Modified Spirulina maxima Pectin Nanoparticles Improve the Developmental Competence of In Vitro Matured Porcine Oocytes

Molecular approaches have been used to determine metabolic substrates involved in the early embryonic processes to provide adequate culture conditions. To investigate the effect of modified Spirulina maxima pectin nanoparticles (MSmPNPs) on oocyte developmental competence, cumulus–oocyte complexes (COCs) retrieved from pig slaughterhouse ovaries were subjected to various concentrations of MSmPNPs (0, 2.5, 5.0, and 10 µg/mL) during in vitro maturation (IVM). In comparison to the control, MSmPNPs-5.0, and MSmPNPs-10 groups, oocytes treated with 2.5 µg/mL MSmPNPs had significantly increased glutathione (GSH) levels and lower levels of reactive oxygen species (ROS). Following parthenogenetic activation, the MSmPNPs-2.5 group had a considerably higher maturation and cleavage rates, blastocyst development, total cell number, and ratio of inner cell mass/trophectoderm (ICM:TE) cells, when compared with those in the control and all other treated groups. Furthermore, similar findings were reported for the developmental competence of somatic cell nuclear transfer (SCNT)-derived embryos. Additionally, the relative quantification of POU5F1, DPPA2, and NDP52 mRNA transcript levels were significantly higher in the MSmPNPs-2.5 group than in the control and other treated groups. Taken together, the current findings suggest that MSmPNP treatment alleviates oxidative stress and enhances the developmental competence of porcine in vitro matured oocytes after parthenogenetic activation and SCNT.

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Relationship between follicle size and oocyte developmental competence in prepubertal and adult pigs

Reproduction Fertility and Development ◽

10.1071/rd07018 ◽

2007 ◽

Vol 19 (7) ◽

pp. 797 ◽

Cited By ~ 69

Author(s):

Melanie A. Bagg ◽

Mark B. Nottle ◽

David T. Armstrong ◽

Christopher G. Grupen

Keyword(s):

In Vitro Maturation ◽

Fold Increase ◽

Cell Number ◽

Developmental Competence ◽

Blastocyst Development ◽

Parthenogenetic Activation ◽

Follicle Size ◽

Low Efficiency ◽

The Relationship

The present study compared the distribution and steroid composition of 3-, 4- and 5–8-mm follicles on the surface of prepubertal and adult ovaries, and determined the relationship between follicle size and developmental competence of oocytes following parthenogenetic activation. The effect of 1 mm dibutyryl cAMP (dbcAMP) for the first 22 h of in vitro maturation (IVM) on the embryo development of prepubertal oocytes from the three follicle size cohorts was also determined. Compared with adult, prepubertal ovaries contained a higher proportion of 3-mm follicles (46 v. 72%, respectively), but a lower proportion of 4-mm (33 v. 22%, respectively) and 5–8-mm follicles (21 v. 6%, respectively). Adult follicular fluid (FF) contained 11-fold higher levels of progesterone (P4) than prepubertal FF, with similar levels observed between all adult follicle sizes. In prepubertal FF, the P4 concentration increased with follicle size from 3 to 4 to 5–8 mm. Rates of blastocyst development following parthenogenetic activation of adult oocytes from all three follicles sizes were similar (approximately 55%), whereas rates from prepubertal oocytes increased with increasing follicle size from 3 (17%) to 4 (36%) to 5–8 mm (55%). Treatment with dbcAMP for the first 22 h of IVM led to a 1.5-fold increase in the rate of blastocyst development for prepubertal oocytes from 3-mm follicles, but had no effect on prepubertal oocytes from the 4 and 5–8 mm classes. Mean blastocyst cell number increased with follicle size in prepubertal ovaries and was similar for all follicle sizes in adult ovaries. The present study demonstrates that the low efficiency of in vitro embryo production observed using prepubertal compared with adult pig oocytes is due to a greater proportion of 3-mm follicles on prepubertal ovaries, which contain oocytes of inferior developmental competence.

