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The objectives of the study were to evaluate the morphological quality of oocytes in repeat breeder and early lactation cows and to determine the possible associations between the quality of oocytes and a range of blood metabolites. Oocyte quality and a range of metabolites were compared between 29 repeat breeder and 13 early lactation cows. The yield of oocytes from the repeat breeders was lower than that from the early lactation cows (4.4 ± 0.2 vs 5.4 ± 0.6, p < 0.05). Percentages of abnormal oocytes for the repeat breeders and the early lactation cows were 52.5% and 37.9%, respectively (p < 0.001). An excess of abnormal oocytes to normal was found in 55.2% of the studied repeat breeders (65.8% vs 34.2%, p < 0.05). Total protein, glucose and aspartate aminotransferase did not differ (p > 0.05) between the repeat breeders with an excess of abnormal oocytes (81 ± 1.0 g/l, 3.5 ± 1.0 mmol/l and 68.5 ± 3.7 U/l), those with the prevalence of normal oocytes (84 ± 1.0 g/l, 3.6 ± 0.1 mmol/l and 73.2 ± 3.5 U/l) and the early lactation cows (83 ± 2.0 g/l, 3.7 ± 0.1 mmol/l and 74.5 ± 3.6 U/I). The repeat breeders with an excess of abnormal oocytes had higher (p < 0.05) urea (5.2 ± 0.2 mmol/l) level than in those with the prevalence of normal oocytes (4.8 ± 0.2 mmol/l) and the early lactation cows (4.7 ± 0.2 mmol/l). A trend for higher total cholesterol and lactate dehydrogenase activity was found in the repeat breeders with an excess of abnormal oocytes. In conclusion, it is suggested that possible causes of repeat breeding in dairy cows may include impaired oocytes. An excess of abnormal oocytes in the repeat breeder cows was associated with elevated blood plasma levels of urea. [ABSTRACT FROM AUTHOR]

Abstract: The numbers of spermatozoa per insemination and the site of semen deposition in the uterine horn appear to interact to influence pregnancy rate. In two experiments, the effect of a single low dose (2×106 spermatozoa) intracornual insemination (LD-ICI) on bovine pregnancy rate was compared with that of intracornual (SD-ICI) and conventional (SD-AI) inseminations of 40×106 spermatozoa. In Experiment 1, 157 cows were treated twice with PGF2α at a 14-day interval and inseminated at a fixed time (80–82h) after the second PGF2α injection using LD-ICI (n =44), SD-ICI (n =61) or SD-AI (n =52). In LD-ICI and SD-ICI groups, semen was deposited in the horn ipsilateral to the ovulatory follicle close to the utero-tubal junction (LD-ICI-UTJ, n =33 and SD-ICI-UTJ, n =41) or in the middle part of the horn (LD-ICI-MH, n =11 and SD-ICI-MH, n =20). Pregnancy rates after LD-ICI-UTJ, LD-ICI-MH, SD-ICI-UTJ and SD-ICI-MH were 27%, 27%, 39% and 35%, respectively (P >0.05). The total pregnancy rate after LD-ICI (27%) did not differ (P >0.05) from that after SD-ICI (37%) or SD-AI (34%). In Experiment 2 (field trial), 362 cows were allotted, at spontaneous estrus, to LD-ICI-UTJ (n =86), LD-ICI-MH (n =97) or SD-AI (n =179). Pregnancy rates after LD-ICI and SD-AI were 47% and 45%, respectively (P >0.05). After LD-ICI-UTJ, the pregnancy rate (54%) did not differ significantly (P >0.05) to that obtained after LD-ICI-MH (41%) and after SD-AI (45%). The results of the study show that the single intracornual insemination of cows with 2×106 spermatozoa at fixed time, 80–82h after the second PGF2α injection or at spontaneous estrus resulted in similar pregnancy percentage as intracornual and conventional inseminations with 40×106 spermatozoa per semen dose. With intracornual insemination using low or standard dose of spermatozoa, the pregnancy rates were not significantly affected by the exact site of semen deposition in the uterine horn, near the utero-tubal junction or in the middle part. [Copyright &y& Elsevier]

Contents The predictability of semen quality of mature sires from measurements at an early age is not well established. The aim of the present study was to determine age-dependent changes in the quality of bull semen from six Estonian Holstein (EHF) bulls, processed when the sires were 3, 5 and 7 years old. Fertility data such as 60-day non-return to oestrus rates (60d-NRRs) were available for 3-year-old bulls. From each batch, semen straws were analysed immediately after thawing [i.e. post-thaw (PT)] (controls) and after a swim-up (SU) procedure. The analyses comprised subjective and computerized measurements of sperm motility using computer-assisted sperm analysis (CASA) as well as estimations of sperm concentration, morphology and membrane integrity. There was a significant (p < 0.05) increase in the percentage of sperm motility (SU), membrane integrity (PT, SU) and normal tail and acrosome morphology (SU) with an increase in the age of the sires. The percentage of total motile spermatozoa PT measured by CASA correlated between 3- and 7-, and between 5- and 7-year-old bulls (p < 0.05). In addition, the proportion of head abnormalities tended to correlate between all three age groups both PT and after SU (p < 0.1). The sperm parameters correlating with fertility were average path velocity (VAP) (p < 0.001), total motility as measured by CASA (p < 0.01), linearly motile spermatozoa (p < 0.05) and CASA-assessed numbers of motile spermatozoa (p < 0.05), all after SU selection. The results showed that overall semen quality examined at 3 years of age is related to the semen parameters later in bulls’ life. Moreover, CASA-assessed motility after SU seems to be a reliable marker for semen quality assessment as it shows correlation not only between the ages, but also to field fertility. [ABSTRACT FROM AUTHOR]

An abstract of the study "Effects of Culture Conditions and Gene Transfection on the Development of Bovine Somatic Cell Nuclear Transfer Embryos," by M. Plaas et al, is presented.

