Efficiency of the zinc chelator 1,10-phenanthroline for assisted oocyte activation following ICSI in pigs.

Context In vitro embryo production in pigs is an important tool for advancing biomedical research. Intracytoplasmic sperm injection (ICSI) circumvents the polyspermy problems associated with conventional IVF in porcine. However, the suboptimal efficiency for ICSI in pigs requires new strategies to increase blastocyst formation rates. Aim To investigate novel methods for assisted activation using the zinc chelator 1,10-phenanthroline (PHEN), and to improve embryo developmental competence and quality of ICSI porcine blastocyst. Methods ICSI embryos were treated with PHEN after or before sperm injection, recording pronuclear formation, blastocyst rate and the expression of SMARCA4, OCT4, SOX2 and CDX2. Key results Neither electrical nor PHEN significantly improves pronuclear formation rates before or after ICSI. Following in vitro culture to the blastocyst stage, no significant differences were observed in developmental rates among the groups. Moreover, the use of PHEN did not alter the total cell number or the expression of OCT4, SOX2 and CDX2 in pig ICSI blastocysts. Conclusions Assisted oocyte activation with PHEN does not affect the preimplantation development of ICSI-derived pig embryos. Implications These results hold significance in refining and advancing the application of assisted oocyte activation techniques. They offer insights into addressing fertility issues and propelling advancements in human and animal reproductive medicine.

Spermatogonial stem cell technologies: applications from human medicine to wildlife conservation†.

Spermatogonial stem cell (SSC) technologies that are currently under clinical development to reverse human infertility hold the potential to be adapted and applied for the conservation of endangered and vulnerable wildlife species. The biobanking of testis tissue containing SSCs from wildlife species, aligned with that occurring in pediatric human patients, could facilitate strategies to improve the genetic diversity and fitness of endangered populations. Approaches to utilize these SSCs could include spermatogonial transplantation or testis tissue grafting into a donor animal of the same or a closely related species, or in vitro spermatogenesis paired with assisted reproduction approaches. The primary roadblock to progress in this field is a lack of fundamental knowledge of SSC biology in non-model species. Herein, we review the current understanding of molecular mechanisms controlling SSC function in laboratory rodents and humans, and given our particular interest in the conservation of Australian marsupials, use a subset of these species as a case-study to demonstrate gaps-in-knowledge that are common to wildlife. Additionally, we review progress in the development and application of SSC technologies in fertility clinics and consider the translation potential of these techniques for species conservation pipelines.

Effect of bicarbonate and polyvinyl alcohol on in vitro capacitation and fertilization ability of cryopreserved equine spermatozoa.

BACKGROUND: Factors contributing to the limited success of in vitro fertilization in horses remain to be studied. In this work, we elucidated the effect of different essential capacitation media components, bicarbonate, and bovine serum albumin or polyvinyl-alcohol, and the incubation microenvironment on sperm parameters associated with capacitation, acrosome reaction, and their ability to activate oocytes via heterologous intracytoplasmic spermatozoa injection in equine cryopreserved spermatozoa. METHODS: Frozen-thawed spermatozoa underwent incubation at different time intervals in either Tyrode's albumin lactate pyruvate medium (non-capacitating; NC) or Tyrode's albumin lactate pyruvate supplemented with bicarbonate, bicarbonate and polyvinyl-alcohol, bicarbonate and bovine serum albumin, polyvinyl-alcohol and bovine serum albumin alone. Protein kinase A-phosphorylated substrates and tyrosine phosphorylation levels, sperm motility, and acrosome reaction percentages were evaluated. After determining the best condition media (capacitating; CAP), heterologous intracytoplasmic spermatozoa injection on pig oocytes was performed and the phospholipase C zeta sperm localization pattern was evaluated. RESULTS: Incubation of frozen-thawed equine spermatozoa with bicarbonate and polyvinyl-alcohol in atmospheric air for 45 min induced an increase in protein kinase A-phosphorylated substrates and tyrosine phosphorylation levels compared to NC condition. Sperm incubation in bicarbonate and polyvinyl-alcohol medium showed an increase in total motility and progressive motility with respect to NC (p ≤ 0.05). Interestingly, three parameters associated with sperm hyperactivation were modulated under bicarbonate and polyvinyl-alcohol conditions. The kinematic parameters curvilinear velocity and amplitude of lateral head displacement significantly increased, while straightness significantly diminished (curvilinear velocity: bicarbonate and polyvinyl-alcohol = 120.9 ± 2.9 vs. NC = 76.91 ± 6.9 µm/s) (amplitude of lateral head displacement: bicarbonate and polyvinyl-alcohol = 1.15 ± 0.02 vs. NC = 0.77 ± 0.03 µm) (straightness: bicarbonate and polyvinyl-alcohol = 0.76 ± 0.01 vs. NC = 0.87 ± 0.02) (p ≤ 0.05). Moreover, the spontaneous acrosome reaction significantly increased in spermatozoa incubated in this condition. Finally, bicarbonate and polyvinyl-alcohol medium was established as CAP medium. Although no differences were found in phospholipase C zeta localization pattern in spermatozoa incubated under CAP, equine spermatozoa pre-incubated in CAP condition for 45 min showed higher fertilization rates when injected into matured pig oocytes (NC: 47.6% vs. CAP 76.5%; p ≤ 0.05). CONCLUSION: These findings underscore the importance of bicarbonate and polyvinyl-alcohol in supporting critical events associated with in vitro sperm capacitation in the horse, resulting in higher oocyte activation percentages following heterologous intracytoplasmic spermatozoa injection. This protocol could have an impact on reproductive efficiency in the equine breeding industry.

