301 INTRACYTOPLASMIC SPERM INJECTION (ICSI) MEDIATED GENE TRANSFER ASSISTED BY ACTIVATION WITH A DOUBLE EXPOSURE TO IONOMYCIN AND 6-DIMETHYLAMINOPURINE OR DEHYDROLEUCODINE

Intracytoplasmic sperm injection-mediated gene transfer (ICSI-MGT) is a powerful technique in the production of transgenic mice. However, it has not yet been exploited in cattle because of ICSI fertilization problems. The objective of this study was to evaluate fertilization rates, transgenesis, blastocysts rates, and enhanced green fluorescent protein (egfp) blastocyst quality parameters (mean cell number, Oct-4 expression, and day of formation) of ICSI-MGT derived bovine embryos treated with 5 different chemical activation protocols. COCs were aspirated from ovaries obtained from local slaughterhouses and IVM was performed using conventional protocols. Sperm samples were frozen/thawed by standard procedures. Coincubation of spermatozoa with DNA construct (pCX-EGFP) was carried out in 2.8% Na citrate, with 0.5 μg of plasmid per million spermatozoa for 5 min at 0°C. Then, spermatozoa were injected into metaphase II (MII) oocytes. Injected oocytes were treated with 5 μM ionomycin (Io) for 4 min, and after 3 h, incubated in 1.9 mM DMAP for 3 h (Io-DMAP); second Io followed by DMAP for 3 h (2Io-DMAP), second Io alone (2Io); 7% ethanol for 5 min (Io-EtOH); or 20 mM SrCl2 for 5 h (Io-SrCl2) immediately after first Io. Embryos were cultured in SOFaa medium. Fertilization efficiency (2 pronuclei and 2 polar bodies) at 16 h after ICSI and condensed sperm head presence at Day 4 were evaluated by Hoechst 33342 staining of ICSI-MGT embryos. Expression of EGFP was observed under the fluorescence microscope (488 nm). Oct-4 expression was detected by immunocytochemistry and confocal laser scanning microscopy. Each experiment was replicated 3 times. Differences between percentages (Fisher test) or means (ANOVA) were significant at P < 0.05. Fertilization rates did not differ between treatments. Transgenic and nontransgenic embryos were obtained by ICSI-MGT. All transgenic embryos were the product of ICSI fertilization (no condensed sperm head present at Day 4). On the other hand, at least 60% of nontransgenic embryos (>4 cells) were the result of parthenogenesis (condensed sperm present). Blastocyst rates after 2Io-DMAP (21/81; 25.9%) were not statistically different from Io-DMAP (15/80; 18.7%) or Io-EtOH (15/102; 14.7%) but they were higher than 2Io (8/85; 9.4%) or Io-SrCl2 (13/119; 10.9%). Transgenesis rates were higher for Io-DMAP (42/80; 52.3%), 2Io-DMAP (43/81; 53%) and Io-SrCl2 (51/119; 42.8%) than for 2Io (24/85; 28.2%) and Io-EtOH (30/102; 29.4%). Over 80% of the blastocysts were transgenic.An explant was derived from an egfp-blastocyst and it proliferated and expressed the transgene for 17 days. Mean cell numbers, Oct-4 expression, and day of formation (≤ Day 7 for all treatments except for 2Io) of the ICSI-MGT egfp-blastocysts were not statistically different neither between ICSI assistance treatments nor to IVF or parthenogenetic (Io-DMAP) controls, indicating similar embryo quality according to these parameters. All treatments proved to be highly efficient to assist ICSI-MGT and to produce transgenic bovine embryos. Moreover, ICSI-MGT with an early expressed marker can help to evaluate ICSI fertilization real efficiency.

