The role of L-carnitine in bovine embryo metabolism. A review of the effect of supplementation with a metabolic modulator on in vitro embryo production.

Early embryo development is driven first by the maternal RNAs and proteins accumulated during the oocyte's cytoplasmic maturation and then after the embryo genome activation. In mammalian cells, ATP generation occurs via oxidative pathways or by glycolysis, whereas in embryonic stem cells, the consumption of glucose, pyruvate, lipids, and amino acids results in ATP synthesis. Although the bovine embryo has energy reserves in glycogen and lipids, the glycogen concentration is deficient. Conversely, lipids represent the most abundant energy reservoir of bovine embryos, where lipid droplets-containing triacylglycerols are the main fatty acid stores. Oocytes of many mammalian species contain comparatively high amounts of lipids stored as droplets in the ooplasm. L-carnitine has been described as a cofactor that facilitates the mobilization of fatty acids present in the oocyte's cytoplasm into the mitochondria to facilitate ß-oxidation processes. However, the L-carnitine effects by addition to media in the in vitro produced embryos on the quality are highly disputed and contradictory by different researchers. This review's objective was to explore the effect that the addition of L-carnitine on culture media could have on the overall bovine embryo production in vitro, from the oocyte metabolism to the modulation of gene expression in the developing embryos.

Mechanism of the adverse effect of hyaluronidase used for oocyte denudation on early development of bovine embryos.

Hyaluronidase is widely used in animal and human assisted reproductive technologies (ARTs) to remove cumulus cells around oocytes. However, adverse effects of hyaluronidase treatment, such as increased rates of degeneration and parthenogenesis, have been found after treatment of human and mouse oocytes. Currently, the mechanism(s) of the detrimental effects are unclear. The present study was initiated to identify the mechanism of adverse responses to hyaluronidase treatment in bovine oocytes and early embryos. Cumulus cells were removed from cumulus-oocyte complexes (COCs) with or without hyaluronidase and the oocytes were subjected to intracytoplasmic sperm injection (ICSI) or in vitro fertilization (IVF). Significantly lower rates of blastocyst formation were obtained in the hyaluronidase treatment group after ICSI (22.4%) and IVF (21.2%) compared with the non-hyaluronidase control groups: 36.1% after ICSI and 30.4% after IVF. Next, we examined the effect of hyaluronidase on parthenogenetic development rates and on the cytoplasmic levels of free calcium ions (Ca2+), reactive oxygen species (ROS) and reduced glutathione (GSH). No differences in parthenogenesis rates were found between treated and untreated groups. Ca2+ levels in oocytes from the hyaluronidase treatment group indicated using mean fluorescence intensity were significantly higher (68.8 ± 5.3) compared with in the control group (45.0 ± 2.5). No differences were found in the levels of ROS or GSH between the treated and untreated groups. We conclude that hyaluronidase might trigger an increase in Ca2+ levels in oocytes, resulting in a decreased potential for normal embryonic development.

Rescue of vitrified-warmed bovine mature oocytes by short-term recovery culture with resveratrol.

Resveratrol, a well-known antioxidant, has been reported to protect mouse metaphase-II (M - II) stage oocytes from vitrification injuries when used as a treatment during a series of vitrification processes. The present study was conducted to investigate whether short-term treatment of post-warm bovine mature oocytes with resveratrol can increase blastocyst formation rate following in vitro fertilization and culture. Bovine denuded M - II oocytes were vitrified-warmed using Cryotop® or nylon mesh (pore size = 37 µm) as a cryodevice. The post-warm oocytes were treated for 2 h with 1 µM resveratrol in recovery culture medium. The resveratrol treatment had no harmful influence on morphological survival and cleavage rate of the oocytes vitrified-warmed with Cryotop® or nylon mesh. In the Cryotop® vitrification series, blastocyst formation rate of resveratrol-treated post-warm oocytes (39.0%) was not significantly different from that of non-treated post-warm oocytes (31.7%). However in the nylon mesh vitrification series, there was a significant increase in the blastocyst yield (42.4% vs. 31.3%, P < 0.05) when post-warm oocytes were treated with resveratrol. Blastocyst yield from fresh control oocytes was 49%. Levels of reactive oxygen species were comparable between post-warm and fresh control M - II oocytes, and decreased in oocytes after recovery culture with resveratrol. Mitochondrial activity of post-warm oocytes was restored to the pre-vitrification level during the recovery culture regardless of resveratrol supplementation. Thus, short-term recovery culture with resveratrol can rescue bovine M - II oocytes vitrified-warmed on a nylon mesh cryodevice.

