Cryopreservation of human failed maturation oocytes shows that vitrification gives superior outcomes to slow cooling

This study investigated whether failed maturation oocytes could be used to evaluate different cryopreservation procedures. A total of 289 failed maturation oocytes (GV and MI stages), obtained from 169 patients undergoing IVF treatment (mean age 33.84 ± 5.0) were divided into two different slow-cooling groups (1.5 mol/l 1,2-propanediol + 0.2 mol/l sucrose in either NaCl (group A) or choline chloride (ChCl) (group B) based cryopreservation solutions) and one vitrification group (15% ethylene glycol + 15% dimethyl sulphoxide). Survival rate, in vitro maturation (IVM) rate, fertilization and developmental rate of cryopreserved oocytes were assessed. Regardless of the stage at which cryopreservation was performed (GV + MI), the slow cooling with ChCl based medium always gave significantly lower survival rate than the slow cooling in NaCl based medium (p = 0.01) and vitrification (p < 0.001). An extended study also showed statistically reduced survival rate between slow-cooling NaCl based medium and vitrification (p < 0.05). Global results of in vitro maturation and fertilization showed worse results between both slow-cooling NaCl and ChCl based media versus vitrification. In conclusion, for oocytes that had failed to mature, vitrification gave better survival, maturation, fertilization and also cleavage rates than the slow-cooling protocols. Four cells embryos were obtained only from vitrified in vitro matured MI oocytes.     

The effect of relaxin supplementation of in vitro maturation medium on the development of cat oocytes obtained from ovaries stored at 4 °C

Relaxin is a member of the insulin-like family of hormones that promotes growth in a number of reproductive tissues, including the granulosa and theca cells. Cat oocytes collected from cold-stored ovaries remain capable of maturing in vitro, but the developmental ability of the oocytes decreases after 24 h of cold storage. To improve the developmental ability of cat oocytes from cold-stored ovaries, we investigated the effect of relaxin supplementation of maturation medium on their meiotic ability and subsequent development. Cat oocytes were collected from ovaries stored at 4 °C for one day and cultured in maturation medium supplemented with different concentrations (0, 10, 20, and 40 ng/ml) of relaxin for 24 h. They were then fertilized in vitro for 12 h with frozen-thawed spermatozoa. After in vitro fertilization, the zygotes were cultured in synthetic oviduct fluid medium for 8 days. There were no significant differences in the maturation rates and glutathione contents of oocytes among the groups, irrespective of relaxin supplementation. The rate of blastocyst formation from oocytes matured with 10 ng/ml relaxin (16.0%) was higher (p < 0.05) than that from oocytes matured without relaxin (5.9%). Our findings indicate that supplementation of 10 ng/ml relaxin into maturation medium may improve blastocyst formation of cat oocytes after in vitro fertilization.     

Effect of the exposure to methyl-β-cyclodextrin prior to chilling or vitrification on the viability of bovine immature oocytes

The present study aimed to evaluate the effect of methyl-β-cyclodextrin (MβCD) as a cholesterol loader to change oocyte plasma membrane and increase its tolerance toward cryopreservation. The first and second experiments were conducted to investigate if MβCD could improve nuclear and cytoplasmic maturation after oocyte exposure to cold stress for 10 or 30 min, respectively. No differences (P > 0.05) in either experiment in the metaphase II (MII) rate of oocytes exposed to MβCD and cold stress; but these oocytes presented lower maturation rates than control groups. In the second experiment, a lower percentage of oocytes showed degenerated chromatin (P < 0.05) after exposure to 2 mg/mL of MβCD compared to the group exposed to 0 mg/mL. However, no differences among treatments were observed in cytoplasmic maturation. Groups exposed to cold stress demonstrated a lower (P < 0.05) capacity for embryonic development compared to the control groups. In the third experiment immature oocytes were exposed to MβCD and then, vitrified (cryotop). After warming, we observed that the ability to reach MII and chromatin degeneration were altered (P < 0.05) by MβCD. The blastocysts rate (P < 0.05) on D7 was higher in the 2 mg/mL MβCD group, but an identical finding was not observed on D8 (P > 0.05). Chromatin degeneration was higher in the vitrification groups. We conclude that MβCD improved nuclear maturation by reducing oocyte degeneration after cold stress or vitrification; however, more studies are required to clarify the usefulness of MβCD use in oocyte cryopreservation.     

