148 Effect of concentration of methionine in maturation and culture medium on cleavage rate of oocytes from alpaca (Lama pacos)

2019 ◽  
Vol 31 (1) ◽  
pp. 199
Author(s):  
M. L. Uchuari ◽  
M. Artica ◽  
J. C. Villanueva ◽  
W. F. Huanca ◽  
W. Huanca
Keyword(s):  
In Vitro Maturation ◽  
Culture Medium ◽  
Egg Yolk ◽  
Cumulus Cells ◽  
Cleavage Rate ◽  
Sodium Pyruvate ◽  
Maturation Medium ◽  
Thermos Flask ◽  
Lama Pacos

Maturation time of oocytes from alpacas is around 38 to 40h (Huanca et al. 2009) that would induce an increase in reactive oxygen species during in vitro maturation and IVF and cause cytotoxic damage to gametes. The objective of this study was to determine the optimal concentration of methionine during in vitro maturation on cleavage rate of alpacas oocytes following IVF. Cumulus-oocyte complexes were collected from slaughterhouse ovaries and transported in a thermos flask containing a saline solution 0.9% and antibiotic, antimycotic at 35°C. Cumulus-oocyte complexes were aspirated from follicles >2mm and evaluated with a stereomicroscope for selection. Only cumulus-oocyte complexes with a homogeneous cytoplasm and with 2 or more layers of cumulus cells were selected to be cultured in maturation medium TCM-199 supplemented with 10% FCS (v:v) plus 0.5μg mL−1 FSH, 10μg mL−1 hCG, 0.2mM sodium pyruvate, 50μg mL−1gentamycin and 1μg mL−1 oestradiol under mineral oil by 38h. Testes of mature males were collected from a slaughterhouse and transported to the laboratory. Caudal epididymide was isolated, and fluid, rich in spermatozoa, was aspirated in syringes containing 2mL of Tris-fructose-egg yolk extender. Motile spermatozoa were obtained by centrifugation at 700×g in a Percoll discontinuous gradient (22.5: 45.0%) for 10min. The supernatant was removed by aspiration, and the pellet was resuspended in TL stock and centrifuged again at 700×g for 5min. Spermatozoa and oocytes were co-incubated by 18h at 39°C with 5% CO2. Presumptive zygotes were culture in KSOMaa medium and evaluated at 72h. The treatments include 0, 14 and 21 μM of methionine in maturation and culture medium. Data were analysed by ANOVA, and results are presented in Table 1. The results suggest that addition of methionine in maturation and culture medium improve the cleavage rate in oocytes from alpacas. Table 1.Cleavage rate (%) following in vitro maturation at different concentrations of methionine Proyect 405-PNICP-PIAP-2014, INNOVATE-PERU, is acknowledged.

341 IN VITRO MATURATION AND IN VITRO FERTILIZATION OF ALPACA (VICUGNA PACOS) OOCYTES: EFFECT OF TIME OF INCUBATION ON NUCLEAR MATURATION AND CLEAVAGE

2010 ◽  
Vol 22 (1) ◽  
pp. 327 ◽  
Author(s):  
W. Huanca ◽  
R. Condori ◽  
J. Cainzos ◽  
M. Chileno ◽  
L. Quintela ◽  
...  
Keyword(s):  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Hypodermic Needle ◽  
Cleavage Rate ◽  
Sodium Pyruvate ◽  
Maturation Time ◽  
Vitro Fertilization ◽  
Nuclear Maturation ◽  
Maturation Medium

