Sheep oocyte activation after intracytoplasmic sperm injection (ICSI)

1998 ◽  
Vol 10 (2) ◽  
pp. 197 ◽  
Author(s):  
M. C. Gomez ◽  
J. W. Catt ◽  
G. Evans ◽  
W. M. C. Maxwell
Keyword(s):  
Calcium Chloride ◽  
Calcium Ionophore ◽  
Fertilization Rate ◽  
Oocyte Activation ◽  
Sham Injection ◽  
Injection Process ◽  
Oviduct Fluid ◽  
Free Media ◽  
Mechanical Injection

The effect of calcium concentration on fertilization and activation was examined in oocytes injected in vitro with sperm. Oocytes were subjected to sperm injection, to sham injection or remained uninjected, and were then cultured for 19 h in bicarbonate-buffered synthetic oviduct fluid (BSOF) without calcium, or containing either calcium chloride or calcium ionophore. There was no difference in fertilization rates after ICSI when oocytes were cultured in vitro in media containing calcium chloride or calcium ionophore but the rate was lower in calcium-free media. There was also no difference in the fertilization rate after ICSI when oocytes were culturedin vivocompared with that observedin vitro in media containing calcium chloride or calcium ionophore. In calcium chloride-treated oocytes, activation was induced by mechanical injection, and in calcium ionophore-treated oocytes, by the ionophore. In uninjected oocytes, calcium itself did not cause oocyte activation. It is concluded that it is possible to induce activation by the injection process, but that manipulation alone is inadequate to cause proper oocyte activation unless calcium is also present. No difference in oocyte activation between ICSI and sham injection was found, indicating that the sperm may play no role in the early events of oocyte activation.

Effect of glucose in the culture medium on development of horse oocytes matured and microfertilized in vitro

1995 ◽  
Vol 7 (5) ◽  
pp. 1067 ◽  
Author(s):  
T Azuma ◽  
YH Choi ◽  
S Hochi ◽  
N Oguri
Keyword(s):  
Calcium Ionophore ◽  
Fertilization Rate ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Cell Stage ◽  
Morula Stage ◽  
Fetal Bovine ◽  
Oviduct Fluid ◽  
Effect Of Glucose

The development of in-vitro matured and microfertilized horse oocytes was examined in vitro. Fertilized oocytes were produced by 20-h insemination of in-vitro matured and partially zona-removed oocytes with frozen spermatozoa that had been treated with caffeine/calcium ionophore A23187 (fertilization rate 34.2%, monospermy rate 76.9%). Embryonic development was assessed by the number of nuclei stained with Giemsa solution. In Experiment 1, a continuous 8-day culture of the microfertilized oocytes in TCM199 or modified synthetic oviduct fluid (m-SOF) supplemented with 10% fetal bovine serum or 0.1% polyvinyl alcohol (PVA) in 5% O2, 5% CO2 and 90% N2 resulted in very few embryos developing beyond the 8-cell stage. In Experiment 2, the effects of different glucose concentrations (0, 0.5, 5.5 mM) in m-SOF/PVA during Days 1-4 and Days 5-8 of culture were examined. Proportions of oocytes having more than one nucleus ranged from 17.7% to 44.7% among the combinations of glucose concentrations. Supplementation with glucose at 0.5 mM during Days 1-4 followed by 5.5 mM during Days 5-8 resulted in the best embryo development; 12/55 (21.8%) nuclei-positive oocytes developed to the 8-16-cell stage, 11 (20.0%) developed to the 17-50-cell stage, and 5 (9.1%) comprised more than 50 cells and were assumed to be at the morula stage.

Activation of bovine oocytes matured in vitro by injection of bovine and human spermatozoa or their cytosolic fractions

Zygote ◽  
2001 ◽  
Vol 9 (1) ◽  
pp. 89-95 ◽  
Author(s):  
Osamu Okitsu ◽  
Shuji Yamano ◽  
Toshihiro Aono
Keyword(s):  
Human Sperm ◽  
Human Spermatozoa ◽  
Oocyte Activation ◽  
Sham Injection ◽  
Bovine Sperm ◽  
Female Pronucleus ◽  
Sperm Factor ◽  
Bovine Spermatozoa ◽  
Bovine Oocytes

The aim of this study was to investigate whether bovine spermatozoa possess so-called sperm factor in the cytosolic fraction (CF) which activates bovine oocytes, and whether bovine oocytes matured in vitro are activated by microinjection of CF extracted from spermatozoa of other species. In the first experiment, bovine and human spermatozoa were microinjected into ooplasm of bovine oocytes matured in vitro. Secondly, CF from bovine and human spermatozoa were injected into bovine oocytes. In the third, CF from human spermatozoa was injected into human unfertilised oocytes obtained 18-20 h after clinical intracytoplasmic sperm injection (ICSI). We found that microinjection of bovine spermatozoa into bovine oocytes induced oocyte activation, as shown by resumption of meiosis and formation of a female pronucleus, at a significantly higher rate than the bovine sham injection (63.0% vs 43.0%; p < 0.05). On the other hand, there was no significant difference in activation rate between the human sperm injection (35.9%) and the human sham injection (22.9%). Furthermore, microinjection of bovine sperm CF into bovine oocytes induced oocyte activation at a significantly higher rate than the human CF injection or sham injection (75.9% vs 14.8%, 20.4%; p < 0.01). Formation of a single female pronucleus and second polar body extrusion was observed in 95.1% of activated oocytes after bovine sperm CF injection. When human sperm CF was injected into human unfertilised oocytes, the activation rate was significantly higher than following sham injection (76.9% vs 44.0%; p < 0.05). These results indicate the presence of sperm factor in bovine sperm CF which activate bovine oocytes, and suggest the possibility that sperm factor has species-specificity at least between bovine and human.

