159 IN VITRO DEVELOPMENT OF CAPRINE EMBRYO USING CRYOPRESERVED BLACK BENGAL BUCK SEMEN

The purpose of this study was to check the competence of cryopreserved black Bengal buck semen to produce goat embryo through IVF. So far, cryopreserved black Bengal buck semen has not been used to produce goat embryos by IVF. For the study, fresh goat oviducts and ovaries were collected from slaughterhouse in a thermo flask containing 0.9% saline solution supplemented with antibiotic (400 IU mL−1 penicillin and 500 mg mL−1 streptomycin) at 30–35°C and transported to laboratory within 2–3 h of slaughter. Cumulus-oocyte complexes were collected from slaughterhouse ovaries, washed 5–6 times, and cultured in maturation media (TCM-199 + 10% FBS + 10 mg mL−1 FSH-P + 0.81 mM sodium pyruvate + 5% follicular fluid + 50 mg mL−1 gentamicin sulfate + 1 μg mL−1 oestradiol + 100 μM cysteamine) for 27 h in a 5% CO2 incubator at 38.5°C with maximum humidity. After 27 h of culture, cumulus cells were separated from matured oocytes by repeated pipetting using a fine pipette in fertilization Bracket and Oliphant’s (BO) medium. After removal of cumulus cells, the oocytes were transferred to acidified Tyrode’s medium for zona thinning for 52 s and were co-incubated with capacitated sperms for fertilization in fertilization BO medium at 38.5°C in 5% CO2 in air with maximum humidity. In the experiment I, freshly collected buck semen was used for IVF after processing for capacitation. In experiment II, cryopreserved buck semen straws were thawed and sperm were capacitated in vitro and used for fertilization. After 5 h of co-incubation, presumptive zygotes were washed thoroughly and cultured in embryo development medium for cleavage. Three different in vitro development media (RVCL, mSOF + 2.5% BSA, and KSOM + 0.5% BSA) were used. After 40 to 42 h, cleavage was observed and embryos were co-incubated with oviducal cells in replacement media for further development. In the fresh group, overall cleavage rates (%) were 37.76 ± 2.98, 39.60 ± 1.75, 29.01 ± 1.74 and morula formation rates (%) were 7.72 ± 3.38, 6.03 ± 1.29, 3.00 ± 3.00 in RVCL, mSOF, and KSOM media, respectively. However, in the cryopreserved group the overall cleavage rates (%) were 29.17 ± 2.56, 27.70 ± 2.31, and 24.17 ± 1.44 in RVCL, mSOF, and KSOM media, respectively, and morula formation (%) was achieved 2.93 ± 0.97 in RVCL media. These results indicate that cryopreserved black Bengal buck semen have competence to produce embryos and could be used for embryo development through IVF.

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212 EFFECT OF CANINE OVIDUCT CELLS AND CUMULUS CELLS CO-CULTURE ON IN VITRO MATURATION OF PORCINE OOCYTES AND EMBRYO DEVELOPMENT

Reproduction Fertility and Development ◽

10.1071/rdv28n2ab212 ◽

2016 ◽

Vol 28 (2) ◽

pp. 237

Author(s):

S. H. Lee ◽

H. J. Oh ◽

G. A. Kim ◽

M. J. Kim ◽

Y. B. Choi ◽

...

Keyword(s):

