Identification and characterization of dairy cows with different backfat thickness antepartum in relation to postpartum loss of backfat thickness: A cluster analytic approach.

The objectives of this study were (1) to characterize the interindividual variation in the relationship between antepartum (ap) backfat thickness (BFT) and subsequent BFT loss during early lactation in a large dairy herd using cluster analysis; (2) to compare the serum concentrations of metabolites (nonesterified fatty acids, ß-hydroxybutyrate), metabolic hormones (leptin and adiponectin), and an inflammatory marker (haptoglobin) among the respective clusters; and (3) to compare lactation performance and uterine health status in the different clusters. An additional objective was (4) to investigate differences in these serum variables and in milk yield of overconditioned (OC) cows that differed in the extent of BFT loss. Using data from a large study of 1,709 multiparous Holstein cows, we first selected those animals from which serum samples and BFT results (mm) were available at d 25 (±10) ap and d 31 (±3 d) postpartum (pp). The remaining 713 cows (parity of 2 to 7) were then subjected to cluster analysis: different approaches based on the BFT of the cows were performed. K-means (unsupervised machine learning algorithm) clustering based on BFT-ap alone identified 5 clusters: lean (5-8 mm BFT, n = 50), normal (9-12 mm, n = 206), slightly fat (SF; 13-16 mm, n = 203), just fat (JF; 16-22 mm, n = 193), and very fat (VF; 23-43 mm, n = 61). Clustering by difference between BFT-ap and BFT-pp (ΔBFT) also revealed 5 clusters: extreme loss (17-23 mm ΔBFT, n = 16), moderate loss (9-15 mm, n = 119), little loss (4-8 mm, n = 326), no loss (0-3 mm, n = 203), and gain (-8 to -1 mm, n = 51). Based on the blood variables measured, our results confirm that cows with greater BFT losses had higher lipid mobilization and ketogenesis than cows with less BFT loss. The serum variables of cows that gained BFT did not differ from normal cows. Milk yield was affected by the BFT-ap cluster, but not by the ΔBFT cluster. Cows categorized as VF had lesser milk yield than other clusters. We further compared the OC cows that had little or no BFT loss (i.e., 2% of VF, 12% of JF, and 31% of SF, OC-no loss, n = 85) with the OC cows that lost BFT (OC-loss, n = 135). Both NEFA and BHB pp concentrations and milk yield were greater in OC-loss cows compared with the OC-no loss cows. The serum concentration of leptin ap was greater in OC-loss than in the OC-no loss cows. Overall, OC cows lost more BFT than normal or lean cows. However, those OC cows with a smaller loss of BFT produced less milk than OC cows with greater losses.

Correction to: Genes associated with survival of female bovine blastocysts produced in vivo.

The first error is on page 5. A sentence lists two genes as SCNA1A and SCNA2A but they should be SCN1A and SCN2A.

Genes associated with survival of female bovine blastocysts produced in vivo.

The objective was to characterize the transcriptome profile of in vivo-derived female embryos competent to establish and maintain gestation. Blastocysts from superovulated heifers were bisected to generate two demi-embryos. One demi-embryo was transferred into a synchronized recipient and the other part was used for RNA-seq analysis. Data on transcript abundance was analyzed for 4 demi-embryos that established and maintained pregnancy to day 60 (designated as PP) and 3 that did not result in a pregnancy at day 30 (designated as NP). Using a false discovery rate of P < 0.10 as cutoff, a total of 155 genes were differentially expressed between PP and NP embryos, of which 73 genes were upregulated and 82 genes were downregulated in the PP group. The functional cluster with the greatest enrichment score for embryos that survived, representing 28 genes (48% of the annotated genes), was related to membrane proteins, particularly those related to olfaction and neural development and function. The functional cluster with the greatest enrichment score for downregulated genes in embryos that survived included terms related to oxidative phosphorylation, mitochondrial function, and transmembrane proteins. In conclusion, competence of in vivo-derived female bovine embryos to survive after transfer is associated with increased expression of genes encoding transmembrane proteins, perhaps indicative of differentiation of the inner cell mass to epiblast, and decreased expression of genes involved in oxidative phosphorylation, perhaps indicative of reduced metabolic activity.

