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ABSTRACT: Our aim was to optimize a cryoprotectant treatment for vitrification of immature porcine cumulus-oocyte complexes (COCs). Immature COCs were vitrified either in 35% ethylene glycol (EG), 35% propylene glycol (PG) or a combination of 17.5% EG and 17.5% PG. After warming, the COCs were in vitro matured (IVM), and surviving oocytes were in vitro fertilized (IVF) and cultured. The mean survival rate of vitrified oocytes in 35% PG (73.9%) was higher (P<0.05) than that in 35% EG (27.8%). Oocyte maturation rates did not differ among vitrified and non-vitrified control groups. Blastocyst formation in the vitrified EG group (10.8%) was higher (P<0.05) than that in the vitrified PG group (2.0%) but was lower than that in the control group (25.0%). Treatment of oocytes with 35% of each cryoprotectant without vitrification revealed a higher toxicity of PG on subsequent blastocyst development compared with EG. The combination of EG and PG resulted in 42.6% survival after vitrification. The maturation and fertilization rates of the surviving oocytes were similar in the vitrified, control and toxicity control (TC; treated with EG+PG combination without cooling) groups. Blastocyst development in the vitrified group was lower (P<0.05) than that in the control and TC groups, which in turn had similar development rates (10.7%, 18.1% and 23.3%, respectively). In conclusion, 35% PG enabled a higher oocyte survival rate after vitrification compared with 35% EG. However, PG was greatly toxic to oocytes. The combination of 17.5% EG and 17.5% PG yielded reasonable survival rates without toxic effects on embryo development.
Journal of Reproduction and Development 05/2013; · 1.46 Impact Factor
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Thanh Quang Dang-Nguyen,
Nguyen Viet Linh,
Rosa Minoia,
Masahiro Kaneda,
Tamas Somfai,
Seiki Haraguchi,
Satoshi Akagi,
Kazuhiro Kikuchi,
Michiko Nakai,
Atsushi Tajima, Takashi Nagai
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ABSTRACT: The purposes of the present study were to examine the effect of naloxone, a mu-opioid receptor (MOR) antagonist, on porcine oocyte maturation and embryo development. MOR gene was expressed in germinal vesicle (GV) and metaphase II (M-II) porcine oocytes, one-, four-cell stage embryos and blastocysts. In blastocysts, MOR gene was mainly expressed in inner cell mass (ICM) cells. Supplementation of 10(-8) mol/L naloxone in in vitro maturation (IVM) medium increased the maturation rate (P < 0.05). However, 10(-4) mol/L naloxone reduced the maturation rate (P < 0.05) compared with the control. The presence of naloxone during IVM had no effects on fertilization status and subsequent embryonic development after in vitro culture (IVC). The addition of 10(-3) mol/L dibutyryl cyclic adenosine monophosphate (dbcAMP), and 10(-8 ) mol/L naloxone together into IVM medium increased nuclear maturation (P < 0.05) compared with the addition of either dbcAMP or naloxone alone. Supplementation with naloxone in IVC medium did not improve embryonic development. However, at the concentrations of 10(-6) mol/L and 10(-8) mol/L, naloxone increased the ratio of ICM to total cells in blastocysts (P < 0.05). In conclusion, at low concentration, naloxone increases maturation rate and the ratio of ICM to total cells in blastocysts. Naloxone and cAMP have a synergistic effect on oocyte maturation.
Animal Science Journal 05/2013; · 0.86 Impact Factor
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Zhao Namula,
Yoko Sato,
Risa Kodama,
Kouta Morinaga,
Vien Viet Luu,
Masayasu Taniguchi,
Michiko Nakai,
Fuminori Tanihara,
Kazuhiro Kikuchi, Takashi Nagai,
Takeshige Otoi
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ABSTRACT: This study investigated the effects of skim milk on the quality and fertility of boar spermatozoa under long-term chilled preservation. Semen samples were stored in Modena solution supplemented with 0 (control) to 50 mg/mL skim milk at 5°C for 4 weeks; spermatozoa stored with 7.5 and 15 mg/mL of skim milk (7.5-SM and 15-SM groups, respectively) exhibited significantly higher motility indices than those of the control group up to 3 weeks (P < 0.05), and the 7.5-SM group showed improved motility indices even after 4 weeks (P < 0.05). In vitro fertilization using spermatozoa in the 7.5-SM and 15-SM groups stored at 5°C for 2 weeks showed significantly higher fertilization rates of spermatozoa and the development rates to blastocyst than the control group (P < 0.05), and the 7.5-SM group showed similar rates of fertilization and blastocyst formation in the fresh non-stored spermatozoa group. After artificial insemination using spermatozoa stored for 2 weeks in the 7.5-SM group, healthy piglets were obtained. Boar spermatozoa can be stored at 5°C in a Modena solution containing skim milk. Supplementation of 7.5 mg/mL skim milk improves boar spermatozoa motility and fertility even after liquid preservation at 5°C for 2 weeks.