Dehydroleucodine (DhL) is a sesquiterpene lactone that inhibits germinal vesicle breakdown in Bufo arenarum oocytes. Its action takes place over early stages of the cdc25 activation cascade (Bühler MI et al. 2007 Zygote 15, 183–187). The aim of this study was to evaluate the potential of DhL to induce parthenogenetic activation by observing nuclear dynamics and second polar body (2PB) extrusion of bovine oocytes, in the presence or absence of Cytochalasin B (CB), comparing these treatments with 6-Dimethylaminopurine (DMAP), an activation agent widely used. Cumulus–oocyte complexes were collected from cow ovaries obtained from a slaughterhouse. They were matured in TCM 199, supplemented with 5% FCS, 10 UI mL–1 penicillin, 10 μg mL–1 FSH, 100 μM cysteamine, 0.3 mm sodium pyruvate and 2 mm glutamine, at 39°C under 6% CO2 in air for 24 h. After removal of cumulus cells, metaphase II (MII) oocytes were selected and treated with 5 μm ionomycin (Io) for 4 min. Afterwards, oocytes were randomly allocated into one of the following treatments: a) incubation with 2 mm DMAP for 3 h (DMAP); b) incubation with 5 μm DhL for 3 h (DhL); and c) incubation with 5 μm DhL and 5 μg mL–1 CB, for 3 h (DhL-CB). A control group was only treated with Io. Activated oocytes were cultured in the maturation medium during 4, 11 or 17 h (Io exposure = 0 h), stained with Hoechst 33342 and analyzed under fluorescence microscope to evaluate nuclear stage and 2PB extrusion. Activation data are presented in Table 1. Oocytes with two extruded polar bodies and a metaphase plate were considered as partially activated (PA) and those exhibiting one pronucleus (PN) or already cleaved, as fully activated (FA). Oocytes that remained arrested at MII were not included in the table. Rates of 2PB emission were 98.3, 4.9, 83.6 and 61.5% for Io, DMAP, DhL and DhL-CB, respectively. These percentages were determined over total number of activated oocytes (PA and FA) within each group, including results from all evaluation times because no differences were found between them. Nuclear evaluation suggests that DhL is as effective as DMAP to induce full activation when combined with CB, and its use does not induce the early PN formation observed with DMAP at 4 h post Io. Most of the oocytes activated with DhL extruded a 2PB; these results were statistically different from those observed for other groups. These results indicate that DhL might be a useful agent to induce parthenogenesis, allowing 2PB extrusion and avoiding early PN formation in bovine oocytes. Table 1.Partial and full activation of bovine oocytes at 4, 11 and 17 h post treatments

The present work was conducted to examine (1) the morphology of dromedary cumulus-oocytes complexes (COCs), (2) to study the incidence of spontaneous development of oocytes in vivo and (3) to assess the ability of in vitro matured dromedary oocytes to chemical parthenogenetic activation compared with in vitro fertilized (IVF) oocytes. COCs were recovered from dromedary ovaries classified according to their morphology into six categories. Oocyte diameter was measured using eye piece micrometer. For chemical activation, COCs with at least three layers of cumulus-cells were in vitro matured (IVM) in TCM 199 + 10 microg/ml FSH + 10 IU hCG/ml + 10% FCS + 50 microg/ml gentamycin. COCs were incubated for 40 h at 38.5 degrees C under 5% CO2 in humidified air. After IVM, matured oocytes with first polar body (first Pb) were divided into two groups. Group 1: activated in 7% ethanol (E) for 5 min followed by culture in 2 mM 6-dimethylaminopurin (6-DMAP, E D, subgroup 1) or 10 microg/ml cycloheximide (CHX, E CHX, subgroup 2) for 3.5 h at 38.5 degrees C under 5% CO2. In group 2, oocytes were activated using 50 microM Ca A23187 (Ca A) for 5 min followed by culture in 2 mM 6-DMAP (Ca D, subgroup 3) or 10 microg/ml CHX(Ca CHX, subgroup 4) for 3.5 h at 38.5 degrees C under 5% CO2. For control group, IVM oocytes were fertilized using frozen-thawed camel spermatozoa separated by swim-up method then suspended in Fert-TALP medium supplemented with 6 mg/ml BSA (FAF) + 10 microg/ml heparin. In all groups, oocytes were in vitro cultured in SOFaa medium + 5% FCS and 5 microg/ml insulin + 50 microg/ml gentamycin. Cleavage rate and embryo development were checked on Days 2, 5 and 8. An average of 11.3 +/- 0.3 COCs were recovered/dromedary ovary. Categories 1 and 2 represented 33.1% and 34.8%, respectively, and were significantly higher (p < 0.01) than the other categories (19.1, 9.2 and 2.6% for categories 3-5, respectively). Category 6 (embryo-like structures) represented 1.2% of the recovered oocytes, staining of these embryo-like structures with orcien dye indicated the presence of divided cells with condensed nuclei. Dromedary oocytes averaged 166.2 +/- 2.6 microm in diameter with black cytoplasm. Chemical activation of IVM dromedary oocyte with first Pb in 7% ethanol or 50 microM Ca A followed by culture in 2 mM 6-DMAP showed significantly higher (p < 0.01) cleavage and developmental rates to the morula stage than oocytes activated using 7% ethanol or 50 microM Ca A followed by 10 microg/ml CHX or in vitro fertilized control group. Higher (p < 0.01) proportion of oocytes sequentially cultured in 10 microg/ml CHX or that in vitro fertilized were arrested at the 2-4-cell stage compared with that cultured in 6-DMAP.

