CHROMOSOMAL ANALYSIS OF 159 BOVINE EMBRYOS COLLECTED 12 to 18 DAYS AFTER ESTRUS

1980 ◽  
Vol 22 (4) ◽  
pp. 615-626 ◽  
Author(s):  
W. C. D. Hare ◽  
Elizabeth L. Singh ◽  
K. J. Betteridge ◽  
M. D. Eaglesome ◽  
G. C. B. Randall ◽  
...  
Keyword(s):  
Embryo Transfer ◽  
Chromosomal Abnormalities ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Chromosomal Analysis ◽  
Bovine Embryos ◽  
Polyploid Cells ◽  
Low Incidence ◽  
In Pregnancy

One hundred and fifty-nine embryos were analysed in conjunction with embryo transfer studies. Chromosome preparations were made from small biopsy fragments of trophoblast, from large portions of trophoblast and the inner cell mass, or from the entire embryo. Results obtained from fragments were similar to those obtained from large portions of trophoblast and inner cell mass. No structural chromosomal abnormalities were observed. Single triploid, diploid-triploid and diploid-hexaploid and 66 diploid-tetraploid embryos were found. The 41.5% incidence of diploid-tetraploid embryos was relatively high, and appeared to be associated with a donor factor. The transfer of 49 biopsied and analysed embryos to recipients resulted in 15 pregnancies, seven with diploid-tetraploid embryos having up to 25% of polyploid cells. The diploid-triploid and diploid-hexaploid embryos were among the transfers that did not result in pregnancy. The low incidence of embryos with chromosomal abnormalities, excluding the diploid-tetraploid embryos, may have been due to the embryos being analysed at 12 to 18 days of age rather than at an earlier age before death or degeneration could have occurred.

scholarly journals Three-Dimensional Live Imaging of Bovine Preimplantation Embryos: A New Method for IVF Embryo Evaluation

2021 ◽  
Vol 8 ◽  
Author(s):  
Yasumitsu Masuda ◽  
Ryo Hasebe ◽  
Yasushi Kuromi ◽  
Masayoshi Kobayashi ◽  
Kanako Urataki ◽  
...  
Keyword(s):  
Embryo Transfer ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Three Dimensional ◽  
Preimplantation Embryos ◽  
Infrared Light ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Cell Stage ◽  
Developmental Potential

Conception rates for transferred bovine embryos are lower than those for artificial insemination. Embryo transfer (ET) is widely used in cattle but many of the transferred embryos fail to develop, thus, a more effective method for selecting bovine embryos suitable for ET is required. To evaluate the developmental potential of bovine preimplantation embryos (2-cell stage embryos and blastocysts), we have used the non-invasive method of optical coherence tomography (OCT) to obtain live images. The images were used to evaluate 22 parameters of blastocysts, such as the volume of the inner cell mass and the thicknesses of the trophectoderm (TE). Bovine embryos were obtained by in vitro fertilization (IVF) of the cumulus-oocyte complexes aspirated by ovum pick-up from Japanese Black cattle. The quality of the blastocysts was examined under an inverted microscope and all were confirmed to be Code1 according to the International Embryo Transfer Society standards for embryo evaluation. The OCT images of embryos were taken at the 2-cell and blastocyst stages prior to the transfer. In OCT, the embryos were irradiated with near-infrared light for a few minutes to capture three-dimensional images. Nuclei of the 2-cell stage embryos were clearly observed by OCT, and polynuclear cells at the 2-cell stage were also clearly found. With OCT, we were able to observe embryos at the blastocyst stage and evaluate their parameters. The conception rate following OCT (15/30; 50%) is typical for ETs and no newborn calves showed neonatal overgrowth or died, indicating that the OCT did not adversely affect the ET. A principal components analysis was unable to identify the parameters associated with successful pregnancy, while by using hierarchical clustering analysis, TE volume has been suggested to be one of the parameters for the evaluation of bovine embryo. The present results show that OCT imaging can be used to investigate time-dependent changes of IVF embryos. With further improvements, it should be useful for selecting high-quality embryos for transfer.

