Resveratrol significantly improves the fertilisation capacity of bovine sex-sorted semen by inhibiting apoptosis and lipid peroxidation.

The aim of this study was to test the effects of five different concentrations (0, 10-3, 10-4, 10-5, and 10-6 M) of resveratrol (Res) supplementation in bull sperm washing and fertilisation medium on levels of reactive oxygen species (ROS), phosphatidylserine (PS) externalisation, mitochondrial membrane potential (Δψm), ATP and malondialdehyde (MDA), acrosomal integrity, blastocyst rate, and blastocyst quality after in vitro fertilisation (IVF). The results for sex-sorted sperm from three bulls showed: (1) ROS and MDA levels in 10-3 M and 10-4 M Res groups were significantly lower than those of controls (P < 0.05); (2) the percentage of viable sperm, percentage of sperm with high Δψm, and the ATP content in 10-3 M and 10-4 M Res groups were significantly higher than those of controls (P < 0.05); (3) the percentage of viable sperm with acrosomal integrity, and the blastocyst percentage and quality of the 10-4 M Res group were significantly higher than those of controls (P < 0.05). In conclusion, 10-4 M Res supplementation in washing and fertilisation medium of sex-sorted bull sperm significantly decreased ROS, PS externalisation, and MDA, and protected mitochondrial function and acrosomal integrity, thereby increasing blastocyst percentage and quality following IVF.

Melatonin improves the fertilization capacity and developmental ability of bovine oocytes by regulating cytoplasmic maturation events.

Melatonin is a well-characterized antioxidant that has been successfully used to protect oocytes from reactive oxygen species during in vitro maturation (IVM), resulting in improved fertilization capacity and development ability. However, the mechanism via which melatonin improves oocyte fertilization capacity and development ability remains to be determined. Here, we studied the effects of melatonin on cytoplasmic maturation of bovine oocytes. In the present study, bovine oocytes were cultured in IVM medium supplemented with 0, 10-7 , 10-9 , and 10-11  mol/L melatonin, and the cytoplasmic maturation parameters of MII oocytes after IVM were investigated, including redistribution of organelles (mitochondria, cortical granules [CGs], and endoplasmic reticulum [ER]), intracellular glutathione (GSH) and ATP levels, expression of endogenous antioxidant genes (Cat, Sod1, and GPx), and fertilization-related events (IP3R1 distribution and expression of CD9 and Juno). Our results showed that melatonin significantly improved the cytoplasmic maturation of bovine oocytes by improving the normal distribution of organelles, increasing intracellular GSH and ATP levels, enhancing antioxidant gene expression levels, and modulating fertilization-related events, all of which resulted in increased fertilization capacity and developmental ability. Meanwhile, melatonin also increased the mRNA and protein expression levels of the Tet1 gene and decreased the Dnmt1 gene mRNA and protein levels in bovine oocytes, indicating that melatonin regulates the expression of the detected genes via demethylation. These findings shed insights into the potential mechanisms by which melatonin improves oocyte quality during IVM.

Calcium ion regulation by BAPTA-AM and ruthenium red improved the fertilisation capacity and developmental ability of vitrified bovine oocytes.

Vitrification reduces the fertilisation capacity and developmental ability of mammalian oocytes; this effect is closely associated with an abnormal increase of cytoplasmic free calcium ions ([Ca2+]i). However, little information about the mechanism by which vitrification increases [Ca2+]i levels or a procedure to regulate [Ca2+]i levels in these oocytes is available. Vitrified bovine oocytes were used to analyse the effect of vitrification on [Ca2+]i, endoplasmic reticulum Ca2+ (ER Ca2+), and mitochondrial Ca2+ (mCa2+) levels. Our results showed that vitrification, especially with dimethyl sulfoxide (DMSO), can induce ER Ca2+ release into the cytoplasm, consequently increasing the [Ca2+]i and mCa2+ levels. Supplementing the cells with 10 µM 1,2-bis (o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM or BAPTA) significantly decreased the [Ca2+]i level and maintained the normal distribution of cortical granules in the vitrified bovine oocytes, increasing their fertilisation ability and cleavage rate after in vitro fertilisation (IVF). Treating vitrified bovine oocytes with 1 µM ruthenium red (RR) significantly inhibited the Ca2+ flux from the cytoplasm into mitochondria; maintained normal mCa2+ levels, mitochondrial membrane potential, and ATP content; and inhibited apoptosis. Treating vitrified oocytes with a combination of BAPTA and RR significantly improved embryo development and quality after IVF.

