Effects of EGF and melatonin on gene expression of cumulus cells and further in vitro embryo development in bovines.

Despite previous research demonstrating the benefits of including growth factors and antioxidants to maturation medium to support embryo production, to date the effect of epidermal growth factor (EGF) and melatonin (Mel) on oocyte competency has not been studied. This study supplemented in vitro maturation (IVM) medium with EGF (10 ng/ml) and Mel (50 ng/ml) alone, or in combination, and evaluated cumulus cell (CC) gene expression and the development and quality of parthenogenetic blastocysts. No differences in CC gene expression levels indicative of developmental potential were found among the treatment groups. Antioxidant gene CuZnSOD was significantly (P < 0.05) decreased in CCs from the Mel group. Moreover, blastocyst rates on day 7 were significantly increased in EGF or Mel (P < 0.05), but not EGF+Mel. Significant decrease (P < 0.05) in GPX1, CuZnSOD, SLC2A1 and HSPA1A (P = 0.07) mRNA levels was observed in blastocysts from the Mel group. OCT4 gene expression was significantly increased (P < 0.05) in EGF+Mel and confirmed using immunofluorescence. Our results indicate that, despite the lack of changes of competence-related genes in CCs, IVM medium supplemented with Mel improved the culture environment sufficiently, resulting in improved blastocysts. Moreover, EGF and Mel combined during maturation increased OCT4 gene and protein expression in blastocysts, indicating its potential for stem cells.

De novo transcription of thyroid hormone receptors is essential for early bovine embryo development in vitro.

Thyroid hormone receptor (THR) α and THRß mediate the genomic action of thyroid hormones (THs) that affect bovine embryo development. However, little is known about THRs in the preimplantation embryo. The aim of the present study was to investigate the importance of THRs in in vitro preimplantation bovine embryos. THR transcripts and protein levels were detected in developing preimplantation embryos up to the blastocyst stage. Embryonic transcription of THRs was inhibited by α-amanitin supplementation, and both maternal and embryonic transcription were knocked down by short interference (si) RNA microinjection. In the control group, mRNA and protein levels of THRs increased after fertilisation. In contrast, in both the transcription inhibition and knockdown groups there were significant (P<0.05) decreases in mRNA expression of THRs from the 2-cell stage onwards. However, protein levels of THRs were not altered at 2-cell stage, although they did exhibit a significant (P<0.05) decrease from the 4-cell stage. Moreover, inhibition of de novo transcripts of THRs using siRNA led to a significant (P<0.01) decrease in the developmental rate and cell number, as well as inducing a change in embryo morphology. In conclusion, THRs are transcribed soon after fertilisation, before major activation of the embryonic genome, and they are essential for bovine embryo development in vitro.

Choosing a culture medium for SCNT and iSCNT reconstructed embryos: from domestic to wildlife species.

Over the past decades, in vitro culture media have been developed to successfully support IVF embryo growth in a variety of species. Advanced reproductive technologies, such as somatic cell nuclear transfer (SCNT), challenge us with a new type of embryo, with special nutritional requirements and altered physiology under in vitro conditions. Numerous studies have successfully reconstructed cloned embryos of domestic animals for biomedical research and livestock production. However, studies evaluating suitable culture conditions for SCNT embryos in wildlife species are scarce (for both intra- and interspecies SCNT). Most of the existing studies derive from previous IVF work done in conventional domestic species. Extrapolation to non-domestic species presents significant challenges since we lack information on reproductive processes and embryo development in most wildlife species. Given the challenges in adapting culture media and conditions from IVF to SCNT embryos, developmental competence of SCNT embryos remains low. This review summarizes research efforts to tailor culture media to SCNT embryos and explore the different outcomes in diverse species. It will also consider how these culture media protocols have been extrapolated to wildlife species, most particularly using SCNT as a cutting-edge technical resource to assist in the preservation of endangered species.

Low levels of exosomal-miRNAs in maternal blood are associated with early pregnancy loss in cloned cattle.

