Lnc5926 is essential for early embryonic development in goats through regulation of ZSCAN4 and EIF1AX.

Long noncoding RNAs (lncRNAs) are abundant in mammalian genomes and have been found to play important roles in many biological events. However, the mechanism by which lncRNAs regulate embryonic development remains to be fully elucidated. Here, we investigated the function of the lncRNA, TCONS_00135926 (referred to as lnc5926), through knockdown and overexpression experiments in goat early embryos. Lnc5926 expression at the eight-cell embryonic stage was significantly higher than that at other stages, which was consistent with the pattern of embryonic genome activation (EGA) gene expression. The blastocyst rate after lnc5926 knockdown in eight-cell embryos was significantly lower than that in the control group (0.2% vs. 17.1%, p < 0.05), whereas the cleavage rate was not affected (71.9% vs. 75.1%, p ˃ 0.05). After knockdown or overexpression of lnc5926 in embryos, we measured expression levels of the potential target genes, STAM, HACD1, UBL5, MIOX, ELF1, and the key EGA genes, ZSCAN4 and EIF1AX. Only ZSCAN4 and EIF1AX were significantly downregulated after lnc5926 knockdown, and this effect was reversed by lnc5926 overexpression. We conclude that lnc5926 plays an essential role in early embryonic development in goats by regulating expression of EGA-associated genes.

Characterization of transcriptional activity during ZGA in mammalian SCNT embryo†.

Developmental arrest of somatic cell nuclear transfer (SCNT) embryos first occurs at zygotic/embryonic genome activation (ZGA/EGA), which is critical for preimplantation development. However, study on transcriptome of SCNT embryos during ZGA/EGA is limited. In the present study, we performed RNA sequencing (RNA-seq) of the eight-cell SCNT embryos in goat and provide cross-species analysis of transcriptional activity of SCNT embryos during ZGA/EGA in mice, human, bovine, and goat. RNA-seq data revealed 3966 differentially expressed genes (DEGs) failed to be reprogrammed or activated during EGA of SCNT embryos in goat. Series test of cluster analysis showed four clusters of DEGs and similar changes of the clusters in the four species. Specifically, genes in cluster 3 were somehow upregulated compared with the donor cells and the in vitro fertilization embryo. Moreover, the histone methylation key players and N6-methyladenosine modifiers (SUV39H1, SETDB1, SETD2, KDM5B, IGF2BP1, and YTHDF2) were differentially expressed in SCNT embryos of all species. Finally, we identified three modules correlated with the development of SCNT embryos in mice and screened 288 genes (such as BTG4, WEE1, KLF3, and USP21) that are likely critical for SCNT reprogramming using weighted gene correlation network analysis. Our data will broaden the current understanding of transcriptome activity during stochastic reprogramming events and provide an excellent source for future studies.

Superovulatory response and embryo quality in Boer does following dietary supplementation with different sources of omega-3 and omega-6 fatty acids during the breeding season.

The purpose of this study was to determine effects of various sources of omega-3 and omega-6 fatty acids on ovarian response and embryo quality in Boer does when there was a superovulation treatment regimen imposed. Pluriparous does were randomly assigned to be treated with 300 g of one of four experimental supplements containing linseed oil (LO), soybean oil (SO), palm oil (PO), or a control supplement without fatty acids (CO), for 15 days. Does were fitted with a controlled internal drug release (CIDR) device containing 0.3 g progesterone for 7 days. At 48 h before CIDR withdrawal, does were treated with 80 mg follicle-stimulating hormone (FSH) administered at 12 h intervals. Embryos were collected 7 days after the last natural mating. Estrous response and interval between CIDR withdrawals to estrous onset were similar between treatments (P > 0.05). Number of ovulations was similar for does in the different groups (10.0, 9.2, 7.0, and 7.0, in LO, SO, PO, and CO, respectively; P > 0.05). There was premature luteal regression in does of the SO, PO, and CO groups, except in LO group. The LO-treated does had a larger (P < 0.05) mean number of ova/embryos recovered than does of SO, PO, and CO groups (7.2, 2.0, 0.2, 0.2, respectively) and transferable embryos (5.1, 1.4, 0.2, 0.2, respectively). These results indicate that including LO in supplements may be a feasible strategy for preventing premature luteal regression and improving embryo quality in goats treated to induce follicular super-stimulation for induction of superovulation.

