155 EFFECTS OF CULTURE WITH OVALBUMIN IN ABSENCE OF FETAL BOVINE SERUM AND BOVINE SERUM ALBUMIN ON IN VITRO PRODUCTION OF CATTLE EMBRYOS

2010 ◽  
Vol 22 (1) ◽  
pp. 236
Author(s):  
T. A. D. Tetzner ◽  
N. Z. Saraiva ◽  
C. S. Oliveira ◽  
S. C. Méo ◽  
M. M. Souza ◽  
...  
Keyword(s):  
Bovine Serum ◽  
Cell Number ◽  
Differential Staining ◽  
Control Group ◽  
Cortical Granule ◽  
In Vitro Production ◽  
Nuclear Maturation ◽  
Fetal Bovine ◽  
Vitro Production

Embryo quality is influenced by culture conditions, which affect IVM, IVF, and IVC rates. One of the most efficient ways to evaluate the embryonic quality of IVP blastocysts is by differential staining of inner cell mass (ICM) and trophoblast (TF). Bovine embryos of superior quality should present the total number of cells close to the number of cell cycles (Neuber et al. 2002 Theriogenology 57, 2193-2202). In this study, we analyzed the effects of fetal bovine serum (F) and bovine serum albumin (B) replacement for ovalbumin (O) on nuclear maturation, cortical granule migration, pronuclear development, blastocyst rates, and differential staining of ICM and TF in Day 7 blastocysts. The treatment groups were named as follows: the first letter is the protein source used for IVM, the second for IVF, and the third for IVC. When 2 protein sources were used in the same step, the plus symbol (+) was used. The oocytes were IVM in TCM-199, supplemented with the following: 10% F, or 4 mg mL-1 B, or 4 mg mL-1 O, and 1.0 Âμg mL-1 of FSH, 50 Âμg mL-1 of hCG, 1.0 Âμg mL-1 of estradiol, 0.2 mM sodium pyruvate, and 83.4 Âμg mL-1 of amikacin. IVF was accomplished in TALP-IVF medium, with 0.2 mM pyruvate, 83.4 Âμg mL-1 of amikacin, and 6 mg mL-1 B or O. IVC was in SOF, with F, B, or O. The control group (CONT) consisted of the treatment FBF + B. Pronuclear development was compared by the chi-square test, whereas the other results were analyzed by ANOVA followed by the Tukey test, using SAS at 5% significance level (SAS Institute Inc., Cary, NC, USA). For IVM, the treatments F, B, O, and B + O did not affect (P > 0.05) nuclear maturation (73.92 to 78.78%) and cortical granule migration rates (58.89 to 66.76%). Regarding pronuclear development, the treatment FO (76.67%) was similar (P > 0.05) to the control group (82.95%), which was superior (P < 0.05) to the treatments BB (56.98%), BO (39.02%), OB (37.36%), and OO (39.24%). Blastocyst rates in FBF (42.8%) and control (45.0%) groups were superior (P < 0.05) to treatment OOO (26.0%) but similar (P > 0.05) to FOF, BBB, BOB, and OBO (32.0 to 35.8%). The average of blastocyst ICM cells of the group OOO (16.79) was inferior (P < 0.05) to the other groups. However, the average of TF cells on blastocysts of the group OOO (38.25) was similar (P > 0.05) to the groups BBB (45.74) and BOB (45.60) and inferior (P < 0.05) to the groups CONT (57.59), FBF (54.41), FOF (56.74), and OBO (47.35). The total average cells in the blastocysts of the group OOO (56.04) was inferior (P < 0.05) to the groups CONT (84.86), FBF (78.96), FOF (81.32), BBB (68.11), BOB (69.55), and OBO (69.82). The total cell number in the treatments, with several sources of protein supplementation, varied from 56.04 to 84.86. Considering the evaluation interval, this average cell number was discreetly inferior to that expected for the chronological age of the blastocysts. We concluded that it is possible to produce bovine embryos in the absence of F and/or B, with the protein source O, although it reduced blastocyst rates when used in all 3 steps of embryo in vitro production and resulted in blastocysts of inferior quality. Financial support: FAPESP 05/60389-2 and CNPq.