Embryo Development ◽

Oocyte Maturation ◽

In Vitro Maturation ◽

Polar Body ◽

Cumulus Cells ◽

In Vitro Development ◽

First Polar Body ◽

Vitro Development ◽

And Control

In oestrus stage, canine oocytes surrounded by cumulus cells undergo maturation in oviduct for 3 days after ovulation. We hypothesised that canine cumulus cells (cCC) and canine oviduct cells (cOC) in oestrus stage might affect the maturation of oocyte and embryo development. Therefore, the present study was aimed to compare the effects of cCC and cOC co-culture system on oocyte in vitro maturation and embryo in vitro development. cCC were separated from cumulus‐oocyte complex (COC) in ovary from bitches in oestrus phase. cOC were collected from oviduct flushing of bitches in oestrus phase. Both cCC and cOC were cultured and cryopreserved until use for co-culture. In the first experiment, the effect of co-culture using cCC and cOC on porcine oocyte in vitro maturation (IVM) were investigated. The porcine COC were randomly cultured in different co-culture groups as follows: 1) co-culturing with cCC for 42 h, 2) co-culturing with cOC for 42 h, and 3) culturing in absence of cCC or cOC. After IVM, extrusion of the first polar body was observed under a microscope. In the second experiment, the matured oocytes with the first polar body derived from each group were activated with electrical stimulus. Parthenotes were cultured in porcine zygote medium-5 (PZM-5) for 7 days at 39°C, 5% CO2 and O2 in a humidified atmosphere. The embryo developmental competence was estimated by assessing the in vitro development under microscope. The third experiment was to evaluate the reactive oxygen species (ROS) levels in each supernatant medium obtained from cCC and cOC co-culture group after IVM using a OxiselectTM ROS ELISA Assay kit. Last, analysis of genes (MAPK1/3, SMAD2/3, GDF9 and BMP15) expression in cCC and cOC co-cultured with porcine COC using real-time PCR is in progress. As results, IVM rate of cOC group (91.19 ± 0.45%) was significantly higher than that of cCC and control group (86.50 ± 0.61% and 79.81 ± 0.82%; P < 0.05). Also, cOC groups expressed the highest efficiency in cleavage rate, blastocyst formation rate, and the total cell number in blastocyst (P < 0.05). In ROS levels, cOC group (555 ± 7.77 nM) were significantly lower than cCC and control groups (596.8 ± 8.52 nM and 657.8 ± 11.34 nM). The present study demonstrated that co-culture with cOC improved the in vitro oocyte maturation and the in vitro development rate of porcine embryos. The ROS level decreased in cOC co-culture would have beneficial influence on oocytes maturation. For further study, we will investigate the relation between gene expression related to oocyte maturation and the co-culture results. This research was supported by a global PhD Fellowship Program through NRF funded by the Ministry of Education (NRF-20142A1021187), RDA (#PJ010928032015), IPET (#311011–05–4-SB010, #311062–04–3-SB010), Research Institute for Veterinary Science, and the BK21 plus program.

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Effect of granulosa and cumulus cells on in vitro development of the bovine follicular oocytes

Asian-Australasian Journal of Animal Sciences ◽

10.5713/ajas.1995.317 ◽

1995 ◽

Vol 8 (4) ◽

pp. 317-320

Author(s):

K. S. Im ◽

H. J. Kim ◽

K. M. Chung ◽

H. S. Kim ◽

K. W. Park ◽

...

Keyword(s):

Cumulus Cells ◽

In Vitro Development ◽

Vitro Development

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Effects of cycloheximide treatment and interval between fusion and activation on in vitro development of pig nuclear transfer embryos

Reproduction Fertility and Development ◽

10.1071/rd01114 ◽

2002 ◽

Vol 14 (4) ◽

pp. 191 ◽

Cited By ~ 10

Author(s):

M. A. Martinez-Diaz ◽

K. Ikeda ◽

Y. Takahashi

Keyword(s):

Nuclear Transfer ◽

Kinase Activity ◽

Short Interval ◽

Cumulus Cells ◽

Cleavage Rate ◽

In Vitro Development ◽

M Phase ◽

Cytostatic Factor ◽

Vitro Development

The effects of cycloheximide (CHX) treatment and the interval between fusion and activation on the development of pig nuclear transfer (NT) embryos constructed with enucleated oocytes and serum-starved granulosa/cumulus cells were examined. One group of couplets was fused and activated simultaneously (FAS) by a single electrical pulse (activation pulse). Another three groups of couplets were fused electricaly 1.5, 2.5 or 4.5 h before being subjected to the activation pulse (FBA). Each group was divided into two subgroups and incubated with or without CHX. The NT embryos treated with CHX showed a high and stable cleavage rate, regardless of the interval between fusion and activation; however, development to blastocysts was improved only when the NT embryos were subjected to FAS with CHX. These results indicate that CHX-sensitive events occurring shortly after FAS may be responsible for the development to blastocysts. Fusion pulse rarely activated M II oocytes, but rapidly dropped the p34cdc2 kinase activity in NT embryos. A pronucleus-like structure was observed 2-2.5 h after the activation pulse with CHX in NT embryos of both the FAS and FBA groups. Therefore, successive inactivation of M-phase promoting factor and cytostatic factor at a certain short interval may also play an important role in the development of NT embryos.

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In vitro development of morulae from immature caprine oocytes

Zygote ◽

10.1017/s0967199400001842 ◽

1994 ◽

Vol 2 (2) ◽

pp. 97-102 ◽

Cited By ~ 10

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.

