60 EFFECT OF CYTOPLASMIC VOLUME ON DEVELOPMENTAL COMPETENCE OF HAND-GUIDED CLONED BUFFALO (BUBALUS BUBALIS) EMBRYOS

2011 ◽  
Vol 23 (1) ◽  
pp. 135
Author(s):  
S. K. Panda ◽  
A. George ◽  
A. P. Saha ◽  
R. Sharma ◽  
N. M. Kamble ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Embryonic Stem ◽  
Cell Types ◽  
Bubalus Bubalis ◽  
Developmental Competence ◽  
Cloning Efficiency ◽  
Cleavage Rate ◽  
Fetal Fibroblasts ◽  
Blastocyst Rate

Despite recent successes in the birth of buffalo calves cloned through SCNT or hand-guided cloning (HGC), the cloning efficiency is very low in this species because of lack of information on factors that influence it. The goal of this study was to examine the effects of cytoplasmic volume on the developmental competence of cloned buffalo embryos produced by HGC. In vitro matured oocytes were stripped of their cumulus investment and zona pellucida using hyaluronidase and pronase, respectively. Protrusion cone-guided bisection of zona-free oocytes was performed to remove the nucleus. For reconstructing control HGC embryos, 2 enucleated oocytes (demi-cytoplasts) were fused with a single somatic cell. For reconstruction of embryos with lower or higher cytoplasmic volume, 1 or 3 demi-cytoplasts were fused, respectively, with the donor somatic cell. 2 different cell types, i.e. buffalo fetal fibroblasts (BFF) between passage 10 and 15 and buffalo embryonic stem cell (ESC)-like cells between passage 22 and 25 were used as nuclear donors in 2 different experiments. Data were analysed by 1-way ANOVA after arcsine transformation of percentage values. For BFF, the blastocyst rate for doublet and triplet embryos were significantly higher (P ≤ 0.01) than that for singlet embryos despite the cleavage rate for the 3 groups being similar. For the ESC-like cells, the cleavage and the blastocyst rate were significantly lower (P ≤ 0.01) for the singlet than that for the doublet embryos. The pregnancies were established only in doublet and triplet embryo groups using BFF cells and in the doublet embryo group using ESC-like cells. These results indicate that increasing the cytoplasmic volume could be helpful in improving cloning efficiency in terms of blastocyst production rate in buffaloes. Table 1.Effect of cytoplasmic volume on the developmental competence of cloned buffalo embryos This work was funded by NAIP grant C 2-1-(5)/2007 to SKS.

18 DEVELOPMENTAL COMPETENCE OF CLONED PORCINE EMBRYOS PRODUCED WITH DIFFERENT CLONING PROCEDURES

2014 ◽  
Vol 26 (1) ◽  
pp. 123
Author(s):  
Y. Liu ◽  
A. Lucas-Hahn ◽  
B. Petersen ◽  
R. Li ◽  
P. Hassel ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Nuclear Transfer ◽  
Cell Number ◽  
Culture Conditions ◽  
Developmental Competence ◽  
Cleavage Rate ◽  
Cell Numbers ◽  
Blastocyst Rate ◽  
Handmade Cloning

