Synthetic Oviduct Fluid Medium Research Articles

Synergistic impact of α-linolenic acid and α-tocopherol on in vitro maturation and culture of buffalo oocytes.

The objective of the current study was to investigate the synergistic impact of α-Tocopherol and α-Linolenic acid (100 µM) on IVM and IVC of Nili Ravi buffalo oocytes. Oocytes were obtained from the ovaries of slaughtered buffaloes within two hours after slaughter and brought to laboratory. Buffalo cumulus oocyte complexes were placed randomly in the five experimental groups included; GROUP 1: Maturation media (MM) + 100 µM ALA (control), GROUP 2: MM + 100 µM ALA + 50μM α-Tocopherol, GROUP 3: MM + 100 µM ALA + 100μM α-Tocopherol, GROUP 4: MM + 100 µM ALA + 200 μM α-Tocopherol and GROUP 5: MM + 100 µM ALA + 300 μM α-Tocopherol under an atmosphere of 5% CO2 in air at 38.5 °C for 22-24 h. Cumulus expansion and nuclear maturation status was determined (Experiment 1). In experiment 2, oocytes were matured as in experiment 1. The matured oocytes were then fertilized in Tyrode's Albumin Lactate Pyruvate (TALP) medium for about 20 h and cultured in synthetic oviductal fluid (SOF) medium to determine effect of α-Linolenic acid (100 µM) and α-Tocopherol in IVM medium on IVC of presumptive zygotes. To study the effect of α-Linolenic acid (100 µM) in IVM media and increasing concentration of α-tocopherol in the culture media on early embryo development (Experiment 3), the presumptive zygotes were randomly distributed into the five experimental groups with increasing concentration of α-tocopherol in culture media. Higher percentage of MII stage oocytes in experiment 1(65.2±2.0), embryos at morula stage in experiment 2 (30.4±1.5) and experiment 3 (22.2±2.0) were obtained. However, overall results for cumulus cell expansion, maturation of oocyte to MII stage and subsequent embryo development among treatments remain statistically similar (P > 0.05). Supplementation of α-tocopherol in maturation media having α-Linolenic acid and/or in embryo culture media did not further enhance in vitro maturation of oocyte or embryo production.

234 Effect of sericin supplementation in synthetic oviductal fluid medium for in vitro maturation of canine oocytes

234 Effect of sericin supplementation in synthetic oviductal fluid medium for in vitro maturation of canine oocytes

In vitro maturation in synthetic oviductal fluid increases gene expression associated with quality and lipid metabolism in bovine oocytes.

Traditionally, in vitro oocyte and embryo culture progresses through a series of varying culture medium. To investigate simplifying the in vitro production of bovine cumulus-oocyte complexes (COCs), this study used synthetic oviductal fluid (SOF) supplemented with conjugated linoleic acid (CLA). Special interest was placed on gene expression linked to lipid metabolism and oocyte maturation. COCs were matured in different media: Medium 199 (M199 group), M199 with 100 μM CLA (M199 + CLA group), SOF (SOF group), and SOF with 100 μM CLA (SOF + CLA group). COCs matured with SOF showed a higher relative abundance of mRNA of quality indicators gremlin 1 (GREM1) and prostaglandin-endoperoxide synthase 2 (PTGS2) in oocytes, and GREM1 in cumulus cells compared with in the M199 group. SOF medium COCs had a higher relative abundance of fatty acid desaturase 2 (FADS2) compared with the M199 group, which is essential for lipid metabolism in oocytes. Furthermore, the abundance of stearoyl-coenzyme A desaturase 1 (SCD1) in oocytes matured with SOF was not influenced by the addition of CLA, whereas the relative abundance of SCD1 was reduced in M199 medium with CLA. We concluded that maturation in SOF medium results in a greater abundance of genes linked to quality and lipidic metabolism in oocytes, regardless of the addition of CLA.

Gene expression at different cell stages of in vitro-fertilized bovine embryos

The birth rate of embryos produced in vitro (IVF) is still lower than that of embryos produced in vivo. Three major steps for the success of the IVF technique are maturation of immature oocytes, fertilization of matured oocytes, and culture of the resulting embryos. Studying mRNA expression in early embryonic development stages is important and can help to assess embryo quality and optimize production protocols in vitro. The current study aimed to determine the expression levels of developmentally important genes in different stages of bovine embryos produced in vitro. Cumulus-oocyte complexes (COCs) were collected from bovine ovaries and cultured in synthetic oviduct fluid (SOF) medium for 7 - 9 days. Embryos were collected at the time-points listed above, and mRNA expression of genes involved in pluripotency (OCT4), DNA methylation (DNMT1), apoptosis (BAX), and metabolism (GLUT1) and a heat shock protein (HSP70) was estimated from the 2-cell stage to the blastocyst stage of embryos. The results showed statistically significant differences in the relative abundance (RA) of OCT4, DNMT1, BAX, and GLUT1 gene transcripts among the different stages, whereas there were non-significant differences in the RA of HSP70 between these stages. In conclusion, gene expression levels differ among the developmental stages of embryos produced in vitro, possibly because of the timing of embryonic genome activation (EGA).

