Preparation and control of culture media

Between Day 10 and 12 of gestation, the pig embryo elongates from a sphere to a long thin, filament. During this time, the embryo increases the production of oestrogen via an increase in steroidogenic transcripts, which is critical for maternal recognition of pregnancy. To date, attempts to elongate porcine embryos in vitro have been unsuccessful. Therefore, the objective of this study was to utilise alginate hydrogels to establish a culture system that promotes in vitro embryo elongation with a corresponding increase in steroidogenic transcripts and oestradiol production. In 3 replicate collections, White crossbred gilts (n = 15) were bred at Day 0 of the oestrous cycle. At Day 9 of gestation, reproductive tracts were collected and flushed with RPMI-1640 containing antibiotics. Embryos were recovered, grouped according to size and washed with RPMI-1640 containing antibiotics and 10% fetal bovine serum (FBS). Embryos were randomly assigned to be encapsulated using a double encapsulation technique (0.7% sodium alginate and 1.5% calcium chloride solution) or used as controls. Encapsulated and control embryos were cultured for 96 h in CO2 -pretreated RPMI-1640 containing antibiotics and 10% FBS at 38°C, 5% CO2 in air and 100% humidity. Every 24 h, the embryos were imaged and half of the media was replaced. The removed media was stored at –20°C and used to assess oestradiol levels by radioimmunoassay. At the end of culture, a subset of encapsulated and control embryos were snap frozen and used to assess the expression level of steroidogenic transcripts (STAR, CYP11 and CYP19) using quantitative PCR. All data were analysed using general linear model (GLM) procedures for ANOVA. Cell survival, assessed by blastocyst fragmentation and confirmed by live/dead staining in representative embryos, was greater (P = 0.01) for encapsulated embryos (60.1 ± 4.8%) compared with controls (33.3 ± 4.8%). Of encapsulated embryos, 27% had some morphological change (minor flattening and tubal formation) and 14% had significant morphological changes (considerable flattening and tubal formation elongating through the gel), consistent with in vivo embryo elongation. In contrast, the control embryos had no morphological changes observed and remained spherical during culture. The expression levels of STAR, CYP11 and CYP19 were significantly (P < 0.05) greater in encapsulated embryos compared with control embryos. Furthermore, a significant (P < 0.01) time-dependent increase in oestradiol levels in the culture media of encapsulated embryos was identified compared with controls and culture media alone. These results illustrate that cultured pig embryos encapsulated in alginate hydrogels undergo limited morphological changes with increased expression of steroidogenic transcripts and oestrogen production. †USDA is an equal opportunity provider and employer.

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Exhaustion of CMV Specific T Cells with Enhanced PD-1 Expression In Persistent Cytomegalovirus Infection After Allogeneic Stem Cell Transplantation

Blood ◽

10.1182/blood.v116.21.3912.3912 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3912-3912 ◽

Cited By ~ 1

Author(s):

Tomonori Kato ◽

Tetsuya Nishida ◽

Miho Murase ◽

Makoto Murata ◽

Tomoki Naoe

Keyword(s):

