151 COMPARISON OF CELL NUMBERS OF ZONA-INTACT AND ZONA-FREE PARTHENOGENETICALLY ACTIVATED PORCINE EMBRYOS CULTURED IN VITRO

2010 ◽  
Vol 22 (1) ◽  
pp. 234 ◽  
Author(s):  
J. Li ◽  
G. Vjata ◽  
H. Callesen
Keyword(s):  
In Vitro Culture ◽  
Zona Pellucida ◽  
Cumulus Cell ◽  
Cell Number ◽  
Culture Conditions ◽  
Developmental Competence ◽  
Fluorescent Light ◽  
Average Cell ◽  
Cell Numbers

Application of an artificial stimulus to activate oocytes and induce development is essential for the successful animal cloning by nuclear transfer (Zhu et al. 2002 Biol. Reprod. 66, 635-641). The embryo’s developmental competence could be further improved with optimal in vitro culture conditions (Du et al. 2007 Theriogenology 68, 1104-1110). Cell number determination is a commonly used and simple criterion to assess developmental quality of pre-implantation stage mammalian embryos (Lagutina et al. 2007 Theriogenology 67, 90-98). Our aim of the study was to investigate porcine embryos activated and cultured in different ways using total cell numbers as the only quality measure. After 43-44 h of in vitro maturation and cumulus cell removal, zona-intact (PAZI) or zona-free oocytes (PAZF; after pronase treatment) were subjected to parthenogenetic activation (Day 0) with a single 80-μs DC pulse of 1.26 kV cm-1 or 0.86 kV cm-1 (Kragh et al. 2005 Theriogenology 64, 1536-1545), followed by a 4-h treatment with 5 μg mL-1 of cytochalasin B and 10 μg mL-1 of cycloheximide. Subsequently, the well of the well system (Vajta in vitro 2000 Mol. Reprod. Dev. 55, 256-264) was used for culture of all PAZF and half of the PAZI embryos (PAZF-WOW and PAZI-WOW groups, respectively), whereas the remaining PAZI embryos were cultured in groups of 25-30 (PAZI group). All cultures were performed in porcine zygote medium 3 (Yoshioka et al. 2002 Biol. Reprod. 66, 112-119). On Day 6, all these in vitro cultured embryos were fixed and stained with Hoechst 33342 and cell numbers were counted on the microscopic pictures taken using fluorescent light. Data analysis was performed using ANOVA. Four replicates were performed with a total of 462 PAZF-WOW, 484 PAZI-WOW, and 467 PAZI group embryos. Embryos of each group were then divided into 5 groups based on their cell number (<5 cells, 5-8 cells, 9-16 cells, 17-32 cells, >32 cells). Percentages of embryos in each group are shown in Table 1. The average cell numbers of zona-intact embryos from PAZI-WOW and the PAZI group were similar to each other (P > 0.05), whereas the cell numbers of PAZF-WOW embryos were significantly different from both PAZI-WOW and PAZI group embryos (P < 0.05), with more embryos having higher cell numbers.The results demonstrate that zona-free parthenogenetically activated embryos cultured in WOW have higher cell numbers than embryos with intact zona pellucida. Accordingly, the presence of zona pellucida may compromise embryo development under certain in vitro culture situations. Table 1.Distribution (in %) of parthenogenetically activated porcine embryos according to their cell number on Day 6 after activation

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

15 HISTONE ACETYLATION PROFILE OF PORCINE EMBRYOS PRODUCED BY 2 CLONING METHODS WITH OR WITHOUT IN VITRO CULTURE

2016 ◽  
Vol 28 (2) ◽  
pp. 137
Author(s):  
Y. Liu ◽  
A. Lucas-Hahn ◽  
B. Petersen ◽  
R. Li ◽  
D. Hermann ◽  
...  
Keyword(s):  
Gene Expression ◽  
In Vitro Culture ◽  
Histone Acetylation ◽  
Mrna Expression ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Number ◽  
Cell Numbers ◽  
Cloning Method

