Synthetic Oviduct Fluid Research Articles

Improvement of bovine in vitro embryo production by fetal calf serum and cysteamine supplementation and investigation of freezability

The aim of this study was to investigate the effects of Cysteamine (Cys) and Fetal Calf Serum (FCS) in Synthetic Oviduct Fluid (SOF) and Charles and Rosenkrans (CR1aa) on the bovine in vitro embryo production and its ability of freeze. The oocytes were divided into two groups and allowed to mature in TCM-199, with and without cysteamine. They were divided into 4 subgroups according to whether they contain Cys and FCS in 2 different culture media (SOF and CR1aa). Accordingly, 8 groups were formed as SOF+FCS+Cys, SOF+Cys, SOF+FCS, S Dulbeco's OF, CR1aa+FCS+Cys, CR1aa+Cys, CR1aa+FCS and CR1aa. Embryos were cultured for 7-9 days and were frozen using vitrification method. Development of embryos was observed during the first 24 hr post-thaw period. The highest rate of compact morula was 20% in SOF+FCS+Cys and 19.4% in CR1aa+FCS+Cys. The rate of blastocyst in SOF+FCS and CR1aa+FCS were found 17.9% and 15.4% respectively and the difference between groups was statistically significant (P<0.05). During the 12 hr vitality assessment, the highest rate was determined in SOF+FCS (47%). The results indicate that FCS has a positive effect in reaching compact morula and blastocyst regardless of the presence of the culture medium or antioxidant used. There was no statistically difference between the values of post-thaw embryos.

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.

Modern achievements of the development of scientific research at obtaining in vitro sheep embryos

New literature data on research aimed at improving the in vitro production of sheep embryos presents in the article. An analysis of the achievements of scientists from different countries to increase the efficiency of the main stages of embryo production in vitro: maturation of oocytes in vitro, their in vitro fertilization and in vitro embryo culture. In the literature experience has shown that the efficiency of oocyte maturation in vitro is significantly influenced by the experience and qualifications of scientists, the age of the egg donor, the improvement of the environment by adding roscovitin to inhibit meiosis, α-linolenic acid, cerium dioxide nanoparticles (CeO2 NPs) and sericin to accelerate nuclear maturation and increase the number of oocytes of the second meiotic metaphase (MII). The main factors influencing the effectiveness of in vitro fertilization have been identified, and the parameters of the limited time of fertilization ability of sperm and the ability of oocytes to fertilize, which is called the “fertile span”, have been determined. The main effective medium that increases the effectiveness of in vitro fertilization – synthetic oviduct fluid (SOF) with the addition of heparin and serum of cattle or sheep. The main parameters of sheep embryo culture in vitro are presented with the definition of the most commonly used media and their influence on embryonic development. Potential ways to improve the production of sheep embryos in vitro with the determination of morphological evaluation of categories of oocytes, methods of synchronization of their maturation in vitro are also highlighted. At the same time, literature data on the synchronization of oocyte-cumulus complexes with the use of a large number of inhibitors of meiotic division are presented, which according to many scientists may be a key factor in improving the efficiency of sheep embryo production in vitro. In addition, the results of studies of many scientists on the expansion of the fertile gap of oocytes of sheep cultured in vitro using certain biologically active substances were analyzed. In conclusion, the prospect of using the technology of in vitro production of sheep embryos in biomedical research is highlighted.

Inhibiting diacylglycerol acyltransferase-1 reduces lipid biosynthesis in bovine blastocysts produced in vitro

