BACKGROUND
the use of in vitro fertilization  in cattle and in other species such as humans has seen rapid increases in the last few decades.
in  <dig> alone, over  <dig>  in vitro-produced bovine embryos were transferred worldwide  <cit> .
in addition  <dig>  infants were born in the united states in  <dig> from assisted reproductive technologies , 99% of which involved ivf  <cit> .
in addition, the international committee for monitoring assisted reproductive technology's  estimated that in  <dig>  approximately  <dig>  babies had been born by art worldwide  <cit> .
although the statistics reflect a striking amount of ivf use, they also depict relatively low success rates.
it is rare to have more than 40% of fertilized in vitro cattle oocytes reach blastocyst stage, and of those transferred pregnancy rates are less than 45% for in vitro techniques  <cit> .
similarly in humans, statistics from  <dig> show an approximate 32-37% live birth rate from transfers  <cit> .
as such, there is increasing pressure to better understand the embryo's biological framework in order to interpret the seemingly low developmental rates.
the reported sensitivity of the embryo to environmental factors, such as ph and ionic stress during the period in which they are manipulated for in-vitro procedures has raised further importance for more in-depth studies  <cit> .
morphologically, distinction between in vivo and ivf pre-implantation embryos have been confirmed by characteristics such as differing cytoplasmic color  <cit> , lipid droplet composition  <cit> , and mitochondrial content  <cit> .
a comparison of in vitro and in vivo blastocysts via light microscopy showed lower microvilli coverage and lipid content in the in vitro-produced embryos  <cit> .
in addition to morphology, previous studies have ascertained differences metabolically  <cit>  and at the chromosomal level, with in vitro embryos incurring higher incidences of abnormalities such as mixiploidy  <cit> .
differential gene expression has gained increasing attention as a mechanism underlying the phenotypic abnormalities between in vivo and in vitro embryos  <cit> .
in addition to cattle, these differences have also been reported in sheep  <cit>  and mice  <cit> , suggesting that the ivf process itself may influence the biological framework of the early developing embryo.
these studies, however, utilized expression microarrays, which provide limited transcriptomic data.
in addition, in vivo embryos were derived from a limited number of super-ovulated donor cows, which presents a confounding variable of potential superovulation effects.
in contrast, this study utilizes in vivo embryos from non-superovulated cows to eliminate potential sources of transcriptomic influence to characterize transcriptomic variations due to the in vitro process itself.
we hypothesize that in vivo and in vitro embryo populations differ in their biological and transcriptomic characteristics and that the identification of these differences can assist in understanding the molecular mechanisms influencing development of ivf embryos.
this study reports a fine scale assessment of the differential gene expression and alternative splicing between bovine in vivo- and in vitro-derived embryos to provide potential transcriptomic characteristics for further investigation.
RESULTS
deep sequencing of the bovine blastocyst transcriptomes
using rna-seq, this study was able to characterize the transcriptomic landscapes of in vivo- versus in vitro-derived bovine blastocysts.
in order to accomplish this, two rounds of linear amplification of mrna were used to ensure that individual embryos produced enough rna input for analysis.
amplified rna from five individual in vitro-derived and five in vivo-derived embryos all with the same sire was pooled, multiplexed, and sequenced on the hiseq <dig>  producing approximately  <dig> million pair-end reads of  <dig> bp in length.
table  <dig> displays the overall results of sequencing read alignments to the bovine reference genome.
analysis of sequencing reads was done using tophat software  <cit>  for alignment with the reference genome .
of the total sequenced fragments, 83% and 85% were mapped to the reference genome for in vitro and in vivo embryos, respectively, and of these, 91% and 92%, respectively, were uniquely mapped to specific regions in the bovine genome .
of those uniquely mapped,  <dig> , <dig> and  <dig> , <dig> were mapped to one of the autosomes or x chromosome for in vitro and in vivo embryos, respectively .
identification and analysis of novel transcribed units
using stringent criteria, our analysis uncovered a total of  <dig> novel transcribed units.
of these,  <dig> show differential expression , with  <dig> transcripts over-expressed in the in vivo embryos and  <dig> over-expressed in the in vitro embryos.
