304 SHORT-PERIOD SPERM - OOCYTE INCUBATION AND FERTILIZATION MEDIA EFFECTS ON THE IN VITRO PRODUCTION OF SWINE EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 267
Author(s):  
M. G. Marques ◽  
A. B. Nascimento ◽  
L. F. Martins ◽  
F. R. O. Barros ◽  
M. D. Goissis ◽  
...  
Keyword(s):  
Incubation Period ◽  
In Vitro Production ◽  
Standard Medium ◽  
Sodium Pyruvate ◽  
Short Period ◽  
Significant Difference ◽  
Incubation Periods ◽  
Chi Squared ◽  
Vitro Production

The aim of this study was to evaluate the effect of 2 sperm–oocyte incubation periods (30 min and 6 h) and 3 IVF media (TCM-199, TCM-199 with Ca-lactate, or mTBM). Cumulus–oocyte complexes (COCs) from abattoir-derived swine ovaries were matured in TCM-199 supplemented with 3.05 mM glucose, 50µg mL−1 gentamycin, 0.91 mM sodium pyruvate, 10% swine follicular fluid, 0.57 mM cysteine, 10 ng mL−1 of epidermal growth factor, 10 IU mL−1 eCG, and 10 IU mL−1 of hCG for 22 h, followed by a 22-h incubation without hormones. After in vitro maturation, oocyteswere allocated into 3 IVF media: TCM-199 (supplemented with 3.05 mM glucose, 0.91 mM sodium pyruvate, 0.57 mM cysteine, 50µg mL−1 gentamycin, 1 mg mL−1 BSA, and 3.06 mM mL−1 caffeine-standard medium);TCM-199 Ca (standard medium with 2.92 mM Ca lactate); or mTBM (supplemented with 5 mM mL−1 sodium pyruvate, 0.57 mM cysteine, 50µg mL−1 entamycin, 1 mg mL−1 BSA, and 2 mM mL−1 caffeine). The refrigerated semen at 15°C was centrifuged and capacitated for 2 h at 38.5°C in an atmosphere of 5% CO2 in air and under high humidity conditions in the respective IVF media. Oocytes were denuded, washed with the respective IVF media, and inseminated with the capacitated spermatozoa at a concentration of 6 × 105 sperm mL−1 in 90-µL microdroplets. After 30 min, a group of oocytes from each fertilization medium was gently washed to remove nonbound spermatozoa and returned to a new medium droplet for another 5.5 h (30-min group). The other group of oocytes remained in the same droplet with spermatozoa for 6 h (6-h group). After IVF, oocytes were cultured in porcine zygote medium-3 for 7 days. Data were analyzed by chi-squared test (P < 0.05).With regard to cleavage rates on the third day of embryo development (Day 3), no significant difference was observed among sperm–oocyte incubation periods; however, there was a difference in the TCM-199 group, compared with the mTBM and TCM-199 Ca groups, at the 30-min incubation period. With 6 h of incubation, no differences were observed among groups. When blastocyst (Day 7) rates were evaluated, no significant differences were observed between the 2 TCM-199 media groups; however, the mTBM groups showed higher blastocyst rates. The sperm–oocyte incubation period of 30 min or 6 h of IVF did not interfere with the blastocyst rate. In conclusion, the IVF medium mTBM was more efficient than the TCM-199 media when considering embryo production. Moreover, 30 min of IVF was sufficient for the spermatozoa responsible for oocyte fertilization to bind to the zona pellucida. Table 1.Cleavage (Day 3) and blastocyst (Day 7) rates of embryos incubated in two sperm–oocyte periods (30 min or 6 h) and cultivated in three IVF media This work was supported by the FAPESP 05/01420-7.

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).

