Synthetic Oviduct Fluid Research Articles

Insulin-like growth factor I enhances the developmental competence of yak embryos by modulating aquaporin 3.

The objective of our present study was to determine the effects of insulin-like growth factor I (IGF-I) on the development of yak (Bos grunniens) embryos after cumulus-oocyte complex (COC) vitrification and warming followed by in vitro fertilization (IVF). In Experiment 1, the yak COCs underwent vitrification and then IVF. Embryos were incubated in synthetic oviductal fluid (SOF) supplemented with four concentrations (0, 50, 100 and 200ng/ml) of IGF-I, while the yak COCs without vitrification or IGF-I supplementation acted as the control group; the BAX, BCL-2, AQP3mRNA and aquaporin 3 (AQP3) protein expression levels in the five groups of blastocysts were evaluated using quantitative real-time PCR and immunofluorescence analyses. In Experiment 2, the groups described above were fertilized and incubated. The cleavage rate, blastocyst rate, total cell count per blastocyst and the rate of growth of the inner cell mass (ICM) and trophectoderm (TE) were evaluated. The results were as follows: (1) the AQP3 gene expression and protein expression in the control and 100ng/ml IGF-I treatment groups were the highest. (2) The BAX gene expression was the lowest and the BCL-2 gene expression was the highest in the control and 100ng/ml IGF-I treatment groups. (3) The rates of cleavage and blastocysts in the control and 100ng/ml IGF-I groups were higher than those in the other three groups. The total cell count per blastocyst in the vitrified and warmed 100ng/ml IGF-I group (106.7±4.9) and the control group (107.3±4.2) was higher than that in the vitrified and warmed 0ng/ml IGF-I (91.2±3.1), 50ng/ml IGF-I (92.3±3.7) and 200ng/ml IGF-I (92.4±3.7) groups. Therefore, we conclude that IGF-I can improve yak blastocyst developmental ability, cytomembrane permeability and formation of the blastocyst cavity after COC vitrification by improving the BAX, BCL-2 and AQP3 expression levels.

Colony-stimulating factor 2 enhances the developmental competence of yak (Poephagus grunniens) preimplantation embryos by modulating the expression of heat shock protein 70 kDa 1A

Colony-stimulating factor 2 (CSF2) is known to promote the development and survival of rodents and ruminants preimplantation embryos; however, the effect of CSF2 on yak embryos has not been reported. The objective of this study was to investigate the effects of CSF2 on the developmental competence of yak embryos cultured in vitro in modified synthetic oviduct fluid (mSOF) medium and on the expression pattern of heat shock protein 70 kDa 1A (HSPA1A). In each experiment, cumulus-oocyte complexes (COCs) were matured in vitro and fertilized with frozen-thawed semen. Zygotes were treated with varying concentrations of CSF2 (0, 10, 50, 100 ng/mL) until day 8 after fertilization. Embryo development was calculated as the percentage of oocytes that formed embryos at the 2-cell, 4-cell, 8-cell, 16-cell, morula and blastocyst stages. The total cell numbers (TCN) per blastocyst and their allocation to the inner cell mass (ICM) and trophectoderm (TE) lineages were determined using differential CDX2 staining. The expression of HSPA1A was examined by quantitative real-time PCR (qRT-PCR) and immunochemistry to determine the mRNA and protein levels. The results showed that treatment with 50 ng/mL CSF2 significantly (P < 0.05) increased the rate of blastocyst formation (19.01% versus 9.93%) and the TCN per blastocyst (96.94 versus 81.41) compared to the control group. However, no significant differences were observed in the other stages of development. qRT-PCR analysis confirmed that treatment with 50 ng/mL CSF2 significantly (P < 0.05) inhibited the expression of HSPA1A mRNA in blastocysts cultured in vitro relative to the control group, but there were no significant differences between the other treatment groups. Immunocytochemical analysis confirmed that HSPA1A protein accumulation was gradually reduced in yak blastocysts cultured in 0, 10, 100 or 50 ng/mL CSF2, however, no significant differences were observed between the 10 and 100 ng/mL treatments (P > 0.05). In conclusion, these findings demonstrate that CSF2 inhibits the expression of HSPA1A to facilitate yak blastocyst formation and increase cell numbers.

Effect of bovine oviductal extracellular vesicles on embryo development and quality in vitro.

The aim of this study was to evaluate the effect of extracellular vesicles (EV) from oviductal fluid (OF), either from the ampulla or isthmus, on the development and quality of in vitro-cultured bovine embryos. Zygotes were cultured in synthetic oviduct fluid (SOF + 3 mg/mL BSA) without calf serum (C- group), in the presence of 3 × 105 EV/mL from ampullary or isthmic OF at either 1 × 104 g (10 K) or 1 × 105 g (100 K), and compared with SOF + 5% FCS (C+ group). OF-EV size and concentration were assessed by electron microscopy and nanotracking analysis system. Embryo development was recorded on Days 7-9, and blastocyst quality was assessed through cryotolerance and gene expression analysis. Lower blastocyst yield was observed on Day 7 in the C- and OF-EV groups (12.0-14.3%) compared with C+ (20.6%); however, these differences were compensated at Days 8 and 9 (Day 9: 28.5-30.8%). Importantly, the survival rate of blastocysts produced with isthmic 100 K OF-EV was higher than that of C+ and C- group at 72 h after vitrification and warming (80.1 vs 34.5 and 50.5% respectively, P < 0.05). In terms of gene expression, blastocysts produced in the presence of 100 K isthmic OF-EV upregulated the water channel AQP3 and DNMT3A and SNRPN transcripts compared with the C+, with the expression in C- being intermediate. The lipid receptor LDLR was downregulated in C+ compared with all other groups. In conclusion, the addition of oviductal fluid extracellular vesicles from isthmus, to in vitro culture of bovine embryos in the absence of serum improves the development and quality of the embryos produced.

