Presumptive Zygotes Research Articles

Embryo co-culture with bovine amniotic membrane stem cells can enhance the cryo-survival of IVF-derived bovine blastocysts comparable with co-culture with bovine oviduct epithelial cells.

Culture conditions have a profound effect on the quality of in vitro-produced embryos. Co-culturing embryos with somatic cells has some beneficial effects on embryonic development. Considering the ability of stem cells to secrete a broad range of growth factors with different biological activities, we hypothesized that bovine amniotic membrane stem cells (bAMSCs) might be superior to bovine oviduct epithelial cells (BOECs) in supporting embryonic development and enhancing their cryo-survival. Bovine abattoir-derived oocytes were matured and fertilized in vitro. The resultant presumptive zygotes were then cultured up to the blastocyst stage in the following groups: (i) co-culture with bAMSCs, (ii) co-culture with BOECs, and (iii) cell-free culture (Con). Embryos that reached the blastocyst stage were vitrified and warmed, and their post-warming re-expansion, survival and hatching rates were evaluated after 72 h culture. Results showed that the cleavage, blastocyst, and 2 h post-warming re-expansion rates of embryos did not differ between groups. However, their survival rates in BOEC and bAMSC groups were significantly higher compared with the control (72.7, 75.6 and 37.5%, respectively, P < 0.05). In conclusion, our results showed that the cryo-survivability of IVF-derived bovine embryos could be improved through co-culturing with bAMSCs. Moreover, considering the possibility to provide multiple passages from bAMSCs compared with BOECs, due to their stemness properties and their ability to produce growth factors, the use of bAMSCs is a good alternative to BOECs in embryo co-culture systems.

Development and ultrastructure of bovine matured oocytes vitrified using electron microscopy grids

The aim of this study was to establish a methodology of cryopreservation of cattle oocytes and the quality assessment of oocytes and subsequent embryos produced in vitro under our laboratory conditions. Previously in vitro matured (IVM) oocytes were vitrified in minimum volume by ultra-rapid cooling technique. The oocytes were put into the equilibration solution (3% ethylene glycol in M199-HEPES + 10% foetal bovine serum) for 12 min, transferred to vitrification solution (30% ethylene glycol + 1 M sucrose in M199-HEPES + 10% foetal bovine serum) at room temperature for 25 s, then placed onto nickel electron microscopy grids and plunged into liquid nitrogen. After warming 75% of the oocytes were assessed as viable. Part of viable oocytes was taken for electron microscopy, the remaining oocytes were fertilized in vitro, and the presumptive zygotes were cultured until the blastocyst stage. Embryo cleavage and blastocyst rates in vitrified group after warming were 64.98% and 17.3%, resp. versus 70.72% and 25.54% in the control group (P < 0.05). No significant differences were found in the blastocyst total cell number, TUNEL and dead cell indexes between both groups. Ultrastructure of vitrified oocytes showed damages in smooth endoplasmic reticulum (SER) vesicles and lipid droplets as well as irregular arrangement of solitary cortical granules. Several mitochondria were damaged and the microtubules around the chromosomes were less occurred compared to the control group. However, the extent of injuries was lower than reported by other authors studying the ultrastructure of vitrified bovine oocytes, what is also supported by the better development of our oocytes after IVF. In conclusion, the designed oocyte vitrification technique ensures obtaining the blastocysts of the quality comparable to the fresh oocytes.

MITOCHONDRIAL SUPPORT OF EMBRYOS FROM WOMEN OF ADVANCED MATERNAL AGE DURING ART

The supplementation of MitoTempo and mitoquinol (MTQ) in mouse embryo culture results in better quality blastocysts by supporting mitochondrial function. Women of advanced maternal age have reduced oocyte quality due in part to dysfunctional mitochondria. The aim of this study was to determine the effect of supplementation of mitochondrially targeted antioxidants (MTQ) during human embryo culture for women of advanced maternal age (≥35 yr). Prospective clinical trial with sibling embryos. MATERIALS AND METHODS:: After oocyte retrieval, cumulus oocyte complexes (COC) were denuded of cumulus cells. Mature oocytes (MII) were fertilized by ICSI and randomly allocated into sequential culture medium, 2/3 to control and 1/3 to medium with MTQ. Zygotes were moved into second step medium on day 3 and cultured for 5-7 days, at which time good quality blastocysts were biopsied for PGT-A and vitrified. MTQ was present throughout the culture period. A total of 11 patients with advanced maternal age (AMA) (average age 39.4 yr, range 35-46) were included in this study. Post ICSI, 143 presumptive zygotes were placed in control medium and 66 in medium supplemented with MTQ. Normal fertilization, determined by presence of 2 pronuclei (2PN), was 83% in control and 91% in MTQ (p=0.11). There were no differences between control and MTQ treatment in D5 (control, 18%; MTQ, 20%) or total (control, 48%; MTQ, 45%) good quality blastocyst (≥grade 3BB) development (per 2PN), or total blastocyst development (control, 63%; MTQ, 62%). There was also no difference in the percentage of euploid blastocysts (control, 33%; MTQ, 30). To date, four euploid blastocysts from the control treatment and one from the MTQ treatment have been transferred individually to a total of 5 patients, all resulting in ongoing pregnancies with fetal heart beat. In this preliminary study, we did not note any improvement in good quality or euploid blastocyst development due to inclusion of MTQ in the culture media. Both treatments resulted in an equal percentage of transferrable euploid embryos per oocyte injected with sperm. One embryo has been transferred after MTQ treatment, resulting in a healthy ongoing pregnancy. These data suggest that MTQ treatment of human embryos in culture is safe, although at this point it does not appear to be effective in increasing blastocyst development for women of AMA. However, further research is required to determine if there are any positive effects of MTQ treatment on implantation and/or pregnancy loss that are not yet evident from this initial trial.