This paper presents the preliminary results of whole genome resequencing of the Holstein cow using the SOLiD 4 System. The aim of this study was to obtain a high-quality Holstein cow genome reference sequence, which could be used as a reference for genomic studies on the Estonian Holstein cattle. Furthermore, the new reference sequence would be made available for other research groups. We generated one mate-paired library and one fragment library from 30 μg of genomic DNA. Libraries were sequenced in 4 flow cells. Colour space fasta files (.csfasta) and appropriate quality files (.qual) were mapped and paired to the reference cow (Bos taurus) genome assembly from Oct. 2007 (Baylor 4.0/bosTau4). Mapping and pairing was performed using the Max Mapper algorithm implemented in the Bioscope Software (version 1.3). Initial sequencing resulted in the 2 842 744 008 fifty-basepair reads. Average mapping efficiency with mismatch penalty -2.00 and clearzone 5 was 73.3%. Altogether 2 065 066 215 reads and 92 778 710 937 bp were successfully mapped, resulting in 35.2 coverage. Pairing indicated that the insert range was 665 to 2195 bp and mean insert size was 1363 bp. Tertiary analysis found 5 472 870 SNP in the cow genome; 3 517 351 were heterozygous and 1 955 519 were homozygous variants. Also, 3 747 199 were transition SNP and 1 093 307 were transversion SNP, with a transition-transversion ratio of 2.17:1.00. Annotation revealed that only 889 901 of all discovered SNP were annotated in the SNP database dbSNP. This means that around 4 582 969 SNP were novel. The number of large indels was 144 035, out of which 68 817 were heterozygous and 75 218 were homozygous variants. The longest deletion was 15 089 bp and there were 18 deletions between 10 000 and 20 000 bp. The largest insertion range was 1000 to 5000 bp and there were 358 insertions falling into this span. Interestingly, the most numerous group of deletions was between 200 and 500 bp and between 100 and 200 bp. Altogether, in these size groups there were 114 578 deletions. Large indels variations accounted for 48 582 675 bp of the entire genome. Analysis of the small indel polymorphisms identified 452 113 small indels, out of which 287 491 were heterozygous and 164 622 were homozygous. Only 1197 small indels were listed in the dbSNP. Most of the small indels were single nucleotide insertions/deletions (261 897). Small indels accounted for the total variation of 1 722 303 nucleotides in the genome. Finally, we identified 287 inversions (largest 151 000 bp) in the genome of the cow. In conclusion, the genome of the cow contains huge amounts of still unknown variations. Better knowledge of these variations could explain significant phenotypic differences (e.g. reproduction) between different breeds. The European Regional Development Fund together with the Archimedes Foundation, target finance grant from the Ministry of Education and Science SF1080045s07, grant from the Estonian University of Life Sciences P8001 and Estonian Science Foundation grant GARFS7479 supported this study. [ABSTRACT FROM AUTHOR]

An abstract of the article "ENDURANCE AND SPEED CAPACITY OF AKHAL-TEKE HORSES" by K. Leisson, K. Alev, P. Kaasik, Ü. Jaakma, and T. Seene is presented.

We analysed the results of insemination (AI) with 2.2 million X-chromosome-bearing frozen–thawed sperm in 2283 Estonian Holstein (EHF) heifers on 7 dairy herds. The heifers of 11 to 18 months of age were inseminated with sexed sperm or unsexed control semen doses (15×106sperm) from 10 different bulls either 1) at fixed time following synchronization of oestrus by 2 injections of PGF2α, 2) at visually detected spontaneous oestrus or 3) at oestrus displayed after a single injection of PGF2α. At AI, the presence and intensity of estrous signs (vulvar edema, hyperemia, discharge of mucus and an ease to pass through the cervix by catheter) were recorded. Pregnancy status of heifers was diagnosed by rectal palpation of the uterus 45–60 days after AI. Statistical analyses were performed using the SAS package (1999; SAS Institute Inc., Cary, NC, USA). The pregnancy rate of heifers after fixed-time AI at synchronized oestrus was 42.7%, which is about 80% of control unsexed semen doses (53.0%, P0.05) from conventional insemination into the uterine body (41.9%). The pregnancy rates after AI of heifers with sexed sperm at spontaneous oestrus (55.9%) or oestrus displayed following a single PGF2αtreatment (50.8%) did not differ between each other, but were higher than at fixed-time AI (42.7%; P<0.05). The pregnancy rates after AI with sexed sperm at spontaneous oestrus and induced oestrus were about 90% and 85% of that of unsexed semen doses, respectively (P<0.05). The pregnancy rates varied among the farms and among the bulls. For some farms and bulls the pregnancy rates with sexed sperm doses were similar to the pregnancy rates with unsexed regular semen doses. The pregnancy rates did not differ between heifers housed in tie-stalls or free-stalls. Pooled across sperm deposition sites, heifers that showed strong estrous signs at fixed-time AI with sexed sperm had 2.7 times higher pregnancy rate than heifers with weak signs of oestrus. The difference in pregnancy rates was 1.2 times higher in heifers with strong estrous signs when unsexed semen was used at fixed-time AI (P<0.05) and when the heifers were inseminated with sexed or unsexed sperm doses at visually detected spontaneous or induced oestrus. The mean age and body weight did not differ between the heifers that conceived and those that failed irrespective of AI treatment. In conclusion, insemination of heifers with sexed sperm at spontaneous oestrus or oestrus induced by PGF2αtreatment resulted in higher pregnancy rates than insemination at fixed time after oestrus synchronization. Intracornual deposition of semen did not improve the pregnancy rate. However, good reproductive status of a herd along with the selection of heifers according to the intensity of oestrus expression improves the efficiency of using sexed sperm for the insemination of dairy heifers. The study was supported by the ESF grant 7814. [ABSTRACT FROM AUTHOR]