Molecular fingerprint of female bovine embryos produced in vitro with high competence to establish and maintain pregnancy†.

The objective was to identify the transcriptomic profile of in vitro-derived embryos with high competence to establish and maintain gestation. Embryos produced with X-sorted sperm were cultured from day 5 to day 7 in serum-free medium containing 10 ng/ml recombinant bovine colony-stimulating factor 2 (CSF2) or vehicle. The CSF2 was administered because this molecule can increase blastocyst competence for survival after embryo transfer. Blastocysts were harvested on day 7 of culture and manually bisected. One demi-embryo from a single blastocyst was transferred into a synchronized recipient and the other half was used for RNA-seq analysis. Using P < 0.01 and a fold change >2-fold or <0.5 fold as cutoffs, there were 617 differentially expressed genes (DEG) between embryos that survived to day 30 of gestation vs those that did not, 470 DEG between embryos that survived to day 60 and those that did not, 432 DEG between embryos that maintained pregnancy from day 30 to day 60 vs those where pregnancy failed after day 30, and 635 DEG regulated by CSF2. Pathways and ontologies in which DEG were overrepresented included many related to cellular responses to stress and cell survival. It was concluded that gene expression in the blastocyst is different between embryos that are competent to establish and maintain pregnancy vs those that are not. The relationship between expression of genes related to cell stress and subsequent embryonic survival probably reflects cellular perturbations caused by embryonic development taking place in the artificial environment associated with cell culture.

Birth of healthy calves after intra-follicular transfer (IFOT) of slaughterhouse derived immature bovine oocytes.

To circumvent the negative impacts of in vitro culture on bovine embryos, we have recently established a new method, the so called intra-follicular oocyte transfer (IFOT), enabling in vivo fertilization and in vivo development of in vitro matured oocytes up to the blastocyst stage as well as to term. In this study, we raised the question whether immature bovine oocytes could also be transferred into a pre-ovulatory follicle to support in vivo maturation prior to subsequent in vivo fertilization, in vivo development as well as to term. To unravel that question, a total of 791 immature oocytes were transferred in groups of ∼50 into pre-ovulatory follicles of 16 recipient heifers. Consequently, we were able to recollect a total of 306 structures 8 days thereafter (38.5%). All in all, 12 heifers (75%) gave embryos developed to the morula or blastocyst stage in addition to the expected native embryos. Among all recollected structures, 40.1% had developed to the morula and/or blastocyst stage, meaning a total efficiency of 17.3% based on all transferred oocytes. Of impact, IFOT-embryos reached significantly higher developmental rates to the Morula and/or blastocyst stage until day 7 compared to in vitro cultured control embryos, despite being derived from the same charge of slaughterhouse ovaries (40.1 vs. 29.3%). This implicates a beneficial effect of the follicular environment for the intrinsic quality of the fertilized embryos during maturation and for subsequent developmental rates up to the blastocyst stage. Finally, the birth of two healthy calves after transfer of frozen-thawed IFOT-derived blastocysts to final recipients established the first proof of principle that IFOT of immature bovine oocytes generates bovine blastocysts bearing developmental capacity to term. Likewise, to the best of our knowledge, these calves are the first calves derived from full in vivo development of immature slaughterhouse derived oocytes. Thus, the results of the present study clearly demonstrate that IFOT of immature slaughterhouse-derived oocytes is now a feasible technique. Since efficiencies following IFOT achieved within the present study were improved compared to previous studies, IFOT now offers an attractive option for designing new scientific experiments.

Cryosurvival of in vitro produced bovine embryos supplemented with l-Carnitine and concurrent reduction of fatty acids.