Changes in acetylation on lysine 12 of histone H4 (acH4K12) of murine oocytes during maternal aging may affect fertilization and subsequent embryo development.

OBJECTIVE: To compare acH4K12 levels in oocytes during mouse aging and then assess how such changes might affect the developmental potential of oocytes. DESIGN: Experimental animal study. SETTING: State key laboratory and university research laboratory. ANIMAL(S): Kunming white strain mice. INTERVENTION(S): Oocytes obtained from TSA treated group or aging mouse group were fertilized and the formation of pronuclei and subsequently developmental potential in vitro or in vivo were assessed. MAIN OUTCOME MEASURE(S): AcH4K12 levels in oocytes were assessed using fluorescence staining, and confocal microscopy and oocyte developmental potentials were determined by in vitro or in vivo methods. RESULT(S): The AcH4K12 levels in oocytes statistically significantly increased during mouse aging. When histone acetylation of oocytes of young mice was artificially increased by trichostatin A (TSA) treatment, the acH4K12 levels in male and female pronuclei in fertilized oocytes showed statistically significant changes. About 38.9% of TSA-treated oocytes failed to form pronuclei or formed morphologically abnormal pronuclei 6 hours after fertilization, which statistically significantly decreased the blastocyst rate of TSA-treated oocytes when compared with the control group (41.5% vs. 60.5%). A similar reduction in blastocyst development was also observed when oocytes collected in older mice were compared with younger mice (17.3% vs. 69.4%). CONCLUSION(S): The AcH4K12 levels in oocytes statistically significantly increased during the aging process in mice, and such changes may affect the acetylation patterns and morphology of pronuclei during fertilization and lead to a reduction in oocyte developmental potential.

Mitochondrial behaviors in the vitrified mouse oocyte and its parthenogenetic embryo: effect of Taxol pretreatment and relationship to competence.

OBJECTIVE: To investigate the effect of Taxol pretreatment on mitochondrial behaviors in vitrified mouse mature oocytes and their parthenogenetic embryos. DESIGN: Experimental animal study. SETTING: University research laboratory and state key laboratory. ANIMAL(S): Sexually mature female Kunming white strain mice. INTERVENTION(S): Taxol before vitrification group (Tax). Oocytes were pretreated with M(2) containing 1 mmol/L Taxol for 2 minutes at 37C and then vitrified-warmed using the OPS vitrification procedure. Both ED solution and EDFS30 solution contained 1 mmol/L Taxol. MAIN OUTCOME MEASURE(S): Mitochondrial behaviors examined by fluorescence microscopy technology and fluorescence recovery after photobleaching (FRAP) technology. RESULT(S): In the control group, mitochondria were homogeneously distributed, in slow movement in oocytes, and perinuclearly distributed in 42.6% (n = 115) of their parthenogenetic two-cell embryos. Mitochondria from the toxicity group showed similar localization and movement to those of the control group, but not in the vitrification group. The perinuclear mitochondrial localization pattern of two-cell embryos was statistically significantly lower in both the toxicity (27.2%) and vitrification groups (19.8%) than in the control group. After parthenogenetic activation, the blastocyst formation rate of oocytes in the treated groups (28.1 to 48.6%) was statistically significantly lower than that of control (61.2%), but the rate of Taxol group (47.9%) was statistically significantly higher than that in the vitrification group (28.1%). CONCLUSION(S): Taxol pretreatment before vitrification helps to reduce the mitochondrial disturbance induced by vitrification in oocytes and their parthenogenetic early-stage embryo.

Effect of vitrification on mitochondrial distribution and membrane potential in mouse two pronuclear (2-PN) embryos.