Two nuclear transfer (NT) techniques are routinely used to produce cloned animals, traditional cloning (TC) and handmade cloning (HMC). The TC embryos keep their zona and can be transferred at early stages, whereas HMC embryos are zona-free and must be cultured to the morula/blastocyst stage before transfer. Some studies have shown that in vitro culture reduces embryo development and quality, but it is not known whether embryos produced by TC or HMC differ because of the NT method or the in vitro culture. Therefore, we investigated the developmental competence and histone acetylation (H3K18ac) of porcine NT embryos produced by TC and HMC with (Day 5 and 6) or without (Day 0) in vitro culture. Nuclear transfer experiments were performed on same day (Day 0), using same batch of porcine oocytes and donor cells and same in vitro culture conditions. Cloning procedures were previously described (TC : Cloning Stem Cells 10 : 355; HMC : Zygote 20 : 61). Parthenogenetically activated embryos (PA) were used as control of activation and culture conditions. Embryos from all groups were collected for immunostaining of H3K18ac on Days 0, 5, and 6. The normalized H3K18ac level was calculated as previously described (Epigenetics 6 : 177). Cell numbers per blastocyst in each group were counted on Days 5 and 6. The cleavage rate (Day 2) and blastocyst rates (Days 5 and 6) between groups were analysed by Chi-squared test, whereas cell number per blastocysts and H3K18ac level between groups and days were analysed by ANOVA (SAS version 9.2; SAS Institute Inc., Cary, NC, USA). Cleavage rate of HMC embryos was lower than that of TC embryos, but blastocyst rate and cell number per blastocyst were higher in the HMC group compared with TC (Table 1). Differences of H3K18ac level between HMC, TC, and PA groups were only observed on Day 6 but not on Day 0 or Day 5. Within HMC and TC groups, there was no difference in H3K18ac level between Day 0 and Day 5, but the level was lower on Day 6 compared with Day 5 in the HMC group, whereas the TC group displayed the opposite pattern. In conclusion, NT embryos produced by HMC show higher blastocyst rate and cell number per blastocyst compared with TC embryos. Both in vitro culture and the NT method result in differences of the normalized H3K18ac levels. Further study is needed to investigate putative differences between NT embryos produced by HMC and TC compared to in vivo embryos also after transfer to recipients. Table 1.Cleavage and blastocyst rate, cell numbers, and normalized H3K18ac level for handmade cloning (HMC), traditional cloning (TC), and parthenogenetically activated (PA) embryos1

60 IMPROVEMENT IN EFFICIENCY AND QUALITY OF CLONED AND PARTHENOGENETIC PORCINE EMBRYOS BY AMINO ACIDS

2007 ◽  
Vol 19 (1) ◽  
pp. 147
Author(s):  
H. T. Lee ◽  
J. M. Jang ◽  
S. H. Lee ◽  
M. K. Gupta
Keyword(s):  
Amino Acids ◽  
Culture Media ◽  
Cell Number ◽  
Control Group ◽  
In Vitro Production ◽  
Cleavage Rate ◽  
Fetal Fibroblasts ◽  
Blastocyst Rate

In vitro production of cloned porcine embryos by somatic cell nuclear transfer (SCNT) has become routine in several laboratories but the efficiency and quality of the resultant blastocysts remains sub-optimal. Cloned porcine blastocysts show low cell number, high fragmentation rate, and apoptosis which results in lower pregnancy rates upon embryo transfer. Earlier we reported that supplementation of culture media with amino acids benefit pre-implantation embryo development of in vivo- as well as in vitro-fertilized porcine embryos (Koo et al. 1997 Theriogenology 48, 791–802). This study evaluated how exogenous amino acids could affect pre-implantation development and quality of cloned or parthenogenetic porcine embryos. The effects of commercially available amino acids, referred to as Eagle's non-essential amino acids (NEAA), added or not added (control) to NCSU23 medium containing fatty acid-free BSA were studied. Oocytes recovered from abattoir-derived prepubertal porcine ovaries were matured in vitro and parthenogenetically activated (PA) or nuclear-transferred with fetal fibroblasts (SCNT), as described earlier (Uhm et al. 2000 Mol. Reprod. Dev. 57, 331–337). At 168 h post-activation, blastocysts were harvested for assessment of embryo quality by TUNEL labeling, Hoechst 33342 staining, and gene expression analysis. Results showed that, in the PA group, the cleavage rate was not affected by the supplementation of NEAA. However, the blastocyst rate was significantly improved when NEAA was present in the medium compared to that of the control group (38.9 ± 0.3 vs. 27.5 ± 0.3%, respectively) throughout the culture period. The supplementation during the pre-compaction period alone gave better results than during the post-compaction period alone (59.5 ± 0.9 vs. 33.4 ± 0.3%, respectively). In the SCNT group, however, both cleavage (73.6 ± 0.2 vs. 64.2 ± 0.4%) and blastocyst rate (18.7 ± 0.2 vs. 13.8 ± 0.3%) were improved by NEAA supplementation. Furthermore, these blastocysts had higher hatching ability (30.0 ± 1.8 vs. 14.6 ± 4.9%) than those of control group (P < 0.05). Supplementation of NEAA also increased the mean nuclei number of PA-derived (76.1 ± 4.9 vs. 66.5 ± 3.3) as well as SCNT-derived (43.1 ± 2.6 vs. 31.8 ± 1.9) blastocysts and reduced the time during which blastocysts formed. TUNEL assay revealed that incidence of nuclear fragmentation and apotosis was reduced by NEAA. Real-time qRT-PCR for Bax and Bcl-XL transcripts revealed that the relative abundance of Bax was reduced while that of Bcl-XL was increased. These effects were more pronounced when NEAA was present during the pre-compaction period alone. Thus, our data suggest that NEAA improves the yield and quality of cloned porcine embryos by enhancing blastocyst expansion and positively modulating the total cell number and apoptosis. These data may have implications for understanding the nutritional needs of cloned porcine embryos produced in vitro and for optimizing the composition of culture media to support their development. This work was supported by the Research Project on the Production of Bio-Organs (No. 200503030201), Ministry of Agriculture and Forestry, Republic of Korea.