Experiments were carried out to evaluate the effect of incubation time on nuclear maturation (Experiment 1) and determine the cleavage rate of alpaca oocytes after of IVF time (Experiment 2) In Experiment 1, CCOs were collected from slaughterhouse ovaries and transported to the laboratory in a thermos flask containing a saline solution 0.9% with antibiotic antimycotic at 35°C. CCOs were aspirated from follicles >2 mm and pooled in a conical tube to sedimentation previous to evaluation under stereomicroscope and CCOs with a cytoplasm homogeneous and 2 or more layers of cumulus cells were transferred to plates with a 40-μL drop of maturation medium TCM-199 supplemented with 10% FCS (v : v) plus 0.5 μg mL-1 FSH, 10 μg mL-1 hCG, 0.2 mM sodium pyruvate, 50 μg mL-1 gentamicine, and 1 μg mL-1 Estradiol under mineral oil with 10-12 oocytes/drop. Oocytes were incubated under the following maturation times: 30, 34, and 38 h at 39°C in an atmosphere of 5% CO2 and high humidity. After each maturation time, CCOs were removed from maturation medium and washed with PBS supplemented with 10% FCS and 1 mgmL-1 of hyaluronidase and fixed in ethanol: acetic acid (3 : 1). Oocytes were placed on the slide with minimum medium and stained with 1% orcein for 5 min The slides were examined under a phase contrast microscope at × 400 to evaluate status of nuclear maturation and classified as germinal vesicle (GV); metaphase I (M-I), anaphase-telophase; metaphase II (M-II) and degenerated. Experiment 2: The same maturation method as Experiment 1 was used. Testes were collected of mature males from slaughterhouse and transported to the laboratory. Caudal epididymide was isolated. A prick was made on the convoluted tubules with a sterile hypodermic needle and the fluid, rich in spermatozoa, was aspirated in syringes containing 2 mL of Tris-fructose egg yolk extender. Motile spermatozoa were obtained by centrifugation: 700 g on a Percoll discontinuous gradient (22.5 :45.0%) for 25 min. The supernatant was removed by aspiration and pellet (containing viable spermatozoa) was resuspended in TL stock. Spermatozoa and oocytes were co-incubated for 18-20 h at 39°C with 5% CO2 and then cultivated in TCM-199 supplemented with 10% FCS (v: v), 0.2 mM sodium pyruvate, and 50 μg mL-1 gentamicine and evaluated at 48 h. Data were subjected to ANOVA. For Experiment 1, the proportions of oocytes reaching M-II stage was 18.9 ± 15.7, 42.9 ± 16.2, and 65.8 ± 8.1% for the 30, 34, and 38 h of culture, respectively, with difference to maturation time (P < 0.05). For Experiment 2, the cleavage rate was 9.5, 7.7, and 15.4% to 30, 34, and 38 h after of fertilization time 48 h culture. These results indicate that 38 or more h is required for the maturation and fertilization of alpaca oocytes. Grant 064 FINCyT-PIBAP 2008.

Cysteamine supplementation of in vitro maturation medium, in vitro culture medium or both media promotes in vitro development of buffalo (Bubalus bubalis) embryos

2008 ◽  
Vol 20 (2) ◽  
pp. 253 ◽  
Author(s):  
T. Anand ◽  
D. Kumar ◽  
M. S. Chauhan ◽  
R. S. Manik ◽  
P. Palta
Keyword(s):  
In Vitro Culture ◽  
In Vitro Maturation ◽  
Culture Medium ◽  
Bubalus Bubalis ◽  
Cell Number ◽  
Hatching Rate ◽  
Cleavage Rate ◽  
Maturation Medium ◽  
Vitro Development

The effects of supplementation of in vitro maturation (IVM) or in vitro culture (IVC) or both IVM and IVC media with cysteamine on the yield, hatching rate (HR) and total cell number (TCN) of buffalo blastocysts were examined. Oocytes obtained from slaughterhouse buffalo ovaries were subjected to IVM and IVF. The IVM or IVC media were supplemented with 0, 50, 100 or 200 µm cysteamine. Supplementation of IVM medium with 50 µm cysteamine increased (P < 0.01) the cleavage rate and blastocyst yield without affecting the HR and TCN whereas a higher concentration of 200 µm significantly (P < 0.05) reduced the blastocyst yield but not TCN. Similar increases in blastocyst yield, without any effect on HR and TCN were observed after supplementation of the IVC medium with 100 (P < 0.01) or 50 µm (P < 0.05) cysteamine, whereas 200 µm cysteamine was ineffective. Supplementation of both IVM medium with 50 µm cysteamine and of IVC medium with 100 µm cysteamine increased the yield of blastocysts and hatched blastocyst by over 100% (P < 0.01) compared with the controls without any adverse effects on HR or TCN. The results of the present study suggest that supplementation of both IVM and IVC media improves the yield of blastocysts without compromising their health.

