Idebenone relieves the damage of heat stress on the maturation and developmental competence of porcine oocytes.

The reproductive function of animals is often affected by climatic conditions. High-temperature conditions can cause damage to oocyte maturation and embryonic development in a variety of ways. The purpose of this study was to prove that supplementation idebenone (IDB) to the maturation medium can improve the maturation and development of porcine oocytes after heat stress (HS). Porcine cumulus-oocyte complexes (COCs) were cultured in the maturation medium with different concentrations of IDB (0, 0.1, 1 and 10 µM) for 44 hr at either 38.5°C or under the HS conditions. The cumulus oophorus expansion, nuclear maturation and blastocyst rate after parthenogenetic activation (PA) were measured. We found that HS (in vitro maturation 20-24 hr, 42°C) exposure significantly reduced cumulus expansion index and maturation rate of oocytes and the blastocyst rate of PA embryos, while IDB supplementation significantly improved oocyte maturation and development to the blastocysts stage after PA. Moreover, the addition of IDB decreased the intracellular level of ROS and increased GSH content, hence enhancing the antioxidant capacity of oocytes under HS. Meanwhile, IDB treatment also obviously improved the mitochondrial membrane potential and ATP synthesis of oocytes under HS conditions. Furthermore, IDB treatment increased the expression of GDF9 and BMP15 in IVM oocytes which attribute to improve the quality and outcome of IVM oocytes and the development competence of PA embryos in pigs. In summary, we demonstrated that IDB supplementation into the maturation medium exerted protective effects and improved the ability of maturation and developmental competence of porcine oocytes exposed to HS.

Effect of L-carnitine on sperm quality during liquid storage of boar semen.

OBJECTIVE: This study was conducted to investigate the effect of L-carnitine on the pig semen characteristics during storage. METHODS: Spermatozoa samples were examined for spermatozoa quality and then randomly divided into 5 groups: 0 (control), 12.5, 25, 50, and 100 mM L-carnitine. Sperm motility, plasma membrane integrity and antioxidant parameters (total reactive oxygen species, total antioxidant capacity, and malondialdehyde) were evaluated after 0, 3, 5, and 10 day cooledstorage at 17°C. Moreover, ATP content, mitochondria activity as well as sperm-binding and in vitro fertilizing ability of preserved boar sperm were also investigated. RESULTS: Supplementation with 50 mM L-carnitine could effectively maintain boar sperm quality parameters such as sperm motility and membrane integrity. Besides, we found that L-carnitine had positive effects on boar sperm quality mainly through improving antioxidant capacities and enhancing ATP content and mitochondria activity. Interestingly, by assessing the effect of L-carnitine on sperm fertility and developmental potential, we discovered that the extender containing L-carnitine could improve sperm quality and increase the number of sperms bounding to zona pellucida, without improving in vitro fertility and development potential. CONCLUSION: These findings suggested that the proper addition of L-carnitine to the semen extender improved boar sperm quality during liquid storage at 17°C.

Protective influence of rosiglitazone against time-dependent deterioration of boar spermatozoa preserved at 17°C.

Spermatozoa are highly specialized cells, and energy metabolism plays an important role in modulating sperm viability and function. Rosiglitazone is an antidiabetic drug in the thiazolidinedione class that regulates metabolic flexibility and glucose uptake in various cell types, but its effects on boar sperm metabolism are unknown. In this study, we investigated the potential effect of rosiglitazone against time-dependent deterioration of boar spermatozoa during liquid preservation at 17°C. Freshly ejaculated semen was diluted with Beltsville Thawing Solution (BTS) containing different concentrations of rosiglitazone, and the motility, membrane and acrosome integrity of sperm were detected. Besides, we measured glucose uptake capacity, l-lactate production level, mitochondrial membrane potential, adenosine triphosphate (ATP) content and mitochondrial reactive oxygen species (mROS) production of sperm after boar semen had been incubated with or without rosiglitazone, iodoacetate (glycolysis inhibitor) and rotenone (electron transport chain inhibitor) for 5 days. The addition of rosiglitazone significantly enhanced sperm quality and had a strong protective effect on the sperm membrane and acrosome integrity during storage. BTS containing 50 µM rosiglitazone maintained the total motility of liquid-preserved sperm above 60% for 7 days. Rosiglitazone improved sperm quality by regulating energy metabolism manner of preserved sperm, protected the sperm mitochondrial membrane potential, enhanced sperm ATP production and in the meanwhile reduced mROS through enhancing glycolysis but not oxidative phosphorylation. The data suggested the practical feasibility of using rosiglitazone for improving boar spermatozoa quality during semen preservation.

Effects of astaxanthin on plasma membrane function and fertility of boar sperm during cryopreservation.