The ability of donkey sperm to induce oocyte activation and mule embryo development after ICSI.

Members of the Equus genus exhibit a fascinating capacity for hybridization, giving rise to healthy offspring. Mules, resulting from the mating of a mare with a jack, represent the most prevalent equid hybrid, serving diverse roles in our society. While in vitro embryo production, particularly through Intracytoplasmic Sperm Injection (ICSI), has rapidly gained significance in domestic horses, the in vitro production in other equids remains largely unexplored. Utilizing donkey sperm for fertilizing horse oocytes not only addresses this gap but also provides an opportunity to investigate donkey sperm's fertilization capability in vitro to further improve donkey ICSI. In this work, we initially studied the localization of donkey sperm Phospholipase C zeta (PLCζ) and assessed the sperm's capacity to induce pronuclear formation and maternal SMARCA4 recruitment upon injection into pig oocytes through ICSI. Subsequently, we investigated the injection of donkey sperm into horse oocytes, evaluating in vitro production up to the blastocyst stage using sperm from different jacks, including frozen and refrigerated samples. Distinct patterns of PLCζ localization were observed for donkey sperm cells compared to their horse counterparts. Additionally, donkey sperm exhibits a reduced ability to induce porcine oocyte activation. However, when injected into horse oocytes, donkey sperm demonstrated sufficient capability to induce oocyte activation as no discernible differences in cleavage or blastocyst rates are observed between in vitro produced mules and horse ICSI embryos. Our study not only delineates PLCζ localization in donkey sperm but also suggests potential differences in the ability to induce oocyte activation in pigs compared to horses while observing no distinctions in pronuclear recruitment of SMARCA4. Interestingly, donkey sperm remains sufficiently capable of inducing horse oocyte activation for in vitro mule blastocyst production.

Selection and Conservation of Gametes and Embryos for Improving Assisted Reproductive Technologies.

We are delighted to present this Special Issue, which is dedicated to the paramount subject of gametes and embryo selection and conservation for improving Assisted Reproductive Technologies (ARTs) [...].

Time-lapse imaging and developmental competence of donkey eggs after ICSI: Effect of preovulatory follicular fluid during oocyte in vitro maturation.

Equus members exhibit very divergent karyotype, genetic plasticity, and significant differences in their reproductive physiology. Despite the fact that somatic cell nuclear transfer and intracytoplasmic sperm injection (ICSI) has gained relevance in the last few years in horses, few reports have been published exploring ovum pick up (OPU) and in vitro maturation (IVM) of cumulus-oocyte complexes (COCs) in donkeys. Yet, some donkey species and breeds are considered endangered, and these assisted-reproductive technologies could help to preserve the genetic of valuable individuals. In this study, we tested the hypothesis that supplementation with jenny preovulatory follicular fluid (PFF) during IVM could improve oocyte developmental competence in the donkey. For this, in vitro nuclear maturation rates, cumulus cell expansion, and embryo development after ICSI of donkey COCs matured in culture media supplemented with fetal bovine serum (FBS) or donkey PFF, with a known metabolomic profile, were assessed. Time-lapse imagining was performed after ICSI of horse and donkey oocytes. Eight OPU sessions were done in five jennies with an average recovery rate of 69.2% (n = 45 COCs). Although lower cumulus cells expansion was observed in oocytes of PFF group (P = 0.0010), no significant differences were described in nuclear maturation rates and preimplantation embryo development between groups. Donkey ICSI embryos showed similar morphokinetics to horse ICSI embryos. Our study shows that supplementing IVM media with FBS or donkey PFF supports nuclear maturation and early preimplantation embryo development after ICSI in donkeys. To our knowledge, the present study is the first report of ICSI, time-lapse imaging and in vitro blastocyst production in donkey.