The present study was designed to investigate the effect of vitrification on mitochondrial distribution, membrane potential (Deltapsi) and microtubule distribution in mouse 2-PN embryos, as well as to document the relationship between mitochondrial distribution and developmental ability of those embryos. Mitochondrial distribution was examined by fluorescence microscopy technology. Results indicated that: (1) The rate of mitochondrial ring formation around pronuclei in vitrified 2-PN embryos was significantly lower than in fresh ones (67.3 +/- 3.0% vs. 84.9 +/- 3.1%) (P < 0.05). (2) Blastocyst development rate of vitrified 2-PN embryos without mitochondrial rings (61.7 +/- 4.5%) was significantly lower than that of vitrified embryos with mitochondrial rings (82.1 +/- 2.8%). (3) Following staining by 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-imidacarbo-cyanine iodide (JC-1), most red-colored mitochondria (high Deltapsi) were distributed peripherally around pronuclei and along cell membranes of fresh 2-PN embryos. Conversely, red-colored mitochondria were greatly diminished in vitrified embryos, with green mitochondria (low Deltapsi) evenly distributed throughout the cytoplasm. The proportion of fresh 2-PN embryos with obvious aggregation of high Deltapsi mitochondria (84.2 +/- 2.2%) was significantly higher than that of vitrified embryos (26.7 +/- 3.0%) (P < 0.05). (4) The proportion of fresh embryos with microtubules distributed around pronuclei (83.5 +/- 3.4%) was similar to that of vitrified embryos (74.7 +/- 2.5%). In conclusion, vitrification affected mitochondrial distribution and decreased the mitochondrial membrane potential in mouse 2-PN embryos, events which may affect subsequent developmental viability of such embryos.

Positive effects of Taxol pretreatment on morphology, distribution and ultrastructure of mitochondria and lipid droplets in vitrification of in vitro matured porcine oocytes.

This study was designed to determine the effects of Taxol pretreatment on the morphology, distribution and ultrastructure of mitochondria and lipid droplets in vitrified porcine oocytes matured in vitro. The result showed that: (1) the rate of normal mitochondria distribution in fresh group (92.85%) was significantly higher (P<0.05) than that in other three groups (toxicity, 72.48%; vitrification, 50.83%; Taxol+vitrification, 69.98%) and Taxol pretreatment significantly (P<0.05) increased the ratio of normal mitochondria distribution in vitrified oocytes; (2) lipid droplets in vitrified oocytes got cracked, resulting in a great number of smaller lipid droplets (diameter <5 microm). The number of lipid droplets (5-10 microm in diameter) in vitrified oocytes pretreated with Taxol was higher (P<0.05) than that in the oocytes without Taxol pretreatment (81.87+/-13.63 vs. 64.27+/-13.72); (3) both toxicity and vitrification cause the difference in the ultrastructure of mitochondria and lipid droplets. Mitochondria were well maintained in the form of typical round and ellipse shape with smooth surface and clear outline and lipid droplets existed in the form of integrity in Taxol pretreatment group. In conclusion, Taxol pretreatment has positive effects on vitrified porcine oocytes matured in vitro in terms of morphology, distribution and ultrastructure of mitochondria and lipid droplets.

OPS vitrification of mouse immature oocytes before or after meiosis: the effect on cumulus cells maintenance and subsequent development.

Cryopreservation can cause cumulus cell damage around the immature oocytes, which may result in poor subsequent development. To evaluate the effect of the meiosis stage on the cumulus cell cryoinjury and determine the suitable stage for cryopreservation in immature oocytes, mouse oocytes at germinal vesicle (GV) and germinal vesicle breakdown (GVBD) stages were vitrified using open pulled straw (OPS) method. Cumulus cells damage was scored immediately after thawing by double-fluorescent staining. The survival rate of the oocytes was evaluated and the subsequent development of oocytes was assessed through in vitro culture (IVC) and in vitro fertilization (IVF) separately. After vitrification, a higher proportion of cumulus cells of GV oocytes were damaged than those of GVBD and untreated control groups. The survival rate of vitrified GVBD oocytes (94.1%) was significantly higher (p < 0.05) than that of GV oocytes (85.4%). Oocytes vitrified at GVBD stage (55.7%) showed similar cleavage rate compared to those at GV stage (49.2%), but significantly higher (p < 0.05) blastocyst rate (40.9% vs. 27.4%). These results demonstrate that oocytes at GVBD stage remain better cumulus membrane integrity and developmental ability during vitrification than those at GV stage, indicating they are more suitable for immature oocytes cryopreservation in mice.

