82 HOW LOW CAN YOU GO? DEFINING THE MINIMAL NUTRIENT REQUIREMENTS FOR BOVINE EMBRYOS IN CULTURE

Recent metabolomic studies in our laboratory have indicated that bovine embryos only use a small amount of the nutrients provided to them in culture media. Our objective was to determine the effects of reducing nutrient concentrations in our media to 75, 50, and 25% (experiment 1) or 25, 12.5, and 6.25% (experiment 2) of those in our control medium (100%) on the development of in vitro-matured/IVF bovine embryos in a serum-free medium. Cumulus–oocyte complexes were matured in a defined maturation medium (0.1 IU mL−1 recombinant human FSH, 50 ng mL−1 recombinant mouse epidermal growth factor, and 2.5 mg mL−1 recombinant human serum albumin) and co-incubated with frozen–thawed spermatozoa (2 × 106 mL−1, 20 h). Embryos were cultured (7.5% CO2, 6.5% O2, 38.7°C) in a sequential media system (0–72 h and 72–168 h). Concentrations of salts, bicarbonate, and protein (2.5 mg mL−1 fatty acid–free BSA) were the same in all treatments. All nutrients (glucose, lactate, pyruvate, amino acids, and vitamins) were diluted to the same extent (e.g. 25%) relative to the control medium for each culture period. Blastocyst formation and hatching (per cleaved embryo) were evaluated on Day 7 of culture. Hatching blastocysts were stained to determine the number of inner cell mass (ICM; SOX2+), trophectoderm (TE; CDX2+), and total cells (ICM+TE) in the embryo. All data were analysed by ANOVA. The proportion of zygotes that cleaved on Day 3 was not affected (P > 0.05) by the concentration of nutrients present. In experiment 1, dilution of nutrients to 25% did not affect (P > 0.05) blastocyst development (40.1 ± 3.7%) or hatching (16.3 ± 2.8%) compared with 100% (45.2 ± 3.8% blastocyst and 24.9 ± 3.3% hatching). In experiment 2, dilution of nutrients to 12.5% tended (P = 0.08) to reduce hatching (12.9 ± 2.6%) compared with 100% (20.0 ± 3.1%) but did not affect (P > 0.05) blastocysts formation (12.5%, 41.7 ± 3.9% v. 100%, 40.0 ± 3.8%). It was not until nutrient concentrations were reduced to 6.25% that blastocyst formation (18.3 ± 3.0%) and hatching (3.0 ± 1.3%) were inhibited (P < 0.05). Hatching blastocysts cultured with 25 or 12.5% nutrients had fewer total (P < 0.05; 150.7 ± 9.7 and 121.6 ± 7.6, respectively), TE (P < 0.05; 124.1 ± 8.5 and 90.5 ± 7.1), and ICM (P = 0.06; 26.6 ± 3.4 and 30.7 ± 4.0) cells compared with control embryos (195.2 ± 15.9 total, 156.1 ± 14.1 TE, and 39.1 ± 4.0 ICM). To determine whether the embryo’s ability to develop with reduced concentrations of nutrients was dependent on lipid metabolism, embryos were cultured with 50, 25, 12.5, and 6.25% nutrients in the presence or absence of an inhibitor of fatty acid oxidation (50 μM etomoxir). The presence of etomoxir reduced (P < 0.05) blastocyst development at all nutrient concentrations, but this effect was more pronounced when nutrients were limited (≤25% nutrients, 28.7 to 40.9% reduction) compared with 50% (12.5% reduction). Although blastocyst cell numbers decrease when nutrient concentrations are reduced to 25% of those in control media, the proportion of embryos reaching the blastocyst stage is not affected until nutrients are reduced to 6.25%. The ability to develop under nutrient-restricted conditions appears to be related to fatty acid metabolism.

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Developmental and molecular response of bovine embryos to reduced nutrients in vitro

Reproduction and Fertility ◽

10.1530/raf-20-0033 ◽

2020 ◽

Vol 1 (1) ◽

pp. 51-65

Author(s):

Jason R Herrick ◽

Sandeep Rajput ◽

Rolando Pasquariello ◽

Alison Ermisch ◽

Nicolas Santiquet ◽

...