Novel Synthetic oviductal fluid for Conventional Freezing 1 (SCF1) culture medium improves development and cryotolerance of in vitro produced Holstein embryos.

In vitro produced (IVP) embryos hold great promise in the cattle industry; however, suboptimal in vitro culture conditions induce metabolic dysfunction, resulting in poor development and low cryotolerance of IVP embryos. This limits the use of IVP embryos in the cattle industry for embryo transfer and commercial scale-up. Previous studies have reported the use of individual metabolic regulators in culture media to improve blastocyst development rates and cryopreservation. In this study, we hypothesized that using a combination of select regulators, chosen for their unique synergistic potential, would alleviate metabolic dysfunction and improve the development of in vitro produced embryos to make them more closely resemble in vivo derived embryos. To test this, we first compared lipid content between Holstein and Jersey embryos produced in vivo and in vitro, and then systematically determined the combination of metabolic regulators that led to the greatest improvements in embryonic development, lipid content, mitochondrial polarity, and cryotolerance. We also tested different slow freezing techniques to further improve cryotolerance and finally validated our results via a clinical trial. Overall, we found that the use of multiple metabolic regulators in one culture media, which we refer to as Synthetic oviductal fluid for Conventional Freezing 1 (SCF1), and an optimized slow freezing technique resulted in improved pregnancy rates for frozen IVP embryos compared to embryos cultured in a synthetic oviductal fluid media. Additionally, there was no difference in pregnancy rate between frozen and fresh IVP embryos cultured in SCF1. This suggests that optimizing culture conditions and slow freezing technique can produce cryotolerance IVP and should allow further dissemination of this assisted reproductive technology.

The Impact of Two Embryo Culture Media, Synthetic Oviduct Fluid and Commercial BO, on pre-and post-Implantation Development of Cloned SAANEN Goat Embryos.

BackgroundSomatic cell nuclear transfer (SCNT) is an approach for the propagation of elite animals. In vitro condi- tion, especially the composition of culture media has a profound effect on the developmental competency of in vitro derived e mbryos. There are limited studies evaluating the effect of culture media on SCNT outcomes. To address this gap, we compare the effect of two culture media synthetic oviduct fluid (SOF) vs. commercial bracket-oliphant (BO) on developmental comptenecy.Materials and MethodsIn this experimental study, embryos derived from in vitro fertilized (IVF) and SCNT were cultured in both BO and SOF media for 7 days. In addition to the assessment of cleavage and blastocyst on day 3, and 7, the quantitative expression of 16 genes in theresultant blastocysts were assessed. The resultant SCNT blastocysts from SOF and BO groups were also transferred to the synchronized recipient for developmental competency to term.ResultsThe blastocyst rate in the BO medium was significantly higher than that of the SOF medium in the SCNT group (P<0.05). All of the examined genes showed increased expression levels in SCNT blastocyst in both media compared to IVF Blastocyst. In the IVF group, Oct4, Bmpr1, and Gcn5 showed significantly higher expression in the SOF medium com- pared to the BO medium while Akt, Fgfr4, Sox2 showed significantly lower expression in the SOF medium compared to the BO medium. In the SCNT group, Fgfr4, Gcn5, Fzd, Ctnnb, Bmpr1, and Fgfr4 showed significantly higher expression in SOF compared to BO derived blastocyst.ConclusionIt appears that in SCNT blastocysts, gene regulation is less controlled compared to IVF ones, irrespective of the type of medium. In addition, there are differences regarding certain genes expressions between IVF and SCNT derived blas- tocysts between SOF and BO, reiterating that culture composition affects developmental competency and gene expression.

Canine IVM With SOF Medium, Insulin-Transferrin-Selenium, and Low O2 Tension Improves Oocyte Meiotic Competence and Decreases Reactive Oxygen Species Levels.

Assisted reproductive technologies in canine species are limited due to the low efficiency of in vitro maturation (IVM). Unlike other mammals, bitches ovulate oocytes in the germinal vesicle stage and complete metaphase II (MII) after 48–72 h in the oviductal environment and become fertilizable. For this reason, we compared two different IVM media, synthetic oviductal fluid (SOF) supplemented with 8% bovine serum albumin (BSA) or a mixture of 8% BSA–2.5% fetal bovine serum (FBS) and TCM-199 with 10% FBS. Additionally, we evaluated the effect of supplementation with insulin-transferrin-selenium (ITS) and low O2 tension in oocyte maturation, reactive oxygen species (ROS) levels, membrane integrity, and embryo development following parthenogenetic activation (PA). After 72 h of culture, SOF + BSA, SOF + BSA + FBS, and TCM-199 + FBS show 5, 7, and 4% of MII, respectively, without a statistical difference. However, SOF + BSA produced significantly higher degeneration rates compared to SOF + BSA + FBS (44 and 23%, respectively). Remarkably, supplementation with 1 μl/ml of ITS under high O2 tension demonstrated a beneficial effect by improving maturation rates up to 20% compared to the other groups. Low O2 tension increased maturation rates to 36.5%, although there were no statistical differences compared to high O2 tension in the presence of ITS. Lower ROS levels and higher integrity of the cytoplasmic membrane were found in the presence of ITS despite no differences in maturation rates under low O2 tension groups. Additionally, after PA, 1% development until the eight-cell stage was obtained after activation of in vitro-matured oocytes in the presence of ITS. Taken together, these results indicate that SOF supplemented with 8% BSA and 2.5% FBS is suitable for IVM of canine oocytes and ITS supplementation was beneficial for both high and low O2 tension. Furthermore, the addition of ITS in the cultured system lowers ROS levels and increases membrane integrity in domestic dog oocytes after IVM.

