Aberrant Expression Levels of Androgen Receptor and SRD5A2 in Epididymal Epithelial Cells of Crossbred Infertile Cattle-Yak.

subfamily and have the same number of chromosomes, hybrid males are sterile because of the inactivation or abnormality of gene expression related to the production of healthy normal sperm. Recently, the analysis of gene expression not only in the testis but also in the epididymis has offered hints about the mechanism of infertility, because the epididymis supports the maturation of sperm in acquiring the capacity of fertilisation. Sperm maturation processes have been thought to be androgen-dependent, and the androgen receptor (AR) can be activated by dihydrotestosterone converted from plasma testosterone by the 5α-reductase isoform 2 (SRD5A2) in epididymal cells. In the present study, we investigated the immuno-expression levels of the AR and SRD5A2 in the epithelial cells of the hybrid cattle-yak epididymal caput in comparison with yak samples using image analysis. Epididymal tissues from yaks (1-3 years of age) and hybrid cattle-yaks (2 years of age) were used in this study. In yaks, AR signal intensity did not show any changes in epididymal epithelial cells during maturation. However, in 2-year-old hybrid cattle-yaks, AR signal intensity was significantly higher in the principal cells of the epididymis compared to that of yaks of the same age, indicating that hybrid sterility is not likely related to AR deficiency in the epididymal epithelium. On the other hand, SRD5A2 signal intensity was stable during maturation in the epithelial cells of the yak epididymis. However, the epididymal SRD5A2 signal intensity in the epithelial cells of the hybrid cattle-yak was lower than that of the yak. This suggests that a deficiency in SRD5A2 production in the epididymis may result in hybrid infertility, as it can subsequently cause incomplete AR signal transduction and altered spermatozoa physiology.

Animals : An Open Access Journal from MDPI


Analysis of myofiber composition in myostatin monoallelic mutant pigs


Effects of Electroporation Timing and Cumulus Cell Attachment on In Vitro Development and Genome Editing of Porcine Embryos.

Pig genome editing using the oviductal nucleic acid delivery (GONAD) method with electroporation would allow the efficient obtention of genetically modified pigs. However, oocytes and zygotes at various stages after ovulation must be targeted, and cumulus cell attachment and mosaic mutations are major obstacles. Therefore, we investigated whether two parameters (electroporation timing and the cumulus cell attachment) influence the effectiveness of multiplex genome editing by electroporation in porcine oocytes or zygotes. Three gRNAs targeting either GGTA1, CMAH or B4GALNT2 were introduced individually into oocytes and zygotes with and without cumulus cells at three different time points, 0 h before in vitro fertilisation (IVF) and 5 h and 10 h after IVF initiation. The introduction of gRNAs into oocytes and zygotes did not significantly affect the rates of blastocyst formation and total mutation of the resulting blastocysts irrespective of cumulus cell attachment and electroporation timing. In conclusion, the electroporation timing and the cumulus cell attachment did not interfere with the efficient delivery of the CRISPR/Cas9 system to the oocytes/zygotes, indicating that porcine genome editing in the oviduct using GONAD method may be possible.

Reproduction in Domestic Animals = Zuchthygiene


Efficient gene editing of pig embryos by combining electroporation and lipofection.

Electroporation combined with lipofection using gRNAs with low mutation rates may improve gene editing efficiency in pig embryos. However, the effects may vary based on the timing of gene editing.


Optimization of embryonic stage for aggregation to generate chimeric pigs using gene-edited blastomeres.

In Vitro Cellular & Developmental Biology. Animal


Genome editing of porcine zygotes via lipofection of two guide RNAs using a CRISPR/Cas9 system.

CRISPR/Cas9-based multiplex genome editing via electroporation is relatively efficient; however, lipofection is versatile because of its ease of use and low cost. Here, we aimed to determine the efficiency of lipofection in CRISPR/Cas9-based multiplex genome editing using growth hormone receptor (GHR) and glycoprotein alpha-galactosyltransferase 1 (GGTA1)-targeting guide RNAs (gRNAs) in pig zygotes. Zona pellucida-free zygotes were collected 10 h after in vitro fertilization and incubated with Cas9, gRNAs, and Lipofectamine 2000 (LP2000) for 5 h. In Experiment 1, we evaluated the mutation efficiency of gRNAs targeting either GHR or GGTA1 in zygotes transfected using LP2000 and cultured in 4-well plates. In Experiment 2, we examined the effects of the culture method on the development, mutation rate, and mutation efficiency of zygotes with simultaneouslydouble-edited GHR and GGTA1, cultured using 4-well (group culture) and 25-well plates (individual culture). In Experiment 3, we assessed the effect of additional GHR-targeted lipofection before and after simultaneous double gRNA-targeted lipofection on the mutation efficiency of edited embryos cultured in 25-well plates. No significant differences in mutation rates were observed between the zygotes edited with either gRNA. Moreover, the formation rate of blastocysts derived from GHR and GGTA1 double-edited zygotes was significantly increased in the 25-well plate culture compared to that in the 4-well plate culture. However, mutations were only observed in GGTA1 when zygotes were transfected with both gRNAs, irrespective of the culture method used. GHR mutations were detected only in blastocysts derived from zygotes subjected to GHR-targeted lipofection before simultaneous double gRNA-targeted lipofection. Overall, our results suggest that additional lipofection before simultaneous double gRNA-targeted lipofection induces additional mutations in the zygotes.

The Journal of Reproduction and Development


Effects of ergothioneine supplementation on meiotic competence and porcine oocyte development.

Supplementing with EGT during IVM leads to better oocyte maturation, quality, and embryonic development due to decreased DNA fragmentation. The present study failed to elucidate the mechanism of DNA fragmentation reduction by EGT. More research needs to be conducted to explore the antioxidant mechanism of EGT.


Effects of centrifugation treatment before electroporation on gene editing in pig embryos.

Genetic mosaicism, characterized by multiple genotypes within an individual, is considered an obstacle to CRISPR/Cas9 genome editing in animal models. Despite the various strategies for minimizing mosaic mutations, no definitive methods exist to eliminate them. This study aimed to enhance gene editing efficiency in porcine zygotes using CRISPR/Cas9, which targets specific genes through centrifugation and zona pellucida removal before electroporation. Centrifugation at 2000 × g did not adversely affect blastocyst formation rates in zygotes electroporated with gRNA targeting the GGTA1 gene; instead, it led to increased total and monoallelic mutation rates compared with control zygotes without centrifugation. However, the groups had no significant differences in biallelic mutation rates. In zygotes electroporated with gRNA targeting the CMAH gene, centrifugation treatments exceeding 1000 × g significantly increased both biallelic mutation rates and mutation efficiency. The combination of centrifugation and zona pellucida removal did not have a detrimental effect on blastocyst formation rates. It led to a higher rate of double biallelic mutations in embryos targeting both GGTA1 and CMAH compared to embryos without centrifugation treatment. In summary, our results demonstrate that pre-electroporation treatments, including centrifugation and zona pellucida removal, positively influenced the reduction of mosaic mutations, with the effectiveness of centrifugation depending on the specific gRNA used.


Evaluation of culture methods and chemical reagent combinations on CRISPR/Cas9 gene editing systems by lipofection in pig zygotes.

The delivery of CRISPR/Cas ribonucleoprotein (RNP) complexes is gaining attention owing to its high cleavage efficiency and reduced off-target effects. Although RNPs can be delivered into porcine zygotes via electroporation with relatively high efficiency, lipofection-mediated transfection appears to be versatile because of its ease of use, low cost, and adaptation to high-throughput systems. However, this system requires improvements in terms of embryo development and mutation rates. Therefore, this study elucidated the effects of culture methods and reagent combinations on the CRISPR/Cas9 gene editing systems by using three lipofection reagents: Lipofectamine™ CRISPRMAX™ Cas9 Transfection Reagent (CM), Lipofectamine™ 2000 Transfection Reagent (LP), and jetCRISPR™ RNP Transfection Reagent (Jet). Porcine zona pellucida-free zygotes were incubated for 5 h with Cas9, a guide RNA targeting CD163, and the above lipofection reagents. When examining the effect of culture methods using 4-well (multiple embryo culture) and 25-well plates (single embryo culture) on the efficiency of CM-mediated zygote transfection, the culture of embryos in 25-well plates significantly increased the blastocyst formation rate; however, there was no difference in mutation rates between the 4-well and 25-well plates. When assessing the effects of individual or combined reagents on the efficiency of zygote transfection, the mutation rate was significantly lower for individual LP compared to individual CM- and Jet-mediated transfections. Moreover, combinations of lipofection transfection reagents did not significantly increase the mutation rate or mutation efficiency.


Programmed cell death-1-modified pig developed using electroporation-mediated gene editing for in vitro fertilized zygotes.

Programmed cell death-1 (PD-1) is an immunoinhibitory receptor required to suppress inappropriate immune responses such as autoimmunity. Immune checkpoint antibodies that augment the PD-1 pathway lead to immune-related adverse events (irAEs), organ non-specific side effects due to autoimmune activation in humans. In this study, we generated a PD-1 mutant pig using electroporation-mediated introduction of the CRISPR/Cas9 system into porcine zygotes to evaluate the PD-1 gene deficiency phenotype. We optimized the efficient guide RNAs (gRNAs) targeting PD-1 in zygotes and transferred electroporated embryos with the optimized gRNAs and Cas9 into recipient gilts. One recipient gilt became pregnant and gave birth to two piglets. Sequencing analysis revealed that both piglets were biallelic mutants. At 18 mo of age, one pig showed non-purulent arthritis of the left elbow/knee joint and oligozoospermia, presumably related to PD-1 modification. Although this study has a limitation because of the small number of cases, our phenotypic analysis of PD-1 modification in pigs will provide significant insight into human medicine and PD-1-deficient pigs can be beneficial models for studying human irAEs.


Generation of allogenic chimera carrying mutations in PDX1 and TP53 genes via phytohemagglutinin-mediated blastomere aggregation in pigs.

The generation of genetically engineered pig models that develop pancreas-specific tumors has the potential to advance studies and our understanding of pancreatic cancer in humans. TP53 mutation causes organ-nonspecific cancers, and PDX1-knockout results in the loss of pancreas development. The aim of the present study was to generate a PDX1-knockout pig chimera carrying pancreas complemented by TP53 mutant cells via phytohemagglutinin (PHA)-mediated blastomere aggregation using PDX1 and TP53 mutant blastomeres, as a pig model for developing tumors in the pancreas with high frequency. First, the concentration and exposure time to PHA to achieve efficient blastomere aggregation were optimized. The results showed that using 300 µg/mL PHA for 10 min yielded the highest rates of chimeric blastocyst formation. Genotyping analysis of chimeric blastocysts derived from aggregated embryos using PDX1- and TP53-edited blastomere indicated that approximately 28.6% carried mutations in both target regions, while 14.3-21.4% carried mutations in one target. After the transfer of the chimeric blastocysts into one recipient, the recipient became pregnant with three fetuses. Deep sequencing analysis of the PDX1 and TP53 regions using ear and pancreas samples showed that one fetus carried mutations in both target genes, suggesting that the fetus was a chimera derived from embryo-aggregated PDX1 and TP53 mutant blastomeres. Two out of three fetuses carried only the PDX1 mutation, indicating that the fetuses developed from embryos not carrying TP53-edited blastomeres. The results of the present study could facilitate the further improvement and design of high-frequency developing pancreatic tumor models in pigs.


