137 EFFECT OF POST-IVF DEVELOPMENTAL KINETICS ON SURVIVAL AND QUALITY OF VITRIFIED - WARMED DOMESTIC CAT BLASTOCYSTS

2007 ◽  
Vol 19 (1) ◽  
pp. 186
Author(s):  
T. Tsujioka ◽  
C. Otzdorff ◽  
J. Braun ◽  
S. Hochi
Keyword(s):  
Survival Rates ◽  
Cell Number ◽  
Domestic Cat ◽  
Minimum Volume ◽  
Cleavage Rate ◽  
Exact Probability ◽  
Cell Staining ◽  
Number Of Cells ◽  
Cooling Procedure

A limited number of reports are available for cryopreservation of IVF-derived cat blastocysts (Karja et al. 2006 Theriogenology 65, 415–423). In the present study, the IVF-derived domestic cat blastocysts exhibiting differences in their developmental kinetics were cryopreserved by vitrification. Pre- and post-warm blastocysts were examined for their cell number, and the survival rate was determined by in vitro culture of the post-warm embryos. Cumulus–oocyte complexes, recovered from the ovaries of post-pubertal queens, were matured for 24 h in TCM-199 supplemented with 3 mg mL−1 BSA, 1 µg mL−1 estradiol, 0.1 IU mL−1 FSH, and 0.0063 IU mL−1 LH. Oocytes were inseminated with 2 × 106 cells mL−1 cauda epididymal spermatozoa for 22 h in TALP solution. Presumptive zygotes were cultured in modified SOF medium at 38.5°C in 5% CO2 in air. Newly formed blastocysts were harvested 6 and 7 days post-IVF and subjected to vitrification (Hochi et al. 2004 Theriogenology 61, 267–275) by a minimum-volume cooling procedure using Cryotop (Kitazato Supply Co., Tokyo, Japan) as a cryodevice. The post-warm blastocysts were cultured for 24 h, and complete re-expansion was considered to be an indicator of survival. Embryos were differentially stained with Hoechst 33342 and propidium iodide to assess the total number of cells and the ICM ratio in the blastocysts. The cleavage rate of oocytes was 47.1% (314/666) and the percentage of oocytes developing to blastocysts on Day 6 and Day 7 was 9.8 and 9.5%, respectively (total blastocyst yield: 19.2%; 128/666). Post-warm in vitro survival rates of blastocysts harvested at Days 6 and 7 were 73.8% (31/42) and 66.7% (18/27), respectively (P = 0.59; exact probability test). There were no significant differences in the total number of cells and the ICM ratio between fresh control and vitrified blastocysts (P ≥ 0.05; ANOVA), although the ICM ratio of surviving Day 7 blastocysts was significantly smaller than that of fresh controls (18.9 vs. 28.9%; P = 0.03), as shown in Table 1. These results indicate that IVF-derived domestic cat blastocysts can survive vitrification by a minimum-volume cooling procedure without a reduction in the parameters studied, as long as the blastocysts are harvested on Day 6. Table 1. Differential cell staining of fresh and vitrified Day 6 and Day 7 cat blastocysts

scholarly journals 160EFFECTS OF EXTRACELLULAR ION CONCENTRATIONS ON IN VITRO DEVELOPMENT OF DOMESTIC CAT IVF EMBRYOS

2004 ◽  
Vol 16 (2) ◽  
pp. 202 ◽  
Author(s):  
W.F. Swanson ◽  
A.L. Manharth ◽  
J.B. Bond ◽  
H.L. Bateman ◽  
R.L. Krisher ◽  
...  
Keyword(s):  
Culture Media ◽  
Ionic Composition ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Domestic Cat ◽  
Embryo Viability ◽  
Blastocyst Development ◽  
In Vitro Development ◽  
Vitro Development