Lipid accumulation is associated with reduced embryonic quality, causing limited survival after cryopreservation. Therefore, in the present study we aimed to reveal the effects of supplementation of a lipid reducing agent, l-carnitine and the removal of fatty acids during in vitro culture on the morphological as well as on the molecular level. To accomplish that, presumptive zygotes were cultured in 4 contrasting groups: namely SOFaa medium supplemented with BSA, (BSA), SOFaa medium supplemented with fatty acid free BSA (FAF), SOFaa medium supplemented with BSA as well as l-Carnitine (BSA + LC) and SOFaa medium concurrently supplemented with fatty acid free BSA and l-Carnitine (FAF + LC). Considering the developmental rates, no impact of different treatments was observed. Conversely, treatment groups clearly affected lipid content, with the lowest amounts detected in embryos derived from FAF and BSA + LC groups, implicating that both removal of fatty acids and supplementation of LC reduces lipid content effectively. Importantly, survival rates after cryopreservation show that LC significantly affects the kinetics of re-expansion, with the highest hatching rates detected for embryos cultured in FAF + LC (p < 0.05). Noteworthy, the highest cryotolerance did not go along with lowest lipid contents. Finally, metabolic alterations between the groups were reflected in different abundances of selected candidate genes related to lipid metabolism and oxidative stress response, like AMPKA1, ACC and PGC1 α or KEAP1 and SOD1. All in all, highly beneficial effects on survival rates after cryopreservation have been detected when embryos were cultured in absence of fatty acids and concurrent presence of l-Carnitine. Highest cryotolerance, however, did not correlate with lowest lipid contents.

Short communication: Telomere lengths in different tissues of dairy cows during early and late lactation.

Telomeres create a protective cap on the ends of chromosomes that shorten with cell division and are influenced by stressful conditions. With the onset of lactation, high-yielding dairy cows are exposed to metabolic stress. In the present study, we aimed to analyze telomere length (TL) in key metabolic organs, such as liver, subcutaneous (sc) adipose tissue (AT), and mammary gland, as well as in peripheral blood cells during early and late lactation in German Holstein cows (n=21). Animals were fed according to their requirement, and biopsies from scAT, liver, and mammary gland as well as blood cells were collected in early and late lactation. The relative quantity of telomere products (qT), which is proportional to the average TL, was determined in genomic DNA by multiplex quantitative PCR. In this study, relative qT varied widely in the investigated tissues and blood. In late lactation, slowly proliferating tissues, such as liver and scAT, had the highest qT, whereas peripheral blood cells and in the mammary gland had the lowest qT. Comparing early with late lactation, relative qT attrition was limited to blood and mammary gland. Relationships between relative qT in blood, mammary gland, scAT, and liver suggest that blood qT might serve as a surrogate marker for tissue-specific qT. Cows with high initial qT in tissues and blood in early lactation had greater qT attrition during the course of lactation than cows with lower qT. The determination of qT could be included when phenotyping dairy cattle to test for associations with performance and fitness traits.

Fateful triad of reactive oxygen species, mitochondrial dysfunction and lipid accumulation is associated with expression outline of the AMP-activated protein kinase pathway in bovine blastocysts.

Low cryotolerance is considered as the major drawback of in vitro-produced bovine embryos and is frequently associated with a triad encompassing increased cytoplasmic lipid accumulation, enhanced levels of reactive oxygen species (ROS) and mitochondrial dysfunction. The aim of the present study was to explore the role of the AMP-activated protein kinase (AMPK) pathway in the process resulting such phenotypes. Comparative analysis under different environmental conditions revealed downregulation of AMP-activated protein kinase cytalytic subunit 1alpha (AMPKA1), peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC1A) and carnitine palmitoyltransferase 1 (CPT1) genes and upregulation of acetyl-CoA carboxylase α (ACC). In contrast, the presence of fatty acids within the culture medium resulted in a distinct molecular profile in the embryo associated with enhanced levels of ROS, mitochondrial dysfunction and elevated lipid accumulation in bovine embryos. Because AMPKA1 regulates PGC1A, CPT1 and ACC, the results of the present study reveal that AMPK in active its form is the key enzyme promoting lipolysis. Because AMPK1 activity is, in turn, controlled by the AMP : ATP ratio, it is possible to speculate that excessive uptake of exogenous free fatty acids could increase cellular ATP levels as a result of the disturbed ß-oxidation of these external fatty acids and could therefore bypass that molecular feedback mechanism. Subsequently, this condition would cause enhanced generation of ROS, which negatively affect mitochondrial activity. Both enhanced generation of ROS and low mitochondrial activity are suggested to enhance the accumulation of lipids in bovine embryos.

Mitochondrial DNA copy number and biogenesis in different tissues of early- and late-lactating dairy cows.