Development of Standardized Fetal Progenitor Cell Therapy for Cartilage Regenerative Medicine: Industrial Transposition and Preliminary Safety in Xenogeneic Transplantation.

Diverse cell therapy approaches constitute prime developmental prospects for managing acute or degenerative cartilaginous tissue affections, synergistically complementing specific surgical solutions. Bone marrow stimulation (i.e., microfracture) remains a standard technique for cartilage repair promotion, despite incurring the adverse generation of fibrocartilagenous scar tissue, while matrix-induced autologous chondrocyte implantation (MACI) and alternative autologous cell-based approaches may partly circumvent this effect. Autologous chondrocytes remain standard cell sources, yet arrays of alternative therapeutic biologicals present great potential for regenerative medicine. Cultured human epiphyseal chondro-progenitors (hECP) were proposed as sustainable, safe, and stable candidates for chaperoning cartilage repair or regeneration. This study describes the development and industrial transposition of hECP multi-tiered cell banking following a single organ donation, as well as preliminary preclinical hECP safety. Optimized cell banking workflows were proposed, potentially generating millions of safe and sustainable therapeutic products. Furthermore, clinical hECP doses were characterized as non-toxic in a standardized chorioallantoic membrane model. Lastly, a MACI-like protocol, including hECPs, was applied in a three-month GLP pilot safety evaluation in a caprine model of full-thickness articular cartilage defect. The safety of hECP transplantation was highlighted in xenogeneic settings, along with confirmed needs for optimal cell delivery vehicles and implantation techniques favoring effective cartilage repair or regeneration.

Analysis of Polycerate Mutants Reveals the Evolutionary Co-option of HOXD1 for Horn Patterning in Bovidae.

In the course of evolution, pecorans (i.e., higher ruminants) developed a remarkable diversity of osseous cranial appendages, collectively referred to as "headgear," which likely share the same origin and genetic basis. However, the nature and function of the genetic determinants underlying their number and position remain elusive. Jacob and other rare populations of sheep and goats are characterized by polyceraty, the presence of more than two horns. Here, we characterize distinct POLYCERATE alleles in each species, both associated with defective HOXD1 function. We show that haploinsufficiency at this locus results in the splitting of horn bud primordia, likely following the abnormal extension of an initial morphogenetic field. These results highlight the key role played by this gene in headgear patterning and illustrate the evolutionary co-option of a gene involved in the early development of bilateria to properly fix the position and number of these distinctive organs of Bovidae.

Estrogen increases KISS1 expression in newly generated immortalized KISS1-expressing cell line derived from goat preoptic area.

Kisspeptin neurons located in the hypothalamic preoptic area (POA) are suggested to be responsible for the induction of the gonadotropin-releasing hormone (GnRH) surge and the following luteinizing hormone (LH) surge to regulate female mammals' ovulation. Accumulating evidence demonstrates that the preovulatory level of estrogen activates the POA kisspeptin neurons (estrogen positive feedback), which in turn induces a GnRH/LH surge. This study aimed to derive a cell line from goat POA kisspeptin neurons as an in vitro model to analyze the estrogen positive feedback mechanism in ruminants. Neuron-derived cell clones obtained by the immortalization of POA tissue from a female Shiba goat fetus were analyzed for the expression of kisspeptin (KISS1) and estrogen receptor α (ESR1) genes using quantitative real-time reverse transcription-polymerase chain reaction and three cell clones were selected as POA kisspeptin neuron cell line candidates. One cell line (GP64) out of the three clones showed significant increase in the KISS1 level by incubation with estradiol for 24 h, indicating that the GP64 cells mimic endogenous goat POA kisspeptin neurons. The GP64 cells showed immunoreactivities for kisspeptin and estrogen receptor α and retained a stable growth rate throughout three passages. Further, intracellular calcium levels in the GP64 cells were increased by the KCl challenge, indicating their neurosecretory ability. In conclusion, we generated a new KISS1-expressing cell line derived from goat POA. The current GP64 cell line could be a useful model to elucidate the estrogen positive feedback mechanism responsible for the GnRH/LH surge generation in ruminants.