Mimicking the temperature gradient between the sow’s oviduct and uterus improves in vitro embryo culture output

This work was designed to determine temperature conditions within the reproductive tract of the female pig and study their impact on ARTs. Temperatures were recorded using a laparo-endoscopic single-site surgery assisted approach and a miniaturized probe. Sows and gilts were used to address natural cycle and ovarian stimulation treatments, respectively. According to in vivo values, IVF was performed at three temperature conditions (37.0°C, 38.5°C and 39.5°C) and presumptive zygotes were cultured in these conditions for 20 h, while further embryo culture (EC) (21-168 h post-insemination) was maintained at 38.5°C. After 20 h, different fertility parameters were assessed. During EC, cleavage and blastocyst stages were evaluated. Sperm membrane fluidity at the experimental temperatures was studied by using differential scanning calorimetry and fluorescence recovery after photobleaching techniques. An increasing temperature gradient of 1.5°C was found between the oviduct and uterus of sows (P < 0.05) and when this gradient was transferred to pig in vitro culture, the number of poly-nuclear zygotes after IVF was reduced and the percentage of blastocysts was increased. Moreover, the temperature transition phase for the boar sperm membrane (37.0°C) coincided with the temperature registered in the sow oviduct, and sperm membranes were more fluid at 37.0°C compared with those of sperm incubated at higher temperatures (38.5°C and 39.5°C). These data suggest that there may be an impact of physiological temperature gradients on human embryo development.

Short term temperature elevation during IVM affects embryo yield and alters gene expression pattern in oocytes, cumulus cells and blastocysts in cattle

Heat stress causes subfertility in cattle by inducing alterations in steroidogenic capacity, follicular function and ovulation defects, which eventually negatively affect oocyte quality and embryo survival. Here, the effects of short, moderate temperature elevation during IVM, on embryo yield, and on the expression of various genes was evaluated. In 8 replicates, cumulus oocyte complexes (COCs) were matured for 24 h at 39 °C (controls n = 605) or at 41 °C from hour 2 to hour 8 of IVM (treated, n = 912), fertilized, and presumptive zygotes were cultured for 9 days at 39 °C. Cleavage and embryo formation rates were evaluated 48 h post insemination and on days 7, 8, 9 respectively. Cumulus cells, oocytes and blastocysts from 5 replicates were snap frozen for the relative expression analysis of genes related to metabolism, thermal and oxidative stress response, apoptosis, and placentation. In treated group, cleavage and embryo formation rates were statistically significantly lower compared with the control (cleavage 86.7% vs 74.2%; blastocysts: day 7, 29.9% vs 19.7%, day 8, 34.2% vs 22.9% and day 9 35.9% vs 24.5%). Relative mRNA abundance of three genes in cumulus cells (HSP90AA1, CPT1B, G6PD) and three genes in blastocysts (DNMT3A, PLAC8, GPX1) indicated significantly different expression between groups (p < 0.05)., The expression of G6PD, SOD2, GXP1 in oocytes and PTGS2 in blastocysts tended to differ among groups (0.05<p < 0.08). Heat stress altered (p < 0.05) the correlation of expression between HSPs and other genes in oocytes (G6PD, GPX1, CCNB1), cumulus cells (LDH, CCNB1) and blastocysts (AKR1B1, PLAC8). These results imply that exposure of oocytes to elevated temperature, even for only 6 h, disrupts the developmental competence of the oocytes, suppresses blastocyst yield and significantly alters the coordinated pattern of gene expressions.

Application of platelet-rich plasma in the in vitro production of bovine embryos.