Mature oocytes are arrested in metaphase II due to the presence of high levels of active maturation promoting factor (MPF). After fertilization, active MPF levels decline abruptly, enabling oocytes to complete meiosis II. One of the first and universal events of oocyte activation is an increase in cytosolic Ca2+ that would be responsible for MPF inactivation. Mature oocytes can also be activated by parthenogenetic activation. The aims of this work are to test the ability of dehydroleucodine (DhL) and its hydrogenated derivative 11,13-dihydro-dehydroleucodine (2H-DhL) to induce chemical activation in amphibian oocytes and to study the participation of calcium in the process. Results indicated that DhL and 2H-DhL induced oocyte activation in a dose-dependent manner. After 90 min of treatment, DhL 36 μM was able to induce 95% activation, while 2H-DhL 36 μM was less active, with only 40% activation. Our results suggest that DhL induced the inhibition of MPF activity, probably by an increase in intracellular Ca2+ concentration. Extracellular Ca2+ would not be significant, although Ca2+ release from intracellular stores is critical. In this sense, IP3Rs and RyRs were involved in the Ca2+ transient induced by lactones. In this species, RyRs appears to be the largest contributor to Ca2+ release in DhL-induced activation. Although more studies are needed on the mechanism of action through which these lactones induce oocyte activation in Rhinella arenarum, the results of this research provide interesting perspectives for the use of these lactones as chemical activators in in vitro fertilization and cloning.

Haploid activation of bovine oocytes is important for reproductive technologies such as intracytoplasmic sperm injection (ICSI) or somatic cell nuclear transfer (SCNT). Nevertheless, it is still a highly inefficient procedure. The aim of this work was to combine different activation drugs, known to have different targets along the activation cascade, to find a more effective activation protocol. Cumulus-oocyte complexes (COC) were aspirated from slaughtered ovaries and in vitro-matured (IVM) for 22 h. Oocytes were activated with 5 µM ionomycin (IO) for 4 min and then randomly allocated into 1 of the following treatments: 50 µM roscovitine (ROSC), 10 µg mL–1 cycloheximide (CHX), ROSC and 10 µM PD0325901 (ROSC/PD), or CHX and PD (CHX/PD) for 5 h; 15 µM dehydroleucodine (DHL) or DHL and ROSC (DHL/ROSC) for 3 h; DHL and CHX for 3 h followed by 2 h with CHX; 5-min exposure to 7% ethanol 4 h post-IO (ET); or ET followed by ROSC (ET-ROSC). Controls were IO followed by 3 h of exposure to 1.9 mM 6-DMAP with or without a previous 3-h culture in TCM-199 (3 h in DMAP and DMAP, respectively). Embryos were cultured in SOF medium. Pronuclear formation (PN) and second polar body extrusion (2PB) were assessed by 5 µg mL–1 propidium iodide oocyte staining, 17 h after IO. Activation was defined as the presence of at least 1 PN, and 2PB extrusion rate was calculated regardless of the nuclear stage. Data were analysed by Fisher’s Test (P &lt; 0.05). Activation (Table 1) was similar in all groups, with the exception of ROSC/PD and ET-ROSC that were the highest and DHL that was the lowest. Although ROSC or CHX seemed to improve 2PB rate when combined with DHL, cleavage decreased significantly, suggesting DHL itself, or its combination with these drugs, negatively affects embryo development. Group ET showed activation rates comparable to other treatments, but it was not reflected on cleavage, suggesting that ET induces PN formation but it might be inefficient to trigger embryo development. Nevertheless, this observation was not made for ET-ROSC, as it showed a higher cleavage rate than ET and ROSC alone. The mitogen-activated protein kinase (MAPK) inhibitor PD showed different effects when combined with ROSC or CHX, despite that they both act on the mammary fat pad (MPF). In ROSC/PD, a slight improvement was observed on activation and cleavage rates compared with ROSC. Group CHX/PD resulted in a slightly higher 2PB percentage, but a lower activation percentage that derived in a significantly lower cleavage than CHX. In conclusion, ROSC and CHX were the most effective single treatments for haploid activation. Moreover, some combined treatments, namely DHL/ROSC and DHL/CHX, proved to be as effective or better at 2PB extrusion rate, which is the defining feature in haploid activation. Table 1.Activation, second polar body extrusion (2PB) and cleavage of bovine oocytes activated with ionomycin followed by single or combined activating agents1

