258 THE PRELIMINARY RESULTS OF IN VITRO PRODUCTION OF THE WISENT HYBRID EMBRYOS

2015 ◽  
Vol 27 (1) ◽  
pp. 219
Author(s):  
A. M. Duszewska ◽  
W. Olech ◽  
P. Trzeciak ◽  
M. Krzysiak ◽  
L. Rapala ◽  
...  
Keyword(s):  
Threatened Species ◽  
Bos Taurus ◽  
Ex Situ ◽  
European Bison ◽  
Bison Bonasus ◽  
In Vitro Production ◽  
Cell Stage ◽  
Culture Development ◽  
Humidified Air

Wisent (Bison bonasus), also called the European bison, is listed as vulnerable on the International Union for the Conservation of Nature Red List of Threatened Species. In Poland, a program for protection in situ and ex situ is being implemented. One new approach is the use of the in vitro embryo production (IVP) procedures to obtain wisent offspring. In contrast to previous successes with cattle IVP, use of IVP with wisent is limited by the small size of the population (only ~5000 individuals in more than 200 herds in Europe) and seasonal reproduction. The aim of this preliminary study was to obtain hybrid embryos (Bison bonasus × Bos taurus) in vitro. Ovaries were isolated from wisent females outside the reproductive season and eliminated from breeding for reasons other than infertility. Cumulus-oocytes complexes (COC) were isolated from all follicles above 2 mm in diameter. All COC were matured in TCM 199 supplemented with 10% fetal bovine serum, 0.02 IU mL–1 of porcine FSH, 17 β-oestradiol, 0.2 mM Na pyruvate, and antibiotics. The COC were cultured for 24 h at 38.5°C and 5% CO2 in humidified air. The matured COC (Bison bonasus) were fertilized in vitro with sperm from Jersey bulls (Bos taurus) in TALP supplemented with 6 mg mL–1 of fatty acid free BSA (BSA FAF), 0.2 mM Na pyruvate, 20 µM penicillamine,10 µM hypotaurine, 1 µM epinephrine, 2 µg mL–1 heparin, and antibiotics. Spermatozoa were used at a final concentration of 1 × 105 per oocyte and were co-cultured for 18 h at 38.5°C and 5% CO2 in humidified air. The hybrid zygotes were cultured in KSOM supplemented with 5 µg mL–1 of MEM Nonessential Amino Acid Solution (100×), 3 mg mL–1 of BSA FAF, and antibiotic for 192 h at 38.5°C and 5% CO2 in humidified air. The medium was partly replaced by fresh medium after 48 and 144 h of culture. Development was evaluated every day. From 25 COC isolated from wisent ovaries, only 18 COC were qualified for in vitro maturation (60%). Of these, 15 COC (83.3%) matured. The percentage of hybrid embryos that cleaved was 80% after 48 h of culture, and the percentage of embryos that developed up to the 8-cell stage was 33% after 96 h of culture. The morula/blastocyst rate was 26.6% after 192 h of culture, as represented by 1 early blastocyst, 2 compact morulae, and 1 morula. The use of the cattle IVP procedure allowed to receive hybrid embryos (Bison bonasus × Bos taurus), but they developed slower than cattle embryos under the same conditions, based on our previous studies. This research will be continued and may make a contribution to the protection of this threatened species.

scholarly journals IN VITRO PRODUCTION OF INTERSPECIES HYBRID EMBRYOS OF CATTLE (Bos taurus) AND WISENT (Bison bonasus)

2016 ◽  
Vol 51 (6) ◽  
pp. 824-829
Author(s):  
G.N. Singina ◽  
◽  
V.A. Bagirov ◽  
S.S. Danch ◽  
T.E. Taradainik ◽  
...  
Keyword(s):  
Bos Taurus ◽  
Bison Bonasus ◽  
In Vitro Production ◽  
Vitro Production ◽  
Interspecies Hybrid