The aim of this study was to replace fetal bovine serum (FBS) with platelet-rich plasma (PRP) for in vitro production of bovine embryos. The maturation media (TCM-199 medium) for the cumulus-oocyte complexes (COCs) was supplemented with 5% (G5) and 10% (G10) PRP or 10% FBS (GC). After fertilization, the presumed zygotes were randomly distributed in culture medium supplemented with 5% (G5) and 10% (G10) PRP or 10% FBS (GC) for 7days. Cumulus cell (CC) expansion was greater (P < 0.05) in the GC (88.9%) group than in G5 (34.1%) or G10 (50.0%). Nevertheless, the expansion of CCs in group G10 was greater than in G5 (P < 0.05). Cleavage was higher in group G5 (86.0%) than in G10 (79.0%) (P < 0.05) and did not differ from group GC (82.0%). The percentage of blastocysts in group G5 (50.0%) was higher than in CG (40.2%) and G10 (34.2%) (P < 0.05). In addition, the number of blastomeres was higher in G5 (159.0 ± 4.18) than in GC (132.4 ± 4.11) and in G10 (127.1 ± 5.88) (P < 0.05). The addition of PRP into the oocytes maturation medium is not beneficial. On the other hand, the PRP addition into the embryo culture medium at 5% concentration is recommended where it increased the quantity and quality of in vitro-produced bovine embryos.

Induction of oxidative stress does not increase the cryotolerance of vitrified embryos

Short-term treatment of mammalian oocytes with different stressors induces stress tolerance of embryos derived from these oocytes. The aims of this study were to evaluate effects on embryo development when there was treatment of oocyte complexes (COCs) used to derive the embryos with hydrogen peroxide (H2O2).The COCs were not incubated with H2O2: control (0 μM), or were incubated with 25, 50, 75, or 100 μM concentrations of H2O2 for 1 h prior to in vitro fertilization, and presumptive zygotes were cultured until day 7. Blastocysts at day 7 of development derived from H2O2-treated (25 μM treatment concentration) COCs were vitrified. Percentage of embryos undergoing cleavage was not affected by any treatment, while percentage of embryos developing to the blastocyst stage was less when there was treatment of COCs with 100 μM of H2O2. Embryo quality was less when COCs used to derive blastocysts were treated with 50, 75, or 100 μM concentrations of H2O2. There were lesser relative abundances of some mRNA transcripts of interest in blastocysts when there was treatment of COCs with H2O2. After vitrification, there were no differences in embryo re-expansion and hatching rates compared with fresh and vitrified blastocysts of the control group and those derived from COCs treated with 25 μM H2O2. In conclusion, treatment of COCs used to derive blastocysts with H2O2 does not induce stress tolerance in vitrified embryos of cattle; however, the viability of these blastocysts is similar to those of the control group.

Individually cultured bovine embryos produce extracellular vesicles that have the potential to be used as non-invasive embryo quality markers

Extracellular vesicles (EVs) are membrane-bound biological nanoparticles (NPs) and have gained wide attention as potential biomarkers. We aimed to isolate and characterize EVs from media conditioned by individually cultured preimplantation bovine embryos and to assess their relationship with embryo quality. Presumptive zygotes were cultured individually in 60 μl droplets of culture media, and 50 μl of media were collected from the droplets either on day 2, 5 or 8 post-fertilization. After sampling, the embryo cultures were continued in the remaining media until day 8, and the embryo development was evaluated at day 2 (cleavage), day 5 (morula stage) and day 8 (blastocyst stage). EVs were isolated using qEVsingle® columns and characterized. Based on EV Array, EVs isolated from embryo conditioned media were strongly positive for EV-markers CD9 and CD81 and weakly positive for CD63 and Alix among others. They had a cup-like shape typical to EVs as analyzed by transmission electron microscopy and spherical shape in scanning electron microscopy, and hence regarded as EVs. However, the NPs isolated from control media were negative for EV markers. Based on nanoparticle tracking analysis, at day 2, the mean concentration of EVs isolated from media conditioned by embryos that degenerated after cleaving (8.25 × 108/ml) was higher compared to that of embryos that prospectively developed to blastocysts (5.86 × 108/ml, p < 0.05). Moreover, at day 8, the concentration of EVs isolated from media conditioned by degenerating embryos (7.17 × 108/ml) was higher compared to that of blastocysts (5.68 × 108/ml, p < 0.05). Furthermore, at day 8, the mean diameter of EVs isolated from media conditioned by degenerating embryos (153.7 nm) was smaller than EVs from media conditioned by blastocysts (163.5 nm, p < 0.05). In conclusion, individually cultured preimplantation bovine embryos secrete EVs in the culture media and their concentration and size are influenced by embryo quality and may indicate their prospective development potential.