Background: Embryonic genome activation (EGA) is a critical step in early embryonic development, as it marks the transition from relying on maternal factors to the initiation of transcription from embryo's own genome. The factors associated with EGA are not well understood and need further investigation. PRD-like (PRDL) homeodomain transcription factors (TFs) are considered to play crucial roles in this early event during development but these TFs have evolved differently, even within mammalian lineages. Different numbers of PRDL TFs have been predicted in bovine (Bos taurus); however, their divergent evolution requires species-specific confirmation and functional investigations., Results: In this study, we conducted molecular cloning of mRNAs for the PRDL TFs ARGFX, DUXA, LEUTX, NOBOX, TPRX1, TPRX2, and TPRX3 in bovine oocytes or in vitro fertilized (IVF) preimplantation embryos. Our results confirmed the expression of PRDL TF genes in early bovine development at the cDNA level and uncovered their structures. For each investigated PRDL TF gene, we isolated at least one homeodomain-encoding cDNA fragment, indicative of DNA binding and thus potential role in transcriptional regulation in developing bovine embryos. Additionally, our cDNA cloning approach allowed us to reveal breed-related differences in bovine, as evidenced by the identification of a high number of single nucleotide variants (SNVs) across the PRDL class homeobox genes. Subsequently, we observed the prediction of the 9aa transactivation domain (9aaTAD) motif in the putative protein sequence of TPRX3 leading us to conduct functional analysis of this gene. We demonstrated that the TPRX3 overexpression in bovine fibroblast induces not only protein-coding genes but also short noncoding RNAs involved in splicing and RNA editing. We supported this finding by identifying a shared set of genes between our and published bovine early embryo development datasets., Conclusions: Providing full-length cDNA evidence for previously predicted homeobox genes that belong to PRDL class improves the annotation of the bovine genome. Updating the annotation with seven developmentally-important genes will enhance the accuracy of RNAseq analysis with datasets derived from bovine preimplantation embryos. In addition, the absence of TPRX3 in humans highlights the species-specific and TF-specific regulation of biological processes during early embryo development., (© 2024. The Author(s).)

Purpose: Ovarian tissue cryopreservation is vital for fertility preservation, yet its effect on ovarian tissue follicle survival and transcriptomic signature requires further investigation. This study delves into the effects of vitrification on tissue morphology, function, and transcriptomic changes, helping to find possibilities for vitrification protocol improvements., Methods: Ovarian cortex from 19 bovine animals were used to conduct pre- and post-vitrification culture followed by histological assessment, immunohistochemistry, and TUNEL assay. Follicles' functionality was assessed for viability and growth within the tissue and in isolated cultures. RNA-sequencing of ovarian tissue was used to explore the transcriptomic alterations caused by vitrification., Results: Follicle density, cell proliferation, and DNA damage in ovarian stroma were unaffected by vitrification. However, vitrified cultured tissue exhibited reduced follicle density of primordial/primary and antral follicles, while freshly cultured tissue manifested reduction of antral follicles. Increased stromal cell proliferation and DNA damage occurred in both groups post-culture. Isolated follicles from vitrified tissue exhibited similar viability to fresh follicles until day 4, after which the survival dropped. RNA-sequencing revealed minor effects of vitrification on transcriptomic signatures, while culture induced significant gene expression changes in both groups. The altered expression of WNT and hormonal regulation pathway genes post-vitrification suggests the molecular targets for vitrification protocol refinement., Conclusion: Vitrification minimally affects tissue morphology, follicle density, and transcriptomic signature post-thawing. However, culture revealed notable changes in vitrified tissue samples, including reduced follicle density, decreased isolated follicle survival, and alteration in WNT signalling and ovarian hormonal regulation pathways, highlighted them as possible limitations of the current vitrification protocol., (© 2024. The Author(s).)