scholarly journals 39 CHROMOSOME ABNORMALITIES IN BOVINE NUCLEAR TRANSFER EMBRYOS PRODUCED BY 'HANDMADE CLONING'

2005 ◽  
Vol 17 (2) ◽  
pp. 169
Author(s):  
A.S. Jakobsen ◽  
G. Vajta ◽  
P.D. Thomsen ◽  
H. Callesen ◽  
T. Greve
Keyword(s):  
Nuclear Transfer ◽  
Chromosomal Abnormalities ◽  
Inner Cell Mass ◽  
False Negative ◽  
Cell Mass ◽  
Developmental Competence ◽  
Additional Parameter ◽  
Chromosomal Variation ◽  
Polyploid Cells ◽  
Handmade Cloning

Embryos produced by nuclear transfer using somatic cells as donors exhibit a lower developmental competence compared to in vivo developed and IVM/IVF/IVC embryos. The so-called handmade somatic cell nuclear transfer (HSCNT or handmade cloning) that was recently presented (Vajta et al. 2003 Biol. Reprod. 68, 571–578) has, however, improved the reconstructed embryo and subsequent blastocyst rate. Further, the high total cell number (n = 216) and the high ratio of cells allocated to the inner cell mass (35%) as well as the initial pregnancy rate of 48% on Day 28 following nonsurgical transfer of HSCNT embryos indicated a high average quality. But still the pregnancy loss of HSCNT embryos was high and resulted in a birth rate of 8%. The aim of this study was to estimate the chromosomal variation in HSCNT embryos using fluorescent in situ hybridization (FISH) and to evaluate this as an additional parameter of embryo quality. Nuclei from 49 Day 7 HSCNT embryos from five independent trials were isolated by hypotonic treatment and fixed. Then the nuclei were hybridized with differentially labelled cJAB8 and p33E39 probes that hybridize specifically to the centromeric region of chromosome 6 and 7 (Viuff et al. 2000 Biol. Reprod. 63, 1143–1148). A total of 6715 nuclei were analyzed for chromosomal abnormalities, and the percentage of nuclei with false negative scores represented 1.8%. Only 4.1% of the embryos (2 of 49) had a completely normal diploid composition, while the remaining 47 embryos exhibited different types of mixoploidy. Of the 47 mixoploid embryos, 87% contained more than one type of chromosomal variation with diploid/triploid/tetraploid being the most frequent constitution (43% of the 47 mixoploid embryos). No pure polyploid embryos were observed. The percentages of HSCNT embryos in the groups of 0%, 1–25%, and 26–100% polyploid cells were 4.1%, 67.5%, and 28.6%, respectively. This is significantly higher than the corresponding figures produced and analyzed by comparable methodology of bovine IVM/IVF/IVC embryos (Viuff et al. 1999 Biol. Reprod. 60, 1273–1278). However, 71.4% of the HSCNT embryos contained less than 25% polyploid cells; this low level may not have compromised the developmental competence of these embryos.

scholarly journals Extended embryo culture is effective for patients of an advanced maternal age

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
R. Sainte-Rose ◽  
C. Petit ◽  
L. Dijols ◽  
C. Frapsauce ◽  
F. Guerif
Keyword(s):  
Live Birth ◽  
Embryo Culture ◽  
Maternal Age ◽  
Chromosomal Abnormalities ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Advanced Maternal Age ◽  
Birth Rates ◽  
Younger Women ◽  
Live Birth Rates

AbstractThe aim of this study was to determine the effectiveness of extended embryo culture in advanced maternal age (AMA) patients (37–43 years). In this retrospective analysis, 21,301 normally fertilized zygotes from 4952 couples were cultured until the blastocyst stage. Blastocyst development, including kinetics and morphology, transfer rate, implantation and live birth rates, were measured. In AMA patients, the blastocyst rate was significantly decreased as compared to that in younger women. On day 5, blastocysts underwent growth retardation in AMA patients, which was highlighted by a decreased rate of full/expanded blastocysts. Organization of the cells (trophectoderm and inner cell mass) was unaffected by age. However, in AMA patients, a ‘good’ morphology blastocyst had a decreased probability to implant compared with an ‘average’ morphology blastocyst in younger women. While the rates of blastocyst transfer and useful blastocysts were similar to younger patients, in AMA patients, both implantation and live birth rates were significantly reduced. Our results support the idea that extended embryo culture is not harmful for AMA patients. However, embryo selection allowed by such culture is not powerful enough to avoid chromosomal abnormalities in the developed blastocysts and therefore cannot compensate for the effect of a woman’s age.