147 THE DEVELOPMENTAL CHARACTERISTICS OF IN VITRO-PRODUCED CATTLE-WISENT (BOS TAURUS-BISON BONASUS) HYBRID EMBRYOS

2017 ◽  
Vol 29 (1) ◽  
pp. 182
Author(s):  
E. N. Shedova ◽  
G. N. Singina ◽  
V. A. Bagirov ◽  
N. A. Zinovieva
Keyword(s):  
Bos Taurus ◽  
Apoptotic Cell ◽  
Cell Number ◽  
Total Cell ◽  
European Bison ◽  
Bison Bonasus ◽  
Epididymal Sperm ◽  
Total Cell Number ◽  
Cell Ratio

Interspecies hybrids are important resources for research and agriculture. Therefore, the aim of this study was to evaluate development, quality, and viability of embryos produced in vitro using cattle (Bos taurus) oocytes and European bison (Bison bonasus) epididymal sperm. The epididymes were obtained following a forced slaughter of one bull aged 7 years. The sperm was collected by scraping the inner surface of the epididymes, diluted with the cryopreservation medium, and equilibrated for 4 h at 4°C. Thereafter, sperm aliquots (0.2 mL) were frozen in liquid nitrogen vapor for 5 min and then plunged into liquid nitrogen for storage. Prior to fertilization, frozen semen was thawed in pre-warmed medium for 1 min at 37°C and prepared by the swim-up method. The frozen-thawed ejaculated sperm from the Russian Black Pied bulls was used as a positive control. Slaughterhouse-derived cumulus-oocyte complexes were matured for 24 h in TCM-199 supplemented with 10% FCS, 0.2 mM sodium pyruvate, 10 μg mL−1 porcine FSH, and 10 μg mL−1 ovine LH. Matured oocytes (35–40 oocytes per group) were co-incubated for 18 h with homologous (n = 266 oocytes) or heterologous (n = 292 oocytes) sperm (spermatozoa/mL) in 500 µL of TALP containing 10 μg mL−1 heparin, 20 μM penicillamine, 10 μM hypotaurine, 1 μM epinephrine, and 0.1% minimal essential medium nonessential amino acids. After IVF, the oocytes were cultured in CR1aa medium (Rosenkrans 1994 J. Anim. Sci. 72, 434–437) to the blastocyst stage. All the cultures were performed at 38.5°C and 5% CO2 in humidified air. At Days 2 and 7 after insemination, the cleavage and blastocyst rates were determined. In addition, a part of obtained blastocysts was fixed with 4% paraformaldehyde, and the total cell number and apoptotic cell ratio were determined by 4’,6-diamidino-2-phenylindole and TUNEL staining. The remaining blastocysts were cultured up to Day 10, and the hatching rates were assessed. The data (3–5 replicates) were analysed by ANOVA. The cleavage rates did not differ among both male species (72.4 and 77.1%). Furthermore, no significant effects of interspecies fertilization on the blastocyst rate or total cell number per blastocyst were found (27.4 ± 1.6% and 77.0 ± 5.7 for cattle embryos and 26.2 ± 1.9% and 83.1 ± 8.9 for cattle-wisent hybrid embryos). On the other hand, the significant differences between homologous and heterologous fertilization were detected in the rate of hatched blastocysts (60.3 ± 5.1 v. 38 ± 2.9, P < 0.05) and apoptotic cell ratio 7.3 ± 0.8 v. 11.6 ± 1.04, P < 0.05). Our findings demonstrate that hybrid embryos produced by IVF of bovine oocytes with the epididymal sperm of European bison can be developed up to advanced blastocyst stages. However, the hybrid embryos have a lower quality and viability than cattle embryos. Research was supported by the Program of Presidium of the Russian Academy of Science, project no. IV.13.3.

scholarly journals Fetal calf serum enhances in vitro production of Bos taurus indicus embryos

2011 ◽  
Vol 75 (3) ◽  
pp. 429-433 ◽  
Author(s):  
F.G. Leivas ◽  
D.S. Brum ◽  
S.S. Fialho ◽  
W.P. Saliba ◽  
M.T.T. Alvim ◽  
...  
Keyword(s):  
Fetal Calf Serum ◽  
Bos Taurus ◽  
Calf Serum ◽  
In Vitro Production ◽  
Bos Taurus Indicus ◽  
Vitro Production

scholarly journals Ex situ conservation of genetic resources of field elm (Ulmus minor Mill) and European white elm (Ulmus laevis Pall)