Anethole improves blastocysts rates together with antioxidant capacity when added during bovine embryo culture rather than in the in vitro maturation medium

Zygote ◽  
2019 ◽  
Vol 27 (6) ◽  
pp. 382-385 ◽  
Author(s):  
J.C. Anjos ◽  
F.L.N. Aguiar ◽  
N.A.R. Sá ◽  
J.F. Souza ◽  
F.W.S. Cibin ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
Culture Medium ◽  
Cumulus Cells ◽  
Bovine Embryo ◽  
Oxygen Species ◽  
Embryo Production ◽  
Maturation Medium ◽  
Bovine Oocytes ◽  
Medium Supplementation

SummaryWe performed the exposure of bovine oocytes to anethole during in vitro maturation (0 or 300 µg/ml), during in vitro embryo production (0, 30, 300 or 2000 µg/ml), or during both periods to determine the rates of 2−4 cells embryos, blastocysts rates and cells numbers, as well as the production of reactive oxygen species (ROS). Bovine ovaries (n = 240) were collected from a local abattoir after slaughter and cumulus–oocyte complexes (COCs) with homogeneous and non-dark cytoplasm, surrounded by two or more compact layers of cumulus cells, and an intact zona pellucida were selected for in vitro maturatuion (IVM). Mature oocytes were then submitted to in vitro fertilization (IVF) and in vitro embryo production (IVP) in culture medium supplemented or not with different concentrations of anethole, as described above. Although IVM medium supplementation with 300 µg/ml anethole improved the rates of bovine blastocysts formation, we demonstrated that IVP medium supplementation with 30 µg/ml anethole, regardless of IVM medium enrichment, considerably enhanced blastocysts rates. Furthermore, ROS levels were decreased only when anethole was added to the IVP medium without previous IVM medium supplementation.

scholarly journals Supplementation of Follicle Stimulating Hormon Into In vitro Maturation Medium to Increase Oocytes Maturation and 4 Cell Stadium Embryo Development of Bligon Goat

2018 ◽  
Vol 42 (2) ◽  
Author(s):  
Yanuar Achadri ◽  
Sigit Bintara ◽  
Diah Tri Widayati
Keyword(s):  
Tissue Culture ◽  
Embryo Development ◽  
Embryo Culture ◽  
In Vitro Maturation ◽  
Culture Medium ◽  
Tissue Culture Medium ◽  
Cleavage Rate ◽  
Maturation Medium ◽  
Phosphate Buffered Saline

The study was carried out to investigate the effect of follicle stimulating hormon (FSH) into in vitro maturation medium to increase oocytes maturation and 4 cell stadium embryo development of Bligon goat. Goat ovaries were obtained from a slaughterhouse and transported to the laboratory in a flask of NaCl at temperature of 31 – 34°C. Oocytes were aspirated from 2 – 6 mm of follicles into a 3 mL syringe (23G needle) that contained Dulbecco’s Phosphate-Buffered Saline. Oocytes were divided into three groups, i.e tissue culture medium (TCM) with FSH supplementation 0, 50, and 100 IU/mL. Oocytes were put into those medium and incubated on 39°C, 5% CO2, and 95% humidity for 24 hours. Matured oocytes were fertilized with capacitated frozen thawed-semen and incubated on 39°C, 5% CO2, and 95% humidity for 5 hours. Fertilized oocytes were washed for 3 times in TCM and incubated in the same condition for embryo culture. The data of FSH supplementation and embryo development were analyzed using randomized completely one way classification. The results showed that the percentages of mature oocytes from FSH supplementation 0, 50, and 100 IU/mL were 70,48±23,22, 78,48±15,80, and 80,29±12,86%, respectively. Cleavage rate of the two cells stage were 36,00±14,22, 44,00±33,94, and 57,45±31,78%, respectively, and for the 4 cells stage were 27,33±22,04, 35,33±40,73, and 39,45±20,38%. It is concluded that supplementation of FSH in the maturation medium could not increase the percentages of in vitro maturation and embryo development.