scholarly journals Base editing in bovine embryos reveals a species-specific role of SOX2 in regulation of pluripotency

2021 ◽  
Author(s):  
Lei Luo ◽  
Yan Shi ◽  
Huanan Wang ◽  
Zizengchen Wang ◽  
Yanna Dang ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Rna Seq ◽  
Bovine Embryos ◽  
Base Editing ◽  
Sox2 Expression ◽  
Nanog Expression ◽  
Species Specific ◽  
Lineage Specific Genes

The emergence of the first three lineages during development are orchestrated by a network of transcription factors, which are best characterized in mice. However, the role and regulation of these factors are not completely conserved in other mammals, including human and cattle. Here, we establish a gene inactivation system by introducing premature codon with cytosine base editor in bovine embryos with a robust efficiency. Of interest, SOX2 is universally localized in early blastocysts but gradually restricted into the inner cell mass in cattle. SOX2 knockout results in a failure of the establishment of pluripotency. Indeed, OCT4 level is significantly reduced and NANOG was barely detectable. Furthermore, the formation of primitive endoderm is compromised with few SOX17 positive cells. Single embryo RNA-seq reveals a dysregulation of 2074 genes, among which 90% are up-regulated in SOX2-null blastocysts. Intriguingly, more than a dozen lineage-specific genes, including OCT4 and NANOG, are down-regulated. Moreover, SOX2 expression is sustained in the trophectoderm in absence of CDX2 in bovine late blastocysts. Overall, we propose that SOX2 is dispensable for OCT4 and NANOG expression and disappearance of SOX2 in the trophectoderm depends on CDX2 in cattle, which are all in sharp contrast with results in mice.

scholarly journals Does Blastocyst Morphology Influence Live Birth Rate after Frozen-thawed Single Euploid Embryo Transfer?

2021 ◽  
Author(s):  
Na Li ◽  
Yichun Guan ◽  
Bingnan Ren ◽  
Yuchao Zhang ◽  
Yulin Du ◽  
...  
Keyword(s):  
Embryo Transfer ◽  
Live Birth ◽  
Birth Rate ◽  
Inner Cell Mass ◽  
Medical Center ◽  
Cohort Analysis ◽  
Cell Mass ◽  
Live Birth Rate ◽  
Poor Quality ◽  
Blastocyst Quality

Abstract Objective To investigate whether the morphologic parameters of euploid blastocyst influence the live birth rate (LBR) following single frozen-thawed embryo transfer (FET) cycles? Methods A retrospective cohort analysis involving autologous single FET cycles after preimplantation genetic testing for aneuploidy (PGT-A) through next generation sequencing (NGS) by a university-based reproductive medical center that was performed from June 2017 to September 2019.Women were divided into three age groups (< 30, 30–34 and ≥ 35 years). The primary outcome measure was LBR. Outcomes were compared to determine the association between different blastocyst quality (Good, Average and Poor), inner cell mass (ICM) grade (A and B), and trophectoderm (TE) grade (A, B and C) and LBR. Results We included 232 single FET cycles for analysis, the total LBR was 48.48%. In the youngest group (< 30 years, n = 86), LBR were compared between cycles with various blastocyst quality (72.22% for good quality, 54.55% for average quality and 34.78% for poor quality; P = 0.019), ICM grade (70.59% for grade A and 42.03% for grade B; P = 0.035) and TE grade (85.71% for grade A,57.58% for grade B and 34.78 for grade C; P = 0.015). Similarly, in the 30–34 years group, LBR ranged from 50.00% for good quality to 45.45% for poor quality (P = 0.870), from 35.29% for ICM grade A to 51.22% for ICM grade B (P = 0.291), from 60.00% for TE grade A to 45.45% for TE grade C (P = 0.634). Likewise, in the oldest group (≥ 35years, n = 47), LBR were also comparable between these subgroups, but no significant differences were seen in blastocyst morphologic parameters and LBR (P > 0.05). Conclusion After single FET cycles, the LBR was associated with morphologic parameters of euploid blastocysts, especially in women < 30 years old. However, these differences were not found in women older than 30 years. We suggested that for older women whose embryos undergoing PGT-A with NGS to be euploid have the same development potential regardless of their blastocyst morphology.