144 PREGNANCY RATES FOLLOWING THE TRANSFER OF IN VITRO FERTILIZED EMBRYOS TO RECIPIENTS ON DAY 6, 7, OR 8 AFTER OESTRUS

2015 ◽  
Vol 27 (1) ◽  
pp. 164
Author(s):  
R. C. Fry ◽  
K. L. Fry ◽  
H. A. McCartney ◽  
W. R. Geddes ◽  
K. Geddes
Keyword(s):  
Pregnancy Rate ◽  
Embryo Transfer ◽  
Pregnancy Rates ◽  
Transfer Program ◽  
In Vitro Production ◽  
Chi Squared ◽  
Grade 3 ◽  
Vitro Production ◽  
Embryo Grade

The aim of this experiment was to investigate the effect of day of synchrony on the pregnancy rate of recipients following the transfer of Day 7 IVF embryos. In addition, the effect of IVF embryo grade and corpus luteum (CL) grade of recipients was determined. A total of 317 cumulus-oocyte complexes collected from 24 dry Brahman cows by TVR were matured, fertilized, and cultured under standard in vitro production procedures (Fry et al. 2003 Theriogenology 59, 446). A total of 89 (44 Grade 1, 43 Grade 2, and 2 Grade 3, IETS classification) in vitro-produced embryos were transferred to parous 4- to 9-year-old dry Brahman cross recipient cattle 7 days after IVF. Two groups of recipient cows were synchronised one day apart with an 8-day CIDR/pg protocol so that oestrous would be concentrated over 3 days with the middle day aligning with the day of IVF (Day 0). Donors that produced a large number of IVF embryos had these divided and transferred into recipients either on Day –1 or Day +1 of synchrony, and those producing less than 4 IVF embryos were transferred into recipients on Day 0. At embryo transfer the ovaries of the recipient were palpated and then scanned by rectal ultrasound and the grade of CL noted (Grade 1 = large distinct CL by palpation, Grade 2 = small distinct CL by palpation, Grade 3 = CL not distinguishable by palpation). Pregnancy was diagnosed by ultrasound scanning on Day 92. Although recipient numbers were low, differences in pregnancy rate between groups were analysed by Chi-squared. Data from the 2 Grade 3 embryos transferred were not included in the analysis (0/2 pregnant). Similar (P > 0.05) pregnancy rates were found when Day 7 IVF embryos were transferred to either Day 6 (17/32 = 53%), Day 7 (9/24 = 38%), or Day 8 (14/31 = 45%) recipients. Furthermore, neither the grade of the embryo (Grade 1: 20/44 = 45%, Grade 2: 20/43 = 47%) nor the grade of recipient CL (Grade 1: 17/45 = 38%, Grade 2: 17/29 = 59%, Grade 3: 6/13 = 46%) effected pregnancy rate (P > 0.05). This experiment demonstrates the flexibility of the IVF embryo to achieve an acceptable pregnancy rate over a range of recipient stages thereby allowing a high usage rate of good-quality recipients in an IVF embryo transfer program.

IN VITRO PRODUCTION OF ERGOT ALKALOIDS BY CULTURES OF CLAVICEPS PURPUREA (FR.) TUL.

1957 ◽  
Vol 3 (1) ◽  
pp. 55-60 ◽  
Author(s):  
W. A. Taber ◽  
L. C. Vining
Keyword(s):  
Liquid Medium ◽  
Incubation Period ◽  
Ergot Alkaloids ◽  
Claviceps Purpurea ◽  
In Vitro Production ◽  
Factors Influencing ◽  
Ammonium Succinate ◽  
Vitro Production ◽  
Selection Of

Stationary cultures of Claviceps purpurea (Fr.) Tul. grown 50 days on a liquid medium containing mannitol, ammonium succinate, and salts produced small amounts of ergot alkaloids.Ergometrine, ergometrinine, ergotamine, ergotaminine, ergocornine, ergocorninine, ergocryptine, agroclavine, and penniclavine were identified by paper chromatography of culture extracts. The selection of strains, the composition of the medium, and the length of the incubation period appeared to be important factors influencing the production of alkaloids.

