In vitro maturation of buffalo oocytes and fertilization by cattle spermatozoa (original) (raw)
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Reproduction, Fertility and Development, 2004
The identification of an optimal in vitro fertilization system is critical in order to improve the in vitro embryo production efficiency in buffalo species. The aim of this work was to evaluate the effects of fertilization media and sperm motility inducing factors (SMIF) on cleavage and blastocyst rates in buffalo species. Cumulus-oocytes complexes (n=516), recovered from slaughtered animals, were matured in vitro in TCM 199+10 % FCS, 0.5μgmL−1 FSH, 5μgmL−1 LH, 1μgmL−1 17β-estradiol and 50μM cysteamine, at 38.5°C under 5% CO2 in humidified air for 24 hours. The mature oocytes were randomly assigned to four groups for fertilization. In particular, IVF was carried out at 38.5°C under 5% CO2 in humidified air in either Tyrode’s modified medium or Brackett Oliphant medium, in the presence of 0.01mM heparin;; each medium was supplemented with either a mixture of 0.2mM penicillamine and 0.1mM hypotaurine or 5mM caffeine. Frozen-thawed sperm from a tested bull was treated by the swim-up pr...
Factors influencing the maturation, fertilization and development of swamp buffalo oocyte in vitro
Research Opinions in Animal & Veterinary Sciences, 2016
Buffalo ovaries (n=610) were collected from the abattoir and the oocytes recovered were matured, fertilized and the resulting zygotes were cultured in vitro. In Experiment 1, the maturation and fertilization rates of O 1-O 2 oocytes were higher than O 3-O 5 oocytes. In Experiment 2, the two batches of TCM-199 medium used with different hormonal supplementation did not influence the maturation and fertilization of the oocytes at 70.7-86.7% and 59.4-66.0% respectively. In Experiment 3, bull variability was observed in terms of their capacity for fertilization. In Experiment 4, higher fertilization rate was observed in response to caffeine (3-10 mg/ml) compared to control. In Experiment 5, 1 × 10 6 sperm/ml concentration during fertilization had a lower penetration rate (43.0%) than 5, 10 × 10 6 sperm/ml concentration (68.4-71.0%). In Experiment 6, development to the blastocyst stage in TCM-199 or mSOF medium with or without cumulus cell monolayer had no significant difference (11.5-22.2%) with total cell count ranging from 84.6±11.2 to 91.4±12.2. Overall, the results showed the importance of cumulus cells in the acquisition of developmental competence of oocytes maturing in vitro and its subsequent fertilization. That, selection of appropriate bull, its optimum sperm concentration, the choice of capacitating agent and its concentration, the type of medium for culture of resulting zygotes is critical for successful application of in vitro maturation, fertilization and culturing for mass production of buffalo embryos.
Theriogenology, 1998
The objective of the present study was to investigate the effects of sperm concentration and presence or absence of cumulus cells on fertilization, cleavage rate and subsequent embryonic development upto the blastocyst stage in buffalo. Cumulus-oocyte-complexes (COCs) obtained from slaughterhouse ovaries were matured in vitro in TCM-199 + 10% FBS + 5 micrograms/mL FSH-P for 24 h. After maturation the COCs were either used as such (cumulus-intact) or freed from attached cumulus cells by repeated pipetting (cumulus-free). Frozen-thawed buffalo spermatozoa were treated with 10 micrograms/mL heparin and 2.5 mM caffeine for sperm capacitation. Oocytes were fertilized in vitro with 1 to 2, 4 to 5 or 9 to 10 million sperm/mL and the cleavage rate was recorded 42 to 44 h post insemination. The cleaved embryos were co-cultured with buffalo oviductal epithelial cells for 10 d post insemination, and the uncleaved oocytes were fixed and stained with aceto-orcein for determination of the penetra...
Acta Veterinaria Brno, 2007
Effects of two maturation media (TCM-199 and Ham's F-12) with and without the addition of oestrus buffalo serum (OBS) and hormones (FSH, LH, E2) on the maturation rate of buffalo follicular oocytes were evaluated. The results revealed a significant (P < 0.05) increase in the maturation rate when the OBS and hormones were added to TCM-199 than in Ham's F-12 medium. The percentage of maturation rates in TCM-199 + hormones + OBS, TCM-199 + hormones, TCM-199 + OBS and TCM-199 were 77.44, 55.17, 62.28 and 26.62 percent, respectively. While in Ham's F-12 + OBS + hormone, Ham'F-12 + hormone, Ham's F-12 + OBS and Ham's F-12 were 32.85, 27.52, 31.38 and 13.46 percent, respectively. A significantly higher (P < 0.05) fertilization rate was recorded for modified Ca2+ free Tyrode's medium (63.72%) than in TALP (10.9%) and IVF-TL (32.18%). Thus, TCM-199 containing hormones and OBS appeared better for in vitro maturation, whereas modified Ca2+ free tyrode's me...