Antioxidant response element activator, BTZO-1, improves the developmental competence of bovine oocytes.

Increased reactive oxygen species (ROS) generation may disrupt the oocytes function and their competence. In this study, we introduced BTZO-1, a new supplement that can regulate the oxidative stress. Addition of BTZO-1 during IVM of bovine oocytes improved their developmental competence in the term of improvement of blastocyst rates. In addition, the quality of the produced embryos was improved by decreasing the apoptosis level by showing a decreased number of TUNNEL positive cells.

Corpora lutea affect in vitro maturation of bovine cumulus-oocyte complexes and embryonic development after fertilization with sex-sorted or conventional semen.

Influence of both the presence of a corpus luteum on the ovary and semen sex-sorting on development following in vitro fertilization is not yet conclusive. To determine the effect of these factors, 376 bovine oocytes were processed in vitro according to luteal presence on the ovary (CL+ and CL-) and type of semen used (sexed or conventional). Maturation rate was higher (P < 0.01) in CL- (136/138; 98.6%) than in CL+ (217/238; 91.2%). Cleavage rate was lower (P < 0.01) in CL+ with sexed semen (60/172; 34.9%) than in CL- with sexed semen (42/71; 59.1%), CL+ with conventional semen (47/66; 71.2%), and CL- with conventional semen (54/67; 85.1%). Compaction was similar (P = 0.69) in CL- (49/99; 49.4%) and CL+ (50/107; 46.7%). Blastulation rate was higher (P < 0.01) in CL- (26/99, 26.2%) than in CL+ (13/107; 12.1%) group. Expansion rate was higher (P = 0.01) in CL- (22/99; 22%) than in CL+ (11/107; 10.2%) group. Compaction rates were similar (P = 0.78) in sex-sorted (50/102; 49.0%) or conventional semen (49/104; 47.1%) groups. Blastulation was also similar (P = 0.91) with sex-sorted semen (19/102; 18.6%) and conventional semen (20/104; 19.2%). The rate of expanded blastocysts was similar (P = 0.89) in sex-sorted (16/102; 15.6%) and conventional (17/104; 16.3%) semen groups. In conclusion, the presence of CL can compromise maturation of the oocytes and their development, as a higher proportion of cleavage-stage embryos can be obtained with non-sexed semen with oocytes from ovaries without a CL.

Usefulness of modified Medium RD as a chemically defined medium for in vitro maturation of bovine oocytes.

PURPOSE: In the present study, I evaluated the usefulness of Medium RD, with mixed RPMI1640 and Dulbecco's modified Eagle's medium (1:1, v/v), as a chemically defined medium for in vitro maturation (IVM) of bovine oocytes. METHODS: In vitro maturation was performed in 10 mmol/L HEPES-buffered TCM199 (mTCM199), 10 mmol/L HEPES-buffered Medium RD (mRD), and mTCM199 supplemented with fetal bovine serum fraction (mTCM199 + FBS fraction) that served as control. Cumulus-oocyte complexes were matured for 24 hours in three different media supplemented with follicle-stimulating hormone, estradiol-17ß, and polyvinylpyrrolidone. Nuclear maturation of oocytes, their developmental competence into blastocysts after in vitro fertilization (IVF) and mitochondrial distribution in oocytes were investigated. RESULTS: There was no difference in the ratio of matured oocytes regardless of IVM media. The percentage of morula stage was higher in mRD than in mTCM199 group (P < .05) at 120-144 hours after IVF, although the blastocyst rates between groups were not significantly different at 168-216 hours. IVM in mRD increased the percentage of oocytes with diffused mitochondrial distribution compared with the immature and mTCM199 and had similar percentage of oocytes in mTCM199 + FBS fraction. CONCLUSIONS: Medium RD would be useful as a chemically defined medium for IVM of bovine oocytes.