180 DEVELOPMENTAL CAPACITY OF SINGLE CULTIVATED EMBRYOS IN SMALL AND BIG DROPLETS

2007 ◽  
Vol 19 (1) ◽  
pp. 206
Author(s):  
I. G. F. Goovaerts ◽  
J. B. P. De Clercq ◽  
M. Nichi ◽  
P. E. J. Bols
Keyword(s):  
Blastocyst Stage ◽  
Developmental Competence ◽  
In Vitro Production ◽  
Significant Difference ◽  
Group Treatments ◽  
Developmental Capacity ◽  
Droplet Sizes ◽  
The Individual ◽  
Vitro Production

An in vitro production system where a single oocyte can be followed from the ovary to the blastocyst stage would be a useful tool for studies concerning developmental competence or follicular environment. Unfortunately, until now, only low blastocyst rates could be obtained after single embryo production, and there is still discussion about the ideal droplet size. The objective of the present experiment was to compare the developmental competence of single cultivated zygotes in 20- and 500-µL droplets. Cumulus–oocyte complexes were obtained from slaughterhouse ovaries and were matured and fertilized in groups of 100 for 22 h; the presumptive zygotes were divided into 4 groups. In treatment 1, 25 zygotes were transferred into 50 µL of SOF medium supplemented with 5% serum under oil, whereas in treatment 2, 25 zygotes were transferred into 500 µL of medium. Zygotes were cultivated separately in treatments 3 and 4: in treatment 3 in 20 µL of medium under oil and in treatment 4 in 500 µL of medium. Cleavage rates and division stages were assessed after 3 days of cultivation (5% CO2, 5% O2, 90% N2); blastocyst rates were determined after 7 days. Statistical analysis was performed by logistic regression using SAS (PROC LOGISTIC). There was no difference in cleavage rates between the 2 group treatments or between the 2 single treatments. Also, the division stages were not different between the 2 single treatments (16-cell: 2.0 vs. 1.3%; 8-cell: 25.8 vs. 31.6%; 4-cell: 41.2 vs. 38.0%; and 2-cell: 31.0 vs. 29.1% for the 20 µL and the 500 µL droplet sizes, respectively). Group cultivation after 7 days in 50 µL was significantly better than in 500 µL; however, both treatments resulted in significantly higher blastocyst rates compared with the individual cultures in 20 or 500 µL, between which no significant difference could be found. Noteworthy, only 4-cell and 8-cell stages on Day 3 resulted in blastocysts on Day 7 of cultivation. In conclusion, these results indicate that cultivation in groups gives higher blastocyst rates, although the same embryo density is used as in individual cultivation (1 embryo 20 µL in treatments 2 and 3). Moreover, no significant difference could be found between single cultivation in small and big droplets. This is confirmed by the cleavage stages on Day 3, which indicate no difference in timing of cleaving between small and big droplets; time of cleaving is indicative of further developmental capacity. Table 1.Cleavage and blastocyst rates after single and group cultivation

289 IN VITRO PRODUCTION OF OVINE EMBRYOS FROM WOOL AND HAIR BREEDS

2006 ◽  
Vol 18 (2) ◽  
pp. 252
Author(s):  
E. A. Ordóñez-León ◽  
J. A. Medrano ◽  
V. O. Mejía ◽  
J. De Lucas ◽  
Y. C. Ducolomb ◽  
...  
Keyword(s):  
Physiological Saline ◽  
Relevant Information ◽  
In Vitro Production ◽  
Concentration Gradients ◽  
Independent Variables ◽  
Genetic Potential ◽  
Significant Difference ◽  
Tropical Areas ◽  
Vitro Production