Keyword(s):

Amino Acids ◽

Fatty Acid ◽

Embryo Development ◽

Cell Mass ◽

Pyruvate Dehydrogenase Kinase ◽

Nutrient Concentrations ◽

Molecular Response ◽

Control Medium ◽

Bovine Embryos ◽

Blastocyst Formation

Recent studies in our laboratory have indicated that bovine embryos only use a small amount of the nutrients available to them in culture. Our objective was to evaluate the developmental and molecular response of bovine embryos when nutrient concentrations in the culture medium were significantly reduced. Following IVM and IVF, embryos were cultured in media containing 75, 50, and 25% (experiment 1) or 25, 12.5, and 6.25% (experiment 2) of the concentrations of nutrients (carbohydrates, amino acids, and vitamins) present in our control medium (100%). Blastocyst formation, hatching, and allocation of cells to the inner cell mass (ICM) and trophectoderm (TE) were evaluated on day 7. Although the number of TE cells was decreased (P < 0.05) when nutrient concentrations were ≤25% (73.8–124.1 cells), it was not until nutrient concentrations were reduced to 6.25% that blastocyst formation (18.3 ± 3.0%) and hatching (3.0 ± 1.3%) were inhibited (P < 0.05) compared to embryos cultured in the control medium (156.1 ± 14.1 cells, 40.0 ± 3.8%, 20.0 ± 3.1%, respectively). Inhibition of fatty acid oxidation (etomoxir) reduced (P < 0.05) blastocyst development, with more pronounced effects at lower nutrient concentrations (≤12.5%). Reducing nutrient concentrations was associated with increased activity of AMPK, decreased activity of mTOR, and altered abundance of transcripts for hexokinase 1 (HK1), carnitine palmitoyl transferase 2 (CPT2), lactate dehydrogenase A (LDHA), and pyruvate dehydrogenase kinase 1 (PDK1), consistent with an increase in glucose and fatty acid metabolism. Reduced nutrient conditions provide a unique perspective on embryo metabolism that may facilitate the optimization of culture media. Lay summary To support early embryo development in the first week after fertilisation, an appropriate mixture of nutrients (carbohydrates, amino acids, and vitamins) is needed in the culturing solution. However, refining these solutions to support optimal embryo health remains challenging. In this study, bovine (cow) embryos derived from abattoir material were used as a model for the development of other mammalian embryos, including humans. These embryos were cultured in the presence of 75, 50, 25, 12.5, or 6.25% of the nutrients present in control conditions (100%), which are similar to those reported for the fluids of the fallopian tubes and uterus. Embryo development was largely unaffected in the 75, 50, and 25% treatments, with some embryos developing in the presence of only 6.25% nutrients. Cow embryos are remarkably resilient to reduced concentrations of nutrients in their environment because they can utilize internal stores of fat as a source of energy.

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Development of bovine embryos cultured in CR1aa and IVD101 media using different oxygen tensions and culture systems

Acta Veterinaria Hungarica ◽

10.1556/avet.58.2010.4.7 ◽

2010 ◽

Vol 58 (4) ◽

pp. 465-474 ◽

Cited By ~ 5

Author(s):

Tamás Somfai ◽

Yasushi Inaba ◽

Yoshio Aikawa ◽

Masaki Ohtake ◽

Shuji Kobayashi ◽

...

Keyword(s):

Inner Cell Mass ◽

Culture Media ◽

Cell Mass ◽

Calf Serum ◽

Bovine Embryos ◽

Blastocyst Formation ◽

Cell Numbers ◽

Culture Systems ◽

Significant Difference

The aim of the present study was to optimise the culture conditions for the in vitro production of bovine embryos. The development of in vitro fertilised bovine oocytes in CR1aa supplemented with 5% calf serum and IVD101 culture media were compared using traditional microdrops and Well of the Well (WOW) culture systems either under 5% or 20% oxygen tension. After 7 days of culture, a significantly higher blastocyst formation rate was obtained for embryos cultured in CR1aa medium compared to those cultured in IVD101, irrespective of O 2 tensions and culture systems. The blastocyst formation in IVD101 was suppressed under 20% O 2 compared to 5% O 2 . Despite their similar total cell numbers, higher rates of inner cell mass (ICM) cells were observed in blastocysts developed in IVD101 medium than in those developed in CR1aa, irrespective of O 2 tensions. There was no significant difference in blastocyst formation, total, ICM and trophectoderm (TE) cell numbers between embryos obtained by microdrop and WOW culture systems irrespective of the culture media and O 2 tensions used. In conclusion, CR1aa resulted in higher blastocyst formation rates irrespective of O 2 tension, whereas IVD101 supported blastocyst formation only under low O 2 levels but enhanced the proliferation of ICM cells.