This study investigated whether metabolomic fingerprints of bovine embryo growth media improve the prediction of successful embryo implantation. In this prospective cohort study, the metabolome from in vitro-produced day 7 blastocysts with successful implantation ( n = 11), blastocysts with failed implantation ( n = 10), and plain culture media without embryos ( n = 5) were included. Samples were analyzed using an AbsoluteIDQ ® p180 Targeted Metabolomics Kit with LC-MS/MS, and a total of 189 metabolites were analyzed from each sample. Blastocysts that resulted in successful embryo implantation had significantly higher levels of methionine sulfoxide ( p < 0.001), DOPA ( p < 0.05), spermidine ( p < 0.001), acetylcarnitine-to-free-carnitine ratio ( p < 0.05), C2 + C3-to-free-carnitine ratio ( p < 0.05), and lower levels of threonine (nep < 0.001) and phosphatidylcholine PC ae C30:0 ( p < 0.001) compared to control media. However, when compared to embryos that failed to implant, only DOPA, spermidine, C2/C0, (C2 + C3)/C0, and PC ae C30:0 levels differentiated significantly. In summary, our study identifies a panel of differential metabolites in the culture media of bovine blastocysts that could act as potential biomarkers for the selection of viable blastocysts before embryo transfer.

Recent advances in preimplantation embryo diagnostics enable a wide range of applications using single cell biopsy and molecular-based selection techniques without compromising embryo production. This study was conducted to develop a single cell embryo biopsy technique and gene expression analysis method with a very low input volume to ensure normal embryo development and to see if there are differences in gene expression profiles between day-5 biopsied bovine embryos that developed into blastocysts and embryos arrested at morula stage. Out of the 65 biopsied morulae, 32 developed to blastocysts (49.2%). Out of the 13,580 successfully annotated genes, 1204 showed a difference in mRNA expression level. Out of these, 155 genes were expressed in embryos developing to blastocysts. The pathway enrichment analysis revealed significant enrichment in "organelle biogenesis and maintenance", "mRNA splicing" and "mitochondrial translation" pathways. These findings suggest principal differences in gene expression patterns and functional networks of embryos able to reach the blastocyst stage compared to embryos arrested in development. Our preliminary data suggest that single blastomere biopsy and selected gene expression profiles at morula stage could offer additional possibilities for early preimplantation embryo selection before transfer.

Mammalian conception involves a multitude of reciprocal interactions via a molecular dialogue between mother and conceptus. Extracellular vesicles (EVs) are secreted membrane-encapsulated particles that mediate cell-to-cell communication in various contexts. EVs, which are present in seminal, follicular, oviductal, and endometrial fluids, as well as in embryo secretions, carry molecular constituents that impact gamete maturation, fertilization, early embryo development, and embryo-maternal communication. The distribution, concentration, and molecular cargo of EVs are regulated by steroid hormones and the health status of the tissue of origin, and thus are influenced by menstrual phase, stage of conception, and the presence of infertility-associated diseases. EVs have been recognized as a novel source of biomarkers and potential reproductive medicine therapeutics, particularly for assisted reproductive technology (ART). There are still many technological and scientific hindrances to be overcome before EVs can be used in clinical diagnostic and therapeutic ART applications. Issues to be resolved include the lack of standardized measurement protocols and an absence of absolute EV quantification technologies. Additionally, clinically suitable and robust EV isolation methods have yet to be developed. In this review, we provide an overview of EV-mediated interactions during the early stages of reproduction from gamete maturation to embryo implantation and then outline the technological progress that must be made for EV applications to be translated to clinical settings., (© The Author(s) 2022. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

While follicular fluid (FF) is known to enhance the functional properties of spermatozoa, the role of FF-derived extracellular vesicles (EVs) in this respect is unknown. We hypothesized that bovine FF EVs convey signals to spermatozoa supporting sperm viability, inducing sperm capacitation and acrosome reaction. In this study, the effects of bovine FF EVs on sperm functions are evaluated. Irrespective of the size of the follicles which FF EVs had originated from, they were capable of supporting sperm viability, inducing capacitation and acrosome reaction. These effects were specific to the source of bovine FF EVs, as human-cell-line-derived or porcine FF EVs did not affect spermatozoa viability or induced capacitation and acrosome reaction. A minimum of 5 × 10 5 EVs/mL was adequate to maintain sperm viability and induce capacitation and acrosome reaction in spermatozoa. Interestingly, with FF EV trypsin treatment, FF EVs lost their ability to support sperm functions. In conclusion, this study demonstrates that bovine FF EVs can support spermatozoa function and may contribute to a favorable periconceptional microenvironment. This is an important aspect of the interactions between different sexes at the earliest stages of reproduction and helps to understand molecular mechanisms modulating processes such as sperm competition and female cryptic choice.

