Advanced Paternal Age Negatively Impacts In Vivo Fertilization and In Vitro Blastocyst Development.

Capsule Male aging effects on aneuploidy rates in embryos. Objective Paternal age is associated with decreasing sperm quality; however, it is unknown if it influences chromosomal abnormalities in embryos. The objective of this study is to evaluate if the aneuploidy rates in embryos are affected by advanced paternal age. Methods A total of 286 embryos, obtained from 32 in vitro fertilization/intracytoplasmic sperm injection cycles with donated oocytes in conjunction with preimplantation genetic diagnosis, were allocated according to paternal age in three groups: Group A: ≤39 years (n = 44 embryos); Group B: 40-49 years (n = 154 embryos); and Group C: ≥50 years (n = 88 embryos). Fertilization rates, embryo quality at day 3, blastocyst development, and aneuploidy embryo rates were then compared. Results There was no difference in the seminal parameters (volume, concentration, and motility) in the studied groups. Fertilization rate, percentages of zygotes underwent cleavage, and good quality embryos on day 3 were similar between the three evaluated groups. The group of men ≥50 years had significantly more sperm with damaged DNA, low blastocyst development rate, and higher aneuploidy rates in embryos compared to the other two evaluated groups ( P < 0.05). Conclusions Our findings suggest that advanced paternal age increases the aneuploidy rates in embryos from donated oocytes, which suggests that genetic screening is necessary in those egg donor cycles with sperm from patients >50 years old.

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Sheep antiluteolytic interferon: cDNA sequence and analysis of mRNA levels

Journal of Molecular Endocrinology ◽

10.1677/jme.0.0020065 ◽

1989 ◽

Vol 2 (1) ◽

pp. 65-70 ◽

Cited By ~ 40

Author(s):

H.J. Stewart ◽

S.H.E. McCann ◽

A.J. Northrop ◽

G.E. Lamming ◽

A.P.F. Flint

Keyword(s):

Amino Acid ◽

Northern Blotting ◽

In Vitro Translation ◽

Major Constituent ◽

Interferon Α ◽

Mrna Levels ◽

Blastocyst Development ◽

Cdna Sequences

ABSTRACT A cloned cDNA has been isolated by probing a sheep blastocyst cDNA library using a synthetic oligonucleotide representing the N-terminal amino acid sequence of the antiluteolytic protein, ovine trophoblast protein-1. Sequence analysis of the cDNA confirms the 70% homology between the antiluteolysin and the interferon-α family of proteins; however, the sequence reported here differs at several points from previously reported amino acid and cDNA sequences for the antiluteolysin. In-vitro translation of day-16 poly(A)+ RNA indicated that antiluteolysin mRNA is a major constituent of total mRNA at this stage of blastocyst development, and Northern blotting confirmed that antiluteolysin mRNA production occurred between days 13 and 22 after oestrus. This is consistent with the stage at which embryonic extracts are antiluteolytic on administration in vivo. These and other data confirm that the ovine trophoblast antiluteolysin is an interferon, and suggest that at least five isoforms of this protein may exist.

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173 EFFECT OF CULTURE SYSTEM FOR IVM-IVF PIG EMBRYOS ON THE ICMS ABILITY TO PRODUCE OUTGROWTHS FOR EMBRYONIC STEM CELL DERIVATION

Reproduction Fertility and Development ◽

10.1071/rdv17n2ab173 ◽

2005 ◽

Vol 17 (2) ◽

pp. 237 ◽

Cited By ~ 3

Author(s):

G. Lazzari ◽

I. Lagutina ◽

G. Crotti ◽

P. Turini ◽

S. Colleoni ◽

...

Keyword(s):

