scholarly journals In vitro production of bovine embryos using flow-cytometrically sexed sperm

2006 ◽  
Vol 49 (2) ◽  
pp. 133-140 ◽  
Author(s):  
L. Kątska-Książkiewicz ◽  
B. Ryńska ◽  
M. Bochenek ◽  
J. Opiela ◽  
J. Jurkiewicz
Keyword(s):  
Bovine Embryos ◽  
In Vitro Production ◽  
Blastocyst Development ◽  
Vitro Fertilization ◽  
Bovine Spermatozoa ◽  
Embryo Cleavage ◽  
And Control ◽  
Vitro Production ◽  
Sperm Freezing

Abstract. The investigation aimed to compare the effect of fresh and frozen-thawed X and Y fractions of flow-cytometrically sorted bovine spermatozoa on in vitro fertilization of bovine in vitro matured oocytes and subsequent blastocyst development. Sperm cells sorted in MoFloSX cytometer were used either for IVF or frozen and stored in liquid nitrogen. Immature oocytes recovered from ovaries of slaughtered animals and matured in vitro in TCM-199 containing 20% estrus cow serum and additional granulosa cells were fertilized in vitro with fresh or frozen-thawed fractions of sorted sperm. Simultaneously, control, fresh or frozen/thawed sperm was used for IVF. A total number of 2712 IVM oocytes were fertilized with sorted and control sperm of 6 bulls. Embryo cleavage rates were significantly affected by bull (P<0.0001), sperm sexing (P<0.0001) and sperm freezing (P<0.01). Blastocysts development was affected by sperm freezing (P<0.04) and sperm sexing (P<0.01). The significant differences were shown between unsorted and sorted sperm, however no differences in embryo cleavage rates and blastocysts rates were observed between X- and Y-sperm fractions, both fresh and frozen/ thawed. There were significant differences in cleavage rates among fresh, control sperm (52.7%), X fraction (26.8%) and Y fraction (24.7%). Similar differences in cleavage rates were shown for frozen/thawed control sperm (52.8%), X fraction (33.9%) and Y fraction (26.2%). The female blastocysts were frozen for further transfer, while the hatched male blastocysts were analysed by PCR revealing 76.2% accuracy. The results suggest that there were significant differences in cleavage rates and blastocyst rates due to sperm sorting in comparison to unsorted sperm and no differences between effectiveness of X and Y fractions of spermatozoa.

222 EFFECT OF THE PRESENCE OR ABSENCE OF PERCOLL CENTRIFUGATION; PENICILLAMINE, HYPOTAURINE, AND EPINEPHRINE; AND HEPARIN ON IN VITRO PRODUCTION OF BOVINE EMBRYOS

2013 ◽  
Vol 25 (1) ◽  
pp. 259 ◽  
Author(s):  
J. F. Hasler ◽  
J. E. Stokes
Keyword(s):  
Percoll Gradient ◽  
Bovine Embryos ◽  
In Vitro Production ◽  
Blastocyst Development ◽  
Vitro Fertilization ◽  
Rat Liver Cells ◽  
Lactate Medium ◽  
Bovine Oocytes ◽  
Percoll Centrifugation

Protocols for the production of bovine embryos in vitro routinely include Percoll centrifugation of semen, usually include heparin, and often include penicillamine, hypotaurine, and epinephrine (PHE) in the fertilization media. This study examined the contribution of each of these components to the success of in vitro fertilization of bovine oocytes and subsequent blastocyst development. Bovine oocytes were aspirated from 2- to 10-mm follicles within 5 h after slaughter of cattle at a local abattoir. Groups of 30 to 40 cumulus–oocyte complexes (COC) were matured in 0.5 mL of TCM-199 with 10% FCS, 4 µg mL–1 of FSH, and 6 µg mL–1 of LH (NOBL Laboratories, Sioux Center, IA, USA) for 24 h (39°C, 4% CO2 in air). The COC were then washed and placed in 0.5 mL of modified Tyrode-lactate medium for IVF with various combinations of 2 µg mL–1 of heparin, 20 µM penicillamine, 10 µM hypotaurine, and 1 µM epinephrine. Each group of COC was inseminated with 0.25 × 106 frozen–thawed sperm from a single bull after 30 min of centrifugation with (Exp. 1) or without (Exp. 2) a 45/90% Percoll gradient with sperm TALP. Oocytes were vortexed to remove the cumulus after 18 h and placed in co-culture wells containing a monolayer of buffalo rat liver cells and 0.5 mL of Menezo’s B2 medium supplemented with 10% FCS. On the fourth day of in vitro culture, cleavage was defined as 2 cells or greater and embryos were transferred to fresh co-culture wells. There were 4 replicates in the first experiment and 6 in the second. Data were analysed by ANOVA. In the first experiment, the use of a Percoll gradient during centrifugation for separation of viable sperm from seminal plasma and cryprotectants resulted in significantly higher cleavage and Day 8 blastocyst rates than did the absence of Percoll when PHE and heparin were used together, and both cleavage and blastocyst rates were lower when only PHE or heparin was used separately compared with when both were used together (Table 1). The absence of Percoll, PHE, and heparin resulted in the lowest rates of cleavage and development. In the second experiment, the absence of either PHE or heparin resulted in lower cleavage rates, but not blastocyst rates, compared with the use of both, and the absence of both resulted in the lowest cleavage and blastocyst rates in spite of the use of Percoll. Table 1.Effects of Percoll; penicillamine, hypotaurine, and epinephrine (PHE); and heparin on cleavage and subsequent embryo development per oocyte