Nylon mesh cryodevice for bovine mature oocytes, easily removable excess vitrification solution.

The aim of this study was to determine pore size of nylon mesh (NM) device suitable for cryosurvival of bovine mature oocytes and to apply the device to vitrification of large quantities of the oocytes. Ten to twelve oocytes were loaded onto an NM device (a square opening 37-, 57- or 77-µm on a side length). After removal of the excess volume of vitrification solution by paper absorption, the oocytes were vitrified-warmed, fertilized and cultured in vitro. Oocyte recovery and morphological survival were comparable among the three groups. However, blastocyst yield in the 37-µm group (39%) was higher than that in the 77-µm group (28%), and the yield in the 57-µm group (31%) was the intermediate. The 37-µm NM device was applicable for increased oocyte number >40 (blastocyst yield, 33%). These results suggest that 37-µm-pore sized NM can serve as cryodevice to vitrify large quantities of in vitro-matured bovine oocytes.

Effect of vitrification on the zona pellucida hardening and follistatin and cathepsin B genes expression and developmental competence of in vitro matured bovine oocytes.

The purpose of our study was to assess the effect of vitrification with or without the presence of calcium in the vitrification solution on the: 1) diameter of oocytes and thickness of the zona pellucida, 2) zona pellucida hardening, 3) expression of mRNA follistatin (FST) and cathepsin B (CTSB) in oocytes and 4) developmental competence of embryos derived from in vitro matured and vitrified oocytes. The results of our study demonstrate, that vitrification did not alter thickness of the zona pellucida and diameter of the oocytes, however it triggered hardening of the zona pellucida. The presence of calcium in the vitrification solutions intensified hardening of zona in immature and mature oocytes (P < 0.04, P < 0.001, respectively) and provoked increased mRNA FST expression in oocytes matured in vitro compared to immature oocytes (P < 0.01) and those vitrified without calcium (P < 0.004). CTSB mRNA expression was increased in immature oocytes and oocytes vitrified with calcium compare to mature oocytes (P < 0.02). The developmental potential of vitrified oocytes was impaired compared to non-vitrified oocytes, being more evident in oocytes vitrified with calcium. In summary, vitrification did not change the oocyte diameter and thickness of the zona pellucida and expression of FST and CTSB mRNA. It diminished developmental potential of the vitrified oocytes. The presence of calcium in the vitrification solutions increased hardening of zona pellucida as well as affected the level of FST and CTSB mRNA in oocytes and developmental potential of these oocytes.

Dynamic changes of histone H3 lysine 9 following trimethylation in bovine oocytes and pre-implantation embryos.

OBJECTIVES: We have examined dynamic changes of histone H3 lysine 9 following trimethylation (H3K9me3), the mRNA expression levels of SUV39H1 and SUV39H2 in bovine oocytes and the role in the development of in vitro fertilization (IVF) pre-implantation embryos. RESULTS: There were strong H3K9me3 signals in germinal vesicle (GV) oocytes but no signals in MII oocytes. H3K9me3 signals were maintained during IVF pre-implantation embryo development. SUV39H1 and SUV39H2 showed significantly higher mRNA expression levels in GV oocytes than MII oocytes (P < 0.01). SUV39H1 showed high mRNA expression level in two-cell embryos, however, SUV39H2 showed high mRNA expression level in four-cell embryos. In other development stage, SUV39H1 and SUV39H2 showed low expression levels. CONCLUSION: Bovine IVF pre-implantation embryos maintain strong H3K9me3 signals and SUV39H1 and SUV39H2 are highly expressed at the early development stage of pre-implantation embryos.

Pretreatment of in vitro matured bovine oocytes with docetaxel before vitrification: Effects on cytoskeleton integrity and developmental ability after warming.

