33 Improved Dog Cloning Efficiency Using Post-Activation with Ro-3306, a Cdk1 Inhibitor

2018 ◽  
Vol 30 (1) ◽  
pp. 156
Author(s):  
M. J. Kim ◽  
H. J. Oh ◽  
E. M. N. Setyawan ◽  
S. H. Lee ◽  
B. C. Lee
Keyword(s):  
Calcium Ionophore ◽  
Donor Cell ◽  
Cumulus Cells ◽  
Cleavage Rate ◽  
Serum Progesterone ◽  
Significance Level ◽  
Chi Squared ◽  
Pregnancy And Delivery ◽  
Developmental Rates

Inactivation of maturation promoting factor requires proteolytic destruction of cyclin B that results in the loss of cyclin-dependent kinase 1 (Cdk1) activity and exit from metaphase. The aim of this study was to investigate that treatment of Ro-3306, a Cdk1 inhibitor, during post-activation could increase the development of somatic cell nuclear transfer (SCNT) embryos in dogs. Mixed breed female dogs aged at 1 to 5 years and weighing 20 to 35 kg were used in this study (approval number: SNU-160602-14-1). Canine cumulus–oocyte complexes were collected surgically by flushing oviducts with HEPES-buffered TCM-199 medium ~72 h after ovulation, which was determined by serum progesterone concentration. After removal of cumulus cells from oocytes by repeated pipetting in hyaluronidase, matured oocytes were selected for the following experiment. In experiment I, oocytes were activated with (1) 10 μM calcium ionophore and then post-activated with 1.9 mM DMAP (control); (2) DMAP along with 10 μM Ro-3306 (10 μM group); or (3) DMAP along with 50 μM Ro-3306 (50 μM group). Parthenotes were cultured in the synthetic oviducal fluid (SOF) medium after post-activation, and in vitro development was evaluated at 48 h (2-4 cell) and 72 h (6-8 cell). In experiment II, SCNT embryos were produced after oocyte enucleation, donor cell injection, fusion, and activation. Only fused cytoplasts were activated with (1) 1.9 mM DMAP (control) or (2) DMAP along with 50 μM Ro-3306 (50 μM group) and transferred to the oviducts of recipients. The day of embryo transfer was regarded as Day 0. Pregnancy diagnosis was performed by ultrasonography after Day 28 and cloned puppies were delivered Day 58 to 60. Embryo developmental rates in experiment I and II were analysed by one-way ANOVA and t-test, respectively, and pregnancy and delivery rate were analysed by chi-squared test using Graph Prism software (GraphPad, San Diego, CA, USA). The significance level was P < 0.05. Results in experiment I showed that cleavage rate of parthenogenetic embryos in the 50 μM group (89.3 ± 6.8%) was significantly higher than that of 10 μM group or control (50.8 ± 9.9% and 55.4 ± 18.8%, respectively). However, embryonic development to 4 cells and 6-8 cells was not different between treatments. In experiment II, pregnancy rates of recipients receiving embryos in 50 μM group (3/5, 60.0%) were significantly higher than that of control (2/6, 33.3%), but the number of healthy cloned puppies delivered in the 50 µM group (n = 6) versus the control (n = 2) was not different. In conclusion, post-activation with 50 μM Ro-3306 may enhance nuclear reprogramming of dog cloned embryos. This study was supported by RDA (#PJ010928032017), Korea IPET (#316002-05-2-SB010), Research Institute for Veterinary Science, Natural Balance Korea and the BK21 plus program.

303 EFFECT OF SEMEN BATCHES AFTER SEXING ON PRODUCTION EFFICIENCY OF BOVINE EMBRYOS IN VITRO

2010 ◽  
Vol 22 (1) ◽  
pp. 307
Author(s):  
T. L. G. Torregrossa ◽  
M. B. Fernandes ◽  
R. B. Prado ◽  
R. A. Vila ◽  
F. P. Elias ◽  
...  
Keyword(s):  
Production System ◽  
Production Efficiency ◽  
Animal Health ◽  
Personal Communication ◽  
Cumulus Cells ◽  
In Vitro Production ◽  
Significance Level ◽  
Sexed Semen ◽  
Developmental Rates