Animal Science Journal 03/2013; · 0.86 Impact Factor
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ABSTRACT: Bovine somatic cell nuclear transfer (SCNT) embryos can develop to the blastocyst stage at a rate similar to that of embryos produced by in vitro fertilization. However, the full-term developmental rate of SCNT embryos is very low, owing to the high embryonic and fetal losses after embryo transfer. In addition, increased birth weight and postnatal mortality are observed at high rates in cloned calves. The low efficiency of SCNT is probably attributed to incomplete reprogramming of the donor nucleus and most of the developmental problems of clones are thought to be caused by epigenetic defects. Applications of SCNT will depend on improvement in the efficiency of production of healthy cloned calves. In this review, we discuss problems and recent progress in bovine SCNT.
Animal Science Journal 03/2013; 84(3):191-199. · 0.86 Impact Factor
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ABSTRACT: This study was conducted to assess the fertilization and development of porcine oocytes matured in a solo follicular fluid (pFF) using different in vitro culture systems and insemination periods. Cumulus-oocyte complexes (COCs), follicular cells (FCs), and pFF were collected from the follicles of ovaries. The pFF was used as a maturation medium (MpFF) after supplementation with follicle stimulating hormone (FSH) and antibiotics. The COCs were matured in a 15 ml test tube containing 3.5 ml of MpFF with FCs (5.2 × 10(6) cells/ml; rotating culture system) or 2 ml of MpFF without FCs in a 35-mm petri dish (static culture system) for 44 to 48 h. After maturation culture, oocytes were co-incubated with frozen-thawed spermatozoa for 5 h and then cultured for 7 days. The total mean rates of sperm penetration, normal fertilization, male pronucleus (MPN) formation, cleavage, and development to the blastocyst stage of oocytes after insemination were significantly higher (P<0.01) in the rotating culture system than in the static culture system. In conclusion, compared with the static culture system, the rotating culture system is adequate for the production of developmentally competent porcine oocytes when MpFF is used as a maturation medium.
Journal of Reproduction and Development 02/2013; · 1.46 Impact Factor
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ABSTRACT: The objective was to determine the effects of adding L-carnitine (an enhancer of lipid metabolism) during IVM, on cryotolerance and developmental competence of bovine oocytes. Oocytes matured in the absence (control) or presence (0.6 mg/mL) of L-carnitine were subjected to IVF and embryo culture after Cryotop vitrification or nonvitrification at the metaphase stage of the second meiotic cell division. Cleavage and blastocyst formation rates, and inner cell mass and trophectoderm cell numbers were determined. Also, ATP content in IVM oocytes was measured and intracellular lipid droplets were observed (Nile red staining and confocal microscopy). L-carnitine had no significant effect on the rate of matured oocytes. Vitrification reduced (P < 0.05) mean (±SEM) rates of live oocytes both in control (80.6 ± 1.9%) and L-carnitine groups (82.7 ± 5.1%) compared with nonvitrified oocytes (100%). After IVF, cleavage rates of vitrified control and L-carnitine groups (56.5 ± 3.9% and 62.8 ± 5.1%, respectively) were significantly lower than those in nonvitrified control and L-carnitine groups (83.9 ± 4.2% and 84.3 ± 1.3%). After vitrification, blastocyst formation rate in the L-carnitine group (54.4 ± 5.2%) was significantly higher compared with the control (34.9 ± 4.4%), and did not significantly differ from those in nonvitrified control and L-carnitine groups (52.1 ± 4.2% and 52.8 ± 3.0%). The numbers and ratio of inner cell mass and trophectoderm cells in blastocysts did not differ significantly among groups. The ATP content in L-carnitine-treated oocytes tended to be higher compared with the control. Vitrification did not reduce ATP content in oocytes, irrespective of L-carnitine treatment. Treatment with L-carnitine dislocated lipid droplets from the peripheral area to the inner cytoplasm. In conclusion, L-carnitine supplementation during IVM redistributed lipid droplets in oocytes; if they survived vitrification, their developmental competence was similar to that of nonvitrified oocytes.