The stabilization of spindle fibersis important for successful vitrification of bovine oocytes because microtubules and other cytoskeleton fibers (CSF) can be damaged during vitrification, resulting in failure of fertilization after thawing. Docetaxel, a stabilizing agent, could potentially reduce CSF damage of bovine oocytes induced during vitrification. However, there have been no reports on the effects of docetaxel on their vitrification. Experiment 1 was conducted to investigate the effects of various doses of docetaxel (0.0, 0.05, 0.5, 5.0 and 50 µM) in preincubation medium of in vitro matured (IVM) bovine oocytes on their developmental ability after in vitro fertilization (IVF). The results show that 0.05 µM docetaxel had no adverse effect on embryo development, while docetaxel at a concentration of ⩾0.5 µM inhibited development. Experiments 2 and 3 were conducted to investigate the effects of preincubation of IVM bovine oocytes with 0.05 µM docetaxel for 30 min prior to vitrification-warming on CSF integrity (Experiment 2), and on oocyte survival and viability after IVF (Experiment 3). When preincubated with 0.05 µM docetaxel for 30 min before vitrification, post-thawed oocytes had less CSF damage and higher survival rates compared with those untreated with docetaxel before vitrification. Surviving oocytes also had higher rates of cleavage and development to the blastocyst stage after IVF. In conclusion, preincubation of IVM bovine oocytes with 0.05 µM docetaxel for 30 min before vitrification was effective at preventing CSF damage during vitrification, and improving oocyte viability after warming and subsequent cleavage and blastocyst formation after IVF.

Mitochondria-Targeted Antioxidant MitoQ Improves In Vitro Maturation and Subsequent Embryonic Development from Culled Cows.

The purpose of this study was to investigate the effects and mechanisms of MitoQ on the IVM of culled bovine oocytes and subsequent embryonic development. The results revealed that in comparison to the control group (0 µmol/L), the IVM rate (p < 0.05) and subsequent blastocyst rate (p < 0.05) of the low-concentration 1 and 5 µmol/L MitoQ treatment group were increased. The level of ROS (p < 0.05) in the MitoQ treatment group was decreased in comparison to the control group. Additionally, the level of GSH, MMP, ATP, and mt-DNA in the MitoQ treatment group was increased (p < 0.05) in comparison to the control group. The expression level of BAX was decreased (p < 0.05) in the MitoQ treatment group, and the BCL2, DNM1, Mfn2, SOD, and CAT were increased (p < 0.05). In conclusion, MitoQ improved mitochondrial dysfunction, increased mitochondrial activity during IVM, and reduced oxidative stress, resulting in increased IVM rates and subsequent embryonic development from culled cows.

Folic acids promote in vitro maturation of bovine oocytes by inhibition of ferroptosis via upregulated glutathione and downregulated Fe2+ accumulation.

Bovine embryos by in vitro fertilization have become the primary source of commercial embryo transfers globally. However, the developmental capacity of in vitro maturation (IVM) oocytes is considerably lower than that of in vivo maturation (IVO) oocytes, owing to the production of reactive oxygen species (ROS) via mitochondrial metabolism, which was higher in IVM oocytes than in IVO oocytes. To avoid the negative effects of ROS on embryo quality, folic acid (FA) was supplemented directly into the IVM medium to antagonize ROS production, however, the mechanisms remain unknown. In the present study, five levels of FA (0, 25, 50, 100, and 200 µM) were supplemented into the bovine oocyte culture medium. The maturation, cleavage, and blastocyst formation rates increased by 8.95 %, 6.94 %, and 4.36 %, respectively, in the 50 µM group compared to the 0 µM group. Moreover, 7904 differential genes were identified between 0 µM and 50 µM groups by transcriptome sequencing, and they were mainly enriched in 8 pathways. The glutathione, ROS, and Fe2+ levels in oocytes were found to be associated with ferroptosis. Our results revealed that 50 µM FA promoted the IVM of bovine oocytes and affected the expression of genes involved in the ferroptosis pathway. The downregulation of TFR1 and STEAP3 led to a decrease in intracellular Fe2+ accumulation, and the upregulation of GCL increased oocyte GSH levels, thereby reducing the production of ROS in the ferroptosis pathway. Our study provides a new insight into the molecular mechanisms by which FA promotes bovine oocyte development in vitro.