Supplementing ±-Linolenic acid in the in vitro maturation media improves nuclear maturation rate of oocytes and early embryonic development in the Nili Ravi buffalo

The present study was conducted to investigate the effect of omega-3 poly unsaturated fatty acid (PUFA), α-linolenic acid (ALA; 18:3 n-3) on the in vitro maturation (IVM) of buffalo oocytes and subsequent embryonic development. Buffalo cumulusoocyte complexes (COCs; n = 2282) were in vitro matured in TCM-199 (0.6% fatty acid free bovine serum albumin, 0.02 Units/ml FSH, 1 µg/ml 17-β-estradiol, 10 µg/ml epidermal growth factor, 50 µg/ml gentamicin) supplemented with 0 (control), 25, 50, 100, 150 or 300 µm ALA under an atmosphere of 5% CO2 in air at 38.5oC for 22-24 h. The matured oocytes were then fertilized in Tyrode’s Albumin Lactate Pyruvate (TALP) medium and cultured in synthetic oviductal fluid (SOF) medium. Concentrations up to 100 μm ALA improves (P ≤ 0.05) the cumulus expansion compared to control. Higher percentage of oocytes reaching MII stage was observed at 50 μm and 100 μm of ALA compared to control (P ≤ 0.05). Concentrations of 150 and 300 µm ALA were detrimental both for cumulus expansion and nuclear maturation rate of buffalo oocytes. Moreover, supplementation with 100 μm ALA improved (P ≤ 0.05) cleavage rate compared to control and treatment with 50 and 100 μm ALA yielded significantly higher morulae compared to control. The results of present study indicate that the supplementation with 100 μm ALA to the IVM medium improves nuclear maturation rate of buffalo oocytes and subsequent early embryonic development.

50 SURVIVAL OF SEXED IVF-DERIVED BOVINE EMBRYOS FROZEN AT DIFFERENT PREIMPLANTATION STAGES OF DEVELOPMENT

The ability to freeze in vitro-produced bovine embryos with a high post-thaw viability is still problematic and hampers logistics of on-farm embryo transfer. The objectives of this experiment were to compare different stages of development, freezing methods, and addition of cytoskeletal stabilisers (cytochalasin-B) before freezing. Ovaries were collected from an abattoir and oocytes aspirated from 2- to 6-mm follicles. Cumulus-oocyte complexes containing compact and complete cumulus cell layers were selected and matured in groups of 50 in 400 µL of M199 medium supplemented with ALA-glutamine (0.1 mM), Na pyruvate (0.2 mM), gentamicin (5 µg mL−1), EGF (50 ng mL−1), ovine FSH (50 ng mL−1), bLH (3 µg mL−1), cysteamine (0.1 mM), and 10% fetal bovine serum (FBS) for 22 to 24 h. Fertilization (Day 0) was done using female sex-sorted semen selected with a discontinuous density gradient and diluted to a final concentration of 1 × 106 sperm/mL. Synthetic oviductal fluid (SOF)-FERT medium was supplemented with fructose (90 µg mL−1), penicillamine (3 µg mL−1), hypotaurine (11 µg mL−1), and heparin (20 µg mL−1). After 18 h, presumptive zygotes were denuded and cultured in groups of 15 to 20 in 50-µL drops of SOF-BSA for 7 days. On Day 3.5 post-fertilization, 3% FBS was added. Low oxygen tension (5% O2) was used for culture. Morulae were selected at Day 5.5–6, blastocysts at Day 6–6.5, and expanded blastocysts at Day 6.5–7. Embryo harvesting for each stage was performed from a dedicated drop/dish and discarded in order to avoid further embryo stage collections. Grade 1 morulae, blastocysts, and expanded blastocysts were selected for freezing and placed randomly into 2 groups: slow-freezing and vitrification. Before freezing, half of the embryos from each stage were exposed to cytochalasin-B for 45 min. The slow freezing protocol consisted of 1.5 M ethylene glycol (EG) + 20% FBS + 0.4% BSA, and the cooling rate was 0.5°C/min. Slow-frozen embryo thawing was performed by exposing the 0.25-mL straws to air (23°C) for 10 s and then underwater at 35°C for 1 min. The vitrification (Cryo-Top) medium was 15% (vol/vol) EG + propylene glycol. Vitrified embryos were thawed in a solution of H199 + 20% FBS and 0.25 M sucrose at 39°C. Thawed embryos from both groups were cultured in SOF-BSA + 10% FBS under cumulus/granulosa cell monolayer co-culture. Embryo assessment involved post-thaw survival (0 h), re-expansion, and hatching of the zona pellucida (72 h). Three replicates were performed for each treatment level. Fisher’s l.s.d. test with Bonferroni correction was used to determine treatment differences (P &lt; 0.05). The post-thaw survival, re-expansion, and hatching results showed that either expanded blastocysts (84.7 ± 3.2%, 74.1 ± 3.9%, and 60.9 ± 4.4%) or blastocysts (81.7 ± 3.5%, 69.6 ± 4.2%, and 55 ± 4.6%) were preferred (P &lt; 0.05) embryo stages for cryopreservation compared with morulae (67.6 ± 4.4%, 52.5 ± 4.6%, and 33.2 ± 4.3%). Vitrification and cytochalasin-B pre-freezing exposure (61.3 ± 3.6% and 56.6 ± 3.8%) provided better (P &lt; 0.05) hatching results compared with slow-freezing and without cytochalasin-B (37.8 ± 3.6% and 42.5 ± 3.7%).