Within an in vitro production system, bulls differ with respect to their semen potential in generating embryos when the variables of maternal effects are minimized (Marquant-le-Guienne and Humblot 1992 Ann. Zootech. 41, 361-370). We have tested the hypothesis that even with this variation among bulls, there is also an intra-bull variation among frozen sexed semen batches (Sexing Technologies, Navasota, TX, USA; personal communication) when used with IVF. In an embryo commercial production system, 5058 viable oocytes obtained by ovum pick-up with ultrasound from 193 Nelore cows (Bos indicus) over a 12-month period were matured in vitro for 24 h in TCM-199 (Gibco, Life Technologies, Carlsbad, CA, USA) containing 0.5 μg mL-1 FSH (Bioniche Animal Health, Belleville, Ontario, Canada), 50 μg mL-1 LH (Intervet, Boxmeer, the Netherlands) and 10% fetal bovine serum (Gibco). Mature oocytes were inseminated in vitro for 18 h in IVF-Talp medium (BSA-FAF 6 mg mL-1; 10 ng of heparin, Sigma, France), using 3 different batches (I, II, III) of frozen-thawed sexed semen from 4 bulls (A, B, C, D), separated with a Percoll gradient (45:90; Sigma, St-Quentin Fallavier, France). Putative zygotes surrounded in cumulus cells were transferred in CR2aa medium droplets (Rosenkrans and First 1994 J. Anim. Sci. 72, 434-437) with 3 mg mL-1 BSA-FV (Sigma) for 163 h in a humidified incubator at 39°C, with an atmosphere of 5% CO2 in air. Total number of oocytes, total number of blastocysts, and embryonic developmental rates for each bull and respective batch are reported in Table 1. The chi-square test was measured with a significance level of P < 0.05 and showed that there is difference between the batches used with respect to developmental rates of blastocysts. Therefore, there is intra-bull variation in the ability to produce in vitro embryos according to the batch of frozen sexed semen. Table 1.Viable oocytes (VO), total blastocysts (TB), and embryo development rate (%E) by bull and batch used in IVF

81 THE EFFECT OF FOLLICULAR AND OVIDUCT OOCYTES ON THE DEVELOPMENT OF RABBIT NUCLEAR TRANSFERRED EMBRYOS IN VITRO

2010 ◽  
Vol 22 (1) ◽  
pp. 199
Author(s):  
L.-Y. Sung ◽  
C.-H. Chen ◽  
T.-A. Lin ◽  
L.-J. Sung ◽  
H.-Y. Su ◽  
...  
Keyword(s):  
Polar Body ◽  
Fluorescent Microscopy ◽  
Donor Cell ◽  
Cumulus Cells ◽  
Fusion Rate ◽  
Adult Rabbit ◽  
Cleavage Rate ◽  
Developmental Potential ◽  
First Polar Body

This study was designed to examine the effect of rabbit oocytes collected from oviducts v. follicles on the developmental potential of nuclear transplant (NT) embryos. Rabbit oocytes were flushed from the oviducts (oviduct oocytes) or collected from the ovarian Graafian follicles(follicular oocytes) of superovulated does at 12 h post-hCG injection (hpi). Cumulus cells were then removed from the oocytes by incubation in 0.5% hyaluronidase and pipetting. Oocyte enucleation was conducted in TCM-199 +10% fetal bovine serum (FBS) and confirmed under fluorescent microscopy. Skin fibroblasts from an adult rabbit were prepared and cultured to passage 8 to 10 before use as nuclear donors. A donor cell with a diameter of approximately 15 to 19 μm was transferred into the perivitelline space of an enucleated oocyte and subsequently fused with the recipient oocyte by applying 3 direct current pulses at 3.2 kV cm-1 for 20 μs per pulse. Fused oocytes were activated by the same electrical stimulation described above, and then cultured in TCM-199 + 10% FBS containing 2.0 mM 6-DMAP and 5 μg mL-1 cycloheximide for 1 h. Cloned embryos were cultured in 2.5% FBS B2 medium in 5% CO2 and 95% humidified air at 38.5°C for 3 d. Embryo development to cleavage (2- to 4-cell), 8-cell, and morula/blastocyst (Mor/BL) stages was evaluated. The data were analyzed by the General Linear Model procedure (SPSS 11.0, SPSS Inc., Chicago, IL, USA).The total number of oocytes collected per animal was 27.6 ± 1.3, with 47.8% from oviducts, and 52.2% from follicles. The percentage of oviduct oocytes that showed the first polar body was 98.3% (n = 150) at the time of collection, whereas follicular oocytes only had 54.8% at collection (n = 93), but it reached 92.4% when immature follicular oocytes were cultured for 3 h in vitro. The enucleation rates were similar between the follicular (82.7%) and the oviduct (79.1%) groups. Table 1 shows that a significantly higher fusion rate was found in follicular oocytes compared with that in the oviduct group (90.8 v. 63.4%; P < 0.05). There was no difference in the cleavage rate and Mor/BL development between the 2 groups, although the 8-cell(78.4 v. 63.9%; P = 0.11) and the overall efficiencies (30.6% v. 17.9%; P = 0.14) appeared higher in the follicular group. These results demonstrated that rabbit follicular oocytes at 12 hpi have potential equivalent or maybe better (fusion) than that with oviduct oocytes for promoting the preimplantational development of NT embryos. Table 1.The effect of follicular and oviduct oocytes on the development of rabbit NT embryos Supported by NIH1R43 RR023774-01A1 and 5R44HL091605-03.