T Cells ◽

Stem Cell ◽

T Cell ◽

Stem Cell Transplantation ◽

Cell Transplantation ◽

Cd8 T Cells ◽

Culture Media ◽

Cmv Infection ◽

Control Serum ◽

And Control

Abstract Abstract 3912 Cytomegalovirus (CMV) is one of the most common pathogens causing morbidity and mortality after allogeneic hematopoietic stem cell transplantation (HSCT), despite preemptive treatments employing antiviral drugs. Cytotoxic T cells are indispensable to control CMV infections. Chronic viral infections with human immunodeficiency virus or hepatitis C virus were shown to be associated with exhausted T cells with high expression of the inhibitory molecule programmed death 1 (PD-1). Recently, it has been reported that PD-1 up-regulation on CMV specific T cells was associated with CMV infection after renal and liver transplantation. PD-1 expression on CMV specific T cells after HSCT has not been well examined. We evaluated the involvements of exhausted CMV specific T cells characterized by high PD-1 expression in persistent CMV infection after allogeneic HSCT. Peripheral blood mononuclear cells (PBMC) and serum were obtained from an HLA-A*2402-positive patient who had received bone marrow transplantation from an HLA-A, B, C and DR matched unrelated donor. This patient failed to eliminate CMV for more than one year after transplantation despite intermittent administration of ganciclovir and foscarnet. Control PBMC and serum were obtained from an HLA-A*2402-positive healthy volunteer because the Japan Marrow Donor Program prohibits blood collection for research use from donor. All blood was collected with written informed consent. We at first analyzed frequencies of CMV-specific CD8+ T cells in patient and control PBMC by flow cytometer using QYDPVAALF/A*2402-specific tetramer and CD8 antibodies. QYDPVAALF is derived from CMV pp65 protein and presented by the HLA-A*2402 molecule. Tetramer stained cells were detected in the patient PBMC but control PBMC (0.11% versus undetectable). Patient and control PBMC were stimulated by a synthetic peptide QYDPVAALF in culture media containing IL-2 for 14 days, and stained with QYD/A*2402-specific tetramer. Remarkably, post-stimulated patient PBMC contained only 0.54% of tetramer stained CD8+ T cells, whereas a more dramatic increase (14.1%) in control PBMC. We analyzed frequencies of IFN-g secreting CD8+ T cells in PBMC after stimulation with a peptide pool covering the whole CMV pp65 protein for 4 hours. Less patient CD8+ T cells produced IFN-g, compared with the control CD8+ T cells (0.5% versus 1.1%) These data demonstrate dysfunction of CMV-specific CD8+ T cells in the patient with persistent CMV infection. To examine the mechanism of dysfunction of CMV-specific CD8+ T cells, we analyzed the expression of PD-1 on CMV-specific CD8+ T cells 14 days after stimulation with QYDPVAALF peptide. Multiparameter flow cytometry and tetramer assay exhibited higher expression of PD-1 on CMV-specific CD8+ T cells generated from patient PBMC, compared with CMV-specific CD8+ T cells generated from control PBMC. To find out whether the engagement of PD-1 to its ligand (PD-L1) leads to T cell exhaustion, we stimulated patient PBMC with QYDPVAALF peptide for 14 days in the presence or absence of anti PD-L1 antibody which blocks PD-1/PD-L1 inhibitory pathway. Blockade of PD-1/PD-L1 pathway resulted in 3.9-fold increase in patient CMV specific T cells. These findings demonstrate that PD-1 is associated with the exhaustion of CMV specific CD8+ T cells during persistent CMV infection in this patient. To examine the effect of patient serum on CMV specific CD8+ T cells, we stimulated patient PBMC with QYDPVAALF peptide for 14 days in culture media with patient or control serum. CMV specific CD8+ T cells increased 4-fold and 55-fold in the presence of patient and control serum, respectively. Patient serum led to higher PD-1 expression on CMV specific CD8+ T cells, compared with control serum (Fig). These findings suggest that patient serum may contain what regulates PD-1 expression level of exhausted T cells. Further investigations to identify factors regulating PD-1 expression in patient serum are in progress. The identification of the factors may provide new strategies to improve exhausted T cell function. Disclosures: No relevant conflicts of interest to declare.

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Identification and Control of Bacterial Contamination in Callus Cultures of Ulmus americana

Journal of Environmental Horticulture ◽

10.24266/0738-2898-14.2.50 ◽

1996 ◽

Vol 14 (2) ◽

pp. 50-52

Author(s):

Lawrence R. Schreiber ◽

Subhash C. Domir ◽

V.M. Gingas

Keyword(s):