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53 EFFECTS OF INSULIN-TRANSFERRIN-SELENIUM IN DEFINED AND PORCINE FOLLICULAR FLUID-SUPPLEMENTED MEDIUM ON IN VITRO PRODUCTION OF PORCINE SCNT EMBRYOS

Reproduction Fertility and Development ◽

10.1071/rdv19n1ab53 ◽

2007 ◽

Vol 19 (1) ◽

pp. 144

Author(s):

Y. U. Kim ◽

D. P. Bhandari ◽

M. S. Hossein ◽

S. M. Park ◽

E. Lee ◽

...

Keyword(s):

Somatic Cell ◽

Inner Cell Mass ◽

Cell Mass ◽

Defined Medium ◽

Culture Conditions ◽

Developmental Competence ◽

Differential Staining ◽

In Vitro Production ◽

Fetal Fibroblasts

Insulin promotes the uptake of glucose and amino acids, and is beneficial for maturation of oocytes in vitro. Transferrin is an iron-transport protein and selenium is an essential trace element. Insulin-transferrin-selenium (ITS) together has been used in some in vitro maturation systems. The present study was designed to evaluate the effects of ITS in defined and porcine folicular fluid (pFF)-supplemented IVM medium on the glutathione (GSH) concentration, and on developmental competence after somatic cell nuclear transfer. ITS liquid media supplement (I-3146) was purchased from Sigma-Aldrich (St Louis, MO, USA). Basic IVM medium was TCM-199 supplemented with 10 ng mL-1 epidermal growth factor, 4 IU mL-1 pregnant mare serum gonadotropin (PMSG) and hCG and either 1% PVA (defined medium) or 10% pFF. Ten �g mL-1 insulin, 5.5 �g mL-1 transferrin, and 5 �g mL-1 selenium was used for the entire 44-h culture period. The GSH content of a gruop of 10 to 20 oocytes was determined by the dithionitrobezoic acid-glutathione disulfide (DTNB-GSSG) reductase recycling assay. Fetal fibroblasts were used as somatic cell donors and reconstructed embryos were cultured in mNCSU-23 medium for 168 h. Cleavage and blastocyst formation was observed at 48 h and 168 h, respectively. The quality of blastocysts was assessed by differential staining of the inner cell mass (ICM) and the trophectoderm (TE) cells. Each experiment was replicated for 5 times. The data were analyzed by one-way ANOVA, and Tukey was used as a posthoc test. The level of GSH production significantly varied in different culture conditions. The highest GSH concentration was observed in the pFF + ITS group (8.2 picomol/oocyte). A total of 116, 125, 126, and 120 reconstructed oocytes were cultured, and 10.1, 15.3, 17.2, and 21.8% blastocysts were observed for PVA, PVA + ITS, pFF, and pFF + ITS groups, respectively (P < 0.05). The numbers of inner cell mass, trophrectoderm cells, and total cells were significantly higher in the pFF + ITS group compared with the other groups. The average number of total cells in blastocysts was 31.9 � 1.8, 43.1 � 3.5, 46.7 � 4.9, and 52.3 � 6.7 for PVA, PVA + ITS, pFF, and pFF + ITS groups, respectively (P < 0.05). ITS supplement improved the developmental competence in both the defined and the pFF supplemented groups. We recommend supplementing porcine IVM medium with 10 �g mL-1 insulin, 5.5 �g mL-1 transferrin, and 5 �g mL-1 selenium.

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58 ISOLATION OF BOVINE TROPHOBLAST AND ITS REPROGRAMMING BY NUCLEAR TRANSFER

Reproduction Fertility and Development ◽

10.1071/rdv23n1ab58 ◽

2011 ◽

Vol 23 (1) ◽

pp. 134

Author(s):

I. M. Saadeldin ◽

B. H. Kim ◽

B. Roibas da Torre ◽

O. J. Koo ◽

G. Jang ◽

...