239 INFLUENCE OF BREED PREDOMINANCE ON MATURATION COMPETENCE, FERTILIZATION, AND DEVELOPMENT IN VITRO OF BOVINE OOCYTES

2013 ◽  
Vol 25 (1) ◽  
pp. 267
Author(s):  
J. Pelaez ◽  
H. Hernandez-Fonseca ◽  
A. Pirela ◽  
F. Baez ◽  
P. Villamediana ◽  
...  
Keyword(s):  
Bos Taurus ◽  
Bos Indicus ◽  
Cumulus Cells ◽  
Fertilization Rate ◽  
Cleavage Rate ◽  
Ethanol Acetic Acid ◽  
Chi Squared ◽  
Development In Vitro ◽  
Bovine Oocytes

The purpose of this research was to compare the competence of bovine oocytes of different breed predominance (Bos taurus v. Bos indicus) to mature and to be fertilized. This was done through the collection, selection, maturation, and fertilization of oocytes from slaughtered cows, predominantly either B. taurus or B. indicus. Only cows that were at least 5/8 B. taurus or 5/8 B. indicus, according to a series of phenotypic characteristics, such as the presence of a hump, dewlap, length of the ears, and others, were selected. To obtain cumulus–oocyte complexes, ovarian follicles (3 to 10 mm in diameter) were aspirated, and only oocytes with 2 or more layers of cumulus cells, an intact zona pellucida, and a homogeneous granular cytoplasm were selected. After selection, oocyte maturation [in vitro maturation (IVM)] and IVF were done. Frozen–thawed semen was used from one Brahman bull (B. indicus). For the evaluation of IVM as for IVF, oocytes were fixed for approximately 24 h at 4°C in a solution of ethanol : acetic acid (3 : 1). They were then stained with 1% acetic orcein. A chi-squared test was performed for all reported rates. The rate of maturation of oocytes from cows with a predominant B. indicus phenotype was 66.93%, whereas cows with a B. taurus phenotype reached 43.10% (P < 0.001). Regarding the fertilization rate, predominantly B. indicus females had 43.68% of oocytes normally fertilized and 41.74% of oocytes were abnormally penetrated. This category included polyspermic and asynchronic (abnormally developed pronucleus) oocytes. In cows with B. taurus predominance, 31.96% of oocytes were normally penetrated and 46.39% were abnormally penetrated by spermatozoa (no significant differences were found). The rate of non-fertilized oocytes was significantly different (P < 0.05) among B. indicus and B. taurus oocytes (6.79 and 17.52%, respectively). A small and nonsignificant proportion of degenerated oocytes resulted in both groups (7.79% for B. indicus and 4.14% for B. taurus). The cleavage rate was not different among phenotypic groups (36.12 and 32.30%, respectively, for B. indicus and B. taurus). In conclusion, the present results indicate that oocytes from predominantly B. indicus cows were more competent than oocytes from cows with a predominance of the B. taurus breed. Nonetheless, this superiority was not evident in terms of cleavage rates. Semen from other B. indicus and B. taurus breeds must be tested to clarify any breed interactions.