Positive effect of partial zona pellucida digestion on in vitro fertilization of mouse oocytes with cryopreserved spermatozoa.

Cryopreservation of mouse spermatozoa has been widely used; however, fertility of frozen spermatozoa in some strains, especially when inseminating cryopreserved oocytes, is low and may be improved by assisted fertilization techniques. The present study was performed to investigate the effect of partial zona pellucida (ZP) digestion on the in vitro fertilization (IVF) capacity of frozen mouse spermatozoa. Mouse oocytes were subjected to partial ZP digestion using acidic Tyrode's solution (pH 3.1). Fertilization rates in digestion groups (30 or 45 s) were higher (P < 0.05) than that of zona-intact control (78.3% or 86.3% vs. 52.5%). The recovery rate at 45 s was lower (P < 0.05) than that at 30 s (84.2% vs. 97.3%). Among vitrified oocytes, the fertilization rate in treatment group (digested for 30 s) was higher (P < 0.05) than that of zona-intact group (50.8% vs. 22.1%). After embryo transfer at the two-cell stage, 17.7% and 11.8% of transferred embryos derived from fresh and vitrified digested oocytes developed to term and showed no significant difference as compared with that from zona-intact oocytes (24.1%, P > 0.05). These results indicate that partial ZP digestion improves IVF efficiency of fresh and vitrified oocytes with frozen mouse spermatozoa, which can provide valuable information for in vitro assisted fertilization using cryopreserved gametes in the re-establishment of mouse colonies.

Open-pulled straw (OPS) vitrification of in vitro fertilised mouse embryos at various stages.

The present study was designed to investigate the cryotolerance of in vitro fertilised (IVF) mouse embryos at various preimplantation developmental stages. IVF mouse embryos were vitrified by the open-pulled straw (OPS) method. After warming, embryos were morphologically evaluated and assessed by their development to blastocysts, hatched blastocysts or term. The results showed that a high proportion (93.3-100.0%) of vitrified embryos at all developmental stages were morphologically normal after recovery. The developmental rate of vitrified 1-cell embryos to blastocyst (40.0%) or hatched blastocyst (32.7%) or term (9.3%) was significantly lower than that from other stages (P < 0.05). Vitrified embryos from 2-cell to early blastocyst stage showed similar blastocyst (71.8-89.5%) and hatched blastocyst rates (61.1-69.6%) and could develop to term without a significant loss of survival compared with those of fresh embryos (P > 0.05). Vitrified 2-cell embryos showed the highest survival rate in vivo (50.6%, 88/174), compared with that from other stages (9.3-30.5%, P < 0.05). The data demonstrate that the OPS method is suitable for the cryopreservation of IVF mouse embryos from 2-cell stage to early blastocyst stage without a significant loss of survival. Embryos at the 2-cell stage had the best tolerance for cryopreservation in the present study.

Production of normal offspring from partially zona-incised vitrified mouse oocytes fertilized with cryopreserved spermatozoa using an optimized protocol.

The present study was designed to investigate the optimized conditions for cryopreservation of Kunming (KM) mice spermatozoa (Experiment 1) and to compare the developmental potential of IVF embryos produced from fresh oocytes (Group 1), vitrified-warmed oocytes without (Group 2) or with partial zona pellucida incised by a piezo manipulator (ZIP) (Group 3) fertilized with frozen-thawed spermatozoa (Experiment 2). In experiment 1, spermatozoa were cryopreserved with the medium containing raffinose and egg yolk with different concentrations (0 to 60 percent) and then followed by fertilization with fresh oocytes after thawing. The highest cleavage (76.2 percent) and blastocysts formation rates (63.6 percent) were obtained when the egg yolk concentration was adjusted to 30 percent. To optimize the equilibration time, the spermatozoa were equilibrated in the optimized medium for 0, 10, 30, 50, 70, 90 min at 40 degree C before plunging into liquid nitrogen. After thawing, the highest cleavage rate (87.4 percent) of IVF embryos was observed when equilibrated for 30 min. In experiment 2, the cleavage and blastocyst rates in Group 1 (81.2 percent, 65.4 percent) and Group 3 (72.5 percent, 45.0 percent) were higher (P less then 0.05) than those in Group 2 (22.2 percent and 13.9 percent), respectively. When 2-cell embryos obtained in Group 1 and 3 were transferred, 32.1 percent and 22.7 percent of embryos in the pregnant receipts developed to term, respectively. In conclusion, the optimized protocol is highly efficient for the cryopreservation of KM mice spermatozoa; the ZIP technique is very useful for improvement of the fertilization efficiency using the cryopreserved gametes and normal offspring can be produced efficiently.