In tropical areas, wool-less or haired breeds of sheep are more prolific than wool breeds. There are no reports about IVF in tropical breeds; therefore, it is not known how these respond under IVF conditions. Developing protocols for in vitro production of embryos in haired breeds could contribute to the preservation and use of their genetic potential in tropical countries where they are economically important. The aim of this study was to determine differences in IVM, IVF, and in vitro (IVD) between wool and hair breeds of sheep. A protocol for IVF (Wani 2002 Small Rum. Res. 44, 89-95) was used in wool (W) and hair (H) breeds. A total of 251 W and 251 H ewes were used. The ovaries were obtained after slaughter and transported to the laboratory in physiological saline (25�C). A total of 411 ovaries from W and 440 from H ewes were used, and 805 follicles of W and 790 of H ewes were aspirated. From these, 663 (82%) cumulus-oocyte complexes (COCs) of Wand 597 (76%) of H ewes were obtained and then used for the procedures of IVM, IVF, and IVD. The average number of COCs recovered per ovary was 1.6 forW and 1.4 for H. The average number of follicles per ovary was 1.9 for W and 1.7 for H. For IVM, COCs were incubated in TCM-199 supplemented with 20% serum from estrous ewe (SEE) for 24 h. All incubations were performed at 38.5�C in a humidified atmosphere of 5% CO2 in air. After this period, COCs were placed in fertilization medium (TALP supplemented with 200�g/mL heparin, 3�g/mL penicillamine, and 1�g/mL hypotaurine). For insemination, frozen-thawed semen from H and W rams was washed by centrifugation in two concentration gradients of a silicone solution. Oocytes and semen from the corresponding breed types were co-incubated for 18 h. For IVD, presumptive zygotes were incubated in SOF medium supplemented with 20% SEE for 7 days. Eight replicates were made. The rates of IVM, IVF, and IVD were analyzed by logistic regression, using as response variables: IVM, IVF, and IVD results, and as independent variables: breed and replicate. The percentage of recovered oocytes was 82% for W and 76% for H. For IVM and IVF, the recovered oocytes produced 535 fertilized oocytes of W and 446 of H. From these, 81% of the W oocytes and 75% of H were fertilized. Oocytes from W showed a higher percentage of IVM and IVF, with a statistically significant difference (P < 0.05). The percentage of division was 63% for W (n = 419) and 52% for H (n = 312). There were no statistically significant differences between the two groups for embryo IVD (P > 0.05). No statistical differences were found between replicates and no interactions were observed for breed � replicate (P > 0.05). It is concluded that IVM, IVF, and IVD procedures used for the development of embryos in W ewes can be used with similar results in H ewes. This is the first report of sheep IVF in Mexico that provides relevant information about the procedures of IVM, IVF, and IVD in hair and wool sheep, andsets a precedent for future investigations on in vitro embryo production in haired sheep breeds in Mexico. Funding for E.A. Ord��ez-Le�n was provided by CONACYT and UNAM.

295 INFLUENCE OF FERTILIZATION MEDIUM ON THE INCIDENCE OF POLYSPERMY IN THE PIG

2007 ◽  
Vol 19 (1) ◽  
pp. 263
Author(s):  
C. Galli ◽  
G. Crotti ◽  
P. Turini ◽  
I. Lagutina ◽  
G. Lazzari
Keyword(s):  
Fertilization Rate ◽  
In Vitro Production ◽  
Domestic Species ◽  
Sperm Suspension ◽  
High Incidence ◽  
Contrast Microscopy ◽  
Chi Squared ◽  
Vitro Production

