140 Effect of addition of different concentrations of L-carnitine during porcine in vitro maturation on embryo quality and development

2021 ◽  
Vol 33 (2) ◽  
pp. 178
Author(s):  
P. R. Cruzans ◽  
M. S. Lorenzo ◽  
G. M. Teplitz ◽  
C. G. Luchetti ◽  
D. M. Lombardo
Keyword(s):  
Rate Control ◽  
Embryo Quality ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Cleavage Rate ◽  
Apoptosis Index ◽  
Significant Difference ◽  
Chi Squared ◽  
Blastocyst Rate ◽  
Number Of Cells

l-Carnitine (LC) plays an important role in the catabolism of lipids and protects cells from the damage caused by reactive oxygen species due to its antioxidant activity. The aim of this study was to evaluate the effect of adding different concentrations of LC during porcine invitro maturation on embryo quality and development. The cumulus–oocyte complexes were obtained by follicular aspiration from ovaries of slaughtered sows and matured invitro for 44h without LC (control) or with different concentrations of LC (0.6 or 1.25mg mL−1) (Sigma-Aldrich) in TCM-199 supplemented with human menopausal gonadotrophin and cyclic AMP (cAMP) during the first 22h. Invitro fertilization was performed with fresh boar semen for 4h in 100-µL drops of TCM-199 with caffeine, bovine serum albumin, sodium lactate, and pyruvate (20 denuded oocytes per drop, 1×106 spermatozoa mL−1). Presumptive zygotes were washed and cultured in NCSU 23 at 39°C, 7% O2, 5% CO2, and humidity. The cleavage rate was registered on Day 2 and the blastocyst rate on Day 7. Embryo quality was assessed by counting the number of cells per blastocyst (Hoescht 33342) and late apoptosis index (TUNEL-positive cells/total cells). Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) was performed according to the kit protocol (Roche). LC significantly decreased the cleavage rate (control: 46.2%; LC0.6: 32.1%; LC1.25: 37.9%; P<0.05, Chi-squared test). No significant differences were detected in the blastocyst rate (control: 19.2%; LC0.6: 17%; LC1.25: 10,2%, Chi-squared test) or in number of cells per blastocyst (control: 51.97±3; LC0.6: 56.11±4; LC1.25: 45.62±4, ANOVA). There was embryo hatching in LC treatments but not in the control (control: 0%, LC0.6: 11%; LC1.25: 7.6%). The apoptosis index decreased in LC1.25 compared with LC0.6 (Control: 7,6±1.3%; LC0.6: 10±1.1%; LC1.25: 5,5±0.8%; P<0.05, ANOVA) but there was no significant difference in the apoptosis index between control and LC treatments. In conclusion, LC treatments decreased the cleavage rate but did not modify the blastocyst rate and allowed embryo hatching.

65 Co-culture of porcine epithelial oviductal cells and invitro-produced embryos: Effect on embryo development and quality

2021 ◽  
Vol 33 (2) ◽  
pp. 139
Author(s):  
M. S. Lorenzo ◽  
G. M. Teplitz ◽  
P. R. Cruzans ◽  
C. G. Luchetti ◽  
J. Ghersa ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Embryo Development ◽  
Bovine Serum ◽  
Embryo Quality ◽  
Culture Conditions ◽  
Sodium Pyruvate ◽  
Apoptosis Index ◽  
Chi Squared ◽  
Blastocyst Rate ◽  
Number Of Cells