Background: The period of time when the embryo and the endometrium undergo significant morphological alterations to facilitate a successful implantation-known as "window of implantation"-is a critical moment in human reproduction. Embryo and the endometrium communicate extensively during this period, and lipid bilayer bound nanoscale extracellular vesicles (EVs) are purported to be integral to this communication., Methods: To investigate the nature of the EV-mediated embryo-maternal communication, we have supplemented trophoblast analogue spheroid (JAr) derived EVs to an endometrial analogue (RL 95-2) cell layer and characterized the transcriptomic alterations using RNA sequencing. EVs derived from non-trophoblast cells (HEK293) were used as a negative control. The cargo of the EVs were also investigated through mRNA and miRNA sequencing., Results: Trophoblast spheroid derived EVs induced drastic transcriptomic alterations in the endometrial cells while the non-trophoblast cell derived EVs failed to induce such changes demonstrating functional specificity in terms of EV origin. Through gene set enrichment analysis (GSEA), we found that the response in endometrial cells was focused on extracellular matrix remodelling and G protein-coupled receptors' signalling, both of which are of known functional relevance to endometrial receptivity. Approximately 9% of genes downregulated in endometrial cells were high-confidence predicted targets of miRNAs detected exclusively in trophoblast analogue-derived EVs, suggesting that only a small proportion of reduced expression in endometrial cells can be attributed directly to gene silencing by miRNAs carried as cargo in the EVs., Conclusion: Our study reveals that trophoblast derived EVs have the ability to modify the endometrial gene expression, potentially with functional importance for embryo-maternal communication during implantation, although the exact underlying signalling mechanisms remain to be elucidated., (© 2021. The Author(s).)

Embryo-derived extracellular vesicles (EVs) may play a role in mediating the embryo-maternal dialogue at the oviduct, potentially carrying signals reflecting embryo quality. We investigated the effects of bovine embryo-derived EVs on the gene expression of bovine oviductal epithelial cells (BOECs), and whether these effects are dependent on embryo quality. Presumptive zygotes were cultured individually in vitro in culture medium droplets until day 8 while their development was assessed at day 2, 5 and 8. Conditioned medium samples were collected at day 5 and pooled based on embryo development (good quality embryo media and degenerating embryo media). EVs were isolated from conditioned media by size exclusion chromatography and supplemented to primary BOEC monolayer cultures to evaluate the effects of embryo-derived EVs on gene expression profile of BOEC. Gene expression was quantified by RNA-seq and RT-qPCR. A total of 7 upregulated and 18 downregulated genes were detected in the BOECs supplemented with good quality embryo-derived EV compared to the control. The upregulated genes included interferon-τ-induced genes, such as OAS1Y, MX1 and ISG15, which have previously been reported as upregulated in the oviductal epithelial cells in the presence of embryos. Of the upregulated genes, OAS1Y and MX1 were validated with RT-qPCR. In contrast, only one differentially expressed gene was detected in BOECs in response to degenerating embryo-derived EVs, suggesting that oviductal responses are dependent on embryo quality. Our results support the hypothesis that embryo-derived EVs are involved in embryo-maternal communication at the oviduct and the oviductal response is dependant on the embryo quality. KEY MESSAGES: • Extracellular vesicles (EVs) released by individually cultured pre-implantation bovine embryos can alter the gene expression of primary oviductal epithelial cells. • The oviductal response, in terms of gene expression, to the bovine embryo-derived EVs varied depending on the embryo quality. • In vivo, the oviduct may have the ability to sense the quality of the pre-implantation embryos. • The observed effect of embryo-derived EVs on oviductal epithelial cells could serve as a non-invasive method of evaluating the embryo quality.

Cell-free RNAs have the potential to act as a means of gene expression regulation between cells and are therefore used as diagnostic markers describing the state of tissue environment. The origin and functions of such RNAs in human ovarian follicle, the environment of oocyte maturation, are unclear. The current study investigates the difference in the microRNA profiles of fertile women and polycystic ovary syndrome (PCOS) patients in three compartments from the same preovulatory follicle: mural granulosa cells (MGC), cell-free follicular fluid (FF), and extracellular vesicles (EV) of the FF by small RNA sequencing. In silico analysis was used for the prediction and over-representation of targeted pathways for the detected microRNAs. PCOS follicles were distinguished from normal tissue by the differential expression of 30 microRNAs in MGC and 10 microRNAs in FF (FDR < 0.1) that commonly regulate cytokine signaling pathways. The concentration of EV-s was higher in the FF of PCOS patients ( p = 0.04) containing eight differentially expressed microRNAs ( p < 0.05). In addition, we present the microRNA profiles of MGC, FF, and EV in the fertile follicle and demonstrate that microRNAs loaded into EVs target mRNAs of distinct signaling pathways in comparison to microRNAs in FF. To conclude, the three follicular compartments play distinct roles in the signaling disturbances associated with PCOS.

The capability of spermatozoa to directly influence maternal gene expression is already established. Indeed, some of the changes induced by spermatozoa may have a direct functional importance in the pre-conceptional period. Although the mechanisms underlying these sperm-maternal interactions are not well characterized, it is possible that they could involve ligands that are released from the spermatozoa. This study therefore aimed to test whether physical contact between bovine spermatozoa and bovine oviductal epithelial cells (BOECs) is a prerequisite for spermatozoa-induced gene expression changes. We used two co-culture models: a contact co-culture model in which spermatozoa interact directly with BOECs, and a non-contact co-culture model in which an insert with the pore size of 0.4 μm was placed between spermatozoa and BOECs. Messenger RNA sequencing analysis of BOECs by RNA-seq revealed ten differentially expressed genes in contact system and 108 differentially expressed genes in the non-contact system after 10 h of co-culture. Retinol metabolism pathway and ovarian steroidogenesis pathway were significantly enriched in the non-contact co-culture system. Q-PCR analysis revealed that transcriptional responses can be rapid, with increased expression of four genes (DHRS3, CYP1B1, PTGS2, and ATF3) detectable within just 90 min of co-incubation, but with expression levels highly dependent on the type of co-culture system. The findings from our study demonstrate that direct contact with spermatozoa is not necessary to induce changes in gene expression of oviductal epithelial cells, suggesting that spermatozoa may be able to signal to maternal tissues in advance of their arrival.