Cell Mass ◽

Embryonic Stem ◽

Farm Animals ◽

Es Cell ◽

Blastocyst Formation ◽

Blastocyst Development ◽

In Vivo Culture

Attempts to derive true embryonic stem cells in large farm animals rely on the supply of good quality embryos. In these species, including the pig, pre-implantation-stage embryos can be produced by in vitro techniques from slaughterhouse ovaries. The objective of this study was to evaluate the ability of the inner cell masses (ICMs) of pig embryos, produced in vitro by different methods, to provide viable initial outgrowths of ICM cells that could be subsequently subcultured and expanded. Porcine oocytes were recovered from slaughtered donors and matured in vitro for 40–44 h in DMEM-F12 supplemented with 10% FCS, 0.05 IU LH and FSH (Menogon, Ferring, Milan, Italy), 0.3 mM cystine, 0.5 mM cysteamine, 50 ng/mL long-EGF, 100 ng/mL long-IGF1, 5 ng/mL bFGF (Sigma-Aldrich, Milan, Italy) in 5% CO2 at 38.5°C. Boar frozen-thawed semen was separated on a percoll gradient and diluted in TALP medium with PHE (penicillamine, hypotaurine, epinefrine) to a concentration ranging from 0.05 to 0.1 million sperm per mL. Oocytes were partially decumulated, co-incubated with sperm for 24 h, and finally denuded and cultured in microdrops of mSOFaa or NCSU. After cleavage, approximately half of the cleaved embryos were surgically transferred into the sheep oviduct for 4 days of in vivo culture and the remaining embryos were left in vitro in the two media. On Day +6 in vivo-cultured embryos were recovered from the sheep oviduct. Blastocyst formation and quality were comparatively evaluated in the three culture groups. Quality specifically referred to the morphology/size of the ICM according to the following criteria: ICM A (large/prominent), ICM B (flat), and ICM C (non-visible). All embryos with a visible inner cell mass were subjected to microdissection with needles to recover the ICMs that were then plated on feeder-layers of mitomycin-treated STO fibroblasts. Attachment and outgrowth was evaluated 48–72 h post-plating. Results are presented in Table 1. Our data indicate that in vivo culture of pig embryos in the sheep oviduct greatly enhance both blastocyst development and ICM quality. As a consequence the efficiency of outgrowth formation, following plating for ES cell derivation, was significantly higher with ICMs derived from IVM-IVF pig embryos cultured in vivo as compared to their in vitro-cultured counterparts. Within the two culture media tested for in vitro culture, SOF and NCSU, the rate of blastocyst formation was similar but the quality of SOF-cultured embryos is higher. In conclusion, embryo/ICM quality represents a fundamental requirement for the derivation of ES cell lines, and in vivo culture in the sheep oviduct provides the most efficient source of high quality IVM-IVF pig embryos. Table 1. Blastocyst development and ICM quality of in vitro-produced pig embryos This work was supported by the Istituto Superiore di Sanità, Programma Nazionale Cellule Staminali, Rome, Italy, grant No. CS 11.

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Phospholipid composition and resistance to vitrification of in vivo blastocyst of a Brazilian naturalized porcine breed

Arquivo Brasileiro de Medicina Veterinária e Zootecnia ◽

10.1590/1678-4162-10249 ◽

2019 ◽

Vol 71 (3) ◽

pp. 837-847

Author(s):

J.F.W. Sprícigo ◽

L.O. Leme ◽

A.L. Guimarães ◽

J.C. Oliveira Neto ◽

P.C.P. Silva ◽

...

Keyword(s):

Phospholipid Composition ◽

Embryo Cryopreservation ◽

Blastocyst Development ◽

Apoptotic Pathway ◽

Maldi Tof ◽

Porcine Embryo ◽

Low Efficiency ◽

Hatching Rates

ABSTRACT Piau porcine blastocysts were submitted to MALDI-TOF to identify the main phospholipids (PL). After that, in vivo blastocysts (D6) were vitrified (n=52), non-vitrified were used as control (n=42). After warming, blastocysts were in vitro cultured to assess re-expansion and hatching at 24 and 48 hours. Finally, at 48 hours, hatched blastocysts were submitted to RT-qPCR searching for BCL2A1, BAK, BAX and CASP3 genes. For MALDI-TOF, the ion intensity was expressed in arbitrary units. Blastocyst development was compared by Qui-square (P< 0.05). Among the most representative PL was the phosphatidylcholine [PC (32:0) + H]+; [PC (34:1) + H]+ and [PC (36:4) + H]+. Beyond the PL, MALDI revealed some triglycerides (TG), including PPL (50:2) + Na+, PPO (50:1) + Na+, PLO (52:3) + Na+ and POO (52:2) + Na. Re-expansion did not differ (P> 0.05) between fresh or vitrified blastocysts at 24 (33.3%; 32.7%) or 48 hours (2.4%; 13.5%). Hatching rates were higher (P< 0.05) for fresh compared to vitrified at 24 (66.7%; 15.4%) and 48 hours (97.6%; 36.0%). BAX was overexpressed (P< 0.05) after vitrification. In conclusion, Piau blastocysts can be cryopreserved by Cryotop. This study also demonstrated that the apoptotic pathway may be responsible for the low efficiency of porcine embryo cryopreservation.

