Improved developmental competence of cloned porcine embryos with different energy supplements and chemical activation

The present study was performed to collect in vivo matured canine oocytes for somatic cell nuclear transfer (SCNT) and to investigate the developmental competence of canine parthenogenetic and SCNT embryos as the preliminary research for producing cloned dog. The day of ovulation as described by Hase et al. (2000 J. Vet. Med. Sci. 62, 243-248) was determined by serum progesterone levels and at that time vaginal cytology was performed to assess the cornified index. In vivo-matured oocytes were recovered by retrograde flushing of the oviducts at around 48 h (n = 20) or 72 h (n = 25) after the estimated time of ovulation. Overall size of each oocyte, as well as ooplasmic diameter, zona pellucida thickness, and perivitelline space width, was determined after removing the cumulus cells by pipetting (Exp. 1). To determine activation protocols, two treatments, (1) chemical activation (10 �M Ca ionophore for 4 min, followed by incubation for 4 h with 1.9 mM 6-dimethylaminopurine) and (2) electrical stimulation (3.1?3.4 kV/cm in 0.25M mannitol solution), were evaluated to induce parthenogenetic activation of oocytes (Exp. 2). Donor cells were obtained from the primary cell culture of a canine ear skin biopsy, and SCNT was performed according to our laboratory procedures (Jang et al. 2004 Theriogenology 62, 512-521). Three voltages (1.7?2.0 kV/cm, 2.1-2.4 kV/cm, and 3.1-3.4 kV/cm) were tested for fusion. The fused couplets were subjected to chemical or electrical stimulation as in parthenogenetic activation and in vitro developmental competence was monitored (Exp. 3). As a result, more in vivo-matured canine oocytes were obtained at 72 h (92%) than at 48 h (15%) after ovulation; the 72-h occytes had progesterone concentrations of 4-8 ng/mL and a cornified index (vaginal cytology) of 83.34. The average number of oocytes recovered was 12 and sizes of ooplasmic diameter, cytoplasm, zona pellucida, and perivitelline space in in vivo canine-matured oocytes (n = 120) were 178.8 � 9.3 �m, 125.0 � 8.2 �m, 21.7 � 3.7 �m, and 12.7 � 3.5 �m, respectively. Parthenogenetically activated oocytes developed to the 16-cell and morula stages, but failed to develop to the blastocyst stage. Among the three voltages, in the highest voltage (75.2%) the number of fused couplets was increased compared to either of the other voltages (33.3% and 44.0%). Cleavage rates (60.9% vs. 58.0%) of cloned embryos were not significantly affected by method of activation. In terms of in vitro developmental competence, cloned embryos developed to the 16-cell or morula stage in vitro after electrical or chemical activation, respectively. In conclusion, in the present study we demonstrated that measurement of progesterone levels, in combination with evaluation of vaginal cytology, can be used to determine the estimated time of ovulation in bitches. In addition, we determined fusion/activation protocols that resulted in in vitro development of a portion of parthenogenetically activated and cloned embryos to the 16-cell and morula stages. This study was supported by grants from the Biogreen 21-1000520030100000.

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320 EFFECTS OF DIFFERENT ACTIVATION REGIMENS ON PRONUCLEAR FORMATION AND PRE-IMPLANTATION DEVELOPMENTAL COMPETENCE OF IN VITRO-MATURED BOVINE OOCYTES AFTER INTRACYTOPLASMIC SPERM INJECTION

Reproduction Fertility and Development ◽

10.1071/rdv27n1ab320 ◽

2015 ◽

Vol 27 (1) ◽

pp. 249

Author(s):

M. E. Arias ◽

R. Sanchez ◽

R. Felmer

Keyword(s):