Diacylglycerol acyltransferase-1 (DGAT1) is one of the DGAT enzymes that catalyzes the final step in the synthesis of triacylglycerol, which is a major component of the lipid droplets in embryos. Intracellular lipids accumulated in embryos produced in vitro have been associated with reduced cryotolerance and quality. The objective of the present study was to investigate the influence of DGAT1 inhibition on embryo development, quality, and post-vitrification survival, in addition to expression profiles of selected lipid metabolism-regulating and oxidative stress genes. Bovine cumulus-oocyte complexes were matured and fertilized in vitro and were cultured in synthetic oviduct fluid (SOF) supplemented with 5% fetal calf serum (FCS) alone (Control) or with 1, 5, 10 or 50 μM DGAT1 inhibitor (A922500®; D1, D5, D10, and D50, respectively) or 0.1% dimethyl sulfoxide (CDMSO: vehicle for DGAT1 inhibitor dilution) from 54 h post-insemination until Day 8 post insemination. No differences were found in blastocyst yield on days 7 and 8 in Control, CDMSO, D10, and D50 groups. Embryos cultured with 10 or 50 μM DGAT1 inhibitor had greater mitochondrial activity (P < 0.01), and increased number of cells (P < 0.05), while the cytoplasmic lipid content was reduced (P < 0.01), the latter associated with altered expression profiles of selected genes regulating lipid metabolism or genes related with oxidative stress (transcript abundance increased for SLC2A1 and SLC2A5 and decreased for DGAT1 and GPX1). Importantly, the survival rate of blastocysts produced with 10 μM DGAT1 was higher than that of Control, CDMSO and D50 groups at 72 h after vitrification and warming (73.8 vs 57.1, 55.9 and 56.1%, respectively, P < 0.001). In conclusion, inhibition of DGAT1 synthesis in bovine embryos produced in vitro abrogates the negative effect of FCS by decreasing their lipid content, increasing mitochondria activity and improving embryo cryotolerance, as well as favoring the expression of lipid metabolism regulating and oxidative stress-related transcripts.

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.

In Vitro Bovine Embryo Production: The Role of FCS and Cysteamine on Cleavage Rate

The aim of this study was to investigate the effects of Cysteamine (C) and Fetal Calf Serum (FCS) on cleavage rates in bovine embryo production. The oocytes were obtained from the slaughtered ovaries by slicing method. They were selected and divided into two groups and maturated in TCM and TCM+Cysteamine for 24 hour at 38.8oC in atmosphere of 5% CO 2 in humidified air. The matured oocytes were transferred to IVF-TALP and allowed to fertilize with frozen bull semen to be 400-600x 10 3/ well which were washed with Sperm-TALP in an incubator containing 5% CO 2 for 18-22 hours at 38.8oC. At the end of this period, the embryos of each group which were denuded by vortexing transferred to Synthetic Oviduct Fluid (SOF) and Charles and Rosenkrans (CR1) media to be developed. These culture media were divided into 4 subgroups according to whether they contain Cysteamine (C) and Fetal Calf Serum (FCS). For this reason Group A1: (SOF C+F+), Group A2: (SOF C+F-), Group A3: (SOF C-F+), Group A4: (SOF C-F-), Group B1: (CR1 C+F+), Group B2: (CR1 C+F-), Group B3: (CR1 C-F+), Group B4: (CR1 C-F-) were formed. Embryos were cultured in an incubator containing 5% CO 2 , 5% N 2 and 90% humidity at 38.8oC for 48 hours. At the end of this period, the clevage rate of the embryos were recorded and there was no statistical difference (P<0.05) between all groups.

Efficient one-step direct transfer to recipients of thawed bovine embryos cultured in vitro and frozen in chemically defined medium

Direct transfer (DT) of cryopreserved embryos to recipients facilitates on-farm application. We analyzed a new freezing/thawing (F/T) procedure for in vitro produced (IVP) embryos, integrating: 1) an ethylene-glycol based system; 2) a culture step without protein; and 3) a synthetic protein substitute (CRYO3) in cryopreservation medium. IVP embryos from abattoir ovaries were cultured in groups in BSA-containing synthetic oviduct fluid with or without 0.1% fetal calf serum (FCS) until Day-6. Morulae and early blastocysts were subsequently cultured without protein from Day-6 onwards. Day 7 and Day 8 expanded blastocysts (EXB) were subjected to F/T or vitrification/warming (V/W). Thawed and warmed EXB were cultured in vitro, and development rates, cell counts and dead cells were analyzed in surviving embryos. V/W improved survival over F/T (live and hatching rates at 2 h, 24 h and 48 h) (P < 0.0001), and FCS before Day 6 did not affect in vitro survival. After F/T, embryos had lower cell counts in the ICM, TE and total cells than after V/W. Day-7 embryos after F/T showed % apoptotic, % pycnotic and % total dead cells higher (p < 0.05) than their Day-8 counterparts, probably because F/T reduced the numbers of ICM cells within Day-8 embryos. Thereafter, Day-7 blastocysts were transferred to heifers in an experimental herd. There were no differences in birth rates with frozen (-FCS [n = 40]: 45%; +FCS [n = 14]: 28%), vitrified (-FCS [n = 47]: 53%; +FCS [n = 11]: 36%) and fresh (-FCS [n = 30]: 47%; +FCS [n = 17]: 53%) embryos. However, frozen embryos produced with FCS showed 5/9 miscarriages after Day-40. Calves born from frozen (n = 22), vitrified (n = 29) and fresh (n = 22) transfers did not differ in birth weight, gestation length and daily gain weight (P > 0.10). Subsequently, transfer of frozen embryos (n = 29) derived from oocytes collected from live, hormonally stimulated cows in experimental herd, led to pregnancy rates of 57% (heifers) and 40% (dry cows). with EXB on Day-62 Finally, embryos produced with BSA were transferred to cows in an on-field trial (frozen [n = 80]; fresh [n = 58]), with no differences in pregnancy rates (days 30–40). Pregnancy and birth rates could not be predicted from in vitro approaches. The new F/T system yields pregnancy and birth rates comparable to vitrified and fresh embryos without birth overweight. The absence of products of animal origin, defined chemical composition, and direct transfer entail sanitary, manufacturing and application advantages.