Culture Media ◽

Leaf Tissue ◽

Callus Cultures ◽

Airborne Spores ◽

Ulmus Americana ◽

Bacillus Macerans ◽

Burner Flame ◽

Ethanol Ethanol ◽

And Control ◽

Latent Bacteria

Abstract Bacillus macerans Schardinger appeared on culture media and forceps used in serial transfers of Ulmus americana callus tissue after several contamination-free transfers and may have originated as an endophyte in the leaf tissue used as an explant. Bacteria remained viable on forceps stored for several weeks in 95% ethanol whether or not the excess was burned off in a flame from an alcohol lamp. Bacteria were eliminated from forceps treated similarily with 85% ethanol. The bacterium on artificially contaminated forceps remained viable after immersion up to 4 hr in either 95%, 85%, 80%, or 70% ethanol with or without flaming. Artificial contamination was eliminated, either by autoclaving for 20 min at 121C (185.8F) or exposure to a bunsen burner flame for 6-8 sec. Bacillus macerans remained viable in both naturally and artificially contaminated ethanol at dilutions of 95%, 85%, 80%, and 70%. Thus, forceps may be contaminated by latent bacteria in callus or contaminated ethanol. Ethanol may become contaminated by storage of nonsterile forceps and airborne spores introduced during routine, septic procedures.

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The Effects Of Estrogen On Von Willebrand Factor Production And Endothelial Cell Number In Culture

10.1055/s-0038-1652001 ◽

1981 ◽

Cited By ~ 1

Author(s):

R L Harrison ◽

P A McKee

Keyword(s):

Endothelial Cell ◽

Cell Cultures ◽

Culture Media ◽

Cell Number ◽

Cell Monolayers ◽

Insoluble Material ◽

Endothelial Cell Cultures ◽

Dose Response Effect ◽

Von Willebrand ◽

And Control

Subconfluent and confluent endothelial cell monolayers derived from human umbilical veins were treated with pharmacologic levels of estrogen and compared functionally to control monolayers. The levels of von Willebrand ristocetin cofactor (vWF) present In media from cell cultures after three days of incubation were assayed by ristocetin-induced platelet aggregation (PA) as well as by a radioreceptor platelet assay (RR) for vWF. DNA content of each monolayer was determined fluorometrically as a measure of cell number. The incorporation of 3H-thymidine into 17β-estradiol (E2)-treated endothelial cell monolayers vs. control cultures was also quantitated. Whether measured by PA or RR, levels of vWF in media from E2-treated cells were reproducibly and significantly greater (1.2±0.33 μg vWF/ml/ μg DNA at 2 ng/ml E2) than in media from control endothelial cells (0.69±0.084 μg vWF/ml/μg DNA, p<0.01) and showed a dose-response effect with respect to the E2 added to the culture media. Compared to control cultures, E2-treated monolayers consistently contained greater DNA (2.0±0.10 μg DNA) than control cultures (1.7±0.12 μg DNA, p<0.001) and incorporated more 3H-thymidine into trichloroacetic acid insoluble material (30.2±6×103 CPM with E2, vs. 21.2±2×103 CPM control, p<0.05). However, differences in cell number between E2-treated and control monolayers did not account for the elevated vWF levels in the presence of exogenous E2. We conclude that estrogen has at least two effects on endothelial cell cultures: 1) estrogen directly stimulates the cells to increase their rate of production of vWF; and 2) estrogen causes an increased rate of endothelial cell replication.

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ANALYSIS OF LYSATE PLATELET-RICH FIBRIN EFFECTS ON HUMAN DENTAL PULP STEM CELL DIFFERENTIATION THROUGH DENTIN SIALOPHOSPHOPROTEIN EXPRESSION

International Journal of Applied Pharmaceutics ◽

10.22159/ijap.2020.v12s2.op-43 ◽

2020 ◽

pp. 34-37

Author(s):

SILVIANA SWASTININGTYAS ◽

ANGGRAINI MARGONO ◽

DINI ASRIANTI ◽

RUNY OKTAYANI ◽

INDAH YULIANTO

Keyword(s):