Using WGCNA (weighted gene co-expression network analysis) to identify the hub genes of skin hair follicle development in fetus stage of Inner Mongolia cashmere goat.

OBJECTIVE: Mature hair follicles represent an important stage of hair follicle development, which determines the stability of hair follicle structure and its ability to enter the hair cycle. Here, we used weighted gene co-expression network analysis (WGCNA) to identify hub genes of mature skin and hair follicles in Inner Mongolian cashmere goats. METHODS: We used transcriptome sequencing data for the skin of Inner Mongolian cashmere goats from fetal days 45-135 days, and divided the co expressed genes into different modules by WGCNA. Characteristic values were used to screen out modules that were highly expressed in mature skin follicles. Module hub genes were then selected based on the correlation coefficients between the gene and module eigenvalue, gene connectivity, and Gene Ontology (GO)/Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. The results were confirmed by quantitative polymerase chain reaction (qPCR). RESULTS: Ten modules were successfully defined, of which one, with a total of 3166 genes, was selected as a specific module through sample and gene expression pattern analyses. A total of 584 candidate hub genes in the module were screened by the correlation coefficients between the genes and module eigenvalue and gene connectivity. Finally, GO/KEGG functional enrichment analyses detected WNT10A as a key gene in the development and maturation of skin hair follicles in fetal Inner Mongolian cashmere goats. qPCR showed that the expression trends of 13 genes from seven fetal skin samples were consistent with the sequencing results, indicating that the sequencing results were reliable.n.

Systematic analyses reveal RNA editing events involved in skeletal muscle development of goat (Capra hircus).

RNA editing is a posttranscriptional molecular process involved with specific nucleic modification, which can enhance the diversity of gene products. Adenosine-to-inosine (A-to-I, I is read as guanosine by both splicing and translation machinery) is the main type of RNA editing in mammals, which manifested as AG (adenosine-to-guanosine) in sequence data. Here, we aimed to explore patterns of RNA editing using RNA sequencing data from skeletal muscle at four developmental stages (three fetal periods and one postnatal period) in goat. We found the occurrences of RNA editing events raised at fetal periods and declined at the postnatal period. Also, we observed distinct editing levels of AG editing across stages, and significant difference was found between postnatal period and fetal periods. AG editing patterns in newborn goats are similar to those of 45-day embryo compared with embryo at 105 days and embryo at 60 days. In this study, we found a total of 1415 significantly differential edited AG sites among four groups. Moreover, 420 sites were obviously clustered into six time-series profiles, and one profile had significant association between editing level and gene expression. Our findings provided some novel insights into understanding the molecular mechanism of muscle development in mammals.

Overexpression of PGC7 in donor cells maintains the DNA methylation status of imprinted genes in goat embryos derived from somatic cell nuclear transfer technology.

