Influences of zinc on fertilisation and development of bovine oocytes in vitro

Zygote ◽  
1999 ◽  
Vol 7 (3) ◽  
pp. 195-201 ◽  
Author(s):  
J.L. Stephenson ◽  
B.G. Brackett
Keyword(s):  
Sperm Motility ◽  
Oocyte Maturation ◽  
Follicular Fluid ◽  
Bovine Embryos ◽  
Sperm Preparation ◽  
Morula Stage ◽  
Zinc Concentrations ◽  
Bovine Oocytes

The effects of zinc (as ZnCl2) on in vitro production of bovine embryos (IVMFC) and components of the procedure, that is in vitro oocyte maturation (IVM), fertilisation (IVF) and embryo development in culture (IVC), and the effect of added zinc on sperm motility were studied. Immature cumulus oocyte complexes (COCs) were aspirated from ovarian follicles (2-5 mm diameter) at slaughter, and matured, fertilised and cultured in chemically defined conditions. The presence of zinc (10, 100 or 1000 μg added per millilitre) throughout IVMFC inhibited fertilisation. After addition of 10 μg zinc per millilitre separately to media for IVM and IVF, fertilisation was inhibited only when zinc was present for IVM. When present for IVF, 80% of oocytes selected for IVM reached 2- to 4-cell stages by 46 h after insemination whereas 67% of control oocytes (inseminated without added zinc) cleaved. Higher zinc concentrations (100 and 1000 μg added per millilitre) for IVF inhibited fertilisation. Sperm motility was reduced with addition of 10 μg per millilitre of zinc for sperm preparation (i.e. capacitation interval). Addition of 1.0 μg zinc per millilitre to media used through IVMFC, or to the IVC medium alone, resulted in inhibition of development after 2- to 4-cell stages. When added to IVM or to both IVM and IVF media 1.0 μg/ml of zinc compromised development to the morula stage and beyond. Maturing bovine oocytes may be more sensitive to 1.0 μg ml of zinc in vitro than in vivo because a concentration of 3.0 μg/ml has been reported for bovine follicular fluid. Fertilisation was not adversely affected by 10 μg/ml of zinc; however, higher concentrations were inhibitory.

scholarly journals Electrolytes, Metabolic And Acid-Base Parameters In Blood And Fluids of The Reproductive Tract During In Vivo Maturation of Bovine Oocytes

2021 ◽  
Author(s):  
Omer F. Gungor ◽  
Saleh Salman ◽  
Saurav Ranjitkar ◽  
Delong Zhang ◽  
Xiuchun (Cindy) Tian
Keyword(s):  
Oocyte Maturation ◽  
Follicular Fluid ◽  
Reproductive Tract ◽  
Venous Blood ◽  
Acid Base ◽  
Time Points ◽  
Base Parameters ◽  
Bovine Oocytes

Abstract Follicular fluid is the microenvironment that supports oocyte maturation and competence. Using Abbott iSTAT1™ and NanoDrop, we determined the dynamics of acid-base, electrolyte, metabolites, and total protein in venous blood, fluids of the dominant follicle (FF), oviduct (OF), and uterus (UF) during the window of oocyte maturation. Holstein heifers (n=36) were synchronized with PGF2α on Days -11 and 0, CIDR during Days -6 to 1, and GnRH given on Day 2 after 2nd PG. Samples were collected at 24h, 48h, 60h, 72h, and 78h after 2nd PG. Most electrolytes analyzed, Cl-, K+, and Ca2+ were significantly affected in blood and FF (P<0.05) by CIDR removal. Similarly, Cl- and Na+ also significantly changed in OF and UF across time. Glucose, lactate, and creatinine significantly changed across time points in FF compared to blood. Moreover, pO2, pCO2, TCO2, and pH significantly changed across time in FF. Most parameters were not significantly correlated between blood and FF across time points except for glucose, Cl- and creatinine. Furthermore, pO2 in FF was nearly 3X higher than blood, suggesting low O2 during in vitro maturation is inappropriate. In conclusion, components of the follicular fluid undergo major changes during the window of oocyte maturation.