Dental Pulp ◽

Culture Media ◽

Stem Cell Differentiation ◽

Alizarin Red ◽

Platelet Rich Fibrin ◽

Dentin Sialophosphoprotein ◽

Human Dental Pulp ◽

Stable Growth ◽

And Control

Objective: In vitro, the culture media in which human dental pulp stem cells (hDPSCs) are grown are supplemented with specific growth factors thatinduce cell cycle entry and differentiation. Lysate platelet-rich fibrin (L-PRF) is a unique and stable growth factor supplement produced from plateletslysed by freezing-thawing. In this study, we aimed to analyze the potential effects of L-PRF on hDPSC differentiation.Methods: We divided hDPSCs isolated from human third molars at the second passage into five culture media groups treated with 1%, 5%, 10%,and 25% L-PRF or 10% fetal bovine serum (control). After 7 days, we evaluated hDPSC differentiation using an enzyme-linked immunosorbent assayspecific for dentin sialophosphoprotein and Alizarin-Red staining.Results: None of our analyses revealed any significant differences between the L-PRF- and control-treated cells.Conclusion: L-PRF could potentially induce the differentiation of hDPSCs in vitro.

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Prostacyclin synthesis in irradiated endothelial cells cultured from bovine aorta

Journal of Applied Physiology ◽

10.1152/jappl.1985.58.2.592 ◽

1985 ◽

Vol 58 (2) ◽

pp. 592-597 ◽

Cited By ~ 47

Author(s):

D. B. Rubin ◽

E. A. Drab ◽

C. H. Ts'ao ◽

D. Gardner ◽

W. F. Ward

Keyword(s):

Endothelial Cells ◽

Culture Media ◽

Cell Detachment ◽

Aortic Endothelial Cells ◽

Endothelial Cell Surface ◽

Ldh Activity ◽

Platelet Adherence ◽

Subendothelial Matrix ◽

60Co Gamma Rays ◽

And Control

Confluent monolayers of bovine aortic endothelial cells were examined 2–72 h after exposure to 0.5–5.0 Gy of 60Co gamma-rays. Accumulation of prostacyclin [PGI2, measured as 6-ketoprostaglandin F1 alpha (6-keto-PGF1 alpha)] in the culture media and PGI2 production stimulated by exogenous arachidonate were correlated with cell detachment and release of lactate dehydrogenase (LDH) activity. Platelet adherence to irradiated and control monolayers also was studied. There were simultaneous time- and dose-dependent increases in cell detachment and in the titers of 6-keto-PGF1 alpha and LDH activity in the culture medium. These changes were evident between 4 and 8 h after 5 Gy or at 24 h after 0.5 Gy. Four hours after 5 Gy, both adherent and detached endothelial cells showed a twofold increase in PGI2 production during a 15-min incubation with arachidonate (10 microM). However, by 72 h this increase was less significant. The accumulation of 6-keto-PGF1 alpha appeared to be related to cell destruction, but radiation also stimulated PGI2 synthesis independent of cell detachment. There was an increased platelet interaction with irradiated monolayers, as a result of platelet adherence to subendothelial matrix exposed after cell detachment. However, irradiation did not alter the nonadherent property of the endothelial cell surface toward platelets.

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118VITRIFICATION OF PRONUCLEAR-STAGE MOUSE EMBRYOS ON ALUMINUM FOIL FLOATING ON LIQUID NITROGEN

Reproduction Fertility and Development ◽

10.1071/rdv16n1ab118 ◽

2004 ◽

Vol 16 (2) ◽

pp. 181 ◽

Cited By ~ 1

Author(s):

H. Sagirkaya ◽

F. Ergin ◽

H. Bagis ◽

S. Arat

Keyword(s):

