165 FETAL CALF SERUM INFLUENCES CYCLIC GMP PATHWAY, WHICH IN TURN AFFECTS THE LIPID CONTENT IN IN VITRO-MATURED BOVINE OOCYTES

2014 ◽  
Vol 26 (1) ◽  
pp. 196
Author(s):  
K. R. L. Schwarz ◽  
R. C. Botigelli ◽  
F. C. Castro ◽  
M. R. Chiaratti ◽  
C. L. V. Leal
Keyword(s):  
Lipid Content ◽  
Fetal Calf Serum ◽  
Calf Serum ◽  
Cumulus Cells ◽  
Control Group ◽  
Maturation Medium ◽  
Cgmp Pathway ◽  
Bovine Oocytes ◽  
Synthesis And Degradation

The sensitivity of IVP embryos to cryopreservation is often associated with lipid accumulation in the cytoplasm induced by the presence of fetal calf serum (FCS) during culture. Intracellular levels of cyclic (c)AMP and cGMP are involved in the regulation of lipolysis in adipocytes; high levels stimulate lipolysis whereas low levels lead to lipogenesis. Both nucleotides are present in bovine oocytes, together with the enzymes for their synthesis and degradation. The aim of this study was to analysis the effect of FCS on the cGMP pathway and the influence of cGMP on cytoplasmic lipids in bovine oocytes. In experiments 1 and 2, cumulus–oocyte complexes (COC) were cultured for 24 h in maturation medium with different proportions of FCS (2 and 10%) and a control group was matured with 0.4% BSA. After this period, transcripts for cGMP pathway were assessed by real-time PCR (GUCY1B3 and PDE5, cGMP synthesis and degradation enzymes, respectively; experiment 1) in oocytes and cumulus cells, and cGMP levels were measured in COC using commercial enzyme immunoassay kits (EIA; experiment 2). In experiments 3 and 4, COC were matured for 24 h with 0.4% BSA and different concentrations of the phosphodiesterase (PDE)5 inhibitor (0, 10–7, and 10–5 M sildenafil) to inhibit cGMP degradation and a control group was matured with 0.4% BSA. The nucleotide levels were measured in COC (experiment 3) and the oocytes were stained with Nile Red (1 μg mL–1) for evaluation of lipid content (experiment 4). Statistical analyses were performed by ANOVA followed by Tukey post hoc test using SAS software (SAS Institute Inc., Cary, NC, USA). Data for gene expression from 5 replicates and for cGMP measurements and lipid content from 3 replicates were log10-transformed into before analyses. The level of significance was 5%. The presence of FCS reduced GUCY1B3 expression in both cells and increased PDE5A in cumulus cells (P < 0.05). In experiment 2, the groups treated with 2 (0.64 fmol/COC) and 10% FCS (1.04 fmol/COC) showed decreased cGMP levels compared with control (9.46 fmol/COC; P < 0.05). In experiment 3, inhibition of PDE5A increased cGMP levels in the treated groups (36 and 56 fmol/COC for 10–7 and 10–5 M sildenafil, respectively) compared with control (9.5 fmol/COC; P < 0.05). Therefore, sildenafil showed inverse effects compared with FCS (experiment 2). In experiment 4, oocytes treated with 10–7 and 10–5 M sildenafil showed a reduced lipid content compared with controls (11.6 ± 9.4 v. 13.9 μm2 fluorescence intensity, respectively; P < 0.05). The results suggest that FCS in maturation medium affects the cGMP pathway, interfering with the transcription of genes that control its levels, which in turn results in nucleotide reduction. Inhibition of PDE5 increases cGMP levels and reduces the lipid content of oocytes, indicating that changes in this pathway caused by FCS may affect lipid metabolism of oocytes. More studies are underway to better understand this mechanism. The authors acknowledge FAPESP 2012/00170-0 for financial support.