Keyword(s):

Nuclear Transfer ◽

Inner Cell Mass ◽

Quantitative Polymerase Chain Reaction ◽

Cell Mass ◽

Donor Cell ◽

Developmental Competence ◽

Control Group ◽

Embryonic Cells ◽

Blastocyst Development

Nuclear transfer (NT) has been used to produce many cloned offspring using several types of cells, including embryonic cells. Even though inner cell mass cells have been used as donor karyoplast for producing cloned animals, there are few studies using trophoblast. In mice, clones were born by nuclear transfer of trophoblasts from the expanded blastocyst into enucleated oocytes as a trial to show the totipotency of both inner cell mass and trophectoderm cells isolated from blastocysts (Tsunoda and Kato 1998 J. Reprod. Fertil. 113, 181–184). However, bovine trophoblast cell (TC) lines have not been used in NT to date. The purpose of this study was to elucidate whether TC as donor cell can be reprogrammed in bovine enucleated oocyte and determine the relative abundance of interferon tau (IFNτ) expression in the resulting cloned preimplantational embryos. Hatched blastocysts produced by IVF were used to isolate TCs on mouse embryonic fibroblasts treated with mitomycin C as feeder cells. TCs and adult fibroblasts (AF, control group for NT) were microinjected to perivitelline space of in vitro mature enucleated oocytes and electrically fused. Reconstructed embryos were chemically activated and cultured in a 2-step chemically defined medium. Levels of IFNτ expression in IVF-, TC-, and AF-derived blastocysts were analyzed using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). IVF produced embryos were used as reference to analyze the linear progressive expression of IFNτ through mid-, expanded, and hatching blastocysts. As a result, TCs expressing IFNτ were successfully isolated and cultured on feeder layers. It grew as cell sheets of cuboidal epithelium with high proliferation capacity as a single colony originated from a small clump of cells measured 0.5 cm within 7 days of culture. TCs were reprogrammed in the enucleated oocytes to blastocyst with similar efficiency to AF (14.5% and 15.6%, respectively; P ≤ 0.05). RT-qPCR studies showed that IFNτ expression was higher in TC-derived blastocysts than IVF- and AF-derived blastocysts. Both IVF- and TC-derived blastocysts, showed progressive increase of IFNτ expression through the advancement of blastocyst development when it was compared to AF-derived blastocysts. In conclusion, using TCs expressing IFNτ as donor cell for bovine NT could increase the developmental competence of cloned embryos as indicated by progressive linear increase in IFNτ expression. This study was supported by grants from IPET (#109023-05-1-CG000), NRF (#M10625030005-10N250300510), MKE (#2009-67-10033839, #2009-67-10033805), and BK21 program. Saadeldin I. M. is supported by Islamic Development Bank (IDB) merit scholarship, Jeddah, Saudi Arabia.

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Tolerance of lamb and mouse oocytes to cryoprotectants during vitrification

Zygote ◽

10.1017/s0967199418000606 ◽

2018 ◽

Vol 27 (1) ◽

pp. 36-45

Author(s):

Jaqueline Sudiman ◽

Alice Lee ◽

Kheng Ling Ong ◽

Wu Zi Yuan ◽

Sarah Jansen ◽

...

Keyword(s):

