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.     

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.     

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.     

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.     

Preovulatory follicular fluid during in vitro maturation decreases polyspermic fertilization of cumulus-intact porcine oocytes in vitro maturation of porcine oocytes

Porcine follicular fluid (pFF), as a supplement of maturation media, has been shown several times to improve the in vitro production (IVP) of porcine embryos. As a transudate of serum, pFF contains locally produced factors in addition to the ones derived from serum. The objective of this study was to determine the additional positive effects of these pFF specific factors on the nuclear and cytoplasmic maturation of porcine oocytes. Follicular fluid and autologous serum were collected from sows in the preovulatory phase of the estrous cycle. Subsequently, oocytes from prepubertal gilts were matured in NCSU23 supplemented with either 10% pFF or 10% autologous serum derived from the same sow. Oocytes were then fertilized and the putative zygotes were cultured for 7 days. Nuclear maturation and cumulus expansion were assessed after the maturation culture. For evaluation of cytoplasmic maturation, oocyte glutathione (GSH) content, fertilization parameters and embryonic development were evaluated. After in vitro maturation (IVM) of the oocytes, both cumulus expansion rate and oocyte GSH content were increased for oocytes matured in pFF (P<0.05). More monospermic penetration was found when cumulus-intact oocytes had been matured in 10% pFF but this effect was lost after fertilization of cumulus denuded oocytes indicating that the pFF was acting through the cumulus. We speculate that the increased cumulus expansion and increased glutathione content, which were prevalent after IVM in pFF, are responsible for the positive effects on fertilization and the pre-implantation development of the embryos.     

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.     

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 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.     

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.     

Improvement in in?vitro fertilization outcome following in?vivo synchronization of oocyte maturation in mice

Synchronization of oocyte maturation in vitro has been shown to produce higher in vitro fertilization (IVF) rates than those observed in oocytes matured in vitro without synchronization. However, the increased IVF rates never exceeded those observed in oocytes matured in vivo without synchronization. This study was therefore designed to define the effect of in vivo synchronization of oocyte maturation on IVF rates. Mice were superovulated and orally treated with 7.5 mg cilostazol (CLZ), a phosphodiesterase 3A (PDE3A) inhibitor, to induce ovulation of immature oocytes at different stages depending on frequency and time of administration of CLZ. Mice treated with CLZ ovulated germinal vesicle (GV) or metaphase I (MI) oocytes that underwent maturation in vitro or in vivo (i.e. in the oviduct) followed by IVF. Superovulated control mice ovulated mature oocytes that underwent IVF directly upon collection. Ovulated MI oocytes matured in vitro or in vivo had similar maturation rates but significantly higher IVF rates, 2–4 cell embryos, than those observed in control oocytes. Ovulated GV oocytes matured in vitro showed similar maturation rates but significantly higher IVF rates than those observed in control oocytes. However, ovulated GV oocytes matured in vivo had significantly lower IVF rates than those noted in control oocytes. It is concluded that CLZ is able to synchronize oocyte maturation and improve IVF rates in superovulated mice. CLZ may be capable of showing similar effects in humans, especially since temporal arrest of human oocyte maturation with other PDE3A inhibitors in vitro was found to improve oocyte competence level. The capability of a clinically approved PDE3A inhibitor to improve oocyte fertilization rates in mice at doses extrapolated from human therapeutic doses suggests the potential scenario of the inclusion of CLZ in superovulation programs. This may improve IVF outcomes in infertile patients.     

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.     

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.     

