Myo-Inositol Safety in Pregnancy: From Preimplantation Development to Newborn Animals

Myo-inositol (myo-Ins) has a physiological role in mammalian gametogenesis and embryonic development and a positive clinical impact on human medically assisted reproduction. We have previously shown that mouse embryo exposure to myo-Ins through preimplantation developmentin vitroincreases proliferation activity and blastocyst production, representing an improvement in culture conditions. We have herein investigated biochemical mechanisms elicited by myo-Ins in preimplantation embryos and evaluated myo-Ins effects on postimplantation/postnatal development. To this end naturally fertilized embryos were culturedin vitroto blastocyst in the presence or absence of myo-Ins and analyzed for activation of the PKB/Akt pathway, known to modulate proliferation/survival cellular processes. In parallel, blastocyst-stage embryos were transferred into pseudopregnant females and allowed to develop to term and until weaning. Results obtained provide evidence that myo-Ins induces cellular pathways involving Akt and show that (a) exposure of preimplantation embryos to myo-Ins increases the number of blastocysts available for uterine transfer and of delivered animals and (b) the developmental patterns of mice obtained from embryos cultured in the presence or absence of myo-Ins, up to three weeks of age, overlap. These data further identify myo-Ins as a possibly important supplement for human preimplantation embryo culture in assisted reproduction technology.

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Developmental and molecular correlates of bovine preimplantation embryos

Reproduction ◽

10.1530/rep.1.01021 ◽

2006 ◽

Vol 131 (5) ◽

pp. 895-904 ◽

Cited By ~ 60

Author(s):

Hakan Sagirkaya ◽

Muge Misirlioglu ◽

Abdullah Kaya ◽

Neal L First ◽

John J Parrish ◽

...

Keyword(s):

Gene Expression ◽

Dna Methyltransferase ◽

Expression Profiles ◽

Expression Patterns ◽

Blastocyst Stage ◽

Culture Conditions ◽

Preimplantation Embryos ◽

Developmental Potential

Expression of embryonic genes is altered in different culture conditions, which influence developmental potential both during preimplantation and fetal development. The objective of this study was to define the effects of culture conditions on: bovine embryonic development to blastocyst stage, blastocyst cell number, apoptosis and expression patterns of a panel of developmentally important genes. Bovine embryos were culturedin vitroin three culture media containing amino acids, namely potassium simplex optimization medium (KSOMaa), Charles Rosenkrans 1 (CR1aa) and synthetic oviductal fluid (SOFaa). Apoptosis in blastocysts was determined by TUNEL assay and expression profiles of developmentally important genes were assayed by real-time PCR.In vivo-produced bovine blastocysts were used as controls for experiments determining gene expression patterns. While the cleavage rates did not differ, embryos cultured in SOFaa had higher rates of development to blastocyst stage (P< 0.05). Mean cell numbers and percentages of apoptotic cells per blastocyst did not differ among the groups. Expression of the heat shock protein 70 (Hsp70) gene was significantly up-regulated in both CR1aa and KSOMaa when compared with SOFaa (P< 0.001). DNA methyltransferase 3a (Dnmt3a) expression was higher in embryos cultured in CR1aa than in those cultured in SOFaa (P< 0.001). Expression of interferon tau (IF-τ) and insulin-like growth factor II receptor (Igf-2r) genes was significantly up-regulated in KSOMaa when compared with CR1aa (P< 0.001). Gene expression did not differ betweenin vivo-derived blastocysts and theirin vitro-derived counterparts. In conclusion, SOFaa supports higher development to blastocyst stage than KSOMaa and CR1aa, and the culture conditions influence gene expression.

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Involvement of Nlrp9a/b/c in mouse preimplantation development

Reproduction ◽

10.1530/rep-19-0516 ◽

2020 ◽

Vol 160 (2) ◽

pp. 181-191 ◽

Cited By ~ 2

Author(s):

Satoko Kanzaki ◽

Shiori Tamura ◽

Toshiaki Ito ◽

Mizuki Wakabayashi ◽

Koji Saito ◽

...