Theriogenology 12/2012; · 1.96 Impact Factor
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ABSTRACT: Abstract Although telomeres are elongated during morula-to-blastocyst transition in cloned embryos, it is still unknown whether donor cell types have any effect on this elongation. In the present study, we examined the changes of telomere length during morula-to-blastocyst transition in cloned porcine embryos using different types of donor cells. Porcine embryonic stem-like cells (pESLCs), porcine cumulus cells (PCs), and porcine embryonic fibroblasts at passages 7 and 10 (PEF7s and PEF10s, respectively) were used as donor cells. Telomere lengths of pESLCs (35.8±1.5 kb), PCs (24.4±0.5 kb), PEF7s (18.7±0.6 kb), and PEF10s (17.2±0.1 kb) were significantly different. In contrast, telomere length in morulae derived from pESLCs (18.2±0.3 kb), PC (17.8±0.7 kb), PEF7 (18.5±0.3 kb), and PEF10 (18.4±0.4 kb) did not differ significantly. Likewise, telomeres in blastocysts derived from pESLCs (22.3±1.5 kb), PCs (23.5±2.6 kb), PEF7s (20.2±1.0 kb), and PEF10s (20.9±1.0 kb) had similar lengths. However, telomeres in blastocysts were significant longer (p<0.05) compared with morulae in each group. Relative telomerase activities of morulae derived from pESLCs (4.2±0.4), PCs (4.0±0.5), PEF7s (5.1±0.4), and PEF10s (4.9±0.4) were significantly lower (p<0.01) than those of blastocysts derived from pESLCs (8.2±1.1), PCs (8.6±0.6), PEF7s (12.5±2.9), and PEF10s (8.3±1.1). In conclusion, the telomere elongation in cloned pig embryos that occurred during morula-to-blastocyst transition may be related to the rise of telomerase activity. The telomere elongation may also be independent of the type and telomere length of the donor cell.
Cellular reprogramming. 12/2012; 14(6):514-9.
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ABSTRACT: Failure of male pronucleus formation has hampered the success of intracytoplasmic sperm injection (ICSI) in swamp buffalo. The aim of the present study was to improve male pronucleus formation by pretreating sperm with various chemicals before ICSI. In Experiments1 and 2, sperm were treated according to one of the following protocols: (1) 0.1% Triton-X 100 (TX) for 1 min, (2) 10 μM calcium ionophore (CaI) for 20 min, (3) freezing and thawing (FT) without any cryoprotectant, or (4) no treatment (control). These sperm treatment groups then either did or did not receive additional sperm treatment with 5 mM dithiothreitol (DTT) for 20 min. Acrosomal integrity (Experiment 1) and DNA fragmentation (Experiment 2) were evaluated in the sperm before ICSI. In Experiment 3, oocytes matured in vitro were subjected to ICSI using pretreated sperm as described above and then were cultured either with or without activation. The TX- and CaI-treated sperm caused an increase in the number of acrosome-loss sperm, whereas the FT treatment and control increased the proportion of acrosome-reacted sperm (P<0.05). The DNA fragmentation did not differ among treatments (P>0.05). At 18 h post-ICSI, pronucleus (PN) formation was found only in activated oocytes. The majority of the activated ICSI oocytes contained intact sperm heads. Normal fertilization was observed in the CaI and FT treatment groups and control group when sperm were treated with DTT before ICSI. In conclusion, DTT treatment of sperm with reacted acrosomes before ICSI together with activation of the ICSI oocytes is important for successful male pronucleus formation.