three transcripts had exclusive expression in the in vivo population while one transcript was exclusive to in vitro-produced embryos.
when compared to known cattle ests from the ucsc genome browser  <cit> , we found that  <dig> novel transcripts were supported by at least five cow ests with total coverage greater than 50%.
twenty of these transcripts showed significant differential expression.
identification and validation of differentially expressed genes
the rna-seq technique allows analysis of the differential expression profile via transcript abundance with a high sensitivity for lowly expressed transcripts that would otherwise be undetected by standard microarrays  <cit> .
overall we tested  <dig>  genes for expression analysis, with  <dig> genes showing differential expression between in vitro and in vivo embryos .
of these,  <dig> genes were exclusively expressed in the in vivo population and  <dig> genes were exclusively expressed in the in vitro population.
of the remaining  <dig> genes,  <dig> showed higher expression in the in vivo embryos versus  <dig> genes that showed higher expression in the in vitro embryos.
table  <dig> shows the breakdown of fold-change among the differentially expressed genes between the two populations.
for the validation of rna-seq results, four genes showing high levels of significance  were selected and normalized to gbg <dig> via qrt-pcr in the individual embryo arna samples.
two genes, cfl <dig> and myl <dig>  showed higher expression in the in vitro embryos with an average of  <dig> - and  <dig> -fold differences, respectively, when measured with qrt-pcr and 18- and 60-fold differences, respectively, using rna-seq .
the other two genes, dus <dig> and b4galt <dig>  showed higher expression in the in vivo embryos, with an average of 15- and  <dig> -fold differences, respectively using qrt-pcr and 21- and 18-fold difference, respectively, with rna-seq .
thus, these four genes showed similar patterns of mrna abundance in rna-seq analysis and qrt-pcr.
differential alternative splicing between in vivo and in vitro embryos
in all, we detected a total of  <dig>  genes expressing more than one splice variant.
for further analysis, we used genes that had multiple isoforms and had a single transcription start site .
of these,  <dig> showed differential alternative splicing between the two embryo populations .
moreover, following wang et al.
<cit> ,  <dig>  isoforms from  <dig>  genes were classified into a total of  <dig>  simple alternative events that fell into six different categories .
it is important to remark that some genes showed more than one variable splicing event.
the rest of the genes  showed more complicated alternative splicing events and it was not possible to classify the events into these simple six categories.
*differential alternative splicing between in vivo- and in vitro-derived embryos
validation of alternative splicing was confirmed for the genes ap2b <dig> and zdhhc <dig>  both of these genes showed evidence for single exon skipping.
as such, primers were designed around this region to confirm the splicing event.
the first primer set for ap2b <dig> had exon inclusion and produced a 134-bp product in comparison to the second primer set excluding the exon which resulted in a 92-bp product .
for zdhhc <dig>  two primers sets were also created, with the first set producing a 97-bp product  and the second set producing a 118-bp product that included the skipped exon .
since both transcripts were present in both embryo samples for each gene, although differing in ratio, they were tested in a pool each of in vivo and in vitro embryo cdna for validation.
overall, rna-seq results for alternative splicing were also validated.
gene ontology analysis
to gain insights into the biological processes that could be regulated differently between in vivo and in vitro embryos, we performed a go analysis.
we found a total of  <dig> pathways significantly enriched  for differentially expressed genes, many of which are related to developmental processes .
the pathway showing the highest level of significance was cholesterol biosynthesis.
eleven genes involved in this pathway showed higher expression in the in vitro embryos compared to the in vivo embryos .
furthermore, after cholesterol biosynthesis, the pathways of system development, sterol synthesis, and cell differentiation showed the highest significance in the go analysis .
all listed genes showed higher expression in the in vitro embryos
discussion
depth of rna-seq analysis
our current knowledge on dynamic changes in gene expression during embryonic development has for the most part been based on results from microarray technology.
although providing insight, microarrays are based on a priori knowledge and oligonucleotide design and may not detect lowly expressed transcripts or provide complete genome coverage due to lack of probe availability.
also, expression microarrays do not provide information regarding alternative splicing and novel transcripts.
therefore, we utilized rna-seq technology in this study to perform an in-depth analysis of the transcriptomic landscape of bovine in vivo- and ivf-derived embryos at the blastocyst stage.