Experiments were conducted to study in vitro maturation of pre-pubertal goat oocytes and their developmental potential after chemical activation. Cumulus-oocyte complexes (n=1170) collected from the ovaries of pre-pubertal goats slaughtered at a local abattoir were matured in TCM-199 supplemented with 0.15mg mL−1 l-glutamine, 0.25mM sodium pyruvate, 0.1mM l-cysteine, 20ng mL−1 epidermal growth factor, 10mg mL−1 FSH, 10mg mL−1 LH, 1μg mL−1 oestradiol and 10% FCS for 24h at 39°C under 5% CO2 in humidified air. In Experiment 1, matured oocytes were activated (r=6) with either 5mM ionomycin (n=85) or 7% ethanol (n=91) followed by culture in 6-DMAP for 4h. All the activated oocytes were then cultured in KSOM supplemented with 3mg mL−1 BSA and were fixed and stained with Hoechst 33342 after 18h of culture to evaluate their activation status. In Experiment 2, oocytes activated with 5mM ionomycin and 6-DMAP were cultured for 7 days (r=6) in 1 of the 4 different culture media [Charles Rosenkrans medium (CR-1), modified TCM-199, KSOM and SOF] to study their developmental potential. All media were supplemented with 3.0mg mL−1 BSA for the first 3 days and 10% FCS for the subsequent 4 days. Of these pre-pubertal oocytes, 59% reached metaphase II stage, and 83% of these oocytes were classified as activated in the group using ionomycin in comparison with 69% in the group using ethanol as an activating agent (P&amp;lt;0.05). No difference was observed in the cleavage rate of activated oocytes cultured in any of the 4 culture media (65.7v. 55.0v. 61.0v. 56.2%, respectively). However, the development to blastocyst stage was observed in only KSOM (16%) and SOF (5%) media. In conclusion, the present study demonstrates that pre-pubertal goat oocytes can mature in vitro and can be activated with 5mM ionomycin, and KSOM, and to a lesser extent SOF, supports development to the blastocyst stage. We plan to use these oocytes as a cytoplast source for interspecies somatic cell NT; however, before that, more studies are needed to evaluate their requirements in culture media to enhance their development to the blastocyst stage.

As has been shown previously, fibroblast growth factor 2 (FGF2), leukaemia inhibitory factor (LIF), and insulin-like growth factor 1 (IGF1) in combination play a positive role in maintaining the quality of mammalian oocytes maturing invitro. In the present work, we studied the effects of these cytokines in optimal concentrations when added in combination to IVM medium on the nuclear status and development competence of bovine oocytes. Slaughterhouse-derived cumulus–oocyte complexes (COC) (n=1107 COC) were cultured for 22h in either IVM medium [TCM-199 supplemented with 10% fetal calf serum (FCS), 0.2mM sodium pyruvate, 10μgmL−1 porcine FSH, and 10μgmL−1 ovine LH] (Control) or the same IVM medium supplemented with FGF2, LIF, and IGF1. The eight combinations of cytokines tested during maturation were (1) 20ngmL−1 LIF/10ngmL−1 IGF1/10ngmL−1 FGF2 (Group 1); (2) 20ngmL−1 LIF/10ngmL−1 IGF1/40ngmL−1 FGF2 (Group 2); (3) 20ngmL−1 LIF/20ngmL−1 IGF1/10ngmL−1 FGF2 (Group 3); (4) 20ngmL−1 LIF/20ngmL−1 IGF1/40ngmL−1 FGF2 (Group 4); (5) 5ngmL−1 LIF/10ngmL−1 IGF1/10ngmL−1 FGF2 (Group 5); (6) 5ngmL−1 LIF/10ngmL−1 IGF1/40ngmL−1 FGF2 (Group 6); (7) 5ngmL−1 LIF/20ngmL−1 IGF1/10ngmL−1 FGF2 (Group 7); and (8) 5ngmL−1 LIF/20ngmL− 1 IGF1/40ngmL−1 FGF2 (Group 8). After IVM, matured and denuded oocytes were activated by culturing in 5μM ionomycin solution for 5min followed by 4h in 2mM 4-dimethylaminopyridine (DMAP) and 10mgmL−1 cyclohexamide. Activated oocytes were cultured in CR1aa medium until Day 5 and then transferred to the same medium supplemented with 5% FCS and cultured until Day 7. All cultures were performed at 38.5°C and 5% CO2 in humidified air. At Days 2 and 7 after activation, the cleavage and blastocyst rates were determined. The data from 6 to 7 replicates (122–181 oocytes per treatment) were analysed by ANOVA. The rate of MII oocytes, assessed by the presence of the first polar body, did not differ between the groups and reached 67.1 to 84.5%. Cleavage rate was higher (84.5±2.9%, P&amp;lt;0.05) in Group 1 compared with Control (68.9±2.7%), Group 4 (67.1±4.9%) and Group 7 (66.6±2.0%), but not compared with Group 2 (76.5±4.8%), Group 3 (76.6±4.8%), Group 5 (78.2±3.1%), or Group 8 (79.9±3.1%). The percentage of blastocyst formation relative to the total number of MII oocytes in the control group was 19.6±2.4%. The culture of COC in Group 1 (20 ngmL−1 LIF/10ngmL−1 IGF1/10ngmL−1 FGF2) and Group 2 (20ngmL−1 LIF/10ngmL−1 IGF1/40ngmL−1 FGF2) caused the blastocyst yield to increase to 33.0±3.8 and 31.2±4.3%, respectively (P&amp;lt;0.05), whereas the culture COC in other cytokine-treated groups had no effect. In Groups 3 to 8, the blastocyst rates were 22.9±4.6, 19.2±3.0, 24.2±3.2, 27.8±1.7, 21.6±1.8, and 25.5±9.0%, respectively, and did not differ (except Group 4) compared with those of Group 1 and Group 2. In conclusion, LIF, FGF2, and IGF1 in optimal combinations can maintain competence for parthenogenetic development of bovine COC during their maturation invitro. This research was supported by RFBR (projects No. 18-29-07089) and the Ministry of Science and Higher Education of Russia.