93 BLASTOCYST BISECTION TO MULTIPLY BIOPSIED AND VITRIFIED BOVINE EMBRYOS

Dairy cattle breeding schemes increasingly integrate embryo-based genomic selection to accelerate genetic gain. In contrast to the single offspring produced with conventional animal-based genomic selection, multifactorial IVF between elite parents increases genotypes for selection. Genetically superior embryos are identified from biopsies, and only those with the desired genotypes are transferred. To manage the logistics of such schemes, and enable seasonally born progeny, the cryo-preservation of embryos after biopsy and before embryo transfer is critical. Here, we compare 2 methods of cryo-preserving biopsied Day 7 blastocysts and report results from bisecting blastocysts to increase the number of selected embryos for transfer. Abattoir-sourced oocytes were matured in vitro and fertilized with sperm from a single sire. Embryos were cultured for 7 days in a modified Synthetic Oviduct Fluid medium. Approximately 15 cells were biopsied from the mural trophectoderm of grade 1 and 2 blastocysts in Embryo Hold medium minus BSA, using a micro-surgical blade (Bioniche Animal Health, Athens, GA, USA). Following biopsy, each blastocyst was cultured in Embryo Hold with 3 mg mL−1 BSA for ~2 h at 38.5°C to allow for re-expansion. In Experiment 1, embryos were randomly assigned to 1 of 2 cryo-preservation treatments: conventional slow freezing or the Cryologic vitrification method (CVM). Slow freezing entailed freezing in 1.5 M ethylene glycol and 0.1 M sucrose. The CVM involved a 2-step vitrification protocol, with 15% of both ethylene glycol and dimethyl sulphoxide in the final solution comprising Embryo Hold, 20% FCS, 1 M sucrose, and 0.1 mM Ficoll (GE Healthcare). Selected embryos were thawed/warmed and transferred in pairs to the uterine horn ipsilateral to the corpus luteum of each synchronized recipient heifer. In Experiment 2, each biopsied blastocyst was individually vitrified using CVM. Following warming, blastocysts were bisected into approximately equal halves. After ~2 h recovery, pairs of demi-embryos were transferred to recipients categorized with either normal (&gt;2.5, &lt;7 ng mL−1) or low (≥2, &lt;2.5 ng mL−1) plasma progesterone concentrations on Day 5 after oestrus. Embryo survival in both experiments was monitored by ultrasonography of fetal heartbeats up to Day 65 of gestation. Statistical significance was determined using Fisher’s exact test. In Experiment 1, embryo survival on Day 65 was significantly greater with CVM than slow freezing (25/54 = 46% v. 9/54 = 17%; P = 0.002). In Experiment 2, there was no significant difference in the number of fetuses as a percentage of original blastocysts, regardless of normal versus low progesterone levels (13/22 = 59% v. 4/9 = 44%, respectively). In conclusion, vitrification is superior for cryo-preserving biopsied blastocysts, possibly reducing cryo-damage compared with conventional slow freezing, and achieves rates of in vivo development similar to fresh IVF embryos. Embryo bisection potentially provides only a modest increase in the probability of generating a calf from each valuable, genomically selected embryo. Improving embryo competency and other methods of multiplication may maximize this likelihood.

Non-invasive metabolomics for improved determination of embryonic sex markers in chemically defined culture medium

Metabolic differences between early male and female embryos can be reflected in culture medium (CM). We used a single bovine embryo culture step (24h) supporting improved birth rates under chemically defined conditions (CDC) to investigate biomarker detection of embryonic sex in contrast to classical BSA-containing medium. In vitro matured slaughterhouse oocytes were fertilized in vitro with a single bull. Embryos were initially cultured in synthetic oviduct fluid with BSA. On day-6, morulae were cultured individually in droplets with (BSA) or without protein (CDC). On day-7, expanded blastocysts were sexed (amelogenin gene amplification) and CM was stored at −145°C until metabolomic analysis by UHPLC-TOF MS. N=10 embryos per group (i.e. male-protein; female-protein; male-non-protein; female-non-protein) were produced. Statistical analysis revealed N=6 metabolites with different concentrations in CM, N=5 in male embryos (methionine, tryptophan, N-stearoyl-valine, biotin and pipecolic acid), N=1 in female embryos (threonine) (P<0.05 in BSA; P<10−7 in CDC). Only the clear threshold between males and females in CDC allowed correct classification of 100% males and 91% females within 5 out of 6 biomarkers (one female outlier showing the male biomarker profile). The use of CDC represents a critical aspect in the efficient detection of embryonic sex biomarkers by metabolomics.

Producción de embriones de búfalo por fertilización in vitro luego de la maduración de los ovocitos durante el transporte prolongado

El búfalo &lt;i&gt;(Bubalus bubalis)&lt;/i&gt; es una especie con excelente adaptación a sectores inundables. El mejoramiento genético a través de superovulación y transferencia embrionaria ha tenido escasos resultados debido a dificultades en la detección de celo, pobre respuesta ovárica y limitada recuperación de embriones post-lavaje. La técnica de fertilización in vitro de embriones (FIV) es una biotecnología de gran impacto en el progreso genético. El objetivo del presente trabajo fue estudiar los eventos tempranos de la FIV, analizando la tasa de maduración y desarrollo embrionario post-fertilización de ovocitos madurados in vitro (IVM) durante el transporte. Ovocitos bovinos y bubalinos fueron obtenidos por punción folicular de ovarios post-mortem e IVM durante el transporte por un período de 18 h. Se realizó la FIV con toros de fertilidad comprobada, con una concentración en microgotas de inseminación de 3-4 x 106 espermatozoides motiles/ml por un período de 6 horas. Los embriones fueron cultivados en medio oviductal sintético SOFaa en incubadora gaseada y ambiente humidificado a 38,5ºC durante 9 días. Se evaluaron las tasas de IVM, clivaje (día 2 post-fertilización) y blastocisto (días 7 a 9). Los resultados fueron analizados estadísticamente utilizando Fischer’s Exact Test (p&lt;0,05). No se observaron diferencias significativas en la tasa de maduración de ovocitos bubalinos de buena calidad respecto al control sin transporte (72 vs 88%), pero se registró una reducción significativa en la maduración de los ovocitos bubalinos de mala calidad (35%). Asimismo, se lograron producir los primeros embriones bubalinos luego de FIV, aunque las tasas de clivaje (34 vs 70 y 78%) y blastocisto (3 vs 27 y 31%) fueron significativamente menores en búfalos que en bovinos con y sin transporte, respectivamente. Los datos del presente trabajo constituirían el primer informe de FIV en búfalos y producción in vitro de embriones luego de IVM de ovocitos durante el transporte.