Energy balance in dairy cows changes during the course of lactation due to alterations in voluntary feed intake and energy required for milk synthesis. To adapt to the demands of lactation, energy metabolism needs to be regulated and coordinated in key organs such as adipose tissue (AT), liver, and mammary gland. Mitochondria are the main sites of energy production in mammalian cells and their number varies depending on age, organ, and physiological condition. The copy number of the mitochondrial genome, the mitochondrial DNA (mtDNA), reflects the abundance of mitochondria within a cell and is regulated by transcriptional and translational factors. Environmental, physiological, and energetic conditions change during lactation and we thus hypothesized that these changes may influence the mtDNA copy number and the abundance of genes regulating mitochondrial biogenesis. Therefore, we aimed to provide an overview of mitochondrial biogenesis in liver, subcutaneous (sc)AT, mammary gland, and peripheral blood cells during early and late lactation in dairy cows. German Holstein cows (n=21) were fed according to their requirements, and biopsies from scAT, liver, mammary gland, and blood were collected in early and late lactation and assayed for relative mtDNA copy numbers and the mRNA abundance of genes regulating mitochondrial biogenesis, such as nuclear-respiratory factor 1 and 2 (NRF-1, NRF-2), mitochondrial transcription factor A (TFAM), and peroxisome proliferator-activated receptor-gamma coactivator 1-α (PGC-1α). The number of mtDNA copies increased from early to late lactation in all tissues, whereas that in peripheral blood cells was greater in early compared with late lactation. Moreover, mitochondrial activity enzymes (i.e., citrate synthase and cytochrome c oxidase) increased from early to late lactation in scAT. Comparing the number of mtDNA copies between tissues and blood in dairy cows, the highest mtDNA content was observed in liver. The mRNA abundance of genes related to mitochondrial biogenesis changed in a tissue-specific manner when comparing early versus late lactation. The mtDNA copy number was associated with transcriptional factors only in AT, suggesting nontranscriptional regulation of mtDNA in the other tissues. We detected strong correlations between peripheral blood mtDNA and tissue mtDNA content in early lactation. Peripheral blood forms an appropriate medium to display the cellular content of mtDNA copy numbers and consequently the cellular energy status of tissues during early lactation.

Changes in endometrial ezrin and cytokeratin 18 expression during bovine implantation and in caruncular endometrial spheroids in vitro.

INTRODUCTION: The feto-maternal interface during bovine implantation was studied in vivo and using three-dimensional bovine endometrial (BCECph) and trophoblast spheroids (CCS), each with underlying fibroblasts. METHODS: The expression of ezrin and cytokeratin 18 (CK18) was analyzed via immunohistochemistry (IHC), RT-PCR and western blotting in bovine endometrium (GD 18-44) with in vivo (VIVO) and in vitro-produced embryos (VITRO). BCECph were stimulated with cotyledon-conditioned media (CCM) and analyzed by TEM/SEM and IHC. CCS were stained (IHC) for TGC markers, to test if spheroidal trophoblast cells had differentiated into TGC. RESULTS: At GD 20, caruncular epithelium (CE) and uterine glands (UG) showed a loss of cytosolic ezrin and CK18 followed by a complete loss of both proteins. At GD 35 both reappeared in CE and UG. The endometrial expression pattern did not differ between VIVO and VITRO. RT-PCR and western blotting confirmed the presence of ezrin and CK18. All spheroids had an outer polarized, cytokeratin and ezrin positive epithelium (CE or trophoblast) with apical microvilli. Stimulation of BCECph with CCM induced similar changes in ezrin expression as observed in endometrial tissue. However, no ultrastructural alterations were found by transmission electron microscopy. Absence of TGC-specific glycoproteins in CCS indicated that TGC differentiation was not induced by three-dimensional culture conditions. DISCUSSION: Ezrin and CK18 are downregulated during implantation in cattle. The expression changes represent a temporal depolarization, which could be important for an establishment of bovine pregnancy. Our in vitro experiments demonstrate that the trophoblast could contribute to this change in vivo.

MicroRNA-regulated molecular mechanism underlying bovine subclinical endometritis.