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178 IMPROVED DEVELOPMENTAL COMPETENCE OF BOVINE EMBRYOS BY PGI2 ANALOG TREATMENT

Reproduction Fertility and Development ◽

10.1071/rdv18n2ab178 ◽

2006 ◽

Vol 18 (2) ◽

pp. 197 ◽

Cited By ~ 2

Author(s):

B. S. Song ◽

J. S. Kim ◽

D. B. Koo ◽

J. S. Park ◽

K. K. Lee ◽

...

Keyword(s):

Inner Cell Mass ◽

Culture Media ◽

Cell Mass ◽

Developmental Rate ◽

Treated Group ◽

Developmental Competence ◽

Bovine Embryos ◽

Frozen Semen ◽

Oviductal Fluid

The microenvironment of the follopian tube, in which the oviductal fluid contains a variety of cytokines and growth factors, affects pre-implantation development of fertilized embryos in mammals. Prostaglandin I2 (PGI2, prostacyclin) exists in oviductal fluid and is synthesized from arachidonic acid by prostacyclin synthetase. PGI2 also enhances the implantation rate of mouse embryos. In this study, the effect of PGI2 analog on the development of bovine embryos was examined. Bovine cumulus oocytes complexes (COCs) were matured in TCM-199 medium supplemented with 10 IU/mL pregnant mare serum gonadotropin (PMSG), 10 IU/mL hCG, and 10 ng/mL epidermal growth factor (EGF) at 39�C, 5% CO2 in air for 20-22 h. Following in vitro maturation, COCs were fertilized in Fert-TALP medium containing 0.6% BSA using frozen semen. Also, oocytes matured in vitro were enucleated, individually reconstructed with bESF cells, fused, and then activated by treatment with 5 �M ionomycin for 5 min and 2 mM 6-DMAP for 4 h. In vitro-fertilized (IVF) and nuclear-transferred (NT) eggs were cultured in 50 ��L drops of CR1-aa medium supplemented with 0.3% BSA in the absence or presence of 1 �M PGI2 analog at 39�C, 5% CO2 in air, respectively. At 3 days of culture, cleaved embryos were further cultured in the same culture media supplemented with 10% FBS for 4 days. Allocations of blastocysts to inner cell mass (ICM) and trophoblast (TE) cells were investigated to assess embryo quality. All experiments were repeated more than three times. All data were analyzed by using the Duncan test of ANOVA by the Statistical Analysis System (SAS Institute, Inc., Cary, NC, USA) and numbers of nuclei in blastocysts were expressed as mean � SE. No difference was detected in the cleaved rate of the eggs between the treated- and nontreated groups. IVF zygotes treated with PGI2 analog represented a higher developmental rate (33%, 122/418) to the blastocyst stage than nontreated controls (24%, 107/456) (P < 0.05). Among IVF-derived blastocysts, interestingly, the proportion (46%, 84/181) of expanded blastocysts was significantly higher in the PGI2 analog-treated group compared with that in the nontreated group (28%, 46/164). The number of nuclei in (165 � 6.1, n = 15) in blastocysts in the PGI2 analog-treated group was higher than that (146.12 � 5.7, n = 18) in the nontreated group (P < 0.05). No difference was detected in the ratio of ICM to total cells between PGI2 analog-treated (42.0 � 3.0%) and nontreated groups (41.9 � 2.9%). Like the IVF embryos, NT embryos in the PGI2 analog-treated group showed a higher in vitro developmental rate (33.6%, 43/128) than the nontreated embryos (24.2%, 32/132) (P < 0.05). Our results indicate that PGI2 analog improves the kinetics of embryo development in cattle.