Cell Number ◽

Blastocyst Stage ◽

Developmental Competence ◽

Cortical Granule ◽

Blastocyst Development ◽

Cleavage Rate ◽

Parthenogenetic Activation ◽

Mouse Oocytes ◽

Different Response

SummaryMouse and lamb oocytes were vitrified with, or exposed to, different cryoprotectants and evaluated for their effects on their survival and developmental competence after in vitro fertilization (IVF) and activation treatments. Control oocytes remained untreated, whilst the remainder were exposed to three different combinations of vitrification solutions [dimethyl sulfoxide (DMSO) + ethylene glycol (EG), EG only, or propanediol (PROH) + EG] and either vitrified or left unfrozen (exposed groups). Oocytes in the control and vitrified groups underwent IVF and developmental competence was assessed to the blastocyst stage. In lambs, survival rate in vitrified oocytes was significantly lower than for oocytes in the exposed groups (P <0.05). Blastocyst development was low in vitrified oocytes compared with controls (<6% vs 38.9%, P <0.01). Parthenogenetic activation was more prevalent in vitrified lamb oocytes compared with controls (P <0.05). No evidence of zona pellucida hardening or cortical granule exocytosis could account for reduced fertilization rates in vitrified lamb oocytes. Mouse oocytes demonstrated a completely different response to lamb oocytes, with survival and parthenogenetic activation rates unaffected by the vitrification process. Treatment of mouse oocytes with DMSO + EG yielded significantly higher survival and cleavage rates than treatment with PROH + EG (87.8% and 51.7% vs 32.7% and 16.7% respectively, P <0.01), however cleavage rate for vitrified oocytes remained lower than for the controls (51.7% vs 91.7%, P <0.01) as did mean blastocyst cell number (33 ± 3.1 vs 42 ± 1.5, P <0.05). From this study, it is clear that lamb and mouse show different tolerances to cryoprotectants commonly used in vitrification procedures, and careful selection and testing of species-compatible cryoprotectants is required when vitrifying oocytes to optimize survival and embryo development.

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Development of Preimplantation Mouse Embryos in vivo and in vitro

Australian Journal of Biological Sciences ◽

10.1071/bi9820187 ◽

1982 ◽

Vol 35 (2) ◽

pp. 187 ◽

Cited By ~ 81

Author(s):

GM Harlow ◽

P Quinn

Keyword(s):

Inner Cell Mass ◽

Cell Mass ◽

Cell Number ◽

Blastocyst Stage ◽

Culture Conditions ◽

Cell Stage ◽

Preimplantation Mouse ◽

Development In Vitro

The culture conditions for the development in vitro of (C57BL/6 X CBA) F2 hybrid two-cell embryos to the blastocyst stage have been optimized. Commercially available pre-sterile disposable plastic culture dishes supported more reliable development than re-usable washed glass tubes. The presence of an oil layer reduced the variability in development. An average of 85 % of blastocysts developed from hybrid two-cell embryos cultured in drops of Whitten's medium under oil in plastic culture dishes in an atmosphere of 5% O2 : 5% CO2 : 90% N2 ? The time taken for the total cell number to double in embryos developing in vivo was 10 h, and in cultured embryos 17 h. Embryos cultured in vitro from the two-cell stage to blastocyst stage were retarded by 18-24 h in comparison with those remaining in vivo. Day-4 blastocysts in vivo contained 25-70 cells (mean 50) with 7-28 (mean 16) of these in the inner cell mass. Cultured blastocysts contained 19-73 cells (mean 44) with 8-34 (mean 19) of these in the inner cell mass. In the uterine environment, inner-cell-mass blastomeres divided at a faster rate than trophectoderm blastomeres and it is suggested that a long cell cycle is associated with terminal differentiation. Although cultured blastocysts and inner cell masses contained the same number of cells as blastocysts and inner cell masses in vivo, the rate of cell division in cultured inner cell masses was markedly reduced.

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Nuclear transfer technique affects mRNA abundance, developmental competence and cell fate of the reconstituted sheep oocytes

Reproduction ◽

10.1530/rep-12-0318 ◽

2013 ◽

Vol 145 (4) ◽

pp. 345-355 ◽

Cited By ~ 29

Author(s):

F Moulavi ◽

S M Hosseini ◽

M Hajian ◽

M Forouzanfar ◽

P Abedi ◽

...

Keyword(s):