Effect of vitrification of in vitro matured prepubertal goat oocytes on embryo development after parthenogenic activation and intracytoplasmic sperm injection

This work studies the effect of vitrification of in vitro matured (IVM) prepubertal goat oocytes on: 1) oocyte damage assessed by reactive oxygen species (ROS) level and apoptosis and 2) embryo development after Intracytoplasmic sperm injection (ICSI) and Parthenogenic Activation (PA). Oocytes were IVM in supplemented TCM-199 for 22–24 h. Control group oocytes matured during 24 h were directly used for the analysis after IVM. Vitrified/warmed IVM-oocytes were vitrified after 22 h of IVM in 15% ethylene glycol (EG), 15% dimethyl sulfoxide (Me2SO) and 0.5 M sucrose and after subjected to warming procedure. Oocyte ROS level was measured by staining denuded IVM-oocytes with 10 μM 2′7′ dichlorodihydrofluorescein diacetate. Apoptosis was analyzed by Annexin V (AV) Apoptosis Detection kit and Propidium iodide (PI) signal and oocytes were classified as: Live (AV− PI−), early apoptotic (AV+ PI−), dead non-apoptotic (AV− PI+) and necrotic (AV+ PI+). Developmental competence of vitrified/warmed oocytes was assessed by PA (5 min in 5 μM Ionomycin plus 4 h in 2 mM 6-Dimethylaminopurine), and by ICSI fertilization. Presumptive zygotes were in vitro cultured for 8 days in commercial media BO-IVC. Vitrified/warmed oocytes showed higher ROS levels (P < 0.0001), lower live oocytes (44 vs. 66%; P: 0.0025) and higher dead non-apoptotic oocytes (33 vs. 13% P: 0.023) compared to control. No differences were found on normal zygote formation (2 PN) (32 vs. 25%) or blastocyst development (0 vs. 4%) after ICSI fertilization. However, after PA, significant differences were found in cleavage rate (59 vs.78%; P < 0.0343) and blastocyst formation (1 vs. 25%; P < 0.0001). In conclusion, vitrification reduced oocyte competence by increasing dead oocytes and ROS levels.

Impacts of oocyte/zygote timing for in vitro fertilization and gene editing in the dog

Previously, we reported the first live births of dogs using in vitro fertilization (IVF), embryo cryopreservation, and transfer. These techniques have potential applications in the conservation of endangered canids, and development of gene editing/repair technologies that could improve animal welfare by restoring normal gene function and removing predisposition to disease. Here, we used IVF as a springboard for initial attempts at genetic modification through gene editing/repair using the Clustered Regularly-Interspaced Short Palindromic Repeat (CRISPR)-CRISPR-associated endonuclease (Cas9) system. We showed previously that timing is critical for successful IVF in that the canine oocyte must be exposed to the oviductal environment beyond simply reaching metaphase II. Others have shown that timing of injection of CRISPR-Cas9 constructs is critical in gene editing, influencing the extent of genetic mosaicism. Therefore, we investigated whether timing of injection of the gene editing/repair constructs might influence the success of embryo production and gene editing in the dog. We achieved similar IVF success to our prior report in generating 2-cell control embryos, and found equally reduced embryo production whether injection was performed in oocytes prior to fertilization, or in presumptive single-cell zygotes already exposed to sperm. We had no success at generating offspring with precise single-nucleotide changes in KRT71 via homology-directed repair (HDR), but did identify mutation of FGF5 using non-homologous end joining (NHEJ). These findings underscore the difficulties inherent to gene repair, but represent important progress on reproducibility of canine IVF, improved techniques of oocyte/embryo handling, and impact of timing of injections on embryo development.