Comparison of chemically mediated CRISPR/Cas9 gene editing systems using different nonviral vectors in porcine embryos.

The transfection efficiency of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas ribonucleoprotein complexes was compared using three nonviral vector transfection reagents: nonliposomal polymeric (TransIT-X2), lipid nanoparticle delivery (CRISPRMAX), and peptide (ProteoCarry) systems. Porcine zona pellucida-free zygotes and embryos were incubated for 5 h with CRISPR-associated protein 9 (Cas9), guide RNA (gRNA) targeting GGTA1, and one of the reagents. In Experiment 1, optimization of Cas9 protein to gRNA molar ratios of 1:2, 2:2, and 4:2, along with single or double doses of reagents, was performed on zygotes at 10 h post-in vitro fertilization. In Experiment 2, optimization of timing was performed at 10 or 29 h post-in vitro fertilization, using optimal molar ratios and reagent doses. Blastocyst formation, mutation rates, and mutation efficiency were measured in each experiment. For each reagent, a 4:2 Cas9:gRNA molar ratio and addition of a double reagent dose exhibited a higher mutation rate; however, blastocyst rate tended to decrease compared with that of control. Moreover, the optimal transfection time varied depending on the reagent, and the proportions of blastocysts carrying mutations were <34%. In conclusion, the above three transfectants allowed gene editing of porcine zygotes and embryos; however, this newly established chemistry-based technology needs further improvement, especially regarding editing efficiency and embryo development.


Mosaic TP53 Mutation on Tumour Development in Pigs: A Case Study.

mutations with truncated amino acids may be related to tumour formation.


GHR-mutant pig derived from domestic pig and microminipig hybrid zygotes using CRISPR/Cas9 system.

Pigs are excellent large animal models with several similarities to humans. They provide valuable insights into biomedical research that are otherwise difficult to obtain from rodent models. However, even if miniature pig strains are used, their large stature compared with other experimental animals requires a specific maintenance facility which greatly limits their usage as animal models. Deficiency of growth hormone receptor (GHR) function causes small stature phenotypes. The establishment of miniature pig strains via GHR modification will enhance their usage as animal models. Microminipig is an incredibly small miniature pig strain developed in Japan. In this study, we generated a GHR mutant pig using electroporation-mediated introduction of the CRISPR/Cas9 system into porcine zygotes derived from domestic porcine oocytes and microminipig spermatozoa. First, we optimized the efficiency of five guide RNAs (gRNAs) designed to target GHR in zygotes. Embryos that had been electroporated with the optimized gRNAs and Cas9 were then transferred into recipient gilts. After embryo transfer, 10 piglets were delivered, and one carried a biallelic mutation in the GHR target region. The GHR biallelic mutant showed a remarkable growth-retardation phenotype. Furthermore, we obtained F1 pigs derived from the mating of GHR biallelic mutant with wild-type microminipig, and GHR biallelic mutant F2 pigs through sib-mating of F1 pigs. We have successfully demonstrated the generation of biallelic GHR-mutant small-stature pigs. Backcrossing of GHR-deficient pig with microminipig will establish the smallest pig strain which can contribute significantly to the field of biomedical research.


Development and Genome Mutation of Bovine Zygotes Vitrified Before and After Genome Editing via Electroporation


Pigs with an INS point mutation derived from zygotes electroporated with CRISPR/Cas9 and ssODN

electroporation-mediated introduction of the CRISPR/Cas9 system into zygotes, thereby avoiding the time-consuming and complicated micromanipulation method.

Frontiers in Cell and Developmental Biology


Efficiency of genetic modification in gene-knockout sperm-derived zygotes followed by electroporation of guide RNA targeting the same gene.

Genetic mosaicism is considered one of the main limitations of the electroporation method used to transfer CRISPR-Cas9/guide RNA (gRNA) into porcine zygotes. We hypothesized that fertilization of oocytes with sperm from gene-deficient boars, in combination with electroporation (EP) to target the same region of the gene in subsequent zygotes, would increase the gene modification efficiency. As myostatin (MSTN) and α1,3-galactosyltransferase (GGTA1) have beneficial effects on agricultural production and xenotransplantation, respectively, we used these two genes to test our hypothesis. Spermatozoa from gene-knockout boars were used for oocyte fertilization in combination with EP to transfer gRNAs targeting the same gene region to zygotes. No significant differences in the rates of cleavage and blastocyst formation as well as in the mutation rates of blastocysts were observed between the wild-type and gene-deficient spermatozoa groups, irrespective of the targeted gene. In conclusion, the combination of fertilization with gene-deficient spermatozoa and gene editing of the same targeted gene region using EP had no beneficial effects on embryo genetic modification, indicating that EP alone is a sufficient tool for genome modification.


Relationship between GnRH-induced LH increase profiles in the serum and vaginal mucus of Japanese Black beef cows.

This study aimed to investigate the relationship between increases in the luteinizing hormone (LH) profiles in the serum and vaginal mucus of cows induced by gonadotropin-releasing hormone (GnRH). Samples for LH determination were collected from Japanese Black beef cows during estrus, which was induced with a controlled internal progesterone-releasing device and the administration of cloprostenol immediately before GnRH administration and every 30 min from the start of GnRH administration until 6.5 h. The peak serum LH concentration was clearly identified at 2.5 h post-GnRH administration, with serum concentrations returning to near-pre-GnRH-administration values after 6.5 h, whereas the peak vaginal mucus LH concentration was identified 4.5 h after GnRH administration. These results indicate that the LH secretion peak in vaginal mucus appeared about 2 h after peak LH secretion in the serum.


Multiple gene editing in porcine embryos using a combination of microinjection, electroporation, and transfection methods.

The combination of CRISPR/Cas9 delivery methods may improve the mutation efficiency of triple-gene edited porcine blastocysts.


Gene editing in porcine embryos using a combination of electroporation and transfection methods.

CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9) technology is growing rapidly and has been greatly influencing the efficiency and effectiveness of genetic modifications in different applications. One aspect of research gaining importance in the development of the CRISPR/Cas9 system is the introduction of CRISPR materials into target organisms. Although we previously demonstrated the efficacy of electroporation- and lipofection-mediated CRISPR/Cas9 gene disruption in porcine zygotes, we still believe that the efficiency of this system could be improved by combining these two methods. The present study was thus conducted to clarify the effects of a combination of electroporation and lipofection for delivering CRISPR/Cas9 components into zona pellucida (ZP)-intact and -free zygotes. The results revealed that electroporation alone significantly increased the biallelic mutation rates in the resulting blastocysts compared to lipofection alone, irrespective of the presence of ZP. None of ZP-intact zygotes treated by lipofectamine alone had any mutations, suggesting that removal of the ZP is necessary for enabling CRISPR/Cas9-based genome editing via lipofection treatment in the zygotes. Additional lipofectamine treatment after electroporation did not improve the rates of total and biallelic mutations in the resulting blastocysts derived from either ZP-intact or -free zygotes.

Reproduction in Domestic Animals = Zuchthygiene


Disruption of cell proliferation and apoptosis balance in the testes of crossbred cattle-yaks affects spermatogenic cell fate and sterility.

The balance between proliferation, differentiation and apoptosis is well-coordinated in spermatogenesis for the timely production of appropriate numbers of sperm in animals. Disruption or decrease in sperm production is due to many conditions, including changes in testicular cell fate balance. Interspecies hybridization of domestic yaks and cattle results in sterility in males because of spermatogenic arrest; however, the underlying mechanisms involved in sterility are still unclear. In the present study, we investigated the proliferation and apoptosis status during the development of yaks and crossbred cattle-yaks using immunohistochemistry of proliferating cell nuclear antigen and terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling assays. Testicular tissues from yaks (immature: 1 year old, mature: 2-3 years old) and backcrossed hybrids (2 year old) were collected and used to investigate the expression of each parameter in testicular cells. During the maturation of yak testes, proliferation and apoptosis became active only in spermatogenic cells, and not in other somatic cells, such as Sertoli cells, myoid cells and Leydig cells. Furthermore, hybrid cattle-yak testes maintained proliferation ability but less apoptotic ability in spermatogenic cells when compared to yaks of the same age, suggesting that normal spermatogenic cell fate control is disrupted by changes in the balance between proliferation and apoptosis. In addition, Leydig cell proliferation rate was higher than apoptosis rate in the cattle-yak testes, indicating an increased number of Leydig cells, which may affect spermatogenesis through changes in steroidogenesis. Although epigenetic changes may be involved in cattle-yak testes, further studies are needed to clarify the modulation of proliferation and apoptosis to elucidate the mechanisms of infertility in hybrid cattle-yak males.


Novel method utilizing bisulfite conversion with dual amplification-refractory mutation system polymerase chain reaction to detect circulating pancreatic β-cell cfDNA.

We developed a novel method for detecting unmethylated insulin DNA in circulation that can be performed using a conventional real-time PCR system. This method would be useful for analyzing dynamic profiles of β-cells in human disease such as type 1 diabetes.


Effects of the timing of electroporation during in vitro maturation on triple gene editing in porcine embryos using CRISPR/Cas9 system

were simultaneously introduced into the oocytes that had been incubated for 40, 42, and 44 h from the start of the IVM culture. Electroporation with three gRNAs at 40 h and 42 h during IVM culture decreased the blastocyst formation rates and did not improve the mutation rates and target number of biallelic mutations in the resulting blastocysts. The blastocyst formation rate, mutation rates, and target numbers in the resulting blastocysts from oocytes treated by electroporation at 44 h of IVM culture were similar to those of control zygotes electroporated at 13 h after the initiation of IVF. In conclusion, multiple gene editing efficiency in the resulting blastocysts was comparable between oocytes electroporated before and after the fertilization, indicating that oocytes with completed maturation time may allow better functioning of materials accepting gene editing application.


Triple gene editing in porcine embryos using electroporation alone or in combination with microinjection.

These results indicate that although a combination of MI and EP does not improve the mutation efficiency or biallelic mutation for triple-gene knockout, MI treatment before EP is better to reduce mortality in porcine zygotic gene editing through a combination of MI and EP.