Developmental competence and gene expression of immature oocytes following liquid helium vitrification in bovine

The objective of this study was to develop an effective ultra-rapid vitrification method and evaluate its effect on maturation, developmental competence and development-related gene expression in bovine immature oocytes. Bovine cumulus oocyte complexes were randomly allocated into three groups: (1) controls, (2) liquid nitrogen vitrification, and (3) liquid helium vitrification. Oocytes were vitrified and then warmed, the percentage of morphologically normal oocytes in liquid helium group (89.0%) was significantly higher (P < 0.05) than that of the liquid nitrogen group (81.1%). When the vitrified–thawed oocytes were matured in vitro for 24 h, the maturation rate in liquid helium group (50.6%) was higher (P < 0.05) than liquid nitrogen group (42.6%). Oocytes of liquid helium vitrification had higher cleavage and blastocyst rates (41.1% and 10.0%) than that of liquid nitrogen vitrification (33.0% and 4.5%; P < 0.05) after in vitro fertilization. Moreover, the expression of GDF9 (growth/differentiation factor-9), BAX (apoptosis factor) and ZAR1 (zygote arrest 1) was analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) when the vitrified–thawed oocytes were matured 24 h. The expression of these genes was altered after vitrification. Expression of GDF9 and BAX in the liquid helium vitrification group was not significantly different from that of the control, however there were significant differences between the liquid nitrogen vitrification group and control. In conclusion, it was feasible to use liquid helium for vitrifying bovine immature oocytes. There existed an association between the compromised developmental competence and the altered expression levels of these genes for the vitrified oocytes.     

Production of channel catfish with sperm cryopreserved by rapid non-equilibrium cooling

This report describes the feasibility of using vitrification for fish sperm. Vitrification can be used to preserve samples in the field and offers an alternative to conventional cryopreservation, although it has not been systematically studied for sperm of aquatic species. The overall goal of the project was to develop streamlined protocols that could be integrated into a standardized approach for vitrification of aquatic species germplasm. The objectives of the present study in channel catfish (Ictalurus punctatus) were to: (1) evaluate the acute toxicity of 5%, 10%, 20% and 30% methanol, N,N-dimethyl acetamide, dimethyl sulfoxide, 1,2-propanediol, and methyl glycol; (2) evaluate a range of devices commonly used for cryopreservation and vitrification of mammalian sperm; (3) compare vitrification with and without cryoprotectants; (4) evaluate the post-thaw membrane integrity of sperm vitrified in different cryoprotectant solutions, and (5) evaluate the ability of vitrified sperm to fertilize eggs. Cryoprotectant concentrations of higher than 20% were found to be toxic to sperm. Methanol and methyl glycol were the least toxic at a concentration of 20% with an exposure time of less than 5 min. We evaluated a method reported for human sperm, using small volumes in loops (15 μl) or cut standard straws (20 μl) with and without cryoprotectants plunged into liquid nitrogen. Cryoprotectant-free vitrification using loops did not yield fertilization (assessed by neurulation), and the fertilization rates observed in two trials using the cut standard straws were low (∼2%). In general, fertilization values for vitrification experiments were low and the use of low concentrations of cryoprotectants yielded lower fertilization (<10%) than the use of vitrification solutions containing high cryoprotectant concentrations (as high as 25%). The highest neurulation obtained was from a mixture of three cryoprotectants (20% methanol + 10% methyl glycol + 10% propanediol) with a single-step addition. This was reflected in the flow cytometry data from which the highest membrane integrity using loops was for 20% methanol + 10% methyl glycol + 10% propanediol (∼50%). We report the first successful sperm vitrification in fish and production of offspring from vitrified sperm in channel catfish. Although the fertilization values were low, at present this technique could nevertheless be used to reconstitute lines (especially in small aquarium fishes), but it would require improvement and scaling up before being useful as a production method for large-bodied fishes such as catfish.     