Domestic cat embryos typically are cultured in media formulated for somatic cells or embryos from rodents or livestock species. Under these conditions, blastocyst development has been inconsistent and delayed relative to embryos grown in vivo, and embryo viability following transfer has been low. Our goal is to systematically define the culture requirements of the feline embryo to improve embryo development and viability. The objective of this study was to determine the ionic (NaCl, KCl, KH2PO4, and CaCl2:MgSO4) preferences of domestic cat IVF embryos. Anestral female cats were injected (i.m.) with 150IU eCG followed 84h later by 100IUhCG. Oocytes were recovered via laparoscopic follicular aspiration approximately 24h post-hCG injection (Day 0). Semen was collected from one of two males by means of an artificial vagina and washed once in HEPES-buffered IVF medium. Mature cumulus-oocyte complexes were co-incubated with 2.5–5×105 motile sperm mL−1 in IVF medium (100mM NaCl, 4.0mM KCl, 1.0mM KH2 PO4, 2.0mM CaCl2, 1.0mM MgSO4-7H2O, 25.0mM NaHCO3, 3.0mM glucose, 0.1mM pyruvate, 6.0mM L-lactate, 1.0mM glutamine, 0.1mM taurine, 1×MEM nonessential amino acids, 50μgmL−1 gentamicin, and 4.0mgmL−1 BSA) for 19 to 22h in 6% CO2 in air (38.7°C). Cumulus cells were removed and embryos cultured (8–11 embryos/50μL drop; 6% CO2, 5% O2, 89% N2, 38.7°C) in media containing 100.0 or 120.0mM NaCl, 4.0 or 8.0mM KCl, 0.25 or 1.0mM KH2PO4, and 1.0mM:2.0mM or 2.0mM:1.0mM CaCl2:MgSO4 (2×2×2×2 factorial design). The remaining components of the culture medium were identical to the IVF medium (but w/o gentamicin). Development to the blastocyst stage by Day 6, metabolism (glycolysis and pyruvate) of each blastocyst, and final cell number (Hoechst 33342 staining) of all embryos were evaluated. Final cell number of cleaved embryos and development to the blastocyst stage were analyzed using analysis of variance in the GLIMMIX macro of SAS. A total of 236 oocytes were inseminated, yielding 128 cleaved embryos (54%), including 6 blastocysts (4.7% of cleaved embryos). Cell number was not (P>0.05) affected by NaCl, KCl, or KH2PO4 concentrations, but tended (P=0.057) to be higher after culture in 2.0mM:1.0mM CaCl2:MgSO4. Treatments did not significantly affect (P>0.05) development to the blastocyst stage, but numerically more blastocysts were produced in 100.0mM NaCl (4/6), 8.0mM KCl (5/6), or 1.0mM KH2PO4 (5/6). Both CaCl2:MgSO4 ratios resulted in 3 blastocysts. Blastocysts contained 61.08±5.1 (mean±SEM, n=6) cells and actively metabolized glucose (glycolysis, 3.7±0.8pmol/embryo/3h or 0.06±0.01pmol/cell/3h) and pyruvate (0.75±0.27pmol/embryo/3h or 0.013±0.005pmol/cell/3h). These results suggest that the ionic composition of culture media influences the in vitro development of cat IVF embryos. (Supported by NIH grant RR15388.)

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.

41 EFFICIENT STRATEGY FOR INTERSPECIFIC CLONING IN FELIDS

2014 ◽  
Vol 26 (1) ◽  
pp. 134
Author(s):  
L. N. Moro ◽  
M. I. Hiriart ◽  
J. Jarazo ◽  
C. Buemo ◽  
A. Sestelo ◽  
...  
Keyword(s):  
Species Conservation ◽  
Total Cell ◽  
Domestic Cat ◽  
Control Group ◽  
Felis Silvestris ◽  
Blastocyst Formation ◽  
Acinonyx Jubatus ◽  
Cell Numbers ◽  
Number Of Cells