Keyword(s):

Asymmetric Cell Division ◽

Culture Conditions ◽

Preimplantation Development ◽

Preimplantation Embryos ◽

Dependent Manner ◽

Blastocyst Development ◽

Triple Mutant ◽

Cell Stage

Nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain-containing proteins (NRLPs) are central components of the inflammasome. Accumulating evidence has shown that a reproductive clade of NRLPs is predominantly expressed in oocyte to cleavage stage embryos and participates in mammalian preimplantation development as a component of a multiprotein complex known as the subcortical maternal complex (SCMC). Nlrp9s belong to the reproductive class of NLRPs; Nlrp9b is unique in acting as an inflammasome against rotavirus in intestines. Here we generated mice carrying mutations in all three members of the Nlrp9a/b/c gene (Nlrp9 triple mutant (TMut) mice). When crossed with WT males, the Nlrp9 TMut females were fertile, but deliveries with fewer pups were increased in the mutants. Consistent with this, blastocyst development was retarded and lethality to the preimplantation embryos increased in the Nlrp9 TMut females in vivo. Under in vitro culture conditions, the fertilized eggs from the Nlrp9 TMut females exhibited developmental arrest at the two-cell stage, accompanied by asymmetric cell division. By contrast, double-mutant (DMut) oocytes (any genetic combination) did not exhibit the two-cell block in vitro, showing the functional redundancy of Nlrp9a/b/c. Finally, Nlrp9 could bind to components of the SCMC. These results show that Nlrp9 functions as an immune or reproductive NLRP in a cell-type-dependent manner.

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Bovine preimplantation embryos with silenced nucleophosmin mRNA are able to develop until the blastocyst stage

Reproduction ◽

10.1530/rep-12-0033 ◽

2012 ◽

Vol 144 (3) ◽

pp. 349-359 ◽

Cited By ~ 3

Author(s):

Tereza Toralová ◽

Veronika Benešová ◽

Kateřina Vodičková Kepková ◽

Petr Vodička ◽

Andrej Šušor ◽

...

Keyword(s):

Ribosome Biogenesis ◽

Blastocyst Stage ◽

Preimplantation Development ◽

Developmental Competence ◽

Preimplantation Embryos ◽

Bovine Embryo ◽

Cell Stage ◽

Embryonic Genome ◽

Genome Activation

This study was conducted to investigate the effect of silencing nucleophosmin in the development of in vitro-produced bovine embryos. Nucleophosmin is an abundant multifunctional nucleolar phosphoprotein that participates, for example, in ribosome biogenesis or centrosome duplication control. We showed that although the transcription of embryonic nucleophosmin started already at late eight-cell stage, maternal protein was stored throughout the whole preimplantation development and was sufficient for the progression to the blastocyst stage. At the beginning of embryogenesis, translation occurs on maternally derived ribosomes, the functionally active nucleoli emerge during the fourth cell cycle in bovines. We found that nucleophosmin localisation reflected the nucleolar formation during bovine preimplantation development. The protein was detectable from the beginning of embryonic development. Before embryonic genome activation, it was dispersed throughout the nucleoplasm. The typical nucleolar localisation emerged with the formation of active nucleoli. At the blastocyst stage, nucleophosmin tended to localise especially to the trophectoderm. To see for how long is maternal nucleophosmin preserved, we silenced the nucleophosmin mRNA using RNA interference approach. Although a large portion of nucleophosmin was degraded in embryos with silenced nucleophosmin mRNA, an amount sufficient for normal development was preserved and we detected only a temporal delay in nucleophosmin relocalisation to nucleoli. Moreover, we observed no defects in nuclear shape or cytoskeleton previously found in somatic cells and only a non-significant decrease in embryonic developmental competence. Thus, our results show that the preserved amount of maternal nucleophosmin is sufficient for preimplantation development of bovine embryo.

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Biological and Clinical Significance of Mosaicism in Human Preimplantation Embryos

Journal of Developmental Biology ◽

10.3390/jdb9020018 ◽

2021 ◽

Vol 9 (2) ◽

pp. 18

Author(s):

Ioanna Bouba ◽

Elissavet Hatzi ◽

Paris Ladias ◽

Prodromos Sakaloglou ◽

Charilaos Kostoulas ◽

...