While follicular fluid (FF) is well known to provide an optimal environment for oogenesis, its functional roles following its release into the oviduct during ovulation are currently elusive. We hypothesized that FF and FF-derived extracellular vesicles (EVs) may be conveyors of signals capable of inducing functionally-relevant transcriptional responses in oviductal cells. The aim of this study was, therefore, to evaluate the effect of FF and FF-derived EVs on the transcriptome of primary bovine oviductal epithelial cells (BOECs). We examined the gene expression of BOECs in three conditions: BOECs cultured with FF, FF-derived EVs, and without supplementations. For each condition, cells were cultured for 6 and 24 h. RNA sequencing results revealed that FF had a stronger effect on BOECs gene expression compared to EVs. We detected 488 and 1998 differentially expressed genes (DEGs) with FF treatment in 6 and 24 h, respectively, whereas only 41 DEGs were detected at 6 h following EV treatment. Pathway analysis of the FF-induced DEGs showed that several pathways were highly enriched, notably oxidative phosphorylation, thermogenesis, arachidonic acid metabolism, and steroid hormone biosynthesis. Some of these pathways have a role in sperm survival, fertilization, and early embryo development. In conclusion, the findings of our study demonstrate for the first time that bovine FF and FF-derived EVs can induce changes in the gene expression of the bovine oviductal cells which, although observed in vitro, may be reflective of in vivo responses which may contribute to a favorable periconceptional microenvironment for sperm survival, fertilization, and early embryo development.

Extracellular vesicles (EVs) are membrane-bound biological nanoparticles (NPs) and have gained wide attention as potential biomarkers. We aimed to isolate and characterize EVs from media conditioned by individually cultured preimplantation bovine embryos and to assess their relationship with embryo quality. Presumptive zygotes were cultured individually in 60 μl droplets of culture media, and 50 μl of media were collected from the droplets either on day 2, 5 or 8 post-fertilization. After sampling, the embryo cultures were continued in the remaining media until day 8, and the embryo development was evaluated at day 2 (cleavage), day 5 (morula stage) and day 8 (blastocyst stage). EVs were isolated using qEVsingle® columns and characterized. Based on EV Array, EVs isolated from embryo conditioned media were strongly positive for EV-markers CD9 and CD81 and weakly positive for CD63 and Alix among others. They had a cup-like shape typical to EVs as analyzed by transmission electron microscopy and spherical shape in scanning electron microscopy, and hence regarded as EVs. However, the NPs isolated from control media were negative for EV markers. Based on nanoparticle tracking analysis, at day 2, the mean concentration of EVs isolated from media conditioned by embryos that degenerated after cleaving (8.25 × 10 8 /ml) was higher compared to that of embryos that prospectively developed to blastocysts (5.86 × 10 8 /ml, p < 0.05). Moreover, at day 8, the concentration of EVs isolated from media conditioned by degenerating embryos (7.17 × 10 8 /ml) was higher compared to that of blastocysts (5.68 × 10 8 /ml, p < 0.05). Furthermore, at day 8, the mean diameter of EVs isolated from media conditioned by degenerating embryos (153.7 nm) was smaller than EVs from media conditioned by blastocysts (163.5 nm, p < 0.05). In conclusion, individually cultured preimplantation bovine embryos secrete EVs in the culture media and their concentration and size are influenced by embryo quality and may indicate their prospective development potential., Competing Interests: Declaration of competing interest The authors have no conflicts of interest., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-seq) has revolutionized our understanding of chromatin-related biological processes. The method, however, requires thousands of cells and has therefore limited applications in situations where cell numbers are limited. Here we describe a novel method called Restriction Assisted Tagmentation Chromatin Immunoprecipitation (RAT-ChIP) that enables global histone modification profiling from as few as 100 cells. The method is simple, cost-effective and takes a single day to complete. We demonstrate the sensitivity of the method by deriving the first genome-wide maps of histone H3K4me3 and H3K27me3 modifications of inner cell mass and trophectoderm of bovine blastocyst stage embryos., Competing Interests: The RAT-ChIP method is subject to a patent application (P1433PC00) by TO, AK and AS.

Background: Successful establishment of pregnancy hinges on appropriate communication between the embryo and the uterus prior to implantation, but the nature of this communication remains poorly understood. Here, we tested the hypothesis that the endometrium is receptive to embryo-derived signals in the form of RNA., Methods: We have utilized a non-contact co culture system to simulate the conditions of pre implantation environment of the uterus. We bioorthogonally tagged embryonic RNA and tracked the transferred transcripts to endometrium. Transferred transcripts were separated from endometrial transcripts and sequenced. Changes in endometrial transcripts were quantified using quantitative PCR., Results: We show that three specific transcripts are transferred to endometrial cells. We subsequently demonstrate a role of extracellular vesicles (EVs) in this process, as EVs obtained from cultured trophoblast spheroids incubated with endometrial cells induced down-regulation of all the three identified transcripts in endometrial cells. Finally, we show that EVs/nanoparticles captured from conditioned culture media of viable embryos as opposed to degenerating embryos induce ZNF81 down-regulation in endometrial cells, hinting at the functional importance of this intercellular communication., Conclusion: Ultimately, our findings demonstrate the existence of an RNA-based communication which may be of critical importance for the establishment of pregnancy.

