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.

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

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

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

Polyphyllin D, a steroidal saponin from Paris polyphylla, inhibits endothelial cell functions in vitro and angiogenesis in zebrafish embryos in vivo

2011 ◽  
Vol 137 (1) ◽  
pp. 64-69 ◽  
Author(s):  
Judy Yuet-Wa Chan ◽  
Johnny Chi-Man Koon ◽  
Xiaozhou Liu ◽  
Michael Detmar ◽  
Biao Yu ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Zebrafish Embryos ◽  
Steroidal Saponin ◽  
Cell Functions ◽  
Paris Polyphylla ◽  
Polyphyllin D

Improving Biomaterials from a Cellular Point of View

2006 ◽  
Vol 925 ◽  
Author(s):  
Venu Gopal Varanasi ◽  
T. Vallortigara ◽  
P. M. Loomer ◽  
E. Saiz ◽  
A. P. Tomsia ◽  
...  
Keyword(s):  
Dissolution Rate ◽  
Cell Number ◽  
Point Of View ◽  
Dissolution Behavior ◽  
Ti Alloy ◽  
Bioactive Glasses ◽  
Culture Studies ◽  
Cell Numbers ◽  
Number Increase

ABSTRACTBioactive glasses (6P55) used for coating Ti/Ti-alloy were tested for their in vitro behavior in a comparative study with commercial Bioglass™ (45S5) and commercial Ti alloy (Ti6Al4V). In vitro testing included pH and dissolution rate determination in simulated body fluid (SBF) along with in vitro cyto compatibility testing. It was seen in this work that 6P55 and 45S5 had similar dissolution behavior, demonstrating t½ dependence and maximum pH of approximately 8.1 after 10 days of immersion. This pH was reduce by 0.2 0.4 pH units when the in vitro V:A ratio was increased from 1 to 3. The dissolution rate of these glasses approached 0 after additional immersion tests after 15 days and the pH stablilized at less than 7.5. Cell culture studies showed that both glasses behaved in similar fashion after 16 hours in culture. Both glasses had an increase in cell numbers of close to 200-250%, whereas Ti6Al4V had a less pronounced cell number increase (∼ 180%)

The human blastocyst: cell number, death and allocation during late preimplantation development in vitro

Development ◽  
1989 ◽  
Vol 107 (3) ◽  
pp. 597-604 ◽  
Author(s):  
K. Hardy ◽  
A.H. Handyside ◽  
R.M. Winston
Keyword(s):  
Cell Death ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Human Embryos ◽  
Cell Numbers ◽  
Human Blastocyst ◽  
Development In Vitro

The development of 181 surplus human embryos, including both normally and abnormally fertilized, was observed from day 2 to day 5, 6 or 7 in vitro. 63/149 (42%) normally fertilized embryos reached the blastocyst stage on day 5 or 6. Total, trophectoderm (TE) and inner cell mass (ICM) cell numbers were analyzed by differential labelling of the nuclei with polynucleotide-specific fluorochromes. The TE nuclei were labelled with one fluorochrome during immunosurgical lysis, before fixing the embryo and labelling both sets of nuclei with a second fluorochrome (Handyside and Hunter, 1984, 1986). Newly expanded normally fertilized blastocysts on day 5 had a total of 58.3 +/− 8.1 cells, which increased to 84.4 +/− 5.7 and 125.5 +/− 19 on days 6 and 7, respectively. The numbers of TE cells were similar on days 5 and 6 (37.9 +/− 6.0 and 40.3 +/− 5.0, respectively) and then doubled on day 7 (80.6 +/− 15.2). In contrast, ICM cell numbers doubled between days 5 and 6 (20.4 +/− 4.0 and 41.9 +/− 5.0, respectively) and remained virtually unchanged on day 7 (45.6 +/− 10.2). There was widespread cell death in both the TE and ICM as evidenced by fragmenting nuclei, which increased substantially by day 7. These results are compared with the numbers of cells in morphologically abnormal blastocysts and blastocysts derived from abnormally fertilized embryos. The nuclei of arrested embryos were also examined. The number of TE and ICM cells allocated in normally fertilized blastocysts appears to be similar to the numbers allocated in the mouse. Unlike the mouse, however, the proportion of ICM cells remains higher, despite cell death in both lineages.