this is the first report of a high-resolution snapshot of transcriptomic differences between in vivo and in vitro embryos beyond expression for any species using rna-seq technology.
another strength of rna-seq technology is the ability to improve genome annotation by the discovery of novel transcripts.
in our study, we identified  <dig>  bp covering novel exonic regions from  <dig> novel transcript units.
of these,  <dig> transcripts showed differential expression between in vivo and in vitro embryos , with  <dig> expressed higher in the in vivo embryos and  <dig> in the in vitro embryos.
in addition, three novel transcripts were exclusive to the in vivo embryos and one to the in vitro.
thus, this is evidence that not only are there uncharacterized regions of the bovine genome, but also that these novel regions may serve important biological roles.
beyond morphology
although the two embryo groups possessed similar morphological appearance and would have received a similar rating for developmental stage and embryo quality, they possessed numerous transcriptomic differences.
of the  <dig> genes showing differential expression,  <dig> had greater than a 10-fold difference in mrna concentrations.
in addition, rna-seq technology provided valuable information regarding alternative and novel splice variants reflecting more complex mechanisms of rna regulation.
a recent study by corcoran et al.
<cit>  found that many genes involved in rna processing and translation were differentially expressed between in vivo and in vitro blastocyst populations.
our study extends these observations by identifying a number of alternatively spliced genes between in vivo and in vitro populations.
overall, our study detected  <dig> genes with alternative splicing, and of these,  <dig> showed differential splicing without showing overall differential gene expression.
this is critical as alternative splicing can influence protein production and biological function.
associations between differential alternative splicing and embryonic development have been reported by zhang et al.
<cit> .
this study reported differential expression levels of alternative transcripts for heat shock proteins in developmentally arrested in vitro bovine embryos in comparison to developed blastocysts, with the suggestion that differential processing of rna is associated with regulation of embryonic development.
thus, our study is presenting transcriptomic information that would otherwise not be detected by standard microarray and yet have key biological importance for development.
beyond individual gene differences, our study also revealed several biological pathways that were significantly enriched or depleted based on substantial differences in embryonic mrna concentrations.
the  <dig> pathways showing the highest level of significance can be found in additional file  <dig>
of particular interest was the cholesterol biosynthesis pathway, which had  <dig> genes with increased expression in the in vitro embryos.
differential expression levels of the cholesterol biosynthesis pathway between our embryo populations could be supported by a study conducted by tint et al.
establishing that the mother provides necessary cholesterol to the in vivo embryo throughout the pre-implantation period  <cit> .
since in vitro embryos lack contact with maternal tissues, and thus maternal sources of cholesterol, there could be increased de novo production of cholesterol.
of the differential genes listed in table  <dig>  many have been shown to be critical to the cholesterol biosynthesis process.
for example, lanosterol synthase  catalyzes - <dig>  oxidosqualene into the parental compounds for cholesterol and ultimate steroid synthesis in mammals  <cit> .
in addition, the gene hmgcr contributes to the major rate-limiting step for conversion of hmg-coa into mevalonate  <cit> .
hmgcr is also regulated through a feedback system, which could be altered by a lack of maternal influence and delivery on the embryo  <cit> .
should the proper levels of these genes be perturbed, it could have significant effects on an organism by altering products of cholesterol .
interestingly it should be noted that another pathway related to steroid production  was also enriched between in vivo and in vitro embryos.
in addition, the cell differentiation pathway was enriched which is intriguing as differentiation is not only critical for the blastocyst formation but to ensure proper implantation and placenta development  <cit> .
this finding supports earlier histological studies showing fewer icm cells in in vitro-produced bovine embryos compared to in vivo  <cit> .
detection of this differential pathway in our study provides further evidence for its potential importance, thus helping to further the ongoing characterization of in vivo and in vitro embryos.
in conclusion, our results suggest that ivf may influence at a larger scale by altering biological pathways critical for early development.
potential limitations
one limitation to this study is the potential shortening of reads due to the rna amplification process, which tends to favor the 3' end of the sequence.
in addition this may cause a lack of complete information on full length alternative splicing.
due to limited amounts of rna in individual embryos, two rounds of linear amplification were necessary.