Liquid Nitrogen ◽

Culture Media ◽

Aluminum Foil ◽

Blastocyst Stage ◽

Culture Conditions ◽

Mouse Embryos ◽

Morphological Appearance ◽

Development Rates ◽

Developmental Rates ◽

And Control

The cryopreservation of pronuclear-stage embryos has a special importance in transgenic technology, cloning, and human-assisted reproductive technology. The objective of this study was to investigate the efficiency of a vitrification method modified in our lab for pronuclear-stage embryos. In experiment I, groups of 10 pronuclear-stage mouse embryos were cultured in 20μL drops of three different culture media (G1.3/G2.3, CZB and M16) covered with mineral oil (Sigma M-8410, St. Louis, MO, USA). Twenty-four hours later, embryos cultured in G1.3 were transferred into G2.3 medium. In experiment II, 25–30 pronuclear-stage embryos were transferred into a 50-μL drop of equilibration medium containing 4% ethylene glycol (EG, Sigma E-9129) in TCM-199 (Sigma M-2520) supplemented with 10% FCS at 37°C for 12–15min; then they were rinsed three times in 30-μL drops of vitrification medium containing 35% EG, 5% polyvinylpyrrolidone (PVP, Sigma P-0930) and 0.4M trehalose (Sigma T-0167) in TCM-199 supplemented with 10% FCS at 37°C for 20–30s. Embryos rinsed in vitrification solution were aspirated into a micropipette as a 1–2-μL drop containing 25–30 embryos and dropped onto aluminum foil floating on liquid nitrogen (LN2). Vitrified droplets were stored in cryovials in LN2. Warming was performed by moving the vitrified droplets into 0.3M trehalose in TCM-199 supplemented with 10% FCS at 37°C. Embryos having normal morphological appearance under stereomicroscope examination were cultured in G1.3/G2.3 medium. Differences in the two experiments were analyzed by one-way ANOVA. In experiment I, development rates to the blastocyst stage were 26%, 10% and 4% for G1.3/G2.3, CZB and M16 media, respectively. The highest development rate in experiment I was obtained in G1.3/G2.3 culture media (P<0.05). Therefore, G1.3/G2.3 media were used for culturing of vitrified-warmed and control embryos. In experiment II, the rate of embryos having normal morphology was 98.5%. There were no significant differences between the development rates of vitrified (13.1%) and control (18.7%) embryos to the blastocyst stage (P>0.05). Although the vitrification method resulted in a high survival rate based on the morphological appearance, developmental rates of vitrified and control embryos were found to be lower than expected and reported previously by other researchers. We believe that the low developmental rates in this study were due to our culture conditions but not our vitrification method. Therefore, it could be concluded that this vitrification method is an efficient one for pronuclear-stage embryo cryopreservation and better development rates could be obtained by improving the culture conditions. This study was supported by a grant from TUBITAK, Turkey (VHAG-1908-102V048). F. Ergin is a young volunteer researcher.

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Calcium Carbonate Nanoparticles Stimulate Cancer Cell Reprogramming to Suppress Tumor Growth and Invasion in an Organ-on-a-chip System

10.21203/rs.3.rs-117446/v1 ◽

2020 ◽

Author(s):

Sandra Lam ◽

Kevin Bishop ◽

Rachel Mintz ◽

Lei Fang ◽

Samuel Achilefu

Keyword(s):

Cancer Cells ◽

Breast Cancer Cells ◽

Culture Media ◽

Metabolic Reprogramming ◽

Cellular Migration ◽

Cell Culture Media ◽

Effects Of Ph ◽

Tumor Viability ◽

And Behavior ◽

And Control

Abstract The acidic microenvironment of solid tumors induces the propagation of highly invasive and metastatic phenotypes. However, simulating these conditions in animal models present challenges that confound the effects of pH modulators on tumor progression. To recapitulate the tumor microenvironment and isolate the effect of pH on tumor viability, we developed a bifurcated microfluidic device that supports two different cell environments for direct comparison. RFP-expressing breast cancer cells (MDA-MB-231) were cultured in treatment and control chambers surrounded by fibrin, which received acid-neutralizing CaCO3 nanoparticles (nanoCaCO3) and cell culture media, respectively. Data analysis revealed that nanoCaCO3 buffered the pH within the normal physiological range and inhibited tumor cell proliferation compared to the untreated control (p < 0.05). Co-incubation of cancer cells and fibroblasts, followed by nanoCaCO3 treatment showed that the nanoparticles selectively inhibited the growth of the MDA-MB-231 cells and reduced cellular migration of these cells with no impact on the fibroblasts. Sustainable decrease in the intracellular pH of cancer cells treated with nanoCaCO3 indicates that the extracellular pH induced cellular metabolic reprogramming. These results suggest that the nanoCaCO3 can restrict the aggressiveness of tumor cells without affecting the growth and behavior of the surrounding stromal cells.