Conditions for in vitro maturation (IVM) of primary oocytes without conventionally used fetal calf serum and hormones were optimized in order to reduce the cost of laboratory produced bufalo embryos. Comparisons were made between oocyte recovery methods (aspiration vs. slicing) and IVM in medium 199 (static culture method vs. lux culture method) supplemented with 4-5 × 106 granulosa cells mL-1 that contained either estrus bufalo serum (EBS) or fetal calf serum (FCS). Recovery methods were compared according to yield, i.e. cumulus oocyte complexes per ovary (COCs/ovary), the expansion rate (% of COCs that expanded), and nuclear maturation rate (% of germinal vesicle breakdown [GVBD]), following IVM for 22-24 h. In vitro maturation methods (static culture with EBS or FCS and Flux culture with EBS or FCS) were compared on the basis of the expansion rate and in vitro fertilization rate (cleavage rate). COC recovery with the slicing method (2.2 COCs/ovary) was better (P < 0.05) than w...
This study was designed to evaluate the efficacy of different somatic cell types and media in supporting in vitro maturation (IVM), in vitro fertilization (IVF) and early embryonic development competence of buffalo follicular oocytes. Cumulus oocyte complexes were collected for maturation from follicles (>6mm) of buffalo ovaries collected at the local abattoir. Oocytes were co-cultured in tissue culture medium (TCM-199) with either granulosa cells, cumulus cells, or buffalo oviductal epithelial cells (BOEC) @ 3x106 cells/ml or in TCM-199 without helper cells (control) at 39°C and 5%CO2 in humidified air. Fresh semen was prepared in modified Ca++ free Tyrode medium. Fertilization was carried out in four types of media: i) Tyrode lactate albumin pyruvate (TALP), ii) TALP+BOEC, iii) modified Ca++ free Tyrode and iv) modified Ca++ free Tyrode+BOEC. Fertilized oocytes were cultured for early embryonic development in TCM-199 with and without BOEC. Higher maturation rates were observed ...
Alexandria Journal of Veterinary Sciences, 2017
In vitro fertilization (IVF) is a necessary procedure in the in vitro production of embryo. Spermatozoa need to undergo capacitation to be able to perform the acrosome reaction which is required for zona pellucida penetration. Different additives are used to aid in that process, such as caffeine and theophylline. In this study, the effects of IVF media supplementation with D-penicillamine, Hypotaurine and epinephrine (PHE) on fertilization and cleavage rates were assessed. Cumulus oocyte complexes (COCs) containing a compact cumulus cells and normal homogenous cytoplasm were selected. In vitro maturation (IVM) was performed in TCM199 supplied with hormones overnight in a culture conditions of 38.5°C and 5% CO 2 in air. After 20-22 hr. of maturation, oocytes were randomly assigned to five groups of sperm-TALP media (control and four different PHE concentrations) then inseminated. The fertilization rate of the expected embryos were assessed after 22 hr. after staining with aceto-orcin 1% or cultured for 48 hr. to estimate the cleavage rate. The current data revealed that, 25 µl/ml PHE significantly (P< 0.05) improved fertilization (55.93 vs 36.96%) and cleavage (46.15 vs 22.22%) rates when compared to control.
IN VITRO MATURATION, FERTILIZATION AND DEVELOPMENT OF PREPUBERTAL AND MATURE BUFFALO OOCYTES
The competence of pre-pubertal buffalo heifers' oocytes for in vitro maturation, fertilization and development were studied and compared with that of the adult buffalo oocytes. Ovaries were collected from local abattoir. The oocytes were recovered from antral follicles (3-8 mm) in diameter by aspiration technique and cultured in TCM-199 medium supplemented with 10% fetal calf serum(FCS), 10 IU/ml PMSG , 20 IU/ml LH , 1 μg/ml Estradiol 17 β and 50 μg/ml Gentamycin sulfate and incubated in 5% CO 2 and high humidity for 24 h at 38.5°C.
STUDY ON In vitro MATURATION OF LOCAL BUFFALO OOCYTES
The present work was conducted to study the in vitro maturation buffalo follicular oocytes. Buffalo ovaries were collected from local abattoir within four hours of slaughter and transported immediately to the laboratory. Follicular oocytes were recovered by aspiration methods. Quality oocytes ovary as it yielded higher number of good quality oocytes 34, 16, 18 oocytes/ ovary, type A, B and C. There was a significant higher (P˂ 0.05) number of grade A (34) oocytes comparative with grade B (16) and C (18) oocytes. Experimental maturation media revealed that tissue culture medium-199 (TCM-199) resulted in significantly better maturation rate (44). The present study concluded the practical, economical and scientific value of establishing a biological bank for oocytes. The possibility of obtaining collection in all grade oocytes by aspiration method from samples slaughterhouse and the possibility maturation oocytes in laboratory.
Animal reproduction science, 2001
The present study was designed to examine the influence of oocyte quality, culture media and gonadotropins on cleavage rate and development of in vitro fertilized buffalo embryos. Three experiments were conducted. In experiment 1, oocytes were classified by number of cumulus cell layers and morphology of the ooplasm as good, fair or poor. Oocytes were cultured for IVM, IVF and IVC in CR1aa medium. In experiment 2, good quality oocytes were cultured for maturation in: (1) CR1aa; (2) CR2aa; (3) TCM-199; (4) MEM or (5) RPMI-1640, and then fertilized using frozen thawed buffalo spermatozoa in CR1aa. The oocytes were cultured in the same medium used for maturation after fertilization. In experiment 3, oocytes were classified into three groups: group (1) was without gonadotropin and serve as a control; group (2) in which IVM medium was supplemented