The efficiency of assisted reproductive technologies is critically dependent on the quality of the oocytes used to produce the embryos. The aim of the present research was to study dose-dependent effects of three cytokines: fibroblast growth factor 2 (FGF2), leukemia inhibitory factor (LIF), and insulin-like growth factor 1 (IGF1) individually on IVM in bovine oocytes and their consecutive development to blastocysts after artificial activation. Slaughterhouse-derived cumulus-oocyte complexes (COC) (n=2052 COC) were cultured for 22h in either standard maturation medium [TCM-199 supplemented with 10% fetal calf serum (FCS), 0.2mM sodium pyruvate, 10μgmL−1porcine FSH, and 10μgmL−1 ovine LH; control] or maturation medium supplemented with different concentrations (5-160ngmL−1) of FGF2, LIF, and IGF1. After IVM, matured oocytes were activated by culturing in 5μM ionomycin solution for 5min followed by 4h in 2mM 4-dimethylaminopyridine (DMAP) and 10mgmL−1 cyclohexamide. Activated oocytes were cultured in CR1aa medium until Day 5 and then transferred to the same medium supplemented with 5% FCS and cultured until Day 7. All cultures were performed at 38.5°C and 5% CO2 in humidified air. At Days 2 and 7 after activation, cleavage and blastocyst rates were determined. In addition, obtained blastocysts were fixed with 4% paraformaldehyde, and the total cell number was determined by 4′,6-diamidino-2-phenylindole (DAPI) staining. The data from 4 to 6 replicates (77-140 oocytes per treatment) were analysed by ANOVA. After 22h of culture, the rate of MII oocytes, assessed by the presence of the first polar body, did not differ between the groups and ranged from 74.4 to 90.7%. No statistical differences were found in the cleavage rate between oocytes matured in cytokine-treated groups compared with the control. Cleavage rates for the LIF experimental groups was 63.5 to 77.2%, for the IGF1 experimental groups was 68.1 to 80.7%, and for the FGF experimental groups 63.7 to 77.0%. Optimal concentrations of LIF (5 and 20ngmL−1), and FGF2 (20 and 40ngmL−1) increased (P&amp;lt;0.05) the blastocyst rate from 21.7±1.5 (control for LIF-treated groups) to 32.7±7.1 and 27.1±3.4 and from 19.6±1.8% (control for FGF-treated groups) to 29.8±1.9 and 31.1±2.1%, respectively. Furthermore, the addition of FGF2 in IVM medium (except at 5ngmL−1) led to an increase in the total cell number in embryos that developed to the blastocyst stage, whereas LIF did not have this effect. The maturation of COC in the presence of IGF1 had no effect on the yield of parthenogenetic blastocysts or on the total cell number in blastocysts compared with the control medium. However, the blastocyst rate was lower in groups with IGF1 at 40 to 80ngmL−1 compared with those with IGF1 at 5 to 20ngmL−1 (P&amp;lt;0.05). Thus, both LIF and FGF2 (each individually) (but not IGF1) are able to maintain competence for parthenogenetic development of bovine COC during their maturation invitro, and FGF2 additionally can improve parthenogenetic blastocyst quality. This research was supported by RFBR (Project no. 18-29-07089).

The aim of this study was to evaluate the potential of dehydroleucodine (DhL), a new drug isolated from a medicinal herb used in Argentina, for activation of bovine oocyte. Several DhL concentrations and exposure times after ionomycin (Io) treatment were tested. The optimal DhL treatment, found for parthenogenetic development, was employed to produce bovine embryos by intracytoplasmic sperm injection (ICSI) and somatic cell nuclear transfer (SCNT). The best parthenogenic embryo developments were observed with 5 μM Io for 4 min followed by 5 μM DhL concentration and after 3-h exposure time (52.3% cleavage; 17.4% morulae; 7.3% blastocyst; n = 109). This treatment generated no significant differences with standard Io plus 6-dimethylaminopurine (DMAP) treatment in preimplantation embryo development. In our conditions, the embryo development reached after ICSI and SCNT assisted by the DhL treatment did not differ in terms of cleavage and blastocyst development from activation with standard Io plus DMAP treatment (p > 0.05). In conclusion, DhL utilization to activate oocytes and induce development of parthenogenotes, ICSI-embryos or SCNT-embryos is reported here for first time.