EVALUATION OF TWO CULTURE MEDIA IN IN VITRO PRODUCTION OF ALPACA (Vicugna pacos) EMBRYOS

The present study aims to evaluate the effect of two culture media on the production of in vitro embryos in alpacas (Vicugna pacos). The ovaries were transported at 10.52° C in 0.9% saline solution supplemented with gentamicin. The ovaries were transported at 10.52° C in 0.9% physiological saline solution supplemented with gentamicin. 492 ovaries were used throughout the experiment. 2142 oocytes of quality I, II and III were recovered. The oocytes were matured in vitro for 32 h and were subsequently fertilized (incubated for 18 h) with sperm obtained from the tail of the epididymis and selected with a 45/90 percoll gradient. Then, the presumed zygotes were denuded from the cumulus cells, to later be cultured in two culture media: synthetic oviductal fluid medium (SOFaa) and simple optimized potassium medium (KSOMaa) and incubated at 38.5 ° C, 5 % CO2, 5%, O2, and 90% relative humidity for 7 days. Morula and blastocyst rate evaluation was performed at the end of embryo culture. The morula rate at 7 days was 41.49 ± 10.52 and 41.51 ± 6.50% for KSOMaa and SOFaa, respectively (P &lt;0.05). The blastocyst rate for the two culture media KSOMaa and SOFaa, was 14.08 ± 5.17 and 11.73 ± 5.69 %, respectively, and there were no statistical differences (P˃0.05). The embryonic quality in KSOMaa and SOFaa media did not show statistical differences. In conclusion, the KSOMaa and SOFaa culture medium can be used in the production of in vitro embryos of alpacas

16 Embryo aggregation and adipose-derived mesenchymal donor cells in bovine somatic cell nuclear transfer

Somatic cell nuclear transfer (SCNT) is a powerful tool, but its efficiency remains low. The use of less differentiated donor cells or the embryo aggregation (EA) strategy improves the SCNT rates in several species. It remains unexplored whether the combined use of both strategies results in a synergistic effect that improves SCNT efficiency in bovine. To evaluate that, we assessed the optimal time of EA using IVF embryos (aim 1) and we evaluated whether the use of adipose-derived mesenchymal stem cells (ASC) as donor for SCNT together with EA improves the blastocyst rates and quality (aim 2). For aim 1, cumulus–oocyte complexes (COCs) were collected from slaughterhouse ovaries, invitro matured (TCM-199), fertilized (16×106 spermatozoa mL−1 for 5h) and cultured (synthetic oviductal fluid media in a humidified gas mixture at 39°C). After IVF, the zona pellucida was enzymatically removed and zona-free (ZF) embryos were cultured individually (1X) or 2 embryos placed together within a microwell (2X) (Day 0, n=70). This procedure was performed at Days 3, 4, 5, 6, or 7 (n=76, 78, 94, 96, 90, respectively) and blastocyst rate was assessed at Day 8. Contribution of both embryos to the 2X blastocyst was confirmed by staining Day 0 IVF embryos either with green or red Mitotracker (ThermoFisher Scientific) before EA. For aim 2, fibroblast (FB) and ASC cells were isolated from the skin and subcutaneous adipose tissue of the same adult animal, respectively. Cloned embryos were produced by ZF enucleation, fusion of one ASC or FB cell, and activation with 5μM ionomycin/6-(dimethylamino)purine (6DMAP). After activation, cloned embryos were aggregated (FB2X or ASC2X) or individually cultured (FB1X or ASC1X). Blastocyst rates were recorded at Day 7 of invitro culture. Three biological replicates were evaluated for each aim. Embryo developmental differences were determined using Fisher’s exact test. Relative expression of OCT4, SOX2, and KRT18 was measured by RTqPCR at the blastocyst stage and analysed by Kruskal–Wallis statistical test. Regarding aim 1, no differences for developmental rates were found for Day 0, 3, 4, and 5 groups (57%, 60%, 61.5%, 61%), but the blastocyst rate was only improved in Day 0 and Day 3 relative to their respective 1X controls (Day 0 2X 54.2% vs. Day 0 1X 25.5% and Day 3 2X 52.6% vs. Day 3 1X 25.3%). No aggregation occurred in Day 6 and Day 7 groups. All blastomeres were homogeneously distributed in the 2X blastocyst. Regarding aim 2, no effect of the donor cell was observed on the blastocyst rate (FB1X 26.8%, n=82; ASC1X 21.7%, n=198; FB2X 39.7%, n=126; ASC2X 33%, n=204), whereas EA improved the blastocyst rate of ASC-derived embryos (ASC1X 21.7% vs. ASC2X 33%). Overall, no synergistic effect of the use of both strategies was observed. Relative expression of KRT18 was significantly different between ASC1X and ASC2X embryos. Although OCT4 and SOX2 expression did not differ between groups, EA tended to bring the values closer to that of an IVF embryo. No effect of the donor cell was observed on the embryo relative expression. Our results suggest that EA at Day 0 improves the blastocyst rate in bovine SCNT and IVF embryos. EA of 2 ASC-derived embryos seemed to normalise the embryo quality and may improve post-implantation development.