Genetika ◽  
2004 ◽  
Vol 36 (3) ◽  
pp. 221-227
Author(s):  
Jelena Aleksic ◽  
Sasa Orlovic
Keyword(s):  
Genetic Resources ◽  
Ex Situ Conservation ◽  
Ex Situ ◽  
In Vitro Production ◽  
Ulmus Minor ◽  
Conservation Of Genetic Resources ◽  
Ulmus Laevis ◽  
Vitro Production

Principles of the conservation of genetic resources of elms (Ulmus spp) do not differ fundamentally from the general principles accepted for the conservation of genetic resources of other common Noble Hardwoods. Efficient conservation can best be achieved through appropriate combination of in situ and ex situ methods, which have distinct advantages. Besides that, ex situ conservation is employed when emergency measures are needed for rare endangered populations and when populations are too small to be managed in situ (e.g. risks of genetic drift and inbreeding). The aim of our research is ex situ conservation of genetic resources of field elm {Ulmus minor Mill) and European white elm (Ulmus laevis Pall) through establishment of field genebanks. Sampling was conducted in one population of field elm and one population of white elm. Plant material (buds) from 8 trees of field elm and 10 trees of white elm was used for in vitro production of clones. Obtained clones will be used for establishment of field genebanks on the experimental estate of the Institute of Lowland Forestry and Environment.

scholarly journals 81 EFFECT OF SUCROSE CONCENTRATION IN WARMING MEDIUM ON THE DEVELOPMENT POTENTIAL OF VITRIFIED BOVINE OOCYTES

2005 ◽  
Vol 17 (2) ◽  
pp. 190
Author(s):  
W.C. Chang ◽  
J. Xu ◽  
S. Jiang ◽  
X.C. Tian ◽  
X. Yang ◽  
...  
Keyword(s):  
Sucrose Concentration ◽  
Control Group ◽  
Bovine Oocyte ◽  
Cell Stage ◽  
Significant Difference ◽  
Humidified Air ◽  
Bovine Oocytes ◽  
Experimental Group ◽  
One Way Anova

The aim of this experiment was to determine the effect of the sucrose concentration (0 to 0.33 M) in the dilution medium on the viability, fertilizability, and development of vitrified bovine oocytes. Bovine oocyte-cumulus complexes were collected from slaughterhouse ovaries and in vitro-matured as reported previously. After 24-h maturation in TCM199-based medium under 5% CO2 humidified air at 39°C, these were exposed to hyaluronidase and carefully pipetted to remove all except the 3–5 innermost layers of cumulus. Oocytes were put into the pre-equilibration medium for 3 min and then into vitrification solution containing HEPES-buffered TCM199 supplemented with 20% FBS, ethylene glycol, and dimethylsulphoxide for 25–30 s; they were then vitrified by modified solid surface vitrification (Dinnyes et al. 2000 Biol. Reprod. 63, 513–518).The oocytes were warmed at 39°C by placing them in holding medium with 0, 0.08, 0.17, 0.25, or 0.33 M sucrose. Non-vitrified oocytes were used as controls. Oocytes were inseminated 30 min after warming, and the presumptive zygotes were cultured in CR1-aa medium supplemented with 6 mg/mL BSA at 39°C in a humidified atmosphere of 5% CO2, 5% O2, and 90% N2 for eight days. Data were analyzed by one-way ANOVA. As shown in Table 1, there was no significant difference in survival rate (P > 0.05) of the vitrified oocytes that were placed in dilution solution containing 0.17, 0.25, or 0.33 M sucrose and the non-treated controls. On Day 2 (fertilized on Day 0), cleavage to the 8-cell stage was similar for the 0.17, 0.25, and 0.33 M dilution groups, but the rates for all three were significantly lower (P < 0.05) than for the control group. The blastocyst rate on Day 8 was significantly higher for the 0.25 M group than for any other experimental group but still significantly lower than for the control. In conclusion, this study suggests that with this vitrification/warming procedure the optimum concentration of sucrose in the dilution solution is 0.25 M. Table 1. Oocyte survival after vitrification/warming and subsequent embryo development The authors would like to thank Ms Colleen Shaffer for the preparation of bovine oocytes.