Abnormal methylation of imprinted genes is commonly observed in the embryos cloned by somatic cell nuclear transfer (SCNT) procedure and is one of the primary reasons for their abnormal development and high mortality. Primordial germ cell 7 (PGC7), a developmentally regulated gene highly expressed in primordial germ cells, maintains the methylation level of imprinted genes by reducing the levels of 5-hydroxy-methylcytosine(5hmC) and increasing the levels of 5-methylcytosine(5 mC) during embryonic development. In this study, we explored the methylation status of H19 differentially methylated regions (DMRs) in the organs of SCNT-cloned goat fetuses. Our results showed abnormal methylation patterns of the imprinted genes in the lungs and placenta of dead cloned goat fetuses than those in normal goat fetuses. The Igf2r DMRs were hypomethylated in the heart, liver, spleen, lungs, kidneys, and placenta of dead cloned goat fetuses compared with normal goat fetuses (P < 0.05). In addition, imprinted gene Igf2r DMRs were hypomethylated in the early-stage SCNT embryos than the IVF embryos. In contrast, imprinted gene Xist DMRs were hypermethylated in SCNT embryos than the IVF embryos. Significantly, the use of PGC7 overexpressing donor cells corrected the abnormal methylation of imprinted genes Igf2r and Xist in SCNT embryos (P < 0.05). Our results suggested that PGC7 plays a vital role in maintaining the methylation of imprinted genes during goat early embryonic development. Moreover, PGC7 overexpression in donor cells may reduce the developmental abnormalities associated with the SCNT embryos, while significantly enhancing both the pregnancy and kids born rates (P < 0.05) thereby increasing SCNT efficiency in livestock.

Transcription profiles of oocytes during maturation and embryos during preimplantation development in vivo in the goat.

RNA sequencing performed on goat matured oocytes and preimplantation embryos generated invivo enabled us to define the transcriptome for goat preimplantation embryo development. The largest proportion of changes in gene expression in goat was found at the 16-cell stage, not as previously defined at the 8-cell stage, and is later than in other mammalian species. In all, 6482 genes were identified to be significantly differentially expressed across all consecutive developmental stage comparisons, and the important signalling pathways involved in each development transition were determined. In addition, we identified genes that appear to be transcribed only at a specific stage of development. Using weighted gene coexpression network analysis, we found nine stage-specific modules of coexpressed genes that represent the corresponding stage of development. Furthermore, we identified conserved key members (or hub genes) of the goat transcriptional networks. Their association with other embryo genes suggests that they may have important regulatory roles in embryo development. Our cross-mammalian species transcriptomic comparisons demonstrate both conserved and goat-specific features of preimplantation development.

Effects of abundances of OCT-4 mRNA transcript on goat pre-implantation embryonic development.

Unlike in mice, the function of pluripotent markers in early embryonic development of domestic animals remains to be elucidated and this may account for the failure to establish embryonic stem cell lines for these species. To study the functions of the OCT-4 protein which has important actions in maintenance of pluripotent and self-renewal processes during early embryonic development, there was induced reduction in relative abundance of OCT-4 mRNA transcript during goat early embryonic development by using RNA interference techniques. The injection of OCT-4 siRNA into goat IVF presumptive zygotes resulted in a decrease in the relative abundance of OCT-4 mRNA transcript; however, there was development of these embryos to the blastocyst stage at the same rate as there was in the control group. The blastocysts from the treated groups had a similar number of TE, ICM, and total cells compared to those from the control group. Although there was a greater relative abundance of NANOG, REX1, and CDX2 mRNA transcript in the embryos injected with siRNA at the 8-16 cell stage, the relative transcript abundances were similar for the control and treatment groups at the blastocyst stage. The relative abundance of SOX2 mRNA transcript was similar for the treatment and control group. It, therefore, is concluded that inhibition of abundances of OCT-4 mRNA transcript to about 20 % of that of the untreated control group did not affect blastocyst formation rate in goats. The functions of OCT-4 in maintaining ICM and TE integrity, however, remains to be assessed.

Trend analysis of the role of circular RNA in goat skeletal muscle development.