scholarly journals Stimulatory Role of Glycated Fetal Bovine Serum Along with Iron on In Vitro Production of Insulin by Differentiated Mouse Embryonic Stem Cells

2011 ◽  
Vol 57 (4) ◽  
pp. 356-361
Author(s):  
Ikuo Nishigaki ◽  
Gowri Rangasamy Gunassekaran ◽  
Panjan Nagappan Venkatesan ◽  
Mandupal Chaco Sabu ◽  
Sabu Priya ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Bovine Serum ◽  
Fetal Bovine Serum ◽  
Embryonic Stem ◽  
In Vitro Production ◽  
Fetal Bovine ◽  
Vitro Production

298 SPERM CAPACITATED WITH CALCIUM IONOPHORE AS A VECTOR FOR IN VITRO PRODUCTION OF BOVINE TRANSGENIC EMBRYOS

2006 ◽  
Vol 18 (2) ◽  
pp. 256
Author(s):  
R. Simões ◽  
M. P. Milazzotto ◽  
C. Yamada ◽  
W. B. Feitosa ◽  
A. R. S. Coutinho ◽  
...  
Keyword(s):  
Cell Monolayer ◽  
Calcium Ionophore ◽  
Control Group ◽  
Sperm Cells ◽  
In Vitro Production ◽  
Successful Technique ◽  
Blastocyst Rate ◽  
Transgenic Embryos ◽  
Vitro Production

Production of transgenic mouse embryos by microinjection is a well established and successful technique. However, when microinjection protocols were used for bovine, the amount of the oocyte lipid content did not allow the production of bovine transgenic embryos. Sperm-mediated gene transfer (SMGT) is an alternative for this species because it has lower cost and does not require microinjection handling. One of the procedures to introduce exogen DNA into oocytes is by means of sperm capacitated with calcium ionophore (CaI). The aim of this work was to evaluate different CaI concentrations ([CaI]), sperm incubation times with CaI (tCa), and incubation times of sperm capacitated with DNA (tDNA) (EYFP; Clontech, Palo Alta, CA, USA) to establish a satisfactory method for IVP of bovine transgenic embryos. Slaughterhouse oocytes with compact cumulus and uniform ooplasm were in vitro maturated in TCM-199 medium + 10% FCS + FSH + hCG + estradiol (E2) + piruvate + gentamicin under 5% CO2 in air, at 39�C and high humidified atmosphere for 24 h. Semen was thawed in a water bath at 37�C for 30 s and separated by Percoll gradient (45/90%) at 600g for 30 min. After this procedure, sperm cells were washed in TALP-semen medium by centrifugation at 200g for 5 min at room temperature. Supernatant was removed and capacitation (5 � 106 spermatozoa/group) was induced with CaI (250 nM or 500 nM for 1 or 5 min). Capacitated sperm cells were incubated with 500 ng/mL DNA for 1 or 2 h. Nontreated spermatozoa were used as control group. Sperm cells (1 � 105) were used to inseminate 20 oocytes/90 mL microdroplets for 18 h. The presumptive zygotes were co-cultured in SOFaa medium with a granulosa cell monolayer under high humidified atmosphere, at 39�C and 5% CO2 in air. Blastocyst rates were analyzed by ANOVA. Independent variables were replicate, [CaI], tCa, tDNA, and the double and triple interactions among the last three variables; when appropriate, means were compared by orthogonal contrasts. There was [CaI] � tCa � tDNA interaction for blastocyst rate (P < 0.02). Treatments with 250 nM ([CaI]), 5 min (tCaI), and 1 h (tDNA) or 500 nM ([CaI]), 1 min (tCaI), and 1 h (tDNA) resulted in 36.1% and 37.4% blastocyst rates, respectively, similar to the control group (30.5%; P > 0.4). These results demonstrated that it is possible to capacitate spermatozoa with CaI to produce transgenic embryos, without alteration of blastocyst rate. This work was supported by FAPESP 03/08542-5 and 03/07456-8.