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156INFLUENCE OF SERUM AND BSA ON BLASTOCYST DEVELOPMENT AND HATCHING IN IVF AND NUCLEAR TRANSFER BOVINE EMBRYOS

Reproduction Fertility and Development ◽

10.1071/rdv16n1ab156 ◽

2004 ◽

Vol 16 (2) ◽

pp. 200

Author(s):

G.F. Mastromonaco ◽

E. Semple ◽

C. Robert ◽

J. Rho ◽

D. Betts ◽

...

Keyword(s):

Fatty Acid ◽

Embryo Development ◽

Nuclear Transfer ◽

Culture Media ◽

Adverse Outcomes ◽

Bovine Embryos ◽

Blastocyst Development ◽

Treatment Groups ◽

Short Term Exposure ◽

Hatching Rates

Important differences exist between in vivo- and in vitro-produced bovine embryos. Studies have shown that various components in culture media affect embryo development, with serum producing some of the more detrimental effects. Efforts to develop a serum-free culture system have included looking at the effects of BSA, polyvinyl pyrrolidone and polyvinyl alcohol on embryo development. In this study, we compare serum and BSA during oocyte maturation and embryo culture of IVF and nuclear transfer (NT) embryos. Experiment A: Oocytes were aspirated from follicles and matured in either collection medium (Hams F-10+2% steer serum (SS); F-10) or in follicular fluid alone (FF). They were subjected to IVM-IVF-IVC as follows: 20–22h maturation in synthetic oviductal fluid +8mgmL−1 fatty acid-free BSA (SOF+BSA-FAF) supplemented with hormones, 18h co-incubation with sperm in IVF-TALP, and culture for 9 days in SOF+BSA-FAF. Experiment B: Oocytes were randomly distributed for IVM-IVF-IVC into the following treatment groups: (i) IVM and IVC in SOF+2% SS (SER), (ii) IVM in SOF+2% SS and IVC in SOF+BSA-FAF (SER-FAF), (iii) IVM and IVC in SOF+BSA-FAF (FAF), and (iv) IVM and IVC in SOF+BSA-FrV (FrV). Experiment C: Oocytes were matured for 18h in either SOF+2% SS (SER) or SOF+BSA-FAF (FAF). Couplets were constructed with adult skin fibroblasts, exposed to a single pulse of 1.5kVcm−1 for 40s and activated using ionomycin and cycloheximide. Embryos were cultured in SOF+BSA-FAF. Three replicates with 100–120 oocytes per treatment group were carried out. Results: Cleavage rates were similar among all treatments in experiments A and B. No differences were observed between oocytes collected in F-10 or FF indicating that short-term exposure to serum does not have long-term effects on embryo development. Although a higher number of blastocysts was observed in the SER group on Day 6 (3.2% v. <0.5%; P<0.05), no differences were seen in blastocyst development among the IVF treatment groups from Day 8 onwards (SER: 29.7%, SER-FAF: 21.1%, FAF: 20.4%, FrV: 19.9%). However, hatching rates on Days 8 and 9 were significantly greater (P<0.05) in groups with serum, with the exception of FAF on Day 9 (SER: 31.1%, 57.2%; SER-FAF: 29.4%, 50.6%; FAF: 23.1%, 46.4%; FrV: 18.5%, 34.2%). In the NT treatment groups, the presence of serum during IVM resulted in a higher proportion of MII oocytes at 18h, better oocyte quality for manipulation, and increased blastocyst development and hatching rates (SER: 31.4%, 18.2%; FAF: 21.7%, 4.8%). These results indicate that both serum and fatty acid-free BSA provide comparable embryo development during IVF. However, development in serum occurs at an accelerated rate as indicated by the shorter nuclear maturation times and blastocyst development on Day 6, which has been associated with adverse outcomes. Despite this, serum may provide the oocyte with factors that are important for membrane flexibility and repair, enabling greater survival after manipulation. Funding from NSERC and OMAFRA. Sperm provided by Gencor.