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.

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.

190 Effect of TCM-199 and synthetic oviductal fluid medium supplemented with varying hormone concentrations on invitro maturation of canine oocytes

Invitro oocyte maturation (IVM) in the domestic canine is yet to be optimized, with low rates of cumulus-oocyte complexes (COCs) reaching MII. This limits the progression of assisted reproductive technologies, which could benefit breeding programs for assistance dogs and endangered Canidae. Canine oocyte maturation differs from that in other mammals, with the ovulation of a COC in the germinal vesicle stage and nuclear maturation occurring in the oviduct. Because of this, the environment in which a canine COC matures is unlike that of other mammals, meaning that IVM protocols cannot be readily adapted. The aim of the current work was to determine (1) the effects of varying concentrations of FSH, human chorionic gonadotrophin (hCG), and oestradiol (E2) during IVM on meiotic resumption and nuclear maturation of canine COCs; and (2) the optimal medium base, either synthetic oviductal fluid (SOF) or tissue culture medium-199 (TCM). Reproductive tracts of bitches (6 months to 7 years of age) were collected from veterinary clinics within 2h of routine spaying. Ovaries were sliced using a scalpel blade, releasing the COCs into aspiration medium. The COCs were randomly allocated to a maturation medium consisting of one of the hormones at two concentrations (FSH: 5 or 10µgmL−1; hCG: 5 or 10IUmL−1; E2: 1 or 5µgmL−1) and for both SOF and TCM base. Each hormone was tested individually for a replicate of eight animals per hormone (total of 12 experimental groups; 24 animals). The COCs were cultured for 72h in their allocated medium and then denuded and stained with Hoechst 33258. Fluorescence microscopy was used to determine nuclear maturation stage. Nuclear maturation rates to MII were analysed using a general linear model with pairwise comparison (SPSS version 25; SPSS Inc./IBM Corp.) with each individual animal acting as a replicate. Canine COCs matured in a SOF-based media had higher rates of meiotic resumption (MI and MII) (SOF: 38.68%, n=515; TCM: 25.78%, n=542; P&amp;lt;0.05) and number reaching MII (SOF: 7.54%; TCM: 4.39%; P&amp;lt;0.05) compared with TCM-based medium. Resumption of meiosis and nuclear maturation to MII did not differ between media with differing E2 or hCG concentrations. The use of FSH at 10µgmL−1 in SOF medium decreased resumption of meiosis (8.57%) and MII rates (0%) compared with 5µgmL−1 FSH in SOF (29.41% and 3.92%, respectively; P&amp;lt;0.05). In summary, our data indicated that higher concentrations of FSH during IVM have a negative effect on meiotic resumption and maturation to MII, whereas canine COCs resume meiosis and mature to MII in higher rates in a SOF-based medium compared with a TCM base. An IVM medium that replicates the invivo environment in which canine COCs mature is beneficial. However, rates of IVM canine oocytes reaching maturity are low, signifying that future research must investigate a greater range of hormone concentrations and combinations to better mimic invivo conditions to assess the possible benefits for canine IVM.

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).