BACKGROUND: Circular RNA (circRNA) is produced during the splicing of mRNA (in addition to linear splicing) and is part of the gene regulatory network. The temporal expression patterns the different developmental stages were inseparable from these molecules' function. RESULTS: Skeletal muscles of Anhui white goat (AWG) across seven fetal to postnatal development stages were sequenced and 21 RNA sequencing libraries were constructed. We thereby identified 9090 circRNAs and analyzed their molecular properties, temporal expression patterns, and potential functions at the different stages. CircRNAs showed complexities and diversity of formation as the same host gene produces multiple isoforms of these nucleic acids with different expression profiles. The differential expression of 2881 circRNAs (DECs, P < 0.05) was identified and four were randomly selected and validated by qPCR. Moreover, 1118 DECs under strict selected (SDECs, |log2FC| > 2 and P-adj value < 0.01) showed 4 expression trends (Clusters 0, 19, 16 and 18). Cluster 0 molecules had increasing expression at all stages with effects on muscle through metabolism, regulation of enzyme activity, and biosynthesis. Cluster 16 circRNAs had high expression in the early and late stages and are involved in "Wnt signaling pathway", "AMPK signaling pathway" and others. Cluster 18 molecules were mainly expressed at F120 and participate in "cytoskeletal protein binding", "Notch signaling pathway" and so on. Cluster 19 circRNAs were down-regulated at all stages and related to muscle structure and development. Lastly, the SDECs divided the period of skeletal muscle development into three transitional stages: stage 1 (F45 to F90), which related to muscle satellite cell proliferation and muscle fiber structure; stage 2 (F90 to B1), in which the attachment of the cytoplasmic surface to the actin cytoskeleton initiates; and stage 3, which involved the "cGMP-PKG signaling pathway". Moreover, the paraffin sections messages also validated that there are three transitional stages of skeletal muscle development. CONCLUSION: Our current study provides a catalog of goat muscle-related circRNAs that can stratify skeletal muscle development fetus 45 days to newborn 90 days into three developmental stages. These findings better our understanding of functional transitions during mammalian muscle development.

Aberrant DNA and histone methylation during zygotic genome activation in goat cloned embryos.

In somatic cell nuclear transfer (SCNT) embryos, developmental defects first appear at the time of zygotic genome activation (ZGA), a process that is under the control of DNA and histone methylation. However, dynamics of 5-mC and 5-hmC during ZGA differ between porcine and bovine SCNT embryos, and histone methylation during ZGA in goat SCNT embryos remains poorly understood. Therefore, in the present study, we investigated the dynamic changes of 5-mC, 5-hmC, H3K4me2/3, and H3K9me3, as well as the expression of key genes related to these epigenetic modifications, during ZGA in goat cloned embryos. Compared with the IVF embryos, the 5-mC signal intensity was significantly increased at the 2- and 4-cell stage SCNT embryos, and the H3K4me3 and H3K9me3 signal intensity was significantly increased at 2- to 8-cell stage SCNT embryos, while the 5-hmC and H3K4me2 signal intensity was significantly lower at the 4- and 8-cell stage SCNT embryos. Of note, the H3K9me3 level was also significantly higher, whereas H3K4me3 signal intensity showed no statistical difference in the pronuclear stage SCNT embryos. Moreover, the expression of TET2, DNMT3B, KDM4A, SUV39H1, G9A, and SETDB1 was significantly increased, while the expression of UHRF1, PCNA, KDM4B, KDM4D, KDM5A, KDM5B, and KDM5C was significantly decreased at the 8-cell stage SCNT embryos. Our data revealed aberrant DNA and histone methylation during ZGA in goat cloned embryos. We further inferred that the abnormally higher level of 5-mC, H3K4me3, and H3K9me3 might serve as epigenetic barriers of the reprogramming and modifying these aberrant modifications might be a promising strategy to improve cloning efficiency in goat.

Molecular Characteristics, Phylogeny and Expression Profile of the PTEN Gene in Goats.