121 DEVELOPMENT OF IN VITRO-PRODUCED BOVINE EMBRYOS CULTURED IN SEQUENTIAL OR CONTINUOUS MEDIA

2008 ◽  
Vol 20 (1) ◽  
pp. 141
Author(s):  
L. S. Amorim ◽  
D. J. Walker ◽  
G. E. Seidel Jr
Keyword(s):  
Culture Media ◽  
Cumulus Cells ◽  
Similar Efficacy ◽  
Defined Medium ◽  
Bovine Embryos ◽  
Cleavage Rate ◽  
Cell Stage ◽  
Developmental Rates ◽  
Effects Of Media

Slaughtered bovine females have different characteristics including age, nutritional status, breed, and management system, all of which may affect the results obtained in in vitro embryo production. Another key consideration is that early embryos move from the oviduct to a slightly different environment in the uterus, which has led to development of sequential embryo culture media (e.g. Lane M et al. 2003 Theriogenology 60, 407–419). However, the benefits and importance of using sequential media are not fully known. Therefore, the aim of the present study was to compare developmental rates of oocytes obtained from slaughterhouse-derived ovaries from cows or heifers after culture in sequential media (CDM-1, CDM-2) or in a continuous medium (C-CDM). The experiment was a 3 × 2 × 2 factorial design [bulls (A, B, or C), source (cows or heifers), and medium (sequential or continuous)]. Cumulus–oocyte complexes were aspirated, within 5 h of slaughter, from 3- to 8-mm ovarian follicles of cows (1482 oocytes) and fattened heifers usually fed melengesterol acetate (2818 oocytes). Embryos were produced in vitro as described by De La Torre-Sanchez et al. 2006 Reprod. Fertil. Devel. 18, 585–596, with slight modifications. Presumptive zygotes were vortexed to remove cumulus cells and cultured for 2.5 d in C-CDM (CDM supplemented with 5.0 mm L-lactate, essential and nonessential amino acids, and 0.5% FAF-BSA, or in CDM-1 (De La Torre-Sanchez et al. 2006 Reprod. Fertil. Devel. 18, 585–596) at 39°C in a humidified incubator under 5% CO2, 5% O2, and 90% N2. Cleavage was assessed after 2.5 d; 2- to 6-cell embryos were considered as cleaved, but were not cultured further. Embryos at the 7- to 8-cell stage were cultured for an additional 4.5 d in fresh C-CDM or CDM-2. The percentage blastocysts per oocyte was assessed after 7 and 8 days of culture. Data were arcsin-transformed and evaluated by ANOVA. There was a significant interaction between bull and ovary source for both 8-cell embryos and cleavage rate (P < 0.05); however, this interaction was no longer significant for blastocysts. No other interactions were significant nor a source of ovaries. Culturing embryos in CDM-C refreshed after cleavage evaluation (continuous) or culturing embryos in CDM-1 early and CDM-2 after cleavage evaluation (sequential) resulted in similar cleavage and blastocyst rates (Table 1). We conclude that bovine embryos can be produced using a single chemically defined medium (+BSA) with similar efficacy as a system using 2 sequential media. Table 1. Effects of media on embryonic development (mean ± SE)

Effects of cycloheximide treatment and interval between fusion and activation on in vitro development of pig nuclear transfer embryos

2002 ◽  
Vol 14 (4) ◽  
pp. 191 ◽  
Author(s):  
M. A. Martinez-Diaz ◽  
K. Ikeda ◽  
Y. Takahashi
Keyword(s):  
Nuclear Transfer ◽  
Kinase Activity ◽  
Short Interval ◽  
Cumulus Cells ◽  
Cleavage Rate ◽  
In Vitro Development ◽  
M Phase ◽  
Cytostatic Factor ◽  
Vitro Development