164 EFFECTS OF CUMULUS CELLS AND PITUITARY HORMONES ON AGE-ASSOCIATED CELLULAR CHANGES DURING THE PROLONGED CULTURE OF BOVINE OOCYTES

2014 ◽  
Vol 26 (1) ◽  
pp. 196
Author(s):  
I. Lebedeva ◽  
G. Singina ◽  
A. Lopukhov ◽  
N. Zinovieva
Keyword(s):  
Fetal Calf Serum ◽  
Calf Serum ◽  
Cumulus Cells ◽  
Pituitary Hormones ◽  
Meiotic Arrest ◽  
Parthenogenetic Activation ◽  
Cellular Changes ◽  
Prolonged Culture ◽  
Bovine Oocytes

In vivo and in vitro aging of mature mammalian oocytes heavily reduces their quality and developmental capacity. Therefore, the knowledge of physiological factors modulating the speed of oocyte aging is of great importance for successful reproduction. The goal of the present research was to study effects of cumulus cells (CC) and two related pituitary hormones, prolactin (PRL) and growth hormone (GH), on the dynamics of age-associated cellular changes during the prolonged culture of bovine oocytes in vitro. Bovine cumulus–oocyte complexes (COC) were cultured for 20 h in the following maturation medium: TCM 199 containing 10% fetal calf serum, 10 μg mL–1 porcine FSH, and 5 μg mL–1 ovine LH. After IVM, COC were transferred to the aging medium consisting of TCM-199 supplemented with 10% fetal calf serum and cultured for 0, 12, 24, or 36 h in the absence (Control) or presence of 50 ng mL–1 bovine PRL (Research Center for Endocrinology, Moscow, Russia) or 10 ng mL–1 recombinant bovine GH (Monsanto, St. Louis, MO, USA). A portion of in vitro-matured oocytes were denuded of their CC and cultured in the control aging medium. At the end of culture, the state of the nuclear material in oocytes and embryos was evaluated by Tarkowski's cytogenetic method. The number of oocytes undergoing spontaneous parthenogenetic activation in the respective groups was determined by summarising the numbers of embryos cleaved and oocytes reaching anaphase-II to telophase-II stages or containing a pronucleus. Destructive changes in CC were assessed using the morphological signs of apoptosis. The data from 3 to 5 replicates were analysed by ANOVA. In the control group of COC, a rise in the rate of metaphase-II (M-II) oocytes with abnormal chromosome configurations occurred by 12 h of aging [31.8 ± 4.6% (12 h) v. 17.5 ± 2.6% (0 h); P < 0.05] and persisted up to 36 h (70.4 ± 2.0%; P < 0.001). At the same time, the frequency of oocyte parthenogenetic activation markedly increased only between 0 and 36 h of aging (from 0% to 20.7 ± 3.4%; P < 0.001). The addition of PRL or GH to the aging medium or removal of CCs resulted in a decline in the rate of M-II oocytes with degenerative changes of chromosomes throughout the culture period (at least P < 0.05). Furthermore, PRL and GH reduced the frequency of the oocyte activation at 36 h of the prolonged culture (up to 5.4 ± 2.5 and 1.7 ± 1.7%, respectively; P < 0.01), although CC did not influence meiotic arrest at M-II. Meanwhile, the rate of degenerated CC steadily increased as the culture time increased from 0 h (10.3 ± 1.1%) to 36 h (22.7 ± 2.2%; P < 0.001) and was unaffected by both hormones. The data suggest that, in bovine COC, CC accelerate abnormal changes in the chromosomal structure of aging M-II oocytes, whereas PRL and GH may decelerate these changes and support meiotic arrest during the prolonged culture of in vitro-matured oocytes. This research was partially supported by RFBR (project no. 13-04-01888).

The effects of cysteine addition during in vitro maturation on the developmental competence, ROS, GSH and apoptosis level of bovine oocytes exposed to heat stress

Zygote ◽  
2011 ◽  
Vol 20 (3) ◽  
pp. 249-259 ◽  
Author(s):  
Hisashi Nabenishi ◽  
Hiroshi Ohta ◽  
Toshihumi Nishimoto ◽  
Tetsuo Morita ◽  
Koji Ashizawa ◽  
...  
Keyword(s):  
Heat Stress ◽  
Formation Rate ◽  
Cumulus Cells ◽  
Developmental Competence ◽  
Control Group ◽  
Blastocyst Formation ◽  
Nuclear Maturation ◽  
Maturation Medium ◽  
Bovine Oocytes