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159 THE EFFECT OF DIFFERENT ZWITTERIONIC BUFFERS AND PBS ON IN VITRO DEVELOPMENT AND MORPHOLOGY OF BOVINE EMBRYOS

Reproduction Fertility and Development ◽

10.1071/rdv18n2ab159 ◽

2006 ◽

Vol 18 (2) ◽

pp. 187

Author(s):

J. De la Fuente ◽

A. Gutiérrez-Adán ◽

P. Beltrán Breña ◽

S. S. Pérez-Garnelo ◽

A. T. Palasz

Keyword(s):

Embryo Development ◽

Cell Number ◽

Apoptotic Cells ◽

Bovine Embryos ◽

Chi Square ◽

Oh Groups ◽

In Vitro Development ◽

Chi Square Analysis ◽

Vitro Development

It is assumed that, contrary to phosphate buffers, zwitterionic buffers are neutral. However, zwitterionic buffers containing hydroxymethyl or hydroxyethyl residues may interact with OH-groups in the media and produce formaldehyde (Shiraishi et al. 1993 Free Radic. Res. Commun. 19, 315-321). Also, it was shown that three zwitterionic buffers tested in this study interact with DNA (Stellwagen et al. 2000 Anal. Biochem. 287, 167-175). Our objective was to evaluate the effect of the following buffers: TES (T), MOPS (M), HEPES (H) (pKa values at 20�C: 7.2-7.5), and PBS on in vitro development and morphology of bovine embryos. Zwitterionic buffers and PBS were prepared at a concentration of 10 mM in TALP medium and the final pH was adjusted to 7.2. Bovine follicular fluid was aspirated from abattoir-derived ovaries and evenly divided into four tubes. Collected oocytes (five replicates) from each tube were processed separately through the entire IVM, IVF, and IVC procedures using washing medium buffered with: PBS (n = 490), Group 1; H (n = 438), Group 2; M (n = 440), Group 3; and T (n = 394), Group 4. All buffers contained 4 mg/mL BSA. Oocytes were matured in TCM-199 + 10% FCS and 10 ng/mL of epidermal growth factor and fertilized in Fert-TALP containing 25 mM bicarbonate, 22 mM sodium lactate, 1 mM sodium pyruvate, 6 mg/mL BSA-FAF, and 10 �g/mL heparin with 1 � 106 spermatozoa/mL. After 24 h, oocytes-sperm co-incubation presumptive zygotes were cultured in SOFaa medium with 8 mg/mL BSA at 39�C under paraffin oil and 5% CO2 in humidified air. Cumulus-oocyte complexes and zygotes were held in designated buffers ?16 min before oocyte maturation, ~7 min after IVM and before IVF, and ~18 min after IVF and before culture. The total time of oocyte/embryo exposure to each buffer was ?41 min. Embryo development was recorded on Days 4, 7, 8, and 9. A total of ten, Day 8 blastocysts were taken randomly from each treatment and fixed in 4% paraformaldehyde for total and apoptotic cells counts, and five blastocysts from each replicate and treatment were frozen for later mRNA analysis. Apoptosis were determined by TUNEL, using commercial In situ Cell Death Detection Kit (Roche Diagnostic, SL, Barcelono, Spain). Embryo development among groups was compared by chi-square analysis. The cleavage rates were not different among the groups: PBS, 70.8%; H, 76.5%; M, 77.5% and T, 73.6%. The number of embryos that developed to d8 cells at Day 4 was higher in M, 36.2%, and PBS, 37.6%, than in H, 30.6%, and T, 29.7%, but was not significantly different. However, more (P < 0.05) blastocysts developed at Days 7, 8, and 9 in H and M than in PBS and T groups (21.9% and 22.9% vs. 16.9% and 14.9%, respectively). No difference was found between groups in total cell number (98.8 � 7, PBS; 111.8 � 11.9, M; 106.8 � 12.9, H; and 104.3 � 9.7, T) and the number of apoptotic cells (9.2 � 1.0, P; 9.2 � 0.8, M; 12.9 � 1.8, H; and 9.7 � 0.9, T). Based on the results of this study, we conclude that within our protocol choice of buffer may affect embryo developmental rates but not morphology.