Keyword(s):

Assisted Reproduction ◽

Basic Research ◽

Blastocyst Stage ◽

Preimplantation Embryos ◽

New Approach ◽

Assisted Reproduction Technology ◽

Clinical Utilization ◽

Art Practices ◽

In Utero Development ◽

Human Preimplantation Embryos

Applications and indications of assisted reproduction technology are expanding, but every new approach is under scrutiny and thorough consideration. Recently, groups of assisted reproduction experts have presented data that support the clinical use of mosaic preimplantation embryos at the blastocyst stage, previously excluded from transfer. In the light of published contemporary studies, with or without clinical outcomes, there is growing evidence that mosaic embryos have the capacity for further in utero development and live birth. Our in-depth discussion will enable readers to better comprehend current developments. This expansion into the spectrum of ART practices requires further evidence and further theoretical documentation, basic research, and ethical support. Therefore, if strict criteria for selecting competent mosaic preimplantation embryos for further transfer, implantation, fetal growth, and healthy birth are applied, fewer embryos will be excluded, and more live births will be achieved. Our review aims to discuss the recent literature on the transfer of mosaic preimplantation embryos. It also highlights controversies as far as the clinical utilization of preimplantation embryos concerns. Finally, it provides the appropriate background to elucidate and highlight cellular and genetic aspects of this novel direction.

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P–804 Morphokinetic development by time-lapse imaging of conceptuses generated from artificial oocytes

Human Reproduction ◽

10.1093/humrep/deab130.803 ◽

2021 ◽

Vol 36 (Supplement_1) ◽

Author(s):

A Trout ◽

P Xie ◽

A Petrini ◽

Z Rosenwaks ◽

G Palermo

Keyword(s):