Cell Fate ◽

Embryo Development ◽

Nuclear Transfer ◽

Inner Cell Mass ◽

Cell Mass ◽

Developmental Competence ◽

Blastocyst Development ◽

Cleavage Rate ◽

Nuclear Remodeling

The effect of technical steps of somatic cell nuclear transfer (SCNT) on different aspects of cloned embryo development was investigated in sheep.In vitro-matured oocytes were enucleated in the presence or absence of zona and reconstituted by three different SCNT techniques: conventional zona-intact (ZI-NT), standard zona-free (ZF-NT) and intracytoplasmic nuclear injection (ICI-NT). Stepwise alterations in nuclear remodeling events and in mRNA abundances, throughput and efficiency of cloned embryo development and cell allocation of the resulted blastocysts were assessed. Early signs of nuclear remodeling were observed as soon as 2 h post-reconstitution (hpr) for fusion-based methods of nuclear transfer (ZI-NT and ZF-NT) but were not observable until 4 hpr with the ICI-NT method. The relative mRNA abundances ofHSP90AA1(HSP90),NPM2andATPasegenes were not affected by i) presence or absence of zona, ii) oocyte enucleation method and iii) nuclear transfer method. After reconstitution, however, the relative mRNA contents ofPOU5F1(OCT4) with the ZI-NT and ZF-NT methods and ofPAPOLA(PAP) with ZF-NT were significantly lower than those for the ICI-NT method. Zona removal doubled the throughput of cloned blastocyst development for the ZF-NT technique compared with ZI-NT and ICI-NT. Cleavage rate was not affected by the SCNT protocol, whereas blastocyst yield rate in ICI-NT technique (17.0±1.0%) was significantly (P<0.05; ANOVA) higher than in ZF-NT (7.1±1.5%) but not in the ZI-NT group (11.2±3.3%). Despite the similarities in total cell number, SCNT protocol changed the distribution of cells in the blastocysts, as ZF-NT-cloned blastocysts had significantly smaller inner cell mass than ZI-NT. These results indicate that technical aspects of cloning may result in the variety of cloning phenotypes.

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Zinc supplementation during in vitro embryo culture increases inner cell mass and total cell numbers in bovine blastocysts1

Journal of Animal Science ◽

10.1093/jas/skz351 ◽

2019 ◽

Vol 97 (12) ◽

pp. 4946-4950 ◽

Cited By ~ 2

Author(s):

Lydia K Wooldridge ◽

Madison E Nardi ◽

Alan D Ealy

Keyword(s):

Embryo Culture ◽

Zinc Supplementation ◽

Inner Cell Mass ◽

Cell Mass ◽

Cell Number ◽

Retention Rates ◽

Bovine Embryo ◽

Blastocyst Development ◽

Cell Numbers

Abstract Deficiencies in current embryo culture media likely contribute to the poor blastocyst development rates and pregnancy retention rates for in vitro produced (IVP) bovine embryos. Of special concern is the lack of micronutrients in these media formulations. One micronutrient of interest is zinc, an essential trace element involved with various enzyme and transcription factor activities. The objective of this work was to describe whether zinc sulfate supplementation during in vitro embryo culture affects bovine embryo development and blastomere numbers. Either 0, 2, 20, or 40 µM zinc sulfate was supplemented to presumptive zygotes cultured in synthetic oviductal fluid containing AAs and bovine serum albumin for 8 d. None of the treatments affected cleavage rates. Percentage of blastocysts on days 7 and 8 postfertilization was not affected by supplementing 2 or 20 µM zinc but were reduced (P < 0.05) with 40 µM zinc. In blastocysts harvested on day 8, inner cell mass (ICM) and total cell number were increased (P < 0.05) with 2 µM zinc supplementation but not with the other zinc concentrations. Numbers of trophectoderm cells were not affected by zinc treatment. In conclusion, supplementing zinc during bovine embryo culture did not impact blastocyst development but improved ICM cell numbers. This improvement in ICM cell number may have implications for improved pregnancy retention rates after IVP embryo transfer as smaller ICM sizes are associated with poor pregnancy success in cattle.

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Melatonin Alleviates the Toxicity of High Nicotinamide Concentrations in Oocytes: Potential Interaction with Nicotinamide Methylation Signaling

Oxidative Medicine and Cellular Longevity ◽

10.1155/2021/5573357 ◽

2021 ◽

Vol 2021 ◽

pp. 1-16

Author(s):

Marwa El-Sheikh ◽

Ahmed Atef Mesalam ◽

Seok-Hwan Song ◽

Jonghyeok Ko ◽

Il-Keun Kong

Keyword(s):