The effects of resveratrol on porcine oocyte in vitro maturation and subsequent embryonic development after parthenogenetic activation and in vitro fertilization

We investigated the effects of resveratrol, a phytoalexin with various pharmacologic activities, on in vitro maturation (IVM) of porcine oocytes. We investigated intracellular glutathione (GSH) and reactive oxygen species (ROS) levels, as well as gene expression in mature oocytes, cumulus cells, and in vitro fertilization (IVF)-derived blastocysts, and subsequent embryonic development after parthenogenetic activation (PA) and IVF. After 44 h of IVM, no significant difference was observed in maturation of the 0.1, 0.5, and 2.0 μM resveratrol groups (83.0%, 84.1%, and 88.3%, respectively) compared with the control (84.1%), but the 10.0 μM resveratrol group showed significantly decreased nuclear maturation (75.0%) (P < 0.05). The 0.5- and 2.0-μm groups showed a significant (P < 0.05) increase in intracellular GSH levels compared with the control and 10.0 μM group. Intracellular ROS levels in oocytes matured with 2.0 μM resveratrol decreased significantly (P < 0.05) compared with those in the other groups. Oocytes treated with 2.0 μM resveratrol during IVM had significantly higher blastocyst formation rates and total cell numbers after PA (62.1% and 49.1 vs. 48.8%, and 41.4, respectively) and IVF (20.5% and 54.0 vs. 11.0% and 43.4, respectively) than the control group. Cumulus-oocytes complex treated with 2.0 μM resveratrol showed lower expression of apoptosis-related genes compared with mature oocytes and cumulus cells. Cumulus cells treated with 2.0 μM resveratrol showed higher (P < 0.05) expression of proliferating cell nuclear antigen than the control group. IVF-derived blastocysts derived from 2.0 μM resveratrol-treated oocytes also had less (P < 0.05) Bak expression than control IVF-derived blastocysts. In conclusion, 2.0 μM resveratrol supplementation during IVM improved the developmental potential of PA and IVF porcine embryos by increasing the intracellular GSH level, decreasing ROS level, and regulating gene expression during oocyte maturation.     

Expression pattern of glucose metabolism genes in relation to development rate of buffalo (Bubalus bubalis) oocytes and in vitro–produced embryos

The expression pattern of glucose metabolism genes (hexokinase, phosphofructokinase, glucose-6-phosphate dehydrogenase [G6PDH], lactate dehydrogenase [LDH], and pyruvate dehydrogenase [PDH]) were studied in buffalo in vitro–matured oocytes and in vitro–produced embryos cultured under different glucose concentrations (0 mM, 1.5 mM, 5.6 mM, and 10 mM) during in vitro maturation of oocytes and culture of IVF produced embryos. The expression of the genes varied significantly over the cleavage stages under different glucose concentrations. Developmental rate of embryos was highest under a constant glucose level (5.6 mM) throughout during maturation of oocytes and embryo culture. Expression pattern of glucose metabolism genes under optimum glucose level (5.6 mM) indicated that glycolysis is the major pathway of glucose metabolism during oocyte maturation and early embryonic stages (pre-maternal to zygotic transition [MZT]) and shifts to oxidative phosphorylation during post-MZT stages in buffalo embryos. Higher glucose level (10 mM) caused abrupt changes in gene expression and resulted in shifting toward anaerobic metabolism of glucose during post-MZT stages. This resulted in decreased development rate of embryos during post-MZT stages. High expression of LDH and PDH in the control groups (0 mM glucose) indicated that in absence of glucose, embryos try to use available pyruvate and lactate sources, but succumb to handle the post-MZT energy requirement, resulting to poor development rate. Expression pattern of G6PDH during oocyte maturation as well early embryonic development was found predictive of quality and development competence of oocytes/ embryos.     

In vitro embryo production in goats: Slaughterhouse and laparoscopic ovum pick up–derived oocytes have different kinetics and requirements regarding maturation media