The effects of cycloheximide (CHX) treatment and the interval between fusion and activation on the development of pig nuclear transfer (NT) embryos constructed with enucleated oocytes and serum-starved granulosa/cumulus cells were examined. One group of couplets was fused and activated simultaneously (FAS) by a single electrical pulse (activation pulse). Another three groups of couplets were fused electricaly 1.5, 2.5 or 4.5 h before being subjected to the activation pulse (FBA). Each group was divided into two subgroups and incubated with or without CHX. The NT embryos treated with CHX showed a high and stable cleavage rate, regardless of the interval between fusion and activation; however, development to blastocysts was improved only when the NT embryos were subjected to FAS with CHX. These results indicate that CHX-sensitive events occurring shortly after FAS may be responsible for the development to blastocysts. Fusion pulse rarely activated M II oocytes, but rapidly dropped the p34cdc2 kinase activity in NT embryos. A pronucleus-like structure was observed 2-2.5 h after the activation pulse with CHX in NT embryos of both the FAS and FBA groups. Therefore, successive inactivation of M-phase promoting factor and cytostatic factor at a certain short interval may also play an important role in the development of NT embryos.

70 NON-PLATED GRANULOSA AND CUMULUS CELLS AND FIRST PASSAGE FIBROBLASTS AS NUCLEUS DONOR FOR GOAT CLONING

2006 ◽  
Vol 18 (2) ◽  
pp. 143
Author(s):  
D. Salamone ◽  
M. Catala ◽  
A. Gibbons ◽  
F. Pereyra Bonnet ◽  
M. Cueto
Keyword(s):  
Granulosa Cells ◽  
Room Temperature ◽  
Uv Light ◽  
Cumulus Cells ◽  
Cleavage Rate ◽  
First Passage ◽  
Chi Square ◽  
Donor Nucleus ◽  
Chi Square Test

Different types of somatic cells have been used as nucleus donors for cloning. Most of them were previously cultured in vitro as a monolayer through several plate passages. The experiment reported here was conducted to study the potential usages of granulosa and cumulus cells for cloning without previous culture as a monolayer. A first-plate-passage fibroblast was also used. Oocytes were aspirated by laparoscopy from Criolla goats and matured in TCM-199 + 5% FCS at 39°C for 24 h. Matured oocytes were denuded by vortexing for 3 min in TL HEPES with 1 mg/mL bovine testis hyaluronidase. Metaphases were assessed and oocytes were enucleated by visualization with Hoechst 33342 (5 μg/mL) under UV light (<6 s). Granulosa and cumulus cells were also recovered by laparoscopy and maintained in maturation medium in cryotube for 20 h at room temperature or 39°C, respectively. Goat adult ear fibroblasts were cultured for 1 or 2 weeks and used 2 days after confluence. All types of donor cells were transferred to the perivitlline space of enucleated oocytes and fused by an electrical pulse. After 2 h, activation was induced by incubation in TL-HEPES with 5 µM ionomycin for 4 min and 2 mM 6-DMAP for 3 h. The oocytes were then washed with TL-HEPES and cultured in SOF medium and atmosphere of 5% CO2 + 5% O2 + 90% N2. Cleavage (Day 2) and development to blastocysts (Day 6) were recorded and analyzed by chi-square test. The cleavage rate for non-plated granulosa cells was higher than for the other treatment goups; cumulus cells had a lower rate of development to blastocysts (Table 1). These results suggest that granulosa cells collected and maintained for 24 h at room temperature could be used to produce cloned blastocysts. Table 1. Effect of non-plated granulosa and cumulus cells and first passage fibroblasts as donor nucleus oocytes in goat cloning

44 IN VITRO DEVELOPMENT OF INTERSPECIES NUCLEAR TRANSFER EMBRYOS GENERATED WITH BOVINE OOCYTES AND EQUINE SKIN FIBROBLASTS

2011 ◽  
Vol 23 (1) ◽  
pp. 128
Author(s):  
J. Lee ◽  
J. Park ◽  
Y. Chun ◽  
W. Lee ◽  
K. Song
Keyword(s):  
Nuclear Transfer ◽  
Polar Body ◽  
Donor Cell ◽  
Skin Fibroblasts ◽  
Developmental Competence ◽  
Cleavage Rate ◽  
Cell Stage ◽  
Bovine Oocytes