Somatic Cell ◽

Histone Deacetylase ◽

Nuclear Transfer ◽

Cumulus Cells ◽

Time Lapse ◽

Culture Conditions ◽

Preimplantation Development ◽

Preimplantation Embryos ◽

Blastocyst Development ◽

Time Lapse Imaging

Abstract Study question What are the ideal culture conditions to enhance full preimplantation development of embryos generated by FVB somatic cell haploidization (SCH) in the mouse model? Summary answer The presence of a histone deacetylase inhibitor yielded the best morphokinetic development of expanded blastocysts generated by FVB SCH, comparable to control blastocysts. What is known already Various culture conditions and medium supplements have been proposed to promote preimplantation development of embryos generated by SCH, including supplementation with trichostatin A (TSA), fasudil, scriptaid, and RAD–51 stimulatory compound–1 (RS–1). TSA and scriptaid, both histone-deacetylase inhibitors, have been found to improve embryo development following nuclear transfer by enhancing histone acetylation and cellular reprogramming. Additionally, fasudil is a Rho-associated kinase inhibitor that has been shown to reduce apoptosis and promote cell proliferation. Finally, RS–1 stimulates RAD51 activity, which promotes the repair of DNA damage and increases the efficacy of somatic cell reprogramming. Study design, size, duration B6D2F1 mouse metaphase II (MII) oocytes underwent enucleation and nuclear transfer, or were ICSI inseminated serving as controls. Reconstituted oocytes showing development of a meiotic-like spindle demonstrated successful SCH, and were ICSI inseminated. SCH conceptuses were cultured in one of three groups: KSOM, KSOM supplemented with TSA (TSA), or KSOM supplemented with fasudil, scriptaid, and RS–1 (Cocktail). ICSI controls (ICSIC) were cultured in KSOM medium. Fertilization and full preimplantation development were compared among all groups. Participants/materials, setting, methods Ooplasts were generated from MII oocytes by removing spindle complexes under OosightÔ visualization and cytochalasin B exposure. A single FVB mouse cumulus cell was transferred into the perivitelline space and fused with the ooplast, facilitated by Sendai virus. Reconstructed oocytes with novel pseudo-meiotic spindles underwent piezo-ICSI and were cultured in different media conditions in a time-lapse imaging system up to 96h. TSA and Cocktail embryos had media changed to regular KSOM 10 hours after insemination. Main results and the role of chance A total of 274 B6D2F1 MII oocytes were enucleated, resulting in a 95.9% survival rate. All ooplasts survived nuclear transfer and 62.1% successfully haploidized after 2 hours. ICSIC and reconstituted SCH oocytes survived piezo-ICSI at rates of 81.5% and 57.0%, respectively (P < 0.01). SCH embryos were then allocated into KSOM, TSA supplied, and Cocktail media. Fertilization rates for ICSIC, KSOM, and TSA embryos were 92.4%, 90.7%, and 94.4%, respectively, while the rate for embryos cultured in Cocktail was only 71.9% (P < 0.03). While embryos cultured in Cocktail had a comparable 2-cell timing to ICSIC, embryos in TSA reached developmental milestones with a closer timing to the ICSIC, having minor delays at the 3-, 4-, and 6-cell stages (P < 0.05). KSOM- and Cocktail-cultured embryos were delayed at most of the stages (P < 0.01), except for the two-pronuclei appearance. Although the TSA group displayed the best embryo developmental pattern, the final rate of blastocyst development was somewhat homogeneous with rates of 15.4%, 23.5%, and 13.0% for the KSOM, TSA, and Cocktail groups, respectively (P < 0.001), and remarkably lower than the ICSIC (81.6%). Limitations, reasons for caution Although live pups have been obtained using BDF cumulus cells, embryos generated by FVB cumulus cells show a remarkably lower blastocyst development, but maintain morphokinetic characteristics similar to ICSIC in the presence of TSA. Wider implications of the findings: While using different strains to enhance genetic variance, the morphokinetic analysis of preimplantation embryos in ideal culture conditions is paramount to the progress of neogametogenesis. The implementation of this technique may soon help create genotyped oocytes for women with compromised ovarian reserve. Trial registration number N/A

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Growth and viability of human blastocysts in vitro

Reproductive Medicine Review ◽

10.1017/s0962279900000351 ◽

2000 ◽

Vol 8 (3) ◽

pp. 241-287 ◽

Cited By ~ 7

Author(s):

GM Jones

Keyword(s):

Blastocyst Stage ◽

Culture Conditions ◽

Cleavage Stage ◽

Embryo Viability ◽

Early Cleavage ◽

Uterine Endometrium ◽

Stage Of Development ◽

Uterine Lavage

The transfer of a blastocyst established the first human clinical pregnancy following in vitro fertilization (IVF). Nine years later Cohen et al. reported pregnancies resulting from the transfer of cryopreserved human blastocysts. However, it was another six years before the first report of births resulting from the transfer of human blastocysts produced in vitro appeared in the medical literature. In the intervening period clinics have opted to transfer embryos at the early cleavage stage to the uterus, despite the fact that in vivo the embryo does not enter the uterus until two to three days later at the morula to blastocyst stage of development. The viability and potential for implantation of blastocysts is high, as indicated by the finding that more than 60% of in-vivo-derived blastocysts, recovered by uterine lavage following artificial insemination of fertile donors, implant and develop into viable fetuses when transferred to recipients. This is in stark contrast to the 10–20% of in-vitro-produced embryos transferred at the early cleavage stage of development that result in a live-birth. This reduction in viability following transfer of in-vitro-derived early cleavage stage embryos may have several possible explanations: (1) a failure of implantation due to poor synchronization between the embryo and the uterine endometrium; (2) a hostile environment in the uterus for early cleavage stage embryos; (3) sub-optimal in vitro culture conditions which result in a reduction in embryo viability; (4) the assumption that all oocytes retrieved in an IVF cycle have an equal ability to develop into viable embryos; and (5) the failure to identify the most viable embryo in a cohort. Certainly, improving culture conditions and laboratory techniques for developing high quality blastocysts routinely in vitro will not only address many of the above questions but will also improve the quality and viability of earlier stages of embryo development.

