Influences of glycine supplementation during vitrification on the developmental potential of vitrified/warmed immature dromedary camel oocytes.

Oocytes experience detrimental osmotic stress during vitrification and warming procedures because of the osmolality imbalance between the vitrification-warming fluids and the intracellular environment. Cellular osmotic homeostasis can be preserved by glycine, a powerful osmolyte with antioxidant properties. We aimed to examine the influences of supplementing glycine to the vitrification solutions (VS) on the developmental potential of vitrified/warmed immature dromedary camel oocytes following IVM/IVF and in vitro embryo culture (IVC). Cumulus oocyte complexes (COCs) were collected from dromedary camel ovaries and randomly allocated into two groups namely control (oocytes subjected directly to IVM) and vitrified (COCs were vitrified into VS supplemented with 0.0, 0.5, 1.0 or 2.0 mM glycine). For vitrification, COCs were equilibrated for 3 min in 12.5% ethylene glycol; EG plus 12.5% dimethyl sulfoxide; DMS and then they were vitrified for 60 s in VS composed of 25% EG + 25% DMSO using solid surface vitrification (SSV). Warming of vitrified oocytes was conducted in decreasing concentrations of trehalose solution. Following vitrification and warming, the morphologically viable oocytes were subjected to IVM for 36 h. Matured oocytes were then fertilized in vitro by epididymal spermatozoa and cultured for seven days. The results showed that the percentage of viable oocytes assessed by trypan blue stain was significantly higher (p ≤ .05) in the 1.0 mM glycine-supplemented group than 0.0- and 2.0-mM glycine-supplemented ones (90.0 % vs. 80.0% and 76.6%, respectively). However, no significant difference was observed between 0.5 mM glycine and other vitrified groups. Nuclear maturation rates, cleavage (48-h post-insemination; pi) and blastocyst rate (7-days pi) were significantly lower in vitrified groups than control ones (p ≤ .05). Among vitrified groups, these parameters were the highest in the 1.0 mM glycine-supplemented group. Taken together, supplementation of vitrification solutions with 1.0 mM glycine could enhance the developmental potential of vitrified/warmed immature dromedary camel oocytes.

Beneficial Effects of L-Carnitine Supplementation during IVM of Canine Oocytes on Their Nuclear Maturation and Development In Vitro.

This study aimed to investigate the effect of L-Carnitine (LC) supplementation during in vitro maturation (IVM) of canine oocytes on nuclear maturation, fertilization status, and preimplantation development. Cumulus-oocyte complexes (COCs) collected from the ovaries of ovariohysterectomized female dogs were matured in vitro for 72 h in a TCM-199 medium supplemented with (0.1, 0.3, 0.6, 1.0, or 2.0 mg/mL) or without (0.0 mg/mL) LC. Matured oocytes were fertilized in vitro with frozen-thawed spermatozoa, and zygotes were cultured in a SOF medium for 7 days. IVM rates were higher (p ≤ 0.05) in 0.3 and 0.6 mg/mL LC supplemented groups than in the control (0.0 mg/mL LC) and other LC groups. Fertilization (18 h postinsemination (pi)) and cleavage (2-16-cell stage at day 3 pi) rates were higher (p ≤ 0.05) in the 0.6 mg/mL LC group than in the control and 0.1, 1.0, and 2 mg/mL LC supplemented groups. Interestingly, 4.5% of fertilized oocytes developed to morula (day 5 pi) in the 0.6 mg/mL LC group, which was higher (p ≤ 0.05) than those developed in the 0.3 mg/mL group (1.0%). No cleaved embryos developed to morula in other groups. In conclusion, LC supplementation at 0.6 mg/mL during IVM of canine oocytes improved their maturation, fertilization, and preimplantation embryo development rates following IVF and in vitro culture (IVC).

Melatonin can improve viability and functional integrity of cooled and frozen/thawed rabbit spermatozoa.