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133 EFFECTS OF FROZEN STORED CULTURE MEDIA ON PRE-IMPLANTATION DEVELOPMENT OF PARTHENOTES AND CLONED EMBRYOS IN PIGS

Reproduction Fertility and Development ◽

10.1071/rdv21n1ab133 ◽

2009 ◽

Vol 21 (1) ◽

pp. 166

Author(s):

Y. H. Zhang ◽

H. T. Xi ◽

Y. Liu ◽

J. Li ◽

A. Pederson ◽

...

Keyword(s):

Culture Media ◽

Polar Body ◽

Frozen Storage ◽

Test Group ◽

Total Cell ◽

Control Group ◽

Cell Numbers ◽

Significant Difference ◽

And Control

The present study was designed to examine if frozen storage of porcine zygote medium (PZM3) plus 3 mg mL–1 BSA (Yoshioka et al. 2002 Bio. Reprod. 66, 112–119) is feasible to culture pig embryos produced by parthenogenetic activation and somatic nuclear transfer. Slaughterhouse-derived sow cumulus–oocyte complexes (COCs) were matured in TCM199 supplemented with 10% porcine follicle fluid, 5% cattle serum, 10 IU mL–1 eCG, 5 IU mL–1 hCG, 0.8 mm L-glutamine and 0.05 mg mL–1 gentamicin at 38.5°C, 100% humidity and 5% CO2 in air. For activation, cumulus cells were removed after 42 to 44 h of maturation, and the denuded oocytes with 1st polar body were activated with a double 160 V mm–1, 100 μs direct pulse followed by culture in PZM3. Each experiment was replicated at least three times. Data were expressed as mean ± SEM and analyzed by using chi-square module in SPSS 11.0, with P < 0.05 denoting significant difference. In Experiment 1, after preparation, liquid PZM3 was aliquoted to 50 mL falcon centrifuge tubes. Randomly, half of the tubes with PZM3 were put into –80°C freezers, and the rest were placed into 4°C refrigerator. Within one week after storage, a tube of frozen PZM3, while that stored at 4°C served as control, was warmed at 38.5°C in CO2 incubator, and more than three 4-well culture dishes were then made with 400 μL PZM3 in each well and balanced for at least 4 h in the incubator before experiment. The results showed that both cleavage (78/93, 83.9 ± 1.2% v. 87/103, 84.5 ± 1.8%, P > 0.05) and blastocyst (60/93, 65.2 ± 2.1% v. 65/103, 63.1 ± 3.8%, P > 0.05) rates were similar between frozen-warmed PZM3 and control, as was total cell numbers per blastocyst (50 ± 7 v. 47 ± 5, P > 0.05) between groups. In Experiment 2, we used somatic cloned embryos to investigate the effect of frozen-warmed PZM3 on pre-implantation development of such embryos. Our results indicated that no significant difference in rates of cleavage (68/95, 71.5 ± 5.1% v. 78/100, 78.1 ± 1.9%, P > 0.05), blastocyst formation (33/95, 34.6 ± 7.6% v. 78/100, 38.2 ± 3.5%, P > 0.05) and total cell numbers per blastocyst (40 ± 11 v. 48 ± 9, P > 0.05) was found between the test and control groups, designed the same as in Experiment 1. In Experiment 3, we tested whether PZM3 in frozen storage for 5 months was able to support in vitro development of parthenotes comparable to freshly-made ones. PZM3 after frozen storage for 5 months was warmed using the same method as Experiment 1, and the newly made PZM3 within 1 week of storage at 4°C acted as control. The results showed that although the cleavage (135/138, 97.8 ± 2.7% v. 117/129, 90.7 ± 3.1%, P > 0.05) and blastocyst (104/138, 75.4 ± 1.6% v. 84/129, 65.1 ± 2.3, P > 0.05) rates in control group were both slightly higher than that in the test group, no statistical differences was observed. We also found no significant difference in total cell numbers per blastocyst (48 ± 7 v. 46 ± 6, P > 0.05) between groups. Taken together, our results imply that frozen storage of PZM3 is feasible, and of practical value for culture pig embryos.