Physiological concentration of atrial natriuretic peptide induces endothelial regeneration in vitro

2003 ◽  
Vol 284 (4) ◽  
pp. H1388-H1397 ◽  
Author(s):  
Hyun Kook ◽  
Hiroshi Itoh ◽  
Bong Seok Choi ◽  
Naoki Sawada ◽  
Kentaro Doi ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Protein Kinase ◽  
Dependent Protein Kinase ◽  
Physiological Concentration ◽  
Cell Numbers ◽  
Cgmp Dependent Protein Kinase ◽  
Dependent Protein ◽  
Regeneration In Vitro

Both nitric oxide (NO) and natriuretic peptides produce apoptosis of vascular smooth muscle cells. However, there is evidence that NO induces endothelial cell proliferation, which suggests that there is a difference in the response of endothelial cells to natriuretic peptides. The purpose of this study was to investigate the effect of atrial natriuretic peptide (ANP) on human endothelial cell survival. ANP within the physiological concentration (10−11mol/l) induced a 52% increase in the number of human coronary arterial endothelial cells and a 63% increase in human umbilical vein endothelial cells at a low concentration of serum. The increase in cell numbers was blocked by pretreatment with RP8-CPT-cGMP (RP8), a cGMP-dependent protein kinase inhibitor, with wortmannin, an Akt/PKB inhibitor, and with PD-98059, an ERK1/2 inhibitor. In a Transwell migration test, ANP also increased the cell migration, and RP8, wortmannin, and PD-98059 blocked this increase. A wound healing assay was performed to examine the effects of ANP on regeneration in vitro. ANP increased both cell numbers and migration, but the effects were blocked by the above three kinase inhibitors. ANP increased the expression of phospho-Akt and of phospho-ERK1/2 within 1.5 h. These results suggest that ANP can potentiate endothelial regeneration by cGMP-dependent protein kinase stimulation and subsequent Akt and ERK1/2 activations.

197 EFFECTS OF HYALURONAN ON OXYGEN CONSUMPTION AND ATP CONTENT IN PIG BLASTOCYSTS PRODUCED IN VITRO

2007 ◽  
Vol 19 (1) ◽  
pp. 215
Author(s):  
K. Yoshioka ◽  
M. Yokoo ◽  
T. Ozawa ◽  
C. Suzuki ◽  
H. Abe ◽  
...  
Keyword(s):  
Oxygen Consumption ◽  
Metabolic Activity ◽  
Formation Rate ◽  
Defined Medium ◽  
Cell Number ◽  
Total Cell ◽  
Chemically Defined Medium ◽  
Atp Content ◽  
Cell Numbers

Hyaluronan (HA), a glycosaminoglycan present in follicular and oviductal fluids, has been related to sperm capacitation, fertilization, and embryo development. We have found that exogenous HA improves cell proliferation of porcine embryos cultured in a chemically defined medium (Yoshioka et al. 2004 Reprod. Fertil. Dev. 16, 264–265). Moreover, mitochondrial maturation was clearly more advanced in blastocysts cultured with HA compared to those cultured without HA, as seen by transmission electron microscopy. In the present study, the effects of HA on oxygen consumption and ATP content of blastocysts, produced in a defined system which reflects metabolic activity, were investigated. Porcine immature oocytes were matured for 44 h in porcine oocyte medium (POM) and subsequently fertilized with frozen–thawed ejaculated semen in porcine gamete medium supplemented with theophylline, adenosine, and cysteine (PGMtac4). Both POM and PGMtac4 were chemically defined media modified from porcine zygote medium (PZM)-5. After IVF, presumptive zygotes were cultured in PZM-5 containing HA (from the microorganism, Nacalai tesque, Kyoto, Japan) at concentrations of 0 [control], 10 [HA10], or 100 [HA100] �g mL-1 until 5 days after IVF. Blastocyst formation rate and total cell numbers/blastocyst at Day 5 were assessed. In addition, oxygen consumption and ATP content of single Day 5 blastocysts were measured. Blastocyst oxygen consumption was quantified using scanning electrochemical microscopy (HV-403; Research Institute for the Functional Peptides, Yamagata, Japan), and embryonic ATP content was determined using a commercial assay based on the luciferin-luciferase reaction (ATPlite; PerkinElmer, Groningen, The Netherlands). Data were statistically analyzed by ANOVA and Fisher&apos;s PLSD test. While the percentage of embryos that developed to the blastocyst stage [30.5% (63/206) to 31.7% (65/206)] did not differ among treatments, blastocyst cell number in the HA100 group [57.9 cells (n = 64)] was greater (P &lt; 0.05) compared to those in the control [48.6 cells (n = 63)] or HA10 [50.0 cells (n = 65)] groups. Blastocyst oxygen consumption rate in the HA100 group [0.629 � 10-14 mol s-1 (n = 15)] was significantly higher than in the control [0.500 � 1-14 mol s-1 (n = 16)] or HA10 [0.464 � 10-14 mol s-1 (n = 14)] groups. ATP content/blastocyst did not differ among treatments [control: 0.645 pmol (n = 38), HA10: 0.727 pmol (n = 42), and HA100: 0.704 pmol (n = 43)]. It is concluded that HA affects the metabolic activity of pig blastocysts developed in a chemically defined medium, enhancing oxygen consumption and their total cell numbers, thus improving the quality of IVP blastocysts.