Most of the 36 species of wild felids are at a level of threat, and interspecific SCNT (iSCNT) comes as a strategy to contribute to these species conservation. The aim of this study was to evaluate the effect of embryo aggregation in cheetah (Ch, Acinonyx jubatus), bengal (Ben, a hybrid between Felis silvestris and Prionailurus bengalensis), and domestic cat (DC, Felis silvestris) embryos generated by cloning. DC oocytes were in vitro matured and zona-free SCNT (with DC fibroblasts) or iSCNT (with Ch or Ben fibroblasts) was performed. The reconstructed embryos were activated with 5 μM ionomycin and 1.9 mM 6-DMAP, and cultured in SOF using microwells. Cloned embryos were cultured individually or as 2-embryo aggregates. The experimental groups were Ch1X, Ch2X, Ben1X, Ben2X, and the control groups were DC1X and DC2X. Embryo development was compared by Fisher's exact test (P ≤ 0.05). Embryo aggregation improved cleavage (Day 2) and blastocyst (Day 7) rates per well in all the groups (87.2% v. 96.7%, 83.8% v. 93.3% and 87.6% v. 98.2% for cleavage; and 13.7% v. 28.6%, 33.3% v. 43.8% and 27.4% v. 47.7% for blastocyst, for Ch1X (n = 102), Ch2X (n = 91), Ben1X (n = 154), Ben2X (n = 105), DC1X (n = 113), and DC2X (n = 109), respectively. Moreover, the Ch2X blastocyst rate was statistically similar as the control group DC1X. The mean total cell numbers of the blastocysts obtained were 264 ± 211 and 400.8 ± 97 for Ch1X and Ch2X, 278 ± 62 and 517 ± 104 for Ben1X and Ben2X, 385 ± 127 and 625 ± 183 for DC1X and DC2X, respectively. Although no statistical differences were obtained between the 1X and 2X groups, the 2X groups nearly doubled the average number of cells compared with the 1X groups. Blastocysts were also classified as grade 1 (expanded blastocysts with a well-defined ICM), grade 2 (expanded blastocysts without a well-defined ICM), and grade 3 (not expanded blastocysts). This classification showed an increase in grade 1 DC2X blastocyst compared with DC1X blastocysts (36.7% v. 16.1%), but no differences were observed in the other species. Expression of OCT-4 was assessed by inmunocytochemistry. The cheetah blastocysts markedly over-expressed this protein: the percentage of cells that expressed OCT-4 in Ch1X, Ch2X, Ben1X, Ben2X, DC1X, and DC2X was 88.2, 80.2, 46.3, 45.4, 51, and 47.4%, respectively, with statistical differences among all the groups except Ben1X and Ben2X. The proportion of OCT-4 expressing cells over total cell numbers was analysed by the difference of proportions test (P ≤ 0.05). In conclusion, iSCNT resulted in high rates of blastocyst formation, especially when embryo aggregation was applied. This strategy has not been previously evaluated in felids or iSCNT procedures, and has been demonstrated to improve blastocyst formation, the number of cells in the 3 groups, and the blastocyst quality in the DC. Other pluripotent genes besides OCT-4 should be studied to determine whether the overexpression of this gene in cheetah embryos is the consequence of an inefficient nuclear reprogramming that prevents a correct regulation. Finally, the iSCNT and embryo aggregation could contribute to species conservation in felids.

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.

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&apos;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 &plusmn; 0.3 vs. 27.5 &plusmn; 0.3&percnt;, 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 &plusmn; 0.9 vs. 33.4 &plusmn; 0.3&percnt;, respectively). In the SCNT group, however, both cleavage (73.6 &plusmn; 0.2 vs. 64.2 &plusmn; 0.4&percnt;) and blastocyst rate (18.7 &plusmn; 0.2 vs. 13.8 &plusmn; 0.3&percnt;) were improved by NEAA supplementation. Furthermore, these blastocysts had higher hatching ability (30.0 &plusmn; 1.8 vs. 14.6 &plusmn; 4.9&percnt;) than those of control group (P &lt; 0.05). Supplementation of NEAA also increased the mean nuclei number of PA-derived (76.1 &plusmn; 4.9 vs. 66.5 &plusmn; 3.3) as well as SCNT-derived (43.1 &plusmn; 2.6 vs. 31.8 &plusmn; 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.