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13 STRATEGIES TO INCREASE OVULATORY FOLLICLE SIZE AND REDUCE OVULATION TIME IN LACTATING DAIRY COWS

Reproduction Fertility and Development ◽

10.1071/rdv20n1ab13 ◽

2008 ◽

Vol 20 (1) ◽

pp. 87

Author(s):

J. O. Giordano ◽

J. L. Edwards ◽

G. M. Schuenemann ◽

N. Rohrbach ◽

F. N. Schrick

Keyword(s):

Oocyte Maturation ◽

Elevated Temperatures ◽

Animal Health ◽

Ovarian Activity ◽

Blastocyst Development ◽

Sterile Saline ◽

Ovulatory Follicle ◽

To Receive

In vitro exposure of oocytes to elevated temperatures hastened oocyte maturation; furthermore, performing IVF of heat-stressed oocytes 5 h earlier than the usual 24 h resulted in blastocyst development similar to that of non-heat-stressed controls (Edwards et al. 2005 J. Dairy Sci. 88, 4326–4333). If elevated ambient temperatures in vivo alter oocyte maturation in a similar fashion, then new strategies are needed to induce earlier release of the oocyte from the ovulatory follicle. Current objectives were to examine follicular growth after FSH administration and examine whether treatment with FSH and an exogenously induced LH surge would hasten ovulation. On Day 0 (8 to 9 days after estrus) of the experimental period, lactating Holstein cows (n = 31; 65–115 days in milk; 1–6 lactations) received an EAZI-BREED CIDR (Pfizer Animal Health, New York, NY, USA) plus 100 µg of gonadotropin-releasing hormone (GnRH, IM; Cystorelin, Merial Ltd, Iselin, NJ, USA). On Day 7, CIDRs were removed and cows were administered 500 µg cloprostenol (IM; Estrumate, Schering-Plough Animal Health, Union, NJ, USA). Concurrently, cows were randomly allocated to receive either 80 mg FSH (FSH; n = 15; Folltropin-V, Bioniche Animal Health, Belleville, ON, Canada) or 4 mL of sterile saline (SAL; n = 16). Forty-eight h later (Day 9), cows within the FSH and SAL groups were randomly subdivided to receive either a 100-µg dose of Cysterolin (GnRH) or 3000 IU of hCG (hCG, IM; Chorulon, Intervet Inc., Millsboro, DE, USA) generating 4 treatment combinations (FSH/GnRH, n = 3; FSH/hCG, n = 7; SAL/GnRH, n = 8; and SAL/hCG, n = 8). Ovarian activity was assessed by ultrasonography to evaluate growth of the ovulatory follicle. Following CIDR removal, frequent ultrasonography was utilized to confirm ovulation (disappearance of the dominant follicle). Data were analyzed using the MIXED procedure of SAS (SAS Institute, Inc., Cary, NC, USA). Five cows from the FSH group were removed from the combination treatment due to ovulation occurring before 48 h post-CIDR removal. Size of the ovulatory follicle at time of GnRH or hCG administration was not different between FSH or SAL groups (16.7 � 0.7 v. 17.5 � 0.6 mm, respectively). Total growth of the ovulatory follicle from CIDR removal to ovulation did not differ between FSH (3.04 � 0.7 mm) and SAL (2.75 � 0.7 mm)-treated cows. As calculated from time of CIDR removal, ovulation occurred earlier in FSH (63.6 � 4.5 h) than in SAL (77.2 � 4.4 h; P < 0.05)-treated cows. Combination of FSH/GnRH produced the earliest ovulation (74 � 1.2 h) which was different only from FSH/hCG (78.6 � 0.8 h; P < 0.05), but not from SAL/GnRH or SAL/hCG (77 � 0.8 and 78 � 0.8 h, respectively). Regardless of FSH or SAL treatment, cows treated with GnRH ovulated earlier than those treated with hCG (75.5 � 0.7 v. 78.3 � 0.6 h, respectively; P < 0.05). In conclusion, while FSH was unable to increase the size of the ovulatory follicle, earlier ovulation occurred when given alone or in combination with GnRH.