In vitro production of zygote from slaughterhouse driven buffalo oocyte

Buffalo is a highly potential animal species in terms of milk and meat production but traditionally they are regarded as poor breeder. In vitro embryos production technology has been introduced in many countries to improve reproductive efficiency of buffalo. Considering the above fact, the present study was undertaken aiming to produce in vitro buffalo embryo in the laboratory. Ovaries of slaughtered buffaloes were collected from abattoir and transported to the laboratory within 4 to 5 hr of slaughter. Cumulusoocyte- complexes (COCs) possessing an even cytoplasm and covered with minimum 3 layers of compact cumulus cells was selected for in vitro maturation (IVM) for 24 hr (5% CO2 in air at 38.5°C with maximum humidity). After IVM, the presumptive matured COCs were co-cultured with capacitated fresh spermatozoa for 18 hr After IVF, the presumptive zygote were denuded, washed and transferred in to in vitro culture medium (IVC 1) for 3 days. After three days cleavage were recorded and 4 cell embryos were transferred in to in vitro culture media II for next 2 days. The development of embryos was evaluated on day 6. A total of 227 buffalo ovaries were collected from the slaughterhouse and categorized into 2 groups based on presence (n=83) or absence (n=144) of corpus luteum (CL). A total of 1464 follicles were counted on the ovarian surface, 1066 being from CL absent and 398 from CL-containing ovaries. A significantly higher (P&lt;0.01) number of follicles, aspirated follicles, normal COCs and total COCs (7.4 ± 0.21, 5 ± 0.00, 1.98 ± 0.77 and 2.98 ± 0.16 respectively) were observed in CL-absent ovaries than those aspirated from CL-containing ovaries (4.80 ± 0.17, 3.92 ± 0.95, 0.88 ± 0.60 and 1.88 ± 0.16 respectively). Total 358 normal COCs were set for in vitro maturation and underwent for IVF and IVC. Results showed that cleavage rates were 56.42%. Among the cleaved embryos, 137 were at 2-cell stage and 65 were at 4-cell stage. Therefore, development rate to 2 cell and 4-cell stage was 38.27% and 18.15% respectively. No embryo developed beyond 4-cell stage. This result indicates that follicle and oocyte numbers and oocyte quality are associated with CL of ovaries and current culture system support in vitro embryo production upto 4-cell stage. The in vitro culture condition may be improved for increasing efficiency of embryo production.&#x0D; Bangladesh J. of Livestock Res. 21-25: 127-132, 2018

In vitro and in vivo development of domestic cat embryos generated by in vitro fertilization after eCG priming and oocyte in vitro maturation

The objective of this study was to evaluate, in the domestic cat, the effect of ovarian stimulation with eCG prior to oocyte in vitro maturation (priming) on in vitro and in vivo development after in vitro fertilization (IVF). For this purpose, oocyte donors were either 1) treated with a single dose of 200 IU eCG four days before oocyte recovery (eCG group), or, 2) given no treatment before oocyte recovery (control group).Ovaries of both groups were collected by ovariohysterectomy (OVH) and cumulus-oocyte complexes (COCs) were recovered by slicing. Immature COCs from both groups were matured in vitro (IVM) for 26–28 h. IVF was done with refrigerated epididymal sperm. After 24 h co-incubation, presumptive zygotes were cultured in vitro for eight days. The rates of cleavage, morulae, blastocyst development and hatching were estimated. Some blastocysts were stained for total cell counting and others were used for gene expression analysis of pluripotency (OCT4, SOX2 and NANOG) and differentiation markers (CDX2 and GATA6). Additionally, to evaluate in vivo development, embryos from the eCG group were transferred at Day 5 and Days 7 or 8 of IVC to synchronized cat recipients. The results showed that, eCG priming increased significantly the rate of blastocyst development as compared to the control group (37.9 and 25.6%, respectively) (P < 0.05). No differences were observed in total cell number of blastocysts and hatching blastocysts (mean ± SD) between the eCG and control groups (420.6 ± 193.6 and 347.0 ± 237.1, respectively) (P > 0.05). In the gene expression analysis, blastocysts generated in the eCG group had higher expression of OCT4 than blastocysts from the control group (P < 0.05). However, no significant differences were observed in the relative expression of SOX2, NANOG, CDX2 and GATA6 (P > 0.05). Additionally, six embryo transfer (ET) procedures were done, three with Day 5 embryos and three with Day 7 or 8 embryos. Recipients from both ET groups delivered live kittens. The total pregnancy rate was 4/6 (67%), meanwhile the live birth rate was 2/6 (33%). In conclusion, eCG priming improved the rate of blastocyst development in vitro and increased relative expression of OCT4. These results demonstrate that eCG priming of oocytes donors before IVM improves oocyte competence, enhance in vitro embryo development and allows live births of healthy offspring after ET.

In vitro Development of Preimplantation Caprine Embryo using Cryopreserved Black Bengal Buck Semen

The aim of the present study was to produce goat embryo in different culture media through in vitro fertilization using cryopreserved black Bengal buck semen. Total 1265 fresh cumulus oocyte complexes (COCs) were collected by aspiration method with a 19 gauge hypodermic needle, washed 5-6 times and cultured in maturation media maintaining 5% CO2 level at 38.5ºC with maximum humidity in a incubator. After 27 h of incubation cumulus cells were stripped off from matured oocytes and transferred to acidified Tyrode’s medium for zona thinning and co-incubated with in vitro capacitated sperms for fertilization in Fert-BO media. In the experiment I, fresh buck semen and in experiment II, frozen buck semen was used for in vitro fertilization after in vitro processing. After 5 h of co-incubation, presumptive zygotes were washed and co-incubated with oviductal cells in three different culture media (RVCL, mSOF, KSOM) for further development. In fresh group cleavage (%) were 37.76 ± 2.98, 39.60 ± 1.75, 29.01 ± 1.74, and morula formation (%) were 7.72 ± 3.38, 6.03 ± 1.29, and 3.00 ± 3.00 in RVCL, mSOF and KSOM media respectively. However, in frozen group cleavage (%) were 29.17 ± 2.56, 27.70 ± 2.31, 24.17 ± 1.44 in RVCL, mSOF and KSOM media respectively and morula formation (%) was 2.93 ± 0.97 only in RVCL media. These results indicate that cryopreserved black Bengal buck semen have competence to produce embryos and could be used for embryo development in RVCL media through in vitro fertilization.