Viability and developmental potential of porcine blastocysts preserved for short term in a chemically defined medium at ambient temperature.

-W [Cell-W] and cell suspension and preservation solution, Cellstor

-S [Cell-S]) for short-term storage. In the first experiment, in vitro-derived blastocyst were stored at 25ºC for 24 hr in Cell-W solution, Cell-S solution and pig embryo culture (PBM) medium. There were no differences in the rates of survival and development of stored blastocysts between the Cell-S and Cell-W solutions, but the total cell number of embryos that survived after storage in Cell-S solution was significantly higher (p < .05) than that in the Cell-W solution. In the second experiment, Cell-S solution was used to store the in vitro-derived blastocysts at 20°C, 25°C and 30°C. Storage at 20°C resulted in a significant decrease in the rates of survival and development of stored blastocysts compared to storage at 25°C or 30°C. No differences in survival and development rates were observed between storage at 25°C and 30°C, but the damage to the embryo quality after storage and culture was significantly lower at 25°C than at 30°C. In the third experiment, Cell-S solution was supplemented with β-mercaptoethanol and curcumin, either alone or in combination, as antioxidant agents. Although the supplementation with curcumin did not improve survival, it significantly increased the development rate of stored blastocysts compared with the control blastocysts stored without antioxidants. In conclusion, when porcine blastocysts were stored at 25°C for 24 hr, a Cell-S solution may be effective for maintaining the survival and development of in vitro embryos.


Short-term preservation of porcine zygotes at ambient temperature using a chemically defined medium.

We aimed to develop a simple method for the short-term preservation of in vitro-produced porcine zygotes at 25°C for up to 2 days. Firstly, we evaluated the efficiency of three storage solutions to preserve porcine zygotes at 25°C for 24 h. Two of these were commercially available defined media for cell storage (Cell-W and Cell-S), and the third was fetal bovine serum (FBS). Thereafter, we examined the effects of storing the zygotes in the Cell-W solution for 24-72 h at 25°C. The Cell-W solution was the most efficient for 24 h storage of porcine zygotes at 25°C, with no apparent effects on blastocyst quality. However, short-term storage of porcine zygotes for 24 h reduced the blastocyst formation rate compared with the fresh control group. As storage duration increased from 24 to 48 or 72 h, blastocyst formation rates were significantly decreased from 11.3% to 4.4% and 0%, respectively. In conclusion, during zygote storage, the developmental competence to the blastocyst stage decreased with time. Storage of zygotes in chemically defined media did not affect blastocyst quality, but the storage in 100% serum had an adverse effect on developing embryos causing apoptosis.


Generation of mutant pigs by lipofection-mediated genome editing in embryos.

The specificity and efficiency of CRISPR/Cas9 gene-editing systems are determined by several factors, including the mode of delivery, when applied to mammalian embryos. Given the limited time window for delivery, faster and more reliable methods to introduce Cas9-gRNA ribonucleoprotein complexes (RNPs) into target embryos are needed. In pigs, somatic cell nuclear transfer using gene-modified somatic cells and the direct introduction of gene editors into the cytoplasm of zygotes/embryos by microinjection or electroporation have been used to generate gene-edited embryos; however, these strategies require expensive equipment and sophisticated techniques. In this study, we developed a novel lipofection-mediated RNP transfection technique that does not require specialized equipment for the generation of gene-edited pigs and produced no detectable off-target events. In particular, we determined the concentration of lipofection reagent for efficient RNP delivery into embryos and successfully generated MSTN gene-edited pigs (with mutations in 7 of 9 piglets) after blastocyst transfer to a recipient gilt. This newly established lipofection-based technique is still in its early stages and requires improvements, particularly in terms of editing efficiency. Nonetheless, this practical method for rapid and large-scale lipofection-mediated gene editing in pigs has important agricultural and biomedical applications.


Effects of individual or in-combination antioxidant supplementation during in vitro maturation culture on the developmental competence and quality of porcine embryos.

The oocyte maturation process requires a high supply of energy, which generates reactive oxygen species (ROS), adversely affecting oocyte and embryo development. Balancing ROS by antioxidant supplementation is essential for maintaining oocyte maturation and embryonic quality in vitro. This study aimed to evaluate the impact of four antioxidants: β-mercaptoethanol (β-ME), chlorogenic acid (CGA), curcumin and sericin, when applied individually or in combinations, during oocyte maturation on development of porcine oocytes. Cumulus-oocyte complexes were collected, cultured in maturation medium supplemented with antioxidants for 44 hr and subsequently subjected to in vitro fertilization (IVF) and culture for 7 days. Combining all four (β-ME + CGA + curcumin + sericin) or three (β-ME + CGA + curcumin) antioxidants increased blastocyst formation rates. However, sericin supplementation alone, or in combination with β-ME or CGA, failed to improve blastocyst formation rates. The total cell numbers of blastocysts from the group supplemented with three antioxidants (β-ME + CGA + curcumin) were significantly higher than those from the other groups, except for the curcumin-supplement group. There were no differences in the maturation rates and proportions of oocytes with fragmented DNA between the antioxidant-supplemented and the non-supplemented control groups. In conclusion, supplementation with three antioxidants (β-ME + CGA + curcumin) during the maturation culture enhanced blastocyst formation and improved blastocyst quality.

Reproduction in Domestic Animals = Zuchthygiene


Aberrant levels of DNA methylation and H3K9 acetylation in the testicular cells of crossbred cattle-yak showing infertility.

Although the interspecies hybridization of bovids, such as cattle-yak (Bos taurus × Bos grunniens), has heterosis benefits, the infertility of hybrid males affects the maintenance of dominant traits in subsequent generations. To achieve reproductive capacity, male germ cell development requires coordinated changes in gene expression, including DNA methylation and generalized histone modifications. Although gene expression-related mechanisms underlying hybrid male sterility have been investigated recently, information on the cell types and stage-specific controls remains limited. Here, we used immunohistochemistry and image analyses to evaluate the 5-methylcytosine (5MC) and acetyl-histone H3 Lys9 (AcK9) expression in all spermatogonia and testicular somatic cell types to determine their roles in cattle-yak spermatogenesis. Testicular tissues from yak (1-3 years old) and backcrossed hybrids (2 years old) were used. In yak, the AcK9 expression levels increased in all cell types during maturation, but the 5MC expression levels did not change until reaching 3 years when they increased in all testicular cell types, except spermatogonia. Cattle-yak hybrids showed higher 5MC expression levels and different AcK9 expression levels in all cell types compared to the same-aged yak. These results suggested that both gene modulation by AcK9 and constant levels of DNA methylation are required for spermatogenesis during maturation in yak. Therefore, inappropriate expression levels of both AcK9 and DNA methylation might be the major factors for disruption of normal germ cell development in cattle-yak. Additionally, various modulations occurred depending on the cell type. Further experiments are needed to identify the stage-specific gene expression modulations in each cell type in yak and cattle-yak to potentially solve the infertility issue in crossbreeding.

Reproduction in Domestic Animals = Zuchthygiene


Development and characterization of Gal KO porcine bone marrow-derived mesenchymal stem cells.

We demonstrated that neonatal porcine bone marrow-derived mesenchymal stem cell (npBM-MSCs) could improve a critical ischemic limb disease in rat model more efficiently compared with human MSCs. However, since porcine MSC presents galactosyl-alpha 1,3-galactose antigen (Gal antigen), MSC could be eliminated by the xenogeneic rejection. Recently, we established Gal knockout (KO) pigs by a technique of the electroporation of the CRISPR/Cas9 system into vitro-fertilized zygotes. In this study, we hypothesized that MSC from the established Gal KO pigs could further improve the efficacy. Before examining the hypothesis, in this study, we have established and characterized bone marrow-derived MSC from the Gal KO adult pigs (apBM-MSCs). Mononuclear cells (MNCs) were isolated from bone marrow cells of both Gal KO adult pigs and wild-type (WT) adult pigs. MNCs were further manipulated to create Gal KO apBM-MSCs and WT apBM-MSCs. Both MSCs were assessed by their surface markers, the capability of differentiation into adipocytes, osteocytes and chondrocytes, grow speed and colony-forming assay. To assess the efficacy of Gal KO apBM-MSCs, angiogenesis-related genes and immunosuppression-related genes were assessed by cytokine stimulation. Gal KO apBM-MSC showed no Gal antigen on their cell surfaces. Both Gal KO apBM-MSCs and WT apBM-MSCs, presented little or no negative surface markers of MSCs, while they presented positive surface markers of MSCs. Furthermore, Gal KO apBM-MSCs were able to differentiate into adipocytes, osteocytes, and chondrocytes as well as WT apBM-MSCs. There was no difference in doubling time between Gal KO apBM-MSCs and WT apBM-MSCs. Interestingly, the colony-forming efficiency of Gal KO apBM-MSCs was about half that of WT apBM-MSC. However, angiogenesis and immunosuppression-related genes were equally upregulated in both Gal KO apBM-MSCs and WT apBM-MSCs by cytokine stimulation. We created and characterized Gal KO apBM-MSCs which showed similar characteristics and cytokine-induced gene upregulation to the WT apBM-MSCs.


Zona pellucida treatment before CRISPR/Cas9-mediated genome editing of porcine zygotes.

Increasing the permeability of the zona pellucida (ZP) of oocytes before CRISPR/Cas9 electroporation may improve the efficiency of gene editing; however, the effects of this approach on subsequent developmental processes are unclear. In this study, the effects of ZP treatment before electroporation on embryonic development and gene editing in porcine embryos were evaluated. The ZP of zygotes was weakened or removed by exposure to 0.5% actinase E, followed by electroporation of the Cas9 protein with guide RNA targeting GGTA1. The blastocyst formation rate of ZP-free zygotes after electroporation was significantly lower (p < 0.05) than that of ZP-intact zygotes. The mutation rate in blastocysts from ZP-weakened zygotes was similar to that in ZP-intact zygotes, whereas ZP removal increased the mutation rate. The mutation efficiency in blastocysts from electroporated zygotes did not differ among ZP treatment groups. Our results indicate that weakening the ZP does not affect the developmental competence, mutation rate, or mutation efficiency of electroporated zygotes, whereas ZP removal has a detrimental effect on embryonic development but may increase the mutation rate.


Timing and duration of lipofection-mediated CRISPR/Cas9 delivery into porcine zygotes affect gene-editing events.