Cell apoptosis and lipid content of in vitro–produced, vitrified bovine embryos treated with forskolin

The presence of fetal calf serum in culture medium influences embryo quality, causing a reduction in postcryopreservation survival. Forskolin has been used to induce lipolysis and increase cryotolerance, functioning as an activator of adenylate cyclase and elevating cAMP levels. In the present experiment, bovine zygotes were cultured in synthetic oviduct fluid with amino acid plus 2.5% fetal calf serum for 6 days, when forskolin was added in three concentrations: 2.5, 5, and 10 μM. Treatment with forskolin lasted for 24 hours. Blastocyst formation rate, quantification of lipid granules, total cell numbers, and apoptosis rate were evaluated. In a second assessment, embryos were vitrified, and warming, re-expansion rate, total cell numbers, and apoptosis rate were also evaluated. There was no difference due to forskolin in blastocyst formation or re-expansion rates after vitrification. However, lipid measurements were lower (control: 136.8 and F 2.5 μM: 128.5; P < 0.05), and number of cells per embryo higher (control: 140.1 and F 2.5 μM: 173.5; P < 0.05) than controls for 2.5 μM forskolin but not for higher forskolin concentrations. The number of intact cells per embryo was higher, and the rate of apoptosis was lower in fresh than in vitrified embryos (number of cells of warmed embryos, control: 104.1, F 2.5 μM: 101.3, F 5 μM: 115.4, F 10 μM: 95.1; apoptotic of fresh cells, control: 12.1%, F 2.5 μM: 16.7%, F 5 μM: 11.1%, F 10 μM: 14.2%; and apoptotic warmed embryos, control: 22.3%, F 2.5 μM: 37.3%, F 5 μM: 33.2%, F 10 μM: 30.3%; P < 0.05). It was concluded that forskolin is an effective lipolytic agent even at low concentrations, leading to formation of blastocysts with a comparatively larger number of cells.

A comparison of in vitro fertilization potential of sperm obtained from refrigerated (4°C) epididymis and fresh ejaculation of buck under different embryo culture media

The present study assessed the fertilization potential of sperm obtained from refrigerated epididymis and fresh ejaculation of buck under 2 culture media, viz. modified synthetic oviductal fluid (mSOF) and research vitro cleave-blastocyst (RVCL-BLAST) media. In the process, the cauda epididymis of slaughtered buck were immediately collected, pre-processed and refrigerated (4°C) for 24 h. The oocytes recovered from slaughter house ovaries were in vitro matured and fertilized with both semen sources. On analysis of embryonic development, ejaculated group showed significantly higher blastocyst rate than epididymal sperm, however, initial cleavage rates of epididymal group was significantly higher than ejaculated semen, irrespective of culture media. Among the culture media, RVCL-BLAST media was significantly efficient than mSOF in terms of cleavage and blastocysts, irrespective of gametes sources. Despite of lower outcome of blastocyst, cold stored epididymal sperm may be considered as an alternative, potent and viable source of gametes for assisted reproduction in case of endangered or other livestock, if immediate facilities for sperm processing are not available.

Crocetin improves the quality of in vitro–produced bovine embryos: Implications for blastocyst development, cryotolerance, and apoptosis

The aim of this work was to assess the effect of supplementation of bovine culture medium with the natural antioxidant crocetin on in vitro blastocyst development and quality. This was evaluated as cryotolerance, apoptosis index, and total cells number and allocation. Abattoir-derived oocytes were matured and fertilized in vitro according to standard procedure. Twenty hours after IVF, presumptive zygotes were cultured in synthetic oviduct fluid medium, supplemented with 0, 1, 2.5, and 5 μM crocetin (experiment 1) at 39 °C under humidified air with 5% CO2, 7% O2, and 88% N2. On Day 7, embryo yields were assessed and the blastocysts were vitrified by Cryotop method in 16.5% ethylene glycol, 16.5% DMSO, and 0.5 M sucrose. Finally, blastocysts produced on Day 8 in the absence (control) and presence of 1 μM crocetin were used for terminal deoxynucleotidyl transferase–mediated dUTP nick end labelling and differential staining to evaluate, respectively, the apoptotic rate and the allocation of cells into inner cell mass (ICM) and trophectoderm (TE) lineages (experiment 2). Embryo development was higher in the 1 μM crocetin group compared to the control, both in terms of total embryo output (37.7 ± 4.2%, 52.9 ± 6.3%, 40.9 ± 7.6%, and 42.4 ± 8.7%, respectively, with 0, 1, 2.5, and 5 μM; P < 0.01) and grade 1 and 2 blastocysts (33.6 ± 4.9%, 46.1 ± 7.3%, 37.8 ± 7.9%, and 39.4 ± 7.9%, respectively, with 0, 1, 2.5, and 5 μM; P < 0.05). Moreover, the percentage of fast-developing embryos increased in 1 μM crocetin group compared to the control (23.4 ± 4.7%, 32.7 ± 6.6%, 27.2 ± 6.6%, and 30.1 ± 7.2%, respectively, with 0, 1, 2.5, and 5 μM; P < 0.05). In addition, the enrichment of culture medium with 1 μM crocetin improved embryo cryotolerance compared to the control, as indicated by higher hatching rates recorded after 48 hours postwarming culture (46.5% vs. 60.4%; P < 0.05). Furthermore, 1 μM crocetin decreased both the average number (9.9 ± 0.4 vs. 7.1 ± 0.3) and the percentage of apoptotic cells (7.1 ± 0.4 vs. 4.2 ± 0.2) in blastocysts compared to the control (P < 0.01). However, no differences were recorded in the average number of ICM, TE, and total cells between 1 μM crocetin and control groups. In conclusion, the enrichment of bovine culture medium with 1 μM crocetin increased both blastocyst yield and quality, as indicated by the improved chronology of embryo development, increased resistance to cryopreservation, and reduced incidence of apoptosis.