The oviduct is involved in many reproductive functions, including early embryo development. The epithelial cells that cover the oviduct produce oviducal fluid and could be used to recreate the invivo environment into which embryo development takes place. This study aimed to evaluate the co-culture of porcine embryos with a monolayer of porcine oviducal epithelial cells (POEC) and its effect on embryo development and quality. The POEC were obtained by pressing the isthmus (from diestrus sow oviducts) using slides and performing 3 cycles of vortexing and decanting in DMEM-F12 medium. Passage 1 cells were used for these experiments (POEC-1). Oocytes were obtained from follicular aspiration of slaughterhouse ovaries. Oocytes were invitro matured for 44h in TCM-199 supplemented with human menopausal gonadotrophin and cyclic AMP during the first 22h. Invitro fertilization was performed with 17°C-refrigerated boar semen for 4h in 100-µL drops of TCM-199 with caffeine, bovine serum albumin, sodium lactate, and sodium pyruvate (20 denuded oocytes per drop, 1×106 spermatozoa mL−1). Presumptive zygotes were washed and randomly assigned to one of the following groups for invitro culture: control (50-µL drop of NCSU-23 with sodium pyruvate and lactate), POEC-1 (same as the control+POEC-1 50 000 cells mL−1), POEC-1+FBS (same as the control+POEC-1 50 000 cells mL−1 and 2.5% of fetal bovine serum). Culture conditions were 7% O2, 5% CO2, 39°C, and humidity. On Day 2, the cleavage rate was recorded, and embryos were transferred to NCSU-23 drops with glucose and without cells. The blastocyst rate was recorded on Day 7. Embryo quality was assessed by counting the number of cells per blastocyst (Hoechst) and the apoptosis index (TUNEL-positive cells/total cells). Co-culture with POEC-1 significantly increased the blastocyst rate (control: 14%; POEC-1+FBS: 10%; POEC-1: 28%; P<0.05 Chi-squared test) and allowed embryo hatching (control: 0; POEC-1+FBS: 22.2%; POEC-1: 7; P<0.05 Chi-squared test). However, there was no significant difference in the number of cells per blastocyst (control: 58.6±6; POEC-1+FBS: 50.3±3.7; POEC-1: 50.6±4.8; nonparametric ANOVA) or in the apoptosis index (control: 8.1; POEC+FBS 8.3; POEC: 7.4; nonparametric ANOVA). The use of POEC-1 during the first 2 days of embryo culture enhanced embryo development and improved culture conditions, allowing embryo hatching. The effect on embryo development could be due to an effect of POEC itself or the effect of feeder cells. Other parameters of embryo quality should be evaluated in the future.

Cryotolerance of in vitro-produced porcine blastocysts is improved when using glucose instead of pyruvate and lactate during the first 2 days of embryo culture

2013 ◽  
Vol 25 (5) ◽  
pp. 737 ◽  
Author(s):  
M. Castillo-Martín ◽  
M. Yeste ◽  
R. Morató ◽  
T. Mogas ◽  
S. Bonet
Keyword(s):  
Survival Rate ◽  
Treatment Group ◽  
In Vitro Culture ◽  
Embryo Development ◽  
Embryo Culture ◽  
Embryo Quality ◽  
Cleavage Rate ◽  
Apoptosis Index ◽  
Number Of Cells

The objective of the present study was to determine the effects of replacing glucose with pyruvate and lactate during the first 48 h of in vitro culture (IVC) in NCSU-23 medium on embryo development, embryo quality and survival of porcine blastocysts after vitrification. To this end, in vitro-produced (IVP) porcine oocytes were cultured with either glucose for 6 days (IVC-Glu) or pyruvate–lactate from Day 0 to Day 2 and then with glucose until Day 6 (IVC-PyrLac). Blastocysts were vitrified on Day 6 using the Cryotop device and, after warming, survival rate and the apoptosis index were evaluated after 24 h incubation in NCSU-23 medium. No significant differences were observed between IVC-Glu and IVC-PyrLac in terms of cleavage rate, blastocyst yield, total number of cells per blastocyst or the apoptosis index (1.82 ± 0.75% vs 3.18 ± 0.88%, respectively) of non-vitrified embryos. However, a significant increase was seen in hatching/hatched blastocysts in the IVC-PyrLac compared with IVC-Glu treatment group (12.71 ± 1.20% vs 3.54 ± 0.47%, respectively). Regardless of treatment, vitrification impaired the survival rate and the apoptosis index. When comparing both treatments after warming, the percentage of apoptotic cells was significantly higher for blastocysts in the IVC-PyrLac compared with IVC-Glu group (18.55 ± 3.49% vs 9.12 ± 2.17%, respectively). In conclusion, under the conditions of the present study, replacement of glucose with pyruvate–lactate during the first 48 h of culture resulted in a lower cryotolerance of IVP porcine embryos.