Effects of abundances of OCT-4 mRNA transcript on goat pre-implantation embryonic development

Unlike in mice, the function of pluripotent markers in early embryonic development of domestic animals remains to be elucidated and this may account for the failure to establish embryonic stem cell lines for these species. To study the functions of the OCT-4 protein which has important actions in maintenance of pluripotent and self-renewal processes during early embryonic development, there was induced reduction in relative abundance of OCT-4 mRNA transcript during goat early embryonic development by using RNA interference techniques. The injection of OCT-4 siRNA into goat IVF presumptive zygotes resulted in a decrease in the relative abundance of OCT-4 mRNA transcript; however, there was development of these embryos to the blastocyst stage at the same rate as there was in the control group. The blastocysts from the treated groups had a similar number of TE, ICM, and total cells compared to those from the control group. Although there was a greater relative abundance of NANOG, REX1, and CDX2 mRNA transcript in the embryos injected with siRNA at the 8–16 cell stage, the relative transcript abundances were similar for the control and treatment groups at the blastocyst stage. The relative abundance of SOX2 mRNA transcript was similar for the treatment and control group. It, therefore, is concluded that inhibition of abundances of OCT-4 mRNA transcript to about 20 % of that of the untreated control group did not affect blastocyst formation rate in goats. The functions of OCT-4 in maintaining ICM and TE integrity, however, remains to be assessed.

Resveratrol treatment during maturation enhances developmental competence of oocytes after prolonged ovary storage at 4 °C in the domestic cat model

Resveratrol (Resv; 3,4,5-trihydroxy-trans-stilbene) is a phytoalexin with antioxidant activity that modulates redox homeostasis in oocytes and improves in vitro embryo production. Cold storage of cat ovaries for a period longer than 24 h alters oxidative status of oocytes after in vitro maturation and reduces their developmental competence. The aim of this study was to evaluate the effect of resveratrol supplementation to the maturation medium on embryo development of oocytes after storage of domestic cat ovaries at 4 °C for 24 h or 48 h. Cumulus–oocyte complexes (COCs) were recovered from ovaries of domestic queens and cultured in maturation medium supplemented with (+) or without (−) 5 μM resveratrol for 24 h. COCs collected from fresh ovaries were matured in vitro (IVM) in standard conditions as control. After IVM, oocytes were in vitro fertilized (IVF) and presumptive zygotes cultured for 7 days. Oocyte nuclear maturation, reactive oxygen species (ROS) and glutathione (GSH) levels as well as cleavage, blastocyst formation and blastocyst cell number were determined. There were no differences in the maturation rates of oocytes between the control and stored groups, irrespective of resveratrol supplementation. Resveratrol treatment during IVM significantly increased the level of GSH and reduced the level of ROS of oocytes recovered from ovaries stored for 48 h as compared to the non-treated group (48 h-). The rate of blastocyst formation from oocytes recovered from ovaries after 48 h storage that underwent IVM with resveratrol was higher (P < 0.05) than that of oocytes matured without resveratrol and similar to that of control oocytes. Resveratrol treatment increased (P < 0.05) cell number in blastocysts from 24 h + and 48 h + groups as compared to their respective counterparts. In conclusion, our results demonstrated that resveratrol supplementation during IVM can reverse the adverse effect of oxidative stress on oocytes, and enhances embryo development after ovary storage at 4 °C for 48 h. These results may provide a basis for improving culture conditions and extend the possibility of storage of cat ovaries for more than 24 h thus ensuring successful in vitro embryo production.