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The roles of pyruvate, lactate and glucose during preimplantation development of embryos from F1 hybrid mice in vitro

Development ◽

10.1242/dev.112.1.99 ◽

1991 ◽

Vol 112 (1) ◽

pp. 99-105 ◽

Cited By ~ 2

Author(s):

J.J. Brown ◽

D.G. Whittingham

Keyword(s):

Inbred Mouse ◽

Blastocyst Stage ◽

Preimplantation Development ◽

F1 Hybrids ◽

Mouse Strains ◽

F1 Hybrid ◽

Morula Stage ◽

Lactate And Pyruvate ◽

Hybrid Mice

Embryos of certain inbred mouse strains, and their F1 hybrids, are able to develop from the 1-cell to blastocyst stage in simple chemically defined media containing lactate (L), pyruvate (P) and glucose (G). The individual roles of these substrates in supporting complete preimplantation development in vitro was examined with 1-cell F2 embryos from B6CBF1 hybrid mice. Embryos collected between 26 and 27 h post hCG were cultured in medium containing L, P, LP or LPG. After 50 h in culture, the proportions developing to the morula stage were 1%, 83%, 94% and 100%, respectively. In combination, lactate and pyruvate appeared to act synergistically and both the rate and level of development to the morula stage were unaffected by the absence of glucose. After a further 46 h in culture, only the embryos grown in the presence of glucose developed into blastocysts. In LP medium, embryos arrested at the compacted morula stage late on day 3 of development. As culture continued in the absence of glucose, embryos decompacted (approximately 82 h post hCG) and subsequently degenerated. Exposure to medium containing glucose for the first, second or third 24 h period in culture was sufficient to support the morula-to-blastocyst transition. Glucose still supported this transition when embryos were transferred to LPG medium 3 h after the completion of compaction (76 h post hCG), but was ineffective 6 h later (82 h post hCG) once decompaction had commenced. We conclude that lactate and pyruvate together are able to support normal development of 1-cell F2 embryos to the morula stage in vitro, but that glucose is an essential component of the culture medium for development to the blastocyst stage.

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86 BIRTH OF HEALTHY CALVES AFTER INTRAFOLLICULAR OOCYTE TRANSFER

Reproduction Fertility and Development ◽

10.1071/rdv27n1ab86 ◽

2015 ◽

Vol 27 (1) ◽

pp. 136

Author(s):

M. Hoelker ◽

A. Kassens ◽

E. Held ◽

C. Wrenzycki ◽

U. Besenfelder ◽

...

Keyword(s):

Lipid Content ◽

Ex Vivo ◽

Survival Rates ◽

Blastocyst Stage ◽

Culture Conditions ◽

In Vitro Production ◽

Negative Effects ◽

Uterine Flushing

The in vitro production (IVP) of bovine embryos is a well-established technique that has been available for nearly 20 years. However, there remain major differences between IVP-derived blastocysts and their in vivo-derived counterparts. Many studies have pointed out that most of these differences are due to the in vitro developmental environment. To circumvent these negative effects due to in vitro culture conditions, a new method – intrafollicular oocyte transfer (IFOT) – was established in the present study. Using modified ovum pick-up (OPU) equipment, in vitro-matured oocytes derived from slaughterhouse ovaries were injected into the dominant preovulatory follicle of synchronised heifers (follicular recipients) enabling subsequent ovulation, in vivo fertilization, and in vivo development. A total of 810 in vitro-matured oocytes were transferred into 14 heifers. Subsequently, 222 embryos (27.3%) were recovered after uterine flushing at Day 7. Based on the number of cleaved embryonic stages, 64.2% developed to the blastocyst stage, which did not differ from the IVP-derived embryos (58.2%). Interestingly, lipid content of IFOT-derived blastocysts did not differ from the fully in vivo-produced embryos, whereas IVP-derived blastocysts showed significantly higher lipid droplet accumulation compared with fully in vivo-derived and IFOT-derived blastocysts (P < 0.05). Accordingly, IFOT blastocysts showed significantly higher survival rates after cryopreservation than complete IVP-derived embryos (77% v. 10%), which might be attributed to a lower degree of lipid accumulation. In agreement, transfer of frozen-thawed IFOT blastocysts to synchronized recipients (uterine recipients) resulted in much higher pregnancy rates compared with transfer of IVP-derived blastocysts (42.1 v. 13.8%) but did not differ from frozen-thawed ex vivo blastocysts (52.4%). Of these presumed IFOT pregnancies, 7 went to term, and microsatellite analysis confirmed that 5 calves were indeed derived from IFOT, whereas 2 were caused by fertilization of the follicular recipient's own oocyte after AI. Taken together, IFOT-derived blastocysts closely resemble in vivo-derived blastocysts, confirming earlier suggestions that the ability to develop to the blastocyst stage is already determined in the matured oocyte, whereas the quality in terms of lipid content and survival rate after cryopreservation is affected by the environment thereafter. However, to the best of our knowledge, this is the first study reporting healthy calves after intrafollicular transfer of in vitro-matured oocytes.