286 INFLUENCE OF REVERSIBLE MEIOSIS INHIBITION ON SWINE EMBRYOS PRODUCED BY IN VITRO FERTILIZATION AND PATHENOGENETIC ACTIVATION

2006 ◽  
Vol 18 (2) ◽  
pp. 250
Author(s):  
M. G. Marques ◽  
A. B. Nascimento ◽  
V. P. Oliveira ◽  
A. R. S. Coutinho ◽  
M. E. O. A. Assumpção ◽  
...  
Keyword(s):  
Culture Medium ◽  
Cell Number ◽  
Control Group ◽  
Cleavage Rate ◽  
Vitro Fertilization ◽  
Electric Pulses ◽  
Cell Numbers ◽  
Blastocyst Rate ◽  
And Control

The present work evaluated the reversible meiosis inhibition effect on the development of swine embryos produced by in vitro fertilization (IVF) or parthenogenetic activation (PA). The efficiency of PZM3 and NCSU23 embryo culture media was also evaluated. Oocytes from ovaries collected at a slaughterhouse were subjected to IVM in two different groups: CHX (cycloheximide 5 µM for 10 h) and control, both with TCM-199 + 3.05 mM glucose + 0.91 mM sodium pyruvate + 10% porcine follicular fluid (pFF) + 0.57 mM cystein + 10 ng epidermal growth factor (EGF)/mL + 10 IU eCG/mL + 10 IU hCG/mL for the initial 22 h. In the remaining period (20 h for CHX and 22 h for control), medium without hormones was utilized. After IVM, oocytes were denuded and fertilized for 6 h (IFV) or the matured oocytes were submitted to activation by electric pulses (PA) (2 DC of 1.5 kV/cm for 30 µs), incubated for 1 h in culture medium with 10 μM of CHX, and again submitted to the same electric pulses for 60 µs. Embryo development was evaluated by cleavage rate on Day 3 and blastocyst rate and blastocyst cell number on Day 7 of culture. Cleavage and blastocyst rates were analyzed by the equality-of-two-ratios test and cell number by the Kruskal-Wallis and Mann-Whitney tests (P < 0.05). In relation to IVF, the PZM3 medium was more efficient than NCSU23 for cleavage rate in the CHX group (PZM3: 68.4%, NCSU23: 44.4%) and had a better blastocyst rate in the control group (PZM3: 13.4%, NCSU23: 5.6%). With reference to PA, NCSU23 presented better cleavage and blastocyst rates than PZM3 in the CHX group (NCSU23: 89.5%, PZM3: 78.5% and NCSU23: 20.4%, PZM3: 13.0%, respectively). In the control group, only the NCSU23 blastocyst rate was higher than that for PZM3 (NCSU23: 22.5%, PZM3: 10.8%). No culture medium effect on cell number mean of IVF and PA blastocysts was observed. Maturation block improved cleavage rates in IVF groups cultured with PZM3 (68.4% and 50.6%, respectively, for CHX and control) and in PA groups cultured with NCSU23 (89.5% and 80.3%, respectively, for CHX and control), but no improvement of blastocyst rates in both groups (IVF and PA) was verified. Table 1 below shows that maturation block decreased the IVF and increased the PA blastocyst cell numbers. As older oocytes are more effectively activated, oocytes blocked with CHX achieved the maturation stage faster than the control group, therefore resulting in high-quality PA blastocysts. In conclusion, PZM3 was more efficient for IVF embryo production in contrast to NCSU23, whereas NCSU23 can be indicated for PA embryo production. Moreover, maturation blockage with CHX influenced blastocyst cell number, decreasing in IVF embryos and increasing in PA embryos. Table 1. Mean (±SD) of blastocyst cell numbers for IVF or PA groups after in vitro maturation without (control) or with cycloheximide (CHX) and cultured in NCSU23 or PZM3 medium This work was supported by FAPESP 02/10747–1.