Correlation between insemination dose and bovine sperm traits on the in vitro embryo production outcomes

In the present study we aimed to test the best insemination dose for in vitro embryo production (IVEP) and to correlate sperm traits in bovine. In vitro matured oocytes were inseminated with three different sperm concentrations of the same bull: G1 (1*106 ), G2 (2*106 ) and G3 (4*106 ) sperm/mL. At 18 h post-insemination (hpi), presumptive zygotes [G1 (n=114), G2 (n=139) and G3 (n=136)] were stained to evaluate the pronuclei numbers, or continued to in vitro culture [G1 (n=102), G2 (n=111) and G3 (n=106)]. Sperm kinetics were analyzed using Computer-Assisted Semen Analysis (CASA). Sperm plasma membrane, acrosome integrity and mitochondrial activity were analyzed using fluorescent probes. In vitro fertilization (IVF) and IVEP data were compared using chisquare (P 0.05) among the groups. In G3, the polyspermy rate was the highest (7.4%; P 0.05) between G1 (0%) and G2 (0%). In G1, the early blastocyst rate was the highest (7.8%; P 0.05) between G2 (1.8%) and G3 (0.9%). The IVF efficiency and total blastocyst rates were positively correlated with curvilinear velocity (VCL) (r≃+1; P<0.05). We concluded that the reduction of insemination dose may negatively affect embryo development and VCL may be used as a parameter to improve the IVEP outcomes.

Ruthenium red improves blastocyst developmental competence by regulating mitochondrial Ca2+ and mitochondrial functions in fertilized porcine oocytes in vitro.

Ruthenium red (RR) inhibits calcium (Ca2+) entry from the cytoplasm to the mitochondria, and is involved in maintenance of Ca2+ homeostasis in mammalian cells. Ca2+ homeostasis is very important for further embryonic development of fertilized oocytes. However, the effect of RR on mitochondria-Ca2+ (mito-Ca2+) levels during in vitro fertilization (IVF) on subsequent blastocyst developmental capacity in porcine is unclear. The present study explored the regulation of mito-Ca2+ levels using RR and/or histamine in fertilized oocytes and their influence on blastocyst developmental capacity in pigs. Red fluorescence intensity by the mito-Ca2+ detection dye Rhod-2 was significantly increased (P < 0.05) in zygotes 6 h after IVF compared to mature oocytes. Based on these results, we investigated the changes in mito-Ca2+ by RR (10 and 20 μM) in presumptive zygotes using Rhod-2 staining and mito-Ca2+ uptake 1 (MICU1) protein levels as an indicator of mito-Ca2+ uptake using western blot analysis. As expected, RR-treated zygotes displayed decreased protein levels of MICU1 and Rhod-2 red fluorescence intensity compared to non-treated zygotes 6 h after IVF. Blastocyst development rate of 20 μM RR-treated zygotes was significantly increased 6 h after IVF (P < 0.05) due to improved mitochondrial functions. Conversely, the blastocyst development rate was significantly decreased in histamine (mito-Ca2+ inhibitor, 100 nM) treated zygotes (P < 0.05). The collective results demonstrate that RR improves blastocyst development in porcine embryos by regulating mito-Ca2+ and MICU1 expression following IVF.

Piglets produced by transfer of embryos obtained by in vitro fertilization of oocytes matured in vitro with thymosin: A case report

The aim of the study was to examine the in vivo viability of in vitro-produced (IVP) porcine embryos obtained from oocytes matured with thymosin. The research material for this study consisted of immature pig oocytes obtained from ovaries after slaughter and ejaculated semen obtained from one boar. The immature oocytes were cultured in vitro until the metaphase II stage in a medium supplemented with thymosin (TMS). The presumptive zygotes obtained were cultured in vitro for 4-40 hours. The presumptive zygotes and 2-4-cell embryos were evaluated in vivo after transferring them to synchronized recipients. After the transfer of embryos from the experimental group into 2 recipients (50 embryos into each gilt) and the transfer of 50 embryos from the control group into 1 recipient, both gilts that had received embryos obtained by in vitro fertilization of oocytes matured with TMS became pregnant and delivered a total of 16 live piglets. After the transfer of embryos from the control group, no pregnancy was achieved. In conclusion, the results of our preliminary study suggest that the maturation of pig oocytes with thymosin supports the in vivo survival of in vitro produced embryos. It is important to note, that this was the first birth of piglets obtained after transfer of IVP embryos in Poland.

97 Loss of aggregation capacity of bovine invitro-produced embryos and blastocyst-derived trophoblasts from Day 6 of development