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92 Culture method for long-distance transport of bovine embryos derived from IVF before blastulation using microtubes

Reproduction Fertility and Development ◽

10.1071/rdv31n1ab92 ◽

2019 ◽

Vol 31 (1) ◽

pp. 172

Author(s):

T. Yamanouchi ◽

H. Matsuda ◽

K. Ogata ◽

Y. Hashiyada

Keyword(s):

Culture Medium ◽

Liquid Paraffin ◽

Calf Serum ◽

Petri Dish ◽

Culture Method ◽

Optimal Number ◽

Blastocyst Development ◽

Long Distance

In vitro-produced (IVP) embryos are more easily damaged by cryopreservation than in vivo-derived embryos. Therefore, transportation of fresh IVP embryos in a manner that can maintain viability is necessary. This study was conducted to determine the preferable culture conditions for transport of embryos at 5 days post-insemination (dpi) in 1.5-mL microtubes. Cumulus-oocyte complexes derived from an abattoir were matured and then inseminated with frozen-thawed semen. Presumptive zygotes were cultured in mCR1aa (CR1)+5% calf serum (CS) until use. In Exp. 1, embryos with 5 blastomeres at 5 dpi were randomly assigned to 1 of 3 groups: 25mM Hepes-CR1aa (H-CR1)+5% CS or 25mM Hepes-M199 (H-M199)+5% CS in air, or CR1 in 5% CO2. Embryos were cultured in microdrops overlaid with liquid paraffin in a petri dish for 48h at 38.5°C. In Exp. 2, the optimal number of embryos to culture per microtube was assessed. Presumptive zygotes were cultured in groups of 20, 40, or 80 in 1mL of CR1 covered with liquid paraffin in microtubes in an incubator at 38.5°C in 5% CO2 until 7 dpi. For Exp. 3, culture of embryos in microtubes in a portable incubator was tested. At 5 dpi, 5-cell embryos (n=17 to 36 per microtube) were statically cultured in 1mL of CR1 or H-CR1 in microtubes in a portable incubator set at 38.5°C for 48h. The CR1 was pre-equilibrated in an incubator in 5% CO2 for 24h before use. Embryos were harvested from microtubes after 48h and were then cultured in microdrops of CR1 overlaid with liquid paraffin in a petri dish in an incubator at 38.5°C in 5% CO2 until 8 dpi. In Exp. 4, embryos (n=29 to 39 five-cell embryos per microtube) were transported in a portable incubator by land for 1000km over a period of 44h using the same conditions as in Exp. 3. Control embryos were statically cultured in microdrops of CR1 in an incubator in 5% CO2. Statistical analyses were carried out by ANOVA (Exp. 1 and 2), t-test (Exp. 3), or Fisher’s exact test (Exp. 4). In Exp. 1, there was no effect (P>0.05) of culture medium on blastocyst development at 7 dpi (27.6±2.3, 25.7±7.2, and 17.3±2.9% for CR1, H-CR1, and H-M199, respectively). In Exp. 2, blastocyst development at 7 dpi was not affected (P>0.05) by the number of presumptive zygotes cultured per microtube (43.6±8.3, 42.4±4.0, and 39.9±2.9% for 20, 40, and 80 presumptive zygotes, respectively). In Exp. 3, blastocyst development at 8 dpi was not affected (P>0.05) by culture medium (60.7±7.4 and 53.1±4.4% for CR1 and H-CR1, respectively); however, the pH of CR1 changed from 7.5 to 8.1 at 48h after culture. In Exp. 4, blastocyst development at 8 dpi was not affected (P>0.05) by transport (57.1, 64.4, and 75.5% for CR1, H-CR1, and control, respectively). These results indicate that IVP embryos harvested at 5 dpi can be transported by portable incubator with no effect on embryo development to the blastocyst stage. This work was supported by grants from the Project of the Bio-oriented Technology Research Advancement Institution, NARO (the special scheme project on advanced research and the development for next-generation technology).