Effect of in-straw thawing on in vitro- and in vivo-development of vitrified mouse morulae.

For the purpose of ascertaining parameters to embryo transfer on some domestic animals, mouse morulae were used as a model to investigate the effect of in-straw thawing on in vitro and in vivo-development of vitrified embryos. Embryos were vitrified in 0.25 ml straws preloaded with dilution solution (0.5 M Sucrose) and thawed in the straw by mixing the vitrification solution (Ethylene glycol + Ficoll 70 + Sucrose) and the dilution solution at 25 degrees C. The embryos were randomly divided into six groups and expelled from the straws after they had been suspended in the in-straw mixture for 3 min, 5 min, 8 min, 12 min, 16 min, and 20 min, respectively, and then they were collected under a microscope for in vitro culture or direct transfer. The in vitro developmental rates of the embryos were 92.3% to 98.4% and hatching rates were 64.1% to 75.6% for the groups of 3 min to 16 min, showing no significant differences with those of nonfrozen controls (100%, 76.2%; P > 0.05). While embryos were suspended in the straw for 20 min, the developmental rate (86.6%) and hatching rate (52.4%) were significant lower than those of the control (100%, 76.2%; P < 0.01). When the 168 frozen-thawed embryos (in-straw thawing for 5 min) and 168 fresh embryos were transferred, respectively, the proportion of live fetuses in the pregnant recipients between them (58.7% vs. 54.5%) showed no significant difference (P > 0.05). The data indicate that vitrification with EFS30 and suspension in the in-straw mixture for 3 min to 16 min, when thawing, did not affect the in vitro developmental rate and hatching rate. Moreover, the in vivo developmental rate between vitrified embryos and fresh embryos did not differ significantly. It can be concluded that this method is fit for nonsurgical embryo transfer in some domestic animals with a suggestion that the operation of embryo transfer should be accomplished within 16 min.

Vitrification solution containing DMSO and EG can induce parthenogenetic activation of in vitro matured ovine oocytes and decrease sperm penetration.

This study was designed to examine the reduced incidence of normal fertilization in vitrified ovine oocytes. After in vitro maturation for 24 h, the oocytes were randomly allocated into three groups: (1) untreated (control), (2) exposed to vitrification solution (VS) without being plunged into liquid nitrogen (toxicity), or (3) vitrified by open-pulled straw method (vitrification). In experiment 1, the treated and control oocytes were matured for another 2 h, and the oocytes were then in vitro fertilized for 12 h to examine sperm penetration. The percentage of monospermy in toxicity group (29.3%) and vitrification group (28.2%) dramatically decreased compared to the control group (45.0%) (P<0.05). To find the mechanism that the VS decreased the monospermy, some treated and control oocytes were used to test the distribution of CG and the resistance of zona pellucida (ZP) to 0.1% pronase E immediately (IVM 24 h), after another 2 h of maturation (IVM 26 h), and after 12 h of in vitro fertilization (IVF 12 h) respectively. Others were used to examine female pronucleus formation after 12 h of culture in fertilization medium with the absence of sperm. The results showed that the percentage of CG completely release in the oocytes (IVM 24 and 26 h) of toxicity group (41.2% and 39.9%) and vitrification group (41.7% and 51.7%) was significantly higher than that of control group (7.1% and 18.4%) (P<0.05). The ZP digestion duration in the oocytes (IVM 26 h) of the toxicity group (435.6 s) and vitrification group (422.3 s) was longer than that of control group (381.6 s) (P<0.05). The percentage of female pronucleus formation in toxicity group (58.7%) and vitrification group (63.9%) was higher than that (8.2%) of control group (P<0.05). The data above demonstrated that the VS containing DMSO and EG could parthenogenetically activate in vitro matured ovine oocytes, resulting in ZP hardening and decreased sperm penetration.