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95 ROLE OF POLYAMINES IN BOVINE PRE-IMPLANTATION DEVELOPMENT

Reproduction Fertility and Development ◽

10.1071/rdv28n2ab95 ◽

2016 ◽

Vol 28 (2) ◽

pp. 177

Author(s):

J. Herrick ◽

A. Greene ◽

W. Schoolcraft ◽

R. Krisher

Keyword(s):

Amino Acids ◽

Inner Cell Mass ◽

Gradient Centrifugation ◽

Cell Mass ◽

Cumulus Cells ◽

Essential Amino Acids ◽

Human Albumin ◽

Bovine Embryo ◽

Bovine Embryos ◽

Blastocyst Formation

Polyamines are involved in trophectoderm attachment and outgrowth, but little is known about their role in earlier stages of development. The objective of this study was to evaluate the effects of an inhibitor of polyamine synthesis (difluoromethylornithine, DFMO) on development (blastocyst formation and hatching) and cell allocation to the trophectoderm (TE, CDX2-positive) and inner cell mass (ICM, SOX2-positive) in the bovine embryo. Cumulus-oocyte complexes (COCs) were recovered from slaughterhouse ovaries and matured for 24 h in a defined maturation medium (5.0 mM glucose, 0.6 mM cysteine, 0.5 mM cysteamine, 0.1 IU mL–1 FSH, 50 ng mL–1 EGF, and 2.5 mg mL–1 recombinant human albumin). Frozen-thawed spermatozoa were processed by gradient centrifugation and co-incubated (2 × 106 mL–1) with COC [10 COC/50 µL; 7.5 µg mL–1 heparin, 2 mM caffeine, and 8.0 mg mL–1 fatty-acid free (FAF) BSA] for 20 to 22 h. After removing cumulus cells, zygotes were cultured (10 embryos/20 µL) in a medium for cleavage stage bovine embryos (0.5 mM glucose, 0.3 mM pyruvate, 6.0 mM lactate, 0.25 mM citrate, 1.0 mM alanyl-glutamine, 0.25 × MEM nonessential and essential amino acids, 5 µM EDTA, and 8.0 mg mL–1 FAF BSA). After 72 h, embryos with >4 cells were randomly allocated (5 embryos/20 µL) to a culture medium for compaction and blastocyst formation (3.0 mM fructose, 0.1 mM pyruvate, 6.0 mM lactate, 0.5 mM citrate, 1.0 mM alanyl-glutamine, 1× MEM nonessential amino acids, 0.5× MEM essential amino acids, 0.075 mM myo-inositol, and 8.0 mg mL–1 FAF BSA) containing 0 (control), 5, or 10 mM DFMO. Embryonic development was evaluated at 192 h post-insemination (96 h in the second medium containing DFMO treatments), and hatching or hatched blastocysts were fixed for analysis of cell allocation. All data were analysed by ANOVA and P < 0.05 was considered significant. Blastocyst formation and hatching (% of embryos cultured in the presence of treatments) were both inhibited (P < 0.05) when embryos (n = 157/treatment) were cultured with 5 (39.5 ± 3.9%, 14.6 ± 2.8%) or 10 (39.5 ± 3.9%, 14.0 ± 2.8%) mM DFMO compared with embryos cultured without DFMO (53.5 ± 4.0%, 26.1 ± 3.5%). The number of TE cells was also reduced (P < 0.05) in the presence of 5 (121.4 ± 7.2) and 10 (123.6 ± 6.7) mM DFMO compared with embryos cultured without DFMO (152.4 ± 9.7), but the number of ICM cells (45.2 to 54.0) and the total number of cells (TE+ICM, 168.8 to 201.1) were not affected (P > 0.05). In a second experiment (n = 163 to 165/treatment), the negative effects of DFMO on hatching (17.0 ± 2.9%; P < 0.05, v. control, 30.7 ± 3.6%) could be partially reversed when embryos were cultured with both 10 mM DFMO and an exogenous polyamine (100 µM putrescine, 23.0 ± 3.3% DFMO+Put; P > 0.05 v. control). The number of TE cells for embryos cultured with DFMO+Put (153.9 ± 8.7) was intermediate between embryos cultured with (138.0 ± 6.9) or without DFMO (control, 161.6 ± 8.7), but these differences were not significant (P > 0.05). These results provide the first evidence of a role for polyamines during blastocyst formation and hatching of bovine embryos, with specific effects on trophectoderm proliferation and hatching.