171 EFFECT OF LONG-TERM TRANSPORTATION OF OVARIES ON THE DEVELOPMENT OF IN VITRO-PRODUCED BOVINE EMBRYOS

2006 ◽  
Vol 18 (2) ◽  
pp. 193
Author(s):  
M. Nakatate ◽  
K. Tsuchiya ◽  
I. Adachi ◽  
K. Takahashi ◽  
A. Aisan ◽  
...  
Keyword(s):  
Bovine Embryos ◽  
Cleavage Rate ◽  
Chi Square ◽  
Vitro Fertilization ◽  
Thermos Flask ◽  
Blastocyst Rate ◽  
Functional Peptides ◽  
Number Of Cells

The transportation of bovine ovaries would allow the shipment of oocytes for research purposes after the slaughter of valuable cows. The objective of this study was to investigate the effect of long-term transportation of ovaries on the development of in vitro-produced bovine embryos. After collection of the ovaries from a slaughterhouse, they were placed inside a thermos flask and transported to the laboratory. The thermos flask was covered with a freezer pack in a foam polystyrene box. The transportation time was 17–18 h, and the temperature of the thermos flask changed from 20°C to 28°C (average 23.8°C) during the transportation. Cumulus–oocyte complexes (COCs) were collected by the aspiration of follicles with a diameter of 2–6 mm. The COCs were matured for 20 h in IVMD101 (RIFP: Research Institute for the Functional Peptides, Yamagata, Japan) containing DM199 supplemented with 5.56 mM glucose, 0.91 mM pyruvate, 5 mM taurine, 5 mM selenium, 5 mM HEPES, and 10 µg/mL gentamicin at 38.5°C under an atmosphere of 5% CO2 in air (Hoshi 2003 Theriogenology 59, 675–685). The matured COCs were inseminated with 5 × 106 sperm/mL in IVF100 (RIFP) medium comprising a modified BO medium supplemented with 1.25 mM sodium pyruvate, 0.5 mM cysteine, 5 mg/mL BSA, 7.5 µg/mL sodium heparin, 5 mM caffeine, and 10 µg/mL gentamicin. After 6 h of gamete co-culture, the presumed zygotes were cultured in IVD101 (RIFP) medium comprising DM199, 2.48 mM lactate, 0.27 mM pyruvate, and 2.22 mM of glucose for 9 days at 38.5°C under an atmosphere of 5% CO2, 5% O2, and 90% N2 in air. As controls, bovine ovaries were transported to the laboratory within 1–1.5 h. Embryo development was evaluated based on the cleavage rate, blastocyst rate, and total number of cells on Days 7–9 after in vitro fertilization. The experiment was replicated five times, and data were analyzed by chi-square test and ANOVA. Results are presented in Table 1. There were no differences in the cleavage rate between the treatments. The blastocyst rate and the number of cells in the blastocyst after long-term transportation of ovaries were significantly lower than those in the controls. These results suggest that the long-term transportation of bovine ovaries does not affect on the cleavage; however, the blastocyst rate and the quality of blastocysts may be affected. Therefore, additional experiments are required to determine suitable conditions for long-term transportation of bovine ovaries. Table 1. Effect of long-term transportation of ovaries on the development of bovine IVM/IVF embryos

141 BIOPSY TECHNIQUE IN BOVINE EMBRYOS PRODUCED IN VITRO AT EARLY STAGES OF DEVELOPMENT: EVALUATION OF QUALITY AND DEVELOPMENT CAPACITY

2008 ◽  
Vol 20 (1) ◽  
pp. 151
Author(s):  
J. Polisseni ◽  
M. O. Guerra ◽  
R. V. Serapião ◽  
M. M. Pereira ◽  
I. M. Folhadella ◽  
...  
Keyword(s):  
Preimplantation Genetic Diagnosis ◽  
Embryo Quality ◽  
Genetic Diagnosis ◽  
Blastocyst Stage ◽  
Chromosome Abnormalities ◽  
Control Group ◽  
Bovine Embryos ◽  
Blastocyst Rate ◽  
Number Of Cells