Wisent (Bison bonasus), also called the European bison, is listed as vulnerable on the International Union for the Conservation of Nature Red List of Threatened Species. In Poland, a program for protection in situ and ex situ is being implemented. One new approach is the use of the in vitro embryo production (IVP) procedures to obtain wisent offspring. In contrast to previous successes with cattle IVP, use of IVP with wisent is limited by the small size of the population (only ~5000 individuals in more than 200 herds in Europe) and seasonal reproduction. The aim of this preliminary study was to obtain hybrid embryos (Bison bonasus × Bos taurus) in vitro. Ovaries were isolated from wisent females outside the reproductive season and eliminated from breeding for reasons other than infertility. Cumulus-oocytes complexes (COC) were isolated from all follicles above 2 mm in diameter. All COC were matured in TCM 199 supplemented with 10% fetal bovine serum, 0.02 IU mL–1 of porcine FSH, 17 β-oestradiol, 0.2 mM Na pyruvate, and antibiotics. The COC were cultured for 24 h at 38.5°C and 5% CO2 in humidified air. The matured COC (Bison bonasus) were fertilized in vitro with sperm from Jersey bulls (Bos taurus) in TALP supplemented with 6 mg mL–1 of fatty acid free BSA (BSA FAF), 0.2 mM Na pyruvate, 20 µM penicillamine,10 µM hypotaurine, 1 µM epinephrine, 2 µg mL–1 heparin, and antibiotics. Spermatozoa were used at a final concentration of 1 × 105 per oocyte and were co-cultured for 18 h at 38.5°C and 5% CO2 in humidified air. The hybrid zygotes were cultured in KSOM supplemented with 5 µg mL–1 of MEM Nonessential Amino Acid Solution (100×), 3 mg mL–1 of BSA FAF, and antibiotic for 192 h at 38.5°C and 5% CO2 in humidified air. The medium was partly replaced by fresh medium after 48 and 144 h of culture. Development was evaluated every day. From 25 COC isolated from wisent ovaries, only 18 COC were qualified for in vitro maturation (60%). Of these, 15 COC (83.3%) matured. The percentage of hybrid embryos that cleaved was 80% after 48 h of culture, and the percentage of embryos that developed up to the 8-cell stage was 33% after 96 h of culture. The morula/blastocyst rate was 26.6% after 192 h of culture, as represented by 1 early blastocyst, 2 compact morulae, and 1 morula. The use of the cattle IVP procedure allowed to receive hybrid embryos (Bison bonasus × Bos taurus), but they developed slower than cattle embryos under the same conditions, based on our previous studies. This research will be continued and may make a contribution to the protection of this threatened species.

In vitro developmental competence of alpaca (Vicugna pacos) and llama (Lama glama) oocytes after parthenogenetic activation

Choice of buffer used for the IVF procedures affects embryo developmental rates (De la Fuente et al. 2006 Reprod. Fertil. Dev. 18, 187). Also, it has been shown that the 3 zwitterionic buffers tested in this study, TES (T), MOPS (M), and HEPES (H) (pKa values at 20°C: 7.2–7.5) interact with DNA (Stellwagen et al. 2000 Anal. Biochem. 287, 167). Our objective was to evaluate the effect of T, M, H, and PBS buffers on the expression of the following genes, Fgf-4 (fibroblast growth factor 4 precursor), Lama1 (laminin alpha 1), Ube2a (ubiquitin-conjugating enzyme), Gsta4 (glutathione S-transferase A4), Il6 (interleukin 6), Sod1 (superoxide dismutase), Prss11 (IGF binding), and Hspb1 (Heat shock protein binding 1), on bovine in vivo and in vitro embryos. Genes were selected based on their sensitivity to adverse in vitro embryo culture conditions by microarray analysis (data not shown). All buffers were prepared at a concentration of 10 mM in TALP medium and the final pH was adjusted to 7.2. Bovine follicular fluid was aspirated from abattoir-derived ovaries and evenly divided into 4 tubes. Collected oocytes (5 replicates) from each tube were processed separately through entire IVM, IVF, and IVC procedures using washing medium buffered with: PBS (n = 490), Group 1; H (n = 438), Group 2; M (n = 440), Group 3; and T (n = 394), Group 4. All buffers contained 4% BSA. Oocytes were matured in TCM-199 supplemented with 10% FCS and 10 ng mL-1 epidermal growth factor, and inseminated in Fert-TALP containing 25 mM bicarbonate, 22 mM sodium lactate, 1 mM sodium pyruvate, 6 mg mL-1 BSA-FAF, and 10 µg mL-1 heparin with 1 × 106 mL spermatozoa. After 24 h of oocytes–sperm co-incubation, presumptive zygotes were cultured in SOFaa medium with 8% BSA at 39°C under paraffin oil and 5% CO2 in humidified air. Cumulus–oocyte complexes and zygotes were held in designated buffers in vivo blastocysts exposed for P in vitro embryos irrespective of buffer used. In addition, higher expression of Hspb1 and lower expression of Ube2a and Lama 1 were identified in PBS and T than in M and H (P in vitro embryos were lower (P in vitro-derived embryos. It can be concluded that mRNA transcription of in vitro-derived embryos is affected by the choice of the buffer used for the IVF procedure.