Study for equine somatic cell nuclear transfer (SCNT) is an attractive field for research, but it has not been a major field of study because it is hard to obtain a sufficient number of ovaries and it takes a lot of time and effort for the recovery of oocytes matured in vivo by ovum pickup. It was reported that the bovine cytoplast could support the remodelling of equine donor cells (Zhou et al. 2007 Reprod. Domest. Anim. 42, 243–247). The objectives of this study are 1) to monitor the early events of equine SCNT by interspecies SCNT (isSCNT) between bovine cytoplast and equine donor cell, and 2) to investigate the developmental competence of isSCNT embryos. Bovine oocytes were recovered from the follicles of slaughtered ovaries, and matured in TCM-199 supplemented with 10 mU mL–1 FSH, 50 ng mL–1 EGF, and 10% FBS at 39°C under 5% CO2 in air for 22 h. Fibroblasts derived from bovine or equine skin tissues were synchronized at G0/G1 stage by contact inhibition for 72 h. After IVM, oocytes with polar body were enucleated and electrically fused with equine or bovine skin fibroblasts (1.0 kV cm–1, 20 μs, 2 pulses). Fused couplets were activated with 5 μM ionomycin for 4 min followed by 5 h culture in 10 μg mL–1 cycloheximide (CHX) and/or 2 mM 6-DMAP, and cultured in modified synthetic oviduct fluid (mSOF) at 39°C under 5% CO2, 5% O2, and 90% N2 for 7 days. All analyses were performed using SAS (version 9.1; SAS Institute, Cary, NC, USA). The cleavage rate of isSCNT embryos derived from equine cell was not different (252/323, 78.7%; P = 0.94) from that of SCNT embryos derived from bovine cell (230/297, 79.2%). However, the rate of isSCNT embryos developed to over 8-cell stage was lower (3.3%; P < 0.0001) than that of bovine SCNT embryos (39.4%), and total cell number of isSCNT embryos developed to over 8-cell stage was lower (17.5, n = 12; P < 0.0001) than that (80.8, n = 110) of bovine SCNT embryos. Also, the rate of blastocyst formation of isSCNT embryos (0/323; 0.0%) was lower (P < 0.0001) than that of bovine SCNT embryos (83/297; 29.3%). Meanwhile, reconstructed oocytes for isSCNT were fixed at 8 h after activation to investigate the formation of pseudo-pronucleus (PPN) after post-activation treatment with CHX or CHX+6-DMAP. The ratio of oocytes with single PPN after treatment with CHX+6-DMAP (26/35; 74.3%) was not different (P = 0.63) from that of oocytes treated with CHX (24/36; 68.1%). Although isSCNT embryos derived from bovine cytoplast and equine donor cell could not develop to more than the 16-cell stage, it is believed that the results of this isSCNT study could be used for the preliminary data regarding the reprogramming of donor cell in equine SCNT.

29 EVALUATION OF DIFFERENT FUSION PARAMETERS IN THE RECONSTRUCTION OF SWINE HANDMADE CLONING EMBRYOS

2008 ◽  
Vol 20 (1) ◽  
pp. 95
Author(s):  
C. Feltrin ◽  
A. S. Lima ◽  
M. Monaco ◽  
S. M. Wilson ◽  
D. Kim ◽  
...  
Keyword(s):  
Uv Light ◽  
Polar Body ◽  
Donor Cell ◽  
Fusion Rate ◽  
Blastocyst Stage ◽  
Cleavage Rate ◽  
Reconstructed Embryos ◽  
Cloning Technique ◽  
Handmade Cloning