Phosphatase and the tensin homologue deleted on chromosome ten (PTEN) has pleiotropic effects on cell growth, organ development, glucose metabolism and insulin resistance in mammals. In the present study, we investigated the molecular characteristics, phylogeny and expression profile of the PTEN gene in different tissues of Jianzhou Daer goats. In this study, eight different tissues from E90, E135 and D90 female goats were collected to quantify the expression pattern of the PTEN gene using quantitative real-time PCR (qPCR), western blotting and FISH. In addition, the dynamic expression of PTEN was also determined during the differentiation of goat precursor adipose cells. A 1212-bp fragment (accession number MG923848), encoding a 403-amino acid protein with a putative molecular weight of 47.14 kDa, was identified in Jianzhou Daer goats by reverse-transcription polymerase chain reaction (RT-PCR). The phylogenetic tree showed that caprine PTEN had a relatively close relationship with ovine PTEN and bovine PTEN. qPCR revealed that PTEN was highly expressed in the liver, lung and spleen, while the lowest expression levels were observed in muscle tissues (P < 0.05). Moreover, the expression of the PTEN gene showed a decreasing trend during the differentiation of goat precursor adipose cells. RNA in situ hybridization yielded a consistent result with the qPCR data. Indeed, low protein expression was found in psoas major muscle and longissimus dorsi muscle, as well as in kidney and liver. However, PTEN protein was expressed at the highest level in the brain. The expression levels of PTEN mRNA and protein were inconsistent with each other, possibly because of post-transcriptional regulation. The findings obtained in our study lay a foundation for further investigations examining the caprine PTEN gene in embryo and organ development.

The effect of anti-müllerian hormone and progesterone concentrations on superovulation response and embryo yield in goats.

We aimed to evaluate the relationship of anti-Müllerian hormone (AMH) and progesterone concentrations with superovulation response in goats and to determine donors exhibiting better superovulation response by measuring AMH concentrations. For this, blood samples were collected from multiparous Angora goats (n = 24) for measuring the progesterone and AMH concentrations on the day the synchronization protocol was initiated (Day 0), on the day of the first FSH administration (Day 9), on the day the progesterone source was removed (Day 11), and on the day of uterine flushing. Descriptive statistics (mean, standard deviation, median, minimum value, maximum value, and percentile) were given for superovulation response and embryo yield. To compare the differences between the two groups, the Student's t-test was used. The relationship between two continuous variables was assessed by the Pearson Correlation Coefficient. The AMH cutoff values in superovulation responses were evaluated by ROC analysis on the day the synchronization protocol was initiated. A strong positive correlation was found between the AMH concentrations measured on the day the synchronization protocol was initiated (Day 0), on the day of the first FSH administration (Day 9), and on the day of removal of the progesterone source (Day 11) and the count of total corpus luteum (CL), total oocyte/embryo, transferable embryo, and Code I quality embryo (P < 0.05). Furthermore, AMH concentration increased on the day the synchronization protocol was initiated, the donor's superovulation response increased as well. The cutoff value was 4.74 ng/ml, as assessed by the ROC curve analysis conducted for selecting donors exhibiting better superovulation responses. The sensitivity and specificity of the selected cutoff value were found to be quite high (P < 0.01). However, a positive correlation was noted between the progesterone concentrations measured on the day of uterine flushing and total CL count, total oocyte/embryo count, transferable embryo count, and Code I quality embryo count (P < 0.01). In conclusion, it was determined that an increase in AMH concentrations in goats led to an increase in the total CL count, embryo count, and embryo quality and that AMH measurement could be used to identify donors that responded better to superovulation. Additionally, a positive correlation was found between the progesterone concentration measured on the day of uterine flushing and the total CL count, transferable embryo count, and embryo quality.

Transcriptome profiling reveals transcriptional and alternative splicing regulation in the early embryonic development of hair follicles in the cashmere goat.