In vitro production of bovine embryos in medium supplemented with a serum replacer: Effects on blastocyst development, cryotolerance and survival to term

2007 ◽  
Vol 68 (9) ◽  
pp. 1316-1325 ◽  
Author(s):  
K. Moore ◽  
C.J. Rodríguez-Sallaberry ◽  
J.M. Kramer ◽  
S. Johnson ◽  
E. Wroclawska ◽  
...  
Keyword(s):  
Bovine Embryos ◽  
In Vitro Production ◽  
Blastocyst Development ◽  
Vitro Production

scholarly journals In vitro production of bovine embryos

2003 ◽  
Vol 57 (3-4) ◽  
pp. 257-264
Author(s):  
Vojislav Pavlovic ◽  
Jelena Aleksic
Keyword(s):  
Pharmaceutical Products ◽  
Bovine Embryos ◽  
In Vitro Production ◽  
Cattle Breeding ◽  
Vitro Fertilization ◽  
Laboratory Conditions ◽  
Female Donor ◽  
Vitro Production ◽  
Bovine Oocytes

Biotechnology is used for the purpose of improving production, and developing animal and pharmaceutical products. In roder to achieve these objectives, it is necessary to manipulate these processes. Reproductive biotechnology can be used independently, or it can be used in connection with other techniques. Thus, for instance, successful culture of embryos in laboratory conditions is a necessary precondition for the production and creation of transgenic and cloned animals. The in vitro process of embryo production is narrowed down to three basic steps: 1. collecting oocytes form a female donor, 2. fertilization of oocytes under laboratory conditions, 3. growth of the embryo in a medium and transfer of the embryo into the recipient. The paper describes the IVP procedure (in vitro production) of bovine embryos; the advantages and shortcomings of this method, as well as possibilities for its application in cattle breeding. This technology is still quite new, so taht both the technique and the mediums are constantly being improved. The technique of fertilizing bovine oocytes, as well as their development in laboratory conditions was discovered in 1980, and the first calf produced using in vitro fertilization (IVF) was born in 1982. IVP implies a series of steps, and if just one of them is not done perfectly, the result is a small number of embryos, or even none at all.

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

scholarly journals Antioxidant activity of oily extract obtained from Lippia origanoides improves the quality of bovine embryos produced in vitro

2019 ◽  
Vol 71 (3) ◽  
pp. 723-731
Author(s):  
N.V. Sollecito ◽  
E.C.M. Pereira ◽  
J.G.V. Grázia ◽  
B.P. Neves ◽  
B.V.R. Couto ◽  
...  
Keyword(s):  
Embryo Culture ◽  
Culture Medium ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Bovine Embryos ◽  
In Vitro Production ◽  
Blastocyst Development ◽  
Embryo Culture Medium ◽  
Vitro Production