15 Blastocysts altered CDX2 and SOX2 gene expression and pregnancy failure after embryo transfer in yak heterospecific somatic cell nuclear transfer

Heterospecific cloning is a tool for the genetic rescue of endangered animals. Our objective was to evaluate the effects of heterospecific yak (Bos grunniens) cloned embryo aggregation on the expression levels of NANOG, OCT4, CDX2, and SOX2 genes, and to compare with IVF, parthenogenetic zona-free (P-ZF), and homospecific bovine cloned embryos (BB1x). Oocytes were recovered from the ovaries of slaughtered cows and invitro matured for 22h. The zona pellucida was removed by protease treatment and then mature oocytes were enucleated by micromanipulation. Enucleated oocytes were placed in phytohemagglutinin to induce adherence with a somatic donor cell followed by electrofusion (with two 30-µs pulses of 1.2 kV/cm, 0.1s apart). Two hours after fusion, reconstructed embryos were activated using ionomycin followed by 6-(dimethylamino)purine (6-DMAP) treatment for 3h and cultured in synthetic oviductal fluid (SOF) medium for 7 days. The experimental groups were IVF, P-ZF, BB1x, heterospecific yak-bovine cloned embryos (1 embryo per microwell, YB1x), and heterospecific yak-bovine cloned embryos aggregated (2 embryos per microwell, YB2x). In all experimental groups, cleavage and blastocyst rates were assessed 7 days after activation. In addition, 5 blastocysts were pooled for each biological replicate, and pluripotency-specific genes (NANOG, SOX2, CDX2, and OCT4) were analysed by quantitative PCR. Data were analysed by the ΔΔCT method using the geometric mean of ACTB (actin) and GAPDH as internal standard followed by one-way ANOVA. Cleavages rates were significantly lower in the YB1x group compared with the other groups. Moreover, blastocyst rates in YB2x (31.34%, n=67) were significantly higher than in YB1x (13.86%, n=101) and BB1x (13.33%, n=45) groups, but there were no significant differences compared with the IVF (43.82%, n=89) and P-ZF (25%, n=68) groups. In contrast, although no significant differences were observed among groups in the expression of NANOG and OCT4 genes, the expression of CDX2 was lower in YB2x and YB1x blastocysts compared with the BB1X, P-ZF, and IVF (control) groups. In addition, a decrease in SOX2 gene expression was observed in the YB2x and YB1x blastocysts compared with the BB1X group. Blastocysts from YB1x (n=5) and YB2x (n=18) groups were transferred to recipient cows (n=23) on Day 7. Forty days after embryo transfer, presence of uterine fluid was detected by ultrasound in 3 recipient cows (from YB2x), suggesting embryo loss. In concordance with our previous reports, yak heterospecific SCNT blastocysts showed underexpression of CDX2 and SOX2 compared with the overexpression observed for these genes in bovine homospecific SCNT blastocysts. Thus, yak heterospecific SCNT blastocysts may have compromised developmental competence associated with altered expression of CDX2 and SOX2 that cannot be rescued by the aggregation of 2 reconstructed embryos.