The effects of EGF and IGF-1 on FSH-mediated in vitro maturation of domestic cat oocytes derived from follicular and luteal stages

The objective of this study was to evaluate the influence of epidermal growth factor (EGF) and insulin like growth factor-I (IGF-1) on the in vitro maturation of cat oocytes recovered from follicular and luteal stage ovaries. Oocytes from follicular (n = 580) and luteal (n = 209) stages were harvested and divided into four groups, which were cultured in FSH-mediated maturation medium supplemented with: (1) EGF alone (25 ng/mL); (2) IGF-1 alone (100 ng/mL); (3) EGF + IGF-1 (25 ng/mL EGF + 100 ng/mL IGF-I); or (4) no growth factor (control). The proportion of follicular stage oocytes reaching the metaphase II stage was significantly higher than that of oocytes obtained at the luteal stage in both control and study groups (p < 0.001). The percentages of oocytes reaching the metaphase II stage during the follicular period were 62.6% in control; 70.9% in EGF; 72.8% in IGF-1, and 78.1% in EGF + IGF-1 groups, whereas the respective values for gametes collected from luteal stage ovaries were 12.5%, 17.5%, 12.5%, and 16.9%. Additionally, the differences between the study and control groups were significant in the case of follicular stage oocytes. Finally, supplementing the maturation medium with EGF and/or IGF-1 significantly enhanced the meiotic maturation of oocytes recovered from follicular stage ovaries. The present study also demonstrated that the combination of EGF and IGF-I provides an additional or synergic effect on meiotic maturation of oocytes recovered from the follicular stage.     

Effects of estrous cycle stage and transport temperature of ovaries on in vitro maturation of canine oocytes

Unlike other domestic animals, in vitro maturation (IVM) of canine oocytes still has limited success. The present study investigated the effects of estrous cycle stage and transport temperature of ovaries on in vitro maturation of canine oocytes. The donor bitches were categorized into three groups based on stage of estrus cycle: follicular (proestrus or estrous), luteal (diestrus) and anestrus. One ovary of each pair collected from 39 mature bitches was transported in Phosphate Buffer Saline (PBS) at 4 °C while the other was transported at 37 °C. A total of 1138 Grade I COCs obtained from all ovaries were grouped and matured in modified synthetic oviduct fluid (mSOF) supplemented with follicle stimulating hormone (FSH), luteinizing hormone (LH), essential and non-essential amino acids at 38.5 °C in a humidified 5% CO₂, 5% O₂, and 90% N₂ atmosphere for 72 h. The nuclear maturation rates were evaluated by aceto–orcein staining.Oocytes harvested from follicular and luteal ovaries have a significantly higher maturation rates (MI + MII) than the oocytes from anestrual ovaries in the 37 °C group (p < 0.05). However, oocytes harvested from anestrual ovaries transported at 4 °C had the highest maturation (MI + MII) rate, and the difference between anestrual and luteal ovary groups was significant (p < 0.05). The oocytes from anestrual ovaries transported at 4 °C have significantly higher maturation rates than those transported at 37 °C (p < 0.0001). However, the transport temperature (37 or 4 °C) did not significantly affect the maturation (MI + MII) rates of oocytes harvested from the luteal (p = 0.61) and follicular (p = 0.48) stage ovaries.It can be concluded from this study that (1) both transport temperature and transport temperature × estrus cycle stage interaction effected the maturation rates, while estrus cycle stage alone did not, and (2) transporting canine ovaries at 4 °C can improve in vitro maturation rates in oocytes harvested from anestrous ovaries.     

Effect of ammonium during in vitro maturation on oocyte nuclear maturation and subsequent embryonic development in pigs

The effects of ammonium in a chemically defined maturation medium on oocyte nuclear maturation and subsequent embryonic development of pigs after in vitro fertilization (IVF) and parthenogenetic activation (PA) were examined. Cumulus–oocyte complexes were matured in Purdue Porcine Medium (PPM) supplemented with 0 mM, 0.02 mM, 0.2 mM, 2 mM, or 20 mM ammonium chloride, or TCM199 with 10% porcine follicle fluid (TCM + pFF; positive control) at 38.7 °C in 7% CO₂ in air for 40–44 h. No significant difference (P > 0.05) in nuclear maturation was found between oocytes matured in TCM + pFF or PPM with 0 mM, 0.02 mM and 0.2 mM ammonium chloride. However, nuclear maturation was decreased (P < 0.05) in oocytes matured in PPM with 2 mM or 20 mM ammonium. After IVF, oocytes matured in PPM with 20 mM ammonium resulted in embryos with reduced (P < 0.05) embryonic cleavage and blastocyst development than all other treatment groups. After PA, oocytes matured in PPM with 20 mM ammonium resulted in embryos with lesser (P < 0.05) embryonic cleavage compared to TCM + pFF. However, PA embryos derived from oocytes matured in PPM with both 2 mM and 20 mM ammonium had reduced (P < 0.05) blastocyst development compared with TCM + pFF. These results demonstrate the detrimental effect of ammonium during in vitro oocyte maturation on nuclear progression to metaphase II. Additionally, the presence of ammonium during in vitro maturation negatively influences subsequent embryonic development, although PA embryos appear to be more sensitive to the negative effects of ammonium during oocyte maturation than do IVF embryos.     