The in vitro production of embryos is well established in most domestic species including cattle, pigs, sheep, and goats. However, a major problem of IVF in the pig is the high incidence of polyspermy. In our laboratory, we investigated the effect of 2 different media, TALP and SOFaa, on the rate of fertilization and polyspermy of pig oocytes. Preliminary experiments indicated that TALP provided the highest fertilization but also the highest polyspermy rates, as reported in the literature (Coy et al. 2002 Reproduction 124, 279–288). By contrast, much lower polyspermy rates but also much lower fertilization rates were obtained in SOFaa. Therefore, we made a direct comparison between the 2 media and a third medium prepared by mixing TALP and SOFaa equally (1 : 1 TALP–SOF) and using 2 different boars for IVF. Porcine oocytes were recovered from slaughtered donors and matured in vitro for 40 to 44 h in DMEM-F12 supplemented with 10% FCS, 0.05 IU of LH and FSH (Menogon; Ferring, Milan, Italy), 0.3 mM cystine, 0.5 mM cysteamine, 50 ng mL−1 of long-EGF, 100 ng mL−1 of long-IGF1, and 5 ng mL−1 of bFGF (Sigma-Aldrich, Milan, Italy) in 5% CO2 at 38.5°C. Boar frozen–thawed semen was separated on a Percoll gradient (45–90%) and diluted in TALP or in SOFaa with PHE (penicillamine, hypotaurine, epinefrine) and heparin (1 µg mL−1) to concentrations ranging from 0.01 to 0.15 million sperm/mL. The concentration was optimized for each boar and medium: For boar A, the concentration was 0.015 million sperm/mL for medium TALP and TALP–SOF and 0.15 million sperm/mL for medium SOF; for boar B, the concentration was 0.1 million sperm/mL for medium TALP and TALP–SOF and 0.15 million sperm/mL for medium SOF. The oocytes were co-incubated with the sperm suspension for 18 h and then were denuded of the surrounding cumulus and fixed in acetic acid–ethanol (1 : 3) for 48 h. Finally, they were stained with lacmoid and observed under phase-contrast microscopy. The data are shown in Table 1 and were compared by a chi-squared test. Our results indicated that TALP was the most efficient medium for pig IVF but over 50% of the oocytes were polyspermic. By contrast, very low polyspermy, but also very low fertilization, was observed in SOF medium for both boars A and B. Interestingly, the empirical approach of mixing the 2 media 50% each provided a dramatic reduction of the polyspermy rate while maintaining the fertilization rate at over 60% in both boars. At present, experiments are ongoing to clarify the role of specific components of the 2 media on the fertilization and polyspermy rates of pig oocytes. Table 1.Effect of different media on fertilization and polyspermy rates with 2 different boars This work was supported by grants from EUROSTELLS-ESF (ERAS-CT-2003-980409).

The In Vitro Production of Thromboxane B2 by Platelets of Diabetic Patients Is Normal at Physiological Concentrations of lonized Calcium

1993 ◽  
Vol 70 (03) ◽  
pp. 389-392 ◽  
Author(s):  
Cristina R Falcon ◽  
Marco Cattaneo ◽  
Alberto Ghidoni ◽  
Pier Mannuccio Mannucci
Keyword(s):  
In Vitro Studies ◽  
Thrombin Inhibitor ◽  
Diabetic Patients ◽  
In Vitro Production ◽  
Significant Difference ◽  
Patients With Diabetes ◽  
Vitro Production ◽  
Induced Aggregation

SummaryPlatelets of patients with diabetes and no evidence of macroangiopathy produce normal amounts of thromboxane (Tx) B2 in vivo, whereas they usually show increased production in vitro. Since in vitro studies have been usually performed in citrated PRP, we tested the hypothesis that the discrepancy between in vivo and in vitro studies is due to the low concentration of plasma ionized calcium ([Ca 2+ ]o) that is present in citrated PRP. In fact, low [Ca 2+ ]o artifactually potentiates the platelet TxB2 production in vitro. Forty patients with diabetes mellitus and 37 matched Controls were studied. Blood was anticoagulated with citrate, the thrombin inhibitor D-phenylalanyl-l-prolyl-l-chloromethylketone (PPACK) or both anticoagulants. Platelet aggregation, release of 14C-serotonin and TxB2 production were induced in platelet rieh plasma (PRP) by several agonists. The following results were obtained: i) Citrated PRP: Arachidonic acid induced aggregation (p <0.01) and TxB2 production (p <0.02) were significantly greater in patients than in Controls. No statistically significant differences were found with other agonists. ii) PPACK PRP: No statistically significant difference was found between diabetic platelets and Controls, iii) PPACK plus citrate PRP: The results were not different from those obtained with citrate alone. Therefore, our results show that diabetic platelets produce normal amounts of TxB2 in vitro when the [Ca2+]o is physiological.