Conventional “Dolly”-based cloned (CNT) embryos maintain zona pellucida and can be transferred early in development. Handmade cloned (HMC) embryos are zona free and are cultured to later stages for transfer. We have shown differences between HMC and CNT embryos (Rep. Fert. Dev. 26, 123), and both in vitro culture and cloning method (NT) are associated with alterations in histone acetylation. More studies are needed to clarify whether CNT and HMC embryos differ in epigenetic profiles due to NT method or culture condition. Here we investigated histone acetylation profile of NT embryos produced by CNT or HMC with or without 5 to 6 days in vitro culture, emphasising quality and gene expression in resulting embryos. Both NT methods were performed on Day 0 (D0) with same oocyte batch, donor cells, and culture medium (CNT in group, HMC in well of well). On D0, 5, and 6 after CNT (Clon. Stem Cells 10, 355) or HMC (Zygote 20, 61), all developed embryos of all morphological qualities were collected for immunostaining of H3K18ac, and on D0 and 6 for mRNA expression of the genes KAT2A/2B, EP300, HDAC1/2, DNMT1o/s, and GAPDH. Embryo quality was evaluated normal (clear inner cell mass, high cell number, no fragments) or bad (no clear inner cell mass, low cell number, fragments). Cell numbers per blastocyst were counted on D5 and 6. Differences in cell number and H3K18ac level between different groups and days were analysed by ANOVA; gene expression data were analysed by GLM (SAS version 9.3, SAS Institute Inc., Cary, NC, USA). Embryo development rates of both NT methods were reported previously (Rep. Fert. Dev. 26, 123). On D5 and 6, all HMC embryos were evaluated as normal, but the CNT group contained both normal and bad embryos. Regarding cell numbers (Table 1), on D5 there was no difference between normal CNT and HMC embryos, but numbers were lower in CNT bad embryos. On D6 the blastocyst cell number was lower in both normal and bad CNT embryos compared with HMC. Regarding H3K18ac levels (Table 1), no differences were found on D5 between normal CNT and HMC embryos, but on D6 both CNT normal and bad embryos had higher H3K18ac level compared with HMC. On D0, no difference was found in mRNA expression of all 8 genes. On D6, KAT2A expression was slight increased (1.8-fold) in CNT compared with HMC embryos (P < 0.05). In conclusion, no differences were found between CNT and HMC embryos after completed NT procedure (D0) or after 5 days in vitro culture. However, differences in quality (cell number and H3K18ac) and gene expression between the 2 NT methods were observed when blastocyst expansion was initiated (D6). Thus, the 2 NT methods seem to produce embryos of similar quality, which is maintained over 5 days in vitro culture, but thereafter gene expression and histone acetylation are more active in CNT embryos. Table 1.Cell number and H3K18ac level1

scholarly journals 301EFFECT OF THE MII-AGE AND ACTIVATION PROTOCOL ON THE PARTHENOGENETIC DEVELOPMENT OF PORCINE OOCYTES

2004 ◽  
Vol 16 (2) ◽  
pp. 270
Author(s):  
I. Lagutina ◽  
G. Lazzari ◽  
C. Galli
Keyword(s):  
Cytochalasin B ◽  
Average Rate ◽  
Polar Body ◽  
Cell Number ◽  
Developmental Competence ◽  
Blastocyst Formation ◽  
Average Cell ◽  
Electric Impulse ◽  
Polar Body Extrusion

The completion of porcine oocyte nuclear maturation (MII) in vitro, characterized by the time of polar body extrusion, starts at about 32h of maturation and lasts more than 12h. This leads to the simultaneous presence in the population of matured oocytes with differing abilities to be activated. We investigated age-dependent changes in pig oocyte maturation, activation and development in SOFaa in response to electric impulse (EL) in the presence of cytochalasin B (CB) and EL in combination with cycloheximide and cytochalasin B (EL+CHX+CB). Oocytes were matured in TCM 199 with 10% FCS, cysteine, LH, FSH (Pergovet, Serono, Geneva, Switzerland) for 36h and then decumulated. Matured oocytes were activated at 40 and 44h by double pulse of 30μs DC 1, 5kVcm−1 and cultured in 5μgmL−1 CB for 4h or by EL followed by incubation in 10μgmL−1 CHX+5μgmL−1 CB for 4h. According to the MII-age before activation oocytes were divided into 2 age classes: 3–7 and 7–11h after polar body extrusion. Embryos were cultured in SOFaa in 5% CO2, 5% O2 at 38.5°C. The rates of cleavage, blastocyst formation and cell number of BL on Day 7 (BLD7) were recorded. Our results showed that the average rate of maturation at 44h was 72% (n=1377). About 50% and 87% of oocytes, that eventually matured, extruded the polar body at 37 and 40h, respectively. The average cell number of BLD7 developed in SOFaa was 80±36 (n=52) and was not affected by activation protocol. Seventy-nine and 27% of BL had more than 50 and 100 cells per BL, respectively. Porcine oocytes activated by EL acquired their developmental competence gradually, achieving the highest rates of cleavage and blastocyst formation 7h after polar body extrusion. By contrast, oocytes activated by EL+CHX+CB showed their maximal developmental competence earlier (3–7h group). In conclusion, we demonstrate that electric impulse in combination with CHX+CB treatment permits earlier efficient activation of porcine oocytes (3–7h after polar body extrusion).