229 From head to tail: A red wolf sperm project

Development of assisted reproductive technologies for the critically endangered red wolf (Canis rufus) is crucial to the maintenance of genetic diversity to support species recovery. Towards this goal, a cryopreservation protocol has previously been developed for red wolf sperm; however, the ability of the gametes to undergo capacitation has not been assessed in this species. Previously, we have shown that oviductal extracellular vesicles (oEVs) improve cat sperm motility and fertilizing ability. The objectives were to (1) compare the effects of culture media on motility and acrosomal integrity of fresh sperm, and select the best medium that can be used in a capacitation protocol; (2) identify potential biomarkers for sperm cryo-tolerance; and (3) determine the influence of canine oEVs on sperm survival and motility post-thaw. In Study 1, sperm were collected by electro-ejaculation from adult red wolves (n=8) and immediately cryopreserved in TRIS-egg yolk buffer with 8% glycerol or incubated for 18h in 5% CO2 and 38.5°C in one of the following media: canine capacitation medium (CCM), FERT-TALP (FERT), NCSU, synthetic oviductal fluid (SOF) and TRIS. At 0, 1, 2, 3, 4, and 18h, sperm were evaluated for total motility and acrosome integrity (FITC-PNA). In Study 2, sperm with high (&amp;gt;80%, HM, n=2 wolves) and low (&amp;lt;15%, LM, n=2 wolves) motility post-incubation at 4°C in the cryopreservation medium for 18h were subjected to proteomic analysis. In Study 3, oviducts were collected from domestic dogs (1-9 years, n=12) after elective spaying, and oEVs from various stages of the oestrous cycle [early follicular (EF), late follicular (LF), early luteal (EL), and late luteal (LL)] were isolated using the Total Exosome Isolation kit (Invitrogen). Frozen-thawed red wolf sperm (n=4 males) were incubated with 30×106 oEVs in non-capacitating CCM, and assessed as in study 1 at 0, 0.5, 1, 2, 3, 4, 6, 8, and 10h. Data were analysed using a paired samples t-test with 95% CI (Prism8, GraphPad Inc.). Sperm incubated in CCM and NCSU had higher motility than those in FERT, SOF, and TRIS after 2h of incubation and onward (2 h: 65±6, 68±6, 42±10, 57±8, and 43±5; 3 h: 60±9, 63±8, 36±11, 46±9, and 34±6; 4 h: 60±9, 60±10, 30±10, 43±8, and 20±5; 18 h: 12±7, 15±7, 9±5, 3±2, and 0, respectively; P&amp;lt;0.05). After 1h of incubation, samples incubated in CCM, NCSU, and SOF had a higher number of sperm with intact acrosomal membranes (P&amp;gt;0.05) than other treatments. A total of 179 proteins were identified, of which 129, including those regulating energy metabolism and mitochondrial mediated apoptosis, were differentially expressed between HM and LM. Preliminary data from Study 3 suggested that thawing and incubating sperm in the presence of LF, EL, and LL oEVs improved sperm motility. In conclusion, CCM and NCSU sustained sperm survival after invitro incubation and could be candidates for invitro fertilization studies in the red wolf. Data generated from sperm proteomic analysis provided insights into cellular pathways regulating sperm cryo-sensitivity. Finally, we demonstrated the potential of oEVs in improving wolf sperm survival post-thawing.

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.

133 CRISPR/Cas9 gene editing of invivo-fertilized bovine embryos via endoscopic oviductal flushing and electroporation of zygotes