scholarly journals In vitro performance of Zebu (Bos indicus) and Taurus (Bos taurus) donor cow embryos

2020 ◽  
Vol 21 ◽  
Author(s):  
Anita Soares Barbosa GUIMARÃES ◽  
Laiara Fernandes ROCHA ◽  
Ronival Dias Lima de JESUS ◽  
Gisvani Lopes VASCONCELOS ◽  
Gabriela ANGHINONI ◽  
...  
Keyword(s):  
Culture Medium ◽  
Bos Taurus ◽  
Bos Indicus ◽  
In Vitro Production ◽  
Embryo Production ◽  
Maturation Medium ◽  
Significant Difference ◽  
Vitro Production ◽  
In Vitro Performance

ABSTRACT In this study, the in vitro production of bovine embryos from zebu and taurine donors was compared. Cumulus-oocyte complexes (COCs) were obtained from 167 Bos taurus and 161 Bos indicus donors by ovum pick-up. COCs were classified based on their morphological quality, matured in incubators for 22 to 24 h in maturation medium, and then fertilized for 18 to 22 h. The zygotes were transferred to the culture medium for seven days. The embryos were classified as morula (OM), initial blastocyst (BI), blastocyst (BL), and expanded blastocyst (BX), before being transferred to synchronized recipient cows. Pregnancy was diagnosed 30-45 days post-transfer. The Bos indicus donors had a higher oocyte yield (n = 2556) than Bos taurus donors (n = 1903) (P = 0.008). The COCs from zebu donors had a better morphological quality than those from taurine donors (n = 689 vs. 444 for grade 1 COC, P < 0.0001; n = 681 vs. 509 for grade 2 COC, P = 0.010, for zebu and taurine donors, respectively). There were differences in embryo production percentages obtained from OM (0.44% from zebu and 6.42% from taurine, P = 0.017), BL (14.18% from zebu and 3.74% from taurine, P < 0.0001), and BX (81.43% from zebu and 75.13% from taurine, P < 0.0001). No significant difference was observed for embryo production from BI and pregnancy rate (P > 0.05). The Bos indicus cows showed greater oocyte recovery, number of viable oocytes, and production of viable embryos than the Bos taurus cows.

scholarly journals 31 Effect of fetal calf serum on production and cryotolerance of in vitro bovine embryos from Ecuadorian Creole heifers

2019 ◽  
Vol 31 (1) ◽  
pp. 141
Author(s):  
M. S. Méndez ◽  
M. E. Soria ◽  
L. R. Galarza ◽  
F. P. Perea ◽  
D. E. Argudo
Keyword(s):  
Calf Serum ◽  
Bovine Embryos ◽  
In Vitro Production ◽  
Sodium Pyruvate ◽  
Embryo Production ◽  
Maturation Medium ◽  
Fetal Bovine ◽  
And Control ◽  
Vitro Production

In the Ecuadorian Andes there is a Creole bovine biotype whose population is disappearing. In vitro embryo production and cryopreservation is an important biotechnology that allows the conservation of animals threatened with extinction. The objective of this study was to determine the in vitro production and cryopreservation of embryos from creole heifers raised in the highlands of Ecuador. Immature cumulus-oocyte complexes were retrieved by ovum pickup from 10 Creole heifers (OPU) and from abattoir ovaries (control). The experiment was completed within 8 replicates. Cumulus-oocyte complexes were cultured in a maturation medium (TCM-199 supplemented with 10% fetal bovine serum, 100µg mL−1 of sodium pyruvate, 0.75mg mL−1 of l-glutamine, 4µg mL−1 of FSH-p, 100µM cysteamine, and 250µg mL−1 of gentamicin) following IVF (SOF medium supplemented with 10µg mL−1 heparin) and in vitro culture (citrate SOF medium). After denudation (Day 1 after IVF), presumptive embryos from each oocyte source (OPU and control) were split into 2 groups: with (FCS+) and without (FCS−) FCS (2.5%), which was added on Day 5 after IVF. On Day 7, embryos were evaluated, and those with quality 1 were vitrified. After warming, embryo re-expansion at 2h and embryo re-expansion and hatching at 24 and 48h were evaluated. Data were analysed by logistic regression in SAS software (SAS Institute Inc., Cary, NC, USA). Results of embryo rate at Day 7 and rates of vitrified, re-expanded, and hatched embryos are shown in Table 1. Regardless of the oocyte source, the addition of 2.5% FCS decreased embryo re-expansion at 2h and reduced embryo hatching at 48h in the OPU group. In conclusion, FCS did not improve embryo production and adversely affected the cryotolerance of embryos produced in vitro from Ecuadorian creole heifers. Table 1.Production and cryotolerance of in vitro bovine embryos