Journal of Reproduction and Development 11/2012; · 1.46 Impact Factor
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ABSTRACT: Telomere is a nucleoprotein structure at the ends of chromosomes that helps to protect the ends of chromosomes from being fused with other chromosomes. Knockout of histone methyltransferases Suv39h1 and Suv39h2 increases the telomere length in murine cells, whereas downregulation of SUV39H1 and SUV39H2 genes decreases the telomere length in human cells, suggesting that telomere biology is different among mammalian species. However, epigenetic regulation of the telomere has not been studied in mammals other than the human and mouse. In the present study, the effect of knockdown of SUV39H1 and SUV39H2 genes on telomere length was examined in porcine embryonic stem-like cells (pESLCs) and porcine embryonic fibroblasts (PEFs). The telomeres in SUV39H1 and SUV39H2 knockdown (SUV39KD) pESLCs (37.1 ± 0.9 kb) were longer (P<0.05) compared with those of the control (33.0 ± 0.7 kb). Similarly, SUV39KD PEFs had longer telomeres (22.1 ± 0.4 kb; P<0.05) compared with the control (17.8 ± 1.1 kb). Telomerase activities were not different between SUV39KD pESLCs (10.4 ± 1.7) and the control (10.1 ± 1.7) or between SUV39KD PEFs (1.0 ± 0.3) and the control (1.0 ± 0.4), suggesting that telomerase activities did not contribute to the telomere elongation in SUV39KD pESLCs and SUV39KD PEFs. Relative levels of trimethylation of histone H3 lysine 9 and expressions of DNMT1, DNMT3A and DNMT3B were decreased in SUV39KD cells, suggesting that telomere lengthening in SUV39KD pESLCs and SUV39KD PEFs might be not only related to the loss of histone modification marks but also linked to the decrease in DNA methyltransferase in pigs.
Journal of Reproduction and Development 09/2012; · 1.46 Impact Factor
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ABSTRACT: The aim of the present study was to compare the efficiency of the solid surface (SSV), cryotop (CT) vitrification methods and cytochalasin B (CB) pretreatment for cryopreservation of immature buffalo oocytes. Cumulus-oocyte complexes (COCs) were placed for 1 min in TCM199 containing 10% dimethylsulfoxide (DMSO), 10% ethylene glycol (EG), and 20% fetal bovine serum, and then transferred for 30 s to base medium containing 20% DMSO, 20% EG and 0.5 mol/L sucrose. CB pretreated ((+)CB) or non-pretreated ((-)CB) COCs were vitrified either by SSV or CT. Surviving vitrified COCs were selected for in vitro maturation (IVM) and in vitro fertilization (IVF). The rate of viable oocytes after vitrification in CT groups (82%) was significantly lower (P < 0.05) than that in a fresh control group (100%), but significantly higher (P < 0.05) than those in SSV groups (71-72%). Among vitrified groups, the highest maturation rate was obtained in the CT (-)CB group (32%). After IVF, the cleavage and blastocyst formation rates were similar among vitrified groups but significantly lower than those of the control group. In conclusion, a higher survival rate of oocytes after vitrification and IVM was obtained in the CT group compared with that in the SSV group, indicating the superiority of the CT method. Pretreatment with CB did not increase the viability, maturation or embryo development of vitrified oocytes.
Animal Science Journal 09/2012; 83(9):630-8. · 0.86 Impact Factor
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Reproduction Fertility and Development 06/2012; · 2.11 Impact Factor
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ABSTRACT: The aim of this study was to examine the effects of bovine follicular fluid (bFF) on mitochondrial activity in in vitro-matured (IVM) oocytes and to assess its importance for fertilisation and embryo development. Bovine follicular oocytes were subjected to IVM in medium supplemented either with polyvinylpyrrolidone, bovine serum albumin, calf serum or bFF. Nuclear maturation, cumulus expansion, mitochondrial distribution and ATP content in oocytes were compared between groups along with subsequent in vitro fertilisation (IVF) and embryo development. Compared with other supplements, bFF generated significantly enhanced re-distribution of active mitochondria in oocytes and this effect was associated with elevated intracellular ATP content. Furthermore, bFF significantly improved cumulus expansion, which was associated with improved fertilisation rates when cumulus-enclosed oocytes were subjected to IVF; however, its promoting effect was neutralised when denuded oocytes were inseminated. Elevating ATP content in oocytes by bFF did not affect maturation or embryo development but promoted fertilisation when mitochondrial electron transport was blocked in oocytes before IVF by Rotenone. In conclusion, supplementation of IVM medium with bFF promotes sperm penetration both by the improvement of cumulus expansion and by enhancing ATP levels in oocytes, which maintains their ability to be fertilised after mitochondrial stress.