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359 ICSI AND ACTIVATION OF OOCYTES RECOVERED FROM TRANSITIONAL AND CYCLING MARES

Reproduction Fertility and Development ◽

10.1071/rdv18n2ab359 ◽

2006 ◽

Vol 18 (2) ◽

pp. 286 ◽

Cited By ~ 3

Author(s):

T. Suh ◽

S. Purcell ◽

G. Seidel Jr

Keyword(s):

Growth Factor ◽

Polar Body ◽

Follicular Development ◽

Transitional Period ◽

Developmental Competence ◽

Blastocyst Development ◽

Cleavage Rate ◽

First Polar Body

Ovarian follicular development in mares during the transitional period before the breeding season leads to an accumulation of antral follicles of various sizes. The quality of oocytes at this stage may be compromized until the first seasonal ovulation. In this study, we evaluated the developmental competence of oocytes recovered from transitional and cyclic mares, and the effect of zygote activation after intracytoplasmic sperm injection (ICSI). A 2 × 2 × 2 factorial experiment consisting of oocytes from transitional and cyclic mares, two follicle sizes (10 to 20 and 20+ mm), and two treatments (control and activated) was conducted. Follicular oocytes of 14 mares were aspirated in March and April (transitional) and May to July (cyclic) five times per each period at 10-day intervals, without use of hCG. Oocytes aspirated from mares were matured in vitro in a defined medium similar to SOF plus FSH, LH, epidermal growth factor (EGF), insulin-like growth factor (IGF), estradiol (E2), prostaglandin (P4) and 10% FCS, for 30 ± 1 h under 5% CO2 in air at 38.5°C; oocytes with a first polar body were used for ICSI. Motile sperm from frozen-thawed semen were used for sperm injection with a piezo-driven pipet. For activation after ICSI, presumptive zygotes were cultured in G1.3 containing 0.02 µM phorbol 12-myristate 13-acetate (PMA) for 2 h, and then in 2 mM 6-dimethylaminopurine (6-DMAP) for 3 h under 6% CO2 in air at 38.5°C. Zygotes were cultured in 50 µL drops of DMEM/F12 containing 10% FCS for 9 days at 38.5°C in 5% CO2/5% O2/90% N2. Medium was replaced every 3 days. Cleavage and blastocyst rates were calculated based on non-degenerating injected oocytes. Data were analyzed by Fisher's exact test. A total of 115 and 78 oocytes were recovered from cyclic and transitional mares. Average maturation rates to MII in the respective groups were 76.5 and 65.4%, respectively (P < 0.07), and those of 10 to 20 and 20+ mm follicle groups were 70.6 and 80.0%, respectively (P > 0.05). The average cleavage rate in cyclic mares was higher than in transitional mares, and that of the activated group averaged over follicle sizes was higher than that of controls (P < 0.05; Table 1); those of 10 to 20 and 20+ mm follicle groups were not different (P < 0.05; Table 1). Blastocyst rates per oocyte within main effects were not different (P < 0.05; Table 1). Oocytes from transitional mares had lower cleavage rates than those of cyclic mares, but blastocyst development was similar. Activation of zygotes clearly improved cleavage rates of in vivo-derived immature equine oocytes after ICSI. Table 1. Main effect means of responses after ICSI

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179 EFFECT OF SERUM DURING CULTURE ON DAY 14 ELONGATED BOVINE EMBRYOS

Reproduction Fertility and Development ◽

10.1071/rdv23n1ab179 ◽

2011 ◽

Vol 23 (1) ◽

pp. 191 ◽

Cited By ~ 1

Author(s):

J. Angulo ◽

G. T. Gentry ◽

R. A. Godke ◽

K. R. Bondioli

Keyword(s):