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180 IDENTIFICATION AND QUANTIFICATION OF DIFFERENTIALLY REPRESENTED TRANSCRIPTS IN PREIMPLANTATION BOVINE EMBRYOS

Reproduction Fertility and Development ◽

10.1071/rdv20n1ab180 ◽

2008 ◽

Vol 20 (1) ◽

pp. 169 ◽

Cited By ~ 1

Author(s):

C. E. McHughes ◽

G. K. Springer ◽

L. D. Spate ◽

R. Li ◽

R. J. Woods ◽

...

Keyword(s):

Relative Abundance ◽

Nuclear Transfer ◽

Developmental Stages ◽

Reproductive Technologies ◽

Blastocyst Stage ◽

Preimplantation Embryos ◽

Cell Stage ◽

Production Techniques

Identification of transcripts that are present at key development stages of preimplantation embryos is critical for a better understanding of early embryogenesis. To that end, this project had two goals. The first was to characterize the relative abundance of multiple transcripts during several developmental stages, including metaphase II-stage oocytes (MPII), and 2-cell-stage (2-cell), precompact morula (PCM), and in vitro-produced blastocyst-stage (IVTBL) embryos. The second was to characterize differences in the relative abundance of transcripts present in in vivo- (IVVBL), in vitro-, and nuclear transfer-produced (NTBL) blastocysts. It was our hypothesis that the identification of differentially represented transcripts from these stages would reveal not only developmentally important genes, but also genes that might be aberrantly expressed due to embryo production techniques. Individual clusters from a large bovine EST project (http://genome.rnet.missouri.edu/Bovine/), which focused on female reproductive tissues and embryos, were compared using Fisher's exact test weighted by number of transcripts per tissue by gene (SAS PROC FREQ; SAS Institute, Inc., Cary, NC, USA). Of the 3144 transcripts that were present during embryogenesis, 125 were found to be differentially represented (P < 0.01) in at least one pairwise comparison (Table 1). Some transcripts found to increase in representation from the MPII to the 2-cell stage include protein kinases, PRKACA and CKS1, as well as the metabolism-related gene, PTTG1. These same transcripts were also found to decrease in representation from the 2-cell to the PCM stage. RPL15 (translation) and FTH1 (immune function) were both more highly represented in the PCM than in the 2-cell stage. From PCM to IVTBL, we saw an increase in RPS11, another translation-related transcript. When comparing blastocyst-stage embryos from different production techniques, several transcripts involved in energy production (e.g., COX7B and COX8A) were found to be more highly represented in the NTBL than in the IVTBL. COX8A was also more highly represented in the IVVBL than in the IVTBL. By investigating these differentially represented transcripts, we will be able to better understand the developmental implications of embryo manipulation. We may also be able to better develop reproductive technologies that lead to in vitro- and nuclear transfer-derived embryos which more closely follow a normal program of development. Table 1. Differentially represented transcripts between developmental stages

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123 OPTIMIZATION OF CULTURE CONDITIONS FOR IN-VITRO-PRODUCED BOVINE EMBRYOS TO ENHANCE BLASTOCYST YIELD AND SURVIVAL FOLLOWING VITRIFICATION