Effects of limonin on oxidative stress and early apoptosis in oocytes during in vitro maturation.

To investigate the effects of limonin (Lim) on oxidative stress and early apoptosis in bovine oocytes during in vitro maturation (IVM), different concentrations of Lim (0, 10, 20, 50 µmol/L) were added to bovine IVM medium. Oocyte maturation rates and development 24 h after in vitro fertilization (IVF) were examined to determine the optimal Lim concentration. The optimal Lim concentration was added to the IVM medium, and 0 µmol/L Lim was used as the control. Immunofluorescence staining was used to detect the abnormal rate of spindle assembly, reactive oxygen species (ROS), glutathione (GSH), mitochondrial membrane potential (MMP) levels, mitochondrial distribution, and the fluorescence intensity of cathepsin B (CB)-active LC3 protein. RT‒qPCR was used to detect the mRNA expression levels of antioxidant-, apoptosis- and autophagy-related genes in oocytes. The total number of blastocysts and the proportion of apoptotic cells among blastocysts were detected. The results showed that the PBI ejection rate, cleavage rate and blastocyst rate of bovine oocytes in the 20 µmol/L Lim group were significantly higher than those in the control group (P < 0.05). Compared with those in the control group, ROS levels, abnormal mitochondrial distribution, the proportion of abnormal spindle assembly, CB activity and LC3 protein fluorescence intensity of oocytes in the 20 µmol/L Lim group were significantly decreased (P < 0.05), and GSH and MMP levels were significantly increased (P < 0.05). The expression of antioxidant genes (Prdx3, Prdx6, Sirt1) and antiapoptotic genes (Bcl-xl, Survivin) were significantly upregulated (P < 0.05), and the expression levels of proapoptotic genes (Caspase-4, BAX) and autophagy-related genes (LC3) were significantly downregulated (P < 0.05). The total number of cells among in vitro fertilized embryos was significantly increased (P < 0.05), and the apoptosis rate of blastocysts was significantly decreased (P < 0.05). Here, we show that Lim exerts positive effects on bovine oocyte IVM by regulating REDOX homeostasis, reducing spindle damage and enhancing mitochondrial function during IVM, thereby inhibiting oocyte apoptosis and autophagy.

Cytokine-Supplemented Maturation Medium Enhances Cytoplasmic and Nuclear Maturation in Bovine Oocytes.

Bovine in vitro oocyte maturation (IVM) is an easy way to obtain oocytes for subsequent assisted reproductive techniques but is inefficient compared to in vivo maturation. Supplementation of three cytokines, fibroblast growth factor 2 (FGF2), leukemia inhibitory factor (LIF), and insulin-like growth factor 1 (IGF1), or FLI, has increased oocyte maturation and embryo development in multiple species, but studies have not explored the oocyte differences caused by FLI IVM supplementation. This study aimed to assess important nuclear and cytoplasmic maturation events in high-quality oocytes. FLI-supplemented oocytes had a decreased GV (3.0% vs. 13.7%, p < 0.01) and increased telophase I incidence (34.6% vs. 17.6%, p < 0.05) after IVM, increased normal meiotic spindles (68.8% vs. 50.0%, p < 0.001), and an increased nuclear maturation rate (75.1% vs. 66.8%, p < 0.001). Moreover, in metaphase II oocytes, the percentage of FLI-treated oocytes with a diffuse mitochondrial distribution was higher (87.7% vs. 77.5%, p < 0.05) and with a cortical mitochondrial distribution was lower (11.6% vs. 17.4%, p < 0.05). Additionally, FLI-supplemented oocytes had more pattern I cortical granules (21.3% vs. 14.4%, p < 0.05). These data suggest that FLI supplementation in bovine in vitro maturation medium coordinates nuclear and cytoplasmic maturation to produce higher-quality oocytes.

Rescue of vitrified-warmed bovine oocytes with rho-associated coiled-coil kinase inhibitor.