82 Invitro embryo production outcomes in adult goats is affected by season

In seasonal breeders, reproductive seasonality can have a substantial influence on the efficiency of assisted reproductive technologies. This study assessed seasonal effects on cleavage and blastocyst rates, as well as on quality of invitro-produced (IVP) goat embryos over 18 months. In total, 2348 (autumn: 811, spring: 404, summer: 639, and, winter: 494) cumulus–oocyte complexes (COC) were recovered from slaughterhouse ovaries during 49 replicates (autumn: 17, spring: 7, summer: 15, and, winter: 10) and matured in TCM-199 with 10ng mL−1 epidermal growth factor and 100µM cysteamine for 22h at 38.8°C (5% CO2 in air). Matured oocytes were fertilized with frozen/thawed semen in synthetic oviductal fluid (SOF) with 10% oestrus sheep serum. Sperm and oocytes were co-incubated for 16h at 38.8°C in a humidified atmosphere of 5% CO2 in air. Presumptive zygotes were cultured in SOF medium supplemented with bovine serum albumin (3mg mL−1) for 8 days at 38.8°C in a humidified atmosphere of 5% O2, 5% CO2 and 90% N2. At Day 2, 10% fetal calf serum was added to the culture droplets. The embryos produced were fixed and stained with Hoechst to count their total number of cells, under an epifluorescence microscope. The results of cleavage and blastocyst rates, including hatching rate, from each routine of IVEP were considered as replicates. These data were tested for normality by the Shapiro-Wilk test, before being subjected to ANOVA, followed by Tukey HSD test. The odds ratio (OR) among seasons (autumn: breeding; spring: anoestrus) were calculated. Values of P&amp;lt;0.05 were considered as significant, and the data reported are mean±s.e.m. Cleavage rate was lower (P&amp;lt;0.05) in spring (51±7.1%) than in either autumn (72±2.1%) or summer (71±2.0%) while winter (66±4.1%) had an intermediate value, being similar (P&amp;gt; 0.05) to all others. Indeed, greater possibility of cleavage was observed in autumn (OR: 2.43) and summer (OR: 2.39) compared with spring. The blastocyst rate from cleaved embryos was greater (P &amp;lt;0.05) in autumn (73±2.7%) than in spring (55±2.6%; OR: 2.18). As a result, the blastocyst formation rate from the initial number of COC entering IVM was greater (P&amp;lt;0.05) in autumn (52±2.5%) than in spring: 28±4.7%, summer: 45±2.3%, and winter: 42±2.1%; indeed, the spring season resulted in the lowest rate (P&amp;lt;0.05), compared with other seasons. Moreover, the OR in the blastocyst rate from initial number of COC was greater (P&amp;lt;0.05) in autumn compared with all other seasons and lower in spring compared with winter (OR: 0.54) and summer (OR: 0.48). There were no differences (P&amp;gt; 0.05) in the embryo hatching (mean: 66±2.0%) and blastocyst cell number (mean: 193±2.0 cells). In conclusion, the breeding season (autumn) leads to improved oocyte developmental competence, resulting in greater cleavage and blastocyst yield, whereas embryo quality remained similar throughout the year. Further studies at the molecular level might indicate the mechanisms involved and provide clues to alleviate the negative effect of season.

In vitro maturation medium supplementation with resveratrol improves cumulus cell expansion and developmental competence of Sanjabi sheep oocytes

The purpose of this study was to evaluate the effect of different concentrations of resveratrol (0, 0.1, 0.25, 0.5, 2.0, and 5.0 μM) which was added to the IVM medium on cumulus expansion, nuclear maturation, and total cell number of embryos in Sanjabi ewes. Meiotic progression was evaluated based on cumulus expansion, the frequency of germinal vesicles, germinal vesicle breakdown and metaphase II (MII). The effects of the resveratrol concentrations in the IVM medium on cleavage and developmental potential to blastocyst stage were also investigated. After maturation, a sample of oocytes were fixed and stained to evaluate nuclear maturation, while the remaining oocytes were fertilized in vitro with fresh semen, then cultured for 8 days in synthetic oviductal fluid (SOF) medium for assessment of oocyte developmental capacity. Addition of 0.25 and 0.5 μM of resveratrol to the maturation medium was shown to significantly increase cumulus expansion (P < 0.05). There was no significant difference in the percentage of MII oocytes between control, and 0.1, 0.25, 0.5 and 2.0 μM resveratrol treatment groups. Furthermore, addition of 0.25 and 0.5 μM of resveratrol to IVM medium also significantly improved blastocyst formation, and addition of 0.5 μM resveratrol in maturation media increased trophectoderm cell numbers, inner cell mass and the total cell number of ovine embryos. However, addition of high resveratrol concentrations (5.0 μM) to IVM medium had a negative effect on the maturation rate and blastocyst formation. Together, these findings show that supplementation of IVM medium with 0.25 and 0.5 μM resveratrol can improve the meiotic competence and early embryonic development of Sanjabi sheep oocytes.

Development of prepubertal goat oocytes after their in vitro maturation and chemical activation.

Experiments were conducted to study in vitro maturation of prepubertal goat oocytes and their developmental potential after chemical activation. In Experiment 1, cumulus-oocytes complexes collected from the ovaries of prepubertal goats slaughtered at a local abattoir were matured in vitro in TCM-199-based medium supplemented with 10 µg/ml luteinizing hormone (LH) (treatment 1) or 10 µg/ml LH + 0.1 mM l-cysteine (treatment 2). In Experiment 2, mature oocytes were activated with either 5 µM ionomycin or 7% ethanol. After 18 h, some oocytes were randomly fixed and stained to evaluate their chromatin status, while others were cultured in embryo culture medium to study their further development. In Experiment 3, oocytes activated with 5 µM ionomycin were cultured for 7 days in one of the four different culture media [Charles Rosenkrans medium (CR-1), TCM-199, potassium simplex optimization medium (KSOM) and synthetic oviductal fluid (SOF)] to study their developmental potential. The maturation rate in control, treatment 1, and treatment 2 media did not differ from each other (P > 0.05). However, the lowest degeneration of oocytes was observed in treatment 3 (P < 0.05) when compared with the other two groups. The proportion of activated oocytes was higher, while non-activated oocytes were lower in ionomycin group when compared with the group activated with ethanol (P < 0.05). The proportions of oocytes cleaved were 65.7, 56.8, 61.0 and 54.4% in CR-1, TCM-199, KSOM and SOF medium, respectively, with no significant difference. However, further development of cleaved oocytes was better in KSOM followed by SOF.