Background: Usage of sexed semen that allows to choose the gender of the calves, is commonly practiced in livestock industry as a profitable breeding alternative, especially in dairy farming. The flow cytometric cell sorting is the only commercially available method for bovine sperm sexing. For validation of the sexing procedure several methods have been developed including sperm fluorescence in situ hybridisation techniques. Latter usually include the use of pre-labelled nucleotides for probe synthesis which is relatively expensive approach compared to combined application of aminoallyl-dUTP and chemical binding of fluorescent dyes. Here a sex determining dual colour bovine sperm fluorescence in situ hybridisation method is presented which is considered more cost-effective technique than the previously reported approaches., Results: The reliability of sex chromosome identifying probes, designed in silico, was proven on bovine metaphase plate chromosomes and through comparison with a commercially available standard method. In the dual colour FISH experiments of unsexed and sexed bovine sperm samples the hybridisation efficiency was at least 98%, whereas the determined sex ratios were not statistically different from the expected. Very few cells carried both of the sex chromosome-specific signals (less than 0.2%)., Conclusions: A protocol for a dual colour bovine sperm FISH method is provided which is cost-effective, simple and fast for sex determination of spermatozoa in bull semen samples.

Selecting high-quality embryos for transfer has been a difficult task when producing bovine embryos invitro. The most used non-invasive method is based on visual observation. Molecular characterisation of embryo growth media has been proposed as a complementary method. In this study we demonstrate a culture medium sampling method for identifying potential embryonic viability markers to predict normal or abnormal embryonic development. During single embryo culture, 20µL culture media was removed at Days 2, 5 and 8 after fertilisation from the same droplet (60µL). In all, 58 samples were analysed using liquid chromatography-mass spectrometry. We demonstrate that it is possible to remove samples from the same culture medium droplets and not significantly affect blastocyst rate (25.2%). Changes in any single low molecular weight compound were not predictive enough. Combining multiple low molecular weight signals made it possible to predict Day 2 and 5 embryo development to the blastocyst stage with an accuracy of 64%. Elevated concentrations of lysophosphatidylethanolamines (m/z=453, 566, 588) in the culture media of Day 8 well-developing embryos were observed. Choline (104m/z) and citrate (215m/z) concentrations were increased in embryos in which development was retarded. Metabolic profiling provides possibilities to identify well-developing embryos before transfer, thus improving pregnancy rates and the number of calves born.

DNA compaction with protamines in sperm is essential for successful fertilization. However, a portion of sperm chromatin remains less tightly packed with histones, which genomic location and function remain unclear. We extracted and sequenced histone-associated DNA from sperm of nine ejaculates from three bulls. We found that the fraction of retained histones varied between samples, but the variance was similar between samples from the same and different individuals. The most conserved regions showed similar abundance across all samples, whereas in other regions, their presence correlated with the size of histone fraction. This may refer to gradual histone-protamine transition, where easily accessible genomic regions, followed by the less accessible regions are first substituted by protamines. Our results confirm those from previous studies that histones remain in repetitive genome elements, such as centromeres, and added new findings of histones in rRNA and SRP RNA gene clusters and indicated histone enrichment in some spermatogenesis-associated genes, but not in genes of early embryonic development. Our functional analysis revealed significant overrepresentation of cGMP-dependent protein kinase G (cGMP-PKG) pathway genes among histone-enriched genes. This pathway is known for its importance in pre-fertilization sperm events. In summary, a novel hypothesis for gradual histone-to-protamine transition in sperm maturation was proposed. We believe that histones may contribute structural information into early embryo by epigenetically modifying centromeric chromatin and other types of repetitive DNA. We also suggest that sperm histones are retained in genes needed for sperm development, maturation and fertilization, as these genes are transcriptionally active shortly prior to histone-to-protamine transition., (© 2017 The Authors.)

The transcriptome analysis of whole-blood RNA by sequencing holds promise for the identification and tracking of biomarkers; however, the high globin mRNA (gmRNA) content of erythrocytes hampers whole-blood and buffy coat analyses. We introduce a novel gmRNA locking assay (GlobinLock, GL) as a robust and simple gmRNA reduction tool to preserve RNA quality, save time and cost. GL consists of a pair of gmRNA-specific oligonucleotides in RNA initial denaturation buffer that is effective immediately after RNA denaturation and adds only ten minutes of incubation to the whole cDNA synthesis procedure when compared to non-blood RNA analysis. We show that GL is fully effective not only for human samples but also for mouse and rat, and so far incompletely studied cow, dog and zebrafish.