158 EFFECTS OF APOTRANSFERRIN ON IN VITRO MATURATION OF CUMULUS - OOCYTES COMPLEXES (COCs) IN HANWOO, KOREAN NATIVE COWS

2006 ◽  
Vol 18 (2) ◽  
pp. 187
Author(s):  
S.-H. Choi ◽  
S.-R. Cho ◽  
M.-H. Han ◽  
H.-J. Kim ◽  
C.-Y. Choe ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
Blastocyst Stage ◽  
Basic Fuchsin ◽  
In Vitro Production ◽  
Control Groups ◽  
Significant Difference ◽  
Native Cattle ◽  
Vitro Development ◽  
Vitro Production

For in vitro production of embryos, animal sera have been used as energy sources, maturation promoters, vitamins, growth factors, and antioxidative compounds. However, the sera had risk of virus and mycoplasma infections which could result in too big offspring and cause dystocia in ovine and bovine. Apotransferrin (apo-Tf) is a component of mammalian sera and has played a role as an antioxidant in media. A study was conducted to investigate the effects of apo-Tf on in vitro maturation of cumulus-oocytes complexes (COCs) in Hanwoo, Korean native cows. Ovaries were collected from a slaughterhouse and COCs were taken from 2-6-mm antral follicles. The collected COCs were washed three times with 0.1M polyvinyl alcohol (PVA)-TCM199 and matured in 0, 1, 10, or 100 �g/mL apo-Tf with TCM-199 at 39�C, 5% CO2, 95% air for 6, 12, or 24 h. Mature COCs were fertilized with frozen-thawed Korean native cattle semen treated with BO medium (Brackett and Oliphants 1975 Biol. Reprod. 12, 260-274) containing 5 mM caffeine and 1 �g/mL heparin for 8 h and developed to the blastocyst stage in 5% FBS and 0.3% BSA in TCM199-IVMD (IFP, Japan). To evaluate the morphology of nuclear types, the matured COCs were fixed in 1:3 acetic acid-ethanol for 30 s and stained with 3% basic Fuchsin. IVM and IVF were replicated three times. All of the results were analyzed by ANOVA using the STATVIEW program. The maturation rates of control were 34.2%, 37.3%, and 45.8% for 6, 12, and 24 h, respectively. There were no differences among the concentrations of apo-Tf, and nuclear types at 78.3-87.0% GVBD for 6 h, 82.8-91.3% MI for 12 h, and 88.9-100.0% MII for 24 h, with 1, 10, and 100 �g/mL apo-Tf, respectively. Conversely, there was significant difference between 1 �g/mL and 10 �g/mL in terms of cleavage rates, although the others did not vary significantly (P < 0.05). There were significant differences among the concentrations of apo-Tf for blastocyst formation (P < 0.05). Blastocysts matured with 1, 10, and 100 �g/mL apo-TF and developed in 5% FBS and 0.3% BSA in TCM199-IVMD showed rates of 8.8-21.6%, 9.4-35.3%, and 9.1-19.1%, respectively. The control groups developed to the blastocyst stage showed rates of 8.6%, 10.8%, and 10.5% in 5% FBS and 0.3% BSA in TCM199-IVMD, respectively. These results suggest that apo-Tf is an important factor for the in vitro maturation and in vitro development of bovine COCs.

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