Effect of serum albumin supplementation on in vitro capacitation and fertilization of caprine oocytes

The aim of the investigation was to study the effect of purity and the type of serum albumin on in vitro fertilization (IVF) and cleavage rate of in vitro matured goat oocytes. Ovaries were collected from the local abattoir and transported within 4 h to the laboratory in warm saline (37 °C) containing 100 IU penicillin-G and 100 μg streptomycin sulfate per ml. A total of 2509 cumulus oocyte complexes (COCs) were collected from 1313 ovaries. Oocytes were matured in TCM-199 medium containing FSH (5 μg/ml), LH (5 μg/ml) and estradiol-17β (1 μg/ml), supplemented with 20% fetal bovine serum at 38.5 °C and 5% CO₂ in an incubator under humidified air for 27 h. After 27 h of in vitro maturation (IVM), oocytes were denuded, washed and randomly divided into 4 groups. Group 1 consisted of in vitro matured oocytes (n = 627) co-incubated with sperm in a 50 μl drop of TALP medium containing a 6 mg/ml crystalline bovine serum albumin (BSA) fraction V and 10 μg/ml heparin. Group 2 was comprised of in vitro matured oocytes (n = 470), co-incubated with sperm in a 50 μl drop of TALP medium containing 3 mg/ml crystalline BSA fraction V, 10% estrous goat serum and 10 μg/ml heparin. Group 3 was comprised of in vitro matured oocytes (n = 489) co-incubated with sperm in a 50 μl drop of TALP medium containing a 6 mg/ml fatty acid free BSA and 10 μg/ml heparin. Group 4 consisted of in vitro matured oocytes (n = 422) co-incubated with sperm in a 50 μl drop of TALP medium containing 20% estrous goat serum and 10 μg/ml heparin. After 18 h of co-incubation, the oocyte–sperm mixture was washed in the culture medium 15–20 times and cultured in 50 μl EDM. Cleavage of the in vitro fertilized oocytes were recorded 48 h post-insemination under an inverted phase contrast microscope. The average oocyte recovery rate/ovary and maturation rate was 1.91% and 80.03%, respectively. The cleavage rate in Group 1, Group 2, Group 3 and Group 4 was 1.59%, 8.93%, 11.86% and 35.30%, respectively. It could be concluded that the use of fatty acid free albumin resulted in a significantly higher (P < 0.05) cleavage rate, compared to unmodified albumin, and the supplementation of 20% estrous goat serum in the fertilization medium, significantly (P < 0.05) increased the cleavage rate of in vitro matured goat oocytes, compared to defatted albumin.     

Effect of vitrification on human oocyte maturation rate during in vitro maturation procedure: A systematic review and meta-analysis

Combination of in vitro maturation (IVM) and cryopreservation offers new opportunities for women with contraindication in ovarian stimulation, and females who desire to postpone the childbearing due to different problems. There are still controversies regarding IVM procedure and its impact on oocytes fertilization capability. This systematic review and meta-analysis were conducted to evaluate the impact of vitrification on human oocyte maturation rate during IVM procedure. In this review, we searched Medline, Embase, Scopus and ISI web of science to identify English-language studies. The last search was implemented on 3 February 2018. The original articles which assessed maturation rate after vitrification of MI or GV oocytes were included. Animal trials and the studies that performed cryopreservation using slow-freeze method were excluded. Bias and quality assessments were performed. 2476 articles were screened primarily. After duplication removing and the application of inclusion and exclusion criteria, 14 studies included for the analysis. All studies compared maturation rate between the oocytes that were vitrified at the GV or MI stage before maturation and oocytes which were matured in vitro without vitrification. Meta-analysis showed that oocyte vitrification at GV stage had a significant negative impact on maturation rate (RR = 0.76, 95% CI: 0.66–0.88); I² = 85.2%; P = 0.000). Finally, based on our results, oocyte vitrification decreases the maturation rate by 24%.     