296 EFFECT OF GROWTH HORMONE ON EMBRYO DEVELOPMENT AND ULTRASTRUCTURE OF BOS TAURUS IVP BOVINE EMBRYOS

2010 ◽  
Vol 22 (1) ◽  
pp. 304
Author(s):  
L. M. C. Pegoraro ◽  
M. N. Dode ◽  
C. F. Weissheimer ◽  
F. G. Leivas ◽  
A. Vieira ◽  
...  
Keyword(s):  
Growth Hormone ◽  
Culture Medium ◽  
Production Systems ◽  
Bos Taurus ◽  
In Vitro Production ◽  
Assisted Reproductive Technique ◽  
Development Rates ◽  
Developmental Rates ◽  
Chi Square Analysis

Bovine in vitro production systems are one of the most used assisted reproductive technique. However, this technique has some limitations especially in Bos taurus breeds, because of the low percentage of viable blastocysts produced (around 40% of oocytes inseminated) and higher cryosensitivity due to higher lipid content. Growth hormone (GH) can be a promising additive to increase in vitro embryo production. The aim of this study was to evaluate the embryo developmental rates (blastocyst/oocytes cleaved and blastocyst/oocytes inseminated) and ultrastructural features in Bos taurus embryos produced in SOFaa medium with or without GH. Cumulus oocyte complexes (COC) were recovered from slaughterhouse-derived ovaries (Angus Red crosses) and by ovum pick-up (OPU) from Jersey donors. After IVM and IVF, the presumptive zygotes were allocated in the SOFaa medium without (control) or with addition of GH (100 ng mL-1), for culture at 39°C in an atmosphere of 5% CO2. The cleavage and viable blastocyst rates were recorded 2 and 8 days after initiation of IVF, respectively. The results were compared by chi-square analysis. Similar (P > 0.05) cleavage rates were found in different culture medium and bovine breeds (61 v. 63% for Jersey control and Jersey GH; 71 v. 72% for cross-breed control and cross-breed GH). The development rates (blastocyst/oocytes cleaved and blastocyst/oocytes inseminated) did not differ in culture medium with or without GH within breeds (35 v. 30% for Jersey control and GH; 52 v. 56% for cross-breed control and GH; 21 v. 20% for Jersey control and GH; 36 v. 41% for cross-breed control and GH, respectively; P > 0.05). However, when breeds were compared, higher development rates were observed in cross-breed obtained from slaughterhouses than Jersey donors obtained by OPU (35 v. 52% for Jersey v. cross breed control; 30 v. 56% for Jersey v. cross-breed GH; 21 v. 36% for Jersey v. cross-breed control; 20 v. 41% for Jersey v. cross-breed GH. P < 0.001). The analyses of ultrastructure demonstrated no difference in the lipid proportion and organelle distribution of embryos produced with or without GH. We concluded that GH addition to SOFaa medium did not increase developmental rates for cross-breed or Jersey IVP embryos.

248 IN VITRO PRODUCTION OF CATTLE × BUFFALO HYBRID EMBRYOS USING BOVINE OOCYTES AND AFRICAN BUFFALO (SYNCERUS CAFFER CAFFER) EPIDIDYMAL SPERM

2007 ◽  
Vol 19 (1) ◽  
pp. 240
Author(s):  
O. D. Owiny ◽  
D. M. Barry ◽  
M. Agaba ◽  
R. A. Godke
Keyword(s):  
Bison Bison ◽  
Abnormal Development ◽  
African Buffalo ◽  
Syncerus Caffer ◽  
Epididymal Sperm ◽  
In Vitro Production ◽  
Cleavage Rate ◽  
Cell Stage ◽  
Bovine Oocytes

