221 EFFECT OF STAGE OF CORPUS LUTEUM DEVELOPMENT ON THE IN VITRO PRODUCTION OF BOVINE EMBRYOS

The objective of this study was to investigate the effect of stage of corpus luteum (CL) development on the in vitro production of bovine embryos. Ovaries were classified according to the expected day of the oestrous cycle based on the morphology of the ovaries. Ovaries with a corpus hemorrhagicum and the remnant of the follicular lumen filled with blood were considered the early luteal stage (Days 2 to 4; Day 0 = day of ovulation, n = 46). Ovaries with a large mass of orange tissue in the CL were classified as the midluteal stage (Days 7 to 10, n = 42). Cumulus–oocyte complexes (COC) were collected by aspiration of 2- to 6-mm follicles. The COC were classified into the following grades: COC with >3 compact layers of cumulus cells and evenly granulated cytoplasm were classified into Grade 1; COC with >3 layers cumulus cells and evenly granulated cytoplasm were classified into Grade 2; COC with partially remaining cumulus cells and abnormal cytoplasm were classified into Grade 3; COC without cumulus cells or those with expanded cumulus cells were classified into Grades 4 and 5, respectively. Grades 1 and 2 COC were in vitro matured for 20 h in TCM-199 supplemented with 5% calf serum and 0.02 mg mL–1 of FSH at 38.5°C under an atmosphere of 5% CO2 in air. Matured COC were inseminated with 5 × 106 sperm for 18 h. Presumptive zygotes were cultured in CR1aa medium supplemented with 5% calf serum at 38.5°C under an atmosphere of 5% O2, 5% CO2, and 90% N2 for 9 days (fertilization = Day 0). The mean number of COC and the proportion of COC classified as Grades 1 and 2 were analysed by ANOVA. Cleavage rates on Day 3 and blastocyst rates on Days 7 to 9 were analysed by a chi-square test. The mean number of recovered oocytes in the early luteal stage (18.7 ± 9.5) was significantly higher (P < 0.05) than the number in the midluteal stage (12.2 ± 5.7). The proportion of Grades 1 and 2 oocytes in the early luteal stage [66.7% (531/789)] was significantly higher (P < 0.01) than that in the midluteal stage [51.6% (252/484)]. The cleavage and blastocyst rates in the early luteal stage [60.9% (181/297) and 32.7% (97/297), respectively] were significantly higher (P < 0.05) than those in the midluteal stage [50.7% (76/150) and 20.7% (31/150) respectively].The present study suggests that the stage of development of the CL in bovine ovaries influences the number of recovered oocytes per ovary and the development of in vitro production of bovine embryos.

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Effects of superstimulation with fsh on follicular population and recovery rate of oocytes in the growing phase of the first and second follicular wave

Veterinární Medicína ◽

10.17221/5800-vetmed ◽

2012 ◽

Vol 47 (No. 2 - 3) ◽

pp. 33-37

Author(s):

S. Čech ◽

V. Havlíček ◽

M. Lopatářová ◽

M. Vyskočil ◽

R. Doležel

Keyword(s):

Czech Republic ◽

Corpus Luteum ◽

Recovery Rate ◽

Dairy Farm ◽

Cumulus Cells ◽

Dominant Follicle ◽

In Vitro Production ◽

Follicular Wave ◽

Vitro Production

Effectiveness of in vitro production of embryos (IVP) is limited among other factors by the recovery rate of oocytes. Gonadotropin superstimulation can improve the recovery rate of oocytes. The effect of FSH treatment on follicular population and recovery rate of oocytes at ovum pick-up (OPU) in the growing phase of the 1st as well as the 2nd follicular wave after superstimulation was the object of our experiment. Twelve unpregnant milking cows (15–20 kg milk per day) housed on a dairy farm were used in the experiment. The cows bearing corpus luteum were synchronized by PGF<sub>2 </sub>(day 0) and they were treated by FSH (Folicotropin inj. sicc. ad us vet., Spofa Prague, Czech Republic, single doses 80, 80, 80, 80, 40 and 40 UI) in 12 h intervals on days 12, 13 and 14. Transvaginal ultrasonographic puncture of oocytes in cows bearing a new corpus luteum was performed on day 7 (OPU 1, various phase of the follicular wave, removal of the dominant follicle) and it was repeated on days 10 (OPU 2, growing phase of the follicular wave – control), 16 (OPU 3, growing phase of the first follicular wave after superstimulation) and 20 (OPU 4, growing phase of the second follicular wave after superstimulation). All follicles > 2 mm were punctured. The ovarian follicles (ultrasonographically) and numbers and qualities of obtained oocytes (microscopically) were evaluated during and immediately after OPU. Follicular population was divided to small (FS, 2–5 mm), medium (FM, 5–9 mm) and large (FL, > 9 mm) follicles. Oocytes were classified as 1st (intact cumulus, > 3 layers of cumulus cells), 2nd (complete 1–3 layers of cumulus cells), 3rd (incomplete layers of cumulus cells, expanded cumulus mass) and 4th (absence of corona cells, degenerated oocytes) classes. Although we found the least of FS (x = 1.0) during OPU 3, significantly more FM (x = 24.7) and FL (x = 3.1) follicles were found at this procedure in comparison with others. Likewise a significantly higher number of oocytes (x = 8.1) was obtained at OPU 3 in comparison with OPU 1 and OPU 2. Significantly higher number of FM (x = 6.1) was found and non-significantly higher number of oocytes was obtained at OPU 4 in comparison with OPU 1 and 2. The results show that administration of FSH increases the number of follicles and the number of collected oocytes in the growing phase of the 1st follicular wave after superstimulation, nevertheless a higher number of follicles and a higher recovery rate of oocytes can be expected in the growing phase of the 2nd follicular wave after superstimulation as well.