302 REPLACEMENT OF FETAL BOVINE SERUM WITH KNOCKOUT SERUM REPLACER AND STEM CELLS CONDITIONED MEDIUM DURING IN VITRO CULTURE OF BOVINE EMBRYOS

2010 ◽  
Vol 22 (1) ◽  
pp. 307
Author(s):  
M. M. Souza ◽  
N. Z. Saraiva ◽  
C. S. Oliveira ◽  
T. A. D. Tetzner-Nanzeri ◽  
R. Vantini ◽  
...  
Keyword(s):  
Stem Cells ◽  
In Vitro Culture ◽  
Bovine Serum ◽  
Fetal Bovine Serum ◽  
Protein Supplementation ◽  
Control Group ◽  
Bovine Embryos ◽  
Fetal Bovine

The use of fetal bovine serum (FBS) as protein supplementation in IVP of bovine embryos has presented difficulties because it can introduce a number of pathogenic components in culture systems, can be related to the birth of calf with abnormal growth and development, and precludes the establishment of the actual nutritional needs of the embryo, because it contains an unlimited variety of substances. This study evaluated the replacement of the FBS in the medium of in vitro culture (IVC) of bovine embryos, using the knockout serum replacer (KSR) as protein supplementation and culture medium conditioned with stem cells. Therefore, bovine oocytes from ovaries of slaughterhouse were selected and matured in vitro in TCM-199 medium supplemented with 10% FBS (Crypion), 1.0 μg mL-1 FSH (Pluset®, Calier, Barcelona, Spain), 50 μg mL-1 hCG (Profasi®, Serono, Geneva, Switzerland), 1.0 μg mL-1 estradiol (Sigma E-2758, Sigma Chemical, St. Louis, MO, USA), 0.2 mM sodium pyruvate, and 83.4 μg mL-1 amikacin for 24 h. After that, 1144 oocytes were fertilized in IVF-TALP medium containing 6 mg mL-1 of BSA. After 18 to 22 h, the zygotes were cultured in SOF + 5% FBS (group 2); SOF + 5% KSR (group 3); SOF (5% FBS) + 10% SOF (5% FBS) conditioned by stem cells (group 4); or SOF (5% KSR) + 10% SOF (5% KSR) conditioned by stem cells (group 5), in an atmosphere of 5% O2 at 38.5°C for 8 days. A control group outside the controlled atmosphere was added, supplemented with 5% FBS (group 1). The SOF medium supplemented with 5% FBS or KSR was conditioned by stem cells and added to SOF medium for the culture of embryo at a concentration of 10%. The rates of cleavage and production of blastocysts were assessed 48 hours and 7 days after IVF, respectively, and analyzed by chi-square test, with a significance level of 5% in the statistical program Minitab® (release 14.1, Minitab, State College, PA, USA). On the eighth day, the TUNEL test for determination of the percentage of apoptosis and the differential staining technique for determination of inner cell mass (ICM) and trophoblast (TF) were performed. The results were submitted to ANOVA, followed by comparing the means by Tukey’s test using the program GraphPad Prism (GraphPad, San Diego, CA, USA). The treatments did not differ in the production of embryos, being similar to the control group: G1 = 31.75% (74/233), G2 = 35.26% (79/224), G3 = 32.70% (74/226), G4 = 28.76% (63/219), and G5 = 26.85% (65/242). With regard to the assessment of embryonic quality, the treatments showed similar results to the control groups. No differences were observed among groups both in color and ICM/TF ratio (G1 = 0.60, G2 = 0.62, G3 =0.65, G4 = 0.60, and G5 = 0.60). Furthermore, the TUNEL showed no significant difference in the percentage of apoptosis among groups (G1 = 7.10%, G2 = 3.76%, G3 = 5.58%, G4 = 4.50%, and G5 = 4.11%). The data obtained so far indicate that it is possible to produce embryos in vitro by replacing the FBS in the culture, achieving results similar to those obtained with serum. Financial support: FAPESP 2007/58506-6.