One of the causes of embryo mortality is chromosome abnormalities that occur during gametogenesis, fertilization, and embryo early development. Thus, a combination of morphological standards and techniques of molecular analyses could identify abnormal embryos. Preimplantation genetic diagnosis (PGD) is an emergent technology for use with farm animal embryos. With this procedure, blastomeres are removed by the biopsy of embryos at the 8- to 16-cell stage to provide cells for analyses of chromosome abnormalities prior to transfer. The aim of this study was to evaluate the effect of biopsy in bovine 8- to 16-cell embryos fertilized in vitro on embryo quality and subsequent development in vitro. A group of 706 oocytes were obtained from slaughterhouse ovaries, matured, and fertilized in vitro at 38.8�C with 95% humidified air and 5% CO2. The zygotes were semi-denuded and cultured in CR2aa medium under the same conditions as for in vitro fertilization. The rate of cleavage was 78.20%. Three days after fertilization, part of the 8- to 16-cell (298/706) embryos were distributed randomly across two groups: control (n = 103) and biopsy (n = 92) of blastomeres, and then returned to in vitro embryo culture to evaluate development until the blastocyst stage and the capacity to hatch. The amount of cells removed was one-fourth of the embryo. The blastocyst rate was evaluated on Day 8 after fertilization and the hatching rate on Day 10. Embryo morphology and quality were evaluated as previously described in the International Embryo Transfer Society manual (1998). To evaluate overall quality, embryos were stained on the 10th day of culture and the blastomeres were counted with the imaging software AxioVision 3.1 (Carl Zeiss, Feldbach, Switzerland). The blastocyst rate was analyzed by treatment groups with the chi-square test and the number of cells/embryo was analyzed by ANOVA with SAS (SAS Institute, Inc., Cary, NC, USA). The percentage of 8- to 16-cell embryos that developed to the blastocyst stage was similar (P > 0.05) between the control (66.0%, 68/103) and the biopsied (53.3%, 49/92) groups. Furthermore, no difference was noted in the hatching rates between the control group and the biopsied group (42.6%, 29/42 v. 44.9%, 22/49, respectively). Overall, no impact was detected on embryo quality from embryo biopsy with no difference in mean (�SE) blastocyst cell number between the control group (blastocysts: 67.1 � 3.1; expanded blastocysts: 100.7 � 6.9; hatched blastocysts: 189.9 � 16.1) and the biopsied group (blastocysts: 61.1 � 5.5; expanded blastocysts: 121.87 � 10.6; hatched blastocysts: 187.3 � 18.5). In conclusion, the biopsy used on 8- to 16-cell bovine IVF-derived bovine embryos does not affect the subsequent embryo development and number of cells/embryo or blastocyst, showing that it can be used to provide genetic material for preimplantation genetic diagnosis without affecting embryo quality. This work was supported financially by FAPEMIG.

scholarly journals Effect of deslorelin acetate treatment in oocyte recovery and in vitro embryo production in domestic cats

2016 ◽  
Vol 19 (10) ◽  
pp. 1091-1095
Author(s):  
Camila Louise Ackermann ◽  
Eduardo Trevisol ◽  
Leticia Ferrari Crocomo ◽  
Tatiana da Silva Rascado ◽  
Rodrigo Volpato ◽  
...  
Keyword(s):  
Treated Group ◽  
Control Group ◽  
Domestic Cats ◽  
Cleavage Rate ◽  
Embryo Production ◽  
Oocyte Recovery ◽  
Significant Difference ◽  
In Vitro Embryo Production ◽  
Blastocyst Rate

Objectives The present study investigated the effect of contraceptive treatment with deslorelin acetate on in vitro embryo production and oocyte recovery in domestic queens. Methods Twenty-one mature domestic cats were used. Eleven queens (treated group) and one tom were kept in an experimental cattery, and 10 queens were privately owned (control group). When in interestrus or diestrus (day 0) a deslorelin acetate implant (Suprelorin, 4.7 mg/animal) was inserted into the subcutaneous tissue of the interscapular region in all queens in the treated group. After 6 months of treatment, all animals were ovariohysterectomized, and the ovaries were used for in vitro embryo production. Percentage of cleavage was determined 18 h after oocyte insemination and blastocyst formation was assessed on the eighth day of culture. The rate of cumulus-oocyte complexes (COCs) recovery was analyzed by an unpaired t-test. The cleavage and blastocyst rates were expressed as percentages and analyzed by Fisher’s exact test. All analyses were performed using GraphPad Prism v5.0, with P <0.05 set as the level of significance. Results In the treated group, we recovered 8.3 ± 1.15 grade I COCs per queen; the cleavage rate was 60% and the blastocyst rate was 36%. In the control group, we recovered 18.4 ± 3.21 grade I COCs per queen; the cleavage rate was 55.97% and the blastocyst rate was 34%. Forty percent of treated females did not produce any blastocysts. In the treated group, we observed a significant decrease in COC recovery. Although there was no significant difference in cleavage and blastocyst rates between groups, 40% of treated females did not produce any blastocysts. Conclusions Recovery of grade I COCs is negatively affected by deslorelin treatment in domestic cats. Regarding embryo production, new studies are still necessary to evaluate the success of this technique owing to the individual effect caused by deslorelin acetate.