Nuclear reprogramming mediated by somatic cell nuclear transfer (SCNT) has many applications in medicine. However, animal clones show increased rates of abortion and reduced neonatal viability. Herein, we used exosomal-miRNA profiles as a non-invasive biomarker to identify pathological pregnancies. MiRNAs play important roles in cellular proliferation and differentiation during early mammalian development. Thus, the aim of this study was to identify exosomal-miRNAs in maternal blood at 21 days of gestation that could be used for diagnosis and prognosis during early clone pregnancies in cattle. Out of 40 bovine-specific miRNAs, 27 (67.5%) were with low abundance in the C-EPL (Clone - Early pregnancy loss) group compared with the C-LTP (Clone - Late pregnancy) and AI-LTP (Artificial Insemination - Late pregnancy) groups, which had similar miRNAs levels. Bioinformatics analysis of the predicted target genes demonstrated signaling pathways and functional annotation clusters associated with critical biological processes including cell proliferation, differentiation, apoptosis, angiogenesis and embryonic development. In conclusion, our results demonstrate decreased exosomal-miRNAs in maternal blood at 21 days of gestation in cloned cattle pregnancies that failed to reach term. Furthermore, the predicted target genes regulated by these 27 miRNAs are strongly associated with pregnancy establishment and in utero embryonic development.

Supplement of autologous ooplasm into porcine somatic cell nuclear transfer embryos does not alter embryo development.

Somatic cell nuclear transfer (SCNT) is considered as the technique in which a somatic cell is introduced into an enucleated oocyte to make a cloned animal. However, it is unavoidable to lose a small amount of the ooplasm during enucleation step during SCNT procedure. The present study was aimed to uncover whether the supplement of autologous ooplasm could ameliorate the oocyte competence so as to improve low efficiency of embryo development in porcine SCNT. Autologous ooplasm-transferred (AOT) embryos were generated by the supplementation with autologous ooplasm into SCNT embryos. They were comparatively evaluated with respect to embryo developmental potential, the number of apoptotic body formation and gene expression including embryonic lineage differentiation, apoptosis, epigenetics and mitochondrial activity in comparison with parthenogenetic, in vitro-fertilized (IVF) and SCNT embryos. Although AOT embryos showed perfect fusion of autologous donor ooplasm with recipient SCNT embryos, the supplement of autologous ooplasm could not ameliorate embryo developmental potential in regard to the rate of blastocyst formation, total cell number and the number of apoptotic body. Furthermore, overall gene expression of AOT embryos was presented with no significant alterations in comparison with that of SCNT embryos. Taken together, the results of AOT demonstrated inability to make relevant values improved from the level of SCNT embryos to their IVF counterparts.

Telomere length and telomerase activity in bovine pre-implantation embryos in vitro.

Telomeres are specialized structures that cap the ends of chromosomes and help to maintain genomic integrity and stability. Telomeres undergo dynamic changes during embryo development, which also represents an important stage for telomere elongation through telomerase enzyme activity. The objectives of this study were to examine changes in telomere length and telomerase activity from the early oocyte, through to the blastocysts stage of development, and the expression of factors with the potential to directly regulate telomeres. In vitro-produced bovine embryos were lysed and analysed for either relative telomere length, or telomerase activity using quantitative real-time PCR protocols. Our results reveal that relative telomere length is the shortest in the presumptive zygote stage of development and gradually increases to the blastocyst stage. We also demonstrate that differences between the mean telomere lengths throughout these stages are statistically significant (p < 0.05). Telomerase activity in the stages examined appears relatively constant until the blastocyst, where the highest level of activity is detected, leading to a significant difference in telomerase activity across embryonic stages (p < 0.005). Bovine telomerase RNA component (bTERC) expression levels were highest in the blastocyst, TERF1 transcripts showed little change in expression, and TERF2 expression decreased in the blastocysts (p < 0.05). Our results suggest that a complex integration of telomere-related RNA and proteins influences the regulatory mechanisms involved in 'reprogramming' of telomeres during early embryonic stages.

Enrichment of Y-chromosome-bearing bull spermatozoa by swim-up through a column.

With the advancement of assisted reproductive biotechnologies, preselecting the sex of offspring has become an important goal for cattle and other livestock breeding as well as for research. The aim of this study was to investigate the feasibility of producing enriched pools of X- or Y-chromosome-bearing sperm by vertical swim-up through a long, narrow column. Sperm recovered from the top portion of the column was predominantly Y-bearing (60%, p < 0.05), which were capable of fertilizing matured oocytes and produce significantly more male embryos compared with standard swim-up protocol.

In vitro development of bison embryos using interspecies somatic cell nuclear transfer.