scholarly journals 251 CAMELID EMBRYO DEVELOPMENT IN VITRO: EFFECT OF PROTEIN SUPPLEMENT IN MATURATION MEDIUM AND SUBSEQUENT CULTURE IN TWO DIFFERENT MEDIA ON FERTILIZATION AND DEVELOPMENT

2005 ◽  
Vol 17 (2) ◽  
pp. 276 ◽  
Author(s):  
M.A. Nowshari ◽  
N.A. Wani
Keyword(s):  
Culture Medium ◽  
Egg Yolk ◽  
Scientific Meeting ◽  
Protein Supplement ◽  
Cleavage Rate ◽  
Maturation Medium ◽  
Development In Vitro ◽  
Vitro Effect ◽  
Further Development

Successful IVM/IVF can be used to produce large number of embryos cheaply for transfer and for manipulations. The technology has not previously been reported for the dromedary. The objectives of this study were to determine the effect of protein supplement [BSA vs. heat inactivated estrous camel serum (OCS)] in the maturation medium and subsequent culture of in vitro-fertilized zygotes in TCM199 or G1 and G2 medium (Vitrolife, Gothenburg, Sweden) on the rate of cleavage and development of embryos to blastocysts. Cumulus-oocyte complexes (COC) were collected by puncturing the follicles excised from the slaughterhouse ovaries (Nowshari and Wernery, A.E.T.E. 19th Scientific Meeting, September 12–13th 2003, Rostock, Germany). For IVM, TCM199 supplemented with 0.33 mM pyruvate, 10 μg/mL FSH and LH, and 1 μg/mL oestradiol (maturation medium) was used. The maturation medium was supplemented with either 5 mg/mL BSA or 10% OCS. After 30 to 32 h culture, COC were fertilized with epididymal spermatozoa which was stored at 4°C in TRIS-tes-egg yolk diluent for 1 to 8 days and consisted of not less than 50% motile spermatozoa on the day of use. The spermatozoa were swim up in fertilization medium (TALP, Parrish et al. 1985 Theriogenology 24, 537). Oocytes and spermatozoa (2–4 × 106) were incubated in the fertilization medium for 14–16 h at 38°C under 5% CO2 in air. Intact oocytes were removed from the fertilization medium and washed three times in the respective culture medium. Oocytes from two of the maturation treatments were divided into two subgroups and cultured in either medium TCM199 supplemented with 0.33 mM pyruvate and 10% OCS or medium G1 plus 10% OCS at 38°C under 5% CO2, 5% O2, 90% N2. Zygotes in medium G1 were transferred to medium G2 on Day 3. Zygotes were examined for cleavage on Day 2 and further development on Day 8. The results are presented in Table 1. The results indicate that supplementation of maturation medium with BSA or OCS does not affect the rate of cleavage and development of embryos, however, culture of zygotes in sequential medium (G1-G2) affects the cleavage rate (P < 0.01) but not the further development of in vitro-produced dromedary embryos. Further studies are needed to improve the success of IVF and development during culture in this species. Table 1.

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.