although it remains unclear as to the degree of transcript shortening that occurs, it should be acknowledged as it may prevent construction of full length transcripts as discussed by huang and khatib  <cit> .
nonetheless, amplification provided a means to utilize individual embryos for validation and pool construction.
a second limitation to this study was the lack of rna-seq replicates.
nonetheless, in a prior rna-seq study by our lab, two pools were utilized and results were consistent with qrt-pcr validations and prior microarray results that used replicates  <cit> .
thus, even with limited number of samples, rna-seq results from this study provide valuable insight into the genomic characteristics of embryo populations.
as such, although there are acknowledged limitations, using rna-seq approach as a hypothesis-generating study provides numerous opportunities for further investigation to better understand early embryonic development.
CONCLUSIONS
use of ivf and in vitro embryo culture has been found to be a valuable tool for both research and industry with applications ranging from infertility treatment, and gamete selection to cloning.
in addition, the bovine embryo model has become an interesting and popular biological model to help in understanding potential problems or optimization procedures for human embryonic practices  <cit> .
as such, results of this study may help elucidate the molecular mechanisms leading to seemingly low success rates of ivf embryos and may help to unlock important factors underlying the early embryo's growth and sensitivity during the pre-implantation period.
overall, our results support the initial hypothesis that in vitro embryos differ from their in vivo counterparts on multiple transcriptomic levels.
in addition, this study highlighted the importance of studying rna processing beyond the levels of expression and the limitations morphology alone has on embryo characterization.
candidate genes and pathways uncovered in this study can be pursued in future studies with the goal of improvement of in vitro fertilization and culture systems and to further our understanding of factors involved in optimal embryonic development.

 the use of in vitro fertilization in cattle and in other species such as humans has seen rapid increases in the last few decades. in alone, over in vitro-produced bovine embryos were transferred worldwide. in addition infants were born in the united states in from assisted reproductive technologies, 99% of which involved ivf. in addition, the international committee for monitoring assisted reproductive technology's estimated that in approximately babies had been born by art worldwide. although the statistics reflect a striking amount of ivf use, they also depict relatively low success rates. it is rare to have more than 40% of fertilized in vitro cattle oocytes reach blastocyst stage, and of those transferred pregnancy rates are less than 45% for in vitro techniques. similarly in humans, statistics from show an approximate 32-37% live birth rate from transfers. as such, there is increasing pressure to better understand the embryo's biological framework in order to interpret the seemingly low developmental rates. the reported sensitivity of the embryo to environmental factors, such as ph and ionic stress during the period in which they are manipulated for in-vitro procedures has raised further importance for more in-depth studies. morphologically, distinction between in vivo and ivf pre-implantation embryos have been confirmed by characteristics such as differing cytoplasmic color, lipid droplet composition, and mitochondrial content. a comparison of in vitro and in vivo blastocysts via light microscopy showed lower microvilli coverage and lipid content in the in vitro-produced embryos. in addition to morphology, previous studies have ascertained differences metabolically and at the chromosomal level, with in vitro embryos incurring higher incidences of abnormalities such as mixiploidy. differential gene expression has gained increasing attention as a mechanism underlying the phenotypic abnormalities between in vivo and in vitro embryos. in addition to cattle, these differences have also been reported in sheep and mice, suggesting that the ivf process itself may influence the biological framework of the early developing embryo. these studies, however, utilized expression microarrays, which provide limited transcriptomic data. in addition, in vivo embryos were derived from a limited number of super-ovulated donor cows, which presents a confounding variable of potential superovulation effects. in contrast, this study utilizes in vivo embryos from non-superovulated cows to eliminate potential sources of transcriptomic influence to characterize transcriptomic variations due to the in vitro process itself. we hypothesize that in vivo and in vitro embryo populations differ in their biological and transcriptomic characteristics and that the identification of these differences can assist in understanding the molecular mechanisms influencing development of ivf embryos. this study reports a fine scale assessment of the differential gene expression and alternative splicing between bovine in vivo- and in vitro-derived embryos to provide potential transcriptomic characteristics for further investigation.  