53 EFFECTS OF INSULIN-TRANSFERRIN-SELENIUM IN DEFINED AND PORCINE FOLLICULAR FLUID-SUPPLEMENTED MEDIUM ON IN VITRO PRODUCTION OF PORCINE SCNT EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 144
Author(s):  
Y. U. Kim ◽  
D. P. Bhandari ◽  
M. S. Hossein ◽  
S. M. Park ◽  
E. Lee ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Defined Medium ◽  
Culture Conditions ◽  
Developmental Competence ◽  
Differential Staining ◽  
In Vitro Production ◽  
Fetal Fibroblasts

Insulin promotes the uptake of glucose and amino acids, and is beneficial for maturation of oocytes in vitro. Transferrin is an iron-transport protein and selenium is an essential trace element. Insulin-transferrin-selenium (ITS) together has been used in some in vitro maturation systems. The present study was designed to evaluate the effects of ITS in defined and porcine folicular fluid (pFF)-supplemented IVM medium on the glutathione (GSH) concentration, and on developmental competence after somatic cell nuclear transfer. ITS liquid media supplement (I-3146) was purchased from Sigma-Aldrich (St Louis, MO, USA). Basic IVM medium was TCM-199 supplemented with 10 ng mL-1 epidermal growth factor, 4 IU mL-1 pregnant mare serum gonadotropin (PMSG) and hCG and either 1% PVA (defined medium) or 10% pFF. Ten �g mL-1 insulin, 5.5 �g mL-1 transferrin, and 5 �g mL-1 selenium was used for the entire 44-h culture period. The GSH content of a gruop of 10 to 20 oocytes was determined by the dithionitrobezoic acid-glutathione disulfide (DTNB-GSSG) reductase recycling assay. Fetal fibroblasts were used as somatic cell donors and reconstructed embryos were cultured in mNCSU-23 medium for 168 h. Cleavage and blastocyst formation was observed at 48 h and 168 h, respectively. The quality of blastocysts was assessed by differential staining of the inner cell mass (ICM) and the trophectoderm (TE) cells. Each experiment was replicated for 5 times. The data were analyzed by one-way ANOVA, and Tukey was used as a posthoc test. The level of GSH production significantly varied in different culture conditions. The highest GSH concentration was observed in the pFF + ITS group (8.2 picomol/oocyte). A total of 116, 125, 126, and 120 reconstructed oocytes were cultured, and 10.1, 15.3, 17.2, and 21.8% blastocysts were observed for PVA, PVA + ITS, pFF, and pFF + ITS groups, respectively (P < 0.05). The numbers of inner cell mass, trophrectoderm cells, and total cells were significantly higher in the pFF + ITS group compared with the other groups. The average number of total cells in blastocysts was 31.9 � 1.8, 43.1 � 3.5, 46.7 � 4.9, and 52.3 � 6.7 for PVA, PVA + ITS, pFF, and pFF + ITS groups, respectively (P < 0.05). ITS supplement improved the developmental competence in both the defined and the pFF supplemented groups. We recommend supplementing porcine IVM medium with 10 �g mL-1 insulin, 5.5 �g mL-1 transferrin, and 5 �g mL-1 selenium.