An impaired uterine environment triggered by the incidence of subclinical endometritis often compromises fertility in the bovine. The uterus is a dynamic organ with tight regulation of specific genes at the transcriptional and translational levels. Herein, we hypothesised that subclinical endometritis alters the expression of uterine microRNAs (miRNAs), which may result in the dysregulation of corresponding target genes and biological pathways. To test this hypothesis, we used a genome-wide RT(2) (Exiqon, Vedbaek, Denmark) miRNA PCR array consisting of 354 miRNA primers and analysed miRNA expression in uterine cytobrush samples taken from cows with and without subclinical endometritis. The results revealed aberrant expression of 23 miRNAs in cows with subclinical endometritis compared with healthy cows. Furthermore, we designed an in vitro endometrial cell culture model challenged by lipopolysaccharide (LPS) to validate the differential regulation of miRNAs in cytobrush samples. Interestingly, we observed similar expression miRNA patterns in cytobrush samples taken from cows with or without subclinical endometritis and in vitro cultured endometrial cells challenged by LPS. To trace signalling pathways and biological functions potentially controlled by the aberrantly expressed miRNAs, we filtered high-ranking target genes from miRBase and analysed them using ingenuity pathway analysis. The gene networks, canonical pathways and biological functions strikingly converged to signalling pathways that mediate inflammatory responses, cellular proliferation, cell movement, the cell cycle and apoptosis in the bovine endometrium. In addition, expression analysis of key genes from the gene networks confirmed their presence and the potential regulation of these genes by uterine miRNAs. Furthermore, luciferase assay data substantiated the primary information from bioinformatic prediction that generated potential target genes for the dysregulated miRNAs in subclinical endometritis. Together, these data suggest the potential regulatory role of uterine miRNAs in the development and progression of bovine subclinical endometritis.

Molecular signatures of bovine embryo developmental competence.

Assessment of the developmental capacity of early bovine embryos is still an obstacle. Therefore, the present paper reviews all current knowledge with respect to morphological criteria and environmental factors that affect embryo quality. The molecular signature of an oocyte or embryo is considered to reflect its quality and to predict its subsequent developmental capacity. Therefore, the primary aim of the present review is to provide an overview of reported correlations between molecular signatures and developmental competence. A secondary aim of this paper is to present some new strategies to enable concomitant evaluation of the molecular signatures of specific embryos and individual developmental capacity.

Expression analysis of regulatory microRNAs in bovine cumulus oocyte complex and preimplantation embryos.

MicroRNAs (miRNAs) are small endogenous molecules that are involved in a diverse of cellular process. However, little is known about their abundance in bovine oocytes and their surrounding cumulus cells during oocyte development. To elucidate this situation, we investigated the relative expression pattern of sets of miRNAs between bovine oocyte and the surrounding cumulus cells during in vitro maturation using miRNA polymerase chain reaction (PCR) array. Results revealed that a total of 47 and 51 miRNAs were highly abundant in immature and matured oocytes, respectively, compared with their surrounding cumulus cells. Furthermore, expression analysis of six miRNAs enriched in oocyte miR-205, miR-150, miR-122, miR-96, miR-146a and miR-146b-5p at different maturation times showed a dramatic decrease in abundance from 0 h to 22 h of maturation. The expression of the same miRNAs in preimplantation stage embryos was found to be highly abundant in early stages of embryo development and decreased after the 8-cell stage to the blastocyst stage following a typical maternal transcript profile. Similar results were obtained by localization of miR-205 in preimplantation stage embryos, in which signals were higher up to the 4-cell stage and reduced thereafter. miR-205 and miR-210 were localized in situ in ovarian follicles and revealed a spatio-temporal expression during follicular development. Interestingly, the presence or absence of oocytes or cumulus cells during maturation was found to affect the expression of miRNAs in each of the two cell types. Hence, our results showed the presence of distinct sets of miRNAs in oocytes or cumulus cells and the presence of their dynamic degradation during bovine oocyte maturation.

Endometrial response of beef heifers on day 7 following insemination to supraphysiological concentrations of progesterone associated with superovulation.