154. EXTRACELLULAR CALRETICULIN ALTERS ENDOTHELIAL CELL AND TROPHOBLAST CELL FUNCTIONS IN VITRO CONSISTENT WITH PRE-ECLAMPSIA

2010 ◽  
Vol 22 (9) ◽  
pp. 72
Author(s):  
N. M. Gude ◽  
K. E. Crawford ◽  
J. L. Stevenson ◽  
S. P. Brennecke
Keyword(s):  
Endothelial Cell ◽  
In Vitro Culture ◽  
Cell Number ◽  
Trophoblast Cell ◽  
Human Trophoblast ◽  
Migratory Activity ◽  
Cell Numbers ◽  
Cell Functions ◽  
Normotensive Pregnancy

Pre-eclampsia is a multisystem disorder of human pregnancy that involves abnormal placentation via insufficient trophoblast cell invasion of the maternal spiral arteries and widespread maternal endothelial cell dysfunction. Factors in plasma of pre-eclamptic women affect both trophoblast and endothelial cell functions during in vitro culture (1). The calcium-binding protein calreticulin is elevated in peripheral blood with pre-eclampsia compared to normotensive pregnancy (2). The aim of this study was to determine the effects of exogenous calreticulin at concentrations relevant to normotensive pregnancy (2 µg/mL) and to pre-eclampsia (5 µg/mL) on human trophoblast cell (HTR8) and microvascular endothelial cell (myometrial) numbers and migratory activity. Cell migration was measured by scratch assay; changes in cell number were measured by MTS assay (Promega). The results showed that calreticulin at 5µg/mL did not affect HTR8 cell number (control 68044+24542 cells, with calreticulin 72810 + 30673 cells, n = 3, P > 0.05) after 48 hours, but significantly inhibited migration of the cells by 48+11% compared to the control at 26 hours (n = 4, P < 0.02). Calreticulin at 5 µg/mL and under conditions that did not change cell number significantly increased migration of the myometrial endothelial cells by 39+7% (n = 4, P < 0.01) at 20 hours. Calreticulin at 5 µg/mL, however, significantly reduced endothelial cell numbers after 3–5 days (control 6213 + 1937 cells, with calreticulin 1937+728 cells, n = 6, P < 0.05). There was no significant change to the functions of either cell type with 2 µg/mL of calreticulin. In conclusion, exogenous calreticulin at a concentration consistent with that found in maternal blood with pre-eclampsia was shown to alter trophoblast and endothelial cell migratory activity and reduce endothelial cell numbers during in vitro culture. These results indicate that elevated circulating calreticulin may contribute to the cellular mechanisms that underlie the development of pre-eclampsia. (1) Harris et al, Reprod Sci, 2009, 16: 1082–90.(2) Gu et al, Molec Human Repro, 2008, 14: 309–15.

scholarly journals Modified Spirulina maxima Pectin Nanoparticles Improve the Developmental Competence of In Vitro Matured Porcine Oocytes

Animals ◽  
2021 ◽  
Vol 11 (9) ◽  
pp. 2483
Author(s):  
Pantu-Kumar Roy ◽  
Ahmad-Yar Qamar ◽  
Bereket-Molla Tanga ◽  
Seonggyu Bang ◽  
Gyeonghwan Seong ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Number ◽  
Culture Conditions ◽  
Developmental Competence ◽  
Spirulina Maxima ◽  
Blastocyst Development ◽  
Parthenogenetic Activation ◽  
Molecular Approaches