Intracytoplasmic Sperm Injection ◽

Standard Procedure ◽

Chemical Activation ◽

Developmental Rate ◽

Fertilization Rate ◽

Developmental Competence ◽

Assisted Reproductive Technique ◽

Pronuclear Formation ◽

Bovine Oocytes

Intracytoplasmic sperm injection (ICSI) is an assisted reproductive technique that has been used with considerable success in humans; however, in the bovine species the efficiency of this technique is far from optimal. The objective of the present study was to evaluate the effect of 4 chemical activation treatments, 6-dimethylaminopurine (DMAP), cycloheximide (CHX), anisomycin (ANY), and ethanol (EtOH) on the pronuclear formation and embryo development of bovine embryos generated by ICSI. Cumulus-oocyte complexes were aspirated from abattoir ovaries, selected, and matured in 400-µL drops of standard TCM-199 maturation medium for 22 h at 38.5°C and 5% CO2. The ICSI was performed by a standard procedure. Injected oocytes were randomly distributed and activated by 5 µM ionomycin for 5 min (Io) followed by i) 5 µg mL–1 CHX for 5 h (Io/CHX), ii) 3 h window followed by a second Io treatment plus 1.9 mM DMAP for 4 h (2Io/DMAP), iii) 1 µg mL–1 ANY for 5 h (Io/ANY), and iv) 3 h window followed by 7% ethanol (Io/EtoH). Embryos were cultured in 50-µL drops of KSOM medium under mineral oil at 38.5°C and 5% CO2, 5% O2, and 90% N2. Cleavage was recorded at 72 h and blastocyst rate at 192 h. Pronuclear formation analysis was carried out at 18 hpa with Hoechst staining. An oocyte was considered fertilized when 2 polar bodies and 1 female and 1 male pronucleus (or a decondensed sperm head) could be observed. The data were transformed to arcsine, analysed by ANOVA, and means were compared using Tukey's test with Statgraphics Plus 2 Software. Results with a total of 431 injected oocytes (114, 104, 101, and 112 for DMAP, CHX, ANY, and EtOH, respectively) showed differences in cleavage (P < 0.01) in DMAP, CHX, and ANY treatments (86, 72, and 78%, respectively), relative to EtOH (12%). Similarly, the rate of blastocysts/injected oocyte at 192 h was higher with DMAP, CHX, and ANY (41, 20, and 32%, respectively), relative to EtOH (4%). Sham-injected oocytes showed cleavage and blastocyst rates of 67, 43, 68, and 12% and 32, 11, 19, and 5%, for DMAP, CHX, ANY, and EtOH, respectively. Despite the higher developmental rate observed with DMAP, pronuclear formation assessment revealed that fertilization rate was higher in CHX (87%) and ANY (75%) treatments relative to DMAP (35%). In conclusion, the results of the present study show that activation of bovine oocytes after ICSI is more efficient with DMAP and ANY, compared with CHX and EtOH.Provision of ovaries by our local slaughterhouse (Frigorifico Temuco, Chile) and funding support from FONDECYT 1120241 CONICYT, Chile, are gratefully acknowledged.

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Effects of disulfide bond reducing agents on sperm chromatin structural integrity and developmental competence of in vitro matured oocytes after intracytoplasmic sperm injection in pigs

Reproduction ◽

10.1530/rep-08-0143 ◽

2009 ◽

Vol 137 (4) ◽

pp. 633-643 ◽

Cited By ~ 11

Author(s):

Wen-Min Cheng ◽

Lei An ◽

Zhong-Hong Wu ◽

Yu-Bo Zhu ◽

Jing-Hao Liu ◽

...

Keyword(s):