In recent years, CRISPR/Cas9 has been used to efficiently edit the genomes of embryos in many animal models. Due to smaller anatomy, lower costs, and multiple ovulations, it is relatively simple to derive large numbers of invivo fertilized zygotes for gene editing experiments in small mammal models. In cattle, however, harvesting invivo fertilized zygotes generally requires a highly invasive surgical procedure. Here, we use the combination of a minimally invasive endoscopic method for harvesting invivo fertilized zygotes by oviductal flushing of superovulated heifers and the subsequent electroporation of zygotes with CRISPR/Cas9 ribonucleoproteins (RNP). After superstimulation of 21 heifers, on average 12 zygotes were flushed per animal with fetal bovine serum, then stored in synthetic oviductal fluid (SOFaa) before electroporation. Targeting exon 1 of the tyrosinase (Tyr) gene, zygotes were electroporated in 1-mm gap cuvettes (Biorad) in groups of ~20 in 20μL of OptiMEM media containing 3μM Cas9 RNP (IDT Cas9 protein pre-incubated with anti-Tyr guide RNA). Electroporation was performed in 3 replicates of 3 electrical potentials, namely 20, 25, and 30V using a Biojet CF 50. The other electroporation parameters were fixed at 5 repetitions of 2-ms square wave pulses at 100-ms intervals. The zygotes were than cultured under standard embryo culture conditions (SOFaa + 0.3% bovine serum albumin, 5% CO2, 5% O2, 39°C, humidified air). Embryo survival, cleavage, and developmental rates to the blastocyst stage were tracked. Statistical significance between groups was determined by pairwise one-way ANOVA using Sidak correction for multiple comparisons. Electroporation of invivo-derived zygotes using 20V yielded significantly higher survival (83.6% vs. 42.8% vs. 20.7% for 20, 25, and 30V, respectively), cleavage (65.6% vs. 37.9% vs. 40.0%), and developmental rates (47.5% vs. 21.4% vs. 16.5%) than 25 or 30V. There was no statistical difference between 25 and 30V. Subsequently, editing rates were determined using the T7 mismatch assay and verified with Sanger sequencing followed by sequence alignment and analysis using Tracking of Indels by Decomposition (TIDE) software (https://tide.nki.nl/). Although there was high variance between electroporation groups, blastocyst editing rates of up to 80.0% were achieved using 30V. To our knowledge, these are the first confirmed gene-edited bovine embryos produced from invivo fertilized zygotes. This method offers the ability to utilise the embryos of high-value cows or cows with known genotypes for genetic engineering experiments. In addition, given that electroporated bovine zygotes can be transferred back to the oviduct endoscopically, our future attempts will focus on genome editing in bovine embryos developed nearly completely within the physiological invivo environment.

83 Validation of propidium iodide dye for live-dead staining of bovine blastocysts: Preliminary results

A widely used criterion routine laboratories utilise for the selection and classification of oocytes and embryos is morphological appearance. In order to make an objective validation of produced embryos, it is necessary to have a method that can help to distinguish viable cells. One simple and easy-to-perform method is the live-dead staining protocol. Usually, ethidium homodimer (EthD) is used to stain the dead cells, in conjunction with Hoechst 33342 for total cell numbers (Stinshoff et al. 2011). However, EthD is a harmful, toxic, and expensive staining dye. Therefore, the aim of this study was to evaluate propidium iodide (PI) as a viable alternative to EthD. To assess the suitability of PI, groups of 20 cumulus-oocyte complexes were collected from abattoir-derived bovine ovaries and matured in tissue culture medium containing bovine serum albumin and equine chorionic gonadotropin/human chorionic gonadotropin for 24h with 5% CO2. The cumulus-oocyte complexes were then fertilised in Fert-TALP (Tyrode's albumin lactate pyruvate) for 19h with 5% CO2 and finally cultured (n=968) in SOFaa (synthetic oviductal fluid with amino acids) with 5% CO2 and 5% O2 with all steps being performed at 39°C. The development rate on Day 8 was on average 25.4%. Expanded Day 8 blastocysts were then utilised to validate the two different staining protocols. Expanded blastocysts were collected from culture drops and washed in PBS containing polyvinyl alcohol. The blastocysts were then stained with the selective dye and finally counterstained to determine live and dead cells. Four alternative parameters were evaluated: (A) PI 1:5 for 15min, (B) PI 1:10 for 15min, (C) PI 1:5 for 5min, and (D) EthD 1:10 for 15min (control). Counterstaining with Hoechst 33342 was performed for 3min in all groups tested. A minimum number of 10 replicate stainings ((A) n=13, (B) n=13, (C) n=12, (D) n=10) was carried out. The evaluation was performed under a fluorescence microscope (Nikon Eclipse Ci) with the NIS-Elements imaging software (Nikon). The statistical analysis was assessed using R. All data were analysed for normal distribution using a Shapiro-Wilk test. Evaluation of the data was made through the application of t-tests. The total cell numbers observed were in the known range for expanded blastocysts: (A) 106.3±15.3, (B) 109.5±18.8, (C) 119.3±15.0, and (D) 113.1±21.8. The number of dead cells did not differ significantly from the control: (A) 13.3±7.7, (B) 8.5±5.0, (C) 11.8±8.7, and (D) 9.3±2.3. The study indicates that PI could be used as an adequate alternative for EthD. Furthermore, it is possible that the concentration or the incubation time could be reduced to generate similar results to EthD. Further replicates are currently being examined in order to corroborate these results. We gratefully acknowledge our colleague M. Vopel.