Study question Can the use of artificial intelligence (AI) enhance the prediction of mature oocytes (MII) from Cumulus-Oocytes-Complexes (COC) significantly surpassing the accuracy of well-trained embryologists? Summary answer The development of a COC classifier, an AI-based tool, improves the prediction of mature oocytes from COCs graded as 3 and 4 before oocyte denudation. What is known already Determining the maturity of Metaphase II oocytes before denudation is a challenge in assisted reproduction due to the presence of a dense cumulus mass surrounding the oocyte and obstructing proper manual scoring, a critical step in intracytoplasmic sperm injection (ICSI) due to risk of granulosa cells removal from immature oocytes. Although there is a certain correlation between COC morphology and nuclear oocyte maturity, it is not 100% accurate. It is reported that approximately 90% of MII oocytes are expected in expanded COCs (grade 1), while in COCs grade 4, more immature oocytes are predicted and only 25% are MII oocytes. Study design, size, duration 931 COC's from 145 patients were submitted to COC grading by both, manual and a new “COC classifier” from October to January 2023. 4 groups were formed according to Ebner criteria, 2008. Grade 1: 192 COC's (suspected mature), fluffy and radiant corona and cumulus; Grade 2: 318 COC's showing fluffy cumulus (suspected mature); Grade 3: 208 COC's with radiant corona (expected mature) and grade 4: 213 COC's (suspected immature) with dense cumulus without visible oocyte. Participants/materials, setting, methods Prospective study included 145 patients (39.3 years ±3.2) from September to December 2023. After COC obtention, excess of granulosa cells, dark and blood cells were removed. 3 hours after egg collection, COCs were photographed and scored manually by 9 well trained embryologists. Before denudation, obtained COCs were classified in 4 morphological stages according to Ebner et al, 2008. Finally, the taken photos from COC's were analysed by the COC classifier, a sypervised deep learning algorithm. Main results and the role of chance After COC's classification as following: Group 1 (suspected mature), fluffy and radiant corona and cumulus with visible oocyte; Group 2, COC's showing dense corona (oocyte clearly visible) but fluffy cumulus (suspected mature); Group 3, COC's with radiant corona (oocyte visible, expected mature); Group 4, COC's (suspected immature) with dense corona and cumulus without visible oocyte. The new software presented an MII oocyte accuracy of 79.16% for group 1, 87.1% for group 2, 83.17% for group 3, and 73.23% for group 4. In comparison, manual COCs grading by nine experienced embryologists yielded accuracy rates of 73.5% MII oocytes in group 1, 79.5% for group 2, 50.3% for group 3, and 46.9% for group 4. Statistical analysis, using the chi-square test, was performed to compare the accuracy rates between the COC classifier and manual grading. The results revealed significant differences in favor of the COC classifier when compared to manual method for the groups 3 and 4 (p &amp;lt; 0.001). These results indicate a superior performance of the COC classifier in predicting MII oocytes for groups with highest rates of inmture oocytes. However, for groups 1 and 2, there were no statistically significant differences between the COC classifier and manual grading. Limitations, reasons for caution Further validation through a large-scale, prospective, randomized study is necessary to confirm the efficacy and reliability of the new COC classifier, particularly in patients with oocyte cohorts enriched with grade 3 and 4 COCs. Ongoing research is currently being conducted in our laboratory. Wider implications of the findings The new COC classifier, an AI-based tool, developed by our group, offers a non-invasive and efficient method in predicting mature oocytes before denudation by identifying more MII oocytes from patients’ cohorts enriched with grade 3 and 4 COC's, postponing denudation and injection timing or destinating them to conventional IVF Trial registration number 5678765