Interspecies somatic cell nuclear transfer (interspecies SCNT) has been explored in many domestic and non-domestic animal species. However, problems arise during the development of these embryos, which may be related to species-specific differences in nuclear-cytoplasmic communication. The objectives of this study were to investigate the possibility of producing bison embryos in vitro using interspecies SCNT and assess the developmental potential of these embryos. Treatment groups consisted of cattle in vitro fertilization (IVF) and cattle SCNT as controls and wood bison SCNT, plains bison SCNT and wisent SCNT as experimental groups. Cleavage and blastocyst rates were assessed, and blastocyst quality was determined using total cell number, apoptotic incidence and relative quantification of mitochondria-related genes NRF1, MT-CYB and TFAM. These results indicate that embryos can be produced by interspecies SCNT in all bison species/subspecies (13.34-33.54% blastocyst rates). Although increased incidence of apoptosis was observed in bison SCNT blastocysts compared to cattle SCNT controls (10.45-12.69 vs 8.76, respectively) that corresponded with significantly lower cell numbers (80-87 cells vs >100 cells, respectively), no major differences were observed in the expression of NRF1, MT-CYB and TFAM. This study is the first to report the production of bison embryos by interspecies SCNT. Blastocyst development in all three bison species/subspecies was greater than the rates obtained in previous studies by IVF, which supports the potential role of SCNT for in vitro embryo production in this species. Yet, further investigation of developmental competence and the factors influencing blastocyst quality and viability is required.

Testis-specific protein Y-encoded copy number is correlated to its expression and the field fertility of Canadian Holstein bulls.

Testis-specific protein Y-encoded (TSPY) is present in varying copy number in both human (20-76 copies) and cattle (37-200 copies), and some studies have linked this variation to semen quality in men. The purpose of this study was to determine if TSPY copy number is associated with fertility in bulls by using adjusted non-return rates, a commonly used measure of field fertility in Canada. In addition, we investigated the associations between TSPY copy number and its expression as well as specific semen parameters, such as average sperm concentration, sperm count, ejaculate volume, and motility. In 2 independent trials, TSPY copy number was shown to be positively correlated to adjusted non-return rates (trial #1: Spearman r = 0.34, p < 0.05; trial #2: Spearman r = 0.77, p < 0.01). Furthermore, TSPY copy number was inversely correlated to TSPY mRNA expression in the testis (Pearson r = -0.71, p < 0.0001). There were no correlations of TSPY copy number or expression with the semen parameters measured. Therefore, TSPY copy number might represent a potential marker of bull fertility, but its mechanism does not appear to be directly related to the semen characteristics analyzed as part of this study.

The effects of substituting glassware for plasticware and the use of an ethanol vector on oocyte maturation in vitro.

The intent of this study was to evaluate specific technical aspects of in vitro oocyte maturation (IVM), which included container material and solvent delivery vector. Oocytes were matured in oil-free, open-well systems contained in either plastic or glass dishes and compared to control oocytes matured in media droplets on plastic dishes overlaid with mineral oil. Open-well experiments were repeated with ethanol in a quantity sufficient for delivery of nonmiscible compounds. Cleavage rates were significantly decreased in the glassware system when compared to controls. The plasticware open-well system did not differ from either the controls or the glassware groups. Cleavage in glassware with ethanol was significantly lower than controls or plasticware with ethanol. Blastocyst rates were only decreased in the glassware-ethanol treatment when compared to plasticware-ethanol treatment. Cell counts and percentage of TUNEL-positive cells did not differ significantly. Unexpectedly, sex ratio was significantly decreased (34% male) from the expected value of 50% male in the glassware group with added ethanol. The current study demonstrates the sensitivity of IVM to subtle technical changes, resulting in significant developmental consequences.

A novel approach to sexing bovine blastocysts using male-specific gene expression.

When examining gene expression profiles for the purposes of assessing embryo quality, it is imperative that sex be considered, because many embryonic transcripts have sex-related expression patterns. The objective of this study was to systematically examine eight Y chromosome linked genes (DDX3Y, EIF1AY, HSFY, SRY, TSPY, USP9Y, ZFY, and ZRSR2Y) to characterize their expression in bovine blastocysts and to examine the usefulness of this expression for the purpose of RNA-based embryo sexing. In order to examine the expression of these genes, pools of blastocysts (groups of 10 and 20) as well as single embryos (N = 50) were analyzed with reverse transcriptase polymerase chain reaction (RT-PCR) and reverse transcriptase quantitative PCR (RT-qPCR). Of the 50 single embryos, 32 were concurrently sexed with DNA-based methods. Transcripts of DDX3Y, EIF1AY, TSPY, USP9Y, ZFY and ZRSR2Y were detected in the pooled and single blastocysts, but no transcripts were detected for HSFY or SRY. After performing DNA-based sexing experiments, we concluded that this expression was restricted to the male embryos. The consistency of the expression varied according to the gene as well as the specific primer set. Three genes were expressed in the full set of male embryos, DDX3Y, USP9Y, and ZRSR2Y and therefore represent good candidates for RNA-based sexing methods.