Preimplantation Developmental Competence of Bovine and Porcine Oocytes Activated by Zinc Chelation.

After sperm-oocyte fusion, intracytoplasmic rises of calcium (Ca) induce the release of zinc (Zn) out of the oocyte (Zn sparks). Both phenomena are known to play an essential role in the oocyte activation process. Our work aimed to explore different protocols for activating bovine and porcine oocytes using the novel zinc chelator 1,10-phenanthroline (PHEN) and to compare developmental rates and quality to bovine IVF and parthenogenetic ionomycin-induced embryos in both species. Different incubation conditions for the zinc chelator were tested, including its combination with ionomycin. Embryo quality was assessed by immunofluorescence of SOX2, SOX17, OCT4, and CDX2 and total cell number at the blastocyst stage. Even though blastocyst development was achieved using a zinc chelator in bovine, bypassing calcium oscillations, developmental rates, and blastocyst quality were compromised compared to embryos generated with sperm-induced or ionomycin calcium rise. On the contrary, zinc chelation is sufficient to trigger oocyte activation in porcine. Additionally, we determined the optimal exposure to PHEN for this species. Zinc chelation and artificial induction of calcium rise combined did not improve developmental competence. Our results contribute to understanding the role of zinc during oocyte activation and preimplantation embryo development across different mammalian species.

State of the art of nuclear transfer technologies for assisting mammalian reproduction.

The transfer of nuclear genomic DNA from a cell to a previously enucleated oocyte or zygote constitutes one of the main tools for studying epigenetic reprogramming, nucleus-cytoplasm compatibility, pluripotency state, and for genetic preservation or edition in animals. More than 50 years ago, the first experiences in nuclear transfer began to reveal that factors stored in the cytoplasm of oocytes could reprogram the nucleus of another cell and support the development of an embryo with new genetic information. Furthermore, when the nuclear donor cell is an oocyte, egg, or a zygote, the implementation of these technologies acquires clinical relevance for patients with repeated failures in ART associated with poor oocyte quality or mitochondrial dysfunctions. This review describes the current state, scope, and future perspectives of nuclear transfer techniques currently available for assisting mammal reproduction.

Aggregation of Leopardus geoffroyi hybrid embryos with domestic cat tetraploid blastomeres.

Heterospecific embryo transfer of an endangered species has been carried out using recipients from related domestic females. Aggregation of an embryo from an endangered species with a tetraploid embryo from the species to be transferred could improve the development of pregnancy to term. The main objective of the present study was to analyze embryo aggregation in domestic cat model using hybrid embryos. For this purpose, we compared in vitro development of synchronic (Sync) or asynchronic (Async) and asynchronic with a tetraploid (Async4n) aggregation of domestic cat IVF embryos. Furthermore, aggregated blastocyst quality was analyzed by evaluation of the total cell number, cell allocation by mitotrackers staining of embryonic cells, expression of Oct4, Nanog, Sox2, Cdx2 genes, number of OCT4+ nuclei, and presence of DNA fragmentation. Additionally, the developmental rates of Async4n aggregation of domestic cat with Leopardus geoffroyi hybrid (hLg) embryos were evaluated. Async aggregation increased blastocyst cell number and the number of OCT4+ nuclei as compared to non-aggregated diploid (2n) and tetraploid (4n) embryos. Moreover, blastocysts produced by Async4n aggregation showed reduced rates of fragmented DNA. No differences were found in the expression of the pluripotent genes, with exception of the Cdx2 expression, which was higher in 4n and aggregated embryos as compared to the control group. Interestingly, hybrids embryos derived by Async4n aggregation with domestic cat embryos had similar rates of blastocysts development as the control. Altogether, the findings support the use of two-cell-fused embryos to generate tetraploid blastomeres and demonstrate that Async4n aggregation generates good quality embryos.