In vitro development of morulae from immature caprine oocytes

Zygote ◽  
1994 ◽  
Vol 2 (2) ◽  
pp. 97-102 ◽  
Author(s):  
Levent Keskintepe ◽  
Gamal M. Darwish ◽  
Abdelmoneim I. Younis ◽  
Benjamin G. Brackett
Keyword(s):  
Oocyte Maturation ◽  
Follicular Fluid ◽  
Glycoprotein Hormones ◽  
Glycoprotein Hormone ◽  
In Vitro Development ◽  
Morula Stage ◽  
Vitro Development ◽  
Hormone Control ◽  
Medium Supplementation

SummaryThe effects of medium supplementation with oestrous goat serum and glycoprotein hormones on caprine oocyte maturation in vitro (IVM) were evidenced by proportions of resulting ova completing in vitro fertilisation (IVF) and development to the morula stage. Oocyte-cumulus complexes (OCCs) were harvested in follicular fluid from 2–5 mm diameter follicles. Oocyte maturation took place during 27 h in TCM-199 supplemented with 20% oestrous goat serum, oestradiol-17β (1.0 μg/ml), and either (a) 0.5 μg FSH/ml, (b) 100 μg LH/ml, (c) 100 μg LH + 0.5 μg FSH/ml, (d) 100 μg hCG + 0.5 μg FSH/ml, (e) 0.5 μg TSH/ml or (f) no added glycoprotein hormone (control). Of 353 immature oocytes cultured in seven experiments, 311 (88.1%) exhibited cumulus expansion at the end of the IVM interval; all normalappearing OCCs were inseminated. In vitro insemination was with ejaculated sperm treated with heparin (10 μg/ml) and caffeine (0.4 μg/ml). Proportions (%) of inseminated ova that were fertilised (cleaved) and that reached the morula stage after IVM with (a) FSH, (b) LH, (c) LH + FSH, (d) hCG + FSH, (e) TSH and (f) no added glycoprotein hormone were (a) 22/52 (42.3%) and 9/52 (17.3%), (b) 25/54 (46.3%) and 14/54 (25.9%), (c) 52/65 (80.0%) and 26/65 (40.0%), (d) 48/78 (61.5%) and 22/78 (28.2%), (e) 14/54 (25.9%) and 4/54 (7.4%), and (f) 11/50 (22.0%) and 1/50 (2.0%), respectively. All treatments yielded better results than IVM with no added glycoprotein hormone. After IVM with added LH + FSH higher proportions of oocytes were fertilised (p<0.05), and higher proportions reached the morula stage (p<0.05) when compared with other treatments.

191 IN VITRO CULTURE DURING OOCYTE MATURATION AND FERTILIZATION INFLUENCES THE TRANSCRIPTOME PROFILES OF BOVINE BLASTOCYSTS

2015 ◽  
Vol 27 (1) ◽  
pp. 186
Author(s):  
A. Gad ◽  
U. Besenfelder ◽  
V. Havlicek ◽  
M. Hölker ◽  
F. Rings ◽  
...  
Keyword(s):  
Lipid Metabolism ◽  
Oocyte Maturation ◽  
Microarray Data ◽  
In Vitro Maturation ◽  
Culture Conditions ◽  
Control Group ◽  
Bovine Embryos ◽  
Vitro Fertilization