Lipofection-mediated introduction of the CRISPR/Cas9 system in porcine zygotes provides a simple method for gene editing, without requiring micromanipulation. However, the gene editing efficiency is inadequate. The aim of this study was to improve the lipofection-mediated gene editing efficiency by optimizing the timing and duration of lipofection. Zona pellucida (ZP)-free zygotes collected at 5, 10, and 15 h from the start of in vitro fertilization (IVF) were incubated with lipofection reagent, guide RNA (gRNA) targeting GGTA1, and Cas9 for 5 h. Lipofection of zygotes collected at 10 and 15 h from the start of IVF yielded mutant blastocysts. Next, ZP-free zygotes collected at 10 h from the start of IVF were incubated with lipofection reagent, gRNA, and Cas9 for 2.5, 5, 10, or 20 h. The blastocyst formation rate of zygotes treated for 20 h was significantly lower (p < 0.05) than those of the other groups, and no mutant blastocysts were obtained. Moreover, the mutation rates of the resulting blastocysts decreased as the incubation time increased. In conclusion, a lipofection-mediated gene editing system using the CRISPR/Cas9 system in ZP-zygotes is feasible; however, further improvements in the gene editing efficiency are required.


Vaginal stimulation enhances ovulation of queen ovaries treated using a combination of eCG and hCG.

Follicular changes throughout the oestrous phase have been poorly documented in queens because of the location and the small size of ovaries. We investigated follicular development in queens treated with a combination of equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG) and evaluated the effects of vaginal stimulation by a tomcat on ovulation induction. A hormonal treatment was administered using a simple crossover design. Four queens were administered 150 IU of eCG (day 1) and 250 IU of hCG on day 5 and 6. Half of the queens were mated with a vasectomised tomcat for 3 days after hCG injection. Ultrasound imaging of the ovaries clamped at a subcutaneous site was performed once a day from day 1 to 7, and on day 13, and the serum concentrations of oestradiol and progesterone were examined on day 1, 5, 7 and 13. The mean number of follicles gradually increased with the eCG treatment and decreased after hCG injection. The ovulation rate of follicles was significantly higher in the vaginal stimulation group (70.0%) than in the control group (42.6%). During the hormonal treatments, the serum concentration of oestradiol and progesterone did not differ between the two groups. Ultrasound imaging of the ovaries clamped at a subcutaneous site showed that eCG and hCG treatment promoted the follicular growth and corpus luteum formation, respectively. The combination of hCG injection with vaginal stimulation by a vasectomised tomcat enhanced the ovulation rate of follicles.


Improvement of the in vitro fertilization and embryo development using frozen-thawed spermatozoa of microminipigs.

This study aimed to compare the quality and the penetration ability of frozen-thawed spermatozoa from three microminipigs and Large White boars and to evaluate the effects of caffeine and heparin as well as the sperm-oocyte co-incubation length on the fertilization and embryonic development in vitro. Results showed that the fertilization rates of spermatozoa from three microminipig boars were significantly lower than those of a Large White boar. In the post-thaw spermatozoa from one of three microminipig boars, the sperm quality, penetration ability, and the oocyte development after in vitro fertilization were significantly lower than those of the spermatozoa from other boars. The caffeine supplementation in the fertilization media increased the rates of fertilization and blastocyst formation for the microminipig spermatozoa with low sperm quality. In addition to caffeine supplementation, the rates of fertilization and blastocyst formation after using microminipig spermatozoa were significantly higher with a 10 h sperm-oocyte co-incubation than with 3 h of co-incubation length. Our results indicate that the differences between the males and the breed influence the quality and fertility of frozen-thawed boar spermatozoa. In conclusion, the presence of caffeine in the in vitro fertilization (IVF) medium and adequate length of sperm-oocyte co-incubation may have beneficial effects for improving IVF results when using microminipig spermatozoa with low quality.


One-Step Generation of Multiple Gene-Edited Pigs by Electroporation of the CRISPR/Cas9 System into Zygotes to Reduce Xenoantigen Biosynthesis.

triple-edited pigs. Immunohistological analyses demonstrated the downregulation of xenoantigens; however, these multiple gene-edited pigs were genetic mosaics that failed to knock out some xenoantigens. Although mosaicism should be resolved, the electroporation technique could become a primary method for the one-step generation of multiple gene modifications in pigs aimed at improving pig-to-human xenotransplantation.

International Journal of Molecular Sciences


Lipofection-Mediated Introduction of CRISPR/Cas9 System into Porcine Oocytes and Embryos.

Liposome-mediated gene transfer has become an alternative method for establishing a gene targeting framework, and the production of mutant animals may be feasible even in laboratories without specialized equipment. However, how this system functions in mammalian oocytes and embryos remains unclear. The present study was conducted to clarify whether blastocyst genome editing can be performed by treatment with lipofection reagent, guide RNA, and Cas9 for 5 h without using electroporation or microinjection. A mosaic mutation was observed in blastocysts derived from zona pellucida (ZP)-free oocytes following lipofection treatment, regardless of the target genes. When lipofection treatment was performed after in vitro fertilization (IVF), no significant differences in the mutation rates or mutation efficiency were found between blastocysts derived from embryos treated at 24 and 29 h from the start of IVF. Only blastocysts from embryos exposed to lipofection treatment at 29 h after IVF contained biallelic mutant. Furthermore, there were no significant differences in the mutation rates or mutation efficiency between blastocysts derived from embryos at the 2- and 4-cell stages. This suggests that lipofection-mediated gene editing can be performed in ZP-free oocytes and ZP-free embryos; however, other factors affecting the system efficiency should be further investigated.

Animals : An Open Access Journal from MDPI


Comparison of the effects of introducing the CRISPR/Cas9 system by microinjection and electroporation into porcine embryos at different stages.

Cytoplasmic microinjection and electroporation of the CRISPR/Cas9 system into zygotes are used for generating genetically modified pigs. However, these methods create mosaic mutations in embryos. In this study, we evaluated whether the gene editing method and embryonic stage for gene editing affect the gene editing efficiency of porcine embryos.

First, we designed five guide RNAs (gRNAs) targeting the B4GALNT2 gene and evaluated mutation efficiency by introducing each gRNA with Cas9 protein into zygotes by electroporation. Next, the optimized gRNA with Cas9 protein was introduced into 1-cell and 2-cell stage embryos by either microinjection or electroporation. The sequence of gRNA affected the bi-allelic mutation rate and mutation efficiency of blastocysts derived from electroporated embryos. Microinjection significantly decreased the cleavage rates in each embryonic stage and blastocyst formation rates in 2-cell stage embryos compared with electroporation (p < 0.05). However, the bi-allelic mutation rate and mutation efficiency of blastocysts from the 1-cell stage embryos edited using microinjection were significantly higher (p < 0.05) than those of blastocysts from the 2-cell stage embryos edited by both methods. These results indicate that the gene editing method and embryonic stage for gene editing may affect the genotype and mutation efficiency of the resulting embryos.


Comparison of blastocyst development between cat-cow and cat-pig interspecies somatic cell nuclear transfer embryos under the treatment of Trichostatin A.

Biology bulletin of the Russian Academy of Sciences


Chlorogenic acid and insulin-transferrin-selenium supplementation during in vitro maturation enhances the developmental competence of interspecies chimera blastocysts following cell injection.


Introduction of a point mutation in the KRAS gene of in vitro fertilized porcine zygotes via electroporation of the CRISPR/Cas9 system with single-stranded oligodeoxynucleotides.

This study aimed to investigate the efficiency of KRAS gene editing via CRISPR/Cas9 delivery by electroporation and analyzed the effects of the non-homologous end-joining pathway inhibitor Scr7 and single-stranded oligodeoxynucleotide (ssODN) homology arm length on introducing a point mutation in KRAS. Various concentrations (0-2 µM) of Scr7 were evaluated; all concentrations of Scr7 including 0 µM resulted in the generation of blastocysts with a point mutation and the wild-type sequence or indels. No significant differences in the blastocyst formation rates of electroporated zygotes were observed among ssODN homology arm lengths, irrespective of the gRNA (gRNA1 and gRNA2). The proportion of blastocysts carrying a point mutation with or without the wild-type sequence and indels was significantly higher in the ssODN20 group (i.e., the group with a ssODN homology arm of 20 bp) than in the ssODN60 group (gRNA1: 25.7% vs. 5.4% and gRNA2: 45.5% vs. 5.9%, p < .05). In conclusion, the CRISPR/Cas9 delivery with ssODN via electroporation is feasible for the generation of point mutations in porcine embryos. Further studies are required to improve the efficiency and accuracy of the homology-directed repair.


Generation of CD163-edited pig via electroporation of the CRISPR/Cas9 system into porcine in vitro-fertilized zygotes.


One-step genome editing of porcine zygotes through the electroporation of a CRISPR/Cas9 system with two guide RNAs.

In the present study, we investigated whether electroporation could be used for one-step multiplex CRISPR/Cas9-based genome editing, targeting IL2RG and GHR in porcine embryos. First, we evaluated and selected guide RNAs (gRNAs) by analyzing blastocyst formation rates and genome editing efficiency. This was performed in embryos electroporated with one of three different gRNAs targeting IL2RG or one of two gRNAs targeting GHR. No significant differences in embryo development rates were found between control embryos and those subjected to electroporation, irrespective of the target gene. Two gRNAs targeting IL2RG (nos. 2 and 3) contributed to an increased biallelic mutation rate in porcine blastocysts compared with gRNA no. 1. There were no significant differences in the mutation rates between the two gRNAs targeting GHR. In our next experiment, the mutation efficiency and the development of embryos simultaneously electroporated with gRNAs targeting IL2RG and GHR were investigated. Similar embryo development rates were observed between embryos electroporated with two gRNAs and control embryos. When IL2RG-targeting gRNA no. 2 was used with GHR-targeting gRNAs no. 1 or no. 2, a significantly higher double biallelic mutation rate was observed than with IL2RG-targeting gRNA no. 3. In conclusion, we demonstrate the feasibility of using electroporation to transfer multiple gRNAs and Cas9 into porcine zygotes, enabling the double biallelic mutation of multiple genes with favorable embryo survival.

In Vitro Cellular & Developmental Biology. Animal


Efficient generation of GGTA1-deficient pigs by electroporation of the CRISPR/Cas9 system into in vitro-fertilized zygotes.

Xenoantigens are a major source of concern with regard to the success of interspecific xenografts. GGTA1 encodes α1,3-galactosyltransferase, which is essential for the biosynthesis of galactosyl-alpha 1,3-galactose, the major xenoantigen causing hyperacute rejection. GGTA1-modified pigs, therefore, are promising donors for pig-to-human xenotransplantation. In this study, we developed a method for the introduction of the CRISPR/Cas9 system into in vitro-fertilized porcine zygotes via electroporation to generate GGTA1-modified pigs. We designed five guide RNAs (gRNAs) targeting distinct sites in GGTA1. After the introduction of the Cas9 protein with each gRNA via electroporation, the gene editing efficiency in blastocysts developed from zygotes was evaluated. The gRNA with the highest gene editing efficiency was used to generate GGTA1-edited pigs. Six piglets were delivered from two recipient gilts after the transfer of electroporated zygotes with the Cas9/gRNA complex. Deep sequencing analysis revealed that five out of six piglets carried a biallelic mutation in the targeted region of GGTA1, with no off-target events. Furthermore, staining with isolectin B4 confirmed deficient GGTA1 function in GGTA1 biallelic mutant piglets. We established GGTA1-modified pigs with high efficiency by introducing a CRISPR/Cas9 system into zygotes via electroporation. Multiple gene modifications, including knock-ins of human genes, in porcine zygotes via electroporation may further improve the application of the technique in pig-to-human xenotransplantation.