Molecular approaches have been used to determine metabolic substrates involved in the early embryonic processes to provide adequate culture conditions. To investigate the effect of modified Spirulina maxima pectin nanoparticles (MSmPNPs) on oocyte developmental competence, cumulus–oocyte complexes (COCs) retrieved from pig slaughterhouse ovaries were subjected to various concentrations of MSmPNPs (0, 2.5, 5.0, and 10 µg/mL) during in vitro maturation (IVM). In comparison to the control, MSmPNPs-5.0, and MSmPNPs-10 groups, oocytes treated with 2.5 µg/mL MSmPNPs had significantly increased glutathione (GSH) levels and lower levels of reactive oxygen species (ROS). Following parthenogenetic activation, the MSmPNPs-2.5 group had a considerably higher maturation and cleavage rates, blastocyst development, total cell number, and ratio of inner cell mass/trophectoderm (ICM:TE) cells, when compared with those in the control and all other treated groups. Furthermore, similar findings were reported for the developmental competence of somatic cell nuclear transfer (SCNT)-derived embryos. Additionally, the relative quantification of POU5F1, DPPA2, and NDP52 mRNA transcript levels were significantly higher in the MSmPNPs-2.5 group than in the control and other treated groups. Taken together, the current findings suggest that MSmPNP treatment alleviates oxidative stress and enhances the developmental competence of porcine in vitro matured oocytes after parthenogenetic activation and SCNT.

87 EFFECTS OF HUMAN RECOMBINATION GRANULOCYTE–COLONY STIMULATING FACTOR (hrG-CSF) ON IN VITRO CULTURE OF PORCINE CLONED EMBRYOS DERIVED FROM THIN CUMULUS CELL LAYER OF OOCYTES MATURED IN VITRO

2017 ◽  
Vol 29 (1) ◽  
pp. 151
Author(s):  
L. Cai ◽  
E. O. Park ◽  
Y.-X. Jin ◽  
K.-C. Hwang ◽  
Y. W. Jeong ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Cell Layer ◽  
Cumulus Cell ◽  
Developmental Competence ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Blastocyst Formation ◽  
Transcript Levels ◽  
Stimulating Factor

Although several cloned pigs have been successfully produced, the developmental competence of cloned embryos in vitro is still very low. Granulocyte colony-stimulating factor receptor (G-CSFR) was founded in the human trophoblastic cell line that is implicated in regulation and proliferation of trophoblast. In the present study, the somatic cell NT embryos derived from oocytes that have more than 3 cumulus cells layer were cultured and supplemented with various concentrations of hrG-CSF (0, 10, 50, and 100 ng mL−1, respectively). Although there were no significant effects on the various concentration of hrG-CSF treatment groups compared with control, the somatic cell NT blastocysts formation tended to increase after 10 ng mL−1 hrG-CSF treatment (24.19 ± 2.90%) compared with control (21.37 ± 2.98%). Moreover, we investigated the effects of 10 ng mL−1 hrG-CSF on in vitro culture of porcine cloned embryos derived from oocytes that were categorized into grade A (cumulus cell layer >10), grade B (10 > cumulus cell layer ≥ 3), and grade C (cumulus cell layer <3). After supplementation of 10 ng mL−1 hrG-CSF on in vitro-culture of different groups, the developmental competence, blastocyst quality, and gene transcript levels were observed. The results showed that 10 ng mL−1 hrG-CSF has no beneficial effects on cloned embryos derived from grade A oocytes (10 ng mL−1 hrG-CSF 25.35 ± 2.53% v. control 25.00 ± 2.66%), but it significantly increased blastocyst formation of embryos derived from grade B oocytes (22.09 ± 2.10%) compared with grade B control (12.09 ± 2.31%, P < 0.05). There were obvious increases in blastocyst formation derived from grade C oocytes after 10 ng mL−1 hrG-SCF treatment (25.74 ± 1.65%) compared with grade C control (16.82 ± 2.30%, P < 0.05). However, there were no significantly differences in cleavage rate and total cell number of blastocysts among each group. Otherwise, the PCNA, POU5F1, Dnmt1, Bcl2, and Bax transcript levels were significantly increased in blastocysts that were derived from grade C oocytes after 10 ng mL−1 hrG-SCF treatment compared with grade C control. In conclusion, supplementation of 10 ng mL−1 hrG-CSF in in vitro-cultured porcine embryos increased blastocyst formation of embryos derived from thin cumulus layer of oocytes by reducing apoptosis while increasing cell proliferation and nuclear reprogramming. These results provide an experimental basis for the use of poor quality oocytes for agricultural production. This work was supported by a grant from Research Program (No. 307–02) Gyeonggi-province project and the Next-Generation BioGreen21 Program [no. PJ01107702], Rural Developmental Administration (RDA), Republic of Korea.

