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.     

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.     

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.     

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.     

Vesicles Cytoplasmic Injection: An Efficient Technique to Produce Porcine Transgene‐Expressing Embryos

The use of vesicles co‐incubated with plasmids showed to improve the efficiency of cytoplasmic injection of transgenes in cattle. Here, this technique was tested as a simplified alternative for transgenes delivery in porcine zygotes. To this aim, cytoplasmic injection of the plasmid alone was compared to the injection with plasmids co‐incubated with vesicles both in diploid parthenogenic and IVF zygotes. The plasmid pcx‐egfp was injected circular (CP) at 3, 30 and 300 ng/μl and linear (LP) at 30 ng/μl. The experimental groups using parthenogenetic zygotes were as follows: CP naked at 3 ng/μl (N = 105), 30 ng/μl (N = 95) and 300 ng/μl (N = 65); Sham (N = 105); control not injected (N = 223); LP naked at 30 ng/μl (N = 78); LP vesicles (N = 115) and Sham vesicles (N = 59). For IVF zygotes: LP naked (N = 44) LP vesicles (N = 94), Sham (N = 59) and control (N = 79). Cleavage, blastocyst and GFP+ rates were analysed by Fisher's test (p < 0.05). The parthenogenic CP naked group showed lower cleavage respect to control (p < 0.05). The highest concentration of plasmids to allow development to blastocyst stage was 30 ng/μl. There were no differences in DNA fragmentation between groups. The parthenogenic LP naked group resulted in high GFP rates (46%) and also allowed the production of GFP blastocysts (33%). The cytoplasmic injection with LP vesicles into parthenogenic zygotes allowed 100% GFP blastocysts. Injected IVF showed higher cleavage rates than control (p < 0.05). In IVF zygotes, only the use of vesicles produced GFP blastocysts. The use of vesicles co‐incubated with plasmids improves the transgene expression efficiency for cytoplasmic injection in porcine zygotes and constitutes a simple technique for easy delivery of plasmids.     

Absence of Sperm Factors as in the Parthenogenesis Does Not Interfere on Bovine Embryo Sensitiveness to Heat Shock at Pre‐Implantation Stage

Oocyte has been considered the major contributor for embryo thermo‐tolerance. However, it was shown that sperm factors can be transferred to the oocyte during fertilization, raising the question of whether the absence of such factors could interfere on embryo thermo‐tolerance. In this study, we used parthenogenesis to generate bovine embryos without spermatozoa in order to test whether the absence of sperm factors could influence their thermo‐sensitiveness at early stages. In vitro fertilized (IVF) and parthenogenetic (PA) embryos at 44 h post‐insemination/chemical activation were exposed to 38.5°C (control) or 41°C (heat shock) for 12 h and then developed for 48 h and up to blastocyst stage. Apoptosis index and expression of PRDX1, GLUT1, GLUT5 and IGF1r genes in blastocysts derived from heat‐shocked embryos were also evaluated. The heat shock decreased the blastocyst rate at day seven (p < 0.05) for IVF embryos and at day eight (p < 0.01) for both IVF and PA embryos. Total cell number was not affected by heat shock in IVF and PA blastocysts, but there was an increased proportion (p < 0.05) of apoptotic cells in heat‐shocked embryos when compared to controls. There was no interaction (p > 0.05) between method of activation (IVF and PA) and temperature (38.5°C or 41.5°C) for all developmental parameters evaluated. Expression of GLUT1 gene was downregulated (p < 0.05) by heat shock in both IVF and PA blastocyst whereas expression of GLUT5 and IGF1r genes was downregulated (p < 0.05) by heat shock in PA blastocysts. Those data show that the heat shock affects negatively the embryo development towards blastocysts stage, increases the apoptotic index and disturbed the expression of some genes in both IVF and PA embryos, indicating that the presence or absence of sperm factors does not influence the sensitivity of the bovine embryo to heat shock.     

