Somatic Cell Nuclear Transfer Research Articles

Epigenetic Iinsights into the Senescence of Porcine Fetal Fibroblasts induced by Passaging

ABSTRACT Epigenetic status of fetal fibroblasts (FFs) is one of the crucial factors accounted for the success of somatic cell nuclear transfer and gene editing, which might inevitably be affected by passaging. But few systematic studies have been performed on the epigenetic status of passaged aging cells. Therefore, FFs from large white pig were in vitro passaged to the 5, 10, and 15 (F5, F10, and F15) passages in the present study to investigate the potential alteration of epigenetic status. Results indicated the senescence of FFs occurs with the passaging, as assessed by the weakened growth rate, increased β-gal expression, and so on. For the epigenetic status of FFs, the higher level both of DNA methylation and H3K4me1, H3K4me2, H3K4me3 was observed at F10, but the lowest level was observed at F15. However, the fluorescence intensity of m6A was significantly higher in F15, but lower (p < 0.05) in F10, and the related mRNA expression in F15 was significantly higher than F5. Further, RNA-Seq indicated a considerable difference in the expression pattern of F5, F10, and F15 FFs. Among differentially expressed genes, not only the genes involved in cell senescence were changed, but also the upregulated expression of Dnmt1, Dnmt3b, Tet1 and dysregulated expression of histone methyltransferases-related genes were detected in F10 FFs. In addition, most genes related to m6A such as METTL3, YTHDF2, and YTHDC1 were significantly different in F5, F10, and F15 FFs. In conclusion, the epigenetic status of FFs was affected by being passaged from F5 to F15.

TGFβ inhibition and mesenchymal to epithelial transition initiation by Xenopus egg extract: first steps towards early reprogramming in fish somatic cell

Xenopus egg extract is a powerful material to modify cultured cells fate and to induce cellular reprogramming in mammals. In this study, the response of goldfish fin cells to in vitro exposure to Xenopus egg extract, and subsequent culture, was studied using a cDNA microarray approach, gene ontology and KEGG pathways analyses, and qPCR validation. We observed that several actors of the TGFβ and Wnt/β-catenin signaling pathways, as well as some mesenchymal markers, were inhibited in treated cells, while several epithelial markers were upregulated. This was associated with morphological changes of the cells in culture, suggesting that egg extract drove cultured fin cells towards a mesenchymal-epithelial transition. This indicates that Xenopus egg extract treatment relieved some barriers of somatic reprogramming in fish cells. However, the lack of re-expression of pou2 and nanog pluripotency markers, the absence of DNA methylation remodeling of their promoter region, and the strong decrease in de novo lipid biosynthesis metabolism, indicate that reprogramming was only partial. The observed changes may render these treated cells more suitable for studies on in vivo reprogramming after somatic cell nuclear transfer.

Dual single guide RNAs-mediating deletion of mature myostatin peptide results in concomitant muscle fibre hyperplasia and adipocyte hypotrophy in pigs

Myostatin (MSTN) is a major gene target for skeletal muscle overgrowth in animals. We hypothesized that deletion of the entire mature peptide encoded by MSTN in pigs would knock out its bioactive form and accordingly stimulate skeletal muscle overgrowth. Thus, we engineered two pairs of single-guide RNAs (sgRNAs) to target exons 1 and 3 of MSTN in primary fetal fibroblasts of Taoyuan black pigs. We found that sgRNAs targeting exon 3, which encodes the mature peptide, had higher biallelic null mutation efficiency than those targeting exon 1. Somatic cell nuclear transfer was conducted using the exon 3 mutation cells as donor cells to generate five cloned MSTN null piglets (MSTN−/−). Growth testing revealed that both the growth rate and average daily weight gain of MST−/− pigs were greater than those of wild-type (MSTN+/+) pigs. Slaughter data demonstrated that the lean ratio of MSTN−/− pigs was 11.3% higher (P < 0.01) while the back-fat thickness was 17.33% lower (P < 0.01) than those of MSTN+/+ pigs. Haematoxylin–eosin staining indicated that the increased leanness of MSTN−/− pigs resulted from muscle fibre hyperplasia rather than hypertrophy.HE staining showed markedly decreased adipocyte size in MSTN−/− pigs. We also critically examined the off-target and random integration by resequencing, which showed that the founder MSTN−/− pigs contained no non-target mutations or exogenous plasmid elements. This study is the first to report the successful knock out of the mature MSTN peptide using dual sgRNA-mediated deletion, leading to the most prominent alteration of meat production traits in pigs published thus far. This new strategy is expected to have a wide impact on genetic improvements in food animals.