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.

60 THE EFFECT OF L-ASCORBIC ACID DURING CULTURE, CRYOPRESERVATION, OR BOTH ON PORCINE EMBRYOS PRODUCED IN VITRO

2013 ◽  
Vol 25 (1) ◽  
pp. 177 ◽  
Author(s):  
M. Castillo-Martín ◽  
M. Yeste ◽  
R. Morató ◽  
T. Mogas ◽  
S. Bonet
Keyword(s):  
Ascorbic Acid ◽  
Embryo Quality ◽  
Cell Number ◽  
Total Cell ◽  
In Vitro Fertilisation ◽  
Blastocyst Formation ◽  
Total Cell Number ◽  
Apoptosis Index ◽  
Number Of Cells

The benefits of adding l-ascorbic acid during the cryopreservation procedure have been reported before in mouse and bovine. In this study, the effects of l-ascorbic acid (AC) supplementation during culture, cryopreservation, or both procedures on the developmental ability and embryo quality of in vitro produced porcine blastocysts were examined. Embryo quality criteria consisted of total cell number, percentage of apoptosis, and cryotolerance. After in vitro fertilisation, presumptive zygotes were randomly assigned to 2 culture treatments in which the culture medium NCSU23 was supplemented with 100 µM AC (n = 1162) or nonsupplemented (n = 1163) for a 144-h period. On Day 6, blastocyst formation was assessed by stereomicroscopy, and a representative fraction of Grade I- and II-blastocysts of each culture treatment was evaluated using 4′,6-diamidino-2-phenylindole-TUNEL co-staining and considered as fresh-control. The remaining fraction of Grade I- and II-blastocysts was vitrified/warmed following the Cryotop® method. To determine the effect of AC supplementation during cryopreservation procedures, each culture treatment was divided into 2 groups: (1) embryos exposed to 100 µM AC, and (2) nonexposed embryos (vitrified-control). Survival was determined according to reexpansion rates after 24 h of recovery in NCSU23 medium. After 24 h, reexpanded blastocysts were co-stained using the 4′,6-diamidino-2-phenylindole-TUNEL technique, and total number of cells and apoptosis indexes were determined. Experiment was replicated 9 times for each group. Data were analyzed by t-test for independent variables and a 2-way ANOVA. Results are expressed as means ± SE, and the significant level was set at 5% (Table 1). After culture, supplementing NCSU23 medium with AC showed no significant differences in blastocyst formation (fresh-control 11.6 ± 7.8 v. AC 11.6 ± 7.7), in number of cells (fresh-control 36.7 ± 15.8 v. AC 36.1 ± 15.9), or in apoptosis index (fresh-control 2.9 ± 5.7 v. AC 3.5 ± 4.7). On the other hand, only when both culture and vitrified media were supplemented with AC was there a significant increase of blastocyst survival. In contrast, no significant differences in embryo survival were observed when only 1 of these 2 media (culture or vitrification) was supplemented. Supplementing culture media or cryopreservation solutions with AC did not affect the total cell number or apoptosis index in vitrified blastocysts. In conclusion, the addition of 100 µM l-ascorbic acid to the culture and cryopreservation solutions improves the cryotolerance of in vitro-produced porcine blastocysts. Table 1.Survival of blastocysts (24 h), total cell number, and percentage of apoptosis after vitrification/warming

253 DEVELOPMENTAL COMPETENCE OF BOVINE OOCYTES MATURED IN SERUM-FREE MEDIUM UNDER LOW OXYGEN TENSION

2009 ◽  
Vol 21 (1) ◽  
pp. 224
Author(s):  
M. M. Pereira ◽  
F. Q. Costa ◽  
P. H. A. Campos ◽  
R. V. Serapiao ◽  
J. Polisseni ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
Embryo Quality ◽  
Cell Number ◽  
Total Cell ◽  
Developmental Competence ◽  
Cleavage Rate ◽  
Total Cell Number ◽  
Blastocyst Rate ◽  
Bovine Oocytes