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Effect of cysteamine supplementation during in vitro culture of early stage bovine embryos on blastocyst rate and quality

Acta Veterinaria Brno ◽

10.2754/avb201281030229 ◽

2012 ◽

Vol 81 (3) ◽

pp. 229-234 ◽

Cited By ~ 4

Author(s):

Martina Lojkic ◽

Iva Getz ◽

Marko Samardžija ◽

Mario Matkovic ◽

Goran Bacic ◽

...

Keyword(s):

Inner Cell Mass ◽

Culture Media ◽

Cell Mass ◽

Cell Number ◽

Total Cell ◽

Hatching Rate ◽

Bovine Embryos ◽

Total Cell Number ◽

Oviductal Fluid

The aim of this study was to evaluate whether the addition of cysteamine to the in vitro culture media enhances the yield, hatching rate, total cell number and inner cell mass/total cell number ratio of bovine embryos. A total of 933 bovine oocytes collected from ovaries of 60 slaughtered donors were subjected to in vitro maturation and in vitro fertilization. Following fertilization, embryos were cultured in synthetic oviductal fluid without glucose. After 24 h embryos were transferred into synthetic oviductal fluid with 1.5 mM glucose and 0 (control), 50, 100 and 200 µM of cysteamine. After 48 h, the embryos were transferred into synthetic oviductal fluid with glucose but without cysteamine and cultured until Day 9. The number of cleaved embryos on Day 2, the total number of blastocysts on Day 7 and the number of hatched blastocysts on Day 9 were calculated. Differential staining of inner cell mass and trophectoderm cells of blastocysts were performed on Day 7 and Day 9 of in vitro culture. Supplementation of in vitro culture media with 100 µM cysteamine increased the blastocyst yield (P < 0.05) without affecting the hatching rate. Furthermore, the embryos cultured in the presence of 100 µM cysteamine had significantly higher number of inner cell mass cells (P < 0.05) and the proportion of inner cell mass cells (P < 0.05) compared with the controls. The results of the present study demonstrated that the addition of 100 µM cysteamine to the in vitro culture media improved blastocyst production rate and enhance embryo quality, which could lead to the improvement of the in vitro culture system for bovine embryos.

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ROCK activity regulates functional tight junction assembly during blastocyst formation in porcine parthenogenetic embryos

PeerJ ◽

10.7717/peerj.1914 ◽

2016 ◽

Vol 4 ◽

pp. e1914 ◽

Cited By ~ 3

Author(s):

Jeongwoo Kwon ◽

Nam-Hyung Kim ◽

Inchul Choi

Keyword(s):

Tight Junction ◽

Inner Cell Mass ◽

Cell Mass ◽

Coiled Coil ◽

Specific Inhibitor ◽

Developmental Competence ◽

Blastocyst Formation ◽

Blastocyst Development ◽

Genes Encoding ◽

Rock Activity

The Rho-associated coiled-coil-containing protein serine/threonine kinases 1 and 2 (ROCK1 and ROCK2) are Rho subfamily GTPase downstream effectors that regulate cell migration, intercellular adhesion, cell polarity, and cell proliferation by stimulating actin cytoskeleton reorganization. Inhibition of ROCK proteins affects specification of the trophectoderm (TE) and inner cell mass (ICM) lineages, compaction, and blastocyst cavitation. However, the molecules involved in blastocyst formation are not known. Here, we examined developmental competence and levels of adherens/tight junction (AJ/TJ) constituent proteins, such as CXADR, OCLN, TJP1, and CDH1, as well as expression of their respective mRNAs, after treating porcine parthenogenetic four-cell embryos with Y-27632, a specific inhibitor of ROCK, at concentrations of 0, 10, 20, 100 µM for 24 h. Following this treatment, the blastocyst development rates were 39.1, 20.7, 10.0, and 0% respectively. In embryos treated with 20 µM treatment, expression levels of CXADR, OCLN, TJP1, and CDH1 mRNA and protein molecules were significantly reduced (P< 0.05). FITC-dextran uptake assay revealed that the treatment caused an increase in TE TJ permeability. Interestingly, the majority of the four-cell and morula embryos treated with 20 µM Y-27643 for 24 h showed defective compaction and cavitation. Taken together, our results indicate that ROCK activity may differentially affect assembly of AJ/TJs as well as regulate expression of genes encoding junctional proteins.