Effect of spermatozoa motility hyperactivation factors and gamete coincubation duration on in vitro bovine embryo development using flow cytometrically sorted spermatozoa.

The aim of the present study was to evaluate the effects of sperm motility enhancers and different IVF times on cleavage, polyspermy, blastocyst formation, embryo quality and hatching ability. In Experiment 1, sex-sorted X chromosome-bearing Bos taurus spermatozoa were incubated for 30min before 18h fertilisation with hyperactivating factors, namely 10mM caffeine (CA), 5mM theophylline (TH), 10mM caffeine and 5mM theophylline (CA+TH); and untreated spermatozoa (control). In Experiment 2, matured B. taurus oocytes were fertilised using a short (8h) or standard (18h) fertilisation length, comparing two different fertilisation media, namely synthetic oviducal fluid (SOF) fertilisation medium (SOF-FERT) and M199 fertilisation medium (M199-FERT). Cleavage and blastocyst formation rates were significantly higher in the CA+TH group (77% and 27%, respectively) compared with the control group (71% and 21%, respectively). Cleavage rates and blastocyst formation were significantly lower for the shortest fertilisation time (8h) in M199-FERT medium (42% and 12%, respectively). The SOF-FERT medium with an 8h fertilisation time resulted in the highest cleavage rates and blastocyst formation (74% and 29%, respectively). The SOF-FERT medium produced the highest embryo quality (50% Grade 1) and hatching rate (66%). Motility enhancers did not affect polyspermy rates, whereas polyspermy was affected when fertilisation length was extended from 8h (3%) to 18h (9%) and in M199-FERT (14%) compared with SOF-FERT (6%). We conclude that adding the motility enhancers CA and TH to sex sorted spermatozoa and Tyrode's albumin lactate pyruvate (TALP)-Sperm can improve cleavage and embryo development rates without increasing polyspermy. In addition, shortening the oocyte-sperm coincubation time (8h) resulted in similar overall embryo performance rates compared with the prolonged (18h) interval.

Developmental competence and cryotolerance of caprine parthenogenetic embryos cultured in chemically defined media

In animal reproduction technologies, the in vitro embryo culture system has advanced over the past few decades. However, in vitro cultured embryos still have reduced functional and physiological abilities compared with those from in vivo conditions, and many factors of oviduct and uterine environments have not yet been revealed. Here, we demonstrated the in vitro culture of domestic goat (Capra hircus) embryos using two types of culture media, modified synthetic oviductal fluid (mSOF) and a two-step chemically defined medium (DI/II). To obtain parthenogenetic goat embryos, oocytes were matured in vitro in tissue culture media-199 supplemented with 10% fetal bovine serum for 22 to 24 hours, and activated with 5 μM, Ca2+ ionomycin for 4 minutes, followed by 1.9 mM, 6-dimethylaminopurine treatment for 4 hours. After 2 days of embryo culture in different culture media, there were no significant differences in cleavage rates (96.6% vs. 95.4% in mSOF vs. DI/II, respectively). However, the DI/II group showed improved development competence to blastocysts (64.6% vs. 82.3% in mSOF vs. DI/II, respectively) and the total cell number of blastocysts (144.3 ± 9.2 vs. 264.4 ± 15.2 in mSOF vs. DI/II, respectively) at Day 7. After the cryopreservation of early-stage blastocysts at Day 6 via the conventional slow-freezing procedure, the surviving embryos were analyzed. The re-expansion rate after freezing and thawing was significantly higher in DI/II (39.66% vs. 67.69% in mSOF vs. DI/II, respectively), but there were no statistical differences in total cell numbers (142.3 ± 12.1 vs. 172.1 ± 11.6 in mSOF vs. DI/II, respectively), apoptotic index (4.9 ± 0.8% vs. 3.8 ± 0.7 in mSOF vs. DI/II, respectively), and the gene expression levels (BAX, GLUT1, MnSOD, and OCT4) among the re-expanded blastocysts. Overall, our data reported that the defined in vitro culture media for goat embryos were established with high efficiency, which will be very useful for goat embryo production.

A novel technique for in vitro maturation of sheep oocytes in a liquid marble microbioreactor.