Production ofin vitrobovine embryos supplemented withl-carnitine in different oxygen tensions and the relation to nitric oxide

SummaryThe aim of this study was to evaluate the production of bovine embryosin vitrowhen supplemented withl-carnitine for 24 h beginning on day 5 (d 5) under two different oxygen tensions (20% or 5%) and the relationship of nitric oxide (NO) inin vitroculture (IVC) medium to embryo development. Cumulus–oocyte complexes (COC;n= 837) were maturedin vitrofor 24 h and fertilization was performed for 18 h. Zygotes were culturedin vitrofor 9 days afterin vitrofertilization in synthetic oviductal fluid (SOF) medium with 5% fetal calf serum. At d 5 the plates were assigned to one of four treatment groups: high (20%) or low (5%) O2tension either with or without the addition of 3.03 mMl-carnitine (High-Cont, High-Lcar, Low-Cont, Low-Lcar). The concentration of NO in the culture medium was evaluated on d 5, d 6 and d 9. On d 7, parts of the embryos were submitted for evaluation of intracellular lipid droplets. The cleavage rate was similar (P&gt; 0.05) between high and low O2tension and the blastocyst rate was similar in all conditions evaluated. The hatching rate was higher (P &lt;0.05) for Low-Cont. The NO concentration was higher at d 9 under low O2tension (P &lt;0.1). The addition of 3.03 mMl-carnitine between d 5 and d 6 of IVC was not efficient in reducing cytoplasmic lipid content of bovine embryos. Additionally, IVC at a low oxygen tension withoutl-carnitine promoted better conditions for embryo development. A higher concentration of NO in medium was observed under low O2tension.

Effect of delipidant agents during in vitro culture on the development, lipid content, gene expression and cryotolerance of bovine embryos.

In vitro produced embryos are still sensitive to the freezing process which can be explained, in part, by the high-lipid accumulation that characterizes these embryos. Therefore, we aimed to evaluate the effect of delipidating agents, L-carnitine and the trans-10 cis-12 conjugated linoleic acid (CLA) isomer, on blastocyst development, lipid content, gene expression and cryotolerance when added to embryo culture media. Embryos were cultured in four different media: T1: control (n=616), synthetic oviduct fluid (SOF) media with 5% foetal bovine serum (FBS); T2: L-carnitine (n=648), SOF medium with 5% FBS and 0.6mg/ml of L-carnitine; T3: CLA (n=627), SOF medium with 5% FBS and 100μM trans-10 cis-12 CLA; and T4: L-carnitine+CLA: (n=597), SOF medium with 5% FBS plus 0.6mg/ml L-carnitine and 100μM trans-10 cis-12 CLA. Supplementation of culture medium with either or both delipidating agents reduced (p<.05) blastocyst rate on D7 (T1=49±3.5; T2=39±3.0; T3=42±3.9 and T4=39±3.9), but did not affected gene expression (p>.05). Although embryos cultured in the presence of L-carnitine contained fewer (p<.05) lipid droplets than the control embryos, they showed a lower re-expansion rate 24hr post-thaw than those (p<.05). In conclusion, although L-carnitine reduced the amount of lipids in cultured embryos, the use of L-carnitine and CLA during in vitro culture was not able to improve the embryo production and the response to cryopreservation.

20 Aggregation of yak heterospecific somatic cell nuclear transfer embryos improves cloning efficiency

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.

96 Evaluation of CRISPR/Cas9 alternative delivery in parthenogenetic porcine embryos

Porcine genetic editing is considered promising in biomedical research, particularly for xenotransplantation. However, invitro porcine embryo production is less efficient than in other animal models. Thus, we aimed to perform a rapid optimization essay by producing parthenogenetic porcine embryos to evaluate transgenesis delivery and CRISPR/Cas9 editing efficiency. First, PCX-enhanced green fluorescent protein (EGFP) plasmid was microinjected (30ng μL−1) into a diploid parthenogenic zygote with (lipo+) or without lipofectamine (lipo−), since it has been shown that this transfection reagent improves transgene delivery and increases its expression in bovine embryos (Vichera et al. 2011 Reprod. Domest. Anim. 46, 214-220; https://doi.org/10.1111/j.1439-0531.2010.01642.x). Briefly, invitro-matured oocytes were electrically activated, followed by incubation in synthetic oviductal fluid medium containing 6-dimethylaminopyridine. Embryos were invitro cultured until Day 7, and EGFP-positive (EGFP+) embryos were corroborated at Day 5. Data were analysed using Fisher's exact test. Cleavage rates were no different among groups (EGFP/lipo−: 40%, n=28; EGFP/lipo+: 45%, n=41; control: 41, 3%, n=45; P&amp;gt;0.05). Also, no significant difference in the percentage of EGFP+ embryos was observed between EGFP/lipo+ (31%, n=13) and EGFP/Lipo− (18%, n=5) groups. Although blastocyst rate showed no statistical difference among groups, a lower blastocyst percentage tendency was observed in the EGFP/Lipo+ group compared with the control group (EGFP/Lipo+: 5%, n=2; control: 20%, n=9; P=0.051), suggesting that the presence of lipofectamine and EGFP plasmid may affect embryo development. Next, two guides (single guide (sg) RNA) were designed for the internal regions of GGTA1, CMAH, and VWF target genes, involved in hyperacute rejection and coagulation in xenotransplantation. A liposome-DNA mixture was used: DNA for sgRNA and Cas9, with and without lipofectamine (10× dilution; CRISPR/lipo+, CRISPR/lipo−), diluted to half concentration with 10% polyvinylpyrrolidone, resulting in a final concentration of 20ng ul−1 for all sgRNA and 40ng ul−1 for Cas9. A total of 2 pL of the mixtures was microinjected into the diploid parthenogenetic zygotes which were invitro cultured until Day 7. Genetic editing was corroborated by analysing the presence of a double cut directed by the two sgRNA designed for the target genes, resulting in an amplicon with lower molecular weight compared to the wild-type PCR fragment. No differences in cleavage or blastocyst rates were observed among groups (blastocyst rates: CRISPR/Lipo−: 12%, n=13; CRISPR/Lipo+: 10%, n=7; control: 15%, n=18; P&amp;gt;0.05). Finally, one embryo showed a single deletion for GGTA1 and another showed a double deletion in GGTA1 and VWF genes, out of seven embryos from the CRISPR/Lipo+ group. No gene deletion was confirmed in any of the embryos from the CRISPR/Lipo− group. These results are preliminary data (more experiments are currently being done) suggesting that microinjection of CRISPR/Cas9 with lipofectamine could be used as an alternative delivery system, since it seems to have no impact on porcine embryo development.