The undercoat fiber of the cashmere goat, from the secondary hair follicle (HF), possesses commercial value. However, very few studies have focused on the molecular details of primary and secondary HF initiation and development in goat embryos. In this study, skin samples at embryonic day 45, 55, and 65 (E45, E55, and E65) were collected and prepared for RNA sequencing (RNA-seq). We found that the HF probably initiated from E55 to E65 by analyzing the functional pathways of differentially expressed genes (DEGs). Most key genes in canonical signaling pathways, including WNT, TGF-ß, FGF, Hedgehog, NOTCH, and other factors showed clear expression changes from E55 to E65. We, for the first time, explored alternative splicing (AS) alterations, which showed distinct patterns among these three stages. Functional pathways of AS-regulated genes showed connections to HF development. By comparing the published RNA-seq samples from the E60, E120, and newborn (NB) stages, we found the majority of WNT/ß-catenin signaling genes were important in the initiation of HF development, while other factors including FOXN1, GATA3, and DLX3 may have a consistent influence on HF development. Our investigation supported the time points of embryonic HF initiation and identified genes that have potential functions of embryonic HF initiation and development. We further explored the potential regulatory roles of AS in HF initiation, which extended our knowledge about the molecular mechanisms of HF development.

Influence of eCG and breed on the number of oocytes collected and the production of in vitro embryos of young goats during the reproductive season.

The objective of this study was to determine the effect of breed and equine chorionic gonadotropin (eCG) on ovarian response and in vitro embryo production from young goats. Thirty-one (12 Alpine, 10 Nubian, and 9 Saanen) were randomly assigned into three treatments of eCG (T1, 0 IU; T2, 500 IU; and T3, 1000 IU). Alpine goats showed the highest amount and largest size of follicles (P = 0.003). The effect of eCG dose 24 h post application was significant (P < 0.05), and was superior in goats undergoing T2. The aspiration rate of cumulus-oocyte complexes (COC) was 34% (P > 0.05), except for percentage of denuded oocytes, which obtained the highest number (P = 0.003) in the Saanen goats. The same difference was found (P = 0.02) in oocytes grade III in T2 and T3, with 42.5 and 37.9% respectively. In vitro embryo production was 80.0% of IVF/cleavage in the Alpine goats (P = 0.003). Embryo production was the greatest for T2 (69.2%; P = 0.004). T3 goats had higher percentage of morula stage (66.6%; P = 0.030). It is concluded that the application of eCG has a significant effect on the ovarian status, and quality and quantity of embryos with a differential response depending on the breed.

Comparison of gene editing efficiencies of CRISPR/Cas9 and TALEN for generation of MSTN knock-out cashmere goats.

The genome editors CRISPR/Cas9 (clustered regularly interspaced short palindromicrepeats/Cas9 nuclease-null) and TALENs (transcription activator-like effector nuclease) are popularly used for targeted modification of the mammalian genome. To date, few comparative studies have been carried out to investigate the differences between the use of CRISPR/Cas9 and TALENs in genome editing for goat breeding. Here, we compared CRISPR/Cas9 and TALEN technologies at multiple levels for generating a knock out (KO) of the Alpas cashmere goat myostatin (MSTN) gene, which negatively regulates the proliferation and differentiation of skeletal muscle cells. The electrotransfection efficiency observed using CRISPR/Cas9 was 8.1% more than that observed using TALEN for generating MSTN KO cells. In addition, the cutting efficiency of CRISPR/Cas9 for editing exon 1 of the MSTN gene was higher than that of TALENs. However, the off-target effects of the CRISPR/Cas9 system were also higher than those of TALENs. Further, we found that the frequency of obtaining MSTN-/- mutations by CRISPR/Cas9 was 8.5 times higher than that by TALEN. The CRISPR/Cas9-edited colonies involved longer deletions (up to 117 bp) than the TALEN-edited colonies (up to 13 bp). Remarkably, when embryos used to generate cloned goat via somatic cell nuclear transfer were compared, we found that the TALEN MSTN KO embryos easily developed to 8 cells and their cleavage rate was significantly higher than that of CRISPR/Cas9-edited embryos. Finally, we produced a MSTN KO lamb using CRISPR/Cas9, which suggested that a high level of targeted gene modification could be achieved in goat using CRISPR/Cas9. Taken together, our study indicates that although TALEN enables a variety of genome modifications and may have some advantages over CRISPR/Cas9, the latter provides a significant advantage by permitting precise and efficient gene editing. Thus, CRISPR/Cas9 has more potential to become a robust gene-engineering tool for application in the breeding of farm animals.