Horse ooplasm supports in vitro preimplantation development of zebra ICSI and SCNT embryos without compromising YAP1 and SOX2 expression pattern.

Several equids have gone extinct and many extant equids are currently considered vulnerable to critically endangered. This work aimed to evaluate whether domestic horse oocytes support preimplantation development of zebra embryos obtained by intracytoplasmic sperm injection (ICSI, zebroid) and cloning, and to study the Hippo signaling pathway during the lineage specification of trophectoderm cells and inner cell mass cells. We first showed that zebra and horse sperm cells induce porcine oocyte activation and recruit maternal SMARCA4 during pronuclear formation. SMARCA4 recruitment showed to be independent of the genetic background of the injected sperm. No differences were found in blastocyst rate of ICSI hybrid (zebra spermatozoon into horse egg) embryos relative to the homospecific horse control group. Interestingly, zebra cloned blastocyst rate was significantly higher at day 8. Moreover, most ICSI and cloned horse and zebra blastocysts showed a similar expression pattern of SOX2 and nuclear YAP1 with the majority of the nuclei positive for YAP1, and most SOX2+ nuclei negative for YAP1. Here we demonstrated that horse oocytes support zebra preimplantation development of both, ICSI and cloned embryos, without compromising development to blastocyst, blastocyst cell number neither the expression of SOX2 and YAP1. Our results support the use of domestic horse oocytes as a model to study in vitro zebra embryos on behalf of preservation of valuable genetic.

Developmentally Programmed Tankyrase Activity Upregulates ß-Catenin and Licenses Progression of Embryonic Genome Activation.

Embryonic genome activation (EGA) is orchestrated by an intrinsic developmental program initiated during oocyte maturation with translation of stored maternal mRNAs. Here, we show that tankyrase, a poly(ADP-ribosyl) polymerase that regulates ß-catenin levels, undergoes programmed translation during oocyte maturation and serves an essential role in mouse EGA. Newly translated TNKS triggers proteasomal degradation of axin, reducing targeted destruction of ß-catenin and promoting ß-catenin-mediated transcription of target genes, including Myc. MYC mediates ribosomal RNA transcription in 2-cell embryos, supporting global protein synthesis. Suppression of tankyrase activity using knockdown or chemical inhibition causes loss of nuclear ß-catenin and global reductions in transcription and histone H3 acetylation. Chromatin and transcriptional profiling indicate that development arrests prior to the mid-2-cell stage, mediated in part by reductions in ß-catenin and MYC. These findings indicate that post-transcriptional regulation of tankyrase serves as a ligand-independent developmental mechanism for post-translational ß-catenin activation and is required to complete EGA.

Time of first polar body extrusion affects the developmental competence of equine oocytes after intracytoplasmic sperm injection.

Assisted reproduction techniques (ARTs) have become widespread in the equine breeding industry. In particular, the combination of oocyte recovery from live mares followed by IVM and intracytoplasmic sperm injection (ICSI) has increased markedly among the ARTs used with valuable or low-fertility animals. There is currently no consensus among research groups regarding the optimal oocyte maturation period to produce high-quality embryos. In this study, we report the maturation dynamics of equine oocytes at different time points, from 20 to 40h (Experiment 1). In addition, in Experiment 2, equine ICSI blastocysts were produced from oocytes that exhibited early (up to 24h) or late (28-30h) extrusion of the first polar body (PB). Blastocyst rates and diameter were recorded and embryo quality was assessed by analysing the number of apoptotic cells and Yes-associated protein 1 (YAP1) expression. By 20h of IVM, 42% of oocytes were mature, and the remaining oocytes matured within the next 17h of IVM. Although no differences were found in cell apoptosis or the number of YAP1-positive cells between groups exhibiting early and late PB extrusion, embryos from the early group (Group I) exhibited an improved total cell number and blastocyst rate compared to embryos from the late group (Group II) (18.60% vs 10.17% respectively).

Targeting epigenetic nuclear reprogramming in aggregated cloned equine embryos.