90 SUCCESSFUL VITRIFICATION OF PARTHENOGENETIC PORCINE BLASTOCYSTS PRODUCED FROM DELIPATED IN VITRO-MATURED OOCYTES

2006 ◽  
Vol 18 (2) ◽  
pp. 153 ◽  
Author(s):  
Y. Du ◽  
P. M. Kragh ◽  
X. Zhang ◽  
H. Yang ◽  
G. Vajta ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Survival Rates ◽  
Cumulus Cells ◽  
Cell Number ◽  
Significant Loss ◽  
Developmental Competence ◽  
Average Cell ◽  
Analogue System ◽  
Hoechst Staining

Cryopreservation of cloned porcine embryos may improve the output of somatic cell cloning considerably by alleviating logistic problems. However, the high lipid content of porcine oocytes and embryos compromises their cryotolerance. Recently a noninvasive procedure was published for delipation of porcine embryos with centrifugation but without subsequent micromanipulation (Esaki et al. 2004 Biol. Reprod. 71, 432-436). This method was applied to our present work with few modifications to compare the cryosurvival of porcine blastocysts produced from delipated vs. intact oocytes with parthenogenetic activation. In four replicates, a total of 192 oocytes were used for the experiments. After in vitro maturation for 44 h, cumulus cells were removed and oocytes were randomly distributed into two groups. For delipation, oocytes were digested with 1 mg/mL pronase in the presence of 50% cattle serum (CS) for 3 min, and washed in HEPES-buffered TCM-199 medium supplemented with 20% CS. Subsequently, 40-50 oocytes were centrifuged (12 000g, 20 min) in HEPES-buffered TCM-199 medium supplemented with 2% CS, 3 mg/mL polyvinyl alcohol and 7.5 �g/mL cytochalasin B (CB). Zonae pellucidae of both centrifuged and intact oocytes were removed completely by further digestion in 2 mg/mL pronase solution. For activation, a single direct current of 85 kV/cm for 80 �s was applied to both groups, followed by 4-h treatment with 5 �g/mL CB and 10 �g/mL cycloheximide. All embryos were then cultured in modified NCSU37 medium. Day 7 blastocysts were vitrified and warmed by using the Cryotop technique (Kuwayama et al. 2005 RBM Online 11, 300-308) at 38.5�C. Survival of vitrified blastocysts was determined according to re-expansion rates after 24 h recovery in culture medium supplemented with 10% CS. Cell numbers of reexpanded blastocysts from both groups were determined after Hoechst staining. Results were compared by ANOVA. Partial zona digestion and centrifugation resulted in successful delipation in 173/192 (90%) of oocytes. The development to blastocysts was not different between delipated and intact oocytes (28 � 7% vs. 28 � 5%, respectively; P > 0.05). However, survival rates of blastocysts derived from delipated oocytes were significantly higher than those developed from intact oocytes (85 � 6% vs. 32 � 7%, respectively; P < 0.01). There was no difference in average cell number of re-expanded blastocysts derived from either delipated or intact oocytes (36 � 7 vs. 38 � 9, respectively; P > 0.05). Our results prove that the simple delipation technique does not hamper the in vitro developmental competence of activated porcine oocytes, and improves the cryosurvival of the derived blastocysts without significant loss in cell number. Future investigations are required to prove the value of the method in an analogue system with blastocysts produced by somatic cell nuclear transfer.

scholarly journals Developmental competence and ultrastructural changes of heat-stressed mouse early blastocysts produced in vitro

2009 ◽  
Vol 55 (1) ◽  
pp. 61-66 ◽  
Author(s):  
Pingping Qu ◽  
Wenru Tian ◽  
Tao Li ◽  
Zhongling Jiang ◽  
Shansong Gao ◽  
...  
Keyword(s):  
Heat Stress ◽  
Normal Development ◽  
Developmental Competence ◽  
Ultrastructural Changes ◽  
Control Group ◽  
Average Cell ◽  
Cell Numbers ◽  
Secondary Lysosomes ◽  
Perivitelline Membrane