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53 EFFECT OF THE NUCLEAR REPROGRAMMING TIME ON IN VITRO DEVELOPMENT OF EQUINE AGGREGATED CLONED EMBRYOS

Reproduction Fertility and Development ◽

10.1071/rdv25n1ab53 ◽

2013 ◽

Vol 25 (1) ◽

pp. 174

Author(s):

R. Olivera ◽

C. Alvarez ◽

I. Stumpo ◽

G. Vichera

Keyword(s):

Embryo Development ◽

Polar Body ◽

Nuclear Reprogramming ◽

Chi Square ◽

In Vitro Development ◽

First Polar Body ◽

Group 3 ◽

Group 2 ◽

Vitro Development

The time allowed for nuclear reprogramming is considered an essential factor for the efficiency of cloning and has not been evaluated in equine aggregated cloned embryos. The aim of our work was to assess the effect of different timing of activation stimulus after fusion of adult equine fibroblast cells to enucleated equine oocytes on embryo development and embryo quality. We processed a total of 1874 equine ovaries, recovering 3948 oocytes, of which 1914 (48.5%) had extruded the first polar body after 24 h of maturation. Oocyte collection, maturation, and the NT procedure were performed as described by Lagutina et al. (2007 Theriogenology 67, 90–98). Reconstructed oocytes (RO) were activated at 3 different times after cell fusion: (1) 1 h, (2) 1.5 h, and (3) 2 h. Activation was performed using 8.7 µM ionomycin for 4 min, followed by a 4-h culture in a combination of 1 mM DMAP and 5 mg mL–1 of cycloheximide. The RO were cultured in the well of the well system, aggregating 3 RO per well. The RO were cultured in DMEM-F12 with 5% fetal bovine serum (FBS) and antibiotics. Cleavage (48 h after activation), blastocyst, and expanded blastocyst rates (8–9 days) were assessed. In vitro development was compared using the chi-square test (P < 0.05). A total of 1608 RO were cultured. Cleavage was significantly lower in group 3 with respect to the other 2 groups [(1): 396/450, 88%; (2): 540/639, 84.5%; (3): 365/519, 70.3%]. There were no significant differences in blastocyst rates within the 3 groups considering the number of total RO [(1): 19/450, 4.2%; (2): 23/639, 3.6%; (3): 15/519, 2.9%] or aggregated RO per well [(1): 12.7%; (2): 10.8%; (3): 8.7%]. However, the rate of blastocyst expansion was higher (P < 0.05) in group 2 than in group 3 [(1): 17/19, 89.5%; (2): 23/23, 100%; (3): 11/15, 73.3%]. In conclusion, the timing of nuclear reprogramming did not affect blastocyst rates but affected cleavage rates and blastocyst quality. This indicates that 1 h before activation stimulus is enough for embryo development of equine aggregated cloned embryos.

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294 IN VITRO DEVELOPMENT OF RAT OOCYTES FOLLOWING MICROINJECTION OF A CUMULUS CELL INTO THE OOPLASM AND CHEMICAL ACTIVATION

Reproduction Fertility and Development ◽

10.1071/rdv19n1ab294 ◽

2007 ◽

Vol 19 (1) ◽

pp. 262

Author(s):

W. Fujii ◽

H. Funahashi

Keyword(s):

Chemical Activation ◽

Formation Rate ◽

Cumulus Cell ◽

Cumulus Cells ◽

Female Rats ◽

Blastocyst Formation ◽

In Vitro Development ◽

Pronuclear Formation ◽

Vitro Development

If diploid zygotes constituted with a somatic and a maternal genome could successfully develop to term, a new reproductive method would be developed to produce animals. However, there appears to be little information on this subject. In the present study, in vitro early development of the constituted zygotes was examined. A cumulus cell was microinjected into a rat non-enucleated oocyte, the reconstructed oocyte was chemically activated, and the pronuclear formation and in vitro development of the embryo was observed. Prepubertal Wistar female rats (21–27 days old) were induced to superovulate with an IP injection of 15 IU of eCG, followed by 15 IU of hCG 48 h later. Cumulus cells were removed from oocytes by pipetting with 0.1% hyaluronidase. Experiment 1: The DNA content of cumulus cells for microinjection was evaluated by flow cytometry. Experiment 2: The optimal concentration of SrCl2 for activation of rat oocytes was examined. Experiment 3: Cumulus cells were injected into mature oocytes in BSA-free HEPES-buffered mKRB containing 0.1% polyvinyl alcohol (PVA) and cytochalasin B (5 �g mL-1), and were then chemically activated by treatment in Ca2+-free mKRB containing 5 mM SrCl2 for 20 min at 0 to 0.5 (A), 1 to 1.5 (B), or 3 to 3.5 h (C) after injection. Activated embryos were cultured in droplets of mKRB in an atmosphere of 5% CO2 in air at 37�C for 9 to 12 h. After being observed for pronuclear formation, the embryos were transferred into mR1ECM-PVA, and the cleavage and blastocyst formation rates were examined 24 and 120 h later, respectively. Results from 3 to 7 replicates were analyzed by ANOVA and Duncan's multiple range test. A total of 90.0 and 9.5% of cumulus cells derived from ovulated oocyte–cumulus complexes contained 2C and 4C DNA contents, respectively. Survival rates did not differ among oocytes stimulated with 0 to 5 mM SrCl2 (96.7–100%) but did differ between those stimulated with 1.25 and 10 mM SrCl2 (100 and 72.9%, respectively). Activation rates of oocytes increased at higher SrCl2 concentrations and were higher at 5 and 10 mM (92.6 and 98.5%, respectively) than at other concentrations. When cumulus-injected oocytes were activated after various periods after the injection, the incidences of pronuclear formation and cleavage did not differ among the periods (A: 95.0 and 81.3%; B: 85.6 and 85.0%; and C: 82.7 and 84.6%, respectively). Although a majority of the embryos developed to the 2- to 4-cell stages (78.7%; 152/208), the blastocyst formation rate was very low (0.8%; 2/208). In conclusion, rat non-enucleated oocytes injected with a cumulus cell can form pronuclei and cleave following chemical activation, but blastocyst formation of the embryos is very limited.