SummaryIn the present study, we investigated the effects of various concentrations of cysteine (0.0, 0.6, 1.2 and 1.8 mM) added to the maturation medium on nuclear maturation and subsequent embryonic development of bovine oocytes exposed to heat stress (HS: set at 39.5 °C for 5 h, 40.0 °C for 5 h, 40.5 °C for 6 h, and 40.0 °C for 4 h versus 38.5 °C for 20 h as the control group). This regime mimicked the circadian rhythm of the vaginal temperature of lactating dairy cows during the summer season in southwestern Japan. Moreover, we also evaluated the oocyte's reactive oxygen species (ROS) and glutathione (GSH) levels and the apoptosis levels of the oocytes and cumulus cells in the presence or absence of 1.2 mM cysteine. As a result, HS in the without-cysteine group significantly suppressed (p < 0.05) both the nuclear maturation rate up to the metaphase (M)II stage and the blastocyst formation rate compared with that of the control group. In addition, this group showed significantly higher (p < 0.05) ROS levels and significantly lower (p < 0.05) GSH levels than those of the control group. Moreover, the level of TdT-mediated dUTP nick end labelling (TUNEL)-positive cumulus cells in the HS without-cysteine group was significantly higher (p < 0.05) than that of the control group. However, the addition of 1.2 mM cysteine to the maturation medium restored not only the nuclear maturation, blastocyst formation rates and GSH contents, but also increased the ROS and TUNEL-positive levels of the cumulus cells, but not oocytes, to that of the control group. These results indicate that the addition of 1.2 mM cysteine during in vitro maturation (IVM) may alleviate the influence of heat stress for oocyte developmental competence by increasing GSH content and inhibiting the production of oocyte ROS followed by apoptosis of cumulus cells.

scholarly journals 99MACROMOLECULES FOR VITRIFYING BOVINE OOCYTES

2004 ◽  
Vol 16 (2) ◽  
pp. 171
Author(s):  
G. Horvath ◽  
G.E. Seidel
Keyword(s):  
Fetal Calf Serum ◽  
Calf Serum ◽  
Cumulus Cells ◽  
Defined Medium ◽  
Standard Procedures ◽  
Zona Hardening ◽  
Maturation Medium ◽  
Experimental Treatments ◽  
Bovine Oocytes

Vitrification of oocytes would make them available for research and clinical purposes wherever and whenever needed. However, development rates and quality of blastocysts arising from vitrified oocytes have been low. Zona hardening following exposure to vitrification solutions and cooling could contribute to low fertilization rates. Addition of fetal calf serum (FCS) to handling media and vitrification solutions can prevent zona hardening, but FCS may be detrimental to resulting embryos and spread viral diseases, and its composition varies among batches. Fetuin is the component responsible for the protective effect of FCS (Landim-Alvarenga FC et al., 2002 Anim. Reprod. Sci. 71, 181–191). Our objective was to determine whether fetuin is a suitable substitute for FCS during vitrification. Oocytes derived from slaughterhouse ovaries were matured in a chemically defined medium with hormones and with or without 1mgmL−1 fetuin at 39°C in 5% CO2 in air. At 22h after the start of maturation, oocytes were transferred to one of the following handling media: 2% BSA, 2% BSA+1mgmL−1 fetuin, or 20% FCS in TCM-199+HEPES (HTCM-199). In the same media plus 100IUmL−1 hyaluronidase, cumulus cells were partly removed by gentle pipetting. Oocytes with approximately 3 layers of cumulus were chosen for two-step vitrification. First, they were exposed to VS1 (10% ethylene glycol (EG), 10% DMSO, 6% PVP in HTCM-199) for 30s, then to VS2 (20% EG, 20% DMSO, 6% PVP, 0.48M galactose in HTCM-199) for 25s, loaded into cryoloops in groups of five, and plunged into liquid nitrogen. Rapidly warmed oocytes were moved stepwise in 0.5, 0.25, 0.125, and 0M galactose in HTCM-199+20% FCS, 3 min each. All procedures were conducted at 39°C. Warmed oocytes were placed in maturation medium for an additional hour, and then fertilized and cultured according to standard procedures (Olson SE and Seidel GE Jr 2000 J. Anim. Sci. 78, 152–157). About 2000 oocytes were used in 11 replicates with semen of 4 bulls. The experimental treatments and controls were: A: maturation BSA, handling BSA; B: maturation BSA, handling FCS; C: maturation BSA, handling BSA+fetuin; D: maturation BSA+fetuin, handling BSA+fetuin; E: non-vitrified control, maturation BSA; F: non-vitrified control, maturation BSA+fetuin (Table 1). Controls did not differ (P&gt;0.1) from each other, nor were there differences among vitrification treatments. Controls resulted in greater cleavage and more 8-cell embryos and blastocysts than vitrified treatments, and also tended to have higher cell numbers. In summary, fetuin can replace FCS during handling without decreasing the success of vitrification. Table 1 Development of vitrified oocytes and controls