deep sequencing of the bovine blastocyst transcriptomes using rna-seq, this study was able to characterize the transcriptomic landscapes of in vivo- versus in vitro-derived bovine blastocysts. in order to accomplish this, two rounds of linear amplification of mrna were used to ensure that individual embryos produced enough rna input for analysis. amplified rna from five individual in vitro-derived and five in vivo-derived embryos all with the same sire was pooled, multiplexed, and sequenced on the hiseq producing approximately million pair-end reads of bp in length. table displays the overall results of sequencing read alignments to the bovine reference genome. analysis of sequencing reads was done using tophat software for alignment with the reference genome. of the total sequenced fragments, 83% and 85% were mapped to the reference genome for in vitro and in vivo embryos, respectively, and of these, 91% and 92%, respectively, were uniquely mapped to specific regions in the bovine genome. of those uniquely mapped,, and, were mapped to one of the autosomes or x chromosome for in vitro and in vivo embryos, respectively. identification and analysis of novel transcribed units using stringent criteria, our analysis uncovered a total of novel transcribed units. of these, show differential expression, with transcripts over-expressed in the in vivo embryos and over-expressed in the in vitro embryos. three transcripts had exclusive expression in the in vivo population while one transcript was exclusive to in vitro-produced embryos. when compared to known cattle ests from the ucsc genome browser, we found that novel transcripts were supported by at least five cow ests with total coverage greater than 50%. twenty of these transcripts showed significant differential expression. identification and validation of differentially expressed genes the rna-seq technique allows analysis of the differential expression profile via transcript abundance with a high sensitivity for lowly expressed transcripts that would otherwise be undetected by standard microarrays. overall we tested genes for expression analysis, with genes showing differential expression between in vitro and in vivo embryos. of these, genes were exclusively expressed in the in vivo population and genes were exclusively expressed in the in vitro population. of the remaining genes, showed higher expression in the in vivo embryos versus genes that showed higher expression in the in vitro embryos. table shows the breakdown of fold-change among the differentially expressed genes between the two populations. for the validation of rna-seq results, four genes showing high levels of significance were selected and normalized to gbg via qrt-pcr in the individual embryo arna samples. two genes, cfl and myl showed higher expression in the in vitro embryos with an average of - and -fold differences, respectively, when measured with qrt-pcr and 18- and 60-fold differences, respectively, using rna-seq. the other two genes, dus and b4galt showed higher expression in the in vivo embryos, with an average of 15- and -fold differences, respectively using qrt-pcr and 21- and 18-fold difference, respectively, with rna-seq. thus, these four genes showed similar patterns of mrna abundance in rna-seq analysis and qrt-pcr. differential alternative splicing between in vivo and in vitro embryos in all, we detected a total of genes expressing more than one splice variant. for further analysis, we used genes that had multiple isoforms and had a single transcription start site. of these, showed differential alternative splicing between the two embryo populations. moreover, following wang et al., isoforms from genes were classified into a total of simple alternative events that fell into six different categories. it is important to remark that some genes showed more than one variable splicing event. the rest of the genes showed more complicated alternative splicing events and it was not possible to classify the events into these simple six categories. *differential alternative splicing between in vivo- and in vitro-derived embryos validation of alternative splicing was confirmed for the genes ap2b and zdhhc both of these genes showed evidence for single exon skipping. as such, primers were designed around this region to confirm the splicing event. the first primer set for ap2b had exon inclusion and produced a 134-bp product in comparison to the second primer set excluding the exon which resulted in a 92-bp product. for zdhhc two primers sets were also created, with the first set producing a 97-bp product and the second set producing a 118-bp product that included the skipped exon. since both transcripts were present in both embryo samples for each gene, although differing in ratio, they were tested in a pool each of in vivo and in vitro embryo cdna for validation. overall, rna-seq results for alternative splicing were also validated. gene ontology analysis to gain insights into the biological processes that could be regulated differently between in vivo and in vitro embryos, we performed a go analysis. we found a total of pathways significantly enriched for differentially expressed genes, many of which are related to developmental processes. the pathway showing the highest level of significance was cholesterol biosynthesis. eleven genes involved in this pathway showed higher expression in the in vitro embryos compared to the in vivo embryos. furthermore, after cholesterol biosynthesis, the pathways of system development, sterol synthesis, and cell differentiation showed the highest significance in the go analysis. all listed genes showed higher expression in the in vitro embryos discussion depth of rna-seq analysis our current knowledge on dynamic