Ovarian stimulation is a routine procedure in assisted reproduction to stimulate the growth of multiple follicles in naturally single-ovulating species including cattle and humans. The aim of this study was to analyze the changes induced in the endometrial transcriptome associated with superovulation in cattle and place these observations in the context of our previous data on changes in the endometrial transcriptome associated with elevated progesterone (P4) concentrations within the physiological range and those changes induced in the embryo due to superovulation. Mean serum P4 concentrations were significantly higher from day 4 to day 7 in superovulated compared with unstimulated control heifers (P < 0.05). Between-group analysis revealed a clear separation in the overall transcriptional profile of endometria from unstimulated control heifers (n = 5) compared with superovulated heifers (n = 5). This was reflected in the number of differentially expressed genes (DEGs) identified between the two groups with 795 up- and 440 downregulated in superovulated endometria. Ten times more genes were altered by superovulation (n = 1,234) compared with the number altered due to elevated P4 within physiological ranges by insertion of a P4-releasing intravaginal device (n = 124) with only 22 DEGs common to both models of P4 manipulation. Fewer genes were affected by superovulation in the embryo compared with the endometrium, (443 vs. 1,234 DEGs, respectively), and the manner in which genes were altered was different with 64.5% of genes up- and 35.5% of genes downregulated in the endometrium, compared with the 98.9% of DEGs upregulated in the embryo. In conclusion, superovulation induces significant changes in the transcriptome of the endometrium which are distinct from those in the embryo.

Transcriptome profile of early mammalian embryos in response to culture environment.

Early embryonic development, the period from maturation until blastocyst formation, is one of the most critical periods of mammalian development involves various morphological, cellular, and biochemical changes related to genomic activity. During the post-fertilization period, several major developmental events occur in the embryo which are regulating by a harmonized expression of genes and strongly influenced by culture conditions. The products of these genes are involved in various biological processes including metabolism, growth factor/cytokine signaling, stress adaptation, transcription and translation, epigenetic regulation of transcription, apoptosis, compaction and blastocyst formation. Post-fertilization culture environment is known to be the most important factor determining the quality of the resulting embryos as indicated in terms of cryo-tolerance and relative abundance of transcripts. However, the exact effect of culture conditions on gene expression and subsequent influences on molecular pathways controlling early development is still unknown. A number of culture environmental factors can influence the gene expression of produced embryos such as media composition, serum supplementation, number of embryos present in the culture drop and gas atmosphere. During the last ten years several studies were concerned with differences in the transcriptome profile of embryos produced under different environmental conditions and its subsequent influence on embryo developmental competence. From these studies, several genes have been determined as candidate genes controlling preimplantation embryo development and affecting its quality. Here we will discuss results of different experiments investigated the effect of different culture conditions on the transcriptome profile of bovine blastocyst. These experiments identified molecular mechanisms and pathways that influenced by culture conditions and this will enable to launch strategies to modify culture conditions to enhance the development of competent blastocyst.

Effects of polymorphonuclear neutrophile infiltration into the endometrial environment on embryonic development in superovulated cows.

Recent studies on bovine uterine disorders have demonstrated that endometrial infiltration with polymorphonuclear neutrophils (PMN) in the postpartum period or at the time of breeding negatively affects reproductive performance. The objective of the present study was therefore to analyze the effect of endometrial PMN infiltration on superovulation outcome. Cows were synchronized and superovulated receiving a total of three artificial inseminations within 24 h. Endometrial cytologic samples were collected by cytobrush technique at first artificial inseminations (AI) (d -1) and before embryo flush (d 7). Embryos were recovered by uterus flushing at Day 7 and evaluated for total cell number and apoptotic cell index. A total of 425 embryos were flushed out of 48 superovulated cows. The PMN dynamics from first AI to flushing had a significant effect on flushing outcome. Significant differences in terms of number of palpable corpora lutea (14.1 vs 7.2) and transferable embryos (8.8 vs 1.9) were found between cows with PMN proportions increasing from zero (0%) at AI to positive proportions (> 0%) at flushing (group PMNZP) and cows with higher endometrial PMN proportions decreasing to lower but still positive proportions from AI to flushing (group PMNHL). Moreover, cows classified to PMN class zero at first AI flushed a significant higher number of total embryos (10.3 vs 6.9) and transferable embryos (6.8 vs 3.7) compared to cows of PMN class positive at first AI (P > 0.05) in our study. Considering a significant interaction effect between PMN class at first AI and flush (P < 0.05), PMN class at first AI (d -1) correlated significantly with number of total flushed and transferable embryos only in combination with a positive PMN class at flush (d 7). Likewise, PMN class at flush (d 7) beard a significant effect on total number of flushed embryos only when classified to PMN class zero at first AI. Collectively, the present work is the first study that demonstrated a significant relationship between endometrial PMN infiltration at first AI as well as PMN dynamic from first AI to time of flush and superovulation outcome.