Overcoming the H4K20me3 epigenetic barrier improves somatic cell nuclear transfer reprogramming efficiency in mice.

Epigenetic reprogramming during fertilization and somatic cell nuclear transfer (NT) is required for cell plasticity and competent development. Here, we characterize the epigenetic modification pattern of H4K20me3, a repressive histone signature in heterochromatin, during fertilization and NT reprogramming. Importantly, the dynamic H4K20me3 signature identified during preimplantation development in fertilized embryos differed from NT and parthenogenetic activation (PA) embryos. In fertilized embryos, only maternal pronuclei carried the canonical H4K20me3 peripheral nucleolar ring-like signature. H4K20me3 disappeared at the 2-cell stage and reappeared in fertilized embryos at the 8-cell stage and in NT and PA embryos at the 4-cell stage. H4K20me3 intensity in 4-cell, 8-cell, and morula stages of fertilized embryos was significantly lower than in NT and PA embryos, suggesting aberrant regulation of H4K20me3 in PA and NT embryos. Indeed, RNA expression of the H4K20 methyltransferase Suv4-20h2 in 4-cell fertilized embryos was significantly lower than NT embryos. Knockdown of Suv4-20h2 in NT embryos rescued the H4K20me3 pattern similar to fertilized embryos. Compared to control NT embryos, knockdown of Suv4-20h2 in NT embryos improved blastocyst development ratios (11.1% vs. 30.5%) and full-term cloning efficiencies (0.8% vs. 5.9%). Upregulation of reprogramming factors, including Kdm4b, Kdm4d, Kdm6a, and Kdm6b, as well as ZGA-related factors, including Dux, Zscan4, and Hmgpi, was observed with Suv4-20h2 knockdown in NT embryos. Collectively, these are the first findings to demonstrate that H4K20me3 is an epigenetic barrier of NT reprogramming and begin to unravel the epigenetic mechanisms of H4K20 trimethylation in cell plasticity during natural reproduction and NT reprogramming in mice.

Reproduction of Sheep through Nuclear Transfer of Somatic Cells: A Bibliometric Approach.

Somatic cell nuclear transfer (SCNT) is a reproductive biotechnology with great potential in the reproduction of different species of zootechnical interest, including sheep. This study aimed to carry out a bibliometric analysis of scientific papers published on the application of SCNT in sheep reproduction during the period 1997-2023. The search involved the Science Citation Index Expanded and Social Sciences Citation Index databases of the main collection of the Web of Sciences with different descriptors. A total of 124 scientific papers were analyzed for different bibliometric indicators using the VOSviewer software. Since 2001, the number of SCNT-related papers that have been published concerning sheep reproduction has increased and it has fluctuated in ensuing years. The main authors, research groups, institutions, countries, papers, and journals with the highest number of papers related to the application of SCNT in sheep reproduction were identified, as well as the topics that address the research papers according to the terms: somatic cell, embryo, oocyte, gene expression, SCNT, and sheep.