Sister chromatid exchange assessment by chromosome orientation-fluorescence in situ hybridization on the bovine sex chromosomes and autosomes 16 and 26.

Mammalian genome replication and maintenance are intimately coupled with the mechanisms that ensure cohesion between the resultant sister chromatids and the repair of DNA breaks. Although a sister chromatid exchange (SCE) is an error-free swapping of precisely matched and identical DNA strands, repetitive elements adjacent to the break site can act as alternative template sites and an unequal sister chromatid exchange can result, leading to structural variations and copy number change. Here we test the vulnerability for SCEs of the repeat-rich bovine Y chromosome in comparison with X, 16 and 26 chromosomes, using chromosome orientation-fluorescence in situ hybridization. The mean SCE rate of the Y chromosome (0.065 ± 0.029) was similar to that of BTA16 and BTA26 (0.065, 0.055), but was only approximately half of that of the X chromosome (0.142). As the chromosomal length affects the number of SCE events, we adjusted the SCE rates of the Y, 16, and 26 chromosomes to the length of the largest chromosome X resulting in very similar adjusted SCE (SCE(adj)) rates in all categories. Our results - based on 3 independent bulls - show that, although the cattle Y chromosome is a chest full of repeated elements, their presence and the documented activity of repeats in SCE formation does not manifest in significantly higher SCE(adj) rates and suggest the importance of the structural organization of the Y chromosome and the role of alternative mitotic DNA repair mechanisms.

Investigation into developmental potential and nuclear/mitochondrial function in early wood and plains bison hybrid embryos.

Studies to date have shown that bison embryo development in vitro is compromised with few embryos developing to the blastocyst stage. The aim of this study was to use bison-cattle hybrid embryos, an interspecific cross that is known to result in live offspring in vivo, as a model for assessing species-specific differences in embryo development in vitro. Cattle oocytes fertilized with cattle, plains bison and wood bison sperm were assessed for various developmental parameters associated with embryo quality, including cell number, apoptosis and ATP content. Decreased development to the blastocyst stage was observed in hybrid wood bison embryos compared with the other treatment groups. Although both wood bison and plains bison hybrid blastocysts had significantly lower cell numbers than cattle blastocysts, only wood bison hybrid blastocysts had a greater incidence of apoptosis than cattle blastocysts. Among the treatment groups, ATP levels and expression profiles of NRF1, TFAM, MT-CYB, BAX and BCL2 were not significantly different in both 8- to 16-cell stage and blastocyst stage embryos. These data provide evidence of decreased developmental competence in the wood bison hybrid embryos, owing to inadequate culture conditions that have increased apoptotic events.

GTG mutation in the start codon of the androgen receptor gene in a family of horses with 64,XY disorder of sex development.

Genetic sex in mammals is determined by the sex chromosomal composition of the zygote. The X and Y chromosomes are responsible for numerous factors that must work in close concert for the proper development of a healthy sexual phenotype. The role of androgens in case of XY chromosomal constitution is crucial for normal male sex differentiation. The intracellular androgenic action is mediated by the androgen receptor (AR), and its impaired function leads to a myriad of syndromes with severe clinical consequences, most notably androgen insensitivity syndrome and prostate cancer. In this paper, we investigated the possibility that an alteration of the equine AR gene explains a recently described familial XY, SRY + disorder of sex development. We uncovered a transition in the first nucleotide of the AR start codon (c.1A>G). To our knowledge, this represents the first causative AR mutation described in domestic animals. It is also a rarely observed mutation in eukaryotes and is unique among the >750 entries of the human androgen receptor mutation database. In addition, we found another quiet missense mutation in exon 1 (c.322C>T). Transcription of AR was confirmed by RT-PCR amplification of several exons. Translation of the full-length AR protein from the initiating GTG start codon was confirmed by Western blot using N- and C-terminal-specific antibodies. Two smaller peptides (25 and 14 amino acids long) were identified from the middle of exon 1 and across exons 5 and 6 by mass spectrometry. Based upon our experimental data and the supporting literature, it appears that the AR is expressed as a full-length protein and in a functional form, and the observed phenotype is the result of reduced AR protein expression levels.