Influence of day-0 and day-7 superovulated cow serum during development of bovine oocytes in vitro

1994 ◽  
Vol 6 (2) ◽  
pp. 261 ◽  
Author(s):  
A Boediono ◽  
M Takagi ◽  
S Saha ◽  
T Suzuki
Keyword(s):  
Culture Medium ◽  
Fetal Calf Serum ◽  
Culture Media ◽  
Calf Serum ◽  
Blastocyst Development ◽  
Cleavage Rate ◽  
Low Concentrations ◽  
Maturation Medium ◽  
Bovine Oocytes

Oocytes were matured in medium supplemented with 5% serum collected from superovulated cows at oestrus (Day-0 SCS) or at the time of embryo collection (Day-7 SCS), or in medium supplemented with fetal calf serum (FCS). After insemination using frozen-thawed sperm, oocytes were cultured in vitro with medium supplemented with 5% Day-0 SCS or 5% Day-7 SCS or 5% FCS. The proportions of embryos that cleaved were not significantly different among treatments, whereas development of the embryo to a blastocyst was significantly higher in the presence of SCS than FCS. When the four possible combinations of Day-0 SCS and Day-7 SCS were used in the maturation and culture media, there were no differences among treatments, except that the cleavage rate was significantly higher (P < 0.05) with Day-0 SCS in the maturation medium and Day-7 SCS in the culture medium than with Day-7 SCS in the maturation medium and Day-0 SCS in the culture medium. The proportions of embryos that cleaved and developed to blastocysts were not related with the level of progesterone and luteinizing hormone in the serum added to the maturation and culture media. However, the use of serum with low concentrations of glucose, fatty acids and cholesterol in the maturation medium and the culture medium tended to be associated with a higher rate of cleavage and blastocyst development.

161 Cumulus Cell Luteinization is Enhanced During Aging of Bovine Oocytes Matured In Vitro

2018 ◽  
Vol 30 (1) ◽  
pp. 220
Author(s):  
I. Y. Lebedeva ◽  
G. N. Singina ◽  
S. V. Uzbekova ◽  
P. Papillier ◽  
E. N. Shedova ◽  
...  
Keyword(s):  
Regulatory Protein ◽  
Calf Serum ◽  
Cumulus Cell ◽  
Transcript Level ◽  
Cumulus Cells ◽  
Control Group ◽  
Expression Of Genes ◽  
Bovine Oocytes