Calcium ionophore enhanced developmental competence and apoptotic dynamics of goat parthenogenetic embryos produced in vitro.

Parthenogenetically developed embryos are efficient sources of in vitro embryo production, having less ethical issue and being useful for investigating culture conditions/treatments, early developmental, genomic studies, and homonymous source of stem cells. Keeping its advantages in mind, we aimed to study the effects of different activating agents on embryo production and its quality and gene expression. In the present study, 1348 immature oocytes recovered were parthenogenetically developed to embryos. Usable-quality immature oocytes were collected by puncturing the surface follicles and matured in in vitro maturation (IVM) medium for 27 h in a humidified 5% CO2 incubator at 38.5°C. The matured oocytes were parthenogenetically activated by exposure to 5 µM calcium ionophore for 5 min or 7% ethanol for 7 min sequentially followed by 4 h incubation in 2 mM 6-DMAP and then in vitro cultured (IVC) in RVCL/G-2 medium for 8 days. Matured oocytes were activated by calcium ionophore, the cleavage rate observed was 76.67 ± 3.47%, and further they developed into 4-cell, 8-16-cell, morula, blastocyst, and hatched blastocyst with 85.30 ± 1.57%, 70.60 ± 2.00%, 45.05 ± 2.66%, 22.89 ± 2.40%, and 5.70 ± 1.97%, respectively. Whereas ethanol-activated oocytes showed cleavage rate of 87.60 ± 1.70% and further culture developed into 4-cell, 8-16 cell, morula, blastocyst, and hatched blastocyst with 86.14 ± 1.03%, 71.56 ± 2.21%, 40.90 ± 2.45%, 19.02 ± 1.26%, and 2.22 ± 0.38%, respectively. Blastocyst developed from calcium ionophore-activated oocytes showed significantly (P < 0.05) higher total cell number (282.25 ± 27.02 vs 206.00 ± 40.46) and a lower apoptotic index (2.42 ± 0.46 vs 4.07 ± 1.44) than blastocyst developed from ethanol-activated oocytes. The relative expression of anti-apoptotic genes (BCL2, BCL2A1, MCL) at different stages of embryos produced by either calcium ionophore or ethanol activation was found to be increased in earlier stages and decreased in later stages of embryonic development. Similarly, when these embryos were subjected to pro-apoptotic genes (BAX, BAD, BAK), expression was found to be slightly higher in blastocysts than other stages. This study shows that calcium ionophore-activated blastocysts were developmentally more competent than the ethanol-activated blastocysts.

The morphological investigations on the heart and some vessels of bovine foetus between the 15th and 25th weeks of gestation.

BACKGROUND: The aim of this study was to define the morphological and morphometric development of the foetus heart obtained from the domestic cattle in the gestation period of 15-25 weeks. MATERIALS AND METHODS: For this purpose, a total of 30 hearts belonging to cattle foetuses (15 males, 15 females) were used. The ages of foetuses were calculated according to the forehead-to-tail length and examined in three different groups. After dissection; biometric, macroanatomic, morphometric and histological findings were obtained from the foetal hearts according to the groups. In addition, mean values of the morphometric findings were determined. RESULTS: As a result of the study, it was found that with the advancing age the convexity of margo ventricularis dexter increased and margo ventricularis sinister transformed from a convex-concave shape to a flat shape. The heart-to-body weight ratio was determined as 0.08% for Group II female foetuses and 0.09% for all other groups. The heart heights for Groups I, II, and III females were identified as 26.21, 41.00, and 46.27 mm, respectively, and for the males 26.45, 34.89, and 47.15 mm, respectively. In the statistical analysis, it was determined that all the morphometric values measured from the heart correlated significantly with the forehead-to-tail length. CONCLUSIONS: The data obtained as a result of the study is thought to help understand the morphological and morphometrical development of the heart, pioneer the attempts to create a foetal cattle heart model, and thus help in the diagnosis of the foetal heart pathologies.acielecka.