A total of 3427 goat oocytes were used in this study to identify possible differences during in vitro embryo production from slaughterhouse or laparoscopic ovum pick up (LOPU) oocytes. In experiment 1, one complex, one semi-defined, and one simplified IVM media were compared using slaughterhouse oocytes. In experiment 2, we checked the effect of oocyte origin (slaughterhouse or LOPU) on the kinetics of maturation (18 vs. 22 vs. 26 hours) when submitted to semi-defined or simplified media. In experiment 3, we determined the differences in embryo development between slaughterhouse and LOPU oocytes when submitted to both media and then to IVF or parthenogenetic activation (PA). Embryos from all groups were vitrified, and their viability evaluated in vitro after thawing. In experiment 1, no difference (P > 0.05) was detected among treatments for maturation rate (metaphase II [MII]; 88% on average), cleavage (72%), blastocyst from the initial number of cumulus oocyte complexes (46%) or from the cleaved ones (63%), hatching rate (69%), and the total number of blastomeres (187). In experiment 2, there was no difference of MII rate between slaughterhouse oocytes cultured for 18 or 22 hours, whereas the MII rate increased significantly (P < 0.05) between 18 and 22 hours for LOPU oocytes in the simplified medium. Moreover, slaughterhouse oocytes cultured in simplified medium matured significantly faster than LOPU oocytes at 18 and 22 hours (P < 0.05). In experiment 3, cleavage rate was significantly greater (P < 0.001) in all four groups of embryos produced by PA than IVF. Interestingly, PA reached similar rates for slaughterhouse oocytes cultured in both media, but improved (P < 0.05) the cleavage rate of LOPU oocytes. Slaughterhouse oocytes had acceptable cleavage rate after IVF (∼67%), whereas LOPU oocytes displayed a lower one (∼38%), in contrast to cleavage after PA. The percentage of blastocysts in relation to cleaved embryos was not affected by the origin of the oocytes (P > 0.05). Therefore, slaughterhouse oocytes developed a greater proportion of blastocysts than LOPU ones, expressed as the percentage of total cumulus oocyte complexes entering to IVM. Vitrified-thawed blastocysts presented similar survival and hatching rates between the oocyte origin, media, or method of activation. In conclusion, slaughterhouse and LOPU derived oocytes may have different IVM kinetics and require different IVM and IVF conditions. Although the IVM and IVF systems still need improvements to enhance embryo yield, the in vitro development step is able to generate good quality embryos from LOPU-derived 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.     

Preliminary assessment of somatic cell nuclear transfer in the dromedary (Camelus dromedarius)

Somatic cloning may enable the maintenance/expansion of the population of camels with the highest potential for milk production or the best racing performances. However, there have been no reports of embryonic or somatic nuclear transfer in camels. The aim of this study was to produce dromedary embryos by nuclear transfer using in vitro matured oocytes and two somatic cells from two sources (adult fibroblasts or granulosa cells). A total of 58 adult females were superstimulated by a single dose of eCG (3500 IU). Ten days later, their ovaries were collected postmortem. Cumulus–oocytes-complexes (COCs) were aspirated from stimulated follicles and were matured in vitro for 30 h. Fibroblasts (from live adult male) and granulosa cells (from slaughtered adult females) were used as donor karyoplasts and injected into mature enucleated dromedary oocytes.The cleavage rate was significantly higher (P < 0.05) for embryos reconstructed with fibroblasts (59%) versus those with granulosa cells (45%). However, there was no difference between the two groups in the proportion of cloned embryos reaching the blastocyst stage (fibroblasts: 14% vs. granulosa cells: 15%) or those that hatched (fibroblasts: 10% vs. granulosa cells: 12%). The viability of reconstructed dromedary embryos from the two sources of donor cells (fibroblasts; n = 5 vs. granulosa cells; n = 7) was examined by transferring them to synchronized recipients. Two females (fibroblasts: 1/5; 20%, granulosa cells: 1/7; 14%) were confirmed pregnant by ultrasonography at 15 and 25 days following transfer. Later, the pregnancies were followed by pregnancy empirical-symptoms. These two pregnancies were lost between 25 and 60 days following transfer, respectively.In conclusion, the present study shows for the first time that the development of dromedary NT embryos derived from either adult fibroblasts or granulosa cells can occur in vitro and the transfer of these cloned embryos to recipients can result in pregnancies.