The quality after culture in vitro or in vivo of porcine oocytes matured and fertilized in vitro and their ability to develop to term

The quality of porcine blastocysts produced in vitro is poor in comparison with those that develop in vivo. We examined the quality of in vitro‐matured and fertilized (IVM/IVF) oocytes, their abilities to develop to blastocysts under in vivo and in vitro conditions, and the potential of the embryos to develop to term after transfer. IVM/IVF oocytes were either transferred and the embryos recovered on Days 5 and 6 (100% and 87.5%, respectively) (‘ET‐vivo’ embryos), or cultured in vitro for 5 or 6 days (‘IVC’ embryos). The proportion of blastocysts differed significantly between the two groups on Day 5 (20.6% and 8.0%, respectively), but not on Day 6 (23.8% and 21.2%, respectively). The mean number of cells in ET‐vivo blastocysts on Days 5 or 6 was significantly higher (72.8 and 78.7, respectively) than that in IVC blastocysts (22.1 and 39.7, respectively). When IVM/IVF oocytes and IVC blastocysts on Day 6 were transferred, all (three and three, respectively) developed to piglets (16 and 16, respectively), without any difference in the rates of development to term (2.1% and 2.6%, respectively). These data suggest that, although blastocyst production differs between the two culture conditions, IVM/IVF oocytes possess the same ability to develop to term.     

Contrasting effects of heat shock during in vitro maturation on development of in vitro‐fertilized and parthenogenetic bovine embryos

This study investigated the influence of heat shock during in vitro maturation on embryo development following in vitro fertilization (IVF) or parthenogenesis (Part). Immature bovine cumulus–oocyte complexes were exposed to heat shock (41.0°C) during the first 12 hr of in vitro maturation (IVM), followed by 12 hr at 38.5°C. Control group consisted of in vitro maturation for 24 hr at 38.5°C. Oocytes were in vitro‐fertilized or activated with ionomycin and cultured in vitro for 192 hr post‐in vitro insemination or parthenogenetic activation (hpia). There was an interaction (p < .01) between temperature of IVM and method of oocyte activation (IVF or Part) for cleavage at 48 hpia. Heat shock had a negative impact (p < .01) on cleavage of IVF embryos, whereas no (p > .05) effect was found in the Part embryos. Embryo development towards blastocyst stage at 168 and 192 hpia decreased in both IVF and Part embryos derived from heat‐shocked oocytes. Heat shock increased (p < .05) the apoptotic index in Part blastocysts, but no effect (p > .05) was found in IVF counterparts. Heat shock also down‐regulated the expression of AQP3 (p < .01) and up‐regulated the expression of HSP70.1 (p < .01) in Part blastocysts, whereas it down‐regulated the expression of ATP1A1 (p < .05) in IVF blastocysts. In conclusion, the effects of heat shock during IVM on early embryo cleavage and blastocyst apoptosis are influenced by the method of oocyte activation and expression of some genes can be disturbed in embryos derived from heat‐shocked oocytes.     

Delay in Cleavage of Porcine Embryos after Intracytoplasmic Sperm Injection (ICSI) Shows Poorer Embryonic Development

In pigs, the embryonic developmental ability after intracytoplasmic sperm injection (ICSI) is inferior to that resulting from in vitro fertilization (IVF). We evaluated the timing of cell division up to blastocyst formation on embryonic development after ICSI using either whole sperm (w-ICSI) or the sperm head alone (h-ICSI) and IVF as a control. At 10 h after ICSI or IVF, we selected only zygotes, and each of the zygotes/embryos was evaluated for cleavage every 24 h until 168 h. We then observed a delay in the 1st and 2nd cleavages of h-ICSI embryos and also in blastocoele formation by w-ICSI embryos in comparison with IVF embryos. The rate of blastocyst formation and the quality of blastocysts in both ICSI groups were inferior to those in the IVF group. In conclusion, the delay in cleavage of porcine ICSI embryos shows poorer embryonic development.     

m‐carboxycinnamic acid bishydroxamide improves developmental competence,reduces apoptosis and alters epigenetic status and gene expression pattern in cloned buffalo (Bubalus bubalis) embryos

Incomplete or aberrant reprogramming of nuclear genome is one of the major problems in somatic cell nuclear transfer. In this study, we studied the effect of histone deacetylase inhibitor m‐carboxycinnamic acid bishydroxamide (CBHA) on in vitro development of buffalo embryos produced by Hand‐made cloning. Cloned embryos were treated with CBHA (0, 5, 10, 20 or 50 μM) for 10 hr from the start of reconstruction till activation. At 10 μM, but not at other concentrations examined, CBHA increased (p < .05) the blastocyst rate (63.77 ± 3.97% vs 48.63 ± 3.55%) and reduced (p < .05) the apoptotic index of the cloned blastocysts (8.91 ± 1.94 vs 4.36 ± 1.08) compared to untreated controls, to levels similar to those in IVF blastocysts (4.78 ± 0.74). CBHA treatment, at all the concentrations examined, increased (p < .05) the global level of H3K9ac in cloned blastocysts than in untreated controls to that observed in IVF blastocysts. Treatment with CBHA (10 μM) decreased (p < .05) the global level of H3K27me3 in cloned blastocysts than in untreated controls but it was still higher (p < .05) than in IVF blastocysts. CBHA (10 μM) treatment increased (p < .05) the relative expression level of pluripotency‐related genes OCT‐4 and NANOG, and anti‐apoptotic gene BCL‐XL, and decreased (p < .05) that of pro‐apoptotic gene BAX than in untreated controls but did not affect the relative expression level of apoptosis‐related genes p53 and CASPASE3 and epigenetics‐related genes DNMT1, DNMT3a and HDAC1. These results suggest that treatment of cloned embryos with 10 μM CBHA improves the blastocyst rate, reduces the level of apoptosis and alters the epigenetic status and gene expression pattern.     