221 EFFECT OF ROCKING ON IN VITRO PRODUCTION OF BOVINE EMBRYOS

2009 ◽  
Vol 21 (1) ◽  
pp. 209
Author(s):  
Y. Serita ◽  
C. Kubota ◽  
T. Kojima
Keyword(s):  
In Vitro Fertilization ◽  
Embryo Development ◽  
Developmental Competence ◽  
In Vitro Production ◽  
Humid Atmosphere ◽  
Vitro Fertilization ◽  
Rate Of Development ◽  
Fetal Bovine ◽  
Vitro Production

This study tested whether embryo development yield using in vitro fertilization (IVF) could be improved by rocking cultures. Bovine ovaries were obtained at a slaughterhouse and transported to the laboratory within 6 h. Cumulus–oocyte complexes were collected and 20–25 were transferred in 100-μL drops of TCM-199 containing 10% fetal bovine serum and antibiotics under paraffin oil. Maturation was for 20–24 h at 38.5°C under 5% CO2 and 95% air in a humid atmosphere (IVM). In vitro fertilization was carried out for 6 h using frozen–thawed sperm from a single bull in modified Brackett and Oliphant (BO) medium. Presumptive zygotes were cultured in CR1aa supplemented with 10 mg mL–1 of BSA or 5% FBS for 9 d at 38.5°C under 5% CO2, 5% O2, and 90% N2 in a humid atmosphere (IVC). Rocking was performed to a height of 6 cm every 7 s using a Profile Rocker (New Brunswick Scientific Co., Edison, NJ, USA) in an incubator. Dishes were placed at a 15-cm distance from the fulcrum of the rocker. The conventional method (no rocking) served as a control, and every experiment was replicated 3 times. For Experiment 1, the effect of the period of rocking on developmental competence was examined when COC or zygotes were subjected to rocking for IVM, IVF, or IVC (IVM-move, IVF-move, and IVC-move). There were no significant differences in rates of oocyte maturation, cleavage, and development for IVM-move v. the control, or for rate of development between IVC-move and the control. However, the rate of fertilization for IVF-move was higher than that of the control (88.9 v. 67.5%; P < 0.01), and the rate of development was higher for IVF-move than for the control (39.0 v. 25.7%; P < 0.05). For Experiment 2, the effect of rocking frequency during IVF on development was determined. The IVF cultures were rocked every 7, 3.5, and 1.5 s (IVF-1move, IVF-2move, IVF-3move). The rates of cleavage on IVF-1move, IVF-2move, IVF-3move, and the control were 74.3, 69.8, 68.8, and 60.4%, and the rates of development were 39.0, 48.3, 26.2, and 25.7%, respectively. The rates of development on IVF-1move and IVF-2move were significantly different from the control and IVF-3move (P < 0.01). These results showed that rocking during IVF improved fertilization and embryo yield, and that frequency of rocking affected embryo development.

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.

282 DIFFERENT CONCENTRATIONS OF FORSKOLIN FOR MEIOSIS BLOCK AND TO IMPROVE IN VITRO PRODUCTION OF BOVINE EMBRYOS

2015 ◽  
Vol 27 (1) ◽  
pp. 230
Author(s):  
D. Paschoal ◽  
R. Maziero ◽  
M. Sudano ◽  
M. Guastali ◽  
L. Crocomo ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Least Squares ◽  
Control Group ◽  
In Vitro Production ◽  
Intact Cells ◽  
Significance Level ◽  
Nuclear Maturation ◽  
Oocyte Meiosis ◽  
Group Control