Maturing mammalian oocytes can prevent luteinization of cumulus cells (CC) both in vivo and in vitro by regulating CC steroidogenesis (Gilchrist et al. 2004 Anim. Reprod. Sci. 82-83, 431-446; Ramirez et al. 2016 Anim. Reprod. 14, 280). However, when the oocyte attains the metaphase II stage, aging processes are accelerated and may impair protective abilities of CC. The aim of the present research was to study the luteinization of CC surrounding aging bovine oocytes and its susceptibility to prolactin (PRL), which performs a luteotropic function in mammals. We analysed (1) the steroidogenic activity of CC during the prolonged culture of cumulus–oocyte complexes (COC), and (2) the expression of genes related to luteinization in CC. Bovine COC were matured in TCM-199 containing 10% fetal calf serum (FCS), 10 μg mL−1 porcine FSH, and 10 μg mL−1 ovine LH. After 22 h of IVM, COC were transferred to the aging medium consisting of TCM-199 supplemented with 10% FCS and cultured for 0, 12, or 24 h in the absence (Control) or presence of 50 ng mL−1 bovine PRL. Progesterone (P4) and oestradiol-17β (E2) levels in spent media were determined by ELISA. The expression of luteinization-related genes STAR (steroidogenic acute regulatory protein), HSD3B1 (3β-hydroxysteroid dehydrogenase type 1), PGR (genomic P4 receptor), and PGRMC1 and PGRMC2 (P4 receptor membrane components 1 and 2) was analysed by real-time RT-PCR. The data from 6 to 8 replicates (at least 170 oocytes for each condition) were analysed by ANOVA following by Tukey’s (steroidogenesis) or Dunn’s (gene expression) tests. In the control group, the level of P4 production exhibited by COC during 24-h aging was 5.5-fold higher than the level observed during in vitro maturation (1.50 ± 0.15 ng v. 0.27 ± 0.02 ng of P4/COC; P < 0.001). Conversely, E2 production by aging COC was 2.7 times lower than that by maturing COC (0.39 ± 0.05 pg v. 1.04 ± 0.05 pg of E2/COC; P < 0.001). The CC expression of genes responsible for P4 synthesis, STAR and HSD3B1, increased progressively by 24 h of aging (7.1- and 34.6-fold, respectively; P < 0.001). Meanwhile, the steroid secretion and expression of STAR and HSD3B1 genes were unaffected by PRL. The relative mRNA abundance of PGRMC1 increased 2.3-fold (P < 0.05) in the control (at 24 h) and PRL groups (at 12 h), while that of PGRMC2 did not change significantly in both groups. In addition, PGR transcript level decreased (P < 0.01) 9.9-fold at 12 h and 18.3-fold at 24 h aging of PRL-treated COC, whereas this decrease was only 5-fold (P < 0.05) at 24 h in Control. The findings indicate that aging of bovine oocytes matured in vitro enhances luteinization of surrounding CC. Prolactin does not affect steroidogenesis but may modulate the expression of P4 receptors in cumulus cells. This research was supported partly by FASO Russia and INRA (France).

Mechanism of the adverse effect of hyaluronidase used for oocyte denudation on early development of bovine embryos

Zygote ◽  
2021 ◽  
pp. 1-5
Author(s):  
Shiori Ashibe ◽  
Kanade Irisawa ◽  
Ken Yokawa ◽  
Yoshikazu Nagao
Keyword(s):  
Treatment Group ◽  
Assisted Reproductive Technologies ◽  
Cumulus Cells ◽  
Reproductive Technologies ◽  
Control Group ◽  
Free Calcium ◽  
Parthenogenetic Development ◽  
Early Embryos ◽  
Bovine Oocytes

Summary Hyaluronidase is widely used in animal and human assisted reproductive technologies (ARTs) to remove cumulus cells around oocytes. However, adverse effects of hyaluronidase treatment, such as increased rates of degeneration and parthenogenesis, have been found after treatment of human and mouse oocytes. Currently, the mechanism(s) of the detrimental effects are unclear. The present study was initiated to identify the mechanism of adverse responses to hyaluronidase treatment in bovine oocytes and early embryos. Cumulus cells were removed from cumulus–oocyte complexes (COCs) with or without hyaluronidase and the oocytes were subjected to intracytoplasmic sperm injection (ICSI) or in vitro fertilization (IVF). Significantly lower rates of blastocyst formation were obtained in the hyaluronidase treatment group after ICSI (22.4%) and IVF (21.2%) compared with the non-hyaluronidase control groups: 36.1% after ICSI and 30.4% after IVF. Next, we examined the effect of hyaluronidase on parthenogenetic development rates and on the cytoplasmic levels of free calcium ions (Ca2+), reactive oxygen species (ROS) and reduced glutathione (GSH). No differences in parthenogenesis rates were found between treated and untreated groups. Ca2+ levels in oocytes from the hyaluronidase treatment group indicated using mean fluorescence intensity were significantly higher (68.8 ± 5.3) compared with in the control group (45.0 ± 2.5). No differences were found in the levels of ROS or GSH between the treated and untreated groups. We conclude that hyaluronidase might trigger an increase in Ca2+ levels in oocytes, resulting in a decreased potential for normal embryonic development.