Low levels of X-inactive specific transcript in somatic cell nuclear transfer embryos derived from female bovine freemartin donor cells.

The present study compared developmental potential, telomerase activity and transcript levels of X-linked genes (HPRT, MECP2, RPS4X, SLC25A6, XIAP, XIST and ZFX) in bovine somatic cell nuclear transfer (SCNT) embryos reconstructed with cells derived from a freemartin (female with a male co-twin) or from normal female cattle (control). The rates of cleavage, development to blastocyst and hatched blastocyst stage, and the mean numbers of total and inner cell mass cells in the freemartin SCNT embryos were not significantly different from those of control SCNT embryos (p > 0.05). The levels of telomerase activity analyzed by RQ-TRAP in the freemartin SCNT embryos were also similar to those of the normal SCNT embryos. Transcript levels of HPRT, MECP2, RPS4X and XIAP, measured by quantitative real-time RT-PCR, were not significantly different between the control and freemartin SCNT embryos (p > 0.05). However, the transcript levels of SLC25A6, XIST and ZFX were significantly decreased in the freemartin SCNT embryos compared to control SCNT embryos (p < 0.05). Transfer of 71 freemartin SCNT embryos to 22 recipient cows resulted in 4 (18%) pregnancies, which were lost between days 28 and 90 of gestation. Taken together, the present study demonstrates that the transcript levels of several X-linked genes, especially XIST, showed an aberrant pattern in the freemartin SCNT embryos, suggesting aberrant X inactivation in freemartin clones which may affect embryo survival.

Disorders of sexual development and abnormal early development in domestic food-producing mammals: the role of chromosome abnormalities, environment and stress factors.

The management of disorders of sexual development (DSD) in humans and domestic animals has been the subject of intense interest for decades. The association between abnormal chromosome constitutions and DSDs in domestic animals has been recorded since the beginnings of conventional cytogenetic analysis. Deviated karyotypes consisting of abnormal sex chromosome sets and/or the coexistence of cells with different sex chromosome constitutions in an individual seem to be the main causes of anomalies of sex determination and sex differentiation. In recent years, a growing interest has developed around the environmental insults, such as endocrine-disrupting compounds (EDC) and heat stressors, which affect fertility, early embryonic development and, in some instances, directly the sex ratio and/or the development of 1 specific sex versus the other. A variety of chemical compounds present in the environment at low doses has been shown to have major effects on the reproductive functions in human and domestic animals following prolonged exposure. In this review, we present an overview of congenital/chromosomal factors that are responsible for the DSDs and link them and the lack of proper embryonic development to environmental factors that are becoming a major global concern.

In vitro developmental potential of nuclear transfer embryos cloned with enucleation methods using pre-denuded bovine oocytes.

Enucleation of a recipient oocyte is an important essential process in the procedure of somatic cell nuclear transfer (SCNT). The present study investigated a method for the improvement of enucleation efficiency. Oocytes were denuded of cumulus cells before the completion of nuclear maturation (pre-denuded) after 12 h of culture at MI stage and subsequently cultured for additional 6 h until the completion of nuclear maturation and extrusion of the first polar body (PB1). The extrusion rate of PB1 was not significantly different in the pre-denuded oocyte group, compared with control oocyte group matured for 18 h. However, the number of oocytes showing the metaphase II (MII) located just underneath the PB1 was significantly higher (p<0.05) in the pre-denuded oocyte group than those in control oocyte group. To test the effect of pre-denuding on the enucleation rate and developmental potential of embryos to blastocyst stage, subsequent somatic cell nuclear transfer comparisons were made with three different methods of enucleation at MII stage using vital dyes (demicoline and Hoescht) or the PB1 (blind enucleation) to localize the chromosome plate. Enucleation rate of the oocytes with demicoline, Hoechst and pre-denuding enucleation groups were significantly higher (p<0.05) than those of blind enucleation groups. However, cleavage rate to two-cell stage and, developmental rate to blastocyst and hatched blastocyst stage, the mean numbers of total and ICM cells in the SCNT embryos with Hoechst enucleation groups were significantly decreased (p<0.05), compared to those of blind, demicoline and pre-denuding enucleation groups. Moreover, the level of telomerase activity was also significantly (p<0.05) decreased in SCNT blastocysts of Hoechst enucleation group, compared to those of blind, demicoline and pre-denuding enucleation groups. Taken together, pre-denuding enucleation group using pre-denuded oocytes was a useful and simple enucleation method for bovine SCNT embryos.