Mammalian sperm is highly susceptible to reactive oxygen species (ROS) during the cryopreservation process. Astaxanthin (AST), a red pigment of the carotenoid family, is recognized as having a variety of beneficial biological activities and effects, including antioxidant, anticancer, anti-diabetic, and anti-inflammatory. The present study aimed to investigate whether the presence of AST protected boar sperm from ROS stress during cryopreservation. Boar sperm was diluted with a freezing medium supplemented with different concentrations of AST (0, 0.5, 1, 2, or 5 µM). The addition of AST, especially at a concentration of 2 µM, exerted positive effects on post-thaw sperm motility parameters. Meanwhile, sperm plasma membrane integrity and acrosome integrity of post-thaw sperm were significantly increased, while lipid peroxidation was inhibited in response to 2 µM AST treatment. Interestingly, compared to the control, supplementation with 2 µM AST increased unsaturated fatty acids (UFAs) levels and decreased saturated fatty acids (SFAs) content in post-thaw sperm, leading to a decreased ratio of SFAs/UFAs in the AST group. In conclusion, the addition of AST to freezing extenders inhibited lipid peroxidation and regulated fatty acid composition of the sperm membrane, improved post-thaw sperm quality, and had no adverse effect on boar sperm in vitro fertilization (IVF) capacity and potential for embryonic development. Our data provide a novel insight into understanding the mechanisms of AST concerning protecting boar sperm quality against ROS damage during cryopreservation.

[Studies on in vitro capacitation of goat spermatozoa by heparin treatment].

Systematical studies are lacking on the influencing factors and mechanisms of the heparin enhanced sperm capacitation, although many studies have shown that heparin enhanced sperm capacitation. The effect of heparin concentration and exposure time, incubation temperature and co-culture with oviductal epithelial cells or cumulus cells on goat sperm capacitation were investigated in this study. The motility, membrane and acrosome integrity and capacitated percentage of goat spermatozoa were assessed after different heparin treatments, and rates of fertilization and embryo cleavage were compared after in vitro insemination of oocytes with spermatozoa capacitated by different heparin treatments. The major results are summarized as follows: 1) When spermatozoa were capacitated with heparin at 5, 10, 25, 50 and 100 microg/mL for 45 min, 50 and 100 microg/mL heparin treatments produced the highest capacitated percentages of 55% and 56%, respectively, but the percentage of spermatozoa with intact acrosomes in the 100 microg/mL heparin treatment decreased significantly (P < 0.05) in comparison with that in the control group, indicating that the optimal heparin concentration for goat sperm capacitation would be 50 microg/mL. 2) Capacitated percentage of spermatozoa increased with extension of treatment time when goat sperm were treated with 50 microg/mL heparin for 0, 10, 20, 30, 45, 60 or 120 min. Although heparin treatments for 45 to 120 min did not differ significantly (P > 0.05) in capacitated sperm percentages, sperm motility and membrane integrity decreased significantly when treated with heparin for 120 min. This suggested that the optimal exposure time of heparin at 50 microg/mL for goat sperm capacitation would be 45 to 60 min. 3) Significantly higher capacitated percentages of spermatozoa were obtained when goat sperm were treated at 42 and 38.5 degrees C than at 15 and 37 degrees C, but sperm motility and acrosome integrity were significantly lower when spermatozoa were treated at 42 degrees C than they were treated at other temperatures. Temperature of 38.5 degrees C would, therefore, be the optimal temperature for goat sperm capacitation. 4) The capacitated percentage of spermatozoa was significantly higher when goat sperm were co-cultured with oviductal epithelial cells than when treated with heparin alone or co-cultured with cumulus cells, but sperm motility and membrane and acrosome integrity did not differ significantly among the three treatments. Rates of fertilization (91.3%) and cleavage (72.2%) were significantly higher in the oviductal epithelial cell co-culture group than those in the heparin alone group. This indicated that co-culture with oviductal epithelial cells significantly enhanced goat sperm capacitation by heparin treatment.

[Effects of sperm and oocyte quality control on intracytoplasmic sperm injection (ICSI) in goats].

ABSTRACT Effects of sperm and oocyte quality control on the efficiency of ICSI of in vitro matured goat oocytes were studied in this paper. The results showed that when injected intracytoplasmically, spermatozoa from caput, corpus and cauda epididymidis resulted in similar rates of fertilization, cleavage and morulae/blastocysts, but when injected subzonally, spermatozoa from caput and corpus gave rise to significantly lower rates of fertilization and embryo development than spermatozoa from the cauda epididymidis and ejaculates. When dead spermatozoa collected from semen that had been preserved in different ways were used for ICSI, those dead from liquid storage at 20 degrees C for 24 h gave rise to the best, but those dead from liquid storage at 5 degrees C for 15 days produced the poorest fertilization and embryo development. When spermatozoa were treated with different concentrations of Triton X-100 before ICSI, significantly higher rates of fertilization, cleavage and morulae/blastocysts were obtained with 0.0005% Triton X-100 than with other concentrations and manual immobilization. Oocytes were classified as of good and poor qualities by treatment in hypertonic sucrose solution, and rates of fertilization and embryo development were significantly higher in the good than in the poor oocytes after ICSI. Post-injection activation of oocytes with either A23187 or ionomycin/6-DMAP significantly increased the rates of fertilization, cleavage and morulae/blastocysts after ICSI. It is therefore concluded that (i) epididymal maturation mainly endowed spermatozoa with the capacity to fuse with the egg plasma membrane; (ii) different methods of semen storage caused different impairment of sperm fertilizing capacity; (iii) pre-injection treatment of spermatozoa with proper concentrations of Triton X-100 might be used to replace manual immobilization for ICSI; (iv) oocyte quality was a major factor influencing the efficiency of ICSI; (v) post-injection activation treatment of oocytes improved fertilization and embryo development after ICSI.