Reproduction Fertility and Development ◽

10.1071/rdv20n1ab123 ◽

2008 ◽

Vol 20 (1) ◽

pp. 142

Author(s):

J. Block ◽

L. Bonilla ◽

P. J. Hansen

Keyword(s):

Bovine Serum ◽

Culture System ◽

Blastocyst Stage ◽

Culture Conditions ◽

Expansion Rate ◽

Hatching Rate ◽

Static System ◽

Cleavage Rate ◽

Static Culture

Objectives were to identify modifications in culture conditions that improve blastocyst yield and cryosurvival. The objective of Experiment 1 was to determine effects of sequential culture and fructose on blastocyst yield. Embryos were cultured in modified SOF with 4 mg mL–1 bovine serum albumin (BSA) and 1.0 mm alanyl-glutamine in 5% (v/v) oxygen with or without 0.5 mm fructose in either a static or sequential culture system. For the sequential system, embryos >4 cells were selected and placed in fresh drops of medium at day 3 after insemination. Culture system and fructose did not affect cleavage rate or the proportion of embryos >4 cells on day 3. The proportion of >4 cell embryos that developed to the blastocyst stage was higher (P < 0.04) for static culture than for sequential culture (41.6 � 1.2 v. 30.6 � 1.2%) and there was a trend (P = 0.1) for the proportion of oocytes that developed to blastocyst at day 7 to be greater for static culture (26.8 � 1.2 v. 20.9 � 1.2%). In both culture systems, fructose increased (P < 0.03) blastocyst yield from embryos >4 cells (32.5 � 1.2 v. 39.7 � 1.2%) and tended (P < 0.06) to improve blastoocyst yield from oocytes (21.8 � 1.1 v. 25.3 � 1.1%). The objective of Exp. 2 was to evaluate whether blastocyst yield and survival after cryopreservation would be enhanced by BSA and hyaluronan. Embryos produced in vitro were cultured in 5% oxygen using a static system of modified SOF with or without 4 mg mL–1 BSA and with 0, 0.1, 0.5, or 1 mg mL–1 hyaluronan. Blastocyst and expanded blastocyst stage embryos on day 7 were vitrified (Campos-Chillon LF et al. 2006 Theriogenology 65, 1200–1214). Vitrified embryos were thawed and then cultured for 72 h in modified SOF containing 10% (v/v) fetal bovine serum and 50 µm dithiothreitol. Re-expansion rate was recorded at 24 and 48 h, and the proportion of embryos that hatched by 72 h of culture was recorded. There was no effect of BSA or hyaluronan on cleavage rate. Blastocyst yield from oocytes was increased (P < 0.0005) by BSA (15.3 � 1.1 v. 20.9 � 1.1%). Addition of hyaluronan at 1 mg mL–1 improved (P < 0.04) blastocyst yield (16.2 � 1.7 v. 21.2 � 1.7%), but there was no effect at lower concentrations. There were no interactions between BSA and hyaluronan. Re-expansion rate at 24 and 48 h after thawing was reduced (P < 0.007) by BSA (24 h: 39.1 � 3.6 v. 17.0 � 3.6%; 48 h: 45.6 � 3.8 v. 18.7 � 3.7%), and BSA tended (P < 0.06) to reduce hatching rate at 72 h (22.3 � 3.0 v. 9.8 � 3.0%). Treatment of embryos with hyaluronan did not affect re-expansion rate at 24 h but tended (P < 0.08) to increase re-expansion at 48 h. Moreover, hyaluronan increased (P < 0.05) hatching rate at 72 h after thawing (0 mg mL–1 – 9.8 � 4.2; 0.1 mg mL–1 – 16.9 � 4.5; 0.5 mg mL–1 – 23.4 � 4.1; 1.0 mg mL–1 – 14.2 � 4.1%). In conclusion, blastocyst yield was improved by addition of fructose, BSA, and hyaluronan to culture medium and by use of a static culture system. Hyaluronan also enhanced cryosurvival, but BSA was detrimental to blastocyst survival after vitrification. Support: USDA NRI 2006-55203-17390, BARD US-3551-04.

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