Insulin influences developmental competence of bovine oocytes cultured in α-MEM plus follicle-simulating hormone

Zygote ◽  
2014 ◽  
Vol 23 (4) ◽  
pp. 563-572 ◽  
Author(s):  
Gustavo Bruno Mota ◽  
Ingrid Oliveira e Silva ◽  
Danielle Kaiser de Souza ◽  
Flavia Tuany ◽  
Michele Munk Pereira ◽  
...  
Keyword(s):  
Basal Medium ◽  
Developmental Competence ◽  
Cleavage Rate ◽  
Serum Free ◽  
Oocyte Competence ◽  
Nuclear Maturation ◽  
Blastocyst Rate ◽  
Defined Culture ◽  
Bovine Oocytes

SummaryThe aim of this study was to evaluate the dose–response effect of insulin, plus follicle-simulating hormone (FSH) at a fixed concentration, in a serum-free defined culture medium (DCM) on the in vitro maturation of bovine cumulus–oocyte complexes (COCs). For oocyte nuclear maturation, the expression levels of GDF9, GLUT1, PRDX1 and HSP70.1 transcripts related to oocyte and embryo developmental competence were analysed. For in vitro maturation (IVM), cumulus–oocyte complexes from slaughterhouse ovaries were distributed into four groups based on insulin concentration added to serum-free DCM, which was composed of alpha minimum essential medium (α-MEM), as basal medium: (1) DCM control: 0 ng/ml; (2) DCM1: 1 ng/ml; (3) DCM10: 10 ng/ml; and (4) DCM100: 100 ng/ml. After IVM, the nuclear status of a sample of oocytes was analysed and the other oocytes were submitted for in vitro fertilization (IVF) and in vitro culture (IVC). Different concentrations of insulin did not affect significantly the nuclear maturation and cleavage rate (72 h post-insemination) across all groups. Blastocyst rate (192 h post-insemination) did not differ in DCM control (24.3%), DCM1 (27.0%) and DCM10 (26.3%) groups, but the DCM100 (36.1%) group showed a greater blastocyst rate (P < 0.05) than the DCM control. Insulin concentrations of 1, 10, or 100 ng/ml decreased the relative levels of GDF9 and HSP70-1 transcripts in oocytes at the end of IVM (P < 0.05). The transcripts levels of PRDX1 decreased (P < 0.05) only when 10 or 100 ng/ml insulin was added to the DCM medium. No difference in levels of GLUT1 transcripts (P > 0.05) was observed at the different insulin concentrations. The results indicated that insulin added to DCM influenced levels of transcripts related to cellular stress (HSP70-1 and PRDX1) and oocyte competence (GDF9) in bovine oocytes and at higher concentrations enhanced blastocyst production.