The aim of this work was to develop a microbioreactor using liquid marble (LM) as a novel system for oocyte in vitro maturation (IVM) in small volumes. Cumulus-oocyte complexes (COCs) obtained from slaughterhouse sheep ovaries were in vitro matured in a LM system prepared by placing a drop (30μl containing 10 COCs) suspended in TCM 199 supplemented with 10% (v/v) oestrus sheep serum (OSS) and 0.1IU FSH and LH onto a polytetrafluoroethylene (PTFE) particle bed (LM group). As a control group (CTRL group), COCs were in vitro matured in standard volume and conditions (600μl of IVM medium in a four-well dish). After 24-h culture at 38.5°C in 5% CO2 in air, COCs were released from LM and the following parameters were evaluated: (a) percentage of MII oocytes, (b) oocyte developmental competence following in vitro fertilization (IVF) or parthenogenetic activation (PA) and embryo culture for 8days in synthetic oviductal fluid (SOF) medium at 38.5°C in 5% O2, 5% CO2, and 90% N2. The results indicated similar percentage of MII oocytes in LM and CTRL groups (88.0 vs. 92.0%). No differences were observed in blastocyst rate after IVF (LM 47.5% vs. CTRL 50.2%, P=0.637) or PA (LM 44.4% vs. CTRL 48.3%, P=0.426). The results indicate that LM microbioreactor is a viable technique that provides a suitable microenvironment to induce oocyte in vitro maturation.

Developmental competence of water buffalo oocytes in a synthetic oviductal fluid base medium

Developmental competence of water buffalo oocytes in a synthetic oviductal fluid base medium

73 PROTEOMIC ANALYSIS OF UTERINE LUMINAL FLUID ON DAY 7 OF PREGNANCY IN CATTLE

In vitro fertilization studies have demonstrated that contact with the female reproductive tract is not necessary in order for the embryo to reach the hatched blastocyst stage. Furthermore, the endometrial transcriptome does not differ between cyclic and pregnant cattle before maternal recognition around Days 15 to 16, suggesting that before that time the cow does not know she is pregnant. Nonetheless, embryo quality is significantly improved by exposure to reproductive tract secretions. Therefore, the aims of this study were to (1) characterise the protein content of uterine luminal fluid (ULF) on Day 7 of pregnancy and the oestrous cycle; (2) identify if differences in ULF composition exist between pregnant and cyclic heifers on Day 7; and (3) determine if blastocyst-derived proteins contribute to ULF composition. Following oestrus synchronization, heifers observed in standing oestrus were randomly assigned to either inseminated (AI: n = 12) or cyclic control (C: n = 6) groups. At slaughter, the uterine horn ipsilateral to the corpus luteum was flushed with 10 mL of PBS. In the AI group, the presence of an appropriately developed embryo was noted (n = 8), and the ULF of both confirmed pregnant and cyclic heifers was clarified by centrifugation at 1000 × g for 15 min at 4°C. Blastocyst-conditioned medium was produced by culturing in vitro-produced blastocysts (n = 300) for 24 h in synthetic oviduct fluid (SOF; groups of 50 blastocysts in 500 µL of SOF). After 24 h the conditioned medium (n = 6 pools), along with contemporaneous blanks (n = 6), was collected and snap frozen. Proteomic analysis of ULF as well as blastocyst-conditioned medium was carried out by nano-LC tandem mass spectrometry (nano-LC-MS/MS). A total of 661 proteins were identified in the ULF of heifers on Day 7 with &gt;95% confidence in at least 4 out of the 6 animals analysed per group. The most abundant proteins were myosin-9, cytoplasmic dynein 1 heavy chain 1-like, fatty acid synthase isoform X1, serum albumin, and aminopeptidase N. No differences were detected between pregnant and cyclic ULF. Of the proteins identified, the biological processes of gene expression (23.8%), small molecule metabolic process (22.6%), and cellular protein metabolic process (17.8%) were most overrepresented. Interestingly, 400 proteins were associated with extracellular exosome as well as extracellular space (87 proteins) and extracellular region (58). Analysis of blastocyst-conditioned medium identified 11 proteins that were not detected in the contemporaneous blanks, of which one (α-1-acid glycoprotein) was also detected in Day 7 ULF. In conclusion, no differences in the ULF composition between pregnant and cyclic heifers were observed on Day 7, consistent with studies on the endometrial transcriptome. A large proportion of the proteins detected are associated with the extracellular space region and may represent candidate proteins that can be added to embryo culture media to enhance early development. Research was funded by Science Foundation Ireland (13/IA/1983).