Melatonin is known to protect sperm against freezing-inflicted damage in different domestic species. The aim of the study was to evaluate the effect of supplementation of semen extender with melatonin on the quality and DNA integrity of cooled and frozen/thawed rabbit spermatozoa. We also investigated whether the addition of melatonin to the semen extender could improve the fertility of rabbit does artificially inseminated with frozen/thawed semen. Semen samples collected from eight rabbit bucks were pooled and then diluted in INRA-82 supplemented either with (0.5, 1.0 or 1.5 mM) or without (0.0 mM) melatonin. Diluted semen was cooled at 5°C for 24 hr. For cryopreservation and based on the first experiment's best result, semen samples were diluted in INRA-82 in the presence or absence of 1.0 mM melatonin and then frozen in 0.25 ml straws. Following cooling or thawing, sperm quality and DNA integrity were evaluated. Furthermore, the fertility of frozen/thawed semen was investigated after artificial insemination. Supplementation of semen extender with 1.0 mM melatonin improved (p < .05) motility, viability, membrane and acrosome integrities in cooled semen compared with other groups. Sperm quality and DNA integrity were higher (p < .05) in frozen/thawed semen diluted in 1.0 mM melatonin-supplemented extender than in the control group. Conception and birth rates were higher in does inseminated with 1.0 mM melatonin treated semen compared with the controls. In conclusion, supplementation of semen extender with 1.0 mM melatonin improved the quality of cooled and frozen/thawed rabbit spermatozoa. Melatonin can preserve DNA integrity and enhance the fertility of frozen/thawed rabbit spermatozoa.

Impact of supplementation of semen extender with antioxidants on the quality of chilled or cryopreserved Arabian stallion spermatozoa.

The aim of the present study was to evaluate the effects of supplementation of semen extender with various non-enzymatic antioxidants on the quality of cooled or cryopreserved Arabian stallion spermatozoa. Semen collected from four pure Arabian stallions was centrifuged at 600g for 15 min. Spermatozoa were then diluted in INRA-82 extender supplemented with bovine serum albumin (BSA; 0, 10, 15 and 20 mg/mL) or trehalose (0, 75, 100 and 150 mM) or zinc sulphate (0, 100, 150 and 200 µM). The diluted semen was then either cooled at 5 °C or cryopreserved in 0.5-ml plastic straws. After cooling or thawing, sperm motility, viability, sperm abnormalities, viability index, and plasma membrane integrity were evaluated. The results showed that supplementation of semen extender with 150 mM trehalose or with 200 µM zinc sulphate significantly (P < 0.05) improved motility, viability, sperm membrane integrity and acrosome status in Arabian stallion spermatozoa after cooling or after freezing and thawing compared with controls (non-supplemented media) or with those supplemented with other concentrations of trehalose or zinc sulphate. Supplementation of semen extender with BSA did not improve sperm motility or cryosurvival of Arabian stallion spermatozoa after cooling or after freezing and thawing. In conclusion, supplementation of semen extender with non-enzymatic antioxidants (trehalose or zinc sulphate) improved the quality of chilled and frozen/thawed Arabian stallion spermatozoa. The most beneficial effects occur when semen diluent was supplemented with 150 mM trehalose or 200 µM zinc sulphate.

l-carnitine supplementation during vitrification of mouse germinal vesicle stage-oocytes and their subsequent in vitro maturation improves meiotic spindle configuration and mitochondrial distribution in metaphase II oocytes.