Interspecies hybridization of bovids occurs between domestic cattle and at least 3 other species: the American bison (Bison bison), yak (Bos grunniens), and banteng (Bos banteng). Birth of a cattle � buffalo hybrid was reported in Russia, but the report was never authenticated. Such hybrids could be important in improving livestock production and managing diseases that impede production in tropical Africa. We investigated hybridization between cattle and their closest African wild relative, the African buffalo (Syncerus caffer caffer). In an attempt to produce pre-implantation cattle � buffalo hybrid embryos in vitro, matured bovine oocytes were subjected to a standard IVF procedure with either homologous (n = 1166 oocytes) or heterologous (n = 1202 oocytes) buffalo epididymal sperm. After IVF, 67.2% of the oocytes inseminated with homologous sperm cleaved. In contrast, insemination with buffalo sperm resulted in a 4.6% cleavage rate. Cleavage was also slower in hybrids than in cattle embryos. Up to 52.2% of cleaved homologous embryos progressed to the morula stage compared with 12.7% for hybrids. No hybrid embryos developed beyond the 16-cell stage, whereas 40.1% of the cleaved bovine embryos developed to the blastocyst stage. Developmental anomalies such as polyspermy, uneven cleavage, vacuolization, and absence of nuclei in some blastomeres were common in the hybrid embryos. We conclude that interspecies fertilization of cattle oocytes with African buffalo sperm occurs in vitro and that the barrier to hybridization is in the early stages of embryonic development. Also, chromosomal disparity is the likely cause of fertilization abnormalities, abnormal development, and subsequent arrest, impairing the formation of pre-implantation hybrid embryos. Investigation into developmental abnormalities, including reciprocal hybridization and genetic studies of the hybrid embryos, are recommended.

257 DIFFERENCES BETWEEN BOS TAURUS AND BOS INDICUS EMBRYOS: TRANSCRIPTS AMOUNTS

2010 ◽  
Vol 22 (1) ◽  
pp. 285
Author(s):  
S. Wohlres-Viana ◽  
M. M. Pereira ◽  
A. P. Oliveira ◽  
J. H. M. Viana ◽  
M. A. Machado ◽  
...  
Keyword(s):  
Production System ◽  
Production Systems ◽  
Bos Taurus ◽  
Bos Indicus ◽  
In Vitro Production ◽  
Embryo Production ◽  
Peroxiredoxin 1 ◽  
Vitro Production

The Zebu breeds (Bos indicus) are different from European breeds (Bos taurus) in some aspects of their reproductive physiology, including follicle recruitment, number of follicular waves, and oocyte ultrastructure. On the other hand, embryos produced in vivo and in vitro show morphological and developmental differences, which can be related to culture environment. The aim of this study was to evaluate the effect of breed (Gyr v. Holstein) within embryo production system (in vivo and in vitro), as well as effect of production systems within breeds on relative abundance of transcripts related to formation, survival, and subsequent development of blastocysts, such as those involved in water and small solutes transport (Aquaporins 3 and 11), blastocoel formation (Na+/K+-ATPase a1 and |52), and cellular stress response (Peroxiredoxin 1). For in vivo embryo production, donors were superstimulated with FSH and inseminated, and embryos were recovered 7 days after AI. For in vitro embryo production, oocytes recovered by ovum pickup were in vitro matured and fertilized and then cultured for 7 days in culture medium under 5% CO2 at 38.5°C. For each group, blastocysts (n = 15) distributed in 3 pools were used for RNA extraction (RNeasy MicroKit, Qiagen, Valencia, CA, USA), followed by RNA amplification (Messageamp II amplification kit, Ambion-Applied Biosystems, Foster City, CA, USA) and reverse transcription (SuperScript III First-Stand Synthesis Supermix, Invitrogen, Carlsbad, CA, USA). The cDNA were submitted to real-time PCR, using the H2a gene as endogenous control, and analyzed by REST© software. To evaluate breed effect within the production systems, 2 comparisons were performed: (1) in vivo: Gyr v. Holstein and (2) in vitro: Gyr v. Holstein, considering Holstein data as 1.00. To evaluate production system effect within breeds, 2 comparisons were performed: (1) Gyr: in vivo v. in vitro and (2) Holstein: in vivo v. in vitro, considering in vivo produced embryo data as 1.00. The results are shown as mean ± SEM. For in vivo comparison between breeds, Aquaporin 3 (1.66 ± 0.77), Na+/K+-ATPase a1 (1.61 ± 0.56), and Peroxiredoxin 1 (1.61 ± 0.66) were up-regulated (P < 0.05) in Gyr embryos when compared with Holstein embryos, whereas for in vitro comparison, no differences (P > 0.05) were found. For comparisons between production systems within breeds, only Peroxiredoxin 1 (0.31 ± 0.39) was down-regulated (P < 0.01) in in vitro produced Gyr embryos when compared with in vivo counterparts. No differences (P > 0.05) were found between production systems for the Holstein breed. In conclusion, these data suggest that there is a difference on gene expression between Bos taurus and Bos indicus blastocysts, but such difference between breeds can be attenuated by the in vitro production system, indicating an embryo adaptation to the in vitro culture conditions. The data also suggest that the in vitro production system can influence the amount of transcripts in Gyr embryos. Other genes should be evaluated for a better understanding of these differences. Financial support was provided by CNPq and FAPEMIG.