G6PDH-activity in equine oocytes correlates with morphology, expression of candidate genes for viability, and preimplantative in vitro development.

Efficiencies for in vitro production of equine embryos are still low due to highly variable developmental competences of equine immature oocytes. In contrast to the equine, in vitro developmental competence of immature oocytes has been predicted successfully by the activity of glucose-6-phosphate dehydrogenase (G6PDH) indicated by brilliant cresyl blue (BCB) dye in a range of different species. Therefore, the aim of the present study was to test the association between G6PDH activity in equine oocytes with: (1) cumulus morphology and oocyte properties in terms of diameter and volume; (2) maturational competence; (3) gene expression of certain molecular markers; and (4) in vitro embryo development after intracytoplasmic sperm injection. Equine oocytes were exposed to BCB stain and were classified as BCB+ or BCB- according to their ability to convert the dye from blue to colorless. Additionally, BCB+ and BCB- oocytes were subclassified as having a compact (Cp) or expanded (Ex) cumulus complex. As a result, BCB+ oocytes had a greater proportion of expanded cumulus oocyte complexes compared with BCB- oocytes (71.2% vs. 49.5%). Moreover, we observed a significant difference in oocyte diameter and volume between BCB+ and BCB- oocytes irrespective of cumulus morphology. BCB+ oocytes reached a higher maturation rate compared with BCB- oocytes (59.0% vs. 28.7%). Regarding the analyzed candidate genes, relative transcript abundance was significantly different for nine genes. The expression of eight genes was significantly higher (P < 0.05) for BCB+ oocytes, including ATPV6E, IF-3, TFAM, DNMT1, STAT3, Aurora-A, ODC1, and CKS2 whereas BCB- oocytes showed higher in expression of COX1. These results are in line with the observed developmental competence. Cleavage rate (45.9% vs. 29.0%) and percentage of embryos that reached the blastocyst stage (9.2% vs. 1.4%) were significantly higher for embryos derived from BCB+ oocytes compared with BCB- oocytes. In conclusion, the present study provides evidence that G6PDH-activity in immature equine oocytes is a useful predictor for subsequent in vitro developmental competence.

Bovine blastocysts with developmental competence to term share similar expression of developmentally important genes although derived from different culture environments.

This study was conducted to investigate the gene expression profile of in vivo-derived bovine embryo biopsies based on pregnancy outcomes after transferring to recipients. For this, biopsies of 30-40% embryos were taken from grade I blastocysts (International Embryo Transfer Society Manual) and the remaining 60-70% of the intact embryos were transferred to recipients. Frozen biopsies were pooled into three distinct groups based on the pregnancy outcome after transferring the corresponding parts, namely those resulting in no pregnancy (NP), pregnancy loss (PL), and calf delivery (CD). Array analysis revealed a total of 41 and 43 genes to be differentially expressed between biopsies derived from blastocysts resulting in NP versus CD and PL versus CD respectively. Genes regulating placental development and embryo maternal interaction (PLAC8) were found to be upregulated in embryo biopsies that ended up with CD. Embryo biopsies that failed to induce pregnancy were enriched with mitochondrial transcripts (Fl405) and stress-related genes (HSPD1). Overall, gene expression profiles of blastocysts resulting in NP and CD shared similar expression profiles with respect to genes playing significant roles in preimplantation development of embryo. Finally, comparing the transcript signatures of in vivo- and in vitro-derived embryos with developmental competence to term revealed a similarity in the relative abundance of 18 genes. Therefore, we were able to present a genetic signature associated with term developmental competence independent of the environmental origin of the transferred blastocysts.

Evaluation of bovine zona pellucida characteristics in polarized light as a prognostic marker for embryonic developmental potential.