Evaluation of multiple gene targeting in porcine embryos by the CRISPR/Cas9 system using electroporation.

The CRISPR/Cas9 system now allows for unprecedented possibilities of genome editing. However, there are some limitations, including achieving efficient one-step multiple genome targeting to save costs, time, and ensure high quality. In the present study, we investigated the efficiency of one-step multiple gene modification by electroporation in porcine zygotes using pooled guide RNAs (gRNAs) targeting CMAH, GHR, GGTA1, and PDX1. We first selected the best-performing gRNA from three different designs for each gene based on the effect on embryo development and mutation efficiency. The three gRNAs showed equivalent effects on the rates of blastocyst formation in each targeted gene; however, gRNAs CMAH #2, GHR #3, GGTA1 #3, and PDX1 #3 showed the highest biallelic mutation rate, although the total mutation rate of PDX1 #3 was significantly lower than that of PDX1 #1. Therefore, CMAH #2, GHR #3, GGTA1 #3, and PDX1 #1 were used as a mixture in electroporation to further clarify whether multiple genes can be targeted simultaneously. Individual sequencing of 43 blastocysts at the target sites of each gene showed mutations in one and two target genes in twenty-four (55.8%) and nine (20.9%) blastocysts, respectively. No mutation was detected in any target gene in ten (23.3%) blastocysts and no blastocysts had a mutation in three or more target genes. These results indicate that electroporation could effectively deliver multiple gRNAs and Cas9 protein into porcine zygotes to target multiple genes in a one-step process. However, the technique requires further development to increase the success rate of multiple gene modification.


Comparative analysis of bilirubin glucuronidation activity in 2D- and 3D-cultured human hepatocellular carcinoma HepG2 cells.


Generation of viable PDX1 gene-edited founder pigs as providers of nonmosaics.

Pancreatic duodenal homeobox 1 (PDX1) is a crucial gene for pancreas development during the fetal period. PDX1-modified pigs have the potential to be used as a model of diabetes mellitus. However, the severe health problems caused by the PDX1 mutation limit phenotypic studies of PDX1-modified pigs as diabetes models. In this study, we generated PDX1-modified pigs by the CRISPR/Cas9 system introduced into zygotes via electroporation and investigated the mosaicism, phenotypes, and inheritance of the resulting pigs. After the embryo transfer of PDX1-modified zygotes, nine mutant piglets were delivered. Two piglets were apancreatic biallelic mutants. For the other seven piglets, the ratio of mutant alleles to total alleles was 17.5-79.7%. Two mutant piglets with high mutation rates (67.7% and 79.7%) exhibited hypoplasia of the pancreas, whereas the other five piglets were healthy. One of the male mutant piglets was further analyzed. The ejaculated semen from the pig contained PDX1-mutant spermatozoa and the pig showed normal reproductive ability. In conclusion, the frequency of the PDX1 mutation is presumed to relate to pancreas formation, and PDX1 mutant founder pigs generated from zygotes introduced to the CRISPR/Cas9 system can serve as providers of nonmosaics to contribute to medical research on diabetes mellitus.


In vitro Development of Zona Pellucida-free Porcine Zygotes Cultured Individually after Vitrification.

Our results suggest that the removal of the ZP does not cause detrimental effects to the development of vitrified-warmed zygotes.


Abnormal functions of Leydig cells in crossbred cattle-yak showing infertility.

In Mongolia, yak (Bos grunniens) are able to live in alpine areas and their products greatly influence the lives of the local people. Increased vigour in hybridized yak and cattle can offer benefits for livestock farmers. However, male hybrids show reproductive defects resulting from spermatogenesis arrest, affecting the conservation and maintenance of dominant traits in the next generation. The underlying mechanisms involved in hybrid cattle-yak infertility have recently been investigated; however, the genetic cause is still unclear. Androgens and androgen receptor (AR) signalling are required for spermatogenesis. We, therefore, evaluated the expression of AR, 3β-hydroxysteroid dehydrogenase (3βHSD) and 5α-reductase 2 (SRD5A2) in Leydig cells to investigate their function in cattle-yak spermatogenesis. Testicular tissues from yaks (1-3 years old) and hybrids (F1-F3, 2 years old) were collected and subjected to immunohistochemistry and image analyses to investigate the expression of each parameter in the Leydig cells. After maturation at 2 years, the expression levels of AR increased and the levels of 3βHSD decreased, but the SRD5A2 levels remained constant in yak. However, the cattle-yak hybrid F2 showed immature testicular development and significantly different expression levels of AR and 3βHSD compared with mature yak. These results suggest that the decreased expression of AR and increased expression of 3βHSD in the Leydig cells of cattle-yak hybrid testes may represent one of the causes of infertility. Our study might help in solving the problem of infertility in crossbreeding.

Reproduction in Domestic Animals = Zuchthygiene


Curcumin supplementation in maturation medium improves the maturation, fertilisation, and developmental competence of porcine oocytes.

This study was conducted to determine the effects of supplementing the maturation medium with the antioxidant curcumin on the in vitro maturation (IVM), fertilisation and development of porcine oocytes. Curcumin supplementation was performed at concentrations of 0, 5, 10, 20, and 40 µM. At concentrations of 5-20 µM, curcumin had significant positive effects (P < 0.05) on maturation and fertilisation rates compared to the non-treated group. Of the groups cultured with 5-20 µM curcumin, the number of oocytes with DNA-fragmented nuclei after IVM was significantly lower than in groups matured without curcumin. Moreover, curcumin supplementation at 10 µM also gave a significantly higher rate of blastocyst formation compared with oocytes matured without curcumin. Increasing the curcumin concentration to 40 µM yielded negative effects on fertilisation and embryonic development compared with the groups treated with lower concentrations of curcumin. Supplementation with 10 µM curcumin had beneficial effects on the oocyte maturation rate and DNA fragmentation index compared to the non-treated group both in the presence and absence of hydrogen peroxide. These results indicate that curcumin supplementation at a suitable concentration (10 µM) is potentially useful for porcine oocyte culture systems, in terms of protecting oocytes from various forms of oxidative stress.


Effects of electroporation treatment using different concentrations of Cas9 protein with gRNA targeting Myostatin (MSTN) genes on the development and gene editing of porcine zygotes.

This study was conducted to investigate the effect of seven concentrations of Cas9 protein (0, 25, 50, 100, 200, 500, and 1,000 ng/µl) on the development and gene editing of porcine embryos. This included the target editing and off-target effect of embryos developed from zygotes that were edited via electroporation of the Cas9 protein with guide RNA targeting Myostatin genes. We found that the development to blastocysts of electroporated zygotes was not affected by the concentration of Cas9 protein. Although the editing rate, which was defined as the ratio of edited blastocysts to total examined blastocysts, did not differ with Cas9 protein concentration, the editing efficiency, which was defined as the frequency of indel mutations in each edited blastocyst, was significantly decreased in the edited blastocysts from zygotes electroporated with 25 ng/µl of Cas9 protein compared with that of blastocysts from zygotes electroporated with higher Cas9 protein concentrations. Moreover the frequency of indel events at the two possible off-target sites was not significantly different with different concentrations of Cas9 protein. These results indicate that the concentration of Cas9 protein affects gene editing efficiency in embryos but not the embryonic development, gene editing rate, and non-specific cleavage of off-target sites.


The relationship between embryonic development and the efficiency of target mutations in porcine endogenous retroviruses (PERVs) pol genes in porcine embryos.

Animals : An Open Access Journal from MDPI


Genome mutation after the introduction of the gene editing by electroporation of Cas9 protein (GEEP) system into bovine putative zygotes

The present study was designed to investigate the effects of voltage strength on embryonic developmental rate and mutation efficiency in bovine putative zygotes during electroporation with the CRISPR/Cas9 system to target the MSTN gene at different time points after insemination. Results showed that there was no significant interaction between electroporation time and voltage strength on the embryonic cleavage and blastocyst formation rates. However, increasing the voltage strength to 20 V/mm to electroporate the zygotes at 10 h after the start of insemination yielded significantly lower blastocyst formation rates (P<0.05) than those of the 10-V/mm electroporated zygotes. Mutation efficiency was then assessed in individual blastocysts by DNA sequence analysis of the target sites in the MSTN gene. A positive correlation between mutation rate and voltage strength was observed. The mutation efficiency in mutant blastocysts was significantly higher in the zygotes electroporated with 20 V/mm at 10 h after the start of insemination (P<0.05) than in the zygotes electroporated at 15 h, irrespective of the voltage strength. We also noted that a certain number of blastocysts from zygotes that were electroporated with more than 15 V/mm at 10 h (4.8-16.7%) and 20 V/mm at 15 h (4.8%) were biallelic mutants. Our results suggest that the voltage strength during electroporation as well as electroporation time certainly have effects on the embryonic developmental rate and mutation efficiency in bovine putative zygotes.

In Vitro Cellular & Developmental Biology. Animal


The effects of electroporation on viability and quality of in vivo-derived bovine blastocysts

The introduction of exogenous molecules into embryos is required for analyses of molecular dynamics and specific gene functions during early embryonic development. Electroporation is an effective method to transport exogenous molecules into cells, but is rarely used in bovine embryos. First, we evaluated the viability of in vivo-derived bovine blastocysts after electroporation with fluorescein (FAM) labeled-oligonucleotides with varying pulse numbers (3, 5, 7, and 10), while keeping the pulse duration at 1 msec and the electric field of 20 V/mm. Next, we examined the effects of zona pellucida status on blastocyst quality after electroporation, by comparing the average diameter of blastocysts before and after electroporation using blastocysts with intact zona pellucida and hatching/hatched blastocysts. Electroporation successfully introduced exogenous molecules into in vivo-derived bovine blastocysts without loss of viability. Moreover, the status of the zona pellucida may be associated with the quality of blastocysts after electroporation.

The Journal of Reproduction and Development


Relationship among ovarian follicular status, developmental competence of oocytes, and anti-M llerian hormone levels: A comparative study in Japanese wild boar crossbred gilts and Large White gilts.