Cryotolerance of matured bovine oocytes is not fully practical even though a promising vitrification procedure with a ultrarapid cooling rate was applied. The present study was conducted to investigate whether recovery culture of vitrified-warmed bovine oocytes with an inhibitor (Y-27632) of Rho-associated coiled-coil kinase (ROCK) can improve the developmental potential after in vitro fertilization (IVF) and in vitro culture. Immediately after warming, almost all oocytes appeared to be morphological normal. Treatment of the postwarming oocytes with 10 µM Y-27632 for 2 h resulted in the significantly higher oocyte survival rate before IVF as well as higher cleavage rate and blastocyst formation rate. Quality analysis of the resultant blastocysts in terms of total cell number and apoptotic cell ratio also showed the positive effect of the Y-27632 treatment. Time-dependent change in mitochondrial activity of the vitrified-warmed oocytes was not influenced by ROCK inhibition during the period of recovery culture. However, the ability of ooplasm to support single-aster formation was improved by the ROCK inhibition. Thus, inhibition of ROCK activity in vitrified-warmed bovine oocytes during a short-term recovery culture can lead to higher developmental competence, probably due to decreased apoptosis and normalized function of the microtubule-organizing center.

Dendrobine enhances bovine oocyte maturation and subsequent embryonic development and quality.

Strategies for improving the quality of oocytes have important theoretical and practical significance for increasing the efficiency of livestock breeding. In this respect, the accumulation of reactive oxygen species (ROS) is a major factor affecting the development of oocytes and embryos. This study investigated the effects of Dendrobium nobile extract (DNE) on the in vitro maturation of bovine oocytes and embryonic development after IVF. DNE is an extract from Dendrobium rhizomes that contains alkaloids with anti-inflammatory, anti-cancer and anti-ageing functions. Various concentrations of DNE (0, 5, 10, 20 and 50 µmol/L) were added during oocyte maturation in vitro, and we found that 10 µmol/L of DNE remarkably increased the oocyte maturation rate, the subsequent blastocyst formation rate and embryo quality. Further, we found that DNE treatment decreased the frequency of spindle/chromosome defects and ROS and increased the oocyte glutathione and mitochondrial membrane potential in oocytes. Moreover, DNE upregulated the expression of oxidative stress-related genes (Sirt1, Sirt2, Sirt3 and Sod1) in oocytes and apoptosis-related genes (Caspase-3, Caspase-4, Bax, Bcl-xl and Survivin) in blastocysts. These results suggest that DNE supplementation can promote oocyte maturation and subsequent embryonic development by regulating redox reactions and inhibiting embryonic apoptosis.

Current Advances in Bovine In Vitro Maturation and Embryo Production Using Different Antioxidants: A Review.

In vitro maturation (IVM) is one of the most important steps in in vitro embryo production (IVEP). It is a complicated procedure in which nuclear and cytoplasmatic changes in oocytes appear. In order to carry out the in vitro maturation procedure correctly, it is necessary to provide the oocytes with as close to a natural (in vivo) environment as possible. Many factors contribute to the overall poor quality of in vitro-matured oocytes. One important factor may be oxidative stress (OS). The generation of oxidants, such as reactive oxygen species, is common under culture conditions. The solution for OC treatment and prevention is antioxidants. In the last 5 years, many studies have examined different antioxidants and their effects on in vitro maturation of oocytes and embryo production. The aim of this systematic review was to present the achievements of scientific research in the last five years, in which the effects of many antioxidants were tested on bovine oocyte maturation and embryo production.

Cryodevices developed for minimum volume cooling vitrification of bovine oocytes.

Unfertilized bovine oocytes can be efficiently cryopreserved only when an extremely rapid cooling rate (>20,000°C/min) is applied to oocytes with a very limited amount of surrounding vitrification solution. This protocol is defined as minimum volume cooling (MVC) vitrification. Various types of cryodevices, such as open pulled straw, Cryoloop, and Cryotop, have been developed to accelerate the cooling efficacy. Furthermore, hollow fibers with nano-scale pores, triangle nylon mesh sheets, and multilayer silk fibroin sheets have been optimized for the loading of large quantities of oocytes and/or the subsequent removal of excess vitrification solution, without requiring skillful operation to transfer individual oocytes using fine capillaries. This article provides an up-to-date review of cryodevices suitable for the MVC vitrification of bovine oocytes at the immature (germinal vesicle-) and mature (metaphase II-) stages.