Co-culture of buffalo (Bubalus bubalis) preantral follicles with antral follicles: a comparative study of developmental competence of oocytes derived from in vivo developed and in vitro cultured antral follicles

Zygote ◽  
2012 ◽  
Vol 21 (3) ◽  
pp. 286-294 ◽  
Author(s):  
G. Taru Sharma ◽  
Pawan K. Dubey ◽  
Amar Nath ◽  
G. Saikumar
Keyword(s):  
Growth Factor ◽  
Bubalus Bubalis ◽  
Developmental Competence ◽  
Cleavage Rate ◽  
Term Survival ◽  
Preantral Follicles ◽  
Antral Follicles ◽  
Epidermal Growth

SummaryThe present study was undertaken to examine whether the presence of antral follicles (AFs) affects the survival, growth and steroidogenesis of preantral follicles (PFs) and compare the maturation and developmental competence of buffalo oocytes derived from in vivo developed and in vitro cultured AFs. Two experiments were carried out. In experiment I, PFs (200–250 μm) were isolated and cultured with or without AFs (3–5 mm) in TCM-199 medium that contained 10% fetal bovine serum (FBS), 1% insulin transferin selenium (ITS), 20 ng/ml epidermal growth factor (EGF), 0.5 μg/ml follicle-stimulating hormone (FSH) and 100 ng/ml insulin-like growth factor (IGF)-I. In experiment II, in vitro developmental competence was compared for the cumulus–oocyte complexes (COCs) recovered from in vivo developed and in vitro cultured AFs. Survival, growth, development of antrum, accumulation of estradiol and progesterone was (P < 0.05) higher when PFs were co-cultured with AFs. Developmental competence of both types of follicular oocytes did not differ significantly in terms of maturation and cleavage rate, but morula and blastocyst production rate were (P < 0.05) higher with in vivo developed AFs as compared with the in vitro cultured antral follicular oocytes. In conclusion, co-culture of PFs with AFs supports long-term survival and growth of buffalo PFs and this co-culture system plays a dual role for in vitro production of embryos as well as understanding the relationship between developing PFs and AFs.

Development and spindle formation in rat somatic cell nuclear transfer (SCNT) embryos in vitro using porcine recipient oocytes

Zygote ◽  
2009 ◽  
Vol 17 (3) ◽  
pp. 195-202 ◽  
Author(s):  
Atsushi Sugawara ◽  
Satoshi Sugimura ◽  
Yumi Hoshino ◽  
Eimei Sato
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Fluorescent Protein ◽  
Cumulus Cells ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Spindle Formation ◽  
Fetal Fibroblasts

SummaryCloning that uses somatic cell nuclear transfer (SCNT) technology with gene targeting could be a potential alternative approach to obtain valuable rat models. In the present study, we determined the developmental competence of rat SCNT embryos constructed using murine and porcine oocytes at metaphase II (MII). Further, we assessed the effects of certain factors, such as: (i) the donor cell type (fetal fibroblasts or cumulus cells); and (ii) premature chromosome condensation (PCC) with normal spindle formation, on the developmental competence of rat interspecies SCNT (iSCNT) embryos. iSCNT embryos that had been constructed using porcine oocytes developed to the blastocyst stage, while those embryos made using murine MII oocytes did not. Rat iSCNT embryos constructed with green fluorescent protein (GFP)-expressing fetal fibroblasts injected into porcine oocytes showed considerable PCC with a normal bipolar spindle formation. The total cell number of iSCNT blastocyst derived from GFP-expressing fetal fibroblasts was higher than the number derived from cumulus cells. In addition, these embryos expressed GFP at the blastocyst stage. This paper is the first report to show that rat SCNT embryos constructed using porcine MII oocytes have the potential to develop to the blastocyst stage in vitro. Thus the iSCNT technique, when performed using porcine MII oocytes, could provide a new bioassay system for the evaluatation of the developmental competence of rat somatic cells.