Disulfide Bond ◽

Intracytoplasmic Sperm Injection ◽

Chemical Activation ◽

Reducing Agents ◽

Developmental Competence ◽

Sperm Chromatin ◽

Dna Integrity ◽

Acridine Orange Staining ◽

Sperm Dna

We recently reported that electrical activation followed by secondary chemical activation greatly enhanced the developmental competence ofin vitromatured porcine oocytes fertilized by intracytoplasmic sperm injection (ICSI). We hypothesized that sperm treatment with disulfide bond reducing agents will enhance the development competence of porcine embryos produced by this ICSI procedure. We examined the effects of glutathione (GSH), dithiothreitol (DTT), GSH or DTT in combination with heparin on sperm DNA structure, paternal chromosomal integrity, pronuclear formation, and developmental competence ofin vitromatured porcine oocytes after ICSI. Acridine orange staining and flow cytometry based sperm chromatin structure assay were used to determine sperm DNA integrity by calculating the cells outside the main population (COMP αT). No differences were observed in COMP αT values among GSH-treated and control groups. COMP αT values in GSH-treated groups were significantly lower than that in DTT-treated groups. Following ICSI, GSH treatments did not significantly alter paternal chromosomal integrity. Paternal chromosomal integrity in sperm treated with DTT plus or minus heparin was also the lowest among all groups. GSH-treated sperm yielded the highest rates of normal fertilization and blastocyst formation, which were significantly higher than that of control and DTT-treated groups. The majority of blastocysts derived from control and GSH-treated spermatozoa were diploid, whereas blastocysts derived from DTT-treated spermatozoa were haploid. In conclusion, sperm treatment with GSH enhanced the developmental capacity of porcine embryos produced by our optimized ICSI procedure.

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24 EFFECT OF TREATMENT WITH TRICHOSTATIN A ON IN VITRO DEVELOPMENT OF BOVINE NUCLEAR-TRANSFERRED EMBRYOS

Reproduction Fertility and Development ◽

10.1071/rdv19n1ab24 ◽

2007 ◽

Vol 19 (1) ◽

pp. 130 ◽

Cited By ~ 1

Author(s):

S. Akagi ◽

K. Fukunari ◽

K. Matsukawa ◽

S. Watanabe ◽

S. Takahashi

Keyword(s):

Trichostatin A ◽

Chemical Activation ◽

Calcium Ionophore ◽

Cell Number ◽

Blastocyst Stage ◽

Developmental Competence ◽

Fibroblast Cells ◽

In Vitro Development ◽

Vitro Development

It has been reported that 5 or 50 nM trichostatin A (TSA) treatment after somatic cell nuclear transfer (NT) improves the success rate of mouse cloning (Kishigami et al. 2006 Biochem. Biophys. Res. Commun. 340, 183–189). In this study, we examined the effect of TSA treatment on the in vitro development of bovine NT embryos. As donor cells for NT, bovine fibroblast cells of passages 3 to 5 were used following culture in serum-starved medium for 5 to 7 days. Oocytes were enucleated after in vitro maturation in TCM-199 supplemented with 10% fetal bovine serum. Enucleated MII oocytes were fused with fibroblast cells by a DC pulse of 25 V/150 µm for 10 µs in Zimmerman mammalian cell fusion medium. Fused oocytes were activated by 10 µM calcium ionophore for 5 min, followed by incubation with 2.5 µg mL−1 cytochalasin D, 10 µg mL−1 cycloheximide, and 5 or 50 nM TSA for 1 h, and then cycloheximide and 5 or 50 nM TSA for 4 h. After chemical activation, NT embryos were cultured in IVD-101 (Research Institute of Functional Peptide Co., Ltd., Yamagata, Japan) with 5 or 50 nM TSA for 10 h and subsequently cultured in IVD-101 without TSA. Control NT embryos were cultured in the same medium without TSA after fusion. After in vitro culture for 8 days, blastocyst formation and cell numbers of blastocysts were examined. The fusion rate of enucleated oocytes with fibroblast cells was 81% (199/247). In vitro development of NT embryos is summarized in Table 1. There were no differences in the cleavage rate and development rate to the blastocyst stage of NT embryos among control, and 5 and 50 nM TSA treatments. The cell number of 50 nM TSA-treated NT embryos at the blastocyst stage was higher than that of control NT embryos without TSA treatment. In conclusion, 50 nM TSA treatment for 15 h after activation did not affect the in vitro developmental competence, but increased total cell number in bovine NT embryos. These results suggest that TSA treatment may improve the quality of blastocysts in bovine NT. Table 1. Effects of TSA treatment on in vitro development of NT embryos derived from fibroblast cells

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48 DEVELOPMENTAL COMPETENCE OF CLONED OR PARTHENOGENETICALLY ACTIVATED PORCINE EMBRYOS: EFFECT OF DIAMETER OF PREPUBERTAL GILT OOCYTES

Reproduction Fertility and Development ◽

10.1071/rdv23n1ab48 ◽

2011 ◽

Vol 23 (1) ◽

pp. 130

Author(s):

J. Li ◽

J. Adamsen ◽

R. Li ◽

H. Pedersen ◽

Y. Liu ◽

...