In vitro maturation (IVM) is a critical step in in vitro bovine embryo production. A number of factors can influence the IVM environment, such as media composition and protein supplementation. Serum and higher O2 tension have been shown to reduce embryo quality; however, little is known about the effects of serum and O2 tension during IVM on embryo quality and development. This study aimed to evaluate the effect of serum and O2 tension on IVM of bovine oocytes. Immature oocytes obtained from slaughterhouse ovaries were randomly distributed in 4 groups of IVM: G1 (n = 253), 0.1% polyvinyl alcohol (PVA) in air; G2 (n = 248), 10% inactivated estrous cow serum (ECS) in air; G3 (n = 270), 0.1% PVA under 5% O2; and G4 (n = 236), 10% ECS under 5% O2. In vitro maturation was performed with TCM-199 culture medium supplemented with 20 μg mL–1 FSH, under 5% CO2 at 38.5°C for 24 h. After maturation, oocytes were in vitro fertilized with 2.0 × 106 sperm mL–1 in Fert TALP medium, supplemented with heparin, for 20 h. Presumptive zygotes were denuded by vortexing and cultured in CR2aa medium with 2.5% fetal calf serum under 5% CO2 and 5% O2 at 38.5°C. Cleavage rate was evaluated 72 h postfertilization, and blastocyst rate and total cell number were evaluated 8 days postfertilization. Morphological classification of embryos was performed at Day 8 according to the International Embryo Transfer Society manual (1998). Cleavage, blastocyst, and grade 1 embryo rates were analyzed by chi-square, and total cell number was analyzed by ANOVA, with means compared by LSD. Results are presented as mean ± SEM. There was no difference (P > 0.05) in cleavage rates among G1, G2, and G4 (61.6, 65.3, and 57.6%, respectively), but cleavage rate was lower (P < 0.05) in G3 (52.5%). Blastocyst rates among G1, G3, and G4 (15.8, 17.7, and 20.3%, respectively) were similar (P > 0.05). However, blastocyst rate in G2 (25.0%) was higher (P < 0.05) than in G1 and G3, but was similar to G4 (P > 0.05). Total cell number was similar (P > 0.05) among G2 (194.1 ± 13.1), G3 (173.3 ± 9.0), and G4 (163.8 ± 8.7), but was lower (P < 0.05) in G1 (124.5 ± 11.4). The grade 1 embryo rate was lower (P < 0.05) in G1 (70.3%) than in G2 (89.5%), but was similar (P > 0.05) to G3 (77.0%) and G4 (83.9%). The results suggest that IVM with PVA in TCM-199 medium under 5% O2 can be performed without reducing embryo development and quality, when compared with ECS. On the other hand, oocyte developmental competence seems to be affected when IVM is performed with PVA under air conditions. Financial support: CNPq, FAPEMIG.

74 Treatment with Melatonin During In Vitro Culture Enhances Porcine Parthenogenetically Activated Embryo Development

2018 ◽  
Vol 30 (1) ◽  
pp. 175
Author(s):  
G. A. Kim ◽  
J.-X. Jin ◽  
S. Lee ◽  
A. Taweechaipaisankul ◽  
B. C. Lee
Keyword(s):  
Oxidative Stress ◽  
Embryo Development ◽  
Developmental Competence ◽  
Free Radical Scavengers ◽  
Control Group ◽  
Cleavage Rate ◽  
Cell Numbers ◽  
Significant Difference ◽  
Blastocyst Rate