The goal of this experiment was to compare different fusion parameters in the handmade cloning technique to produce cloned swine embryos. After in vitro maturation of 618 oocytes, 431 (69.8%) presented a visible polar body and were used in the experiment. The next step was the removal of the cumulus oophorus cells and the digestion of the zona pellucida using pronase (5 mg mL–1) in HEPES TCM199. Oocytes were then exposed to a medium containing cytochalasin B (5 µg mL–1) for 15 min before being bisected with a hand-held blade. The bisected oocytes (cytoplasts) were then placed in medium supplemented with Hoechst 33342 and exposed to UV light to select cytoplasts without metaphase II plates. Next, two cytoplasts and a mesenchymal stem cell (nucleus donor) were pushed together in a phytohemagglutinin (550 µg mL–1) solution. Once adhered, these structures were divided into 3 groups (G) to be fused using different parameters: (G1) 2 pulses (DC) of 0.6 kV cm–1 for 30 µs, (G2) 2 pulses (DC) of 0.9 kV cm–1 for 30 µs, and (G3) 2 pulses (DC) of 1.2 kV cm–1 for 30 µs. For all three groups, 0.3 m of mannitol solution (without calcium) was used in the fusion chamber, and an initial pre-pulse (AC) of 10V for 15 s was performed to permit the alignment of 100% of the cytoplast-donor cell structures. After fusion, reconstructed embryos were activated in 0.3 m mannitol and 0.1 mm calcium in the fusion chamber using 2 pulses of 0.9 kV cm–1 for 30 µs followed by incubation in 10 µg mL–1 of cycloheximide solution for 4 h. Afterwards, the reconstructed embryos were transferred to NCSU23 medium supplemented with amino acids (nonessential and essential) and 0.4% bovine serum albumin. The embryos were cultured at 39�C in a 100% humidified atmosphere containing 5% CO2, 5% O2, and 90% N2. Cleavage rates were evaluated after 48 h of culture. For G1, the fusion rate was 43% (25/58) with 72% cleavage (18/25), the G2 fusion rate was 87% (56/64) with 80% cleavage (45/56), and the G3 fusion rate was 79% (53/67) with 69% cleavage (37/53). Statistical analysis was performed using the chi-square test. There were no significant differences in fusion rates between groups G2 and G3, but the fusion rate of these groups was significantly different from that of G1 (P < 0.05). No significant differences in cleavage rate were observed among the three groups. In conclusion, fusion using 2 pulses at either 0.9 or 1.2 kV cm–1 for 30 µs was more efficient for embryo reconstruction in the handmade cloning technique compared to that using 2 pulses at 0.6 kV cm–1 for 30 µs. Further studies need to be performed to improve cleavage rates and assess development to the blastocyst stage.

256 EFFECT OF SPERM SELECTION ON THE RATE OF IN VITRO FERTILIZATION IN ALPACA (VICUGNA PACOS)

2015 ◽  
Vol 27 (1) ◽  
pp. 217
Author(s):  
E. Mellisho ◽  
V. Rivas ◽  
J. Ruiz ◽  
G. Mamani
Keyword(s):  
Extended Period ◽  
Cumulus Cells ◽  
Sperm Selection ◽  
Cleavage Rate ◽  
Slow Cooling ◽  
Vitro Fertilization ◽  
Progressive Motility ◽  
Frozen Semen ◽  
Washing Method

In alpacas, improvement of reproductive efficiency of male camelids is limited by the small size of the testes, extended period of ejaculation, and low quality of semen. This study was designed to determine the effect of 2 sperm preparation treatments before IVF on the cleavage rate. The sperm was obtained by slicing the head of the epididymis of slaughtered male alpacas (n = 8), diluting in Tris-yolk-glycerol, and freezing with the slow-cooling method. Frozen semen straws per each male were thawed in a water bath at 37°C for 15 s and evaluated for percentage of progressive motility (32 ± 8.6%) and concentration (66.5 ± 24 × 106 sperm mL–1) post-thawing. Sperm selection by the swim-up method was performed by centrifugation at 1077 × g for 5 min with washing sperm medium eliminating the supernatant; sperm were settled in inclined tube with fertilization medium (without capacitating agent) for 60 min, after which 100 μL from the surface was recovered for use in IVF. The washing method consisted in repeated washing (twice) of sperm in washing sperm medium and fertilization medium by centrifugation at 1077 × g for 5 and 3 min, respectively, and recovery of 50 μL from the bottom of the tube for use in IVF. Sperm selected by swim-up or washing methods had similar characteristics of progressive motility (18 and 23%); however, the concentration was higher for the washing v. swim-up method (52 v. 14 × 106 sperm mL–1, respectively). Cumulus-oocyte complexes (COC) were recovered from 278 ovaries of alpacas killed at abattoirs and classified (Grade 1 and 2) for in vitro maturation (38.5°C at 5% CO2 in air for 27 h in 50 μL of 10 COC per drop). A total of 839 oocytes cultured for 27 h in maturation medium were partially stripped out of cumulus cells by gentle aspiration with a pipette. Sperm suspensions in Fert TALP medium (5 μL) from each treatment group were added to each fertilization drop with 10 oocytes per drop of 45 μL obtaining a final concentration of 10 × 106 sperm mL–1 and cultivated for 72 h until their evaluation. The data for the 13 repetitions of the rate of cleavage (2 to 8 cells) were converted to angular values (angle = arcsin √%) with the object of normalizing the distribution of the data; the analysis of variance was performed (complete randomised design with sub-sampling, P < 0.05) using SAS® version 8.0 for Windows. The rate of cleavage (cell division) did not show statistical differences (P = 0.67) for the swim-up method (37%; 155/421) v. washing method (35%; 147/418). The methods of sperm selection (swim-up and washing) did not affect the rate of IVF.