194 Extracellular vesicles derived from ampullary oviductal fluid improve developmental competence of bovine oocytes

Invivo, oocytes mature in the presence of follicular fluid (FF) and ampullary oviductal fluid (AOF), starting from premature to final maturation stages (3-6h postovulation). Extracellular vesicles (EVs) have been identified as important mediators of gamete/embryo-maternal interactions. They carry regulatory molecules, such as microRNAs and mRNAs. So far, the functionality of EVs derived from FF and AOF has not yet been determined. The aim of this study was to evaluate the effect of EVs derived from FF and AOF during invitro oocyte maturation (IVM) on the development and quality of resulting bovine embryos. Follicular fluid of a preovulatory follicle was collected by ovum pickup, and AOF was collected from the oviducts of slaughtered cows in early luteal phase. Then, EVs from FF and AOF were isolated by size exclusion chromatography and confirmed by western blot. The concentration of EVs was determined by NanoDrop and Nanoparticle tracking analysis. Integrity and size of EVs were assessed by electron microscopy. Bovine oocytes (n=1331) were allocated at random to five groups. In the control group (C), oocytes (n=347) were cultured for 22.5h in maturation medium (TCM-199 and epidermal growth factor 20ngmL−1) (MM). In the negative control group (NC), oocytes (n=331) were cultured for 18h in MM, and then transferred into fresh MM for 4.5h. In the FF group, oocytes (n=162) were cultured in MM supplemented with FF EVs (12.5µgmL−1) for 18h, and then transferred to MM for 4.5h. In the AOF group, oocytes (n=328) were cultured for 18h in EV-free MM, and then transferred to MM supplemented with AOF EVs (1.7µgmL−1) for 4.5h. In the FF+AOF group, oocytes (n=163) were cultured in MM supplemented with FF EVs for 18h, and then transferred into MM supplemented with AOF EVs for 4.5h. At 22.5h post-incubation, mature oocytes were fertilized invitro. At 21h post-insemination, presumptive zygotes were denuded and cultured in synthetic oviductal fluid with insulin-transferrin-selenium for 8 days. Cleavage rate and blastocyst rate were recorded at 48h and 8 days post-insemination, respectively. Blastocyst quality was assessed by differential apoptotic staining. The data were analysed using one-way ANOVA. Cleavage rate was not affected by the treatment, whereas blastocyst yield in the AOF group (48.75%) was significantly higher than that in the C (42.91%) and NC (37.72%) groups. In addition, embryos produced in the AOF group showed the highest number of trophectoderm cells and inner cell mass cells, and lowest number of apoptotic cells (P&amp;lt;0.05). Trophectoderm and inner cell mass cells were higher and apoptotic cells were lower in FF and FF+AOF compared with the C and NC groups. In conclusion, adding AOF EVs to oocyte MM in the last 4.5h of IVM improves quantity and quality of blastocysts. In addition, although FF EVs showed no effect on blastocyst yield, they improved blastocyst quality by increasing cell number.

88 MicroRNA profile of invitro bovine embryos cultured in the presence of amniotic extracellular vesicles shifts toward invivo-collected blastocysts