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35 EFFECTIVENESS OF MICROWELL-BASED IN VITRO CULTURE SYSTEMS FOR BOVINEZONA-FREE CLONED EMBRYOS

Reproduction Fertility and Development ◽

10.1071/rdv23n1ab35 ◽

2011 ◽

Vol 23 (1) ◽

pp. 124

Author(s):

C. Feltrin ◽

M. Machado ◽

L. M. V. Queiroz ◽

M. A. S. Peixer ◽

P. F. Malard ◽

...

Keyword(s):

In Vitro Culture ◽

Embryo Development ◽

Zona Pellucida ◽

Blastocyst Stage ◽

Aggregation State ◽

Developmental Potential ◽

In Vitro Development ◽

Vitro Development

In vitro embryo production by handmade cloning (HMC) usually requires individual embryo culture, because zona-free embryos cannot be grouped in standard in vitro culture (IVC) protocols. The aim of this study was to evaluate the developmental potential of bovine embryos produced by HMC (Ribeiro et al. 2009 Cloning Stem Cells 11, 377–386) after in vitro culture (IVC) in 3 microwell (WOW) systems. After in vitro maturation, oocytes were denuded and incubated in demecolcine (Ibáñez et al. 2003 Biol. Reprod. 68, 1249–1258), followed by zona pellucida removal, oocyte bisection, embryo reconstruction, electrofusion, and chemical activation. Cloned embryos were allocated to 1 of 3 IVC groups: cWOW: conventional microwells (250 μm, round; Vajta et al. 2000 Mol. Reprod. Dev. 55, 256–264); mWOW: modified microwells (130 μm, conical; Feltrin et al. 2006 Reprod. Fert. Dev. 18, 126); and WOW-PDMS: microwells in polydimethylsiloxane chips (170 μm, cylindrical with microchannels); IVF embryos were used as controls (Bertolini et al. 2004 Reproduction 128, 341–354). Cleavage (Day 2), blastocyst (Day 7), and pregnancy (Day 30) rates were analysed by the chi-square test, for P < 0.05. Results are shown in Table 1. Cleavage rates were similar between groups, but development to the blastocyst stage was higher in IVF controls than cloned embryo groups. Among cloned embryo groups, blastocyst rate was higher in the mWOW group than the conventional and the PMDS-based microchannels. Nevertheless, in vivo development to Day 30 of pregnancy was not different between cloned groups. Our results for in vitro embryo development indicated that the mWOW provided more suitable conditions for embryo development to the blastocyst stage when compared with cWOW or even WOW-PDMS. Among some possible reasons include the physical advantage of a smaller microwell that may better mimic the constraining effect of the zona pellucida on the developing embryo. That may also provide greater blastomere stability, favouring the aggregation state during the first rounds of cleavages, also aiding compaction and subsequent cavitation. The narrower microwell system appeared to have promoted better in vitro development than the conventional and the DMPS-based microwell systems, with no impact on subsequent in vivo development. However, the IVC in the WOW-PDMS system supported reasonable rates of development, in accordance with the current literature. Table 1.In vitro development of bovine IVF and cloned embryos produced after the in vitro culture in distinct IVC systems

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

Indian Journal of Animal Research ◽

10.18805/ijar.b-3722 ◽

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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