199 Strategies to improve canine oocyte invitro maturation

Canine oocyte invitro maturation (IVM) is one of the challenges of animal reproduction because of low maturation and high degeneration rates. In the bitch, after ovulation, oocytes remain in an immature stage and acquire their competence in the intra- and extrafollicular (oviductal) environments. Oxidative stress and reactive oxygen species affect canine oocytes, which can be related to the high amount of lipids they contain. Therefore, the use of antioxidants such as insulin-transferrin-selenium (ITS) and lower oxygen tension during IVM could be beneficial for oocyte maturation and survival. The purpose of this study was to determine an optimum IVM culture medium and to evaluate the effect of ITS and lower oxygen tension in canine IVM. In experiment 1, TCM-199 and synthetic oviductal fluid (SOF) media were evaluated for their ability to promote nuclear maturation at 72 and 48h of culture. Also, two protein sources were used: 8% bovine serum albumin (BSA) and 2.5% fetal bovine serum (FBS), and media were supplemented with hormones. The results revealed that SOF with FBS and BSA had similar results to TCM-199 supplemented with FBS after 72 and 48h of IVM (MII rates of 7% and 4% for the 72-h group, and 4% and 10% for the 48-h group). Synthetic oviductal fluid supplemented with BSA but without FBS produced significantly higher degeneration rates compared with SOF with FBS and BSA (44% and 23%, respectively). Forty-eight hours of IVM decreased degeneration rates, with higher MII rates compared with 72h of IVM. In experiment 2, SOF medium supplemented with FBS and BSA was chosen. Oocytes were cultured in SOF with FBS and BSA supplemented at two concentrations of ITS (1 and 10μLmL−1 ITS). Supplementation with 1μLmL−1 ITS demonstrated a beneficial effect by improving maturation rates up to 20%, compared to control and 10μLmL−1 supplemented group (4% and 6% MII, respectively) after 72h of IVM. For experiment 3, oocytes were cultured in SOF medium with or without ITS (0 and 1μLmL−1 ITS) under two oxygen tensions (5% and 20% O2) for 48h. Results from this experiment demonstrated that the combination of low oxygen tension and ITS (5% O2 and 1μLmL−1 ITS) improved maturation rates up to 26.2%, although there were no statistically significant differences compared with high oxygen and ITS (20% O2 and 1μLmL−1 ITS) and low oxygen without ITS (5% O2 and 0μLmL−1 ITS) groups. These treatments were able to increase MII rates compared with the control group (20% O2 and 0μLmL−1 ITS). Parthenogenetic activation was performed on the low oxygen with or without ITS supplemented groups. The untreated group generated higher degeneration rates after 7 days of culture, and cleavage rates were low for both groups. Nevertheless, an oocyte at the 8-cell stage was obtained in the ITS-supplemented group. Taken together, these results indicate that ITS supplementation and low oxygen tension during IVM improve canine oocyte maturation.