The aim of this study was to investigate the ovarian follicular development, developmental competence of oocytes, and plasma anti-M llerian hormone (AMH) levels of Japanese wild boar crossbred (wild hybrid) gilts, whose litter size is inferior to that of European breeds. Ovary and plasma samples were collected from two different breeds of gilts (wild hybrid and Large White breeds). The ovaries from the wild hybrid gilts had a lower average numbers of secondary follicles and vesicular follicles in ovarian cross-sections and of good quality oocytes collected from ovarian follicles as compared with those from Large White gilts (p < 0.05). The development rate to the blastocyst stage of good quality oocytes after in vitro maturation, fertilization and culture was also lower (p < 0.05) in wild hybrid gilts than in Large White gilts. Plasma AMH levels with >0.16 ng/ml were detected in 8.3% of the examined wild hybrid gilts and 33% of the Large White gilts. These results indicate that the low reproductive performance of wild hybrid breed may result in part from low numbers of vesicular follicles and good quality oocytes, and low developmental competence of oocytes. Moreover, plasma AMH levels may support low number of vesicular follicles in ovaries of wild hybrid gilts.


Hypothermic storage of porcine zygotes in serum supplemented with chlorogenic acid.

The current study was conducted to investigate the effects of 100% foetal bovine serum (FBS) and 100% porcine follicular fluid (pFF) as a storage medium on the developmental competence of porcine zygotes stored at 25 C for 24 hr. Moreover, we evaluated the additive effects of chlorogenic acid (CGA) in the storage medium. When in vitro-produced zygotes were stored at 25 C for 24 hr in tubes containing either tissue culture medium (TCM) 199 supplemented with 1 mg/ml bovine serum albumin (BSA), 100% of FBS or 100% of pFF, the rate of blastocyst formation was significantly higher in 100% of FBS than in BSA-containing TCM 199. When the effects of CGA supplementation in 100% of FBS on the development of zygotes stored at 25 C for 24 hr was evaluated, more zygotes stored with 50 M CGA developed to blastocysts compared with the other concentrations of CGA. When the formation date and quality of blastocysts derived from zygotes stored in 100% of FBS supplemented with 50 M CGA were investigated, the highest ratio of blastocysts formation in the storage group appeared 1 day later than in the non-stored control group. However, a higher proportion of blastocysts with apoptotic nuclei was observed in the stored group as compared to the non-stored group. In conclusion, 100% of FBS is available for a short storage medium of porcine zygotes. The supplementation of 50 M CGA into the storage medium improves the rates of blastocyst formation of zygotes after storage, but the quality of embryos from the stored zygotes remains to be improved.

Reproduction in Domestic Animals = Zuchthygiene


Effects of Tris (hydroxymethyl) aminomethane on the quality of frozen-thawed boar spermatozoa.

Tris (hydroxymethyl) aminomethane (Tris) has been used as a pH regulator for buffering the pH of dilution extenders for boar semen, such as the Modena extender. The purpose of the present study was to assess the effects of Tris supplementation at different concentrations (0, 8, 24 and 72 M) into the freezing extender on the quality and fertilising capacity of frozen-thawed boar spermatozoa. The results showed that the supplementation of 24 M of Tris gave significantly higher percentages of sperm viability and plasma membrane integrity than those of the control group at any time point of assessment (0 h and 3 h post-thawing) (P < 0.05). However, there were no significant differences in the acrosome integrity parameter among the groups. Higher percentages of sperm motility were observed in the spermatozoa cryopreserved with 24 M of Tris compared to the control groups when the samples were analysed 0 h after thawing (P < 0.05). However, an increase of the Tris concentration to 72 M did not enhance the sperm motility parameters. The total numbers of fertilised oocytes and blastocysts obtained with spermatozoa frozen with 24 M Tris were significantly higher than those of the control group without Tris (P < 0.05). In conclusion, the supplementation of 24 M Tris into the freezing extender contributes to a better boar sperm quality and fertilising capacity after the process of freezing and thawing.


Genome mutation after introduction of the gene editing by electroporation of Cas9 protein (GEEP) system in matured oocytes and putative zygotes.

The application of CRISPR/Cas9 strategy promises to rapidly increase the production of genetically engineered animals since it yields stably integrated transgenes. In the present study, we investigated the efficiency of target mutations after electroporation with the CRISPR/Cas9 system using sgRNAs to target the MSTN or FGF10 genes in porcine-matured oocytes and putative zygotes. Effects of pulse number (3-7 pulse repetitions) during electroporation on the embryonic development and mutation efficiency were also investigated. Our results showed that the cleavage rate of matured oocytes with electroporation treatment significantly decreased as compared with electroporated putative zygotes (p < 0.05). Moreover, the rates of blastocyst formation from oocytes/zygotes electroporated with more than 5 pulses decreased. Mutation efficiency was then assessed after sequencing the target sites in individual blastocysts derived from oocytes/zygotes electroporated by 3 and 5 pulses. No bi-allelic mutations in all examined blastocysts were observed in this study. There were no differences in the mutation rates (50-60%) between blastocysts derived from matured oocytes electroporated by 3 and 5 pulses, irrespective of targeting gene. In the targeting MSTN gene, however, the mutation rate (12.5%) of blastocysts derived from putative zygotes electroporated by 3 pulses tended to be lower than that (60%) from 5-pulsed electroporated putative zygotes. These data indicate that the type of eggs may influence not only their development after electroporation treatment but also the mutation rate in the resulting blastocysts.


Effects of concentration of CRISPR/Cas9 components on genetic mosaicism in cytoplasmic microinjected porcine embryos.

Cytoplasmic microinjection (CI) of the CRISPR/Cas9 system enabled the induction of site-specific mutations in porcine zygotes and resulting pigs. However, mosaicism is a serious problem for genetically modified pigs. In the present study, we investigated suitable timing and concentration of CRISPR/Cas9 components for introduction into oocytes/zygotes by CI, to reduce mosaicism in the resulting blastocysts. First, we introduced 20 ng/ l of Cas9 protein and guide RNA (gRNA), targeting the -1,3-galactosyltransferase (GalT) gene in oocytes before in vitro fertilization (IVF), in zygotes after IVF, or in oocytes/zygotes before and after IVF, twice. CI treatment had no detrimental effects on blastocyst formation rates. The highest value of the rate of mutant blastocysts was observed in zygotes injected after IVF. Next, we injected Cas9 protein and gRNA into zygotes after IVF at a concentration of 20 ng/ l each (20 ng/ l group) or 100 ng/ l each (100 ng/ l group). The ratio of the number of blastocysts that carried mutations to the total number of blastocysts examined in the 100 ng/ l group was significantly higher (P < 0.05) than that in the 20 ng/ l group. Although no blastocysts from the 20 ng/ l group carried a biallelic mutation, 16.7% of blastocysts from the 100 ng/ l group carried a biallelic mutation. In conclusion, increasing the concentration of Cas9 protein and gRNA is effective in generating biallelic mutant blastocysts. To reduce mosaicism, however, further optimization of the timing of CI, and the concentration of CRISPR/Cas9 components, is needed.


Generation of PDX-1 mutant porcine blastocysts by introducing CRISPR/Cas9-system into porcine zygotes via electroporation.

Recently, we established the GEEP ("gene editing by electroporation of Cas9 protein") method, in which the CRISPR/Cas9 system, consisting of a Cas9 protein and single guide RNA (sgRNA), is introduced into pig zygotes by electroporation and thus induces highly efficient targeted gene disruption. In this study, we examined the effects of sgRNA on the blastocyst formation of porcine embryos and evaluated their genome-editing efficiency. To produce an animal model for diabetes, we targeted PDX-1 (pancreas duodenum homeobox 1), a gene that is crucial for pancreas development during the fetal period and whose monoallelic disruption impairs insulin secretion. First, Cas9 protein with different sgRNAs that targeted distinct sites in the PDX-1 exon 1 was introduced into in vitro-fertilized zygotes by the GEEP method. Of the six sgRNAs tested, three sgRNAs (sgRNA1, 2, and 3) successfully modified PDX-1 gene. The blastocyst formation rate of zygotes edited with sgRNA3 was significantly (p < 0.05) lower than that of control zygotes without the electroporation treatment. Our study indicates that the GEEP method can be successfully used to generate PDX-1 mutant blastocysts, but the development and the efficiency of editing the genome of zygotes may be affected by the sgRNA used for CRISPR/Cas9 system.


Generation of a TP53-modified porcine cancer model by CRISPR/Cas9-mediated gene modification in porcine zygotes via electroporation.

TP53 (which encodes p53) is one of the most frequently mutated genes in cancers. In this study, we generated TP53-mutant pigs by gene editing via electroporation of the Cas9 protein (GEEP), a process that involves introducing the Cas9 protein and single-guide RNA (sgRNA) targeting exon 3 and intron 4 of TP53 into in vitro-fertilized zygotes. Zygotes modified by the sgRNAs were transferred to recipients, two of which gave birth to a total of 11 piglets. Of those 11 piglets, 9 survived. Molecular genetic analysis confirmed that 6 of 9 live piglets carried mutations in TP53, including 2 piglets with no wild-type (WT) sequences and 4 genetically mosaic piglets with WT sequences. One mosaic piglet had 142 and 151 bp deletions caused by a combination of the two sgRNAs. These piglets were continually monitored for 16 months and three of the genome-edited pigs (50%) exhibited various tumor phenotypes that we presumed were caused by TP53 mutations. Two mutant pigs with no WT sequences developed mandibular osteosarcoma and nephroblastoma. The mosaic pig with a deletion between targeting sites of two sgRNAs exhibited malignant fibrous histiocytoma. Tumor phenotypes of TP53 mosaic mutant pigs have not been previously reported. Our results indicated that the mutations caused by gene editing successfully induced tumor phenotypes in both TP53 mosaic- and bi-allelic mutant pigs.


Follicular development of canine ovaries stimulated by a combination treatment of eCG and hCG.

Ovarian follicular dynamics is not well known in dogs. Imaging of ovaries is technically difficult; however, ovaries clamped at a subcutaneous site can more easily be monitored using ultrasound imaging. This study investigated the follicular development of canine ovaries stimulated by hormone treatment using ultrasound imaging of the ovaries clamped at a subcutaneous site. Oestrus was induced using subcutaneous administration of 500 IU equine chorionic gonadotropin (eCG) and 1000 IU human chorionic gonadotropin (hCG) (eCG/hCG). Five bitches were given 1000 IU hCG 11 days after eCG/hCG administration. Examinations with ovarian ultrasonography using a 7.5-MHz sector transducer, vaginal cytology, and assays of serum oestrogen and progesterone were performed daily until 20 days after eCG/hCG administration. Serosanguineous vaginal discharges and vaginal cytology of two of the bitches were observed. New follicular growth (>1.0 mm in diameter) was observed in all bitches from 2 to 8 days after eCG/hCG administration. The mean diameter of follicles and maximum numbers of follicles per ovary ranged from 2.8 to 5.5 mm and 4 to 16, respectively. The elevation in oestrogen concentrations after eCG/hCG administration was observed in all bitches, and elevation in progesterone concentration (>2 ng mL ) was observed in three bitches. However, no follicles ovulated until 9 days after hCG administration. In conclusion, although the number of examined bitches were limited, follicular growth in ovaries clamped at a subcutaneous site can be monitored using ultrasound imaging. Ovarian ultrasonography showed that eCG/hCG administration induced new follicular growth and hCG administration induced increases in oestrogen concentrations but not ovulation by hCG administration.