STUDY QUESTION: How does l-carnitine (LC) supplementation during vitrification and in vitro maturation (IVM) of germinal vesicle stage (GV)-oocytes improve the developmental competence of the resultant metaphase II (MII) oocytes? SUMMARY ANSWER: LC supplementation during both vitrification of GV-oocytes and their subsequent IVM improved nuclear maturation as well as meiotic spindle assembly and mitochondrial distribution in MII oocytes. WHAT IS KNOWN ALREADY: Vitrification of GV-oocytes results in a lower success rate of blastocyst development compared with non-vitrified oocytes. LC supplementation during both vitrification and IVM of mouse GV-oocytes significantly improves embryonic development after IVF. STUDY DESIGN, SIZE, DURATION: GV-oocytes were collected from (B6.DBA)F1 and B6 mouse strains and subjected to vitrification and warming with or without 3.72 mM LC supplementation. After IVM with or without LC supplementation, the rate of nuclear maturation and the quality of MII oocytes were evaluated. At least 20 oocytes/group were examined, and each experiment was repeated at least three times. All experiments were conducted during 2013-2014. PARTICIPANTS/MATERIALS, SETTING, METHODS: Extrusion of the first polar body in IVM oocytes was observed as an indication of nuclear maturation. Spindle assembly and chromosomal alignment were examined by immunostaining of α-tubulin and nuclear staining with 4,6-diamidino-2-phenylindole (DAPI). Mitochondrial distribution and oxidative activity were measured by staining with Mitotracker Green Fluorescence Mitochondria (Mitotracker Green FM) and chloromethyltetramethylrosamine (Mitotracker Orange CMTMRos), respectively. ATP levels were determined by using the Bioluminescent Somatic Cell Assay Kit. MAIN RESULTS AND THE ROLE OF CHANCE: LC supplementation during both vitrification and IVM of GV-oocytes significantly increased the proportions of oocytes with normal MII spindles to the levels comparable with those of non-vitrified oocytes in both mouse strains. While vitrification of GV-oocytes lowered the proportions of MII oocytes with peripherally concentrated mitochondrial distribution compared with non-vitrified oocytes, LC supplementation significantly increased the proportion of such oocytes in the (B6.DBA)F1 strain. LC supplementation decreased the proportion of oocytes with mitochondrial aggregates in both vitrified and non-vitrified oocytes in the B6 strain. The oxidative activity of mitochondria was mildly decreased by vitrification and drastically increased by LC supplementation irrespective of vitrification in both mouse strains. No change was found in ATP levels irrespective of vitrification or LC supplementation. Results were considered to be statistically significant at P < 0.05 by either χ(2)- or t-test. LIMITATIONS, REASONS FOR CAUTION: It remains to be tested whether beneficial effect of LC supplementation during vitrification and IVM of GV-oocytes leads to fetal development and birth of healthy offspring after embryo transfer to surrogate females. WIDER IMPLICATIONS OF THE FINDINGS: This protocol has the potential to improve the quality of vitrified human oocytes and embryos during assisted reproduction treatment. STUDY FUNDING/COMPETING INTEREST: Partially supported by the Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant and Mitacs Elevate Postdoctoral Fellowship, Canada.

L-carnitine supplementation during vitrification of mouse oocytes at the germinal vesicle stage improves preimplantation development following maturation and fertilization in vitro.

Oocyte cryopreservation is important for assisted reproductive technologies (ART). Although cryopreservation of metaphase II (MII) oocytes has been successfully used, MII oocytes are vulnerable to the damage inflicted by the freezing procedure. Cryopreservation of germinal vesicle stage oocytes (GV-oocytes) is an alternative choice; however, blastocyst development from GV-oocytes is limited largely due to the need for in vitro maturation (IVM). Herein, we evaluated the effects of l-carnitine (LC) supplementation during vitrification and thawing of mouse GV-oocytes, IVM, and embryo culture on preimplantation development after in vitro fertilization (IVF). We first compared the rate of embryonic development from the oocytes that had been collected at the GV stage from three mouse strains, (B6.DBA)F1, (B6.C3H)F1, and B6, and processed for IVM and IVF, as well as that from the oocytes matured in vivo, i.e. ovulated (IVO). Our results demonstrated that the rate of blastocyst development was the highest in the (B6.DBA)F1 strain and the lowest in the B6 strain. We then supplemented the IVM medium with 0.6 mg/ml LC. The rate of blastocyst development improved in the B6 but not in the (B6.DBA)F1 strain. Vitrification of GV-oocytes in the basic medium alone reduced the rate of blastocyst development in both of those mouse strains. LC supplementation to the IVM medium alone did not change the percentage of blastocyst development. However, LC supplementation to both vitrification and IVM media significantly improved blastocyst development to the levels comparable with those obtained from vitrified/thawed IVO oocytes in both of the (B6.DBA)F1 and B6 strains. We conclude that LC supplementation during vitrification is particularly efficient in improving the preimplantation development from the GV-oocytes that otherwise have lower developmental competence in culture.