Early embryonic development, the period from oocyte maturation until blastocyst formation, is the most critical period of mammalian development. It is well known that in vitro maturation, fertilization, and culture of bovine embryos is highly affected by culture conditions. However, the stage-specific effect of culture environment is poorly understood. Therefore, we aimed to examine the effect of in vitro culture conditions during oocyte maturation and fertilization on the transcriptome profile of the resulting blastocysts. Bovine oocytes were matured in vitro and then either directly transferred to synchronized recipients, fertilized, and cultured in vivo (Vitro_M), or transferred after in vitro fertilization (Vitro_F), or at zygote stage (Vitro_Z) and blastocysts were collected at Day 7 by uterine flushing. For in vivo or in vitro fertilization, the same frozen-thawed commercial bull semen has been used. Complete in vitro (IVP) and in vivo produced blastocysts were used as controls. Gene expression patterns between each blastocyst group and in vivo produced blastocyst group were compared using EmbryoGENE's bovine microarray (EmbryoGENE, Québec, QC, Canada) over six replicates of each group (10 blastocyst/replicate). Microarray data were statistically analysed using the Linear Models for Microarray Data Analysis (LIMMA) package under the R program (The R Project for Statistical Computing, Vienna, Austria). Results showed that, the longer the embryos spent under in vitro conditions, the higher was the number of differentially expressed genes (DEG, fold-change = 2 with adjusted P-value = 0.05) compared with in vivo control group. The Vitro_M group showed the lowest number of DEG (149); in contrast IVP group represented 841, DEG, respectively compared to in vivo control group. Ontological classification of DEG showed that lipid metabolism was the most significant function influenced by in vitro maturation conditions. More than 55% of DEG in the Vitro_M group were involved in the lipid metabolism process and most of them showed down-regulation compared to in vivo control group. On the other hand, Vitro_F and Vitro_Z groups showed nearly similar numbers of DEG (584 and 532, respectively) and the majority of these genes in both groups were involved in cell-death- and cell-cycle-related functions. Pathway analysis revealed that retinoic acid receptor activation pathways were the common ones in the Vitro_M and Vitro_F groups. However, different signalling pathways were commonly dominant in the Vitro_F and Vitro_Z groups. This study provides the transcriptome elasticity of bovine embryos exposed to different environments during maturation, fertilization, and culture periods of development.

169 Nuclear Maturation Kinetics and In Vitro Fecundation of Immature Bovine Oocytes Injected into Preovulatory Follicles

2018 ◽  
Vol 30 (1) ◽  
pp. 224
Author(s):  
L. M. S. Simoes ◽  
A. P. C. Santos ◽  
E. A. Lima ◽  
R. E. Orlandi ◽  
M. P. Bottino ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
Germinal Vesicle ◽  
Causative Factor ◽  
Nuclear Maturation ◽  
Preovulatory Follicles ◽  
Fertilization Capacity ◽  
Bovine Oocytes ◽  
Metaphase I

The objective was to evaluate in vitro nuclear maturation and fecundation kinetics of oocytes injected into preovulatory follicles of synchronized cows using the intra-follicular oocyte injection (IFOI) technique. In experiment 1, 438 immature abattoir-bovine cumulus–oocyte complexes (COC) of grades I, II, and III were randomly allocated to 1 of 3 groups: Matvitro (n = 111), COC matured in vitro for 22 h; Matvivo20 (n = 172) and Matvivo30 (n = 155), 30 oocytes were injected into each preovulatory follicle of pre-synchronized recipients. In Matvivo20, oocytes were matured for 19.8 ± 0.1 h and in Matvivo30, for 28.3 ± 0.1 h. All cows received 12.5 mg of LH (Lutropin, Bioniche, Canada) at IFOI (Matvivo20) or 10 h after IFOI (Matvivo30). Oocytes from Matvivo20 and Matvivo30 were aspirated 20 h after LH injection for assessment of oocyte maturation and recovery rates. Oocytes were evaluated according to maturation kinetics as germinal vesicle, metaphase I, anaphase I, telophase I, metaphase II, parthenogenetically activated, and degenerated (chromosomal aberrations, presence of diffuse or indefinite chromatin). In experiment 2, immature abattoir-bovine COC (n = 202) of grades I, II, and III were randomly distributed into 2 groups: Matvitro (n = 103), COC were matured and fertilized in vitro; Matvivo (n = 99), same as Matvivo20 protocol, and COC fertilized in vitro. Presumptive zygotes were evaluated as fertilized, unfertilized, or polyspermic. Statistical analyses were performed by the GLIMMIX procedure of SAS (SAS Institute Inc., Cary, NC, USA). Recovery rate was lower (P < 0.001) in Matvivo20 (52.9%, 91/172) compared with Matvivo30 (72.9%, 113/155). Germinal vesicle (P = 0.94), metaphase I (P = 0.98), anaphase I (P = 0.99), and telophase I (P = 0.20) rates were similar. However, there were differences in metaphase II [Matvitro: 81.0% (90/111)a, Matvivo20: 74.5% (35/47)a, and Matvivo30: 41.6% (32/77)b; P = 0.001], degenerate [Matvitro: 5.4% (6/111)c, Matvivo20: 21.3% (10/47)b and Matvivo30: 48.1% (37/77); P = 0.001] and parthenogenetically activated [Matvitro: 0.0% (0/111)b, Matvivo20: 0.0% (0/47)b and Matvivo30: 9.1% (7/77)a; P = 0.001]. Polyspermic (P = 0.18) and abnormal (P = 0.98) rates were similar. However, there was a higher rate (P = 0.05) of fertilized oocytes in Matvivo (60.6%, 60/99) than in Matvitro (46.6%, 48/103). In conclusion, oocyte maturation in vivo after IFOI for 20 h does not alter maturation kinetics and increases in vitro oocyte fertilization capacity. However, the 10-h increase in intra-follicular oocyte permanence decreased the proportion of viable oocytes. Thus, the oocyte maturation phase is not the limiting causative factor for the low IFOI-embryo production rates.