Efficiency of equine oocyte recovery from post mortem ovaries or by ovum pick up

Most oocytes used for cloned embryo production are obtained from abattoir ovaries even though in some countries, slaughtering horses is not allowed. The advantage of scraping post mortem ovaries is the high number of oocytes available in a short period of time to perform somatic cell nuclear transfer. However, mare age, reproductive and health status or ovary manipulation by abattoir staff are not controlled before slaughter and the effect on oocyte quality and early embryo development is uncertain. Ovum pick up (OPU) is an alternative technique to obtain oocytes ex vivo from controlled mares and embryos obtained from those oocytes potentially have better development. Cortez et al. (Journal of Equine Veterinary Science 113 2022; 14) reported higher cleavage and blastocyst rates in cloned embryos from OPU-derived rather than post mortem derived ovaries. The aim of this retrospective study was to determine and compare the oocyte recovery rates from abattoir-sourced ovaries or collected by OPU. Data was collected between September and November 2022 in Argentina. For statistical analysis, generalized linear models were used. A total of 3476 ovaries were collected from the abattoir and 16% (557/3476) were discarded since antral follicles were not visible. Using the scraping technique, 8392 oocytes were recovered in 24 sessions obtaining an averageof 350 oocytes per session. Mean (± SD) oocyte recovered per scraped ovary was 2.9±0.4 and for all collected ovaries per session was 2.4±0.3. On average the scraping time calculated per mare (pair of ovaries) for all collected ovaries per session was 1 min and 36 sec (59 sec-2 min). OPU was performed in 44 mixed breed healthy mares during the southern hemisphere breeding season in which a mean of 16.7 (7-29) follicles were aspirated per mare in an average of 27 min and 7 sec (6 min and 3 sec- 58 min). Oocyte recovery rate per aspirated follicle was 63.3% and a mean of 9.7 oocytes were retrieved per OPU. Mean oocyte retrieval per mare was higher using OPU than scraping (9.7 ± 4.8 vs. 5.8 ± 0.9; p<0.0001). Although oocyte recovery per follicle could not be compared in this study these results could be influenced by mares’ selection for OPU although there was not a minimum number of follicles set as a criteria to perform OPU. The data show that the OPU procedure is a suitable technique to obtain oocytes for equine cloning programs. In some circumstances, the procedures to obtain oocytes by OPU are more expensive than those recovered by scraping, however published data suggest that fewer oocytes recovered by OPU are needed to obtain a cloned embryo.

Forgotten Clones: The Birth of Cloning and the Biological Revolution. By NathanCrowe. Pittsburgh, PA: University of Pittsburgh Press. 2021. pp. 336. $60 (Hardcover).

Forgotten Clones: The Birth of Cloning and the Biological Revolution. By NathanCrowe. Pittsburgh, PA: University of Pittsburgh Press. 2021. pp. 336. $60 (Hardcover).

Deciphering the placental abnormalities associated with somatic cell nuclear transfer at single-nucleus resolution.

Deciphering the placental abnormalities associated with somatic cell nuclear transfer at single-nucleus resolution.

Production of Somatic Cell Nuclear Transfer derived cloned calves in the United Arab Emirates.

Despite the tremendous advancement in somatic cell nuclear transfer (SCNT) in camels, the generation of cloned calf using SCNT in cows is yet to be reported in the United Arab Emirates (UAE). The present study intended to produce cloned calves in the local environment of the UAE. We surgically collected ear tissue from two high-performance Holstein cows (average production of 71 liter/ day) and established the fibroblast cell line to perform SCNT. Fifty- seven (18.48%) blastocysts were derived from 330 fused oocytes for cell donor A, and 51 (19.14%) blastocysts were generated from 289 fused oocytes for cell donor B. We used 102 recipients for embryo transfer; among those, 53 and 49 recipients received embryos reconstructed with cell donor A and B, respectively. A single blastocyst in transfer media was transferred deep into the horn of the uterus of natural cycling recipient cows by trans cervical method on Day 7; where estrus was considered as Day 0. Non-return to estrus (up to 42 days), early pregnancy, and the live birth rate did not differ significantly for the two donor cells. Four (26.67%) and 1 (7.69%) live calves were produced for cell donor A and B, respectively. Microsatellite analysis of 18 bovine-specific loci confirmed the perfect match of cell donors and cloned calves. This is the first reported case of cloned calves using SCNT in the UAE. Our results could benefit the UAE dairy industry by supporting the locally produced high- performance replacement heifers. Keywords: Bovine; Cloning; In vitro maturation; Oocytes; UAE

First gene-edited calf with reduced susceptibility to a major viral pathogen.