Melatonin and its metabolites are powerful antioxidants and free radical scavengers. Because porcine embryos are vulnerable to oxidative stress in vitro, the addition of various protective chemicals to the culture medium, including melatonin, has been explored. The aim of this study was to investigate the effect of melatonin on in vitro developmental competence of porcine parthenogenetically activated (PA) embryos. Immature cumulus–oocyte complexes (COC) were collected and cultured in medium comprising TCM-199 supplemented with 10 ng mL−1 epidermal growth factor, 0.57 mM cysteine, 0.91 mM sodium pyruvate, 5 μL mL−1 insulin, transferrin selenium solution 100×, 10% porcine follicular fluid, 10 IU mL−1 eCG, and 10 IU mL−1 hCG for 44 h. Then, COC were denuded and PA with electrical stimulation, and PA embryos were cultured in porcine zygote medium 5 (PZM-5) supplemented with melatonin at increased concentrations (10−9, 10−7, 10−5 M) at 39°C in a humidified atmosphere of 5% O2, 5% CO2, and 90% N2 for 7 days. Subsequent embryo development, including cleavage rate, blastocyst rate, and blastocyst cell numbers, was compared between groups (mean no. of embryos; control, 27.14; 10−9 M, 28.86; 10−7 M, 27.71; 10−5 M, 26.43). The experiments were repeated 7 times for each treatment group. Statistical analyses of all data were performed using one-way ANOVA with Dunn’s multiple comparison test. Results are expressed as the mean ± SEM and all differences were considered significant at P < 0.05. No apparent effect on cleavage rate of melatonin treatment of various concentrations was noted. Blastocyst cell number did not show any significant difference between groups. However, the potential of PA oocytes to develop into blastocysts was significantly higher in the group supplemented with 10−9 M melatonin compared with the control group (35.44 ± 3.84 v. 24.71 ± 1.59) and other melatonin treated groups (10−5 M, 21.35 ± 2.82; 10−7 M, 24.01 ± 2.31; P < 0.05). These indicated that treatment with 10−9 M melatonin in embryo culture might reduce the oxidative stress properly compared with other concentrations, which results in improvement of blastocyst rate formation. In conclusion, treatment with 10−9 M melatonin positively promoted the blastocyst formation rate of porcine PA embryos with no beneficial effects on their blastocyst cell numbers or cleavage rate. This study was supported by the National Research Foundation (#2015R1C1A2A01054373; 2016M3A9B6903410), Research Institute for Veterinary Science and the BK21 PLUS Program.

In vitro developmental competence of pig nuclear transferred embryos: effects of GFP transfection, refrigeration, cell cycle synchronization and shapes of donor cells

Zygote ◽  
2006 ◽  
Vol 14 (3) ◽  
pp. 239-247 ◽  
Author(s):  
Yun-Hai Zhang ◽  
Deng-Ke Pan ◽  
Xiu-Zhu Sun ◽  
Guo-Jie Sun ◽  
Xiao-Hui Liu ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Fusion Rate ◽  
Cleavage Rate ◽  
Preparation Methods ◽  
Transfected Cells ◽  
Cell Cycle Synchronization ◽  
Donor Cells ◽  
Significant Difference ◽  
Blastocyst Rate

SummaryThe present study was designed to evaluate the feasibility of producing pig transgenic blastocysts expressing enhanced green fluorescent protein (GFP) and to examine the effects of shape and preparation methods of donor cells on in vitro developmental ability of pig nuclear transferred embryos (NTEs). In experiment 1, the effect of GFP transfection on development of pig NTEs was evaluated. The cleavage and blastocyst rates showed no significant difference between NTEs derived from transfected and non-transfected donors. In experiment 2, the effect of different nuclear donor preparation methods on in vitro development of NTEs was examined. The cleavage rate showed no statistically significant differences among three preparation methods. The blastocyst rates of donor cells treated once at −4 °C and those of freshly digested cells were similar to each other (26.3% vs 17.9%). The lowest blastocyst rates (5.88%) were observed when cells cryopreserved at −196 °C were used as donors. In experiment 3, the effect of different cell cycle synchronization methods on the in vitro development potential of pig NTEs was evaluated. The cleavage rate of NTEs derived from cycling cells was much better than that of NTEs derived from serum-starved cells (64.4% vs 50.5%, p < 0.05), but no significant difference was observed between the the blastocyst rates of the two groups. In experiment 4, the effect of different shapes of cultured fibroblast cells on the in vitro development of pig NTEs was examined. The fusion rate for couplets derived from rough cells was poorer than that observed in couplets derived from round smooth cells (47.8% vs 76.8%, p < 0.05). However, there were no significant differences observed in the cleavage rate and blastocyst rate. In conclusion, the present study indicated that (i) refrigerated pig GFP-transfected cells could be used as donors in nuclear transfer and these NTEs could be effectively developed to blastocyst stage; (ii) serum starvation of GFP-transfected cells is not required for preimplantation development of pig NTEs; and (iii) a rough surface of GFP-transfected donor cells affects fusion rate negatively but has no influence on the cleavage rate or blastocyst rate of pig NTEs.

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