Inner Cell Mass ◽

Active Form ◽

Cell Mass ◽

Potential Interaction ◽

Developmental Competence ◽

Ros Scavenging ◽

Vitamin B3 ◽

Blastocyst Development ◽

Preimplantation Embryo Development

Despite the numerous studies on melatonin and nicotinamide (NAM, the active form of vitamin B3), the linkage between these two biomolecules in the context of signaling pathways regulating preimplantation embryo development has not yet been investigated. In this study, we used bovine oocyte model to elucidate the effect of melatonin on the developmental competence of oocytes under the stress of high NAM concentrations. Results showed that NAM (20 mM) administration during in vitro maturation (IVM) significantly reduced oocyte maturation and actin distribution, while induced reactive oxygen species (ROS) accumulation and mitochondrial dysfunction, the multiple deleterious effects that were alleviated by melatonin (10−7 M). The RT-qPCR and/or immunofluorescence showed upregulation of the apoptosis (Caspase-3, Caspase-9, and BAX), autophagy (Beclin-1, LC3A, LC3B, ATG7, LAMP1, and LAMP2), cell cycle (P21, P27, and P53), and DNA damage (COX2 and 8-OxoG) specific markers in oocytes matured under NAM treatment, compared to NAM-melatonin dual-treated and the untreated ones. In addition, the total cleavage and blastocyst development rate, as well as the total number of cells and the inner cell mass (ICM) per blastocyst, were reduced, while DNA fragmentation was induced, in the group of NAM sole treatment than NAM-melatonin cotreatment and control. Inspecting the underlying mechanisms behind NAM-associated toxicity revealed an increase in transcription pattern of NAM methylation (NNMT and AHCY) genes in NAM-treated oocytes while the opposite profile was observed upon melatonin supplementation. In conclusion, to our knowledge, this is the first study reporting that melatonin can protect oocytes and embryos from NAM-induced injury through its ROS-scavenging activity together with potential interaction with NAM methylation signaling.

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Enhancement of histone acetylation by trichostatin A during in vitro fertilization of bovine oocytes affects cell number of the inner cell mass of the resulting blastocysts

Zygote ◽

10.1017/s0967199409005279 ◽

2009 ◽

Vol 17 (3) ◽

pp. 209-215 ◽

Cited By ~ 7

Author(s):

Shuntaro Ikeda ◽

Atsuhiro Tatemizo ◽

Daisaku Iwamoto ◽

Shunji Taniguchi ◽

Yoichiro Hoshino ◽

...

Keyword(s):

Histone Acetylation ◽

Inner Cell Mass ◽

Trichostatin A ◽

Cell Mass ◽

Reproductive Technologies ◽

Cell Number ◽

Preimplantation Embryos ◽

Blastocyst Development ◽

Bovine Oocytes

SummaryHistone acetylation is one of the major mechanisms of epigenetic reprogramming of gamete genomes after fertilization to establish a totipotent state for normal development. In the present study, the effects of trichostatin A (TSA), an inhibitor of histone deacetylase, during in vitro fertilization (IVF) of bovine oocytes on subsequent embryonic development were investigated. Cumulus-enclosed oocytes obtained from slaughterhouse bovine ovaries were matured in vitro and subjected to IVF in a defined medium supplemented with 0 (control), 5, 50, and 500 nM TSA for 18 h. After IVF, presumptive zygotes were cultured in modified synthetic oviductal fluid (mSOF) medium until 168 h postinsemination (hpi). Some oocytes were immunostained using antibody specific for histone H4-acetylated lysine 5 at 10 hpi. Cleavage, blastocyst development and cell number of inner cell mass (ICM) and trophectoderm (TE) of blastocysts were assessed. TSA treatment enhanced histone acetylation that was prominent in decondensed sperm nuclei. TSA did not affect the postfertilization cleavage, blastocyst rates, and TE cell number. However, it significantly enhanced ICM cell number (p < 0.05). These results indicate that TSA treatment during IVF of bovine oocytes does not affect blastocyst development but alters the cell number of ICM, suggesting that overriding epigenetic modification of the genome during fertilization has a carryover effect on cell proliferation and differentiation in preimplantation embryos. Thus, further environmental quality controls in assisted reproductive technologies are needed in terms of factors which affect chromatin remodelling.

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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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