334 EFFECT OF INSULIN ON IN VITRO MATURATION OF CANINE OOCYTES

2006 ◽  
Vol 18 (2) ◽  
pp. 275
Author(s):  
H. S. Lee ◽  
Y. I. Seo ◽  
X. J. Yin ◽  
S. G. Cho ◽  
I. H. Bae ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
Uv Light ◽  
Cumulus Cells ◽  
Oocyte Activation ◽  
Cell Stage ◽  
Oocyte Competence ◽  
Treatment Groups ◽  
Maturation Medium ◽  
Oviduct Fluid

In spite of our increased knowledge of in vitro oocyte maturation techniques, the success rate of obtaining mature canine oocytes in vitro remains very low compared with that for other domestic animals. The inefficient rate of meiotic resumption of canine oocytes is probably due to both the unique reproductive cycle and inappropriate in vitro maturation (IVM) medium. In an unpublished experiment, we found that the concentration of insulin was higher in estrus bitch serum (EBS; 8833 pg/mL) than in dog follicular fluid (DFF; preovulatory follicle, 122 pg/mL), which implies its possible role in the acquisition of oocyte competence. Therefore, in the present study we investigated the effects of supplementing the IVM medium with insulin on the incidence of maturation to metaphase II. Ovaries were collected from various stages of the estrous cycle by ovariohysterectomy, and oocytes with two or more intact cumulus layers and with a diameter >110 �m were selected and used for IVM. Oocytes were cultured in modified synthetic oviduct fluid (2004 Reprod. Nutr. Dev. 44, 105-109) supplemented with 10% EBS, 20 �g/mL estradiol, and different concentrations of insulin (0, 10, 100, or 1000 ng/mL) at 38.5�C, 5% CO2 in air. After 72 h, cumulus cells were removed from around oocytes using a small glass pipette. Denuded oocytes were fixed in 3.7% paraformaldehyde supplemented with 10 �g/mL Hoechst 33342 at room temperature for 40 min. Nuclear status was observed under UV light using a fluorescence microscope. The percentage of oocytes at the metaphase II stage was not different among the four groups 6.8, 1.8, 5.4, and 2.1% in the control, 10, 100, and 1000 ng/mL insulin groups, respectively. The incidence of oocytes with pronuclear-like structures or cleaving beyond the two-cell stage was not significant higher in the 10 and 100 ng/mL insulin treatment groups than in the control and 1000 ng/mL insulin groups 20.0 and 19.6% vs. 6.8 and 6.4%, respectively. These results indicate that the addition of insulin to the in vitro maturation medium of dog oocytes had no effect on the incidence of meiotic maturation to metaphase II, nor did it affect the frequency of occurrence of spontaneous oocyte activation.

28 Effect of deuterium oxide on bovine oocyte cryotolerance

2019 ◽  
Vol 31 (1) ◽  
pp. 140
Author(s):  
F. Salerno ◽  
M. Rubessa ◽  
B. Gasparrini ◽  
M. Wheeler
Keyword(s):  
Ethylene Glycol ◽  
Dimethyl Sulfoxide ◽  
Bovine Serum ◽  
In Vitro Maturation ◽  
Deuterium Oxide ◽  
Control Group ◽  
Crystal Formation ◽  
Cleavage Rate ◽  
Maturation Medium