Keyword(s):

Chemical Activation ◽

Cell Number ◽

Visual Observation ◽

Total Cell ◽

Developmental Competence ◽

Cleavage Rate ◽

Total Cell Number ◽

Mean Diameter ◽

Blastocyst Rate

One of the primary factors influencing the developmental ability of cloned embryos is the oocyte′s diameter (Hirao et al. 1994 J. Reprod. Fertil. 100, 333–339). However, the oocyte donor's age (i.e. its sexual maturity) is also important to consider, because a high proportion of immature oocytes can be expected (Ikeda and Takahashi 2003 Reprod. Fertil. Dev. 15, 215–221). The present study was to investigate the effect of diameter of oocytes collected from prepubertal gilts weighing 100 to 120 kg on the developmental ability of cloned and parthenogenetically activated (PA) embryos. Cumulus–oocyte complexes collected from ovaries of prepubertal gilts were in vitro matured for 42 to 44 h as described for sow oocytes (Li et al. 2008 Theriog 70, 800–808). After removal of the cumulus cells, the matured oocytes were sorted into 2 groups based on visual inspection: large (L) and small (S) oocytes, whereas non-sorted oocytes were used as control (C). In addition, 1 batch from each of the 3 groups of oocytes had their mean size measured. Subsequently, all 3 groups were used for handmade cloning (HMC; Li et al. 2009 Reprod. Domest. Anim. 44, 122–127) or parthenogenetic activation (PA; Kragh et al. 2005 Theriogenology 64, 1536–1545). Then a chemical activation with 5 μg mL–1 cytochalasin B and 10 μg mL–1 cycloheximide in PZM-3 medium was applied for 4 h on both HMC and PA embryos. Finally, the activated embryos were washed and cultured in PZM-3 medium using the WOW system. The embryo development was evaluated by cleavage rate (Day 2), blastocyst rate (Day 6), and total cell number in blastocysts. Data were analysed by ANOVA with single factor in Excel (Microsoft Excel 2007, Redmond, WA, USA). The results showed (Table 1) that by simple visual observation, oocytes could be easily sorted into the following groups: L group (mean diameter 110 μm, from 105 to 116 μm), S group (mean diameter 101 μm, from 93 to 106 μm) and C group (mean diameter 107 μm, from 93 to 116 μm). Cleavage rates and total cell number were similar in the 3 groups. However, the blastocyst rate in L group either for HMC or PA was higher than S group. The data confirm that prepubertal gilt oocytes are useful for cloning and PA, but developmental rates can be increased by selection of large oocytes by simple visual observation. Table 1.Data analysis results

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376 FAILURE OF SPERM DECONDENSATION AFTER INTRACYTOPLASMIC SPERM INJECTION IN SWAMP BUFFALO (BUBALUS BUBALIS)

Reproduction Fertility and Development ◽

10.1071/rdv22n1ab376 ◽

2010 ◽

Vol 22 (1) ◽

pp. 344

Author(s):

V. Chankitisakul ◽

A. Tharasanit ◽

K. Thaseephoo ◽

M. Techakumphu

Keyword(s):

