number of blastocyst were enhanced in ALA treatment group during early-embryogenesis. These findings indicated that ALA supplement enhance the nuclear maturation of oocyte and embryo development, however, excessive ALA could negatively influence. Therefore, we suggest that ALA is used for improvement of in vitro production of mammalian embryo and further study regarding with functional mechanism of ALA is needed.