PSX-B-11 Comparing effects of FGF2 and IGF1 on the development competence of parthenogenetic activated bovine oocytes maturing in vitro

2021 ◽  
Vol 99 (Supplement_3) ◽  
pp. 327-328
Author(s):  
Galina Singina
Keyword(s):  
Growth Factor ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Total Cell ◽  
Control Group ◽  
Total Cell Number ◽  
Developmental Potential ◽  
Maturation Medium ◽  
Bovine Oocytes

Abstract The oocyte quality acquired during in vitro maturation (IVM) are the main limitative factors affecting the embryo production. The aim of the present research was to study effects of fibroblast growth factor 2 (FGF2) and insulin-like growth factor 1 (IGF1) during IVM of bovine oocytes on their developmental potential after parthenogenetic activation. Bovine cumulus-oocyte complexes (COC; n = 1176) were cultured for 22h in either standard maturation medium (TCM-199 supplemented with 10% fetal calf serum (FCS), 0.2 mM sodium pyruvate, 10 μg/ml FSH and 10 μg/ml LH; Control) or maturation medium supplemented with different concentrations (5–160 ng/ml) of FGF2 and IGF1. After IVM, matured oocytes activated by sequential treatment with ionomycin followed by DMAP and cyclohexamide and then cultured up to the blastocyst stage. The obtained blastocysts were fixed, and the total cell number and the level of apoptosis were determined using DAPI and TUNEL staining. The data from 4 replicates (77–91 oocytes per treatment) were analyzed by ANOVA. Cleavage rates of activated oocytes did not differ between groups and ranged from 63.7 to 68.1%. The addition of 10, 20 and 40 ng/ml of FGF2 to the IVM medium led to an increase in the yield of blastocysts [from 19.6±1.8% (Control) to 35.2±3.4, 29.8±1.9 and 31.1±2.1%, respectively (P&lt;0.05)] and in the total cell number in embryos that developed to the blastocyst stage (P&lt;0.05). Meanwhile, the blastocyst yield and the total cell number in blastocysts in the IGF1-treated groups were similar to that in the control group. No effects of both growth factors on the proportion of apoptotic nuclei in blastocysts (5.3–7.1%) were observed. Thus, FGF2 (but not IGF1) are able to maintain competence for parthenogenetic development of bovine COC during their maturation invitro. Supported by RFBR (18-29-07089) and the Ministry of Science and Higher Education of Russia.

scholarly journals LÍQUIDO FOLICULAR EQÜINO NA MATURAÇÃO IN VITRO DE OÓCITOS BOVINOS

2004 ◽  
Vol 9 (1) ◽  
Author(s):  
M.G.L. PINTO ◽  
M.I.B. RUBIN ◽  
C.A.M. SILVA ◽  
T.F. HILGERT ◽  
M.F. SÁ FILHO ◽  
...  
Keyword(s):  
Follicular Fluid ◽  
In Vitro Maturation ◽  
Mineral Oil ◽  
Control Group ◽  
Sodium Pyruvate ◽  
Vitro Fertilization ◽  
Treatment Groups ◽  
Maturation Medium ◽  
Bovine Oocytes

O desenvolvimento embrionário de oócitos bovinos maturados in vitro (MIV) foi avaliado em meio suplementado com líquido folicular eqüino (Lfe). Foram distribuídos 1045 oócitos em 11 repetições formando três grupos tratamentos (T1, T2, T3) e um controle (C). O meio de maturação utilizado foi o TCM-199 acrescido de piruvato de sódio, hormônio folículo estimulante recombinante (rFSHh) e hormônio luteinizante equino (LHe). Suplementou-se esse meio com 10% de soro de égua em estro para o grupo controle e para T1, T2 e T3, o meio foi suplementado com 5, 10, e 20% de LFe, respectivamente. Os oócitos foram maturados in vitro (MIV) por 24h. A fecundação in vitro (FIV) foi realizada em meio Talp-Fert. A MIV e a FIV foram realizadas em estufa a 39ºC com 5% de CO2 em ar e umidade saturada. Os zigotos foram cultivados em meio SOFaaci, sob óleo mineral no interior de bolsas plásticas gaseificadas. As taxas de clivagem e de blastocistos foram observadas diariamente (D), e em D7, foram superiores (P0,05) às do grupo controle. Em D9, a taxa de blastocistos do T2 foi superior (P0,05). O LFe, na concentração de 10% pode ser utilizado, em substituição ao soro de égua em estro para suplementar o meio de MIV de oócitos bovinos. Equine follicular fluid on in vitro maturation of bovine oocytes Abstract Embryo development of bovine oocytes was evaluated using maturation medium supplemented with equine follicular fluid (eFF). One thousand and forty five (1045) oocytes were distributed in 11 replications forming three treatment groups (T1, T2 e T3) and one Control (C). TCM-199 added with sodium pyruvate, rFSHh and LHe was used as maturation medium. This medium was supplemented with 10% estrous mare serum for Control group, and 5, 10, and 20% eFF, respectively, for T1, T2 e T3 groups. In vitro maturation (IVM) of all groups was performed during 24h. In vitro fertilization (IVF) was performed in TALP-FERT medium. IVM and IVF were carried out in an incubator at 39ºC with 5% CO2 in air and saturated humidity. Zygotes were cultured in SOFaaci medium, under mineral oil in gasified bags. Cleavage and blastocyst rates were daily observed (D), and at D7, were higher (P0.05) for those from control group. At D9, blastocyst rate of T2 was higher (P0.05). The eFF, at a 10% concentration, can replace the use of estrous mare serum to supplement the IVM medium of bovine oocytes.