Bovine viral diarrhea virus (BVDV) is one of the most important viruses affecting the health and well-being of bovine species throughout the world. Here, we used CRISPR-mediated homology-directed repair and somatic cell nuclear transfer to produce a live calf with a six amino acid substitution in the BVDV binding domain of bovine CD46. The result was a gene-edited calf with dramatically reduced susceptibility to infection as measured by reduced clinical signs and the lack of viral infection in white blood cells. The edited calf has no off-target edits and appears normal and healthy at 20 months of age without obvious adverse effects from the on-target edit. This precision bred, proof-of-concept animal provides the first evidence that intentional genome alterations in the CD46 gene may reduce the burden of BVDV-associated diseases in cattle and is consistent with our stepwise, in vitro and ex vivo experiments with cell lines and matched fetal clones.

Intracellular Strain Evaluation-Based Oocyte Enucleation and Its Application in Robotic Cloning

Since the first cloned sheep was produced in 1996, cloning has attracted considerable attention because of its great potential in animal breeding. Somatic cell nuclear transfer (SCNT) is widely used for creating clones. However, SCNT is very complicated to manipulate and inevitably causes intracellular damage during manipulation. Typically, only less than 1% of reconstructed embryos develop into live cloned animals. This low success rate is considered to be the major limitation in the extensive application of cloning techniques. In this study, we proposed an intracellular strain evaluation-based oocyte enucleation method to reduce potential intracellular damage in SCNT. We first calculated the intracellular strain based on the intracellular velocity field and then used the intracellular strain as a criterion to improve the enucleation operation. We then developed a robotic batch SCNT system to apply this micromanipulation method to animal cloning. Experimental results showed that we increased the blastocyst rate from 10.0% to 20.8%, and we successfully produced 17 cloned piglets by robotic SCNT for the first time. The success rate of cloning was significantly increased compared to that of traditional methods (2.5% vs 0.73% on average). In addition to the cloning technique, the intracellular strain evaluation-based enucleation method is expected to be applicable to other biological operations and for establishing a universal cell manipulation protocol to reduce intracellular damage.

Sperm-borne microRNA-34c regulates maternal mRNA degradation and preimplantation embryonic development in mice

BackgroundStudies have shown that sperm-borne microRNAs (miRNAs) are involved in mammalian preimplantation embryonic development. In humans, spermatozoan miR-34c levels are correlated with in vitro fertilization outcomes, such as embryo quality and the clinical pregnancy and live birth rates. In rabbits and cows, miR-34c improves the developmental competence of embryos generated by somatic cell nuclear transfer. However, the mechanisms underlying the regulation of embryonic development by miR-34c remain unknown.MethodsFemale C57BL/6 mice (6–8 weeks old) were superovulated, and pronucleated zygotes were collected and microinjected with an miR-34c inhibitor or a negative-control RNA. The embryonic development of the microinjected zygotes was evaluated, and the messenger RNA (mRNA) expression profiles of the embryos at the two-cell, four-cell and blastocyst stages (five embryos per group) were determined by RNA sequencing analysis. Gene expression levels were verified by reverse transcription–quantitative polymerase chain reaction. Cluster analysis and heat map visualization were performed to detect differentially expressed mRNAs. Pathway and process enrichment analyses were performed using ontology resources. Differentially expressed mRNAs were systematically analyzed using the Search Tool for the Retrieval of Interacting Genes/Proteins database to determine their biological functions.ResultsEmbryonic developmental potential was significantly reduced in zygotes microinjected with the miR-34c inhibitor compared with those microinjected with a negative-control RNA. Two-cell stage embryos microinjected with an miR-34c inhibitor presented altered transcriptomic profiles, with upregulated expression of maternal miR-34c target mRNAs and classical maternal mRNAs. Differentially expressed transcripts were mainly of genes associated with lipid metabolism and cellular membrane function at the two-cell stage, with cell-cycle phase transition and energy metabolism at the four-cell stage; and with vesicle organization, lipid biosynthetic process and endomembrane system organization at the blastocyst stage. We also showed that genes related to preimplantation embryonic development, including Alkbh4, Sp1, Mapk14, Sin3a, Sdc1 and Laptm4b, were significantly downregulated after microinjection of an miR-34c inhibitor.ConclusionsSperm-borne miR-34c may regulate preimplantation embryonic development by affecting multiple biological processes, such as maternal mRNA degradation, cellular metabolism, cell proliferation and blastocyst implantation. Our data demonstrate the importance of sperm-derived miRNAs in the development of preimplantation embryos.