146 Quercetin supplementation during boar semen thawing and incubation improves the in vitro production of pig embryos

2019 ◽  
Vol 31 (1) ◽  
pp. 198
Author(s):  
E. Hicks ◽  
E. Winn ◽  
B. Whitaker
Keyword(s):  
Oxidative Stress ◽  
Embryonic Development ◽  
Developmental Stages ◽  
Membrane Damage ◽  
In Vitro Production ◽  
Cell Stage ◽  
Stage Of Development ◽  
Boar Semen ◽  
Morphological Deformities

Elevated levels of reactive oxygen species in the in vitro environment cause oxidative stress, which leads to membrane damage, decreased fertility, and morphological deformities of spermatozoa. Antioxidants, such as quercetin (a polyphenol flavonoid), are often supplemented to reduce the effects of oxidative stress on spermatozoa. Supplementing frozen-thawed boar semen with quercetin improves sperm forward progressive motility, viability and lipid peroxidation up to 10h after thawing. However, the effects of fertilizing with quercetin-supplemented sperm are unknown. Therefore, the objective of this study was to determine the effects of supplementing quercetin (0.25, 0.50, 0.75mM) during the thawing and incubation of frozen-thawed boar semen on oocyte fertilization characteristics (n=400) and subsequent embryonic development (n=1340) at 48 and 144h for cleavage and blastocyst formation, respectively. Oocytes from aspired aspirated mature follicles (3-6mm diameter) were obtained from a local abattoir and matured in medium 199 for 40 to 44h at 38.5°C in an atmosphere of 5% CO2. Fertilization was performed using pooled frozen-thawed semen from 3 different boars, and co-incubation of the sperm (2×105 sperm mL−1) and oocytes (30 oocytes/well) lasted for 6 to 8h at 38.5°C in an atmosphere of 5% CO2. Data were analysed using ANOVA with the main effects including treatment, well and replicate. Chi-squared analysis was used to determine percentages of embryos reaching the different developmental stages for each treatment. There were no differences in penetration rates and male pronuclear formation between treatment groups; however, supplementation of 0.25 (18.18±10.63%), 0.50 (20.93±9.89%) and 0.75mM (18.07±12.02%) quercetin significantly decreased (P&lt;0.05) polyspermic penetration rates compared with no supplementation (40.00±11.34%). Embryos produced from frozen-thawed boar sperm supplemented with 0.25 and 0.50mM quercetin had a significantly higher percentage (P&lt;0.05) of embryos reaching the 2-cell stage of development by 48h after IVF (75.00±7.89%, 68.75±2.23%, respectively) compared with 0.75mM quercetin supplementation (64.62±3.88%) and no supplementation (62.97±4.11%). Supplementation of 0.25 (44.12±6.23%), 0.50 (43.75±7.02%) and 0.75mM (43.08±2.98%) quercetin to the sperm significantly increased (P&lt;0.05) the percentage of embryos reaching the blastocyst stage of development by 144h after IVF compared with no supplementation (28.27±8.07%). These results indicate that supplementing frozen-thawed boar semen with quercetin decreases the incidence of polyspermic penetration and improves early embryonic development in pigs.

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