Effects Of Acetyl-L-Carnitine On Buffalo Oocytes In Vitro Maturation

Oocyte quality is one of the important factors in female fertility and the process of oocyte maturation requires high energy.The beta oxidation of fatty acids is one of the important energy sources for oocytes maturation and subsequent embryonic development.Acetyl-L-carnitine(ALC)can promote the beta oxidation of fatty acids.At present,there are few studies on the relationship between ALC and mammalian reproductive performance.The available data confirm that ALC can alleviate oxidative stress in early mice embryos and promote lipid metabolism in lamb oocytes,However,the research of ALC on buffalo oocytes in vitro maturation is deficient and the mechanism is unclear.In this study,the effects of ALC on buffalo oocytes in vitro maturation and subsequent embryonic development were investigated.The changes of lipid fingerprints and metabolite fingerprints of buffalo oocytes affected by ALC were analyzed by mass spectrometry,the main findings of this research are as follows:1.The effects of ALC on buffalo oocyte maturation and subsequent embryo development in vitro were investigated.To determine the optimal level of ALC supplementation,different concentration groups of 0 mM,1.25 mM,2.5 mM and 5 mM were designed.Results showed that 2.5 mM ALC had significantly higher blastocyst rate than that of other groups(P<0.05).In the following studies,2.5 mM was supplemented to detect various markers of oocytes and cumulus cells.The results showed that the cumulus cells had significant proliferative ability(P<0.05),oocytes had lower concentrations of ROS(P<0.05)and a higher rate of diffuse mitochondrial distributions in the ALC groups than in unsupplemented groups(P<0.05).Additionally,the mtDNA was higher in the ALC-treated oocytes and cumulus cells than that in the untreated cells(P<0.05).The expression of P450scc and GDF9 in oocytes and cumulus cells of ALC group was significantly higher than that of control group(P<0.05).Therefore,in buffalo,our results suggest that ALC reduces ROS content,regulates mitochondrial function and oocyte-derived paracrine factors,and improves the production of steroid hormones,leading to increased quality of matured oocytes and improved embryonic development in vitro.2.The effect of ALC on the metabolomics profile of spent mature medium for buffalo cumulus-oocyte complexs was investigated by Ultra Performance Liquid Chromatography Mass Spectrometry(UPLC-MS)using OPLS-DA to analyze mass spectrometry data,seven significant difference metabolites were identified.Dopaxanthin(related to antioxidant activity),O-acetyl-ADP-ribose(metabolic intermediates of sirtuins)and Aldosterone 18-glucuronide(steroids and steroid derivatives)significantly higher abundance in ALC-treated medium than in un-treated medium(P<0.05),(S)-a-Amino-2,5-dihydro-5-oxo-4-isoxazolepropanoic acid N2-glucoside,Sudachiin A(related to glycometabolism,involvement of galactose metabolism and energy metabolism signal pathway),Lactose 6-phosphate(related with glucose metabolism),Fumaric acid(three carboxylic acid intermediates,participate in the three carboxylic acid cycle and oxidative phosphorylation pathway)and D-Glucurono-6,3-lactone(glucose metabolite)significantly lower abundance in ALC-treated medium than in un-treated medium(P<0.05).Therefore,during buffalo oocytes maturation in vitro,adding ALC may decrease the glucose metabolism level,improve the levels of antioxidant metabolism and steroid production in cumulus-oocyte complex.3.The effect of ALC on the lipid fingerprint of mature oocytes was investigated by Matrix-assisted laser desorption time of flight mass spectrometry(MALDI-TOF MS).Using OPLS-DA to analyze mass spectrometry data,three lipid ions(m/z 728.7[PC(32:3)],768.8[PC(P-36:3)]and 894.9[TAG(58:8)])in mature oocytes of ALC group were significantly lower than those of the un-treated group(P<0.05).The content of total lipids in the oocyte of each group was determined by Nile Red.The results showed that the content of total lipids in the ALC group was significantly lower than that in the un-treated group(P<0.05).Therefore,during buffalo oocytes maturation in vitro,adding ALC significantly reduced the total lipid content,and reduced the phospholipid and triglyceride content in mature oocytes.4.The effect of ALC on the vitrification of mature oocytes was investigated by MALDI-TOF MS technology.Mass spectrometry data were analyzed by 02PLS-DA multivariate analysis.The results showed that ALC-supplemented vitrified group significantly increased the cleavage rate,morula rate,blastocyst rate and mitochondrial membrane potential than that of unsupplemented vitrified group(P<0.05),and five phospholipid ions(m/z 728.7[PC(32:3)],746.9[PC(32:5)],760.6[PC(34:1)],768.8[PC(P-36:3)],and 782.6[PC(36:4)];P<0.05)were identified as significantly more abundant in fresh oocytes than in unsupplemented vitrified oocytes.Meanwhile,three phospholipid ions(m/z 734.6[PC(32:0)],760.6[PC(34:1)],and 782.6[PC(36:4)];P<0.05)were more abundant in ALC-supplemented vitrified oocytes than in unsupplemented vitrified oocytes.Therefore,supplementation with ALC during IVM may improve buffalo oocyte quality after vitrification by enhancing mitochondrial function and altering the phospholipid composition of the vitrified oocyte.In conclusion,the results suggest that ALC promotes buffalo oocytes in vitro maturation and subsequent embryonic development by regulating mitochondrial function,paracrine factors,steroid hormone expression and lipid metabolism.The results of this study can provide a reference for further interpretation of the influence of ALC on reproductive biology function of buffalo oocytes maturation in vitro.