Gene Expression ◽

Messenger Rna ◽

Culture Media ◽

Calf Serum ◽

Blastocyst Development ◽

Cleavage Rate ◽

Expression Levels ◽

The Mean

It has been reported that the addition of serum to embryo culture media alters gene expression and triggers the development of large offspring syndrome. The objectives of this study were to determine gene expression levels in embryos cultured in the absence or presence of 5% calf serum and in vivo-derived (IVD) embryos and to determine the effects of serum on the length of elongated embryos. Abattoir-derived oocytes were obtained from a commercial provider and fertilized at 24 h of maturation with semen from a bull previously used for IVF. At 18 h post-insemination (hpi), embryos were denuded and groups of 15 presumptive zygotes were cultured in 30-μL drops of modified SOF medium with amino acids and 6 mg mL–1 of BSA (mSOFaa). At 72 hpi, cleavage rate was assessed and embryos were randomly allocated into 2 treatments: mSOFaa without and with 5% calf serum. Embryos were then cultured to 168 hpi and blastocyst rates were assessed and recorded. Blastocysts (n = 5 to 10) from each treatment were transferred into synchronized recipients, and Day 14 embryos were recovered 7 days post-transfer. Embryos were photographed, measured, and immediately stored at –80°C in a minimal volume of PBS + 0.1% polyvinyl alcohol. Messenger RNA was isolated using a Dynabeads mRNA Direct Kit™ (Invitrogen, Carlsbad, CA), and reverse transcription was performed using an iScript™ cDNA Synthesis Kit (Bio-Rad Laboratories, Inc., CA). Quantitative PCR was performed to determine the transcript abundance for COX6A, IFNT1a, PLAC8, IGF2R, and GAPDH for each sample. The GAPDH was used as a reference gene, and gene expression was calculated as a ratio of expression levels between each gene of interest and GAPDH. Expression levels for each gene were determined from standard curves generated by serial dilutions of PCR amplicons starting with 0.4 pg/reaction. Blastocyst development rates were higher in embryos cultured with serum compared with the nonserum treatment (14.9 and 7.4% respectively; chi-square, P < 0.001). Lengths of elongated embryos from the serum (3395.3 ± 414.7 μm) and nonserum (2784 ± 741.8 μm) culture treatments differed from the IVD (6297.7 ± 677.2 μm) treatment (mean ± SE; ANOVA, P < 0.0052). There were no differences in the mean expression levels for COX6A, IFNT1a, PLAC8, and IGF2R across treatment groups, but in the serum treatment, 3 out 11 overexpressed IFNT1a, 4 out of 11 overexpressed IGF2R, and 2 out of 11 overexpressed PLAC8, defined as being 2 standard deviations above the mean of the IVD treatment for each respective gene. In the in vitro-produced nonserum and IVD treatments, overexpression by this definition was not observed. Although mean expression levels were not affected by culture with serum under these conditions, very high expression of IFNT1a, IGF2R, and PLAC8 was observed in some embryos cultured with serum, but not in embryos cultured without serum or IVD embryos.

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282 GENE EXPRESSION PATTERNS AND QUALITY OF BOVINE EMBRYOS PRODUCED UNDER DIFFERENT OXYGEN CONCENTRATIONS

Reproduction Fertility and Development ◽

10.1071/rdv19n1ab282 ◽

2007 ◽

Vol 19 (1) ◽

pp. 256

Author(s):

W. J. Son ◽

M. K. B. ◽

Y. J. Jeong ◽

S. Balasubramanian ◽

S. Y. Choe ◽

...

Keyword(s):

Gene Expression ◽

Embryo Development ◽

Expression Patterns ◽

Cell Number ◽

Blastocyst Stage ◽

Total Cell ◽

Blastocyst Development ◽

Glut 1

Various factors are known to influence the survival and development of in vitro-produced embryos, including co-culture with somatic cells, antioxidants, and O2 tension. Studies in several species report that embryo development and quality were enhanced at low O2 concentrations. This study compared the effects of 2 O2 concentrations on IVP embryo development, embryo quality, and gene expression to those of in vivo counterparts. Cumulus–oocyte complexes were matured in vitro in TCM-199 with hormones and 10% FCS, and inseminated in TALP medium. Presumptive zygotes were cultured in SOF medium under either 5% or 20% O2 in air. In triplicate, sets of 5 embryos at the 2-cell, 4-cell, 8-cell, 16-cell, morula, and Day 7 blastocyst stages were used for analyzing the expression patterns of apoptotic (Bax and Bcl2), metabolism (Glut-1 and Glut-5), stress (Sox, Hsp70, and G6PDH), compaction (Cx43), oxidation (PRDX5, NADH, and MnSOD), and implantation (VEGF and IFN-tau) genes using real-time quantitative PCR. The expression of each gene was normalized to that of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Statistical analysis was performed with Bonferroni and Duncan tests by ANOVA (P < 0.05). Cleavage rates did not differ among groups. Blastocyst and hatched blastocyst development in 5% O2 was significantly (P < 0.05) higher than in 20% O2. Total cell number of in vivo blastocysts was significantly (P < 0.05) higher than that of IVP blastocysts. ICM ratio and apoptosis of in vivo blastocysts were significantly (P < 0.05) lower than for IVP blastocysts. The relative abundances (RAs) of Glut-1, Glut-5, MnSOD, NADH, PRDX5, Cx43, Bcl2, and IFN-τ were significantly (P < 0.05) higher in in vivo embryos, whereas the RAs of Sox, G6PDH, Hsp70, Bax, and VEGF were significantly (P < 0.05) lower than for IVP counterparts. In conclusion, culture at 5% O2 concentration resulted in higher rates of development to the blastocyst stage, higher total cell numbers, and decreased apoptosis. Furthermore, differences in expression of genes including Glut-1, Glut-5, Sox, G6PDH, Hsp70, Bax, Bcl2, Cx43, PRDX5, NADH, MnSOD, VEGF, and IFN-τ may prove useful in determining optimal culture conditions. This work was supported by ARPC (204119-03-SB010), Republic of Korea.