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149 Uptake of C18:0 from culture media during invitro culture decreases cryosurvival rates of bovine embryos

Reproduction Fertility and Development ◽

10.1071/rdv33n2ab149 ◽

2021 ◽

Vol 33 (2) ◽

pp. 183

Author(s):

I. Bertijn ◽

B. M. Gadella ◽

H. T. A. van Tol ◽

A. Rijneveld ◽

P. L. A. M. Vos ◽

...

Keyword(s):

Embryo Culture ◽

Negative Binomial ◽

Culture Media ◽

Negative Binomial Regression ◽

Terminal Deoxynucleotidyl Transferase ◽

Bovine Embryos ◽

Multiple Ovulation ◽

Binomial Regression ◽

Post Hoc ◽

And Control

Cryosurvival of invitro-produced bovine embryos is lower than that of invivo-produced embryos, limiting their usability in the field. Previous work showed that the embryo’s lipid composition relates to its quality and cryosurvival. The present study aimed to investigate the effects of free fatty acid (FA) additions to embryo culture media during the oviduct phase of embryonic development on the improvement of cryosurvival of invitro-produced blastocysts. Bovine cumulus–oocyte complexes (n=1675, 3 replicates) were harvested from slaughterhouse ovaries, invitro matured (23h), and subsequently fertilized (18–20h). Embryos were cultured until Day 5 post-fertilization in synthetic oviductal fluid (SOF) with (1) bovine serum albumin (BSA; control, n=253); (2) delipidified BSA (>96% FA free, n=460); (3), delipidified BSA complexed with 25µM unsaturated oleic acid (C18:1, n=455); or (4) with saturated stearic acid (C18:0, n=507) with a stoichiometry of 5:1. At Day 5, SOF was refreshed and embryos were cultured without supplementation. At Days 7 and 8, blastocyst rates were determined. Blastocysts were LD540 stained for lipid droplets (LD), and the LD number and size were analysed by ANOVA. Cryosurvival%, defined by re-expansion of the blastocoel, was analysed by logistic regression. Additionally, fresh and frozen–thawed blastocysts were stained for apoptosis (terminal deoxynucleotidyl transferase dUTP nick-end labelling, TUNEL), necrosis (EthD-1), and DNA (Hoechst 33342) and analysed using negative binomial regression. Group differences were tested using a post hoc Tukey test. Statistical analysis was performed in R Studio (version 3.4.2), and P-values <0.05 were considered significant. FA-free culture delayed and decreased blastocyst rates to 19% compared with any FA supplementation: 35%, 27%, and 29% for control, C18:1, and C18:0, respectively (P<0.04). Cryosurvival doubled with culture in FA-free SOF (58%) and C18:1 (63%) compared with C18:0 (23% P=0.01 and P<0.01, respectively) and control (29%; P=0.15 and P<0.02, respectively), approaching cryosurvival rates of donated multiple ovulation embryo transfer (MOET) embryos (CRV Company; 67%). C18:0 exposure also resulted in elevated necrosis levels after cryopreservation (5–8% of cells), compared with all groups (2–4%; P<0.016). The LD size increased in blastocysts cultured with C18:1 compared with all groups (3.1µm2 vs. 2.4–2.7µm2; P<0.016). C18:0 addition to SOF during embryo culture invitro, as well as a mixture of FA in control SOF (including C18:0), caused a reduction of ∼50% in blastocyst cryosurvival compared with MOET blastocysts. Interestingly, either C18:1 addition or the complete omission of FA in SOF during embryo culture invitro restored the cryosurvival of blastocysts to the level of MOET blastocysts. Currently, we are investigating whether the free FA conditions in the oviduct endorse the distinct quality between invivo- and invitro-produced embryos.

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