Effect of Supplementation of Cryoprotectant Solution with Hydroxypropyl Cellulose for Vitrification of Bovine Oocytes.

The technology of successful cryopreservation is a very important factor in research and commercial applications. However, the survival and development of the vitrified-thawed (VT) oocytes are lower than those of non-vitrified-thawed (non-VT) oocytes. This study investigated the effect of the addition of hydroxypropyl cellulose (HPC) to a vitrification solution of bovine oocytes. For the vitrification, bovine metaphase II oocytes were pretreated with a solution containing 10% ethylene glycol supplemented with 0, 10, 50, or 100 µg/mL HPC for 5 min, then exposed to a solution containing 30% ethylene glycol supplemented with 0, 10, 50, or 100 µg/mL HPC for 30 sec, and then directly plunged into liquid nitrogen. Oocytes exposed to 0, 10, 50, and 100 µg/mL HPC were named the 0, 10, 50, and 100 HPC groups, respectively. Samples were thawed via sequential incubation in Dulbecco's phosphate-buffered saline (D-BPS) supplemented with 10% fetal bovine serum and decreasing concentrations of sucrose (1, 0.5, 0.25, and 0.125 M) for 1 min each time. After thawing, VT oocytes were treated at 0.05% hyaluronidase, and cumulus cells were removed by mechanical pipetting. The oocytes were washed with HEPES-buffered Tyrode's medium and incubated in a droplet of previously cultured in vitro maturation medium for 1 h to recover. The survival rate of the oocytes was significantly higher in the 50 HPC group (84.2%) than in the 0 (75.4%), 10 (80.4%), and 100 (75.5%) HPC groups. The reactive oxygen species (ROS) levels of the non-VT and 50 HPC groups were lower than the 0, 10, and 100 HPC groups. The mRNA levels of proapoptotic genes (Bax) were lower in the non-VT, 0, and 50 HPC groups than in the other groups. The mRNA expression levels of antiapoptotic genes (BCl2) was higher in the non-VT than in the other groups. The mRNA level of a stress-related gene (Hsp70) was lower in the 50 HPC than in the other groups. At day 8, the developmental capacity of embryos obtained via parthenogenetic activation (PA) was determined in the non-VT, 0 HPC, and 50 HPC groups. The cleavage rate of the non-VT group was significantly higher, but the blastocyst development rate and total cell number per blastocyst did not significantly differ between the non-VT and 50 HPC groups. The mRNA levels of proapoptotic genes (Bax and Caspase-3) and a stress-related gene (Hsp70) were higher in the 0 HPC group than in the non-VT and 50 HPC groups. In conclusion, supplementation of vitrification solution with HPC improves the survival rate of VT bovine oocytes and the development capacity of embryos derived from these oocytes via PA.

Evaluation of fertilization capability of frozen-thawed completely immotile spermatozoa collected from a white bengal tiger after interspecific ICSI with bovine oocytes.

The objective of this study was to evaluate the fertilization capability of White Bengal Tiger frozen-thawed completely immotile spermatozoa after interspecific intracytoplasmic sperm injection (ICSI) with bovine oocytes. The fertilization status of presumptive zygotes was assessed 18 h after ICSI by immunofluorescence staining and confocal microscopy. The fertilization rate was 34.8% (8/23), as confirmed by the extrusion of two polar bodies, or male and female pronuclei formation. For unfertilized oocytes (65.2%, 15/23), one activated oocyte had an activated spermatozoon but most were unactivated oocytes with unactivated spermatozoa (1/15, 6.7% vs 10/15, 66.7%, respectively, p < 0.05). These results showed that White Bengal Tiger frozen-thawed completely immotile spermatozoa retained the capacity to fertilize bovine oocytes after interspecific ICSI. This is the first report of in vitro produced zygotes using tiger immotile sperm with bovine oocytes by interspecific ICSI technique, which provides an efficient and feasible method for preservation and utilization of endangered feline animals.