scholarly journals Versican G1 Fragment Establishes a Strongly Stabilized Interaction with Hyaluronan-Rich Expanding Matrix during Oocyte Maturation

2020 ◽  
Vol 21 (7) ◽  
pp. 2267 ◽  
Author(s):  
Eva Nagyova ◽  
Antonietta Salustri ◽  
Lucie Nemcova ◽  
Sona Scsukova ◽  
Jaroslav Kalous ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Oocyte Maturation ◽  
Follicular Fluid ◽  
Cell Function ◽  
Ovarian Follicle ◽  
Rt Pcr ◽  
Positive Form ◽  
And Function

In the mammalian ovary, the hyaluronan (HA)-rich cumulus extracellular matrix (ECM) organized during the gonadotropin-induced process of oocyte maturation is essential for ovulation of the oocyte-cumulus complex (OCC) and fertilization. Versican is an HA-binding proteoglycan that regulates cell function and ECM assembly. Versican cleavage and function remain to be determined in ovarian follicle. We investigated versican expression in porcine ovarian follicles by real-time (RT)-PCR and western blotting. The aims of the present work were to determine whether 1) versican was produced and cleaved by porcine OCCs during gonadotropin stimulation; 2) these processes were autonomous or required the participation of mural granulosa cells (MGCs); and 3) versican cleavage was involved in the formation or degradation of expanded cumulus ECM. We demonstrate two cleavage products of G1 domain of versican (V1) accumulated in the HA-rich cumulus ECM. One of them, a G1-DPEAAE N-terminal fragment (VG1) of ~70 kDa, was generated from V1 during organization of HA in in vivo and in vitro expanded porcine OCCs. Second, the V1-cleaved DPEAAE-positive form of ~65 kDa was the only species detected in MGCs. No versican cleavage products were detected in OCCs cultured without follicular fluid. In summary, porcine OCCs are autonomous in producing and cleaving V1; the cleaved fragment of ~70 kDa VG1 is specific for formation of the expanded cumulus HA-rich ECM.

237 CHROMOSOMAL ABNORMALITIES IN IN VITRO-PRODUCED EARLY BOVINE EMBRYOS: USE OF HOMOLOGOUS FOLLICULAR FLUID SUPPLEMENTATION IN THE OOCYTE MATURATION MEDIA

2013 ◽  
Vol 25 (1) ◽  
pp. 266
Author(s):  
S. Demyda-Peyrás ◽  
M. Hidalgo ◽  
J. Dorado ◽  
L. De Luca ◽  
E. Genero ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
Follicular Fluid ◽  
Chromosomal Abnormalities ◽  
Culture Media ◽  
Control Group ◽  
Bovine Embryos ◽  
Humid Atmosphere ◽  
Serum Replacement ◽  
Laboratory Techniques