Embryo Development ◽

Intracytoplasmic Sperm Injection ◽

Chemical Activation ◽

Formation Rate ◽

Calcium Ionophore ◽

Developmental Competence ◽

Ionophore A23187 ◽

Blastocyst Formation ◽

Swamp Buffalo ◽

Pronuclear Formation

Intracytoplasmic sperm injection (ICSI) has been intensively used to examine the early events of gamete activation, but few studies have been reported for swamp buffalo. The first objective (Exp. 1) was to compare the developmental competence of oocytes after ICSI using either live or dead frozen-thawed spermatozoa. Matured oocytes were fertilized by ICSI using live (n = 148) or dead (n = 151) spermatozoa, followed by chemical activation using calcium ionophore (A23187) and cyclohexamide (CHX) in SOF medium. In vitro fertilization (n = 149) served as thecontrol. Cleavage rate was recorded on Day 2 and blastocyst formation rate was evaluated on Day 7. The second objective (Exp. 2) was to examine the effects of ICSI and activation regime on the decondensation of buffalo spermatozoa. A total of 148 matured oocytes were subjected to ICSI. The sperm-injected oocytes (n = 87) were then activated using the activation protocol as described in Exp. 1. The ICSI oocytes without activation (n = 61) and sham-injected oocytes with activation (n = 35) were used as controls. Nuclear changes of presumptive zygotes were mor- phologically evaluated for pronuclear formation using 4′,6-diamidino-2-phenylindole procedure and epifluorescent microscopy at 18 h post-ICSI. Statistical differences were determined among the groups using chi-square test. In Exp. 1, the results showed that the percentages of cleavage and blastocyst formation rate were 79.7, 77.0, and 41.6% and 33.8, 30.5, and 14.8% in the live sperm, dead sperm, and IVF groups, respectively. Embryo development rates did not significantly differ between ICSI groups; however, these rates were significantly higher than in the IVF group (P < 0.05). In Exp. 2, the pronuclear formation rate was significantly higher in the ICSI with chemical (70.1%) and sham injection with chemical (60.6%) groups than in the ICSI without chemical group (3.2%; P < 0.01). However, most of the presumptive zygotes with pronuclear formation from the ICSI with chemical activation group showed only intact sperm heads instead of the full male pronuclear formation. Our study suggests that the chemical activation directly affected the female pronuclear formation and embryo development but that it was not associated with the male pronuclear formation. It is postulated that ICSI oocytes that developed to cleavage and blastocyst stages underwent parthenogenesis after chemical activation. This work was supported by TRF-MAG (MRG-WII515S056) and CHE-TRF Senior Research Fund (RTA5080010).

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Developmental competence of cat (Felis domesticus) oocytes and embryos after parthenogenetic stimulation using different methods

Zygote ◽

10.1017/s0967199418000011 ◽

2018 ◽

Vol 26 (2) ◽

pp. 119-126 ◽

Cited By ~ 3

Author(s):

Joanna Kochan ◽

Agnieszka Nowak ◽

Wojciech Niżański ◽

Sylwia Prochowska ◽

Anna Migdał ◽

...

Keyword(s):

Mechanical Activation ◽

Chemical Activation ◽

Experimental Studies ◽

Negative Control ◽

Developmental Competence ◽

Group Iii ◽

Oocyte Collection ◽

Artificial Activation ◽

Parthenogenetic Embryos

SummaryThe aim of this study was to compare the effects of various activating factors on feline oocytes. The study included activation within the ovary (natural), activation during in vitro maturation (spontaneous activation), chemical activation (ionomycin + 6-DMAP), activation by spermatozoa and injection (ICSI) and mechanical activation (sham ICSI). According to our results, parthenogenetic embryos could emerge at every step of in vitro embryo production (IVP) procedures. After oocyte collection, 6% of parthenogenetic embryos were observed, mainly at the 2–4-blastomere stages. After 24 h of in vitro maturation, parthenogenetic activation was observed in 7% of oocytes. Using ionomycin and 6-DMAP to artificially activate oocytes, 53% of cleaved embryos were obtained. The results after ICSI (54% cleaved embryos) were not significantly different from the results in Group III using chemical activation (53% cleaved embryos). But only after ICSI were blastocysts obtained (5/73.7%) as a result of in vitro culture. Moreover, embryos after ICSI were of the best morphological quality with minor levels of fragmentation evident in the embryos. After sham mechanical activation, ‘sham ICSI’, 8% of cleaved embryos were noted. Therefore, it is advised to maintain a negative control in parallel with each step of IVP techniques, to avoid misleading results. Chemical methods for artificial activation of feline oocytes are the most promising for application to the cloning and production of parthenogenetic embryos for experimental studies.

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