changes in gene expression during embryonic development has for the most part been based on results from microarray technology. although providing insight, microarrays are based on a priori knowledge and oligonucleotide design and may not detect lowly expressed transcripts or provide complete genome coverage due to lack of probe availability. also, expression microarrays do not provide information regarding alternative splicing and novel transcripts. therefore, we utilized rna-seq technology in this study to perform an in-depth analysis of the transcriptomic landscape of bovine in vivo- and ivf-derived embryos at the blastocyst stage. this is the first report of a high-resolution snapshot of transcriptomic differences between in vivo and in vitro embryos beyond expression for any species using rna-seq technology. another strength of rna-seq technology is the ability to improve genome annotation by the discovery of novel transcripts. in our study, we identified bp covering novel exonic regions from novel transcript units. of these, transcripts showed differential expression between in vivo and in vitro embryos, with expressed higher in the in vivo embryos and in the in vitro embryos. in addition, three novel transcripts were exclusive to the in vivo embryos and one to the in vitro. thus, this is evidence that not only are there uncharacterized regions of the bovine genome, but also that these novel regions may serve important biological roles. beyond morphology although the two embryo groups possessed similar morphological appearance and would have received a similar rating for developmental stage and embryo quality, they possessed numerous transcriptomic differences. of the genes showing differential expression, had greater than a 10-fold difference in mrna concentrations. in addition, rna-seq technology provided valuable information regarding alternative and novel splice variants reflecting more complex mechanisms of rna regulation. a recent study by corcoran et al. found that many genes involved in rna processing and translation were differentially expressed between in vivo and in vitro blastocyst populations. our study extends these observations by identifying a number of alternatively spliced genes between in vivo and in vitro populations. overall, our study detected genes with alternative splicing, and of these, showed differential splicing without showing overall differential gene expression. this is critical as alternative splicing can influence protein production and biological function. associations between differential alternative splicing and embryonic development have been reported by zhang et al.. this study reported differential expression levels of alternative transcripts for heat shock proteins in developmentally arrested in vitro bovine embryos in comparison to developed blastocysts, with the suggestion that differential processing of rna is associated with regulation of embryonic development. thus, our study is presenting transcriptomic information that would otherwise not be detected by standard microarray and yet have key biological importance for development. beyond individual gene differences, our study also revealed several biological pathways that were significantly enriched or depleted based on substantial differences in embryonic mrna concentrations. the pathways showing the highest level of significance can be found in additional file of particular interest was the cholesterol biosynthesis pathway, which had genes with increased expression in the in vitro embryos. differential expression levels of the cholesterol biosynthesis pathway between our embryo populations could be supported by a study conducted by tint et al. establishing that the mother provides necessary cholesterol to the in vivo embryo throughout the pre-implantation period. since in vitro embryos lack contact with maternal tissues, and thus maternal sources of cholesterol, there could be increased de novo production of cholesterol. of the differential genes listed in table many have been shown to be critical to the cholesterol biosynthesis process. for example, lanosterol synthase catalyzes - oxidosqualene into the parental compounds for cholesterol and ultimate steroid synthesis in mammals. in addition, the gene hmgcr contributes to the major rate-limiting step for conversion of hmg-coa into mevalonate. hmgcr is also regulated through a feedback system, which could be altered by a lack of maternal influence and delivery on the embryo. should the proper levels of these genes be perturbed, it could have significant effects on an organism by altering products of cholesterol. interestingly it should be noted that another pathway related to steroid production was also enriched between in vivo and in vitro embryos. in addition, the cell differentiation pathway was enriched which is intriguing as differentiation is not only critical for the blastocyst formation but to ensure proper implantation and placenta development. this finding supports earlier histological studies showing fewer icm cells in in vitro-produced bovine embryos compared to in vivo. detection of this differential pathway in our study provides further evidence for its potential importance, thus helping to further the ongoing characterization of in vivo and in vitro embryos. in conclusion, our results suggest that ivf may influence at a larger scale by altering biological pathways critical for early development. potential limitations one limitation to this study is the potential shortening of reads due to