In vitro production of bovine embryos in medium supplemented with a serum replacer: Effects on blastocyst development,cryotolerance and survival to term

In this study, we evaluated a serum replacer (SR; Knockout SR^®, Invitrogen) in our in vitro culture systems. We hypothesized that SR would benefit bovine embryo development, since SR supported survival of embryonic stem cells (which originate from embryos). Experiment 1 compared oocyte maturation with SR versus fetal bovine serum (FBS). Following fertilization, blastocyst development was lower for oocytes matured with SR (21.5 versus 34.1, P < 0.05). Experiment 2 evaluated SR for culturing embryos. Following fertilization, embryos were cultured for 3 days in KSOM, and then assigned to treatments: (1) KSOM static culture (KNM); (2) fresh KSOM (KD3); (3) KSOM + SR or (4) KSOM + FBS and cultured to Day 7 (fertilization = Day 0). Blastocyst development in FBS or SR was higher than either KNM or KD3 (48.2, 47.2, 32.7, and 35.5, respectively, P < 0.05). Experiment 3 evaluated cryosurvival of embryos cultured in the same manner as Experiment 2. On Day 7, embryos were vitrified and upon warming, embryos cultured in SR had greater 24 h survival rates (70.6%) than all other treatments (P < 0.05). Finally, Experiment 4 evaluated effects of SR on pregnancy rate and development to term. Culture in SR was not detrimental to pregnancy or calving rates (50 and 50%, respectively), and SR calves had normal birth weights (mean = 38.8 kg ± 1.5). In conclusion, the use of SR for maturation of oocytes was not beneficial; however, SR enhanced embryo culture by improving development in vitro, cryotolerance and survival, effectively replacing serum in culture.     

Cumulus cell layers as a critical factor in meiotic competence and cumulus expansion of ovine oocytes

A critical stage in the optimization of in vitro maturation (IVM) is the selection of good quality oocytes. There exists a relationship between the size of the cumulus investment and the in vitro developmental ability of the cumulus–oocyte complex (COC), which provides a basis for the selection of the COCs. This study was designed to evaluate the effect of the number of cumulus cell layers which enclose the oocytes, on the in vitro maturation, cytoplasm quality and cumulus expansion of the ovine oocytes. Ovaries were obtained from an abattoir and transported to the laboratory within 1–2 h, at 37 °C. Oocytes (n = 535) were recovered by means of an aspiration pump (set at a flow rate of 10 mL H₂O/min), with a disposable 20 G needle attached. Oocytes were divided into four classes (classes I to IV – with more than 5, 3–4, 1–2 and no cumulus cell layers, respectively) and separately cultured in a TCM199 medium for 24 h. The morphology of oocytes was evaluated following in vitro culture (IVC) to assess cumulus expansion, cytoplasm quality (score I with a homogenous cytoplasm and II with granulated cytoplasm) and nuclear maturation stage. The percentage of maximum cumulus expansion for classes I to III oocytes were 53.0 ± 1.0, 36.3 ± 2.2 and 16.3 ± 1.8% respectively. The rate of meiotic resumption of oocytes in classes I to IV were 77.0 ± 2.7, 77.2 ± 1.9, 53.0 ± 2.1 and 2.7 ± 1.1% respectively. The proportion of oocytes with a cytoplasm quality I in oocyte classes I to IV were 62.8 ± 1.5, 59.4 ± 1.2, 36.4 ± 2.1 and 0.5 ± 1.1%, respectively. Results showed that the presence of ≥3 cumulus cell layers in the COC prior to IVM led to a better (p < 0.05) cumulus expansion, meiotic resumption and cytoplasmic maturation rate. Thus the morphological grading of immature ovine oocytes may be an appropriate selection criterion regarding their developmental ability.     