53 TARGETED SCREEN FOR AMINO ACIDS THAT REGULATE BOVINE INNER CELL MASS DEVELOPMENT

Pluripotency relies on species-specific amino acid (AA) metabolism. In the mouse, inner cell mass (ICM) and ICM-derived pluripotent stem cells (PSCs) need threonine, which is catabolized by threonine dehydrogenase (TDH) into acetyl–CoA and glycine. Depleting (Δ) the culture medium of threonine (ΔT) or blocking TDH activity induces PSC death. By contrast, human PSCs do not survive without lysine (ΔK), leucine (ΔL), or methionine (ΔM). Since isolated bovine PSCs cannot be propagated in vitro, we screened for AAs that selectively support pluripotent ICM cells in intact bovine embryos. Five days (D5) post-IVF, embryos were transferred into glutamine-free synthetic oviduct fluid (gSOF) with Eagle’s nonessential (NE) and essential (E) AAs (gSOF_AA) plus BSA. Embryos were individually cultured until D8 under different conditions. Statistical significance was determined using Fisher’s exact test for blastocyst development (morphological grading to IETS standard) and t-tests for cell numbers (differential stain) and gene expression (quantitative or qPCR). Removal of BSA reduced grade 1–3 blastocyst (B1–3) development (37% v. 25%, n = 3; P &lt; 0.001). Depleting NEAAs from gSOF_AA did not significantly decrease B1–3, but depleting all 12 EAAs did (25% v. 8%, n = 6; P &lt; 0.001). Because ΔEAA was most effective, we focused on this. Experiments were conducted in gSOF+NEAA and compared with gSOF_AA as a positive control (n = 2–6 replicates). One (ΔT, ΔM), two (ΔMT, ΔCM, ΔCT; ΔIL, ΔIK, ΔKL), three (ΔCMT, ΔIKL), or six (ΔHPRVWY) EAA drop-out did not affect blastocyst formation, even when NEAAs were also removed for ΔT and ΔM groups (n = 3). However, depleting another six (ΔCIKLMT), nine (+CMT, +IKL), or eleven EAAs (+T, +M) increasingly compromised B1–3 (P &lt; 0.05). Because no clear EAA candidates emerged from the screen, we focused on TDH. TDH mRNA was present at similar levels in microsurgically isolated (by microblade) trophectoderm (TE) and chemically isolated (by Triton X-100) ICM, but undetectable in five adult tissues. Despite ΔT medium showing no effect, exposure to the TDH inhibitor QC1 (50 µM) reduced B1–3 and B1–2 compared with a dimethylsulfoxide (DMSO) solvent control (25% v. 37% and 8% v. 19%, n = 8; P &lt; 0.005). ICM and TE cell numbers were equally reduced in QC1 v. DMSO-treated blastocysts (10 v. 19 and 37 v. 67 with N = 21 and N = 29 embryos, respectively, n = 3; P &lt; 0.005). Yet TDH, hypoblast (PDGRFα), epiblast (NANOG, FGF4, SOX2), and trophoblast (CDX2, KRT8) markers were not consistently affected by QC1. We next applied 3-hydroxynorvaline (3-HNV), which TDH hydrolyses into glycine and propionyl-CoA instead of acetyl-CoA. Compared with solvent controls, 3-HNV (300 µM) killed all embryos and bovine fetal fibroblasts within 3 days in ΔT medium. This toxic effect was fully rescued by &gt;10-fold T-supplementation. Thus, 3-HNV protein incorporation, rather than acetyl-CoA reduction, may nonspecifically impair cellular function. In summary, we found that bovine ICM formation did not specifically depend on metabolizing threonine or any other single EAA. Research was supported by AgResearch Core Funding.

249 OPTIMIZING DONOR EFFICIENCY WITH FOLLICLE STIMULATING HORMONE SUPERSTIMULATION IN A COMMERCIAL IN VITRO FERTILIZATION PROGRAM IN HOLSTEINS

Commercial IVF programs in Holstein (HO) donors in North America require that not only quantity, but also quality of embryos be increased. These programs are intended to produce the maximal number of offspring from elite females and males over a short period of time. Optimal embryo quality is needed for cryopreservation (for embryo trade and a recurrent problem with a shortage of recipients) and to maximize the number of pregnancies. Preliminary studies in our laboratory showed a significant increase in blastocyst rates following superstimulation with FSH (42 v. 22%; P &lt; 0.0001). The objective of this presentation is to describe parameters of practical importance that affect donor efficiency in an IVF program that involves superstimulation. Data from commercial IVF records were retrospectively analysed. All in-house HO donors admitted since 2013 and at least 12 months of age at the time of collection were included. In total, 1387 ultrasound-guided follicle aspirations (ovum pickup) were performed on 376 HO donors following a constant dose, 6 injections, pFSH (Folltropin-V, Vetoquinol) protocol with a 30- to 43-h coasting period. The IVF system included maturation of cumulus-oocyte complex in TCM-199 based medium, fertilization with frozen‐thawed unsexed, sexed, and reverse-sorted semen, as well as culture in modified synthetic oviduct fluid under mineral oil in a humidified atmosphere containing 6.9% CO2, 5% O2, and 88.1% N2 at 38.5°C for 7 days. Antral follicular counts (AFC), assessed with a 7.5-MHz endovaginal ultrasound probe at random stages during diestrus, were correlated with the number of follicles stimulated. Average number of follicles stimulated was 13.9 (median = 12, maximum = 111) with 74.6% of follicles between 7 and 15 mm at the time of ovum pickup. Average recovery rate was 73.2% and tended to decrease as size of the follicles increased. Transferable embryos rate (TER), defined as the number of IETS quality 1 and 2 embryos produced out of the number of oocytes placed in maturation per ovum pickup session, was 42.1% (median = 43.7%, maximum = 100%). Nearly two-thirds of the transferable embryos produced met our freezability criteria. When these frozen embryos were transferred, over 60% of the recipients were pregnant at 30 days. No difference was observed in efficiency of pregnant versus open donors for TER. In most donors, OPU were repeated every 2 weeks, regardless of pregnancy status. The effect of multiple stimulations on the ability of a donor to maintain its efficiency was assessed by comparing number of follicles stimulated and TER in the first and last 3 collections in donors that had at least 10 collections, with no significant difference. Individual variation appeared very high, as approximately one-third of the donors produced two-thirds of the embryos. When defining a poor responder based on the number of follicles available for ovum pickup following superstimulation, TER was lower in donors with &lt;6 follicles and &gt;18 follicles. Data suggest that HO donors with high AFC are more efficient in an IVF program following superstimulation because of a possible greater oocyte competence.

28 DOUBLING OOCYTE CYTOPLASM VOLUME INCREASES BLASTOCYST QUALITY FOLLOWING INTERSPECIES SOMATIC CELL NUCLEAR TRANSFER IN ARGALI SHEEP (OVIS AMMON)