Reproduction Fertility and Development 06/2012; 24(5):743-52. · 2.11 Impact Factor
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ABSTRACT: Interspecies/intergeneric mitochondrial heteroplasmy can occur in interspecies/intergeneric hybrid embryos or following nuclear transfer. In the present study, intergeneric buffalo (Bubalus bubalis) mitochondria (WB-mt) or interspecies murine (Mus spretus) mitochondria (M-mt) were injected into bovine (Bos taurus) oocytes, and the subsequent embryonic development was characterized. Fibroblast mitochondria (WB-mt or M-mt) were microinjected into in vitro matured bovine oocytes followed by oocyte activation by a combination of electrical stimulation and 6-dimethylaminopurine treatment. After seven days of culture, embryo development was evaluated. The copy number of specific mtDNA populations (introduced and native mtDNA) from heteroplasmic oocytes was estimated using real-time PCR. The results illustrated that oocytes injected with either WB-mt or M-mt can develop to the blastocyst stage (20.6% and 19.6%). Cleavage division rates and development to the morula stage in oocytes injected with WB-mt were lower (76.2% and 45.9%, respectively) in comparison with uninjected oocytes (89.2% and 59.1%, respectively) (P<0.05). However, no differences were found in comparing M-mt injected oocytes and controls (P>0.05). An increase in bovine mtDNA copy number was observed at the expanded blastocyst stage of injected embryos (P<0.01), while the number of injected mtDNA was stable throughout development. This study demonstrates that interspecies/intergeneric mitochondrial injected bovine oocytes have the ability to develop to the blastocyst stage after parthenogenetic activation and that injected mtDNA was neither selectively destroyed nor enhanced through development. Moreover, injected intergeneric mitochondria had a demonstrated influence on bovine parthenogenetic development and mtDNA replication.
Journal of Reproduction and Development 03/2012; 58(3):323-9. · 1.46 Impact Factor
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ABSTRACT: Recent improvements in cryopreservation of mammalian eggs enable the long-term preservation of female germ cells in several mammalian species. Nevertheless, cryopreservation of porcine oocytes is still considered as a challenge. Although the use of vitrification techniques result in reasonable survival rates, developmental competence of vitrified oocytes has been compromised. Alterations of zona characteristics, cytoskeleton, mitochondrial functions and antioxidant-defense ability caused by vitrification are among the most frequently observed malformations which may be responsible for the low developmental competence of cryopreserved porcine oocytes. Furthermore, in vitro maturation, fertilization and embryo culture technologies, which are indispensable for generating embryos from cryopreserved oocytes, generate high rates of abnormal fertilization (polyspermy) and additional stress in resultant embryos further compromising their developmental competence. As a result, embryo development of porcine cryopreserved oocytes is still at low level and to date no piglet has been produced from such oocytes. The aim of the present review is to summarize knowledge on viability and developmental competence of vitrified porcine oocytes and to give ideas for future perspectives for the improvement of porcine oocyte cryopreservation technology.