351 POLARIZED LIGHT MICROSCOPY: DETECTION OF MICROTUBULES AND ITS EFFECTS ON THE VIABILITY OF IN VITRO-MATURED PORCINE OOCYTES

2010 ◽  
Vol 22 (1) ◽  
pp. 332 ◽  
Author(s):  
I. Molina ◽  
M. Muñoz ◽  
C. Díez ◽  
E. Gómez ◽  
E. A. Martínez ◽  
...  
Keyword(s):  
Light Microscopy ◽  
Polarized Light ◽  
Polarized Light Microscopy ◽  
Developmental Competence ◽  
Meiotic Spindle ◽  
Cleavage Rate ◽  
Cell Numbers ◽  
Oocyte Developmental Competence ◽  
Blastocyst Rate

The meiotic spindle in the oocyte is composed of microtubules and plays an important role during chromosome alignment and separation at meiosis. Polarized light microscopy (PLM) is used as a tool in human and, recently, in farm animals assisted reproductive technologies. PLM could be useful for a non-invasive evaluation of the meiotic spindle. The objectives of the present study were to assess the efficiency of PLM to detect microtubule-polymerized protein within in vitro-matured porcine oocytes and to examine the effects of PLM on the oocyte developmental competence. Cumulus-oocyte complexes from slaughterhouse ovaries were matured in vitro for 42 h as described by Gil et al. (2004 Theriogenology 62, 544-552). In the first experiment, a total of 97 oocytes from 6 replicates were placed individually in 10-μL drops of TCM-199-Hepes-FCS in a glass Petri dish. PLM was used to detect the presence of polymerized protein which could be forming a meiotic spindle. The presence of polymerized protein and a meiotic spindle was confirmed in individual oocytes by inmunostaining and chromatin detection as described by Morató et al. (2008 Mol. Reprod. Dev. 75, 191-201). In the second experiment, a total of 160 oocytes from 4 replicates were exposed or not (controls) to PLM for 10 minutes. Thereafter, the oocytes were parthenogenetically activated and cultured in vitro. Cleavage rate, total blastocyst rate, expanded blastocyst rate on Day 7 and total cell numbers in expanded blastocysts were assessed. Data were analyzed by GLM procedure of SAS. There was a positive correlation (r = 1; P < 0.0001) between the signal obtained by PLM and the presence of microtubule-polymerized protein as confirmed by inmunostaining. A positive PLM signal was detected in 98.9% of the oocytes. A barrel-shape spindle was observed in 94.8% of the individual samples by inmunostaining and all of these oocytes were positive to PLM. Moreover, oocytes exposed to PLM did not differ significantly from controls on cleavage rate (83.7 ± 1.5 v. 84.4 ± 1.5), total blastocyst rate (36.9 ± 3.6 v. 41.2 ± 3.6) and expanded blastocyst rate on Day 7 (21.9 ± 1.7 v. 26.2 ± 1.7), respectively. There were also no differences in total cell numbers counted in expanded blastocysts (32.8 ± 2.6 v. 35.6 ± 2.5). These results indicate that polarized light microscopy did not exert detrimental effects on porcine oocyte developmental competence and it seems an efficient system to detect polymerized protein in in vitro-matured porcine oocytes. Grant support: INIA: RZ2007-00013-00-00. I. Molina, M. Muñoz, B. Trigal and D. Martín are sponsored by INIA, RYC08-03454, Cajastur and PTA2007-0268-I, respectively.

49 IMPROVED PORCINE CLONING EFFICIENCY WITH CELLS CULTURED FOR SEVERAL GENERATIONS AFTER A SINGLE TREATMENT WITH XENOPUS EGG EXTRACT

2011 ◽  
Vol 23 (1) ◽  
pp. 130
Author(s):  
Y. Liu ◽  
O. Østrup ◽  
J. Li ◽  
G. Vajta ◽  
P. M. Kragh ◽  
...  
Keyword(s):  
Developmental Competence ◽  
Cloning Efficiency ◽  
Blastocyst Development ◽  
Cleavage Rate ◽  
Chi Square ◽  
Xenopus Egg Extract ◽  
Transcriptional Reprogramming ◽  
Egg Extract ◽  
Fetal Fibroblasts ◽  
Chi Square Test