158 IMPACT OF MATURED CATTLE OOCYTES AT HIGHER INCUBATION TEMPERATURE ON IN VITRO EMBRYO PRODUCTION

2016 ◽  
Vol 28 (2) ◽  
pp. 209
Author(s):  
M. Nkadimeng ◽  
E. van Marle-Koster ◽  
K. P. M. Lekola ◽  
M. L. Mphaphathi ◽  
M. M. Seshoka ◽  
...  
Keyword(s):  
Incubation Temperature ◽  
Cell Types ◽  
Cell Number ◽  
Cleavage Rate ◽  
Embryo Production ◽  
Developmental Potential ◽  
Cell Numbers ◽  
Significant Difference ◽  
In Vitro Embryo Production

Heat stress during IVF is associated with reduced fertility in cattle oocytes. It may, however, enhance thermo-tolerance or cause detrimental effects on a variety of cell types or organisms, depending on the duration and intensity of the thermal challenge. The aim of this study was to evaluate the developmental potential of cumulus-oocyte complexes (COC) matured for 18 or 24 h and incubated at 39°C or 41°C. A total of 1000 immature oocytes were collected at slaughter from indigenous South African cow ovaries. The COC were randomly allocated (100/treatment) into 2 maturation times (18 or 24 h) and cultured in M199 + FSH-LH-estradiol medium under oil at 100% humidity and 5% CO2 at 39°C or 41°C. Post maturation, oocytes were subjected to normal subsequent embryo conditions. The Bracket and Oliphant medium was used for IVF. All matured oocytes were fertilised for 6 h with frozen-thawed Nguni bull semen at a concentration of 265 × 106. The presumptive zygotes from each treatment were cultured into SOF-BSA medium under oil and incubated at 39°C for assessment of cleavage rate 48 h post IVF. After Day 7 of culture, blastocyst were stained (Hoechst 33323) for nuclei cell count. Statistical analyses was performed using Genstat® software of SAS (SAS Institute, Cary, NC, USA; P < 0.05). Oocytes that were matured for 18 h in 41°C resulted in more 8-cell embryos (41%) compared with those incubated at 39°C (21.6%). However, no difference was observed for cleavage rate at both maturation times and incubation temperatures (41 or 39°C). There was more morula formation from oocytes matured for 18 h (19.6%) and 24 h (19.0%) at 41°C compared to 39°C (8.4%) group. The results further showed more blastocyst formation during 18 h at 41°C (15.2%) than at 39°C (7.4%) and during 24 h at 41°C (11.2%), 39°C (11.4%). However there was no difference in the nuclei cell number during 18 h at 41°C (45.2), 24 h (45.8), and 18 h at 39°C (43.4) of maturation. Thus, there was a significant difference in the nuclei cell numbers at 24 h on 39°C (n = 133.2) and 41°C (n = 45.8). In conclusion, oocytes that were matured for 18 and 24 h at 41°C or for 18 h at 39°C developed further to blastocyst stage on in vitro embryo production, however, with low nuclei cell numbers due to accelerated maturation temperature or shortened maturation period.

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