Abstract Mouse early blastocysts were exposed to temperatures of 39°C and 41°C for 2 h, respectively, to determine their developmental competence and ultrastructural changes. The results showed that heat stress at 41°C for 2 h, significantly reduced the percentages of expanded and hatched blastocysts, but not at 39°C for 2 h. The average cell numbers in expanded blastocysts, which developed from early blastocysts heat-stressed at temperatures of 39°C and 41°C, were significantly reduced. The average cell numbers in hatched blastocysts subjected to heat stress were no different from those in the control group cultured at 37°C. The mitochondria of the early blastocysts heat-stressed at 39°C for 2 h, were slightly swollen, but they had recovered after culturing at 37°C for 2 h. However, the mitochondria in the blastocysts heat stressed at 41°C for 2 h were severely swollen, and their number increased. The ribosomes shed from the rough endoplasmic reticulum, and the number of secondary lysosomes in the plasma increased. The integrity of desmosomes was disrupted. The space between the nuclear envelope and the perivitelline membrane enlarged. The fibre fraction and the particulate fraction of nucleoli were separated. The heterochromatin in nucleoli was also increased in its quantity. There were some lamellar-shape structures and heterogeneous dense materials exhibiting in the cytoplasm. The ultrastructural changes induced by heat shock at 41°C for 2 h were not reversible. In conclusion, the damage of heat stress to mitochondria, lysosomes, ribosomes and cell nucleus, may be one of the most important factors that inhibit the normal development of mouse early blastocysts.

scholarly journals Developmental Competence of Early Stage Porcine Embryos Cultured in Medium with Different Energy Substrate in vitro

2015 ◽  
Vol 11 (1) ◽  
Author(s):  
Ni Wayan Kurniani Karja ◽  
Kazuhiro Kikuchi ◽  
Mokhamad Fahrudin
Keyword(s):  
In Vitro Culture ◽  
Early Stage ◽  
Energy Requirement ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
State University ◽  
Energy Substrate ◽  
The Mean

To elucidate the effect of energy requirement during the early embryonic development on their developmentalability to develop to blastocyst stage, in vitro fertilized (IVF) porcine one-cell embryos were cultured in modifiedNorth Carolina State University (NCSU)-37 supplemented with different energy substrate. Result indicated that thecleavage rate of embryos in Pyr-Lac and Gluc-Pyr-Lact groups was significantly higher than in those in Gluc groupand Gluc-Rib group (P < 0.05). At Day 6 of culture, the highest proportion of embryos develop to the blastocyst stagewas obtained in the presence of pyruvate-lactate only. In the medium with glucose, the addition of pyruvate-lactateor ribose slightly increased the proportion of embryos develop to the blastocyst stage, however the value were notsignificantly different form those obtained in the presence of glucose only. The mean cell number in blastocystsderived from Pyr-Lac and Gluc-Pyr-Lact groups were significantly higher than those in the Gluc group (P < 0.05).These results indicated that the presence of glucose only, as energy substrate, during the first 2 days of in vitro culture(IVC) caused a decrease in development of in vitro produced (IVP) porcine embryos to the blastocyst stage and meancell number in blastocysts .Keywords: porcine embryos-energy substrate-in vitro culture

Characterization of isolated bovine preantral follicles based on morphology, diameter and cell number

Zygote ◽  
2020 ◽  
Vol 28 (2) ◽  
pp. 154-159
Author(s):  
Juliana I. Candelaria ◽  
Anna C. Denicol
Keyword(s):  
Granulosa Cells ◽  
Developmental Stages ◽  
Assisted Reproductive Technologies ◽  
Reproductive Technologies ◽  
Cell Number ◽  
Culture Conditions ◽  
Preantral Follicles ◽  
Secondary Stage

SummaryPreantral follicles are a potential reservoir of oocytes to be used in assisted reproductive technologies. With the increasing interest in developing techniques to grow preantral follicles in vitro, and as the bovine emerges as an appropriate model species to understand human folliculogenesis, the establishment of an accurate classification of developmental stages is needed. Classification of bovine preantral follicles has been mostly based on histological analysis and estimation models, which may not translate well to correctly characterize preantral follicles isolated from the ovary. In this study, we classified bovine preantral follicles by morphology upon isolation, determined diameter and number of granulosa cells by direct counting, and compared our results with previous studies reporting bovine preantral follicle classification. Follicles were isolated via homogenization of ovary tissue and classified into primary, early secondary and secondary stage based on morphology and number of layers of granulosa cells. Diameter was individually measured and Hoechst 33342 was used as a nuclear stain to count granulosa cells. We found that follicles classified by morphology into primary, early secondary, and secondary had different mean diameter and cell number (P < 0.01); cell number and diameter were positively correlated, as were cell density and cell number in each developmental stage (P < 0.01). Results obtained here were mostly in agreement with previous classifications based on histological sections and on isolated follicles, with some discrepancies. The present data add accuracy to classification of bovine preantral follicles that is critical to optimize culture conditions to produce developmentally competent oocytes.

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