79 MicroRNAs of extracellular vesicles secreted by embryos as an early biomarker of competence

Extracellular vesicles (EVs), including exosomes and microvesicles, are secreted by different cell types and participate in cellular communication by carrying molecules as microRNAs (miRNAs) that can interfere with gene expression of target cells. Extracellular vesicles have become relevant as a mechanism of embryo-maternal communication. The aim of this study was to evaluate miRNA content in EVs secreted after embryonic genome activation, by bovine embryos with different developmental potential. Bovine embryos were produced invitro and cultured in group until Day 3.5 in synthetic oviductal fluid (SOF) medium. Only 8-16-cell embryos were cultured individually in EVs-depleted SOF until Day 5. The SOF was EV depleted by ultrafiltration. Culture media (CM) were collected at Day 5 and embryos continued in culture until Day 7 with fresh SOF. Collected media were conserved individually and identified with the corresponding embryo. Then, CM were classified according to capacity of its embryo to reach blastocyst stage at Day 7: G1-CM (blocked embryos in 8-16 cell) and G2-CM (embryos that reach blastocyst stage). The EV isolation was carried out using the protocol described by Mellisho et al. (2017). Recovered EVs were evaluated by nanoparticle tracking analysis (NTA), Transmission electron microscopy and the presence of surface markers (CD9, CD63, CD81, and CD40L). After NTA, individual CM were pooled to organise 3 replicates of 10CM each, for G1 and G2. The whole miRNA isolation, library preparation, and sequencing was performed by Norgen Biotek facilities (Canada). The quality of libraries was analysed using the FastQC program platform followed by Trimmomatic to remove remnant adapters. For the miRNA library it accepted reads with value above 30 Phreads and 22 to 30bp length. The reads were mapped against the reference genome ARS-UCD1.2 using Bowtie2 software and miRDeep2 mapper, and the gene counts were calculated using HTSeq. Differential expression analysis was performed in EdgeR package. To expand this information, principal component analysis, Heatmap, and Volcano plot were plotted and pathway enrichment analysis was conducted. The NTA, transmission electron microscopy, and flow cytometry confirmed the presence of exosomes and microvesicles in isolated EVs. According to NTA, the mean size of EVs was 102.1-176.2nm and concentration of 8.4×107-8.6×108 particlesmL−1 in G1 and G2, respectively. We identified 96 miRNAs significantly expressed across the samples. Only eight miRNAs in EVs were differentially expressed between groups (G2 vs. G1). The bta-miR-103, bta-miR-502a, bta-miR-100, and bta-miR-1 were up-regulated (Log2 fold-change&amp;gt;1), whereas bta-miR-92a, bta-miR-140, bta-miR-2285a, and bta-miR-222 were down-regulated (Log2 fold-change&amp;lt;1). The more significant (P-value&amp;lt;0.01) up-regulated Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were fatty acid biosynthesis and metabolism, lysine degradation, gap junction, and signaling pathways regulating pluripotency of stem cells. The EVs secreted by embryos to culture environment carry miRNAs that can reflect the molecular state of their parental cell. This lets us suggest culture media derived-EVs and their miRNA cargo as early biomarkers to select more competent bovine embryos. This research was supported by FONDECYT, Chile (1170310).

87 Analyzing metabolomic profile of bovine IVF and somatic cell nuclear transfer embryos through Raman spectroscopy

While knowledge of early embryo development and viability has continually increased, there is still a need for noninvasive methods to identify embryos with the highest chances of development to term when transferred. The most widely used technique, morphological assessment, is highly subjective and limited by personnel experience. Spent media from invitro culture could be used as a valuable noninvasive marker for embryo quality assessment. Raman spectroscopy has proven to be a powerful tool for identifying the molecular characteristics of spent culture media by measuring vibration of chemical bonds, allowing for metabolomic profiling of varying stages and quality of embryos. It is well documented that embryos produced through somatic cell nuclear transfer (SCNT) result in lower pregnancy rates and higher incidences of pregnancy loss when compared with embryos produced through IVF (Heyman et al. 2002 Biol. Reprod. 66, 1-13). This study was designed to examine differences in spent media between bovine embryos produced by IVF and SCNT. The SCNT embryos with a metabolomic profile more similar to IVF embryos may have a higher developmental competence. Bovine cumulus-oocyte complexes (COCs) were retrieved from abattoir-derived ovaries and matured for 21h in maturation medium. After IVM, COCs were either used for IVF or SCNT and cultured in 50μL droplets of synthetic oviductal fluid medium + fetal bovine serum in groups of 45 from Day 0-5. On Day 5, embryos that had reached morula stage were placed in individual droplets of 13μL of synthetic oviductal fluid + bovine serum albumin until Day 7. All embryos were cultured at 38.5°C and 5% CO2. On Day 7 embryos were assessed for developmental stage and quality and 10μL of medium from individual culture drops was collected for Raman spectroscopy. Samples were loaded on an MgF2 optical window, dried, and analysed using a 785nm near-infrared laser in the spectral range of 600 to 1800cm−1. Raw Raman data were first pre-processed by baseline correction (asymmetric least-squares smoothing) and normalization. Principal component analysis and partial least squares were then applied to reduce data dimensions. The score of the most significant principal components from principal component analysis and the optimum number of scores from partial least squares were used for linear discriminant analysis. Spent media samples from 4 SCNT embryos, 3 IVF embryos, and 3 empty media samples were analysed, with 50 spectra obtained from each sample. Preliminary data showed grouping of medium containing embryos developed to blastocyst from medium containing embryos arrested at morula or empty medium. We also saw grouping of medium containing SCNT embryos from medium containing IVF embryos within both the morula and blastocyst stage from empty medium. This shows evidence of metabolomic differences between embryos of different developmental potential and embryos produced by IVF and SCNT. Further investigation of the Raman profile of these groups can display specific differences in chemical components and help to identify candidate genes causing differing metabolism of these groups.