ABSTRACT The aim of this study was to evaluate the supplementation of embryo culture medium with antioxidant obtained from oily extract of Lippia origanoides on in vitro blastocyst development and quality. Oocytes collected from slaughterhouse ovaries were matured and fertilized in vitro following standard laboratory procedures. Zygotes were cultured in SOF medium supplemented according to the following treatments: T1 embryo culture medium without antioxidant supplementation; T2)50μM/mL Cysteamine; T3)2.5μg/mL; T4)5.0μg/mL and T5)10.0μg/mL of antioxidant obtained from oily extract of Lippia origanoides. On the seventh day of culture, the blastocysts were fixed and evaluated for apoptosis rates, number of total cell and inner cell mass cells by means of the TUNEL Test. The use of antioxidants during cultivation did not increase (P> 0.05) the final blastocyst production rate. The treatments T2, T3, T4 and T5 had the lowest (P< 0.05) apoptotic indexes (4.5±1.1%, 8.4±2.5%, 3.4±1.1% and 5.5±0.9%, respectively) when compared to T1 treatment (10.0±1.4%). The number of inner cell mass did not differ (P> 0.05) among embryos from different treatments. The addition of antioxidant obtained from oily extract of Lippia origanoides reduces the apoptosis rate and improves the quality without increasing the total in vitro production of bovine embryos.

135 RECOMBINANT BOVINE SOMATOTROPIN ON THE IN VITRO PRODUCTION OF BOVINE EMBRYOS

2014 ◽  
Vol 26 (1) ◽  
pp. 181
Author(s):  
L. Berté ◽  
L. Vasconcelos ◽  
L. Hatamoto-Zervoudakis ◽  
W. Yamazaki ◽  
L. Yamazaki ◽  
...  
Keyword(s):  
Bovine Embryos ◽  
In Vitro Production ◽  
Blastocyst Development ◽  
Cleavage Rate ◽  
Recombinant Bovine Somatotropin ◽  
Group 4 ◽  
Group 3 ◽  
Group 2 ◽  
Vitro Production

Bovine growth hormone (bGH) has been used to improve the results for in vitro production of bovine embryos. Inclusion of bGH in the maturation medium increases both rate of cleavage and frequency of blastocyst development. Thus, the purpose of the present study was to evaluate the effect of recombinant bovine somatotropin (rBST) on cleavage and blastocyst development of bovine embryos when included during in vitro maturation (IVM) only (Group 1), during both IVM and in vitro culture (IVC; Group 2), during IVC only (Group 3), or not included during either IVM or IVC (Group 4). Specifically, in Group 1, oocytes were matured in TCM 199 (Earle's salts) supplemented with 10% FCS, LH, FSH, oestradiol, and amikacin (IVM medium), plus 100 ng mL–1 of rBST and cultured in SOFaaci supplemented with essential amino acids, tri-sodium citrate, myo-inositol, and 5% FBS. In group 2, oocytes were matured in IVM medium containing 100 ng mL–1 of rBST and cultured in SOFaaci supplemented with essential amino acid, tri-sodium citrate, myo-inositol, 5% FBS; on Day 5, rBST (50 ng mL–1) was added. In Group 3, oocytes were matured in IVM medium without rBST; on Day 5, rBST (50 ng mL–1) was added. Group 4 was the control, without rBST supplementation. The treatment groups were analysed using the SAS® (SAS Institute Inc., Cary, NC, USA) in a completely randomised design (P < 0.05). Somatotropin has receptors in cumulus cells and in the zona pellucida acting directly in the oocyte; however, the increase in cleavage rate seen in previous studies after rBST treatment was not observed in the present study. Supplementation of culture medium with rBST during Day 2 to 6 of IVC has been shown to increase the number of trophoblast and subsequent pregnancy rate following transfer. However, in the present study, addition of 50 and 100 ng mL–1 of rBST to the maturation or culture medium did not affect the cleavage rate of embryos and blastocyst production. Table 1.Analysis of the meaning and percentages related to cleavage and production of embryos

scholarly journals 144 EFFECT OF ANTRAL FOLLICLE COUNT IN BEEF HEIFERS ON IN VITRO FERTILIZATION/PRODUCTION

2017 ◽  
Vol 29 (1) ◽  
pp. 180
Author(s):  
C. C. Chase ◽  
R. A. Cushman ◽  
A. K. McNeel ◽  
E. C. Wright-Johnson ◽  
G. A. Perry ◽  
...  
Keyword(s):  
Antral Follicle ◽  
Antral Follicle Count ◽  
Blastocyst Stage ◽  
In Vitro Production ◽  
Blastocyst Development ◽  
University Of Florida ◽  
Vitro Fertilization ◽  
Frozen Semen ◽  
Vitro Production