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75 EPIGENETIC EFFECTS OF VITRIFICATION ON PRONUCLEAR DOMESTIC CAT EMBRYOS

Reproduction Fertility and Development ◽

10.1071/rdv27n1ab75 ◽

2015 ◽

Vol 27 (1) ◽

pp. 131 ◽

Cited By ~ 1

Author(s):

J. H. Galiguis ◽

C. E. Pope ◽

M. N. Biancardi ◽

C. Dumas ◽

G. Wang ◽

...

Keyword(s):

Dna Methylation ◽

Genetic Material ◽

Domestic Cat ◽

Blastocyst Development ◽

In Vitro Development ◽

Epigenetic Effects ◽

Donor Cells ◽

Vitro Development

Vitrification remains a promising technique in the preservation of valuable genetic material; however, in the cat, success has varied. Live kittens have been produced from embryos vitrified at early cleavage stages, but phenotypic abnormalities in some kittens suggest possible epigenetic effects of the vitrification process. It has been reported that cryopreservation alters epigenetic events in somatic donor cells, which indirectly influences physical status of cloned offspring. However, extending post-warming in vitro culture of donor cells corrects these epigenetic modifications, resulting in normal embryos/clones. Accordingly, in the present study, vitrification was performed at the pronuclear stage to lengthen pretransfer culture time, and vitrified cat zygotes were assessed by analysing (1) histone acetylation/methylation, (2) global DNA methylation, (3) pluripotent gene expression, (4) in vitro development, and () in vivo viability. In vivo matured/IVF oocytes were vitrified in 15% dimethyl sulfoxide, 15% ethylene glycol, and 0.5 M sucrose at 16 h post-insemination (PI). After warming in 1.0 M sucrose at 38°C, embryos were fixed at 18 h or 40 h PI, and the nuclear intensity of either acetyl/dimethyl-H3K9 or 5-methylcytosine was determined by immunofluorescence. Results showed that at 18 h PI, mean H3K9ac intensity of vitrified embryos (11.8; n = 6) was higher than that of corresponding nonvitrified (fresh) controls (4.5; n = 6) and the fresh (3.2; n = 11) and vitrified (0.6; n = 7) 40-h groups (2-way ANOVA; P < 0.05). H3K9me2 in the fresh (36.9) and vitrified (32.5) 18-h embryos was similar but increased relative to both fresh (10.7) and vitrified (9.2) 40-h groups (P < 0.05). Mean DNA methylation (5MeC) in the fresh (31.6; n = 1) and vitrified (24.7; n = 3) 18-h groups was similar to that of the fresh 40-h group (19.8; n = 4) but higher than that of the vitrified 40-h group (15.0; n = 5; P < 0.05). To assess expression of POU5F1 and Nanog, qRT-PCR was performed on Day 8 blastocysts. Relative to controls (n = 9), mean POU5F1 and Nanog levels in vitrified blastocysts (n = 24) were 1.38- and 1.98-fold higher, respectively (one-way ANOVA; P > 0.05). In terms of in vitro development, Day 2 cleavage of vitrified zygotes (59%; n = 508) was similar to that of controls (66%; n = 340), but Day 8 blastocyst formation was reduced (9 v. 31%; t-test; P < 0.05). In vivo viability was assessed by oviducal transfer of 41 Day 1 embryos into 2 recipients. One pregnancy was established (50%), with 3 live kittens weighing 70, 79, and 131 g delivered without assistance on Day 65 of gestation. The 2 smaller kittens died within a few hours of birth, with the smallest exhibiting an umbilical hernia and organ exteriorization. The third kitten developed into a normal, healthy adult. In summary, mean H3K9me2, 5MeC, and POU5F1/Nanog expression of vitrified zygotes was similar to corresponding controls. H3K9ac increased at 18h PI as a result of vitrification, but was reduced after culture to 40 h PI. Although vitrified zygotes cleaved in vitro at rates similar to controls, blastocyst development was reduced. In vivo viability was demonstrated; however, postnatal survival of kittens produced was low.

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