Insulin‐like growth factor I enhances the developmental competence of yak embryos by modulating aquaporin 3

The objective of our present study was to determine the effects of insulin‐like growth factor I (IGF‐I) on the development of yak (Bos grunniens) embryos after cumulus–oocyte complex (COC) vitrification and warming followed by in vitro fertilization (IVF). In Experiment 1, the yak COCs underwent vitrification and then IVF. Embryos were incubated in synthetic oviductal fluid (SOF) supplemented with four concentrations (0, 50, 100 and 200 ng/ml) of IGF‐I, while the yak COCs without vitrification or IGF‐I supplementation acted as the control group; the BAX, BCL‐2, AQP3mRNA and aquaporin 3 (AQP3) protein expression levels in the five groups of blastocysts were evaluated using quantitative real‐time PCR and immunofluorescence analyses. In Experiment 2, the groups described above were fertilized and incubated. The cleavage rate, blastocyst rate, total cell count per blastocyst and the rate of growth of the inner cell mass (ICM) and trophectoderm (TE) were evaluated. The results were as follows: (1) the AQP3 gene expression and protein expression in the control and 100 ng/ml IGF‐I treatment groups were the highest. (2) The BAX gene expression was the lowest and the BCL‐2 gene expression was the highest in the control and 100 ng/ml IGF‐I treatment groups. (3) The rates of cleavage and blastocysts in the control and 100 ng/ml IGF‐I groups were higher than those in the other three groups. The total cell count per blastocyst in the vitrified and warmed 100 ng/ml IGF‐I group (106.7 ± 4.9) and the control group (107.3 ± 4.2) was higher than that in the vitrified and warmed 0 ng/ml IGF‐I (91.2 ± 3.1), 50 ng/ml IGF‐I (92.3 ± 3.7) and 200 ng/ml IGF‐I (92.4 ± 3.7) groups. Therefore, we conclude that IGF‐I can improve yak blastocyst developmental ability, cytomembrane permeability and formation of the blastocyst cavity after COC vitrification by improving the BAX, BCL‐2 and AQP3 expression levels.     

Optimizing injection time of GFP plasmid into sheep zygote

Microinjection of exogenous DNA into the cytoplasm of matured oocytes or zygotes is a promising technique to generate transgenic animals. However, the data about the microinjection time and procedure in sheep are limited and have not treated in detail. To obtain more in-depth information, the Sarda sheep oocytes from abattoir-derived ovaries were subjected to IVM and IVF. Then, the GFP plasmid as a reporter gene was injected into the cytoplasm of MII oocytes (n: 95) and zygotes at different post-insemination intervals (6–8 hpi, n: 120; 8–10 hpi, n: 122; 10–12 hpi, n: 110 and 12–14 hpi, n: 96). There were no significant differences in the cleavage rates between the groups. However, blastocyst rate of injected zygotes at all-time intervals was significantly lower than injected MII oocytes and control group (p < 0.05). Interestingly, the proportion of GFP-positive embryos was higher at 8–10 hpi compared with other injected groups (4 % versus 0 %, p  < 0.01). Among these, the proportion of mosaic embryos was high and two of those embryos developed to the blastocyst stage. In conclusion, we settled on the cytoplasmic microinjection of GFP plasmid at 8–10 hpi as an optimized time point for the production of transgenic sheep and subsequent experiments.     