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Antioxidant activity of oily extract obtained from Lippia origanoides improves the quality of bovine embryos produced in vitro

Arquivo Brasileiro de Medicina Veterinária e Zootecnia ◽

10.1590/1678-4162-10323 ◽

2019 ◽

Vol 71 (3) ◽

pp. 723-731

Author(s):

N.V. Sollecito ◽

E.C.M. Pereira ◽

J.G.V. Grázia ◽

B.P. Neves ◽

B.V.R. Couto ◽

...

Keyword(s):

Embryo Culture ◽

Culture Medium ◽

Inner Cell Mass ◽

Cell Mass ◽

Bovine Embryos ◽

In Vitro Production ◽

Blastocyst Development ◽

Embryo Culture Medium ◽

Vitro Production

ABSTRACT The aim of this study was to evaluate the supplementation of embryo culture medium with antioxidant obtained from oily extract of Lippia origanoides on in vitro blastocyst development and quality. Oocytes collected from slaughterhouse ovaries were matured and fertilized in vitro following standard laboratory procedures. Zygotes were cultured in SOF medium supplemented according to the following treatments: T1 embryo culture medium without antioxidant supplementation; T2)50μM/mL Cysteamine; T3)2.5μg/mL; T4)5.0μg/mL and T5)10.0μg/mL of antioxidant obtained from oily extract of Lippia origanoides. On the seventh day of culture, the blastocysts were fixed and evaluated for apoptosis rates, number of total cell and inner cell mass cells by means of the TUNEL Test. The use of antioxidants during cultivation did not increase (P> 0.05) the final blastocyst production rate. The treatments T2, T3, T4 and T5 had the lowest (P< 0.05) apoptotic indexes (4.5±1.1%, 8.4±2.5%, 3.4±1.1% and 5.5±0.9%, respectively) when compared to T1 treatment (10.0±1.4%). The number of inner cell mass did not differ (P> 0.05) among embryos from different treatments. The addition of antioxidant obtained from oily extract of Lippia origanoides reduces the apoptosis rate and improves the quality without increasing the total in vitro production of bovine embryos.

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Biological differences between in vitro produced bovine embryos and parthenotes

Reproduction ◽

10.1530/rep-08-0220 ◽

2009 ◽

Vol 137 (2) ◽

pp. 285-295 ◽

Cited By ~ 36

Author(s):

Enrique Gómez ◽

Alfonso Gutiérrez-Adán ◽

Carmen Díez ◽

Pablo Bermejo-Alvarez ◽

Marta Muñoz ◽

...

Keyword(s):

De Novo ◽

Inner Cell Mass ◽

Relative Proportion ◽

Cell Mass ◽

Blastocyst Stage ◽

Bovine Embryos ◽

Blastocyst Development ◽

Cell Counts ◽

Biological Differences

Parthenotes may represent an alternate ethical source of stem cells, once biological differences between parthenotes and embryos can be understood. In this study, we analyzed development, trophectoderm (TE) differentiation, apoptosis/necrosis, and ploidy in parthenotes andin vitroproduced bovine embryos. Subsequently, using real-time PCR, we analyzed the expression of genes expected to underlie the observed differences at the blastocyst stage.In vitromatured oocytes were either fertilized or activated with ionomycin +6-DMAP and cultured in simple medium. Parthenotes showed enhanced blastocyst development and diploidy and reduced TE cell counts. Apoptotic and necrotic indexes did not vary, but parthenotes evidenced a higher relative proportion of apoptotic cells between inner cell mass and TE. The pluripotence-relatedPOU5F1and the methylationDNMT3Agenes were downregulated in parthenotes. Among pregnancy recognition genes,TP-1was upregulated in parthenotes, whilePGRMC1andPLAC8did not change. Expression ofp66shcandBAX/BCL2ratio were higher, andp53lower, in parthenotes. Among metabolism genes,SLC2A1was downregulated, whileAKR1B1,PTGS2,H6PD, andTXNwere upregulated in parthenotes, andSLC2A5did not differ. Among genes involved in compaction/blastulation,GJA1was downregulated in parthenotes, but no differences were detected withinATP1A1andCDH1. Within parthenotes, the expression levels ofSLC2A1,TP-1, andH6PD, and possiblyAKR1B1, resemble patterns described in female embryos. The pro-apoptotic profile is more pronounced in parthenotes than in embryos, which may differ in their way to channel apoptotic stimuli, throughp66shcandp53respectively, and in their mechanisms to control pluripotency andde novomethylation.