The inhibition of nuclear maturation allows time for the oocyte to accumulate molecules that are important for embryonic development. It was suggested that the inhibition of spontaneous nuclear IVM might allow for more time to accumulate the molecules important for embryonic development. The objective of this work was to evaluate blocking oocyte meiosis with the addition of forskolin. Slaughterhouse-derived bovine Zebu ovaries were collected and carried to the laboratory. Oocytes (n = 584) with at least 3 intact layers of cumulus cells and homogeneous cytoplasm were selected for IVM. The oocytes were transferred to drops of TCM 199 plus 10% FCS and hormones. The oocytes remained in IVM medium in 3 different concentrations of forskolin (6886), 0.1, 0.05, 0.025 mM, and a control group (withouth forskolin), for 6 h. Then they were maturated for an additional 18 h in forskolin-free medium. The first period above was an attempt to block (Block) and the second to resume (Res) the oocyte meiosis. The oocytes were incubated in a humidified atmosphere with 5% CO2 at 38.5°C in an air incubator. The oocytes were assessed for the stage of nuclear maturation, to see if they were in M II. Then oocytes were in vitro fertilized (IVF) with frozen Nelore bull semen (Bos taurus indicus). Presumptive zygotes (20–30/group) were cultured in SOFaa (synthetic oviducal fluid) supplemented with 5 mg mL–1 of BSA; the embryos were kept in an incubator with 5% CO2, 5% O2, and 90% N2 at 38.5°C and absolute humidity. On Day 7 (Day 0 = IVF) the blastocyst, the number of viable cells, and apoptosis rate (terminal deoxynucleotide transferase uridine nick-end labelling) were observed. Data were analysed with ANOVA using SAS PROC GLM (SAS Inst. Inc., Cary, NC, USA). Sources of variation in the model, including treatment and replication, were respectively considered fixed and random effects. If ANOVA was significant, the contrasts of means were performed using the least-squares difference. Data are presented as the mean and the standard error of least-squares. For all analyses, we used a significance level of 5%. No differences were observed for the stage of nuclear maturation of the oocyte (N = 336; control: 67.7 ± 8.3; F 0.025 mM, Block/Res: 67.7 ± 8.9; F 0.05 mM, Block/Res: 65.9 ± 9.8; F 0.1 mM, Block/Res: 50.2 ± 8.9), the blastocyst rate (N = 584; Control: 36.7 ± 3.7; F0.025 mM, Block/Res: 32.6 ± 3.7; F0.05 mM, Block/Res: 29.2 ± 3.7; F0.1 mM, Block/Res: 25.1 ± 3.7), and total number of intact cells (N = 10–15 embryos/group; Control:140.1 ± 13.0; F0.025 mM, Block/Res: 129.9 ± 13.0; F0.05 mM, Block/Res: 139.0 ± 13.0; F0.1 mM, Block/Res: 104.4 ± 13.0; P > 0.05). However, a higher rate of apoptosis was observed in the blastocysts produced from oocytes blocked for 6 h with the higher concentration of forskolin (N = 10–15 embryos/group): Control: 12.1 ± 2.5a; F 0.025 mM, Block/Res: 12.9 ± 2.5a; F0.05 mM, Block/Res: 13.5 ± 2.5a; F 0.1 mM, Block/Res: 30.2 ± 2.5b (P < 0.05). We conclude that all the experimental groups reached the stage of M II after the addition of forskolin and the highest concentration of forskolin caused cellular degeneration without harming embryo production on the seventh day.