Production of MSTN Gene-Edited Embryos of Buffalo Using the CRISPR/Cas9 System and SCNT.

The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system and somatic cell nuclear transfer (SCNT) have been used to produce genome-edited farm animal species for improved production and health traits; however, these tools are rarely used in the buffalo and can play a pivotal role in milk and meat production in tropical and subtropical countries. In this study, we aimed to produce myostatin (MSTN) gene-edited embryos of the Murrah buffalo using the CRISPR/Cas9 system and SCNT. For this, fibroblast cells were electroporated with sgRNAs carrying all-in-one CRISPR/Cas9 plasmids targeting the first exon of the MSTN gene. Following puromycin selection, single-cell clonal populations were established and screened using the TA cloning and Sanger sequencing methods. Of eight single-cell clonal populations, one with a monoallelic and another with a biallelic heterozygous gene editing event were identified. These two gene-edited clonal cell populations were successfully used to produce blastocyst-stage embryos using the handmade cloning method. This work establishes the technical foundation for generation of genome-edited cloned embryos in the buffalo.

Methylation Genome-Wide Profiling in Lowly and Highly Efficient Somatic Cell Nuclear Transfer in Pigs

Swine is a common model organism for biomedical research. Epigenetic reprogramming in somatic cell nuclear transfer (SCNT) embryos does not fully recapitulate the natural DNA demethylation events at fertilisation. This study aimed to conduct genome-wide methylation profiling to detect differentially methylated regions (DMRs) responsible for epigenetic differences in stem cells that displayed high and low efficiency of SCNT and to elucidate the low efficiency of cloning rate in pigs. Adipose tissue mesenchymal stem cells (AMSC)s lines were isolated from adipose tissue of adult male pigs (n = 20; high-efficiency cells = 10; and low-efficiency cells = 10). Reduced representation bisulfite sequencing (RRBS) was performed on an Illumina HiSeq1500. Paired-end reads were filtered to remove the adapter contamination, and low-quality reads using TrimGalore! Filtered reads were mapped to the reference genome using Bismark. MethylKit was used to identify differentially methylated regions (DMRs) (bases and tiles), showing statistically significant differential methylation between high and low-efficiency AMSCs. Hierarchical cluster analysis according to methylation patterns clearly defined groups with low and high cloning efficiency. We report 3704 bases with statistically significant differences in methylation and 10062 tiles with statistically significant differences in methylation. Most differentially methylated sites are intergenic 62%, 31% are intronic, 4% are in exons, and 4% in promoters. Moreover, 37% of differentially methylated sites are located in known CpG islands (CGIs), and 4% in CpG island shores (CGSs).