Extracts from eggs of Xenopus laevis frogs can induce nuclear remodelling or increase transcriptional reprogramming in somatic cells. However, it is not known if this effect is passed on from one cell generation to another, or how it affects somatic cell nuclear transfer in porcine cells. This study aimed to investigate the effect of extract-treated cells over several generations on porcine cloning. Extracts were prepared from 2 frogs (B1 and B2) by the same protocol (Higa et al. 2006 Methods 39, 284–290). Fetal fibroblasts grown on poly-L-lysine coated coverslips were permeabilized by digitonin (7 μg mL–1, 2 min, 4°C) and incubated with 1 extract batch at 37°C for 30 min. After resealing the membrane in DMEM supplemented with 2 mM CaCl2 at 37°C for 2 h, the remaining cells were cultured in ES medium (Vejlsted et al. 2005 Mol. Reprod. Dev. 70, 445–454) for 7 to 8 days when they formed colonies. The colonies were trypsinized and divided onto 2 coverslips for subculture, defined as Experimental Passage 1 (XP1). New subcultures were made every 7 to 8 days when 70 to 80% clusters become colonies until XP15. Colonies from XP3, 8 and 15 were isolated and trypsinized before being used in handmade cloning. Nontreated cells grown in DMEM were used as controls (no colony formation was observed). On each cloning day, cells from different XP number and controls were used. Rates of cleavage (Day 2) and blastocyst development (Day 6) were analysed with chi-square test (SAS version 9.2, SAS Institute Inc., Cary, NC, USA). Results are summarised in Table 1. No difference was observed in cleavage rate between groups. Blastocyst rates of all XP colony cells were significantly higher than their controls. For the same XP number and their controls, blastocyst rates were similar between the colony cells from the 2 extract batches, and there was no difference between their controls, either. In conclusion, the cloning efficiency in porcine cells could be increased with extract-treated cells used for several generations, and this effect was present at XP3, 8, and 15. Table 1.Developmental competence of cloned porcine embryos with extract-treated cells from different batches of extract (B1 and B2) and Experimental Passage (XP) numbers

55 Donor age has the least influence on recovery, quality, and in vitro developmental competence of ovum pickup–based Holstein Friesian oocytes under subtropical conditions

2021 ◽  
Vol 33 (2) ◽  
pp. 134
Author(s):  
M. Yaseen ◽  
M. Saleem ◽  
M. Nawaz ◽  
N. Ahmad ◽  
A. Riaz
Keyword(s):  
Transvaginal Ultrasound ◽  
Developmental Competence ◽  
Donor Age ◽  
Adult Group ◽  
Cleavage Rate ◽  
Holstein Friesian ◽  
Frozen Semen ◽  
Chi Squared ◽  
Blastocyst Rate