Use of chemically defined system for the direct comparison of inner cell mass and trophectoderm distribution in murine, porcine and bovine embryos

Zygote ◽  
1997 ◽  
Vol 5 (4) ◽  
pp. 309-320 ◽  
Author(s):  
Rabindranath de la Fuente ◽  
W. Allan King
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Number ◽  
Total Cell ◽  
Differential Staining ◽  
Similar Proportion ◽  
Bovine Embryos ◽  
Total Cell Number ◽  
Cell Numbers

SummaryThe mammalian blastocyst comprises an inner cell mass (ICM) and a trophectoderm cell layer. In this study the allocation of blastomeres to either cell lineage was compared between murine, porcine and bovine blastocysts. Chemical permeation of trophectoderm cells by the Ca2+ ionophore A23187 in combination with DNA-specific fluorochromes resulted in the differential staining of trophectoderm and ICM. Confocal microscopy confirmed the exclusive permeation of trophectoderm and the internal localisation of intact ICM cells in bovine blastocysts. Overall, differential cell counts were obtained in approximately 85% of the embryos assessed. Mean (±SEM) total cell numbers were 72.2 ± 3.1 and 93.1±5 for in vivo derived murine (n = 41) and porcine (n = 21) expanded blastocysts, respectively. Corresponding ICM cell number counts revealed ICM/total cell number ratios of 0.27 and 0.21, respectively. Comparison of in vivo (n = 20) and in vitro derived bovine embryos on day 8 (n = 29) or day 9 (n = 29) revealed a total cell number of 195.25±9.9, 166.14±9.9 and 105±6.7 at the expanded blastocyst stage with corresponding ICM/total cell ratios of 0.27, 0.23 and 0.23, respectively. While total cell numbers differed significantly among the three groups of bovine embryos (p<0.05), the ICM/total cell ratio did not. These results indicate that a similar proportion of cells is allocated to the ICM among blastocysts of genetically divergent species.

178 IMPROVED DEVELOPMENTAL COMPETENCE OF BOVINE EMBRYOS BY PGI2 ANALOG TREATMENT

2006 ◽  
Vol 18 (2) ◽  
pp. 197 ◽  
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.

scholarly journals The incompletely fulfilled promise of embryo transfer in cattle—why aren’t pregnancy rates greater and what can we do about it?

2020 ◽  
Vol 98 (11) ◽  
Author(s):  
Peter J Hansen
Keyword(s):  
Embryo Transfer ◽  
Artificial Insemination ◽  
Blastocyst Stage ◽  
Pregnancy Rates ◽  
Bovine Embryos ◽  
Uterine Receptivity ◽  
Stage Of Development ◽  
Stage Embryo ◽  
Repeat Breeder ◽  
In Pregnancy

Abstract Typically, bovine embryos are transferred into recipient females about day 7 after estrus or anticipated ovulation, when the embryo has reached the blastocyst stage of development. All the biological and technical causes for failure of a female to produce a blastocyst 7 d after natural or artificial insemination (AI) are avoided when a blastocyst-stage embryo is transferred into the female. It is reasonable to expect, therefore, that pregnancy success would be higher for embryo transfer (ET) recipients than for inseminated females. This expectation is not usually met unless the recipient is exposed to heat stress or is classified as a repeat-breeder female. Rather, pregnancy success is generally similar for ET and AI. The implication is that either one or more of the technical aspects of ET have not yet been optimized or that underlying female fertility that causes an embryo to die before day 7 also causes it to die later in pregnancy. Improvements in pregnancy success after ET will depend upon making a better embryo, improving uterine receptivity, and forging new tools for production and transfer of embryos. Key to accelerating progress in improving pregnancy rates will be the identification of phenotypes or phenomes that allow the prediction of embryo competence for survival and maternal capacity to support embryonic development.