Embryo aggregation consists of placing more than one zona-free (ZF) embryo in contact during development to obtain a unique structure. It has been reported in different species that aggregated cloned embryos show certain benefits compared with nonaggregated embryos. One way to obtain these benefits in IVF embryos would be to generate a transient chimera by the introduction of trophoblastic cells. Bovine trophoblastic cells can be obtained by embryo bisection of blastocysts, cutting asymmetrically to use trophoblasts (Tr) for aggregation and leaving aside the portion that contains the inner cell mass (ICM). Taking all this into account, the objectives of this work are to study the aggregation of Tr at different days of development and to determine the appropriate time of aggregation. To this aim, cumulus-oocyte complexes (COCs) collected from slaughterhouse ovaries 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 24h, at 6.5% CO2 in humidified air and 38.5°C. We performed IVF with 16×106 spermatozoa per mL for 5h. Afterwards, presumptive zygotes were cultured in synthetic oviductal fluid (SOF) for 7 days in a humidified atmosphere at 38.5°C, 5% O2, 5% CO2, and 90% N2. In Experiment 1, embryo bisection of Day 7 blastocysts was performed manually under stereoscopic observation with a microblade to obtain Tr. These were aggregated, with the bisected part containing the ICM (n=22) or with ZF embryos of Days 4 (n=23), 5(n=25), or 6 (n=22) and blastocysts (n=25), and placed in microwells in a 100-μL SOF drop covered by mineral oil (Gambini et al. 2012 Biol. Reprod. 87, 15; https://doi.org/10.1095/biolreprod.112.098855). In Experiment 2, ZF synchronous whole embryos were aggregated in microwells at different developmental days: Day 3 (n=18), 4 (n=18), 5 (n=47), 6 (n=48), and 7 (n=45). In both experiments, aggregation was assessed at Day 8. In Experiment 1, no aggregation was observed between the Tr and the embryos or the bisected ICM. Experiment 2 showed embryo aggregation on Days 3 (55%), 4 (27%), and 5 (61%), whereas on Days 6 and 7 no aggregation was observed. According to these results, we can conclude that, in our culture conditions, Tr obtained by blastocyst bisection have no capacity for aggregation. Day 6 and 7 whole ZF embryos also do not aggregate. As a general conclusion, there is a period from Days 0-5 of the invitro development of bovine embryos in which aggregation is possible. Aggregation of blastocyst-derived Tr to cloned or high-value IVF embryos, aiming for quality improvement, is not an effective strategy.

92 Using physical parameters of bovine zygotes to predict invitro development success

The objective of this study was to determine if physical parameters of bovine zygotes are correlated with invitro development success. We examined the relationship between a zygote's outer diameter (OD), cell area, and zona pellucida (ZP) thickness on blastocyst development and blastomere number. Bovine cumulus-oocyte complexes, from abattoir-derived ovaries, were matured in tissue culture medium 199-based maturation medium for 21-23h. Invitro fertilisation was performed with frozen thawed semen containing a pool of 4 Holstein bulls. After incubation with sperm for 16-18h, presumptive zygotes (n=875) were denuded and placed in 5µL droplets of synthetic oviductal fluid-bovine embryo 1 medium (SOF-BE1) under oil to be individually photographed using an inverted scope with a digital camera. Zygotes were then group-cultured in 50µL of synthetic oviductal fluid-BE1 droplets in a polyester micromesh to identify individuals. Development to the blastocyst stage was recorded on Day 7 and Day 8 of culture, and Day 8 embryos were fixed and stained with Hoechst 33342 (n=87). ImageJ was used to measure the OD, area, and ZP from images. Data were analysed with a logistic regression using the lme4 package in R with Day 7 or Day 8 blastocyst development as the response; OD, area, and ZP as predictors; and study replicate (n=11) and culture droplet as random effects. Residual estimates of area and ZP calculated from OD were used to account for collinearity. Average OD was 151.2µm, ranging 130.4-171.6µm. Average ZP was 11.8µm, ranging 7.2-17.5µm. Area averaged 9856.7µm2, ranging from 6421.9 to 13 814.8 µm2. There was a tendency for an effect of OD on probability of development on Day 8 (P=0.08) but not Day 7. There was an effect of ZP on the probability of development on Day 8 (P=0.04) but not Day 7. Blastocyst rates on Day 8 for zygotes with ZP &amp;lt;11.8µm were 24.3%, whereas zygotes with ZP &amp;gt;11.8µm were 21.7%. There was an interaction (P&amp;lt;0.05) between OD and ZP thickness on Day 7 and Day 8. Estimated probability of development of zygotes with ZP &amp;lt;10.7µm and OD &amp;lt;146.9µm averaged 28.5%, whereas those with ZP &amp;gt;12.7µm and OD &amp;gt;155.8µm averaged 43.3%. The observed blastocyst rates for these two groups were 26.2% and 25%, respectively. Zygotes with OD between 146.9-155.8µm and ZP between 10.7-12.7µm had an estimated development probability of 22.2%. The obtained Day 8 blastocyst rate for this group was 22.5%. Area did not affect probability of development, but was positively correlated with total blastomere number at Day 8 (P=0.01; marginal R2=0.09). The observed interaction between ZP thickness and OD may be indicative of an optimum ooplasm area before maturation or fertilisation that may enhance development. Area assessment after fertilisation is not correlated with development success. In addition, these data in comparison with our previous data generated in parthenogenetic embryos indicate that artificial activation of oocytes is not an ideal model in place of IVF for studying development. We continue to demonstrate that physical parameters of zygotes may have potential as a noninvasive, objective selection tool of embryos.