Viability of canine ovaries autografted to different peripheral sites

The International Journal of Applied Research in Veterinary Medicine


Effects of chlorogenic acid and caffeic acid on the quality of frozen-thawed boar sperm.

Chlorogenic acid (CGA) and caffeic acid (CA) are potent antioxidants that are mostly found in coffee beans. This study aimed to investigate the effects of CGA and CA supplementation during semen freezing on the quality of frozen-thawed boar spermatozoa. The antioxidants CGA and CA were added to a semen extender to achieve final concentrations of 50, 100, 200 and 400 µM. Supplementation of 100 µM CGA and CA yielded a significantly higher percentage of sperm viability (increased by 8%-10%) and plasma membrane integrity (increased by 4%-6%) than the control groups without the antioxidants at 0 and 3 hr after thawing (p < 0.05). At a concentration of 100 µM, CGA and CA also yielded beneficial effects on total and progressive sperm motility. Increases of CGA and CA concentrations to more than 200 µM did not enhance any sperm quality parameters. When the sperm penetrability and oocyte development by spermatozoa frozen with CGA and CA were evaluated, CGA and CA supplementations had no positive effects on the percentages of total fertilization, monospermic fertilization, cleavage and blastocyst formation. In conclusion, the supplementation of 100 µM CGA and CA during sperm freezing improved certain sperm parameters including motility, viability and plasma membrane integrity.

Reproduction in Domestic Animals = Zuchthygiene


Comparative Effects of Different Dosages of hCG on Follicular Development in Postpartum Dairy Cows With Cystic Ovarian Follicles.

= 0.07) for group hCG-1 compared to group hCG-C on day 5. The preliminary findings of this study suggest that multiple smaller doses of hCG might be equally effective as a single large dose of hCG in modulating ovarian follicular development in dairy cows with COFs.


Effects of chlorogenic acid (CGA) supplementation during in vitro maturation culture on the development and quality of porcine embryos with electroporation treatment after in vitro fertilization.

Electroporation is the technique of choice to introduce an exogenous gene into embryos for transgenic animal production. Although this technique is practical and effective, embryonic damage caused by electroporation treatment remains a major problem. This study was conducted to evaluate the optimal culture system for electroporation-treated porcine embryos by supplementation of chlorogenic acid (CGA), a potent antioxidant, during in vitro oocyte maturation. The oocytes were treated with various concentrations of CGA (0, 10, 50, and 100 μmol/L) through the duration of maturation for 44 hr. The treated oocytes were then fertilized, electroporated at 30 V/mm with five 1 msec unipolar pulses, and subsequently cultured in vitro until development into the blastocyst stage. Without electroporation, the treatment with 50 μmol/L CGA had useful effects on the maturation rate of oocytes, the total cell number, and the apoptotic nucleus indices of blastocysts. When the oocytes were electroporated after in vitro fertilization, the treatment with 50 μmol/L CGA supplementation significantly improved the rate of oocytes that developed into blastocysts and reduced the apoptotic nucleus indices (4.7% and 7.6, respectively) compared with those of the untreated group (1.4% and 13.0, respectively). These results suggested that supplementation with 50 μmol/L CGA during maturation improves porcine embryonic development and quality of electroporation-treated embryos.


Effect of ferulic acid supplementation on the developmental competence of porcine embryos during in vitro maturation.

The value of laboratory and genetically-modified pigs is becoming increasingly clear; however, their in vitro development remains inefficient. Trans-ferulic acid (trans-FA) is an aromatic compound that is abundant in plant cell walls, and which exhibits antioxidant effects in vitro. Trans-FA is known to improve sperm viability and motility; however, its effects on porcine oocytes are unknown. Our aim was to investigate the effects of trans-FA supplementation during in vitro maturation on the meiotic and developmental competence of porcine oocytes. Oocytes were matured either without (control) or with trans-FA (10, 100 and 1,000 µM), fertilized, and cultured in vitro for 7 days. The maturation rate of oocytes cultured with 10 µM trans-FA (81.6%) was significantly higher than that of controls (65.0%; P<0.05). The fertilization rate of oocytes matured with 10 µM trans-FA (57.4%) was also significantly higher than that of controls (32.7%) and oocytes cultured with other concentrations (33.1% and 22.7% for 100 and 1,000 µM, respectively; P<0.05). Moreover, the blastocyst formation rate of oocytes matured with 10 µM trans-FA (6.9%) was significantly higher than that of controls (2.3%; P<0.05). Our results suggest that in vitro maturation with 10 µM trans-FA is beneficial for the in vitro production of porcine embryos and has the potential to improve production system.

The Journal of Veterinary Medical Science


Effects of Antifreeze Protein Supplementation on the Development of Porcine Morulae Stored at Hypothermic Temperatures.

Supplementation of AFP type III (1.0 microgram per mL) maintained the quality of embryos stored at 25C, but did not have beneficial effects on the development of embryos stored at hypothermic temperatures.


Effects of Orvus Es Paste (OEP) on the viability of bull spermatozoa after double freezing and thawing.


Effects of voltage strength during electroporation on the development and quality of in vitro-produced porcine embryos.

This study was conducted to determine suitable conditions for an experimental method in which the CRISPR/Cas9 system is introduced into in vitro-produced porcine zygotes by electroporation. In the first experiment, when putative zygotes derived from in vitro fertilization (IVF) were electroporated by either unipolar or bipolar pulses, keeping the voltage, pulse duration and pulse number fixed at 30 V/mm, 1 msec and five repeats, respectively, the rate of blastocyst formation from zygotes electroporated by bipolar pulses decreased compared to zygotes electroporated by unipolar pulses. In the second experiment, the putative zygotes were electroporated by electroporation voltages ranging from 20 V/mm-40 V/mm with five 1-msec unipolar pulses. The rate of cleavage and blastocyst formation of zygotes electroporated at 40 V/mm was significantly lower (p < .05) than that of zygotes electroporated at less than 30 V/mm. Moreover, the apoptotic nuclei indices of blastocysts derived from zygotes electroporated by voltages greater than 30 V/mm significantly increased compared with those from zygotes electroporated by voltages less than 25 V/mm (p < .05). When zygotes were electroporated with Cas9 mRNA and single-guide RNA (sgRNA) targeting site in the FGF10 exon 3, the proportions of blastocysts with targeted genomic sequences were 7.7% (2/26) and 3.6% (1/28) in the embryos derived from zygotes electroporated at 25 V/mm and 30 V/mm, respectively. Our results indicate that electroporation at 25 V/mm may be an acceptable condition for introducing Cas9 mRNA and sgRNA into pig IVF zygotes under which the viability of the embryos is not significantly affected.


Chlorogenic acid supplementation during in vitro maturation improves maturation, fertilization and developmental competence of porcine oocytes.

Chlorogenic acid (CGA) is a quinic acid conjugate of caffeic acid, and a phytochemical found in many fruits and beverages that acts as an antioxidant. The present study investigated the effects of CGA supplementation during in vitro maturation (IVM), on in vitro development of porcine oocytes, to improve the porcine in vitro production (IVP) system. Oocytes were matured either without (control) or with CGA (10, 50, 100 and 200 μM). Subsequently, the matured oocytes were fertilized and cultured in vitro for 7 day. The rates of maturation, fertilization and blastocyst formation of oocytes matured with 50 μM CGA were significantly (p < .05) higher than those of the control oocytes. Hydrogen peroxide (H2 O2 ) is one of the reactive oxygen species and induces DNA damage in porcine oocytes. When oocytes were matured with 1 mM H2 O2 to assess the protective effect of CGA, 50 μM CGA supplementation improved the maturation rate and the proportion of DNA-fragmented nuclei in oocytes compared with control oocytes matured without CGA. Moreover, when oocytes were matured with either 50 μM CGA (control) or caffeic acid (10, 50 and 100 μM), the rates of maturation, fertilization and the blastocyst formation of oocytes matured with 50 μM CGA were similar to those of oocytes matured with 10 and 50 μM caffeic acid. Our results suggest that CGA has comparable effects to caffeic acid, and IVM with 50 μM CGA is particularly beneficial to IVP of porcine embryos and protects oocytes from DNA damage induced by oxidative stress. Supplementation of CGA to the maturation medium has a potential to improve porcine IVP system.


Sensitivity of the meiotic stage to hyperthermia during in vitro maturation of porcine oocytes.

The present study was conducted to clarify whether the meiotic stage of porcine oocytes has the highest sensitivity to hyperthermia during in vitro maturation by evaluating meiotic competence and DNA damage. Oocytes were exposed to 41 °C for 12 h at various intervals during 48 h of maturation culture. When the oocytes were exposed to 41 °C from 12 to 24 h of the maturation culture, the proportion of oocytes reaching metaphase II (MII) decreased as compared to the control oocytes cultured at 38.5 °C (P < 0.05). Moreover, the proportions of DNA fragmentation in all oocytes exposed to 41 °C in each culture period after 12 h from the start of maturation culture were significantly higher (P < 0.05) than for the control oocytes. When the meiotic stage of oocytes cultured at 38.5 °C between 12 and 24 h was examined, the majority of oocytes remained at the germinal vesicle (GV) stage at 12 h and approximately half of the oocytes reached metaphase I (MI) at 24 h. These results indicate that the meiotic stage of porcine oocytes having the highest sensitivity to hyperthermia during in vitro maturation is a transition period from the GV stage to the MI stage.


Somatic cell reprogramming-free generation of genetically modified pigs.

Genetically modified pigs for biomedical applications have been mainly generated using the somatic cell nuclear transfer technique; however, this approach requires complex micromanipulation techniques and sometimes increases the risks of both prenatal and postnatal death by faulty epigenetic reprogramming of a donor somatic cell nucleus. As a result, the production of genetically modified pigs has not been widely applied. We provide a simple method for CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 gene editing in pigs that involves the introduction of Cas9 protein and single-guide RNA into in vitro fertilized zygotes by electroporation. The use of gene editing by electroporation of Cas9 protein (GEEP) resulted in highly efficient targeted gene disruption and was validated by the efficient production of Myostatin mutant pigs. Because GEEP does not require the complex methods associated with micromanipulation for somatic reprogramming, it has the potential for facilitating the genetic modification of pigs.