Equine disorders of sexual development in 17 mares including XX, SRY-negative, XY, SRY-negative and XY, SRY-positive genotypes.

We described the clinical, cytogenetic and molecular findings of 17 clinical equine cases presented for abnormal sexual development and infertility. Six horses with an enlarged clitoris had an XX, SRY-negative genotype, which displayed male-like behavior (adult individuals). Bilateral ovotestes were noted in 2 of those cases, while another case showed increased levels of circulating testosterone. Six horses with a female phenotype, including normal external genitalia, had an XY, SRY-negative genotype. These individuals had small gonads and an underdeveloped internal reproductive tract. Four horses with normal appearing external genitalia had an XY, SRY-positive genotype, 3 of them had hypoplastic testes and male-like behavior. In addition, one young filly with enlarged clitoris and hypoplastic testes had the same genotype but did not show male-like behavior due to her age. Three of these horses were related with 2 being siblings. These findings demonstrate the diversity of disorders of sexual development seen in the horse. Furthermore, they emphasize the need for further research to identify genes involved in abnormal sex determination and differentiation in the horse.

Incidence of chromosomal abnormalities in bovine blastocysts derived from unsorted and sex-sorted spermatozoa.

The aim of the present study was to examine the incidence of chromosomal abnormalities in bovine blastocysts produced by IVF with unsorted, X-sorted or Y-sorted spermatozoa. In Experiment 1, individual blastocysts were processed to examine the incidence of mixoploidy using fluorescent in situ hybridisation. Overall, 80% (44/55) of blastocysts were mixoploid (10/15, 14/15 and 20/25 for X-sorted, Y-sorted and unsorted spermatozoa, respectively; P > 0.05). However, the prevalence of abnormal XY chromosome complements was relatively low in all groups; on average, only a small fraction of the total nuclei per embryo appeared polyploid (1.64%, 5.62% and 6.0% for X-sorted, Y-sorted and unsorted spermatozoa, respectively). Interestingly, 20% (5/25) of blastocysts derived from unsorted spermatozoa were found to be chimeric (XX/XY). In Experiment 2, chimeric embryos were detected among the blastocysts derived from two of five sires tested. In addition, one chimeric blastocyst was detected among nine in vivo-derived blastocysts obtained following AI. In conclusion, based on the results of the present study, the incidence of chromosomal abnormalities did not different between blastocysts derived from sex-sorted or unsorted spermatozoa. In addition, the occurrence of mixed sex chimeras was not limited to a single sire and was not unique to blastocysts derived from IVF.

Thyroid hormone supplementation improves bovine embryo development in vitro.

BACKGROUND: Early embryo development (EED) forms the basis of assisted reproductive technologies (ARTs), which are used to treat human infertility and to propagate other mammalian species. Thyroid hormones (THs) play an important role in the post-implantation development of the embryo in mammals; however, the effects of THs on pre-attachment embryos are not known. Currently utilized in-vitro embryo production media are devoid of THs and hence our main objective was to examine whether THs affected EED in a bovine model. METHODS: To determine if THs are present at the site of fertilization and EED in cattle, we evaluated the presence of the hormones in oviductal and uterine horn tissues. To assess the outcome of free TH supplementation (50 ng/ml of each hormone: triiodothyronine-T3 and thyroxin-T4), embryos were followed through standard and TH-supplemented in-vitro procedures, and evaluated for the cleavage rates, blastocyst formation rate and hatching rates. Embryo quality was assessed using TUNEL assay and post-cryopreservation survival was also evaluated. RESULTS: Although TH levels in in-vitro culture media were found to be approximately 60% of the administered doses, the TH-treated embryos exhibited significant increases in blastocyst formation and hatching rates (P < 0.05). Embryo quality was significantly improved in the treated groups as demonstrated by greater total cell counts and reduced proportions of apoptotic cells (P < 0.05). Finally, TH supplementation was associated with improved post-cryopreservation viability, defined by blastocyst re-expansion and hatching rates after frozen embryos had been thawed and cultured (P < 0.05). CONCLUSIONS: These findings not only provide a way of optimizing ART efficiency, but also further our understanding of how THs influence embryonic development in mammals.