The absence of maternal-embryo signals could be an important cause of the poor pregnancy rates of invitro-produced embryos, compared with those collected invivo. In the context of paracrine communication, co-culture of embryo with amniotic-derived extracellular vesicles (EVs) improved their quality compared with control (CTR) (Perrini and Lange Consiglio 2018 Reprod. Fertil. Dev. 30, 658-671), and after cryopreservation, provided higher invitro embryo hatching and recipient pregnancy rate (Lange-Consiglio et al. 2019 Reprod. Fertil. Dev. 31, 155). After these results, the aim of this study was to evaluate microRNA (miRNA) profiling of invitro-produced blastocysts with or without EV supplementation, using invivo-produced blastocysts as CTR. Invitro embryos were produced based on our protocol (Perrini and Lange Consiglio 2018 Reprod. Fertil. Dev. 30, 658-671) with or without 100×106 EVsmL−1 in synthetic oviductal fluid with amino acids (SOFaa) on Day 5 post-fertilisation (Perrini and Lange Consiglio 2018 Reprod. Fertil. Dev. 30, 658-671). Grade 1 blastocysts (B7) were immediately snap frozen in liquid nitrogen for genomic study. These embryos were obtained from three replicates. Invivo embryos were obtained from three cows superovulated by Folltropin and inseminated by the same cryopreserved semen. After flushing, only B7 were snap frozen for genomic study. Samples for RNA isolation were obtained from 3 pools of 10 embryos each for each condition (vivo, vitro-CTR, and vitro+EVs). Total RNA was isolated by a NucleoSpin1 miRNA kit. Concentration and quality of RNA were determined by an Agilent 2100 Bioanalyzer. Libraries were prepared using TruSeq Small RNA Library Preparation kits (Illumina). Differential expression analyses between samples were run with the Bioconductor edgeR package (false discovery rate&amp;lt;0.05). MicroRNA cluster analysis was performed with Genesis. The average quantity of total RNA extracted from each pool was 3.5ng. Our results show that the miRNAs identified were 1.74E5, 2.3E5, and 3.6E5 for vivo, vitro-CTR, and vitro+EVs, respectively. Principal component analysis calculated on differentially expressed miRNAs showed a separation of the three groups with a distinctive miRNA trait. The miRNAs differentially expressed among three comparisons (vivo vs. vitro-CTR, vivo vs. vitro+EVs, and vitro-CTR vs. vitro+EVs) were 20, 15, and 2, respectively. Principal component 1, which explains 62.4% of the variance, clearly separates invivo- and invitro-produced embryos even if EV addition seems to ameliorate the effect of invitro production, and this agrees with the embryo quality and pregnancy rate after EV supplementation (Perrini and Lange Consiglio 2018 Reprod. Fertil. Dev. 30, 658-671; Lange-Consiglio et al. 2019 Reprod. Fertil. Dev. 31, 155). Indeed, vitro-CTR and vitro+EVs embryos differ significantly for two miRNAs (miR-130a, miR-181b) that are found to be higher in our vitro-CTR embryos compared with vitro+EV ones. The miR-181b was also found to be higher in degenerate bovine embryos compared with good blastocysts (Kropp et al. 2014 Front. Genetics 24, 91). In conclusion, this is the first study reporting the complete miRNA profiling of invitro blastocysts compared with those obtained invivo. The addition of EVs during invitro production seems to influence the expression of specific miRNAs involved in the success of embryo implantation.

12 Assessment of oocyte and embryo production in senescent beef cows

Fertility associated with age has been poorly documented in cattle. There is evidence that more than 50% of cows became infertile (failure to bear a calf for two successive years) at the age of 15. The causes of infertility in cows have been attributed to poor oocyte quality and/or follicle depletion. The present study aimed to compare the rates of oocyte and invitro embryo production of fertile cows older than 15 years. Sixteen Nellore cows (Bos taurus indicus) 200.3±4.9 months old were submitted to two ovum pickup series (OPU1 and OPU2) at a 2-year (2017 and 2019) interval. Before the OPU1 of each series, the cows were prepared with an IM injection of 2mg estradiol benzoate and 0.15mg prostaglandin F2α analogue and a progesterone-releasing intravaginal device (1g). After five days, the progesterone-releasing intravaginal device was withdrawn and the OPU1 was performed. The second OPU (OPU2) was performed 5 days later to aspirate only the growing follicles. The number and morphologic quality of recovered cumulus-oocyte complexes were registered. After IVM (tissue culture medium 199) and fertilization (Tyrode's solution) of cumulus-oocyte complexes, the presumptive zygotes were cultured in synthetic oviductal fluid with amino acids to assess developmental rates to blastocyst. Data were analysed by chi-squared and Wilcoxon test. The number of oocytes recovered at OPU1 was higher in 2107 than in 2019 (27.8±6.6 vs. 10.8±4.2; P&amp;lt;0.01), whereas in OPU2 the production of oocytes was not different (14.7±3.3 vs. 10.9±3.1). In OPU1, eight cows in 2017 and four in 2019 produced more than 20 oocytes, compared with only one cow in OPU2 of both years (P=0.02). Although there was no significant difference in the proportion of viable oocytes in OPU1 (83.6% and 87.2%) and OPU2 (85.5 and 68.0%), more viable oocytes were produced at OPU1 of 2017 (23.2±5.8) compared with OPU1 of 2019 (9.4±4.1), OPU2 of 2017 (12.6±2.7) and OPU2 of 2019 (7.4±2.4), respectively (P&amp;lt;0.05). There was no significant difference in the proportion of cleaved oocytes in OPU1 (74.2 and 75.1%) and OPU2 (68.9 and 79.8%). Two cows in 2017 (12.5%) and five in 2019 (31.2%) did not produce embryos in both OPUs. Cows produced more embryos (blastocysts) in OPU1 at 2017 (7.3±1.6) than 2019 (2.8±0.6; P&amp;lt;0.05), whereas in OPU2 there was no difference (4.1±0.8 and 3.1±0.8). In conclusion, senescent fertile cows decreases the number of oocytes and embryos as they approach 20 years of age; however, the quality of oocytes does not appear to be compromised. Financial support was provided by FAPESP (proc. 2018/07450-5).