52 HYALURONIC ACID IMPROVES CRYOTOLERANCE OF BUFFALO (BUBALUS BUBALIS) IN VITRO-DERIVED EMBRYOS

2012 ◽  
Vol 24 (1) ◽  
pp. 138
Author(s):  
L. Boccia ◽  
M. Rubessa ◽  
M. De Blasi ◽  
S. Di Francesco ◽  
G. Albero ◽  
...  
Keyword(s):  
Hyaluronic Acid ◽  
In Vitro Culture ◽  
Developmental Stage ◽  
Production Efficiency ◽  
Embryo Quality ◽  
Survival Rates ◽  
Blastocyst Development ◽  
Cleavage Rate ◽  
Morphological Criteria

Although in vitro embryo production efficiency in buffalos has greatly improved over the years, the in vitro-produced embryos show lower viability and resistance to cryopreservation. Therefore, it is necessary to optimize the in vitro culture conditions to improve embryo quality. Hyaluronic acid, a glycosaminoglican present in oviducal and uterine fluids, has been shown to successfully support in vitro development of bovine embryos (Stojkovic et al. 2002 Reproduction 124, 141–153). The aim of this study was to evaluate the influence of high concentrations of hyaluronic acid (HA) during late in vitro culture on blastocyst development, as well as on their cryotolerance after cryotop vitrification in buffalos. In vitro matured and fertilized buffalo oocytes (n = 1007) from slaughterhouse ovaries were cultured for 4 days in SOFaa supplemented by 8 mg mL–1 of BSA in a controlled gas atmosphere consisting of 5% CO2, 7% O2 and 88% N2, in humidified air, at 38.5°C. On Day 4, cleavage rate was assessed (75.2%) and all of the cleaved elements were divided into 3 different late culture groups: 8 mg mL–1 of BSA (n = 244; group A), 8 mg mL–1 of BSA supplemented by 6 mg mL–1 of HA (n = 251; group B) and 1 mg mL–1 of BSA supplemented by 6 mg mL–1 of HA (n = 262; group C). On Day 7 after IVF, embryo outcome was assessed and all of the embryos were vitrified by cryotop [De Rosa et al. 2007 Ital. J. Anim. Sci. 6 (Suppl 2), 747–750] and cultured for 24 h. The resistance to cryopreservation was evaluated by assessing the survival rate on the basis of morphological criteria and the percentage of embryos reaching a more advanced developmental stage after 24 h culture. Data were analysed by the chi-square test. No differences in blastocyst rate were recorded among groups (43.9, 44.3 and 40.0%, respectively in A, B and C groups). However, out of the total embryos, a higher percentage of Grade 1 hatched blastocysts (Robertson and Nelson 1998 Manual of the International Embryo Transfer Society 9, 103–16) was observed in group C (P < 0.05) than in groups A and B (14.3, 18.8 and 25.5% in A, B and C groups, respectively). Although the supplementation with HA did not improve the survival rates following vitrification-warming (51.1, 59.4 and 58.4% in A, B and C groups, respectively), the percentage of vitrified-warmed embryos that resumed development and reached a more advanced developmental stage after culture increased (P < 0.01) in group C (20.7, 27.7 and 37.6% in A, B and C groups, respectively). In conclusion, the addition of 6 mg mL–1 of HA, together with a limited protein source (i.e. 1 mg mL–1 of BSA), during late culture improved buffalo embryo quality, indicated by both the greater percentage of advanced-stage embryos and by the resumption of development after post-warming culture.