Cloning endangered species has the limitation that generally the number of available oocytes is limited. Reprogramming the nuclei heterospecifically using an enucleated oocyte from a different species is an alternative. Aggregation of SCNT (somatic cell nuclear transfer) embryos from the same specie results in improved embryo development. However, after aggregation of heterospecific SCNT embryos from different genera, no effects were observed (Moro et al. 2015 Reproduction 50, 1-10). The objective of this study was to evaluate the influence of aggregation of yak (Bos grunniens) embryos produced by heterospecific SCNT using enucleated oocytes from an animal from the same genus Bos taurus. As control homospecific SCNT of Bos taurus, parthenogenic zone-free embryos and IVF embryos were used. Cumulus-oocyte complexes were recovered from bovine slaughterhouse ovaries by follicular aspiration. The cumulus-oocyte complexes were matured in tissue culture medium 199 containing 10% fetal bovine serum, 10μgmL−1 FSH, 0.3mM sodium pyruvate, 100mM cysteamine, and 2% antibiotic-antimycotic for 22h, at 6.5% CO2 in humidified air and 38.5°C. After denudation, mature oocytes were stripped of the zona pellucida using a protease and then enucleated by micromanipulation. Staining was performed with Hoechst 33342 to observe MII. Enucleated oocytes were placed in phytohemagglutinin to induce adherence with the donor cell followed by electrofusion. All reconstituted embryos were activated using ionomcine. This was followed by a treatment with 6-dimethylaminopurine for 3h. Zona-free reconstituted cloned embryos were cultured in the wells of the well system, placing one (1×) or two (2×) per microwell, in synthetic oviductal fluid medium. The experimental groups were parthenogenic zone free; IVF; reconstituted embryos bull fibroblast-enucleated oocyte from cow (BC1×); reconstituted embryos yak fibroblast-enucleated oocyte from cow (YC1×); and reconstituted embryos aggregated yak fibroblast-enucleated oocyte from cow (YC2×). In all experimental groups, cleavage of at least one embryo in the wells and blastocyst formation at Day 7 were assessed. The effect of cloned embryo aggregation on blastocyst rates was analysed using Fisher exact tests (GraphPad Prisma 8), and results are shown on Table 1. Results demonstrated that aggregation of two SCNT heterospecific embryos increased the blastocyst formation rate of yak (P&amp;lt;0.05). In conclusion aggregation in yak heterospecific SCNT embryos from species of the same genus (Bos) can improve development to blastocyst. Table 1.Aggregation of yak heterospecific somatic cell nuclear transfer embryos Experimental group1 No. of embryos No. of embryos-wells2 Cleavage (%) Blastocyst (%) PZF 68 68 66 (97.06%)a 17 (25.00%)acd IVF 89 - 81 (91.01%)ab 39 (43.82%)b BC1× 45 45 41 (91.11%)b 6 (13.33%)cd YC1× 101 101 77 (76.24%)c 14 (13.86%)c YC2× 134 67 61 (91.04%)ab 21 (31.34%)ab a-dDifferent superscripts in the same column indicate significant difference (Fisher's exact test, P&amp;lt;0.05). 1PZF, parthenogenetic zone free; IFV, IVF fecundation; BC1×, clone of bovine; YC1×, clone of yak-bovine; YC2×, clone of yak-bovine added. 2Wells used with embryos.

In this work, Dehydroleucodine (DhL) was evaluated as a chemical activator of bovine oocytes and somatic cell nuclear transfer (SCNT) reconstituted embryos. Oocytes were activated with 5 microM Ionomycin (Io) and exposed for 3 h to 1 or 5 microM DhL alone (Io-Dhl1 or Io-DhL5) or combined with Cytochalasin B (Io-DhL1/CB; Io-DhL5/CB). Control groups were Io (Io), Io followed by 1.9 mM 6-Dimethylaminopurine (Io-6DMAP), and embryos produced by in vitro fertilization (IVF). Pronuclear formation and development to blastocysts of activated oocytes were evaluated. Embryos obtained by the DhL concentration that induced the highest blastocyst rates (1 microM) were karyotyped. An additional treatment based in Io-DhL1 plus lengthened (6-h) exposure to CB (Io-DhL1/long CB) was included to improve the proportion of diploid blastomeres. Finally, DhL combined with CB was employed to assist cloning by intracytoplasmic injection of whole cumulus cells. Results showed that DhL induces a pronuclear formation dynamic that was more similar to IVF-produced embryos than DMAP. Development to blastocyst stage was higher after activation with 1 microM DhL than with 5 microM DhL, either for groups combined or not with CB (19.15; 21.74 vs. 6.82; 0%, respectively) (p < 0.05). Io-DhL1 and Io-DhL1/CB treatments induced blastocyst-cleaved embryo ratios not statistically different from those of Io-DMAP (35.85%) and IVF (33.33%) groups (p > 0.05). Io-DhL1/long CB induced higher diploid blastomere rates than Io-Dhl1, Io-DhL1/CB and Io-DMAP (63.8 vs. 36.8; 40 and 31.6%, respectively) (p < 0.05). Moreover, all DhL treatments resulted in polyploidy rates that were lower than Io-DMAP (5.2, 12.0, 10.6, and 31.6%, respectively) (p < 0.05). Io-DhL1/CB and Io-DhL1/long CB induced cloned embryo blastocyst rates that were not significantly different from Io-DMAP (6.1, 9.4, and 18.3%, respectively) (p < 0.05). Our results indicate that Io-DhL1/long CB protocol could be useful for SCNT programs.