The use of oocytes obtained from younger donors for invitro fertilization followed by embryo transfer represents an opportunity to accelerate genetic gain by reducing generation interval. The present study was designed to evaluate the effect of age of donor on the follicular population, recovery, quality and invitro developmental competence of ovum pickup based Holstein Friesian oocytes under subtropical conditions. A total of eight (n=8) Holstein Friesian (with proper oestrus cyclicity) were selected for the study and divided into 2 groups based on animal age: (1) heifers (n=4), 1.5 to 2 years of age, and (2) adults (n=4), 5 to 6 years of age. The study was conducted near Lahore (31°33′ N, 74°19′ E), Punjab, Pakistan, from November 2019 to February 2020. The animals were wave synchronized using the physiological method of wave synchronization. After 4 days of second dominant follicle puncture, the first ovum pickup was carried out and a total of nine (n=9) OPU sessions were held for each group. The COCs from the follicles were aspirated using a transvaginal ultrasound–guided needle. Following searching and grading, COCs of grade A, B and C were processed for IVM in 100-µL droplets of BO-IVM under mineral oil at 37°C, 5% CO2, and 95% humidity for 24h. The frozen semen of a high-pedigree bull was thawed at 37°C and observed for post-thaw sperm motility. The semen samples of the same bull having motility &gt;50% were processed using the sperm swim-up method throughout the study. The IVF was carried out by placing the COCs and required amount of sperm in 100-µL droplets of BO-IVF at similar conditions for a maximum of 18h. The presumptive zygotes were denuded by gentle pipetting and cultured for a period of 7 days after placing in 100-µL drops of BO-IVC at 37°C, 5% CO2, 5% O2, and maximum humidity. The presumptive zygotes were observed for cleavage rate and blastocyst rate on Days 2 and 7 following COCs-sperm co-incubation. Data on the follicular population, oocytes recovered, and viable oocytes were analysed by the PROC GLIMMIX of SAS (SAS Institute Inc.), and proportional data were analysed by the Chi-squared method using SAS 9.1. COCs of grade AB (35.2 vs. 25.4%) were higher (P&gt;0.05) in the adult group than in the heifer group, respectively. Similarly, COCs with grade CD (57.5 vs. 71.9%) were lower (P&lt;0.05) in the adult group compared with the heifer group, respectively. However, the total follicles (6.55±0.42 vs. 6.39±0.39), number of COCs recovered (3.33±0.32 vs. 3.17±0.41), viable oocytes (3.08±0.29 vs. 3.08±0.39), cleavage rate (60.3 vs. 68.7%), and blastocyst rate (38.7 vs. 48.8%) did not differ (P&gt;0.05) between the groups. To conclude, donor age up to third lactation, under subtropical conditions, does not affect invitro embryo production in Holstein Friesian undergoing repeated OPU.

scholarly journals Progress and Future Challenges of Human Induced Pluripotents Stem Cell in Regenerative Medicine

2011 ◽  
Vol 3 (2) ◽  
pp. 76
Author(s):  
Anna Meiliana ◽  
Andi Wijaya
Keyword(s):  
Stem Cells ◽  
Somatic Cell ◽  
Expression Profiles ◽  
Human Fibroblasts ◽  
Embryonic Stem ◽  
Cell Types ◽  
Differentiation Potential ◽  
Human Ipscs

BACKGROUND: Less than a decade ago the prospect for reprogramming the human somatic cell looked bleak at best. It seemed that the only methods at our disposal for the generation of human isogenic pluripotent cells would have to involve somatic cell nuclear transfer (SCNT). Shinya Yamanaka in August 2006 in his publication (Cell) promised to change everything by showing that it was apparently very simple to revert the phenotype of a differentiated cell to a pluripotent one by overexpressing four transcription factors in murine fibroblasts.CONTENT: Mouse and human somatic cells can be genetically reprogrammed into induced pluripotent stem cells (iPSCs) by the expression of a defined set of factors (Oct4, Sox2, c-Myc, and Klf4, as well as Nanog and LIN28). iPSCs could be generated from mouse and human fibroblasts as well as from mouse liver, stomach, pancreatic, neural stem cells, and keratinocytes. Similarity of iPSCs and embryonic stem cells (ESCs) has been demonstrated in their morphology, global expression profiles, epigenetic status, as well as in vitro and in vivo differentiation potential for both mouse and human cells. Many techniques for human iPSCs (hiPSCs) derivation have been developed in recent years, utilizing different starting cell types, vector delivery systems, and culture conditions. A refined or perfected combination of these techniques might prove to be the key to generating clinically applicable hiPSCs.SUMMARY: iPSCs are a revolutionary tool for generating in vitro models of human diseases and may help us to understand the molecular basis of epigenetic reprogramming. Progress of the last four years has been truly amazing, almost verging on science fiction, but if we can learn to produce such cells cheaply and easily, and control their differentiation, our efforts to understand and fight disease will become more accessible, controllable and tailored. Ability to safely and efficiently derive hiPSCs may be of decisive importance to the future of regenerative medicine.KEYWORDS: iPSCs, ESC, reprogramming factor, reprogramming efficiency, somatic cell

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