248 SUPERSTIMULATION STRATEGIES FOR OVUM PICKUP IN HOLSTEIN DONORS

2016 ◽  
Vol 28 (2) ◽  
pp. 256
Author(s):  
L. M. Vieira ◽  
G. A. Bó ◽  
R. J. Mapletoft
Keyword(s):  
Oocyte Quality ◽  
Control Group ◽  
Limiting Factor ◽  
Total Dosage ◽  
In Vitro Production ◽  
Total Period ◽  
Expected Time ◽  
Follicular Wave ◽  
Vitro Production

In vitro embryo production (IVP) is an important tool to enhance genetic gain in cattle. However, oocyte quality is a limiting factor for the success of IVP programs in high-producing donors. A series of studies using protocols for follicular wave synchronization and superstimulation before ovum pickup were performed to improve the efficiency of ovum pickup and in vitro production in dairy cattle. The first study evaluated superstimulation with FSH (Folltropin-V®) before ovum pickup in lactating (n = 15) and non-lactating (n = 15) Holstein donors in a crossover design. Cows underwent synchronization of follicle wave emergence (FWE) and at the expected time of FWE, the FSH group received a total dosage of 200 mg of FSH in 4 decreasing doses 12 h apart; controls received no FSH, and ovum pickup was conducted 72 h after the expected FWE in all cows. The FSH-treated cows had a higher (P < 0.01) percentage of medium-sized follicles (6 to 10 mm) at the time of ovum pickup (55.1%) than control cows (20.8%) as well as lower cumulus‐oocyte complexes (COC) recovery rates (60.0 v. 69.8%, respectively; P = 0.002). However, FSH-treated cows had a higher blastocyst production rate (34.5 v. 19.8%; P < 0.01) and more transferable embryos per ovum pickup session (3.0 ± 0.5 v. 1.8 ± 0.4; P = 0.02). Subsequent trials evaluated plasma FSH profiles in 23 heifers and in vitro production following ovum pickup in 90 non-lactating Holstein donors superstimulated with a single IM injection of FSH in 0.5% hyaluronan (HA; MAP-5®, 50 mg). Controls received no treatment, while the F200 group received 200 mg of FSH in 4 decreasing doses 12 h apart. The F200HA and F300HA groups received 200 or 300 mg of FSH in 5 or 7.5 mL, respectively, of 0.5% HA by a single IM injection. Circulating FSH area under curve (AUC) in FSH-treated animals was greater than in the control group (P = 0.02). Although the AUC for F200 group did not differ from HA groups (P = 0.56), the total period of time plasma FSH levels were elevated was greater than in the HA groups (P < 0.01). In the IVP trial, FSH-treated cows had a greater proportion of medium-sized (6–10 mm) follicles than controls (P < 0.001). Also, numbers of follicles (P = 0.01) retrieved (control: 13.1 ± 1.0; F200: 16.5 ± 1.2; F200HA: 19.5 ± 2.1; F300HA: 15.4 ± 1.4; P = 0.01) and blastocysts produced per ovum pickup session (control: 2.4 ± 0.5; F200: 3.7 ± 0.7; F200HA: 4.7 ± 0.7; F300HA: 3.1 ± 0.6; P = 0.06) were greater in cows receiving FSH, regardless of treatment. Cows in the F200HA group had a greater recovery rate (P = 0.009), number of COC cultured (P = 0.04), and blastocysts per ovum pickup session (P = 0.06) than cows in the F300HA group. In conclusion, superstimulation of Holstein donors before ovum pickup increased the efficiency of in vitro production. Additionally, a single IM dose of FSH in 0.5% HA resulted in similar plasma FSH profiles to twice-daily FSH treatment. Non-lactating donors treated with FSH produced more embryos per ovum pickup session regardless of FSH treatment. Lastly, all in vitro-produced endpoints were greater following a single dose of 200 mg of FSH in 0.5% HA than 300 mg of FSH in 0.5% HA.