Growth and antrum formation of bovine primary follicles in long-term culture in vitro

Successful antral formation in vitro from bovine preantral follicles (145–170 μm) has been described previously, but antrum formation from the primary follicle (50–70 μm) has not yet been achieved in vitro. The aim of the study was to establish an optimal culture system supporting the growth and maturation of bovine primary follicles (50–70 μm) in vitro. Bovine primary follicles were cultured in a three-dimensional culture system for 13 or 21 days in alpha-minimum essential medium. Various treatments including follicle stimulating hormone (FSH), luteinizing hormone (LH), 17β-estradiol (E₂), basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) were tested. The follicular diameter and antrum formation rate were recorded, and follicular maturation markers (P450 aromatase, CYP19A1; anti-Mullerian hormone, AMH; growth differentiation factor-9, GDF9; bone morphogenetic protein-15, BMP15; and type III transforming growth factor β receptor, TGFβR3) were analyzed by real-time RT-PCR. After 21 days of culture under each treatment condition, the follicular diameter was significantly enlarged in the presence of FSH + LH + E₂ + bFGF or FSH + LH + E₂ + bFGF + EGF (p < 0.05). An addition of 50 ng/ml bFGF or bFGF + 25 ng/ml EGF initiated antrum formation by day 19 and day 17 of culture, and the antral cavity formation rate was 16.7% and 33.3% by 21 days of culture, respectively. The expression of follicular maturation markers (CYP19A1, AMH, GDF9, BMP15 and TGFβR3) was significantly altered. We conclude that addition of 50 ng/ml bFGF + 25 ng/ml EGF to media containing FSH + LH + E₂ turned out to be the most effective optimized culture conditions to support the growth and maturation of bovine primary follicles in vitro.     

Development to the blastocyst stage of immature pig oocytes arrested before the metaphase-II stage and fertilized in vitro

In vitro fertilization (IVF) and embryonic development of mature and meiotically arrested porcine oocytes were compared in the present study. After in vitro maturation (IVM) of cumulus-oocyte complexes for 48 h, 75.4% of them extruded a visible polar body (PB). Most of the oocytes with a first polar body (PB+ group) were at the metaphase-II (M-II) stage (91.4%). Most of the oocytes without a visible polar body (PB− group) appeared to be arrested at the germinal vesicle (GV) (41.6%) and metaphase-I (M-I) (34.0%) stages. After IVF of oocytes (day of IVF = Day 0), there was no difference between PB⁺ and PB⁻ groups in rates of sperm penetration, mono-spermy, however oocyte activation rate after penetration was greater in the PB+ than in the PB− group (P < 0.05). On Day 2, there was no difference between rates of embryos cleaved at the 2–4 cell stages in PB+ and PB− groups (42.1 ± 48.8% and 33.6 ± 2.1%, respectively). On Day 4, the rate of PB+ embryos developing beyond the 4-cell stage was greater than that of PB− embryos (P < 0.05, 31.7 ± 3.9% and 14.1 ± 1.5%, respectively), and PB+ embryos had more cells than the PB− embryos (P < 0.05, 8.3 ± 0.4 and 6.0 ± 0.8 cells, respectively). On Day 6, a greater proportion of PB+ embryos developed to the blastocyst stage than did PB− embryos (P < 0.05, 34.6 ± 2.4% and 20.7 ± 2.8%, respectively). However, when the GV oocytes of the PB− group were not included in recalculations, there was no difference in blastocyst rates between M-I arrested and M-II oocytes (35.3 and 34.6%, respectively). The number of blastomere nuclei in embryos obtained from the PB+ group (52.0 ± 2.5) was greater than that from the PB− group (P < 0.05, 29.1 ± 2.8). The proportion of degenerated parts in the blastocysts, as determined by morphological appearance, was the same in the PB+ and PB− groups. Although the quality of PB+ embryos was enhanced as compared with that of the PB− group, the proportion of inner cell mass and trophectoderm cells in PB+ and PB− blastocysts did not differ (1:1.9 and 1:2.2, respectively). Chromosome analysis revealed that PB+ blastocysts had more diploidy (P < 0.05, 69.7%) than did PB− blastocysts (44.0%), whereas PB− blastocysts had more triploid cells (P < 0.05, 34.0%) than did PB+ oocytes (8.4%). These results indicate that pig oocytes arrested before the M-II stage (M-I oocytes) undergo cytoplasmic maturation during maturation culture and have the same ability to develop to blastocysts after IVF as M-II oocytes, but some of them resulted in degeneration or delayed development with poor embryo quality.     