Journal of Reproduction and Development 01/2012; 58(1):17-24. · 1.46 Impact Factor
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Satoshi Sugimura,
Shuji Kobayashi,
Yutaka Hashiyada,
Masaki Ohtake,
Masahiro Kaneda,
Tadayuki Yamanouchi,
Hideo Matsuda,
Yoshio Aikawa,
Shinya Watanabe, Takashi Nagai,
Eiji Kobayashi,
Kazuyuki Konishi,
Kei Imai
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ABSTRACT: We examined the influence of recipient oocytes on in vitro development, oxygen consumption, and gene expression in the resulting cloned bovine embryos. Oocytes derived from slaughterhouse ovaries and ovum pickup (OPU)-derived oocytes were used as recipient cytoplasts for the production of cloned embryos. A series of OPU sessions was conducted on Holstein cows without follicular growth treatment (FGT). In the same cows, we then performed dominant follicle ablation and subsequently administered follicle-stimulating hormone and prostaglandin F(2α) with controlled internal drug release device before a second series of OPU. Cumulus cells collected from single Holstein cows were used as donor cells. After measurement of oxygen consumption at the blastocyst stage with modified scanning electrochemical microscopy, analysis of 10 genes (CDX2, IFN-tau, PLAC8, OCT4, SOX2, NANOG, ATP5A1, GLUT1, AKR1B1, and IGF2R) was performed with real-time RT-PCR. Rates of fusion, cleavage, and blastocyst formation were not different among the treatment groups. Levels of oxygen consumption in cloned blastocysts derived from slaughterhouse ovaries or OPU without FGT were significantly lower than in blastocysts derived from artificial insemination (AI). However, oxygen consumption was increased in cloned blastocysts derived from OPU with FGT, depending on the individual oocyte donor. Furthermore, gene expression of IFN-tau and OCT4 in cloned blastocysts derived from OPU with FGT was similar to that in AI-derived blastocysts, whereas expression of those genes in cloned blastocysts derived from slaughterhouse ovaries or OPU without FGT was significantly different from that in AI-derived blastocysts. Thus, recipient oocytes collected by OPU in combination with manipulation of follicular growth in donor cows are suitable for producing cloned embryos.
Cellular reprogramming. 12/2011; 14(1):29-37.
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Tamás Somfai,
Kei Imai,
Masahiro Kaneda,
Satoshi Akagi,
Shinya Watanabe,
Seiki Haraguchi,
Eiji Mizutani,
Thanh Quang Dang-Nguyen,
Yasushi Inaba,
Masaya Geshi, Takashi Nagai
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ABSTRACT: Since BSE testing of slaughtered cattle is obligatory in Japan, storage of ovaries at 15-20 C overnight in phosphate buffered saline has become a routine protocol in in vitro production (IVP) of cattle embryos. Ovary storage is known to reduce developmental competence of oocytes; however, its effects on oocyte gene expression have not been clarified yet. This study compared oocytes collected from stored slaughterhouse-derived ovaries with those collected by Ovum Pick-Up (OPU) in terms of the expression of 20 selected genes to determine if ovary storage affects cellular processes at the molecular level. Expression of mRNA in oocytes was assayed before and after in vitro maturation (IVM) by real-time quantitative PCR. Maternal mRNA levels of genes were investigated in 2-cell stage embryos obtained from slaughterhouse oocytes to assess their roles for blastocyst formation. In immature OPU oocytes, genes related to metabolism (GAPDH), transporters (GLUT8, ATP1A1) and stress resistance protein (HSP70) showed significantly higher expression compared with oocytes derived from stored ovaries. During IVM, the expression of GDF9, GLUT8, CTNNB1 and PMSB1 was significantly decreased irrespective of oocyte source. Two-cell stage embryos cleaving at 22-25 h after in vitro fertilization (IVF) showed a significantly higher blastocyst formation rate and ATP1A1 gene expression level compared with those cleaving at 27-30 h after IVF. Our results reveal that storage of ovaries alters mRNA levels in oocytes. Correlation of Na/K ATPase ATP1A1 expression in IVP embryos at the 2-cell and 8-cell stages with their developmental ability to the blastocyst stage may suggest the importance of maternal mRNA of this gene during blastulation in embryos derived from slaughterhouse oocytes.
Journal of Reproduction and Development 09/2011; 57(6):723-30. · 1.46 Impact Factor
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ABSTRACT: The aim of the present study was to assess the effects of L-carnitine, an enhancer of lipid metabolism and mitochondrial activity, during in vitro maturation (IVM) on nuclear maturation and in vitro fertilisation of porcine follicular oocytes and subsequent embryo development. Mitochondrial functions, intracellular lipid content and reactive oxygen species (ROS) levels in oocytes were also investigated. L-carnitine supplementation in 0.6-5mgmL(-1) concentration during IVM significantly improved (P<0.05) the rates of metaphase-II (MII) stage oocytes compared with the control; however, fertilisation rates and monospermy were not improved. Although supplementation of IVM medium with L-carnitine significantly increased oocyte cleavage (P<0.05), further development to the blastocyst stage was not improved. The density of active mitochondria was significantly higher and the density of lipid droplets was significantly lower (P<0.05) in L-carnitine-treated oocytes compared with the control. Furthermore, the ROS levels in L-carnitine-treated oocytes were significantly lower than those in the control. In conclusion, enhancing mitochondrial functions by L-carnitine improved oocyte maturation and cleavage underlining the importance of lipid metabolism for nuclear and cytoplasmic maturation of porcine oocytes.