It has previously been demonstrated that zona pellucida imaging of human oocytes using polarized light microscopy is a clinically applicable method for the noninvasive assessment of oocyte quality. This study was designed to investigate whether zona pellucida characteristics of bovine oocytes and zygotes in polarized light may similarly serve as a useful marker for developmental competence in bovine reproductive biotechnologies. Zona birefringence intensity parameters of 2862 oocytes/zygotes were objectively evaluated with an automatic analysis system and correlated with oocyte/zygote quality. In detail, immature oocytes of good quality assessed with brilliant cresyl blue staining showed significantly lower zona birefringence than poor-quality counterparts (P<0.001). After in vitro maturation and classification according to maturational status, the birefringence intensity parameters were significantly different in those oocytes that reached metaphase II compared with arrested stages (P<0.001). Following either parthenogenetic activation or IVF with subsequent in vitro culture in a well-of-the-well system until day 9, superior development as determined by cleavage, blastocyst formation, and hatching ability was associated with lower zona birefringence intensity parameters. When early zygote-stage embryos were selected and assorted in groups based on zona birefringence (high/medium/low), the group of embryos derived from high-birefringence zygotes displayed a significantly compromised developmental potential compared with low-birefringence zygotes. These results clearly show that developmentally competent bovine oocytes/zygotes exhibit lower zona birefringence intensity parameters. Therefore, birefringence imaging of zona pellucida is a suitable technique to predict bovine preimplantation embryo development.

Transcriptome dynamics and molecular cross-talk between bovine oocyte and its companion cumulus cells.

BACKGROUND: The bi-directional communication between the oocyte and its companion cumulus cells (CCs) is crucial for development and functions of both cell types. Transcripts that are exclusively expressed either in oocytes or CCs and molecular mechanisms affected due to removal of the communication axis between the two cell types is not investigated at a larger scale. The main objectives of this study were: 1. To identify transcripts exclusively expressed either in oocyte or CCs and 2. To identify those which are differentially expressed when the oocyte is cultured with or without its companion CCs and vice versa. RESULTS: We analyzed transcriptome profile of different oocyte and CC samples using Affymetrix GeneChip Bovine Genome array containing 23000 transcripts. Out of 13162 genes detected in germinal vesicle (GV) oocytes and their companion CCs, 1516 and 2727 are exclusively expressed in oocytes and CCs, respectively, while 8919 are expressed in both. Similarly, of 13602 genes detected in metaphase II (MII) oocytes and CCs, 1423 and 3100 are exclusively expressed in oocytes and CCs, respectively, while 9079 are expressed in both. A total of 265 transcripts are differentially expressed between oocytes cultured with (OO+CCs) and without (OO-CCs) CCs, of which 217 and 48 are over expressed in the former and the later groups, respectively. Similarly, 566 transcripts are differentially expressed when CCs mature with (CCs+OO) or without (CCs-OO) their enclosed oocytes. Of these, 320 and 246 are over expressed in CCs+OO and CCs-OO, respectively.While oocyte specific transcripts include those involved in transcription (IRF6, POU5F1, MYF5, MED18), translation (EIF2AK1, EIF4ENIF1) and CCs specific ones include those involved in carbohydrate metabolism (HYAL1, PFKL, PYGL, MPI), protein metabolic processes (IHH, APOA1, PLOD1), steroid biosynthetic process (APOA1, CYP11A1, HSD3B1, HSD3B7). Similarly, while transcripts over expressed in OO+CCs are involved in carbohydrate metabolism (ACO1, 2), molecular transport (GAPDH, GFPT1) and nucleic acid metabolism (CBS, NOS2), those over expressed in CCs+ OO are involved in cellular growth and proliferation (FOS, GADD45A), cell cycle (HAS2, VEGFA), cellular development (AMD1, AURKA, DPP4) and gene expression (FOSB, TGFB2). CONCLUSION: In conclusion, this study has generated large scale gene expression data from different oocyte and CCs samples that would provide insights into gene functions and interactions within and across different pathways that are involved in the maturation of bovine oocytes. Moreover, the presence or absence of oocyte and CC factors during bovine oocyte maturation can have a profound effect on transcript abundance of each cell types, thereby showing the prevailing molecular cross-talk between oocytes and their corresponding CCs.