Effects of Addition of Tissue‐Type Plasminogen Activator in In Vitro Fertilization Medium on Bovine Embryo Development and Quality

Plasminogen activators/Plasmin system plays pivotal role in regulating reproductive functions of mammals. Here, we examined the effects of modification of in vitro fertilization medium (IVF medium) with the addition of tissue‐type plasminogen activator (t‐PA), on bovine embryo development and quality, assessed by quantification of expression of various genes related to metabolism, oxidation, implantation and apoptosis. In addition, plasminogen activator activity (PAA) and plasminogen activator inhibition (PAI) were measured in the spent media. After conventional IVM, 2016 cumulus‐oocyte complexes (COCs) were divided into four groups with modified composition of the IVF medium containing t‐PA and/or its inhibitor epsilon‐aminocaproic acid (control, t‐PA, t‐PA+ε‐ACA, ε‐ACA). Presumptive zygotes were cultured for 8 days in synthetic oviductal fluid (SOF) medium; gene expression studies were carried out on morulae and blastocysts. t‐PA alone significantly suppressed cleavage and blastocyst formation rates, but this effect was neutralized by the addition of ε‐ACA. PAA in the treated group was significantly reduced by ε‐ACA, but without total elimination. Significant differences were detected in the expression of genes related to apoptosis and/or cell cycle arrest (BAX, BCL2L1, KAT2B) between embryos produced in t‐PA‐modified media and controls, giving an overall notion that the inferior developmental competence of treated embryos may be attributed to apoptotic phenomena induced by t‐PA. In conclusion, it appears that excessive t‐PA content in the IVF media, suppresses blastocyst formation rate, possibly due to induction of apoptotic phenomena.     

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 µM 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 µM 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 µM 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.     

Buffalo SCNT embryos exhibit abnormal gene expression of ERK/MAPK pathway and DNA methylation

Inhibition of ERK/MAPK pathway has been shown to decrease DNA methylation via down‐regulation of DNA methyltransferases (DNMTs) in several studies suggesting that this pathway plays an important role in regulation of DNA methylation. We examined the relative expression level of seven important genes related to ERK/MAPK pathway and DNMTs (DNMT1, DNMT3a and DNMT3b) by quantitative real‐time PCR in buffalo blastocysts produced by Hand‐made cloning and compared it with that in blastocyst‐stage embryos produced by in vitro fertilization (IVF). The expression level of six of seven genes related to ERK/MAPK pathway examined i.e., p21RAS, RAF1, AKT1, ERK2, PIK3R2 and c‐Myc was significantly higher (p < 0.05) in cloned than in IVF embryos. However, the expression level of FOS was lower (p < 0.005) in cloned than in IVF embryos. The relative expression level of DNMT3a and DNMT3b but not that of DNMT1 was significantly higher (p < 0.05) in cloned than in IVF embryos. These results indicate that the cloned embryos exhibit an abnormal expression of several important genes related to ERK/MAPK pathway and DNMTs. Although a direct link between ERK/MAPK pathway and DNMTs was not examined in the present study, it can be speculated that ERK/MAPK pathway may have a role in regulating the expression of DNMTs in embryos, as also observed in other tissues.     

Beneficial Effect of Two Culture Systems with Small Groups of Embryos on the Development and Quality of In Vitro‐Produced Bovine Embryos

Currently, in vitro‐produced embryos derived by ovum pick up (OPU) and in vitro fertilization (IVF) technologies represent approximately one‐third of the embryos worldwide in cattle. Nevertheless, the culture of small groups of embryos from an individual egg donor is an issue that OPU‐IVF laboratories have to face. In this work, we tested whether the development and quality of the preimplantation embryos in vitro cultured in low numbers (five embryos) could be improved by the addition of epidermal growth factor, insulin, transferrin and selenium (EGF‐ITS) or by the WOW system. With this aim, immature oocytes recovered from slaughtered heifers were in vitro matured and in vitro fertilized. Presumptive zygotes were then randomly cultured in four culture conditions: one large group (LG) (50 embryos/500 μl medium) and three smaller groups [five embryos/50 μl medium without (control) or with EGF‐ITS (EGF‐ITS) and five embryos per microwell in the WOW system (WOW)]. Embryos cultured in LG showed a greater ability to develop to blastocyst stage than embryos cultured in smaller groups, while the blastocyst rate of WOW group was significantly higher than in control. The number of cells/blastocyst in LG was higher than control or WOW, whereas the apoptosis rate per blastocyst was lower. On the other hand, the addition of EGF‐ITS significantly improved both parameters compared to the control and resulted in similar embryo quality to LG. In conclusion, the WOW system improved embryo development, while the addition of EGF‐ITS improved the embryo quality when smaller groups of embryos were cultured.     