171 Effect of progesterone and prolactin on the developmental competence of bovine oocytes during the terminal phase of in vitro maturation

2019 ◽  
Vol 31 (1) ◽  
pp. 210
Author(s):  
G. Singina ◽  
E. Shedova ◽  
T. Taradajnic ◽  
V. Konnova ◽  
E. Tsyndrina
Keyword(s):  
Cell Number ◽  
Total Cell ◽  
Developmental Competence ◽  
Terminal Phase ◽  
Cleavage Rate ◽  
Total Cell Number ◽  
One Step ◽  
Group 2 ◽  
The One

To date, considerable progress has been achieved in in vitro production (IVP) technologies in cattle; however, developmental potentials of oocytes matured in vitro remain low compared with in vivo-matured oocytes. Thus, a better understanding of different aspects of oocyte maturation may allow us to increase the embryo development rate. Our study was aimed to assess the effects of progesterone (P4) and prolactin (PRL) on the bovine oocyte developmental competence. Bovine cumulus-enclosed oocytes (CEO) were matured using either one-step or two-step maturation conditions. For the one-step protocol, CEO were cultured for 24h in TCM-199 supplemented with 10% fetal calf serum (FCS), 10μg mL−1 porcine FSH, and 10μg mL−1 ovine LH (standard medium). For the two-step procedure, CEO were first cultured for 16h in the standard medium (n=1263) and then transferred to 1 of 3 experimental media and cultured for additional 8h in either absence or presence of either P4 (50 ng mL−1) or bovine PRL (50ng mL−1). The 3 media tested in the two-step maturation were (1) TCM-199 containing 10% FCS (group 1), (2) TCM-199 containing 3mg mL−1 BSA (group 2), or (3) Fert-TALP medium supplemented with 6mg mL−1 BSA (group 3). Fert-TALP was selected because it can potentially be used throughout maturation and fertilization. Following in vitro maturation, all oocytes underwent an IVF/in vitro culture procedure as described previously (Singina et al. 2014 Reprod. Fertil. Devel. 26, 154). The embryo development was evaluated at Days 2 and 7 for cleavage and blastocyst rates. In addition, obtained blastocysts were fixed, and the total cell number and the level of apoptosis were determined using 4′,6-diamidino-2-phenylindole (DAPI) and TUNEL staining. The data from 4 to 5 replicates (113-159 oocytes per treatment) were analysed by ANOVA. For oocytes matured for 24h in the one-step culture, the cleavage rate, blastocyst rate, total cell number, and apoptotic nuclei per blastocyst were 66.1±1.1, 23.7±2.0, 71.4±9.1, and 4.8±1.2%, respectively. For the two-step culture, the cleavage rate did not differ from that of the one-step culture system, ranging from 64.8 to 76.5%. Also, no effects of the two-step systems were observed on total cell number (63.0-78.8) or the proportion of apoptotic nuclei (3.3-5.3%) at the blastocyst stage. The culture of CEO in group 1 (without the supplements) had a reduced blastocyst rate (17.4±0.4%; P&lt;0.05) compared with the standard one-step maturation group, and the addition of P4 (but not PRL) improved the blastocyst yield (P&lt;0.05). Furthermore, when P4 (but not PRL) was added to group 2 and group 3 media, blastocyst rates increased significantly (32.9±3.1 and 32.8±2.7%, respectively) compared with those of the one-step group (P&lt;0.05), but did not differ from those of untreated groups 2 and 3 (26.2±2.7 and 30.0±3.0%, respectively). Our data indicate that P4 supplementation during the terminal phase of two-step IVM can enhance the developmental competence of bovine oocytes and that the nature of this effect depends on the composition of IVM medium, whereas no effect of PRL supplementation was observed. The study was supported by RFBR (No. 17-29-08035).

47 EFFECT OF L-CARNITINE TREATMENT DURING OOCYTE MATURATION ON THE POST-THAW DEVELOPMENT OF PORCINE EMBRYOS VITRIFIED AT THE PRONUCLEAR STAGE

2016 ◽  
Vol 28 (2) ◽  
pp. 153 ◽  
Author(s):  
C. G. Grupen ◽  
T. Somfai ◽  
K. Kikuchi
Keyword(s):  
Lipid Droplets ◽  
In Vitro Maturation ◽  
Survival Rates ◽  
Cell Number ◽  
Blastocyst Formation ◽  
Porcine Embryo ◽  
Porcine Oocyte ◽  
Proportional Data ◽  
Bovine Oocytes