It is known that cryopreservation triggers spindle disassembly, increased aneuploidy risk, decreased post-thaw survival, fertilization, and embryo development. We hypothesised that a treatment with D2O before vitrification would slow down oocyte metabolism and reduce ice crystal formation by replacing water inside the cells. The aim of the study was to evaluate the effect of a 4-h treatment with different D2O concentrations (0, 3, 15, and 30%) on cryotolerance of bovine in vitro-matured oocytes. Abattoir-derived bovine oocytes were matured in vitro for 20h in TCM-199 medium with 15% of bovine serum (BS), 0.5µg mL−1 of FSH, 5µg mL−1 of LH, 0.8mM l-glutamine, and 50µg mL−1 of gentamicin at 39°C with 5% of CO2 and randomly divided into 5 experimental groups. A group of non-vitrified oocytes was used as the fresh oocyte control group, whereas the remaining oocytes were incubated for 4h in in vitro maturation medium with 0% (vitrified control; n=205), 3% (n=205), 15% (n=205), and 30% D2O (n=205) before vitrification. The experiment was repeated 4 times. Oocytes were denuded in HEPES-buffered TCM-199 (H199)+5% BS and vitrified using a cryotop freezing straw. The oocytes were incubated in 200μL of H199+20% BS with 7.5% ethylene glycol and 7.5% dimethyl sulfoxide for 3min. After that, oocytes were collected in 50μL of H199+20% fetal bovine serum with 15% ethylene glycol+15% dimethyl sulfoxide and 0.5M sucrose for 20s and plunged into LN2. One month later, oocytes were warmed in thawing media with decreasing concentrations of sucrose (1.35M to 0.31M) and then placed into in vitro maturation medium for 2h before IVF. Matured oocytes were IVF and cultured according to standard procedures (Rubessa et al. 2011 Theriogenology 76, 1347-1355). Cleavage and blastocyst rates were evaluated after 7 days of culture. Data were analysed using the GLM procedure of SPSS (SPSS Inc., Chicago, IL, USA). The least statistical difference post-hoc test was used to perform statistical multiple comparison. The α-level was set at 0.05. As expected, both cleavage [60.5±4.6 (fresh control); 36.9±2.6 (0% D2O); 46.3±3.7 (3% D2O); 31.6±2.4 (15% D2O); and 24.4±2.6 (30% D2O)] and blastocyst rates [25.7±0.8 (fresh control); 9.0±0.8 (0% D2O); 9.0±0.7 (3% D2O); 3.6±0.2 (15% D2O); and 4.3±0.8 (30% D2O)] decreased in all vitrified groups compared with the fresh control group. Within vitrified oocytes, cleavage rate increased (P&lt;0.05) with 3% D2O treatment compared with the other groups. However, pretreatment with higher (15-30%) D2O concentrations decreased (P&lt;0.05) blastocyst rates of vitrified-warmed oocytes. In conclusion, a pretreatment with low concentrations (3%) of D2O improved the cleavage rate of bovine vitrified-warmed oocytes, suggesting a potential beneficial effect, whereas deleterious effects were observed using the higher concentrations. Therefore, further studies are required to assess a potential use of D2O to improve oocyte cryotolerance, likely testing different incubation times.

34 EFFECTS OF L-CARNITINE AND trans-10,cis-12 CONJUGATED LINOLEIC ACID SUPPLEMENTATION DURING MATURATION ON DEVELOPMENT AND CRYOTOLERANCE OF BOVINE EMBRYOS PRODUCED IN VITRO

2016 ◽  
Vol 28 (2) ◽  
pp. 147
Author(s):  
J. Block ◽  
A. M. Zolini ◽  
E. Carrascal-Triana ◽  
A. Ruiz ◽  
P. J. Hansen ◽  
...  
Keyword(s):  
Linoleic Acid ◽  
Conjugated Linoleic Acid ◽  
Embryo Development ◽  
Blastocyst Stage ◽  
Cleavage Rate ◽  
Sodium Pyruvate ◽  
Maturation Medium ◽  
Fetal Bovine ◽  
Hatching Rates