The combination of rolipram and cilostamide improved the developmental competence of cloned porcine embryos

In vitro maturation of porcine oocytes is characterized by asynchronous cytoplasmic and nuclear maturation, leading to less competent oocytes supporting embryo development. The purpose of this study was to evaluate the combined effect of rolipram and cilostamide as cyclic Adenine monophosphate (cAMP) modulators to find the maximum cAMP levels that temporarily arrest meiosis. We determined the optimal time to maintain functional gap junction communication during pre-in vitro maturation to be four hours. Oocyte competence was evaluated by the level of glutathione, reactive oxygen species, meiotic progression, and gene expression. We evaluated embryonic developmental competence after parthenogenetic activation and somatic cell nuclear transfer. The combined treatment group showed significantly higher glutathione and lower reactive oxygen species levels and a higher maturation rate than the control and single treatment groups. Cleavage and blastocyst formation rates in parthenogenetic activation and somatic cell nuclear transfer embryos were higher in two-phase in vitro maturation than in the other groups. The relative levels of BMP15and GDF9 expression were increased in two-phase in vitro maturation. Somatic cell nuclear transfer blastocysts from two-phase in vitro maturation oocytes showed a lower level of expression of apoptotic genes than the control, indicating better pre-implantation developmental competence. The combination of rolipram and cilostamide resulted in optimal synchrony of cytoplasmic and nuclear maturation in porcine in vitro matured oocytes and there by enhanced the developmental competence of pre-implantation embryos.

Farrerol directly activates the deubiqutinase UCHL3 to promote DNA repair and reprogramming when mediated by somatic cell nuclear transfer

Farrerol, a natural flavanone, promotes homologous recombination (HR) repair to improve genome-editing efficiency, but the specific protein that farrerol directly targets to regulate HR repair and the underlying molecular mechanisms have not been determined. Here, we find that the deubiquitinase UCHL3 is the direct target of farrerol. Mechanistically, farrerol enhanced the deubiquitinase activity of UCHL3 to promote RAD51 deubiquitination, thereby improving HR repair. Importantly, we find that embryos of somatic cell nuclear transfer (SCNT) exhibited defective HR repair, increased genomic instability and aneuploidy, and that the farrerol treatment post nuclear transfer enhances HR repair, restores transcriptional and epigenetic network, and promotes SCNT embryo development. Ablating UCHL3 significantly attenuates farrerol-mediated stimulation in HR and SCNT embryo development. In summary, we identify farrerol as an activator of the deubiquitinase UCHL3, highlighted the importance of HR and epigenetic changes in SCNT reprogramming and provide a feasible method to promote SCNT efficiency.

Mechanical strain treatment improves nuclear transfer reprogramming efficiency by enhancing chromatin accessibility.

Cellular mechanical properties are considered to be important factors affecting cell fate transitions, but the links between cellular mechanical properties and transition efficiency and chromatin structure remain elusive. Here, we predicted that mechanical strain treatment could induce signatures of cellular dedifferentiation and transdifferentiation, and we validated this prediction by showing that mechanical strain-treated mouse cumulus cells (CCs) exhibit significantly improved somatic cell nuclear transfer (SCNT) reprogramming efficiency. We found that the chromatin accessibility of CCs was globally increased by mechanical straintreatment and that this increase was partially mediated by the induction of the YAP-TEAD interaction. Moreover, using mechanical strain-treated CCs could prevent transcriptional dysregulation in SCNT embryos. Taken together, our study results demonstrated that modulating cell mechanical properties to regulate epigenetic status is a promising approach to facilitate cell fate transition.

Optimization of Electroporation and Adeno-Associated Virus-Mediated Generation of 2.7 kb Knock-In Livestock Blastocysts