The extreme cryo-sensitivity of porcine oocytes and embryos is attributed to their endemically high content of cytoplasmic lipid droplets. In attempts to improve the cryo-tolerance of porcine embryos, various strategies have been used to reduce the amount of lipid droplets present in the cytoplasm before vitrification. Recently, the cryo-tolerance of bovine oocytes vitrified at the metaphase II stage was improved by supplementing in vitro maturation (IVM) medium with l-carnitine (LC), a stimulator of lipid metabolism (Chakitisakul et al. 2013 Theriogenology 79, 590–598). The objective of this study was to determine the effect of supplementing IVM medium with LC on the post-thaw development of porcine embryos vitrified at the pronuclear stage. Oocytes recovered from the ovaries of prepubertal gilts were matured in modified porcine oocyte medium supplemented with 0 (control) or 12 mM LC during the final 22 h of IVM. Following IVF, presumptive zygotes were cultured in porcine zygote medium-3. At the pronuclear stage, cohorts of embryos from each group were either vitrified using a solid surface vitrification procedure (Somfai et al. 2009 Biol. Reprod. 80, 42–49) or cultured for 7 d without being vitrified. Vitrified zygotes were subsequently warmed and cultured for 7 d. The rates of cleavage, blastocyst formation, and hatching were recorded, and all blastocysts were stained to determine the total cell numbers. Three replicates were performed. Proportional data were arcsine transformed and subjected to ANOVA, and cell number data were analysed by t-test. The post-thaw survival rates of the embryos that were vitrified did not differ between the groups (control: 95.7%; LC: 97.1%; P > 0.05). There were no significant effects of LC treatment or vitrification on the rates of cleavage, blastocyst formation, and hatching (Table 1). Vitrified embryos derived from LC-treated oocytes produced blastocysts with fewer cells than vitrified embryos derived from untreated oocytes (Table 1; P < 0.05). In contrast to previous findings in other species, the results indicate that supplementing IVM medium with LC did not enhance the post-thaw development of porcine embryos vitrified at the pronuclear stage. Table 1.Effect of l-carnitine (LC) treatment and vitrification on porcine embryo development C. Grupen was supported by an OECD Fellowship.

The effect of steer sera generating low or high concentrations of ammonia in culture on bovine embryo development and cell number in vitro

1999 ◽  
Vol 1999 ◽  
pp. 2-2 ◽  
Author(s):  
M. Kuran ◽  
M.E. Staines ◽  
G.J. McCallum ◽  
A.G. Onal ◽  
T.G. McEvoy
Keyword(s):  
Embryo Development ◽  
Cell Number ◽  
Early Embryo ◽  
Bovine Embryo ◽  
Cleavage Rate ◽  
Cell Monolayers ◽  
High Concentrations ◽  
Oviduct Fluid ◽  
Bovine Oocytes

Ovine embryos produced in synthetic oviduct fluid (SOF) medium or in coculture with granulosa cell monolayers supplemented with low (A; 120 μmol/l) and high (B; 190 μmol/l) ammonia-producing steer sera caused different degrees of fetal oversize (Carolan et al., 1998). The objective of the present study was to determine whether the effects on fetal growth induced by these sera were associated with alterations in early embryo development.A total of 911 bovine oocytes, used in 8 replicates to test the effect of three culture treatments on embryo development, were matured and fertilized in vitro (IVF= Day 0). Presumptive zygotes were allocated on Day 1 to culture in SOF supplemented with 10% v/v steer serum (SOF+A, n=308; SOF+B, n=302) or with amino acids plus 0.4% w/v crystalline BSA (SOFaaBSA, n=301). All cultures were in 20 μl droplets under oil (38.5°C; 5% CO2, 5% O2; 4 zygotes per drop) and droplets were renewed every 48 h. Cleavage rate was recorded on Day 3. On Days 7 and 8, blastocyst yields, grade 1 and 2 blastocysts, their cell numbers (by staining with Hoechst 33342) and their stage and diameter were determined.

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