76 Extracellular vesicles from oviduct and uterus in sequential culture improve the quality of bovine embryos produced invitro

Extracellular vesicles (EVs) are released by cells and transport cargo that affect functions of other cells. Oviductal fluid (OF) and uterine fluid (UF) have been shown to improve quality of embryos during invitro culture (Hamdi et al. 2017 Reprod. Fertil. Dev. 30, 935-945) which may be due to their content of EV (Lopera-Vásquez et al. 2017 Reproduction 153, 461-470). Thus, the aim of this study was to evaluate the effect of EVs from OF and UF on a sequential invitro culture system on the development and quality of bovine embryos. Zygotes were cultured in synthetic oviduct fluid (SOF) supplemented with 3mgmL−1 bovine serum albumin (BSA; n=1228) or 5% EV-depleted fetal calf serum (dFCS, n=1261) in the presence (BSAEV, n=1265 and dFCSEV, n=1253) or absence of 3×105 EVmL−1 from OF (Day 1 to Day 4) and UF (Day 4 to Day 9), mimicking invivo conditions. The EVs pooled from 5 oviducts (early luteal phase) and 5 uterine horns (middle luteal phase) from slaughtered heifers were isolated by a Size Exclusion Chromatography kit (Hansa BioMed). The EV size and concentration were assessed by the nanotracking analysis system and morphology by transmission electron microscopy. Embryo development was recorded on Days 7/9. Day 7/8 blastocysts were assessed for quality by staining with (a) Hoechst 33342 (10 µgmL−1, 30min) for total cell number, (b) Bodipy 493/503 (20 µgmL−1, 1h) for lipid content (lipid droplet area in µm2), and (c) for survival rate after vitrification/warming. Data were analysed by one-way ANOVA and Tukey test. The EV concentration was 2.97 and 7.98×1010 particlesmL−1, and mode size 137.2 and 151.2nm for OF and UF, respectively. Transmission electron microscopy confirmed EV presence and size, showing typical cup-shaped morphology. Blastocyst yield was lower (P&amp;lt;0.05) on Day 7 in the BSA groups (BSA: 15.7±1.9 and BSAEV: 15.2.4%) compared with serum groups (dFCS: 28.1±2.6 and dFCSEV: 30.1±2.9%) irrespective of EV supplementation; however, these differences were compensated at Days 8 and 9 (range: 30.0±3.2-40.8±3.9%). The EVs increased (P&amp;lt;0.05) blastocyst total cell number in dFCSEV (152.6±2.9) and BSAEV (140.5±1.5) compared with dFCS (117.9±2.0) and BSA groups (122.4±1.1). However, lipid content was decreased (P&amp;lt;0.05) in the presence of EVs only in dFCSEV (0.231±0.05µm2) compared with BSA (0.393±0.03µm2) and BSAEV (0.379±0.03µm2) groups. The dFCS did not differ from any group (0.371±0.05µm2; P&amp;gt;0.05). Blastocyst survival after vitrification/warming was high in all groups up to 72h (range: 80.0±3.8-100%; P&amp;gt;0.05). In conclusion, mimicking physiological conditions using EV from OF and UF during invitro culture does not affect development but improves embryo quality by increasing blastocyst total cell numbers and decreasing lipid contents. These results provide evidence of the association of the reproductive tract environment and developing embryo, confirming embryo-maternal communication. Funding was provided by MINECO-Spain AGL2015-70140-R; Y. N. Cajas, SENESCYT-Ecuador; C. L. V. Leal, FAPESP-Brazil 2017/20339-3.