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62 Sequential nutrient restriction and provision during bovine in vitro embryo culture differentially affect blastocyst development and quality with oocytes from varied sources

Reproduction Fertility and Development ◽

10.1071/rdv31n1ab62 ◽

2019 ◽

Vol 31 (1) ◽

pp. 156

Author(s):

R. Pasquariello ◽

Y. Yuan ◽

D. Logsdon ◽

J. Becker ◽

L. Yao ◽

...

Keyword(s):

Fatty Acids ◽

Embryo Culture ◽

Inner Cell Mass ◽

Cell Mass ◽

Cell Number ◽

Total Cell ◽

Nutrient Concentrations ◽

Blastocyst Development ◽

Total Cell Number

We have demonstrated that bovine blastocyst development was improved after culture in medium with only 6.25% of standard carbohydrate and amino acid concentrations, supplemented with fatty acids. However, these blastocysts had lower cell numbers. We hypothesised that this was due to deficiencies in embryo metabolism at the time of blastocyst formation. Thus, our objectives were to (1) determine whether using a sequential combination of nutrient concentrations could rescue blastocyst cell number; and (2) investigate the efficacy of reduced nutrient medium in 2 sources of oocytes. Oocytes were in vitro matured in identical medium either in our laboratory or during shipment from a commercial supplier. Oocytes in our laboratory were derived from feedlot heifers while purchased oocytes were obtained from culled cows. Zygotes were cultured using sequential medium with fraction V BSA. In step 1/step 2, embryos were cultured using 100% (glucose 0.5 mM/fructose 3.0mM, pyruvate 0.3/0.1mM, lactate 10.0/6.0mM, NEEA 1×/1× MEM, EAA 0.25×/0.5× MEM), 25% or 6.25% of standard nutrient concentrations. On Day 3, embryos were moved to step 2 as follows: 100% to 100%, 25% to 25%, 25% to 100%, 6.25% to 25%, or 6.25% to 100%. Lipid content of single mature oocytes from both sources was determined using gas chromatography coupled to an ISQ-LT MS/MS (GC-MS; Thermo Scientific, Waltham, MA, USA). Data (mean±s.e.m.) were analysed using ANOVA (P<0.05). When oocytes from feedlot heifers were used, blastocyst development and cell number did not differ between treatments. When oocytes from culled cows were used, blastocyst development was improved after embryo culture in 25-25% (45.1±3.3%) and 6.25-25% (46.6±3.2%) compared with 100-100% (34.2±3.2%). However, inner cell mass number of blastocysts cultured in 25-25% (25.6±2.5) and 6.25-25% (26.0±2.6) was reduced compared with 100-100% (41.4±4.5); TE and total cell number did not differ. Embryos cultured in 100-100%, 25-100%, and 6.25-100% were equivalent. Metabolomics revealed that 10 lipid compounds (polyunsaturated fatty acids, glycosyldiacylglycerols, and glycerophospholipids) differed in abundance between the two sources of oocytes. These results show that oocytes from different sources lead to different experimental outcomes, likely due to a combination of age, body condition, diet, and hormone treatment of the female. Oocytes from culled cows result in embryos that develop to blastocysts better in a reduced nutrient environment, although these embryos have fewer inner cell masses, suggesting that quality may be reduced. Embryos from feedlot heifer oocytes are relatively immune to nutrient fluctuations. Different endogenous fatty acid reserves in the oocyte may lead to differing metabolic strategies in the subsequent embryo, altering their response to substrate availability during in vitro culture. These results also demonstrate that reduction of nutrients during culture has no detrimental effect on blastocyst development or total cell number in either oocyte source, but that inner cell mass formation requires increased nutrient provision.

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