The presence of cumulus cells on nuclear maturation of sheep oocytes during in vitro maturation

This study was carried out to investigate the role of maturation by cumulus cells and the initial bond between the cumulus cells and the oocyte on nuclear maturation of sheep oocytes. Ovaries collected from the local abattoir were transported to the laboratory in saline at 30–35 °C within 1–3 h after collection. The oocytes of follicles, 2–6 mm in diameter, were recovered by aspiration and collected in a pre-incubated (at 38.6 °C, 5% CO₂, and 100% humidity) Hepes-modified TCM 199 medium. After preliminary evaluation, the oocytes with evenly granulated cytoplasm and which were surrounded with at least two layers of cumulus cells (good quality oocytes) were selected and subjected to culture in pre-incubated bicarbonate-buffered TCM 199 supplemented with 0.05 IU/ml recombinant human follicle stimulating hormone (rhFSH), 1 IU/ml human chorionic gonadotropin (hCG), and 1 μg/ml estradiol (OCM: oocyte culture medium). Before culturing, the selected oocytes were randomly divided into four treatment groups: Group 1, cumulus enclosed oocytes cultured in OCM; Group 2, denuded oocytes cultured in OCM; Group 3, denuded oocytes co-cultured with a cumulus cell monolayer in OCM; Group 4, denuded oocytes cultured in OCM in the presence of cumulus cells-conditioned medium. After an incubation period (26–27 h), the nuclear status of the oocytes in each treatment group was assessed using a 2% orcein stain. The rate of oocytes reaching the metaphase II (MII) stage (metaphase of second stage of meiosis division) was 82%, 5%, 11%, and 47% for Groups 1, 2, 3, and 4, respectively. The differences between groups were significantly (P < 0.05) different. The percentage of MII oocytes in Group 4 (47%) was higher than that obtained in Group 3 (11%), indicating a higher efficiency in a cumulus cell-conditioned medium, compared to the cumulus cells monolayer in providing the proper condition for sheep oocyte nuclear maturation. The results suggest the ability of sheep oocytes to resume meiosis in the absence of gap junctional communication (GJC) between the cumulus cells and oocyte being drastically interrupted while for optimum oocyte nuclear maturation, the intact physical contact between the oocyte and cumulus cells is essential.     

Development and subsequent cryotolerance of domestic cat embryos cultured in serum-free and serum-containing media

The objectives of this study were to examine the effects of the presence or absence of serum during the in vitro culturing period of domestic cat embryos on their developmental potential into blastocysts as well as their tolerance to cryopreservation using a slow-freezing method. In vitro-fertilized cat oocytes were incubated in a modified synthetic oviduct fluid (mSOF) containing 4 mg/mL bovine serum albumin (BSA) throughout culturing (BSA group) or in mSOF containing 4 mg/mL BSA for the first 3 days followed by mSOF containing 5% fetal bovine serum (FBS group). The developmental potential of the embryos to the blastocyst and expanded blastocyst stages was evaluated 7 days after in vitro fertilization. The blastocysts were frozen-thawed by the slow-freezing method and cultured for 3 days to examine their viability in vitro. There were no differences in the formation rates of blastocysts or expanded blastocysts, or number of cells in the embryos between the two groups. After cryopreservation, the hatching rates of the expanded blastocysts in the BSA group were significantly higher (P < 0.05) than those of the FBS group. The postthaw viability of blastocysts was lower than that of expanded blastocysts irrespective of culture medium. These results indicate that the developmental potential of cat embryos cultured in serum-free medium is comparable to those cultured in serum-containing medium. Furthermore, expanded blastocysts produced without serum exhibit better postthaw viability than those produced with serum.     

Measurement of the size of intracellular ice crystals in mouse oocytes using a melting point depression method and the influence of intracellular solute concentrations

Characterization of intracellular ice formed during the cooling procedures of cells significantly benefits the development and optimization design of cryopreservation or cryosurgery techniques. In this study, we investigated the influence of the concentration of extracellular non-permeable and permeable solutes on the melting points of the intracellular ice in mouse oocytes using cryomicroscopy. The results showed that the melting points of the intracellular ice are always lower than the extracellular ice. Based on this observation and the Gibbs–Thomson relation, we established a physical model to calculate the size of intracellular ice crystals and described its relationship with the concentrations of intracellular permeating solutes and macromolecules. This model predicts that the increased concentration of macromolecules in cells, by increasing the extracellular non-permeating solute concentration, can significantly lower the required concentration of permeable solutes for intracellular vitrification. The prediction was tested through the cryomicroscopic observation of the co-existence of intracellular vitrification and extracellular crystallization during cooling at 100 °C/min when the extracellular solutions contain 5 molal (m) ethylene glycol and 0.3 to 0.6 m NaCl.