Effects of duration of electric pulse on in vitro development of cloned cat embryos with human artificial chromosome vector.

Reproduction in Domestic Animals = Zuchthygiene


Characteristics and fertility of sumatran tiger spermatozoa cryopreserved with different sugars.

Cryopreservation of semen is one of the most important methods for the preservation of endangered tigers. This study evaluated the effects of sugar supplementation on the cryosurvival of spermatozoa from Sumatran tigers (Panthera tigris sumatrae). The post-thaw characteristics and fertility of spermatozoa cryopreserved with different sugars (glucose, lactose, and trehalose) were evaluated using heterologous in-vitro fertilisation with cat oocytes. All parameters of post-thaw spermatozoa significantly decreased as compared with those of fresh spermatozoa. The index of sperm motility for semen cryopreserved with lactose was significantly higher than that for semen cryopreserved with trehalose. The percentage of total fertilisation for tiger spermatozoa cryopreserved with trehalose was significantly lower than that for control cat spermatozoa. Our findings indicated that supplementation with lactose or glycerol as the main sugar in the egg yolk extender resulted in a better motility and fertility potential for post-thawed spermatozoa.


Treatment with protein kinase C activator is effective for improvement of male pronucleus formation and further embryonic development of sperm-injected oocytes in pigs

To assist the process of oocyte activation, which is essential for promotion of fertilization events, i.e., resumption of meiosis, extrusion of the second polar body and formation of the pronucleus (PN), artificial stimuli such as an electrical pulse have been applied to porcine oocytes after injection of sperm. However, the efficiency of fertilization and embryonic development remains low. It is well known that in vertebrates, inactivation of mitogen-activated protein (MAP) kinase is required for oocyte activation. We have hypothesized that even after electrical stimulation of sperm-injected oocytes, MAP kinase may not be inactivated. As it has been reported that MAP kinase activity is regulated by protein kinase C, we examined the effectiveness of phorbol 12-myristate 13-acetate (PMA), a protein kinase C activator, for improvement of fertilization and embryonic development of sperm-injected porcine oocytes. First, we examined the concentrations (0, 0.01, 0.1, 1, and 10 μM) and durations (0, 1, 3, 5 hours) of PMA treatment that were efficient for the extrusion of two polar bodies and formation of two PNs (2PB+2PN) and embryonic development. When the sperm-injected oocytes were treated with 0.01-μM PMA for 3 hours after electrical stimulation, the rates of 2PB+2PN and embryonic development were higher than those in the other treatment groups. We then examined the effect of PMA treatment (0.01 μM, 3 hours) on MAP kinase activity. Unexpectedly, after electrical stimulation, the activity remained low until PN formation, irrespective of whether or not the oocytes had been treated with PMA. On the other hand, transformation of the injected sperm nucleus into the male PN was accelerated after the PMA treatment. Our present results suggest that the low efficiency of fertilization and embryonic development in sperm-injected oocytes is not due to high activity of MAP kinase but due to poor transformation of the injected sperm nucleus into the male PN. Furthermore, a combination of electrical stimulation and PMA is a fairly effective artificial protocol for promoting 2PB+2PN and embryonic development in sperm-injected porcine oocytes.


The optimal period of Ca-EDTA treatment for parthenogenetic activation of porcine oocytes during maturation culture

The changes triggered by sperm-induced activation of oocytes, which are required for normal oocyte development, can be mediated by other agents, thereby inducing the parthenogenesis. In this study, we exposed porcine oocytes to 1 mM Ca-EDTA, a metal-ion chelator, at various intervals during 48 hr of in vitro maturation to determine the optimum period of Ca-EDTA treatment for parthenogenetic activation. When the oocytes were cultured with or without Ca-EDTA from 36 hr (post-12), 24 hr (post-24), 12 hr (post-36) and 0 hr (post-48) after the start of maturation culture, the blastocyst formation rates were significantly higher (P<0.05) in the post-24, post-36 and post-48 groups (3.3%, 4.0%, and 2.6%, respectively) than those in the control group without treatment (0%). Furthermore, when the oocytes were cultured with Ca-EDTA for 0 hr (control), 12 hr (pre-12), 24 hr (pre-24), 36 hr (pre-36) and 48 hr (pre-48) from the start of maturation culture, the oocytes formed blastocysts only in the pre-36 and pre-48 groups (0.4% or 0.8%, respectively). Pronuclei (<66.7%) were observed only when the periods of Ca-EDTA treatment were more than 12 hr during maturation culture. In the control group, no pronuclei were detected. Our findings demonstrate that porcine immature oocytes can be parthenogenetically activated by Ca-EDTA treatment for at least 24 hr to 36 hr during maturation culture, leading to pronucleus formation followed by the formation of blastocysts.

The Journal of Veterinary Medical Science


Effects of parity and season on pregnancy rates after the transfer of embryos to repeat-breeder Japanese Black beef cattle.

<jats:p>Abstract. Repeat-breeder (RB) cows are a major source of economic waste due to their decreased fertility. Embryo transfer (ET) is an alternative tool to improve the fertility of RB cows. The aims of the present study were to evaluate the effects of recipient parity and the season on pregnancy rates following ET in RB Japanese Black beef cattle. Embryos were transferred nonsurgically to recipients, consisting of 155 heifers (< 2 years old) and 172 cows (< 8 years old), which were defined as RB cattle. Of the recipients that were presented for ET, 57 recipients received a fresh embryo and 270 recipients received a frozen embryo. There were no differences in the pregnancy rates between cattle that received fresh embryos or frozen embryos. The rates of recipients with pregnancy, abortion, stillbirth, and normal calving were similar between heifers and cows. In cows, the pregnancy rates were lower (P < 0.05) in summer (June to August) than in spring (March to May) and winter (December to February). In heifers, however, there were no differences in the pregnancy rates among the seasons. Our findings indicate that in RB Japanese Black beef cattle, the parity of the recipients does not have an effect on the pregnancy rates following the transfer of fresh and frozen embryos. However, heat stress may affect reproductive performance in RB Japanese Black cows. </jats:p>


Effects of dibutyryl cyclic adenosine monophosphate and human chorionic gonadotropin on the formation of antral follicle-like structures by bovine cumulus-oocyte complexes.

This study evaluated the effect of dibutyryl cyclic adenosine monophos-phate (dbcAMP) and human chorionic gonadotropin (hCG) on the formation of antral follicle-like structures (AFLSs) and on the meiotic status of bovine cumulus- oocyte complexes (COCs) embedded in collagen gel. Supplementation with dbcAMP increased the mean diameter of AFLSs during days 4-8 of culture compared with that of control COCs, irrespective of the concentration of dbcAMP used (0.5-2.0 mM). When the embedded COCs were cultured for 8 days with hCG, the diameters of AFLSs after 4 days of culture tended to be lower in the supplemented COCs than in the control COCs without hCG, irrespective of the concentration used (1-100 IU/mL). Supplementation with 10 IU/mL hCG increased the concentrations of anti-Müllerian hormone but not progesterone and oestradiol in the culture medium after 4 days of culture. Almost all oocytes collected from AFLSs had resumed meiosis by the end of culture, irrespective of supplementation of dbcAMP and hCG. These results indicate that although dbcAMP had a positive effect on AFLS formation and development, supplementation with hCG was detrimental. Moreover, hCG supplementation did not influence the luteinisation of granulosa cells in the AFLS for 4 days after the start of culture.


Melatonin Supplementation During In Vitro Maturation and Development Supports the Development of Porcine Embryos.

Melatonin has been reported to improve the in vitro development of embryos in some species. This study was conducted to investigate the effect of melatonin supplementation during in vitro maturation (IVM) and development culture on the development and quality of porcine embryos. In the first experiment, when the in vitro fertilized embryos were cultured with different concentrations of melatonin (0, 10, 25 and 50 ng/ml) for 8 days, the blastocyst formation rate of embryos cultured with 25 ng/ml melatonin (10.7%) was significantly increased (p < 0.05) compared to the control embryos cultured without melatonin (4.2%). The proportion of DNA-fragmented nuclei in blastocysts derived from embryos cultured with 50 ng/ml melatonin was significantly lower (p < 0.05) than that of embryos cultured without melatonin (2.1% vs 7.2%). In the second experiment, when oocytes were cultured in the maturation medium supplemented with different concentrations of melatonin (0, 10, 25 and 50 ng/ml), fertilized and then cultured with 25 ng/ml melatonin for 8 days, there were no significant differences in the rates of cleavage and blastocyst formation among the groups. However, the proportions (2.7-5.4%) of DNA-fragmented nuclei in blastocysts derived from oocytes matured with melatonin were significantly decreased (p < 0.05) compared to those (8.9%) from oocytes matured without melatonin, irrespective of the concentration of melatonin. Our results suggest that supplementation of the culture media with melatonin (25 ng/ml) during IVM and development has beneficial effects on the developmental competence and quality of porcine embryos.


Formation of an Antral Follicle-Like Structure by Bovine Cumulus-Oocyte Complexes Embedded with Fragmin/Protamine Microparticles.

Fragmin/protamine microparticles (F/P MPs) approximately 0.5-1 µM in diameter were prepared by the simple mixing of fragmin with protamine. This study investigated the effects of F/P MP-containing collagen gels as a hormone carrier on the formation of antral follicle-like structures and on the development of growing bovine oocytes. The supplementation of F/P MPs in collagen gels contributed to the beneficial effects of follicle stimulating hormone (FSH) on the formation and size of antral follicle-like structures. The F/P MPs may serve as potential hormone carriers for the growth of cultured bovine oocytes from early antral follicles.


EFFECTS OF ORVUS ES PASTE ON THE MOTILITY AND VIABILITY OF YAK (BOS GRUNNIENS) EPIDIDYMAL AND EJACULATED SPERMATOZOA AFTER FREEZING AND THAWING.

The addition of the detergent Orvus ES Paste (OEP) to semen freezing extenders has been observed to improve the post-thaw survival and longevity of spermatozoa from various species but has never been evaluated for yak spermatozoa. This study evaluated the effects of OEP on the post-thaw motility and viability of epididymal and ejaculated yak spermatozoa. Semen samples were frozen and thawed in semen freezing extender supplemented with 0 %, 0.375 %, 0.75 % or 1.5 % OEP. The motility and viability of frozen-thawed spermatozoa were evaluated before and after 3 h of incubation. The addition of 0.75 % OEP to the freezing extender significantly improved the mean motility and viability values of both the epididymal and ejaculated spermatozoa immediately after thawing, but the beneficial effects on motility disappeared after 3h of incubation. Our findings indicate that the addition of 0.75 % OEP is effective for the preservation of yak spermatozoa.