Epigenetic perturbations during the reprogramming process have been described as the primary cause of the low efficiency of somatic cell nuclear transfer (SCNT). In this study, we tested three strategies targeting nuclear reprogramming to investigate effects on equine SCNT. First, we evaluated the effect of treating somatic cells with chetomin, a fungal secondary metabolite reported to inhibit the trimethylation on histone 3 lysine 9 (H3K9 me3). Second, caffeine was added to the culture medium during the enucleation of oocytes and before activation of reconstructed embryos as a protein phosphatase inhibitor to improve nuclear reprogramming. Third, we tested the effects of the histone deacetylase inhibitor trichostatin A (TSA) added during both activation and early embryo culture. Although none of these treatments significantly improved the developmental rates of the invitro aggregated cloned equine embryos, the first equine cloned foal born in Australia was produced with somatic cells treated with chetomin. The present study describes the use of chetomin, caffeine and TSA for the first time in horses, serving as a starting point for the establishment of future protocols to target epigenetic reprogramming for improving the efficiency of equine cloning. Cloning is an expensive and inefficient process, but has gained particular interest in the equine industry. In this study we explored different strategies to improve cloning efficiency and produced the first cloned foal born in Australia. Our data serve as a starting point for the establishment of future protocols for improving equine cloning efficiency.

Mediator complex component MED13 regulates zygotic genome activation and is required for postimplantation development in the mouse.

Understanding factors that regulate zygotic genome activation (ZGA) is critical for determining how cells are reprogrammed to become totipotent or pluripotent. There is limited information regarding how this process occurs physiologically in early mammalian embryos. Here, we identify a mediator complex subunit, MED13, as translated during mouse oocyte maturation and transcribed early from the zygotic genome. Knockdown and conditional knockout approaches demonstrate that MED13 is essential for ZGA in the mouse, in part by regulating expression of the embryo-specific chromatin remodeling complex, esBAF. The role of MED13 in ZGA is mediated in part by interactions with E2F transcription factors. In addition to MED13, its paralog, MED13L, is required for successful preimplantation embryo development. MED13L partially compensates for loss of MED13 function in preimplantation knockout embryos, but postimplantation development is not rescued by MED13L. Our data demonstrate an essential role for MED13 in supporting chromatin reprogramming and directed transcription of essential genes during ZGA.

A journey through horse cloning.

Interest in equine somatic cell nuclear transfer technology has increased significantly since the first equid clones were produced in 2003. This is demonstrated by the multiple commercial equine cloning companies having produced numerous cloned equids to date; worldwide, more than 370 cloned horses have been produced in at least six different countries. Equine cloning can be performed using several different approaches, each with different rates of success. In this review we cover the history and applications of equine cloning and summarise the major scientific advances in the development of this technology in horses. We explain the advantages and disadvantages of different procedures to produce cloned equine embryos and describe the current status of equine clone commercialisation, along with observations of differences in regional breed association registration regulations.

Embryo aggregation does not improve the development of interspecies somatic cell nuclear transfer embryos in the horse.

The low efficiency of interspecies somatic cell nuclear transfer (iSCNT) makes it necessary to investigate new strategies to improve embryonic developmental competence. Embryo aggregation has been successfully applied to improve cloning efficiency in mammals, but it remains unclear whether it could also be beneficial for iSCNT. In this study, we first compared the effect of embryo aggregation over in vitro development and blastocyst quality of porcine, bovine, and feline zona-free (ZF) parthenogenetic (PA) embryos to test the effects of embryo aggregation on species that were later used as enucleated oocytes donors in our iSCNT study. We then assessed whether embryo aggregation could improve the in vitro development of ZF equine iSCNT embryos after reconstruction with porcine, bovine, and feline ooplasm. Bovine- and porcine-aggregated PA blastocysts had significantly larger diameters compared with nonaggregated embryos. On the other hand, feline- and bovine-aggregated PA embryos had higher blastocyst cell number. Embryo aggregation of equine-equine SCNT was found to be beneficial for embryo development as we have previously reported, but the aggregation of three ZF reconstructed embryos did not improve embryo developmental rates on iSCNT. In vitro embryo development of nonaggregated iSCNT was predominantly arrested around the stage when transcriptional activation of the embryonic genome is reported to start on the embryo of the donor species. Nevertheless, independent of embryo aggregation, equine blastocyst-like structures could be obtained in our study using domestic feline-enucleated oocytes. Taken together, these results reported that embryo aggregation enhance in vitro PA embryo development and embryo quality but effects vary depending on the species. Embryo aggregation also improves, as expected, the in vitro embryo development of equine-equine SCNT embryos; however, we did not observe positive effects on equine iSCNT embryo development. Among oocytes from domestic animals tested in our study, the feline ooplasm might be the most appropriate recipient to partially allow preimplantation embryo development of iSCNT equine embryos.