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

Reproduction Fertility and Development ◽

10.1071/rdv31n1ab31 ◽

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

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298 THE INFLUENCE OF DISTINCT CENTRAL BULL STATIONS ON THE IN VITRO EMBRYONIC DEVELOPMENT OF NELORE BULLS

Reproduction Fertility and Development ◽

10.1071/rdv22n1ab298 ◽

2010 ◽

Vol 22 (1) ◽

pp. 305

Author(s):

A. Renzi ◽

F. P. Elias ◽

R. A. Vila ◽

R. B. Lôbo

Keyword(s):

Calf Serum ◽

Cumulus Cells ◽

In Vitro Production ◽

Freezing And Thawing ◽

Chi Square ◽

Frozen Semen ◽

Chi Square Test ◽

Vitro Production ◽

In Vitro Embryonic Development

Reproductive biotechnology is growing worldwide as one of the most important tools of cattle breeding because it accelerates the process of genetic improvement. Most of the embryos produced are obtained using frozen semen from different AI centers. During freezing and thawing of semen, the sperm can be damaged by the rapid and dramatic changes in the physicochemical conditions that occur during cooling and ice formation. It has previously been described that bad management of frozen semen can result in reduced fertilization. This study investigated the influence of different central bull stations on the development of in vitro-produced bovine embryos. We compared semen of 154 Nelore bulls, used for IVF, from 8 different central bull stations (all of which used the same cryopreservation protocol) in the development of blastocysts. The in vitro production of embryos was performed as described: oocytes were collected from the slaughterhouse and matured in TCM-199 + 10% fetal calf serum (FCS) +0.5 μg mL-1 FSH + 50 μg mL-1 LH+ 1 μg mL-1 estradiol, for 24 hat 38.5°C in 5%CO2 in atmospheric air. Viable spermatozoids were obtained by centrifugation in Percoll gradient (45 and 90%), and used for IVF in a concentration of 2 million spermatozoa per mL in TALP + 10 μg mL-1 of heparin medium. After 12 h, the presumptive zygotes were transferred to a CR2+ 10% FCS medium and co-cultured with cumulus cells. After 168 h of IVF, we evaluated the number and stage of cleaved embryos produced with the semen of each bull. Statistical analyses were performed by using the chi-square test. Our results suggest that there are differences among distinct central bull stations in the proportion of embryos that developed into blastocysts and the different stages they hatched. FAPESP, CNPq, PROEX, FAEPA.

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226 IN VITRO PRODUCTION OF BOVINE EMBRYOS USING CHEMICAL PACKETS THAT REGULATE CO2 AND O2

Reproduction Fertility and Development ◽

10.1071/rdv27n1ab226 ◽

2015 ◽

Vol 27 (1) ◽

pp. 202

Author(s):

K. Saeki ◽

Y. Fujiki

Keyword(s):