26 Drugs that Modify Epigenetics...What do they do to Porcine Clones?

2018 ◽  
Vol 30 (1) ◽  
pp. 152
Author(s):  
C. P. Buemo ◽  
A. Gambini ◽  
L. N. Moro ◽  
N. Canel ◽  
D. F. Salamone
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Electric Pulse ◽  
Donor Cell ◽  
T Test ◽  
Control Group ◽  
Developmental Rates ◽  
Student’S T ◽  
Student’S T Test

Although somatic cell nuclear transfer (SCNT) technology was developed more than 20 years ago, cloning efficiency remains low. Failures in the reprogramming of the donor cell result in embryos with aberrant epigenetic patterns and low developmental rates. In this study, we assessed whether the use the inhibitor of DNA (cytosine 5) methyltransferase 5-azacitidine (5Aza) combined with the MEK inhibitor in the MAPK pathway PD0325901 (PD) could improve SCNT efficiency in pigs. In vitro maturation of cumulus–oocyte complexes was performed in TCM for 44 h at 39°C under 5% CO2. Cumulus cells and zona pellucida was removed from matured oocytes, followed by enucleation of the metaphase plate previously stained with Hoëchst 33342. Each enucleated oocyte was attached to a donor cell by phytohemagglutinin treatment followed by an electric pulse of 80V for 30 μs. After fusion, reconstituted embryos were activated by an electric pulse followed by an incubation in 2 mM 6-DMAP for 3 h. Cloned embryos were cultured in vitro in a modified well-of-well system in SOF medium, where 3 cloned embryos were placed per microwell (3X). The experimental group 3X + drugs was exposed for the first 3 days to 1 μM PD and 1 μM 5Aza in SOF medium. After washing, embryos were cultured until Day 7 in regular SOF medium. The control group (3X) was cultured in regular SOF medium for 7 days. In vitro embryo developmental rates, gene expression, histone acetylation, and DNA methylation status were studied. The use of epigenetic modifying drugs significantly increased blastocyst rates (40.9% v. 29%; Fisher’s test, P < 0.05) and embryo size (41.46% v. 28.56%; Student’s t-test, P < 0.05) compared with the control group. Regarding gene expression, an increase of the relative expression of genes related to cell differentiation (Igf2 and Cdx2), antiapoptotic pathways (Bcl-xl) and DNA methylation modulation (Mapk1) was observed (P < 0.05). Pluripotency genes Oct4 and Nanog did not show differences between groups. The Bax proapoptotic gene significantly decreased its expression after drug treatment, as did the Klf4 gene (P < 0.05). Results were analysed by Student’s t-test. According to Histone H3K27ac, which is associated with enhancers or gene promoters, its marker was located mainly in the nuclear periphery respect to the control group with a uniform dispersion, indicating that the treatment could be activating certain genes by locating them near the periphery. Histone H3K4me1 was more uniformly localised throughout the nucleus in both groups. The intensity of the fluorescence was measured by quantitative confocal microscopy using a histogram produced by the ImageJ program (National Institutes of Health, Bethesda, MD, USA). Regarding DNA methylation by bisulphite sequencing, the 2 genes studied (Oct4 and DNMT1) showed a higher demethylation status for the treated group. Our results indicate that the combination of 5Aza+PD during early pre-implantation development dramatically increase blastocyst rates and embryo quality. This novel combination could be used as a strategy to improve the efficiency of SCNT in pigs and potentially other animals.

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