Interspecies somatic cell NT (SCNT) can be used in the conservation of endangered animals but only when there is an abundant source of compatible oocytes and recipients. The objective of this study was to compare the efficiency of intra- and interspecies SCNT in sheep using zona-free embryo reconstruction methods. Skin fibroblasts from either an argali (Ovis ammon) or control (Ovis aries) ram were used as donor cells for SCNT between passages 2 to 5 and following culture in medium containing 0.5% FCS for 4 to 6 days. Single cells were electrically fused to cytoplasts prepared following enucleation of in vitro-matured zona-free metaphase II-arrested oocytes obtained from domestic ewes. In an additional experiment with argali, a double cytoplast (DC-SCNT) procedure was used whereby a second cytoplast was fused to the first reconstruct within 1 h. Reconstructs were artificially activated ~25 h after the start of maturation using ionomycin and 6-DMAP. Zona-free parthenogenote (PG) control oocytes were activated around the same time. In each treatment, 10 to 12 zona-free embryos where cultured in microwells formed in 20-μL drops of modified synthetic oviduct fluid under oil. Half the medium was replaced on Day 3, and developing embryos were transferred to individual 5-μL drops on Day 6. Development on Day 7 was expressed as a percentage of cleaved embryos. Statistical significance was determined using Fisher’s exact test for embryo development and two-tailed t-test for embryo cell numbers. Total embryo development on Day 7 was significantly greater with intraspecies sheep SCNT compared with interspecies argali SCNT (34/157 = 21.7% v. 34/363 = 9.4%, respectively; P &lt; 0.001). Moreover, SCNT embryo development was significantly less than PG controls (122/360 = 33.9%; P &lt; 0.001). Although argali DC-SCNT had no effect on total embryo development compared with SCNT (9/69 = 13.0% v. 7/56 = 12.5%, respectively), doubling cytoplasm volume increased the proportion of grade 1 and 2 embryos on Day 7 (8.7 v. 0%; P &lt; 0.05). Consequently, DC-SCNT blastocysts had greater numbers of nuclei compared with SCNT (108 ± 47 v. 65 ± 9; n = 6, n = 6, respectively; P = 0.054). In comparison, PG blastocysts possessed on average 122 ± 27 nuclei (n = 7). A sample of 14 argali cloned blastocysts were all confirmed to have been derived from the respective ram after genotyping ~6000 ovine single nucleotide polymorphisms on a custom-made chip (Illumina, San Diego, CA, USA). Chromosome spreads of argali embryos revealed a modal number of 4 bi-armed autosomes as opposed to 6 in Ovis aries. In conclusion, in vitro development following interspecies SCNT in argali was about half that compared with domestic sheep. Blastocyst quality was improved by increasing oocyte cytoplasmic volume facilitated by zona-free cloning. Alternative sources of cytoplasm may further improve development. Confirmation that Ovis aries cytoplasm can fully reprogram a differentiated argali nucleus remains to be determined. This research was supported by AgResearch Core Funding.

180 ASSESSMENT OF BULL SEMEN QUALITY LOADED IN NEW SensiTemp STRAWS USING SEMEN AND IN VITRO PRODUCTION TECHNOLOGIES

SensiTemp, a new in vitro maturation (IMV) bull straw concept, presents the advantage of colour changing while the straw is thawed. The colour of frozen straws is blue and straws start to become white when the temperature reaches 33°C, with a complete change of colour at 37°C. The objective of this study is to assess sperm quality after thawing of semen frozen in SensiTemp from 2 bulls, by analysing, in experiment 1, sperm motility and membrane integrity using computer-assisted semen analysis (CASA) and flow cytometry (FC), and, in experiment 2, the in vitro embryo production (IVP) using IVP technologies [IVM, IVF, and in vitro culture (IVC)]. The ejaculates of 2 bulls, selected during preliminary experiments on high in vitro fertility, were harvested at CIA L’Aigle, France, and split ejaculates were frozen in experimental (SensiTemp) and conventional (control) straws. In experiment 1 after thawing semen from the 2 types of straws (5 pooled straws each; 2 replicates), motility was assessed using the IVOS CASA system (Hamilton Thorne Inc., Beverly, MA, USA) and membrane integrity was evaluated through FC with Cytosoft software (Millipore-Guava Technologies Inc., Hayward, CA, USA). In experiment 2, IVF was used to evaluate the non-toxicity of SensiTemp and control straws. Cumulus-oocyte complexes (COC; n = 1178; 4 replicates) collected from slaughterhouse ovaries were matured in IVM medium (TCM-199 with bicarbonate, Sigma-Aldrich, Saint Quentin Fallavier, France; 10 µg mL–1 FSH-LH, Reprobiol, Liège, Belgium; and 10% FCS, Thermo Fisher, Illkirch, France) for 22 h. After fertilization, presumptive zygotes of each group (SensiTemp and control for each bull) were cultured in synthetic oviduct fluid medium (SOF, Minitube, Tiefenbach, Germany) with 1% estrous cow serum (ECS) and 0.6% BSA (Sigma-Aldrich, France) up to 8 days. All cultures were conducted at 38.5C in 5% CO2, and 5% O2. The cleavage and blastocysts rates were evaluated on Days 3 and 7, respectively, for each group. Embryo quality was recorded on Day 7 according to the IETS evaluation. Data from each bull were analysed separately using the chi-squared test (P &lt; 0.05). In experiment 1, neither sperm motility from bull 1 (61.2 and 60.5%) and bull 2 (66.2 and 66.5%) nor membrane integrity from bull 1 (58.6 and 52.2%) and bull 2 (61.0 and 61.9%) were different between SensiTemp and control, respectively. Results from experiment 2 showed no difference (P &gt; 0.05) in cleavage rate between SensiTemp and control for the 2 bulls: 92.1 and 91.7% for bull 1 and 94.2 and 94.6% for bull 2 respectively. The blastocysts rate on Day 7 did not differ (P &gt; 0.05) among groups (47.5, 47.1 and 51.3, 50.4% for SensiTemp and control bull 1 and bull 2, respectively) nor the quality of embryos retrieved in the different groups: 25.4, 23.3, and 30.8, 29.6% in grade 1 embryo for SensiTemp and control bull 1 and bull 2, respectively. Those results demonstrate, in vitro, that the new SensiTemp straws were non-toxic and did not affect the semen quality after thawing nor did the SensiTemp straws affect the ability of sperm cells to fertilize oocytes and produce 8-day-old embryos.