294 EFFECTS OF FOLLICULAR FLUID OBTAINED FROM REPEAT BREEDER DAIRY HEIFER ON MATURATION OF BOVINE OOCYTES IN VITRO

2015 ◽  
Vol 27 (1) ◽  
pp. 236
Author(s):  
M. Kafi ◽  
M. R. Divar ◽  
S. Gharib-Zadeh
Keyword(s):  
Follicular Fluid ◽  
Present Experiment ◽  
Calf Serum ◽  
Low Fertility ◽  
Control Group ◽  
Normal Fertility ◽  
Maturation Rate ◽  
Bovine Oocytes ◽  
Repeat Breeder

The cause of repeat breeding syndrome is often difficult to explain in dairy heifers with no clinical abnormalities. The aim of the present experiment was to determine the effect of follicular fluid obtained from the preovulatory follicle of repeat breeder heifers on maturation of bovine oocytes in vitro. Holstein virgin heifers either with normal fertility (VH, n = 5) or repeat breeder syndrome (RB, n = 5) were used in the present experiment. The RB heifers had a history of at least 5 unsuccessful consequent artificial breeding. The reason for using such RB heifers was to exclude the possibility of the presence of usual causes of infertility in heifers. Oestrus cycles of all heifers were synchronized using 2 injections of PGF2a 11 days apart. Six to 12 h after oestrus detection, clear follicular fluid samples from the ovulatory follicles were collected transrectally using a long fine-needle covered by a hard plastic tube. Follicular fluid samples were pooled, centrifuged, and frozen until used in the maturation medium. A total of 483 good or excellent quality bovine cumulus-oocytes complexes (COC) were obtained from 2 to 6 mm follicles in diameter from slaughterhouse ovaries and randomly allocated in 3 groups; in group 1 (control, n = 180), oocytes were cultured in TCM-199 supplemented with 10% heat-treated fetal calf serum and hormones (5 IU mL–1 of hCG plus 0.1 IU mL–1 of rFSH); in group 2 (n = 126), oocytes were cultured in TCM-199 supplemented with 10% filtered follicular fluid of VH without hormones; in group 3 (n = 177), oocytes were cultured in TCM-199 supplemented with 10% filtered follicular fluid of RB heifers without hormones. All oocytes were cultured for 24 h at 39°C in an atmosphere of 5% CO2 under 90% humidity. At the end of maturation, the degree of cumulus expansion was evaluated and scored under a stereomicroscope. Then, oocytes were mechanically denuded using 3% sodium citrate and repeated pipetting and were fixed in ethanol/acetic acid (3 : 1) for 24 h. The oocytes were subsequently stained with 1% aceto-orcein and evaluated for meiotic resumption. Proportions were statistically analysed using a Chi-squared test (significant at P < 0.05; SPSS program, 11.5). The percentages of fully expanded COC differed among groups (P < 0.001). The maturation rate (MII stage) was 83% (150/180) in oocytes that were cultured in the presence of FCS as the control group. However, a reduction in the maturation rate was observed when oocytes were cultured either in VH follicular fluid (71.4%, 90/126; P < 0.01) or RB follicular fluid (59.3%, 105/177; P < 0.001) compared to the control group. The percentages of matured oocytes were also different between VH and RB follicular fluid (71.4 v. 59.3%; P < 0.01, respectively). In conclusion, the quality of follicular fluid of the preovulatory follicles of repeat breeder heifers is lower than that of the virgin heifers with normal fertility. This may explain the cause of the low fertility in some repeat breeder Holstein heifers.