Gas Phase ◽

Subsequent Development ◽

Cumulus Cells ◽

Bovine Embryo ◽

Bovine Embryos ◽

In Vitro Production ◽

Catalog Number ◽

Co2 Level ◽

Vitro Production

Bovine embryos are now routinely produced with oocytes collected from slaughterhouse ovaries or by transvaginal ovum pickup. The oocytes are matured, fertilized, and cultured in a water-jacketed CO2/O2 incubator. Gas phase in incubators is usually maintained at 5% CO2 in air for in vitro maturation (IVM) and IVF of oocytes and at 5% CO2, 5% O2, and 90% N2 for in vitro culture (IVC) of embryos. Here we investigated whether two chemical packets that regulate CO2 and O2 for culturing bacteria (Mitsubishi Gas Chemical, Tokyo, Japan) could be used to control the gas phase in vitro production (IVP) of cattle embryos. One packet (Anaero Pack-CO2) was maintained at a CO2 level of ~5% in a 2.5-L container and the other (Anaero Pack-MicroAnaero) was maintained at a CO2 level of 5–8% and an O2 level of 6 to 12%. Bovine cumulus-oocyte complexes (COC, n = 970) were collected from slaughterhouse ovaries, matured in HEPES-buffered TCM-199 (catalog number 12340–030, Invitrogen) supplemented with 10% FCS, 0.02 Armour unit mL–1 FSH and 1 µg mL–1 E2 for 22 h, and fertilized in medium IVF100 [Research Institute for the Functional Peptides Co. Ltd. (IFP), Yamagata, Japan] with frozen-thawed sperm (4 × 106 cells mL–1) for 6 h. Sperm and cumulus cells were removed from the oocytes. The denuded oocytes were cultured in IVD101 (IFP, 20 to 30 embryos/50 μL) for 8 days (Day 0 = IVF). Culture was carried out at 39°C with maximum humidity. Five different combinations of gas conditions were used for incubation (Table 1). Experiments were repeated 3 times. Cleavage and blastocyst rates were assessed on Day 8. Data were analysed by ANOVA followed by Fisher's PLSD test. In the five conditions, rates of matured oocytes (oocytes at MII, n = 210) were 70 to 73% and rates of normal fertilized oocytes (oocytes with 2 pronuclei, n = 310) were 67 to 75%. Cleavage rates of embryos after 8 days of culture (n = 450) were 68 to 75%, and rates of blastocysts from cleaved embryos were 25 to 40%. None of the above measures were significantly different among the 5 conditions (P > 0.05). These results indicate that gas phase control is not needed for IVM and IVF of bovine oocytes for their subsequent development. Anaero Pack-MicroAnaero (5–8% CO2, 6–12% O2) can be used for IVC of bovine embryos. The CO2-generating and deoxidizing packets can be successfully used to control the gas phase during bovine embryo production. Table 1.Five different combinations of gas conditions used for incubation

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In Vitro Production Of Goat Embryos In Bangladesh

Bangladesh Journal of Animal Science ◽

10.3329/bjas.v37i1.9859 ◽

1970 ◽

Vol 37 (1) ◽

pp. 1-9

Author(s):

A Mondal ◽

MAMY Khandoker ◽

MA Mondal ◽

AHMS Rahman ◽

AS Apu ◽

...

Keyword(s):

Corpus Luteum ◽

Culture Condition ◽

In Vitro Maturation ◽

Calf Serum ◽

Type I ◽

Type Ii ◽

In Vitro Production ◽

Vitro Fertilization ◽

Vitro Production

The present study was undertaken to collect the quality cumulus-oocyte-complexes (COCs) from ovaries of goat from slaughterhouse by aspiration to establish the suitable culture condition for in vitro maturation (IVM), in vitro fertilization (IVF) and in vitro culture (IVC). Follicular COCs were collected from follicles of 2-6 mm diameter, categorized by microscopic observation and cultured for 22 h in TCM-199 medium supplemented with 5% fetal calf serum (FCS) to determine the success rate of in vitro maturation in a condition of 5% CO2 in air at 38.5°C. The collected ovaries were classified as type-I (corpus luteum absent) and type-II (corpus luteum present). The average numbers of follicles aspirated per ovary were 3.15 and 2.57 in type-I and type-II, respectively. The collected COCs were classified into normal COCs (grade A and B) and abnormal COCs (grade C and D). The number of normal and abnormal COCs collected from two type of ovaries were significantly (P<0.01) differed. Average number of normal COCs per ovary obtained from type-I (1.30) was significantly (P<0.01) higher than that of type-II (0.68). Within the normal COCs significantly (P<0.01) higher maturation was obtained in grade A COCs (71.70%) than that of grade B (51.52%). The matured COCs were cultured for 5 h with fresh buck semen in Brackett and Oliphant (BO) medium and assumed that the COCs were fertilized successfully. In progress, IVC was practiced in TCM-199 supplemented with FCS and bovine serum albumen (BSA) at 38.5°C with 5% CO2 for 6-7 days. The rate of development to compact morula was found significantly (P<0.01) higher in grade A (25.64%) compared to grade B COCs (6.89%) and similar trend of blastocyst was found in grade A COCs (12.82%) than that of grade of B (3.45%). The results suggested that culture condition for IVM, IVF and IVC was found optimum and grade A COCs might be suitable for in vitro production (IVP) of goat embryos.DOI: http://dx.doi.org/10.3329/bjas.v37i1.9859 BJAS 2008; 37(1): 1-9

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