The production of genome-edited livestock has traditionally relied on either somatic cell nuclear transfer cloning of an edited cell or microinjection of gene-editing reagents into developing embryos. Electroporation of gene-editing reagents into zygotes offers an alternative that is both high throughput and easy to use. Transfer of large (>1 kb) homology-directed repair (HDR) donor nucleic acid templates into mammalian zygotes is hindered by the thick extracellular zona pellucida (ZP) glycoprotein layer. A potentially scalable approach to deliver HDR templates of up to 4.9 kb into zygotes is to use recombinant adeno-associated virus (rAAV) vectors. In this study, electroporation conditions were optimized to efficiently deliver CRISPR-associated protein 9 (Cas9):single guide RNA (sgRNA) ribonucleoprotein (RNP) complexes to bovine and ovine zygotes, while retaining embryo viability. rAAV vectors were then used to transduce a 3.9-kb HDR template in combination with electroporation of Cas9:sgRNA RNP editing reagents 6 h postinsemination to generate bovine blastocysts with a targeted 2.7 kb knock-in (KI) at the H11 safe harbor locus. With this approach, there was no need to remove or weaken the ZP of the blastocysts that developed, and a KI rate of up to ∼38% was observed. However, to circumvent transduction of the cumulus cells, the oocytes were denuded before transduction that decreased the blastocyst development rate. In addition, genetic mosaicism was observed in the blastocysts. These results highlight that further optimization of editing approaches will be required to achieve nonmosaic embryos with a targeted KI at scale for livestock applications.

Developmental competence of chimeric porcine embryos through the aggregation of parthenogenetic embryos and somatic cell nuclear transfer embryos

The efficiency of somatic cell nuclear transfer (NT) in pigs is low and requires enhancement. We identified the most efficient method for zona pellucida (ZP) removal and blastomere aggregation in pigs and investigated whether the aggregation of NT and parthenogenetic activation (PA) of blastomeres could reduce embryonic apoptosis and improve the quality of NT-derived embryos by investigating. Embryonic developmental competence after ZP removal using acid Tyrode's solution or protease (pronase E). The embryonic developmental potential of NT-derived blastomeres was also investigated using well-of-the-well or phytohemagglutinin-L. We analyzed apoptosis in aggregate-derived blastocysts. The aggregation rate of protease-treated embryos was lower than that of Tyrode’s solution-treated embryos (69.2% vs. 88.3%). No significant difference was observed between phytohemagglutinin-L and well-of-the-well (35.7%–38.5%). However, 2P1N showed a higher number of blastocysts compared to 3N (73.8% vs. 24.3%) and an increased blastocyst diameter compared to the control and 1P2N (274 μm vs. 230–234 μm). In blastomeres aggregated using phytohemagglutinin-L, the apoptotic cell ratio was significantly higher in 1P2N and 3N than in 3P (5.91%–6.46% vs. 2.94%, respectively). Our results indicate that aggregation of one NT embryo with two PA embryos improved the rate of blastocysts with increased blastocyst diameter.

Factors affecting superovulation induction in goats (Capra hericus): An analysis of various approaches.

Goats are generally called a "poor man's cow" because they not only provide meat and milk but also other assistance to their owners, including skins for leather production and their waste, which can be used as compost for fertilizer. Multiple ovulation and embryo transfer (MOET) is an important process in embryo biotechnology, as it increases the contribution of superior female goats to breeding operations. The field of assisted reproductive biotechnologies has seen notable progress. However, unlike in cattle, the standard use of superovulation and other reproductive biotechnologies has not been widely implemented for goats. Multiple intrinsic and extrinsic factors can alter the superovulatory response, significantly restricting the practicability of MOET technology. The use of techniques to induce superovulation is a crucial step in embryo transfer (ET), as it accelerates the propagation of animals with superior genetics for desirable traits. Furthermore, the conventional superovulation techniques based on numerous injections are not appropriate for animals and are labor-intensive as well as expensive. Different approaches and alternatives have been applied to obtain the maximum ovarian response, including immunization against inhibin and the day-0 protocol for the synchronization of the first follicular wave. While there are several studies available in the literature on superovulation in cattle, research on simplified superovulation in goats is limited; only a few studies have been conducted on this topic. This review describes the various treatments with gonadotropin that are used for inducing superovulation in various dairy goat breeds worldwide. The outcomes of these treatments, in terms of ovulation rate and recovery of transferrable embryos, are also discussed. Furthermore, this review also covers the recovery of oocytes through repeated superovulation from the same female goat that is used for somatic cell nuclear transfer (SCNT).