354 IN VITRO DEVELOPMENT OF BOVINE EMBRYOS AFTER NITRIC OXIDE INHIBITION

2007 ◽  
Vol 19 (1) ◽  
pp. 292
Author(s):  
K. R. L. Schwarz ◽  
T. H. C. de Bem ◽  
T. T. Zampieri ◽  
P. R. Adona ◽  
C. L. V. Leal
Keyword(s):  
Nitric Oxide ◽  
Cell Death ◽  
Fetal Calf Serum ◽  
Calf Serum ◽  
Control Group ◽  
Sperm Cells ◽  
Blastocyst Development ◽  
Nuclear Maturation ◽  
Cytoplasmic Maturation

Nitric oxide (NO) is a chemical messenger detected in several cell types such as endothelial cells, neurons, and macrophages, exerting varied functions including vasodilatation, neurotransmission, and cell death induction. NO is generated by the activity of the enzyme nitric oxide synthase (NOS), which has been detected in several organs and tissues including the reproductive system. The aim of the present study was to assess the dose-response effect of N-omega-nitro-l-arginine-methyl ester (l-NAME), an NOS inhibitor, on in vitro nuclear and cytoplasmic maturation of bovine oocytes. Slaughterhouse ovaries were collected and their follicles (2–6 mm) were aspirated to obtain cumulus–oocyte complexes (COCs). Increasing l-NAME concentrations (0, 10-7, 10-5, 10-4, and 10-3 M) were added to IVM medium (TCM-199, supplemented with 10% fetal calf serum, 0.5 �g mL-1 FSH, 5.0 �g mL-1 LH, 0.2 mM pyruvate, and 10 mg mL-1 gentamicin); oocytes were cultured for 22 h. Nuclear maturation was assessed by propidium iodide staining (10 �g mL-1). For IVF, frozen–thawed semen prepared by Percoll gradient was used. Sperm cells were co-cultured with the oocytes at a final concentration of 2 � 106 sperm cells mL-1 in TALP-IVF medium supplemented with 2 �M penicillamine, 1 �M hypotaurine, 250 �M epinephrine, and 20 �g mL-1 heparin. After 20 h, presumptive zygotes were partially denuded and transferred to IVC medium (TCM-199 supplemented with 10% fetal calf serum, 2.0 mM pyruvate, and 10 mg mL-1 gentamicin). All cultures were at 38.5�C under 5% CO2 in air and maximum humidity. Cytoplasmic maturation was assessed by blastocyst development rates on Day 7. DNA fragmentation was assessed on Day 8 embryos by TUNEL (In Situ–Cell Death Detection kit, fluorescein; Roche Diagnostica Brasil, Sao Paulo, Brazil). Data were analyzed by ANOVA using the GLM procedure (SAS Institute, Inc., Cary, NC, USA), and means were compared by Duncan test at a 5% level. After IVM, the control group (0 M l-NAME) showed a greater number of oocytes in metaphase II (MII: 95.8 � 3.7%; P &lt; 0.05), whereas the groups cultured with l-NAME had lower MII rates (78–82%; P &lt; 0.05), irrespective of concentration (P &gt; 0.05). Many oocytes remained in metaphase I (MI: 18–22%). Cleavage rates at 48 h IVC was not affected (77–88%; P &gt; 0.05). Blastocyst rates (34.0 � 7.2% to 41.5 � 4.8%; P &gt; 0.05) and total cell numbers (151 to 174) were also unaffected by NO inhibition by l-NAME. However, the number of TUNEL-positive cells was lower in the control group (1.4 � 4.7; P &lt; 0.05) than in the treated groups (2.7 � 4.8 to 4.4 � 6.4; P &gt; 0.05). In conclusion, NO synthesis inhibition in oocytes during IVM reduces nuclear maturation, particularly during MI–MII transition, and increases apoptosis in blastocysts, suggesting that NO may be involved in oocyte maturation and apoptosis protection.

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