Vitrification of bovine matured oocytes and blastocysts in a paper container

In the present study, we aimed to determine the applicability of a paper container for the vitrification of in vitro matured (IVM) bovine oocytes. In experiment 1, IVM oocytes were exposed to vitrification solution (20% dimethylsulfoxide (DMSO), 20% ethylene glycol (EG), and 5 mol/L sucrose), using a two‐step method, for 30 s; loaded onto either a paper container or Cryotop; and stored in liquid nitrogen. No significant difference (P < 0.05) in the survival and blastocyst formation rates after in vitro vitrification was observed between the paper container and Cryotop. In experiment 2, IVM oocytes were exposed to either a two‐ or three‐step vitrification solution. The three‐step vitrification solution was not significantly different from the two‐step solution in terms of oocyte survival, cleavage and blastocyst rates. In experiment 3, in vitro produced blastocysts were graded according to the manual of the International Embryo Transfer Society (grades 1 and 2) and vitrified using the two‐ and three‐step methods. For grade 2 blastocysts, the three‐step method showed significantly higher (P < 0.05) survival and hatched blastocyst rates than the two‐step method, whereas for grade 1 blastocysts, no significant difference was observed. In conclusion, the paper device and three‐step technique are suitable for oocytes and embryo vitrification.     

Optimization of parthenogenetic activation of rabbit oocytes and development of rabbit embryo by somatic cell nuclear transfer

The present study explored a suitable parthenogenetic activation (PA) procedure for rabbit oocytes and investigated the developmental potential of somatic cell nuclear transfer (SCNT) embryos using rabbit foetal fibroblasts (RFFs). The electrical activation had the optimal rate of blastocyst (14.06%) when oocytes were activated by three direct current (DC) pulses (40 V/mm, 20 μs each) followed by 6‐dimethylaminopurine (6‐DMAP) and cycloheximide (CHX) treatment; the blastocyst rate of ionomycin (ION) + 6‐DMAP + CHX (12.07%) activation was higher than that of ION + 6‐DMAP (8.6%) activation or ION + CHX (1.24%) activation; there was no significant difference in blastocyst rate between ION + 6‐DMAP + CHX and DC + 6‐DMAP + CHX groups. The blastocyst rate of ION + 6‐DMAP + CHX‐activated oocytes in the basic rabbit culture medium (M‐199) + 10% foetal bovine serum (FBS; 14.28%) was higher than that in buffalo conditioned medium (5.75%) or G1/G2 medium (0), and the blastocyst rate was increased when M‐199 + 10% FBS was supplemented with amino acids. Refreshing culture medium every day or every other day significantly increased the blastocyst rate. Treatment of donor cells with 0.5% FBS for 3–5 days increased blastocyst rate of SCNT embryos (33.33%) than no serum starvation (22.47%) or 0.5% FBS treatment for 6–9 days (23.61%); the blastocyst rate of SCNT embryos derived from nontransgenic RFFs was higher than that derived from transgenic RFFs by electroporation. The blastocyst development ability of SCNT embryos derived from RFFs by electroporation (32.22%) was higher than that of liposome (19.11%) or calcium phosphate (20.00%) transfection, and only the embryos from electroporation group have the EGFP expression (24.44%). In conclusion, this study for the first time systematically optimized the conditions for yield of rabbit embryo by SCNT.     

Effect of varying glucose concentrations during in vitro maturation and embryo culture on efficiency of in vitro embryo production in buffalo

This study was aimed to optimize glucose level at different stages of buffalo in vitro embryo production procedure. Three glucose levels (1.5, 5.6 and 10 mm ) along with a control (0 mm ) were used at three phases of in vitro fertilisation (IVF) procedure viz. in vitro maturation (IVM), in vitro culture (IVC‐I) (12–72 hpi) and IVC‐II (72 hpi to 7 dpi). Maturation rate of oocytes was found different under different glucose concentrations, and significantly more number of oocytes reached to MII under 5.6 mm glucose. The glucose levels at each phase (IVM, IVC‐I and IVC‐II) individually had significant effect on blastocyst rate, and the level used at one phase had significant effect on the outcome of next phase. Complete withdrawal of glucose from any of these stages irrespective of concentrations used at subsequent stage/s resulted in significantly lower number of blastocysts. However, the changing levels of glucose had differential effects during different phases of IVF steps. The most prominent effect of glucose level was observed during IVM. The presence of 5.6 mm glucose at all stages was most effective to yield highest blastocyst rate in buffalo IVF system.