A significant proportion of mammalian fertilization is mediated through the proteomic composition of the sperm surface. These protein constituents can present as biomarkers to control and regulate breeding of agricultural animals. Previous studies have addressed the bovine sperm cell apical plasma membrane (PM) proteome with nitrogen cavitation enrichment. Alternative workflows would enable to expand the compositional data more globally around the entire sperm's surface. We used a cell surface biotin-labeling in combination with differential centrifugation to enrich sperm surface proteins. Using nano-LC MS/MS, 338 proteins were confidently identified in the PM-enriched proteome. Functional categories of sperm-egg interaction, protein turnover, metabolism as well as molecular transport, spermatogenesis, and signal transduction were represented by proteins with high quantitative signal in our study. A highly significant degree of enrichment was found for transmembrane and PM-targeted proteins. Among them, we also report proteins previously not described on bovine sperm (CPQ, CD58, CKLF, CPVL, GLB1L3, and LPCAT2B) of which CPQ and CPVL cell surface localization was further validated. A descriptive overview of the bovine sperm PM integral and peripheral proteins is provided to complement future studies on animal reproduction and its relation to sperm cell surface. All MS data have been deposited in the ProteomeXchange with identifier PXD001096 (http://proteomecentral.proteomexchange.org/dataset/PXD001096)., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In the present study, we describe the deep sequencing and structural analysis of the Holstein breed bull genome. Our aim was to receive a high-quality Holstein bull genome reference sequence and to describe different types of variations in its genome compared to Hereford breed as a reference. We generated four mate-paired libraries and one fragment library from 30 μg of genomic DNA. Colour space fasta were mapped and paired to the reference cow (Bos taurus) genome assembly from Oct. 2011 (Baylor 4.6.1/bosTau7). Initial sequencing resulted in the 4,864,054,296 of 50-bp reads. Average mapping efficiency was 71.7 % and altogether 3,494,534,136 reads and 157,928,163,086 bp were successfully mapped, resulting in 60 × coverage. This is the highest coverage for bovine genome published so far. Tertiary analysis found 6,362,988 SNPs in the bull's genome, 4,045,889 heterozygous and 2,317,099 homozygous variants. Annotation revealed that 4,330,337 of all discovered SNPs were annotated in the dbSNP database (build 137) and therefore 2,032,651 SNPs were novel. Large indel variations accounted for the 245,947,845 bp of the variation in entire genome and their number was 312,879. We also found that small indels (number was 633,310) accounted for the total variation of 2,542,552 nucleotides in the genome. Only 106,768 small indels were listed in the dbSNP. Finally, we identified 2,758 inversions in the genome of the bull covering in total 23,099,054 bp of genome's variation. The largest inversion was 87,440 bp in size. In conclusion, the present study discovered different types of novel variants in bull's genome after high-coverage sequencing. Better knowledge of the functions of these variations is needed.

The aim of our study was to create a high-quality Holstein cow genome reference sequence and describe the different types of variations in this genome compared to the reference Hereford breed. We generated one fragment and three mate-paired libraries from genomic DNA. Raw files were mapped and paired to the reference cow (Bos taurus) genome assemblies bosTau6/UMD&#95;3.1. BioScope (v1.3) software was used for mapping and variant analysis. Initial sequencing resulted in 2,842,744,008 of 50-bp reads. Average mapping efficiency was 78.4 % and altogether 2,168,425,497 reads and 98,022,357,422 bp were successfully mapped, resulting in 36.7X coverage. Tertiary analysis found 5,923,230 SNPs in the bovine genome, of which 3,833,249 were heterozygous and 2,089,981 were homozygous variants. Annotation revealed that 4,241,000 of all discovered SNPs were annotated in the dbSNP database and 1,682,230 SNPs were considered as novel. Large indel variations accounted for 48,537,190 bp of the entire genome and there were 138,504 of them. The largest deletion was 18,594 bp and the largest insertion was 13,498 bp. Another group of variants, small indels (n = 458,061), accounted for the total variation of 1,839,872 nucleotides in the genome. Only 92,115 small indels were listed in the dbSNP and therefore 365,946 small indels were novel. Finally, we identified 1,876 inversions in the bovine genome. In conclusion, this is another description of the Holstein cow genome and, similar to previous studies, we found a large amount of novel variations. Better knowledge of these variations could explain significant phenotypic differences (e.g., health, production, reproduction) between different breeds.

In present study we describe the sequencing and annotated analysis of the individual genome of Estonian. Using SOLID technology we generated 2,449,441,916 of 50-bp reads. The Bioscope version 1.3 was used for mapping and pairing of reads to the NCBI human genome reference (build 36, hg18). Bioscope enables also the annotation of the results of variant (tertiary) analysis. The average mapping of reads was 75.5% with total coverage of 107.72 Gb. resulting in mean fold coverage of 34.6. We found 3,482,975 SNPs out of which 352,492 were novel. 21,222 SNPs were in coding region: 10,649 were synonymous SNPs, 10,360 were nonsynonymous missense SNPs, 155 were nonsynonymous nonsense SNPs and 58 were nonsynonymous frameshifts. We identified 219 CNVs with total base pair coverage of 37,326,300 bp and 87,451 large insertion/deletion polymorphisms covering 10,152,256 bp of the genome. In addition, we found 285,864 small size insertion/deletion polymorphisms out of which 133,969 were novel. Finally, we identified 53 inversions, 19 overlapped genes and 2 overlapped exons. Interestingly, we found the region in chromosome 6 to be enriched with the coding SNPs and CNVs. This study confirms previous findings, that our genomes are more complex and variable as thought before. Therefore, sequencing of the personal genomes followed by annotation would improve the analysis of heritability of phenotypes and our understandings on the functions of genome., (Copyright © 2011 Elsevier B.V. All rights reserved.)