Chromosomal abnormalities were described as a possible cause of embryo failures in cattle, even more so when they are in vitro produced. It has been widely demonstrated that the post-fertilization culture environment affects the frequency of blastomeric aneuploidies. However, the literature concerning the effect of the oocyte maturation techniques on in vitro-produced embryos is scarce. The aim of this study was to determine the effect of homologous bovine follicular fluid (BFF) as a possible replacement for commercial sera in the appearance of chromosomal abnormalities on early IVF-produced embryos. Cumulus–oocyte complexes obtained from ovarian puncture were maturated in modified bicarbonate-buffered TCM-199 media, supplemented with glutamine, sodium pyruvate, FSH, LH, oestradiol, and gentamicin in three different groups. Two treatments were performed: 1) base media supplemented with BFF, obtained aseptically from follicles between 4 and 10 mm in diameter (10 and 20%), and 2) a control group, with base media supplemented with 10% FCS without BFF. After 20 h of culture at 38.5°C in a 5% CO2 humid atmosphere, cumulus–oocyte complexes from both treatments were fertilized in IVF media and then cultured for 72 h in SOF media, according to our laboratory techniques. A total of 152 early embryos were cytogenetically evaluated following our standard laboratory techniques. Developed embryos were individually fixed onto a slide, disaggregated into blastomeres with acetic acid, and stained with Giemsa solution. Chromosomal numerical abnormalities were evaluated in each embryo by direct observation at 1250× magnification using a bright field microscope. Results were statistically compared among treatments by the expected proportion test. No significant differences (P > 0.05) were found between different culture media on the percentages of normal diploid embryos or each kind of numerical abnormality. According to our results (Table 1), the use of homologous follicular fluid as a supplement on the oocyte maturation media did not influence the appearance of abnormal complements in the embryos produced compared with the use of FCS. In conclusion, homologous follicular fluid may be considered a valid serum replacement in the maturation media on IVF-produced bovine embryos. Table 1.Analysis of chromosomal complements of Day 3 in vitro-produced bovine embryos derived from oocytes maturated in culture media with different serum supplementation1

145 CHANGES IN OXYGEN COMSUMPTION RATES OF EMBRYOS IN KOREAN CATTLE

2010 ◽  
Vol 22 (1) ◽  
pp. 231
Author(s):  
C. Y. Choe ◽  
S. R. Cho ◽  
J. K. Son ◽  
S. H. Choi ◽  
C. Y. Cho ◽  
...  
Keyword(s):  
Oxygen Consumption ◽  
Embryo Quality ◽  
Blastocyst Stage ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Cell Stage ◽  
Significant Difference ◽  
Morula Stage

Oxygen consumption has been regarded as a useful indicator for assessment of mammalian embryo quality. This study was carried out to identify whether oxygen consumption rates measured in bovine embryos using SECM can be used as a standard criteria to evaluate bovine embryo quality. Oxygen consumption of bovine embryos at various developmental stages was measured and analyzed using SECM and ANOVA analysis, respectively. We found that the oxygen consumption significantly increased in blastocyst-stage embryos compared to other stage embryos (from 2-cell stage to morula stage), indicating that oxygen consumption reflects the cell number (5.2-7.6 × 1014 mol-1 s-1 v. 1.2-2.4 × 1014 mol-1 s-1, P < 0.05). There was no significant difference between 2-cell-stage embryos and 8-cell-stage embryos. In the morula-stage embryos, the oxygen consumption of in vivo derived embryos was significantly higher than that of in vitro produced embryos (4.0 × 1014 mol-1 s-1 v. 2.4 × 1014 mol-1 s-1, P < 0.05). However, there was no significant difference in consumption of oxygen by in vivo and in vitro derived bovine blastocyst-stage embryos (P > 0.05). Good-quality embryos with grade 1 or 2 showed significantly higher oxygen consumption than grade 3 or 4 embryos. These results showed that SECM could measure oxygen consumption in bovine embryos and the oxygen consumption could reflect embryonic development stage and embryo quality.