28 Short equilibration improves vitrification of invitro-produced bovine embryos using VitTrans device

2020 ◽  
Vol 32 (2) ◽  
pp. 140
Author(s):  
I. Martínez-Rodero ◽  
T. García-Martínez ◽  
M. López-Béjar ◽  
T. Mogas
Keyword(s):  
Ethylene Glycol ◽  
Dimethyl Sulfoxide ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Calf Serum ◽  
Field Conditions ◽  
Bovine Embryos ◽  
Fluid Medium ◽  
Apoptosis Index ◽  
Vitrification Solution

For the successful application of vitrification technology to field conditions, the procedures for the warming and transfer of the cryopreserved bovine embryos should be as simple as possible. The device VitTrans, designed by our group, enables warming/dilution of embryos and their transfer directly to recipient females in field conditions (Morato and Mogas 2014 Cryobiology 68, 288). VitTrans vitrification protocol consists of an incubation in equilibration solution during 12min followed by an exposure of 40s to vitrification solution. However, there are other reports using similar vitrification devices where equilibration length is shorter than ours. This study aimed to improve VitTrans methodology by comparing two vitrification protocols: short equilibration (SE) and long equilibration (LE). A total of 63 invitro-produced Day 7 blastocysts (IETS stage code 7) were randomly placed in an equilibration solution with 7.5% ethylene glycol + 7.5% dimethyl sulfoxide in holding medium (tissue culture medium-199 HEPES + 20% fetal calf serum) for either 3min (SE) or 12min (LE). Then, blastocysts were transferred to vitrification solution (15% ethylene glycol + 15% dimethyl sulfoxide + 0.5M sucrose in holding medium) for 40s, loaded onto the VitTrans device, plunged into liquid nitrogen, and covered with a 0.5mL straw. Fresh nonvitrified blastocysts (n=30) were set as control. For warming, the VitTrans was quickly submerged into a water bath at 45°C, while a syringe containing 0.3mL of diluting solution (0.5M sucrose in holding medium) at 45°C was injected through the hollow of the device. Blastocysts were then transferred to synthetic oviductal fluid medium and cultured for 24h at 38.5% in a 5% CO2 and 5% O2 environment in a humidified atmosphere. Re-expansion rates were recorded 3 and 24h after warming. Blastocysts were fixed and stained with SOX2 (Invitrogen) for inner cell mass (ICM) count, TUNEL (Roche) for apoptosis index assessment, and DAPI (Vector Laboratories) for total cell count (TCC). Images were captured using a Leica TCS SP5 confocal microscope (Leica Microsystems) and examined with Imaris 9.2 software (Oxford Instruments). Blastocyst survival rates were compared between groups using chi-squared test. Blastocyst TCC, ICM count, and apoptosis indices were analysed using analysis of variance. Significance was set at P ≤ 0.05. No differences were observed in re-expansion rate at 3h postwarming (61.3 and 59.4% for SE and LE, respectively). However, significantly higher re-expansion rates were found after 24h of culture for the blastocysts of the SE group (74.2%) when compared with the blastocysts of the LE group (65.7%). Blastocysts vitrified using the LE protocol produced the lowest TCC (115±5.9; P ≤ 0.05), whereas TCC of the SE (152±9.7) and fresh control (138±8.6) treatments were similar. No differences were found in ICM count among groups. Nevertheless, apoptosis index was higher (P ≤ 0.05) in both vitrification groups when compared with fresh control. These results indicate that short equilibration vitrification not only improves VitTrans outcomes but adds efficiency by taking less time to perform. Supported by MCIU, Spain (Project AGL2016-79802-P and Grant BES-2017-081962).

95 ROLE OF POLYAMINES IN BOVINE PRE-IMPLANTATION DEVELOPMENT

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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