Reproduction Fertility and Development 09/2011; 23(7):912-20. · 2.11 Impact Factor
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ABSTRACT: Dolly, the first mammal cloned from a somatic cell, had shorter telomeres than age-matched controls and died at an early age because of disease. To investigate longevity and lifetime performance in cloned animals, we produced cloned cows with short telomeres using oviductal epithelial cells as donor cells. At 5 years of age, despite the presence of short telomeres, all cloned cows delivered multiple healthy offspring following artificial insemination with conventionally processed spermatozoa from noncloned bulls, and their milk production was comparable to that of donor cows. Moreover, this study revealed that the offspring had normal-length telomeres in their leukocytes and major organs. Thus, cloned animals have normal functional germ lines, and therefore germ line function can completely restore telomere lengths in clone gametes by telomerase activity, resulting in healthy offspring with normal-length telomeres.
Journal of Reproduction and Development 08/2011; 57(5):636-42. · 1.46 Impact Factor
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ABSTRACT: We have shown in pigs that oocytes denuded of cumulus cells at 24 h of in vitro maturation culture and subsequently matured for a total of 46 h (DO24 oocytes) have lower cytoplasmic maturity than those matured with cumulus cells for 46 h and then denuded (DO46 oocytes). In the present study, DO24 zona-free oocytes were fused with one (1C) or two (2C) cytoplasmic fragments produced by serial centrifugation ("centri-fusion") of DO46 oocytes (DO24+1C and DO24+2C oocytes, respectively). Groups of (1) DO46 (a control), (2) DO24, (3) DO24+1C and (4) DO24+2C oocytes were partheno-activated by an electrical pulse or fertilized in vitro and subsequently cultured for 6 days. In the fused groups, female pronucleus (FPN) formation rates were higher than that in the DO24 group after parthenogenetic activation (PA); however, the blastocyst rates were intermediate between those of the control and DO24 groups. After in vitro fertilization, the male pronucleus (MPN) formation rates in the fused groups were similar to that in the control group and higher than that in the DO24 group; the normal fertilization rate in the DO24+2C group was higher than that in the DO24 group and similar to that in the control group, resulting in significantly higher blastocyst rates in the DO24+2C and control groups than that in the DO24 group. These results suggest that centri-fusion using ooplasm from fully matured DO46 oocytes can offer a potentially novel approach for restoration of cytoplasmic maturity to oocytes with low developmental ability and subsequent improvement of fertilization and developmental competence.
Journal of Reproduction and Development 06/2011; 57(5):620-6. · 1.46 Impact Factor
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ABSTRACT: The reproductive ability, milk-producing capacity, survival time and relationships of these parameters with telomere length were investigated in 4 groups of cows produced by somatic cell nuclear transfer (SCNT). Each group was produced using the same donor cells (6 Holstein (1H), 3 Holstein (2H), 4 Jersey (1J) and 5 Japanese Black (1B) cows). As controls, 47 Holstein cows produced by artificial insemination were used. The SCNT cows were artificially inseminated, and multiple deliveries were performed after successive rounds of breeding and conception. No correlation was observed between the telomere length and survival time in the SCNT cows. Causes of death of SCNT cows included accidents, accident-associated infections, inappropriate management, acute mastitis and hypocalcemia. The lifetime productivity of SCNT cows was superior to those of the controls and cell donor cows. All SCNT beef cows with a relatively light burden of lactation remained alive and showed significantly prolonged survival time compared with the cows in the SCNT dairy breeds. These results suggest that the lifetime productivity of SCNT cows was favorable, and their survival time was more strongly influenced by environmental burdens, such as pregnancy, delivery, lactation and feeding management, than by the telomere length.
Journal of Reproduction and Development 06/2011; 57(5):572-8. · 1.46 Impact Factor