Embryo Aggregation in Pig Improves Cloning Efficiency and Embryo Quality.

In this study, we analyzed the effects of the cloned embryo aggregation on in vitro embryo development and embryo quality by measuring blastocyst diameter and cell number, DNA fragmentation levels and the expression of genes associated with pluripotency, apoptosis, trophoblast and DNA methylation in the porcine. Zona-free reconstructed cloned embryos were cultured in the well of the well system, placing one (1x non aggregated group) or three (3x group) embryos per microwell. Our results showed that aggregation of three embryos increased blastocyst formation rate and blastocyst diameter of cloned pig embryos. DNA fragmentation levels in 3x aggregated cloned blastocysts were significantly decreased compared to 1x blastocysts. Levels of Oct4, Klf4, Igf2, Bax and Dnmt 1 transcripts were significantly higher in aggregated embryos, whereas Nanog levels were not affected. Transcripts of Cdx2 and Bcl-xl were essentially non-detectable. Our study suggests that embryo aggregation in the porcine may be beneficial for cloned embryo development and embryo quality, through a reduction in apoptotic levels and an improvement in cell reprogramming.

The aggregation of four reconstructed zygotes is the limit to improve the developmental competence of cloned equine embryos.

Embryo aggregation has been demonstrated to improve cloning efficiency in mammals. However, since no more than three embryos have been used for aggregation, the effect of using a larger number of cloned zygotes is unknown. Therefore, the goal of the present study was to determine whether increased numbers of cloned aggregated zygotes results in improved in vitro and in vivo embryo development in the equine. Zona-free reconstructed embryos (ZFRE's) were cultured in the well of the well system in four different experimental groups: I. 1x, only one ZFRE per microwell; II. 3x, three per microwell; III. 4x, four per microwell; and IV. 5x, five ZFRE's per microwell. Embryo size was measured on day 7, after which blastocysts from each experimental group were either a) maintained in culture from day 8 until day 16 to follow their growth rates, b) fixed to measure DNA fragmentation using the TUNEL assay, or c) transferred to synchronized mares. A higher blastocyst rate was observed on day 7 in the 4x group than in the 5x group. Non-aggregated embryos were smaller on day 8 compared to those aggregated, but from then on the in vitro growth was not different among experimental groups. Apoptotic cells averaged 10% of total cells of day 8 blastocysts, independently of embryo aggregation. Only pregnancies resulting from the aggregation of up to four embryos per microwell went beyond the fifth month of gestation, and two of these pregnancies, derived from experimental groups 3x and 4x, resulted in live cloned foals. In summary, we showed that the in vitro and in vivo development of cloned zona-free embryos improved until the aggregation of four zygotes and declined when five reconstructed zygotes were aggregated.

Equine cloning: in vitro and in vivo development of aggregated embryos.

The production of cloned equine embryos remains highly inefficient. Embryo aggregation has not yet been tested in the equine, and it might represent an interesting strategy to improve embryo development. This study evaluated the effect of cloned embryo aggregation on in vitro and in vivo equine embryo development. Zona-free reconstructed embryos were individually cultured in microwells (nonaggregated group) or as 2- or 3-embryo aggregates (aggregated groups). For in vitro development, they were cultured until blastocyst stage and then either fixed for Oct-4 immunocytochemical staining or maintained in in vitro culture where blastocyst expansion was measured daily until Day 17 or the day on which they collapsed. For in vivo assays, Day 7-8 blastocysts were transferred to synchronized mares and resultant vesicles, and cloned embryos were measured by ultrasonography. Embryo aggregation improved blastocyst rates on a per well basis, and aggregation did not imply additional oocytes to obtain blastocysts. Embryo aggregation improved embryo quality, nevertheless it did not affect Day 8 and Day 16 blastocyst Oct-4 expression patterns. Equine cloned blastocysts expanded and increased their cell numbers when they were maintained in in vitro culture, describing a particular pattern of embryo growth that was unexpectedly independent of embryo aggregation, as all embryos reached similar size after Day 7. Early pregnancy rates were higher using blastocysts derived from aggregated embryos, and advanced pregnancies as live healthy foals also resulted from aggregated embryos. These results indicate that the strategy of aggregating embryos can improve their development, supporting the establishment of equine cloned pregnancies.