Octylphenol, an environmental oestrogen, affects oocyte maturation in cattle

2001 ◽  
Vol 2001 ◽  
pp. 64-64 ◽  
Author(s):  
P Pocar ◽  
R Augustin ◽  
F Gandolfi ◽  
B Fischer
Keyword(s):  
Oocyte Maturation ◽  
Nonionic Surfactants ◽  
Cumulus Cells ◽  
Model System ◽  
Animal Cells ◽  
Nuclear Maturation ◽  
Maturation In Vitro ◽  
Bovine Oocytes

4-tert-octylphenol (OP) is an alkylphenolic compound formed as metabolite of some nonionic surfactants that are widely used in industrial detergents, as plastic additives, dispersant for insecticides, etc. (Naylor et al., 1992). OP accumulates in adipose tissue. Micromolar concentrations of these compounds may constitute health hazards to animal cells. Furthermore, it has previously been shown to exert oestrogenic activity in vivo and in vitro (White et al., 1994). A growing concern about “endocrine disruptors” and their impact on oestrogen-dependent phenomena led us investigate the effects of OP on oocyte maturation. For variuos reasons bovine oocytes were chosen as the model system. We examined the effects of OP exposure on oocyte nuclear maturation in vitro and on the expression of oestrogen receptors in cumulus cells.

scholarly journals 103A NEW PAPER CONTAINER FOR THE VITRIFICATION OF BOVINE EMBRYOS

2004 ◽  
Vol 16 (2) ◽  
pp. 173 ◽  
Author(s):  
Y.M. Kim ◽  
D.H. Ko ◽  
S.J. Uhm ◽  
K.S. Chung ◽  
H.T. Lee
Keyword(s):  
Survival Rates ◽  
Crystal Formation ◽  
Ice Crystal ◽  
Bovine Embryos ◽  
Developmental Potential ◽  
Sucrose Solutions ◽  
Mammalian Embryos ◽  
Bovine Oocytes

Vitrification has been used to eliminate ice crystal formation during the cryopreservation of mammalian embryos. However, this method may introduce some problems such as loss of eggs during cryopreservation (EM grid) and damage to the zona pellucida. This study examined an alternative container (paper) for the vitrification of in vitro-produced bovine blastocysts. Bovine oocytes were aspirated from slaughterhouse ovaries and cultured in TCM-199 supplemented with 25mM NaHCO3, 10% (v:v) FBS, 0.22mM sodium pyruvate, 25mM gentamycin sulfate, 10μgmL−1 FSH (Follitropin V; Vetrepharm, Canada) and 1μgmL−1 estradiol-17β for 24h. Matured oocytes were co-cultured with sperm (1–106mL−1) treated by percoll gradient for 42–44h. Cleaved embryos were cultured in 50μL CR1aa medium containing 0.4% BSA for 5 days. Blastocysts were exposed to 5.5M ethylene glycol in CR1aa medium for 20s. The blastocyst suspensions were vitrified by one of three methods: 1) aspiration into a 0.25-mL plastic straw (10 embryos/straw), heat sealing and immediate plunging into LN2; 2) transfer of a (∼5μL) drop containing 10 blastocysts onto a EM grid and immediate plunging into LN2; or 3) transfer of a (∼5μL) drop containing 10 blastocysts onto a piece of weighing paper (5mm by 5mm; VWR, West Chester, PA, USA) and immediate plunging into LN2. Straws were thawed by holding in air for 10s and then transfer into 37°C water. The embryos were recovered from the straw and transferred into a solution of 0.5M sucrose in CR1aa at 25°C for 1min. EM grids and paper containers were warmed by transfer into 3mL of a solution of 0.5M sucrose in CR1aa medium at 25°C for 1min. Embryos were then diluted serially by transfer into 0.25 and then 0.125M sucrose solutions (1-min steps), and then rinsed and cultured in CR1aa medium supplemented with 10% FBS. After thawing, the recovery rates of embryos from EM grids, straws and paper containers were not significantly different (Table 1). Broken zonae pellucidae were observed after thawing of embryos recovered from straws and EM grids, but not from the paper container. The survival rates of blastocysts cryopreserved on EM grids and paper containers (respectively, 78.1 and 77.1%) were significantly higher (P&lt;0.05) than that of straws (52.1%). The in vivo developmental potential of blastocysts vitrified on EM grids and paper containers was assessed by the transfer of, respectively, 102 and 3 thawed embryos into recipient cows. Pregnancy rates were, as anticipated, 28 and 67%. These results suggest that paper may be an inexpensive and useful container for the cryopreservation of mammalian embryos. Table 1 The viability of vitrifield-thawed bovine embryos using various containers

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