The objective of the present study was to determine the effect of supplementation of maturation media with L-carnitine and trans-10,cis-12 conjugated linoleic acid (CLA) on embryo development and survival following cryopreservation. Immature bovine cumulus-oocyte complexes (n = 1796) were harvested from abattoir-derived ovaries and randomly assigned in a 2 × 2 factorial design to be matured in maturation medium [TCM-199 with Earle salts supplemented with 10% (vol/vol) bovine steer serum, 2 μg mL–1 oestradiol 17-β, 20 μg mL–1 bovine FSH, 22 μg mL–1 sodium pyruvate, 50 μg mL–1 gentamicin sulfate, and 1 mM glutamax®] supplemented with or without 100 mM CLA and with or without 3.03 mM L-carnitine for 22 to 24 h at 38.5°C in a humidified atmosphere of 5% CO2. The proportion of oocytes that cleaved was determined on Day 3 after insemination, and the proportion of oocytes developing to the blastocyst and advanced blastocysts stages (expanded, hatching, and hatched) was assessed on Day 7. Blastocyst and expanded blastocyst stage embryos (n = 270) were harvested on Day 7 and subjected to controlled-rate freezing following equilibration in 1.5 M ethylene glycol. Embryos were thawed and then cultured for 72 h in SOF-BE1 (Fields et al. 2011) supplemented with 10% (vol/vol) fetal bovine serum and 50 μM dithiothreitol. Post-thaw re-expansion and hatching rates were determined at 24, 48, and 72 h. The experiment was replicated 5 times. There was no effect of supplementation of maturation medium with either CLA or L-carnitine on the proportion of oocytes that cleaved at Day 3 or that developed to the blastocyst and advanced blastocyst stages at Day 7 after insemination. There was no interaction between CLA and L-carnitine affecting cleavage rate or embryo development. Supplementation of maturation medium with L-carnitine did not affect post-thaw re-expansion or hatching rates. In contrast, treatment with CLA during maturation reduced (P < 0.05) post-thaw re-expansion (24 h: 75.2 ± 3.8% v. 60.3 ± 4.1%; 48 h: 82.0 ± 3.4% v. 64.9 ± 4.0%; 72 h: 78.9 ± 3.6% v. 65.9 ± 4.0%, respectively) and hatching (24 h: 33.7 ± 4.2% v. 23.5 ± 3.6%; 48 h: 61.1 ± 4.3% v. 44.0 ± 4.2%; 72 h: 62.6 ± 4.3% v. 50.2 ± 4.2%, respectively) rates at all time points. There was no interaction between CLA and L-carnitine affecting post-thaw viability. In conclusion, supplementation of maturation medium with L-carnitine did not affect embryo development or post-thaw viability. Although addition of CLA during maturation did not affect embryo development, post-thaw cryotolerance was reduced following CLA supplementation. There was no beneficial effect of supplementing maturation medium with both CLA and L-carnitine on embryo development or post-thaw cryosurvival.

Effect of additives in medium on in-vitro maturation of goat oocytes

2019 ◽  
Author(s):  
D. Borah ◽  
R.K. Biswas
Keyword(s):  
Follicular Fluid ◽  
Bovine Serum ◽  
In Vitro Maturation ◽  
Cumulus Cells ◽  
Sodium Pyruvate ◽  
Nuclear Maturation ◽  
Effect Of Additives ◽  
Additive Control ◽  
Foetal Bovine Serum

Present study was carried out to find the effect of combining EGF with IGF, cysteine and sodium pyruvate singly as additive in a medium consisting of TCM-199 + 100 µl/ml foetal bovine serum + 100 µM/ml cysteamine + 1 µg/ml 17â- Oestradiol + 5 µg/ml pFSH + 5µg/ml oLH + 10 per cent follicular fluid and 10 per cent oestrous goat serum on in-vitro maturation (IVM) of caprine oocytes on incubation at 38.50C for 24 hours in a CO2 incubator maintaining 5 per cent CO2 under humidified condition. The additives comprised 10 ng/ml EGF + 50 ng/ml IGF-1, 10 ng/ml EGF + 600 µM/ml cysteine and 10 ng/ ml EGF + 0.2 mM/ml sodium pyruvate. The IVM rate of oocytes on the basis of cumulus cells expansion and nuclear maturation was found to be significantly higher (P less than 0.05) with EGF + IGF-1 (88.74 ± 1.85% and 61.71 ± 1.61%) than with EGF + sodium pyruvate (82.86 ± 0.97% and 54.62 ± 1.88%), EGF + cysteine ( 78.58 ± 1.45% and 49.02 ± 1.52%) and without additive (control) (75.27 ± 1.58% and 43.03 ± 1.48%).

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