the rna amplification process, which tends to favor the 3' end of the sequence. in addition this may cause a lack of complete information on full length alternative splicing. due to limited amounts of rna in individual embryos, two rounds of linear amplification were necessary. although it remains unclear as to the degree of transcript shortening that occurs, it should be acknowledged as it may prevent construction of full length transcripts as discussed by huang and khatib. nonetheless, amplification provided a means to utilize individual embryos for validation and pool construction. a second limitation to this study was the lack of rna-seq replicates. nonetheless, in a prior rna-seq study by our lab, two pools were utilized and results were consistent with qrt-pcr validations and prior microarray results that used replicates. thus, even with limited number of samples, rna-seq results from this study provide valuable insight into the genomic characteristics of embryo populations. as such, although there are acknowledged limitations, using rna-seq approach as a hypothesis-generating study provides numerous opportunities for further investigation to better understand early embryonic development.  use of ivf and in vitro embryo culture has been found to be a valuable tool for both research and industry with applications ranging from infertility treatment, and gamete selection to cloning. in addition, the bovine embryo model has become an interesting and popular biological model to help in understanding potential problems or optimization procedures for human embryonic practices. as such, results of this study may help elucidate the molecular mechanisms leading to seemingly low success rates of ivf embryos and may help to unlock important factors underlying the early embryo's growth and sensitivity during the pre-implantation period. overall, our results support the initial hypothesis that in vitro embryos differ from their in vivo counterparts on multiple transcriptomic levels. in addition, this study highlighted the importance of studying rna processing beyond the levels of expression and the limitations morphology alone has on embryo characterization. candidate genes and pathways uncovered in this study can be pursued in future studies with the goal of improvement of in vitro fertilization and culture systems and to further our understanding of factors involved in optimal embryonic development. 
we detected expression for a total of genes, with genes showing differential expression between the two embryo populations with false discovery rate <. it is rare to have more than 40% of fertilized in vitro cattle oocytes reach blastocyst stage by day of culture, and pregnancy rates are reported as less than 45% for in vitro produced embryos. in addition, genes showed evidence of alternative splicing, with genes displaying differential alternative splicing between the two pools. a valuable tool for both research and industry, in vitro fertilization has applications range from gamete selection and preservation of traits to cloning. to investigate potential influences in-vitro fertilization has on embryonic development, this study compares in vivo- and in vitro-derived bovine blastocysts at a similar stage and quality grade to determine the degree of transcriptomic variation beyond morphology using rna-seq. thus, our results support that ivf may influence at the transcriptomic level and that morphology is limited in full characterization of bovine preimplantation embryos. using go enrichment analysis, multiple biological pathways were found to be significantly enriched for differentially expressed genes, including cholesterol and sterol synthesis, system development, and cell differentiation. in vivo embryoin vitro fertilizationrna-seqtranscriptomealternative splicing
 a valuable tool for both research and industry, in vitro fertilization has applications range from gamete selection and preservation of traits to cloning. although ivf has achieved worldwide use, with approximately bovine embryos transferred in alone, there are still continuing difficulties with efficiency. it is rare to have more than 40% of fertilized in vitro cattle oocytes reach blastocyst stage by day of culture, and pregnancy rates are reported as less than 45% for in vitro produced embryos. to investigate potential influences in-vitro fertilization has on embryonic development, this study compares in vivo- and in vitro-derived bovine blastocysts at a similar stage and quality grade to determine the degree of transcriptomic variation beyond morphology using rna-seq.  a total of, and, fragments were sequenced for in vitro and in vivo embryo pools, respectively. we detected expression for a total of genes, with genes showing differential expression between the two embryo populations with false discovery rate <. there were also novel transcribed units found, of which were differentially expressed. in addition, genes showed evidence of alternative splicing, with genes displaying differential alternative splicing between the two pools. using go enrichment analysis, multiple biological pathways were found to be significantly enriched for differentially expressed genes, including cholesterol and sterol synthesis, system development, and cell differentiation.  thus, our results support that ivf may influence at the transcriptomic level and that morphology is limited in full characterization of bovine preimplantation embryos. in vivo embryoin vitro fertilizationrna-seqtranscriptomealternative splicing 