Effect of protein supplementation on development to the hatching and hatched blastocyst stages of cat IVF embryos

2002 ◽  
Vol 14 (5) ◽  
pp. 291 ◽  
Author(s):  
N. W. Kurniani Karja ◽  
Takeshige Otoi ◽  
Masako Murakami ◽  
Minori Yuge ◽  
Mokhamad Fahrudin ◽  
...  
Keyword(s):  
Bovine Serum ◽  
Culture Medium ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Early Embryo ◽  
Protein Supplementation ◽  
Vitro Fertilization ◽  
Fetal Bovine ◽  
The Mean

The effects of protein supplementation in culture medium on development to the hatching and hatched blastocyst stages of cat in vitro-fertilized embryos were investigated. In the first experiment, presumptive zygotes derived from in vitro maturation and in vitro fertilization (IVF) were cultured in modified Earle's balanced salt solution (MK-1) supplemented with 0.4% bovine serum albumin (BSA) or 5% fetal bovine serum (FBS) for 9 days. There were no significant differences between the BSA and FBS groups with respect to the proportion of cleavage and development to the morula and blastocyst stages of zygotes. However, the presence of FBS in the medium enhanced development to the hatching blastocyst stage of zygotes compared with the BSA group (31.4% v. 7.8%). Moreover, 2.9% of zygotes cultured with FBS developed to the hatched blastocyst stage. The mean cell number of blastocysts derived from zygotes cultured with FBS was significantly higher (P&lt;0.01) than that from zygotes cultured with BSA (136.6 v.101.5). In the second experiment, embryos at the morula or blastocyst stage, which were produced by culturing in MK-1 supplemented with 0.4% BSA after IVF, were subsequently cultured in MK-1 with 0.4% BSA or 5% FBS. Significantly more morulae developed to the blastocyst (P&lt;0.05) and hatching blastocyst stages (P&lt;0.01) in the FBS group than in the BSA group (71.5% and 53.6% v. 44.9% and 6.0%, respectively). Although none of the morulae cultured with BSA developed to the hatched blastocyst stage, 11.5% of morulae cultured with FBS developed to the hatched blastocyst stage. Moreover, the proportion of development to the hatching blastocyst stage of blastocysts was significantly higher (P&lt;0.01) in the FBS group than in the BSA group (68.7% v. 9.8%). None of the blastocysts cultured with BSA developed to the hatched blastocyst stage, whereas 7.3% of blastocysts cultured with FBS developed to the hatched blastocyst stage. The results of the present study indicate that supplementation with FBS at different stages of early embryo development promotes development to the hatching and hatched blastocyst stages of cat IVF embryos.

scholarly journals Use of Melatonin in the In Vitro Production of Bovine Embryos

2020 ◽  
Vol 21 ◽  
Author(s):  
Alan da Silva LIRA ◽  
Ricardo de Macedo CHAVES ◽  
Felipe de Jesus MORAES JUNIOR ◽  
Sergio Henrique COSTA JUNIOR ◽  
Brenda Karine Lima do AMARAL ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Control Group ◽  
Bovine Embryos ◽  
In Vitro Production ◽  
Maturation Medium ◽  
Significant Difference ◽  
Maturation Rate ◽  
Vitro Production

ABSTRACT We aimed to assess the effects of melatonin in the in vitro production of bovine embryos. Our experiment was conducted at the Laboratório de Reprodução Animal of the Universidade Estadual do Maranhão. The cumulus-oocyte complexes (COCs) were distributed among treatments at concentrations of 0, 10-1, 10-3 and 10-5 µMol/L melatonin. Our experiment was further divided into two: the first was to assess the effect of different concentrations of melatonin (treatments) on the maturation rate of COCs, and the second was to assess the effects of melatonin treatments on the in vitro production of bovine embryos. The results from the first experiment demonstrated no significant difference between the in vitro maturation rate of the cultivated COCs in treatments with melatonin. In the second experiment, however, melatonin treatments yielded statistically higher cleavage, morula and blastocyst rates in the 10-5 µM group (52.9%, 52.9%, and 35.3%, respectively), and lower rates in the 10-1 µM group (19.5%, 19.5% and 7.8%, respectively), compared to the others. The control group (no melatonin) and the 10-3 µM group showed similar results. We concluded that supplementation of melatonin in the in vitro maturation medium resulted in no improvement in the oocyte maturation rate, but in the in vitro production of embryos at different concentrations, the 10-5 µM group displayed better results, but with no improvement in the variables (P < 0.05).

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