Our objective has been to compare the IVF and in vitro production (IVP) of embryos from low and high antral follicle count (AFC) heifers. This is the fourth year of the study with years 1 to 3 reported individually. For this report, we add data for the fourth year and present a combined analysis (years 1 to 4) for the first time. Each year, AFC was determined on ~120 Angus heifers using transrectal ultrasonography. Ten heifers with the lowest AFC and 10 heifers with the highest AFC and all with evidence of oestrous cyclicity were synchronized with two 5-mL injections of PGF2α 11 days apart. Half were harvested on Day 5 to 6 and half on Day 15 to 16 of the oestrous cycle. The IVF procedure was slightly modified each year. For year 4, the IVF procedure included protocols for semi-defined media and was as described (IVP Protocol, P. J. Hansen’s Laboratory, University of Florida). Cumulus-oocyte complexes (COC) from follicles less than 8 mm in diameter were cultured in maturation medium (5% CO2; 38.5°C) for 24 h. Matured COC were fertilized using thawed frozen semen from a bull that was purified using isolate. Motile spermatozoa were added to COC in fertilization medium at a final concentration of 1 × 106 spermatozoa per mL. About 24 h later, presumptive zygotes were placed in micro drops of development medium under oil, and cultured (5% CO2; 5% O2; balance N2; 38.5°C). On Day 3 and 8 after fertilization, cleavage and blastocyst development rates, respectively, were assessed. Data were analysed using the MIXED procedure of SAS (SAS Institute Inc., Cary, NC, USA) and the model included the effects of year (1 to 4), group (high or low AFC), and their interaction. The year × group interaction was not significant (P > 0.10). Low AFC heifers, compared with high AFC heifers, had fewer numbers of COC (P < 0.0001; 12.8 ± 1.83 v. 31.9 ± 1.86), fewer numbers of COC that cleaved (P < 0.0001; 8.0 ± 1.38 v. 21.6 ± 1.40), and fewer numbers of COC that developed to the blastocyst stage (P < 0.0001; 1.7 ± 0.58 v. 5.7 ± 0.58). Year affected the numbers of COC that cleaved (P < 0.003) and the numbers of COC that developed to the blastocyst stage (P < 0.0001). Year also influenced the percentage of COC that cleaved (P < 0.0002) and the percentage of COC that developed to blastocysts (P < 0.0001). Group (AFC) did not influence (P > 0.19) the percentage of COC that cleaved (61.2 ± 2.83 v. 66.4 ± 2.83%, for low v. high AFC, respectively). Low AFC heifers had a lower (P < 0.002) percentage of COC that developed to blastocysts (10.3 ± 1.52%) than high AFC heifers (17.6 ± 1.52%). These results indicate that high AFC heifers, compared to low AFC heifers, have more COC recovered, more COC cleaved, and more COC developed to the blastocyst stage. The percentage of COC cleaved did not differ between AFC groups; however, the percentage of COC that developed to the blastocyst stage was greater for high than low AFC heifers. This suggests a potential advantage in maternal to embryonic transition for high compared with low AFC heifers.

scholarly journals In Vitro Production of Embryos from Prepubertal Holstein Cattle and Mediterranean Water Buffalo: Problems, Progress and Potential

Animals ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 2275
Author(s):  
Luke Currin ◽  
Hernan Baldassarre ◽  
Vilceu Bordignon
Keyword(s):  
Water Buffalo ◽  
Holstein Cattle ◽  
In Vitro Production ◽  
Blastocyst Development ◽  
Follicular Aspiration ◽  
Current State ◽  
Widespread Implementation ◽  
Vitro Production ◽  
Mediterranean Water

Laparoscopic ovum pick-up (LOPU) coupled with in vitro embryo production (IVEP) in prepubertal cattle and buffalo accelerates genetic gain. This article reviews LOPU-IVEP technology in prepubertal Holstein Cattle and Mediterranean Water Buffalo. The recent expansion of genomic-assisted selection has renewed interest and demand for prepubertal LOPU-IVEP schemes; however, low blastocyst development rates has constrained its widespread implementation. Here, we present an overview of the current state of the technology, limitations that persist and suggest possible solutions to improve its efficiency, with a focus on gonadotropin stimulations strategies to prime oocytes prior to follicular aspiration, and IVEP procedures promoting growth factor metabolism and limiting oxidative and endoplasmic reticulum stress.

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