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.

376 EFFECT OF SPERM TREATMENT ON THE ABILITY TO ACTIVATE OOCYTES AFTER ICSI IN PIGS

2007 ◽  
Vol 19 (1) ◽  
pp. 303
Author(s):  
M. Nakai ◽  
N. Kashiwazaki ◽  
N. Maedomari ◽  
M. Ozawa ◽  
J. Noguchi ◽  
...  
Keyword(s):  
Freeze Drying ◽  
Oocyte Activation ◽  
Sham Injection ◽  
Oocyte Meiosis ◽  
Freeze Dried ◽  
Sperm Penetration ◽  
Sperm Factor ◽  
Pre Treatment ◽  
Pronuclear Formation

During fertilization, sperm penetration (gamete membrane fusion and exposure of sperm cytoplasm) allows oocyte activation (resumption of oocyte meiosis, pronuclear formation, etc.) by inducing an elevation of the intracellular free Ca2+ concentration. So a spermatozoon ought to be able to fully activate an oocyte. However, in pig ICSI oocytes, although a spermatozoon is injected successfully into ooplasm, complete activation is deficient in some of the oocytes. A variety of sperm pre-treatments before ICSI have been reported; however, there is a possibility that the treatment affects the ability to activate oocytes after the injection. We examined the effect of sperm treatments (freezing, freeze-drying, and sonication) on the ability to activate oocytes. Ejaculated boar semen was centrifuged (10 min, 600g) and the supernatant was discarded. The sperm pellet was resuspended in Modena solution (Weitze 1991 Reprod. Domest. Anim. (Suppl. 1), 231–253). The sperm were then treated with or without sonication for 10 s (fresh whole and sonicated sperm, respectively). The freezing of sperm was carried out as was described (Kikuchi et al. 1998 Theriogenology 50, 615–623). Frozen–thawed spermatozoa were then treated with or without sonication (frozen–thawed sonicated and whole sperm, respectively). The fresh whole and sonicated sperm were subjected to a freeze-drying system and the sperm were then re-hydrated (freeze-dried whole and sonicated sperm, respectively). A whole sperm or 1 or 3 sonicated sperm heads were then injected into in vitro-matured oocytes, as described previously (Nakai et al. 2003 Biol. Reprod. 68, 1003–1008; 2006 Reproduction 131, 603–611). Sham injection was also performed. No artificial stimulation was added to the injected oocytes. The oocytes with more than one pronucleus(i) at 10 h after the injection were defined as being activated. As shown in Table 1, the rates of activated oocytes after injection of one sonicated head or sham injection were significantly lower than those of the oocytes injected with whole sperm or 3 sonicated sperm heads in each sperm source (P &lt; 0.05 by ANOVA and Duncan's multiple range test). Furthermore, the rates of activated oocytes for each injection category were not different among the 3 sperm sources. These results suggest that sonication before ICSI may reduce the quantity of activation-inducing sperm factor. It is also suggested that sperm pre-treatment such as freezing or freeze-drying does not affect the ability for oocyte activation. Table 1. Effect of sperm treatment on oocyte activation after ICSI

scholarly journals Penambahan Glutathione pada Medium Fertilisasi Efektif Mendukung Pembentukan Pronukleus dan Perkembangan Awal Embrio Sapi (SUPPLEMENTATION OF GLUTATHIONE IN FERTILIZATION MEDIUM EFFECTIVELY SUPPORT NORMAL PRONUCLEUS FORMATION AND EARLY BOVINE EMBRYONIC DE

2017 ◽  
Vol 18 (3) ◽  
pp. 327
Author(s):  
Aras Prasetiyo Nugroho ◽  
Iman Supriatna ◽  
Mohamad Agus Setiadi
Keyword(s):  
Tissue Culture ◽  
Embryonic Development ◽  
Culture Medium ◽  
Fertilization Rate ◽  
Tissue Culture Medium ◽  
Randomized Design ◽  
Oviduct Fluid ◽  
And Control ◽  
Bovine Oocytes

The objective of this study was to determine fertilization rate effectiveness and early embryonic development competency with glutathione (GSH) supplementation in fertilization medium and culture This study consisted of two experiments comprising each of the four treatment and six repetitions with completely randomized design (CRD) using 651 oocytes. In the first experiment, a total of 317 bovine oocytes were matured in tissue culture medium (TCM) 199 at incubator 5% CO2 with temperature 39 ºC for24 h, then fertilized with sperm separated by swim up technique. Oocyte and sperms were incubated in fertilization medium supplemented with 0.25 mM, 0.50 mM, 1.00 mM GSH. In the second experiment, bovine oocytes were matured in maturation medium and fertilized with same procedure as mentioned before, then cultured in modified synthetic oviduct fluid (mSOF) with the following treatment: supplementation GSH only in fertilization medium (T1), supplementation GSH only in culture medium (T2), and supplementation GSH in both fertilization and culture medium (T3), while control not supplementation GSH (T0). Result of the first experiment showed that supplementation 1.00 mM GSH in fertilization medium can increase higher normal pronucleus (2PN) formation (86,9%) compared to other treatments, 0.50 mM (80.3%), 0.25 mM (73.8%), and control (58.9%) (P<0.05). In the second experiment showed that early bovine embryonic development on 2nd day cultured which reached 5-8 cell on treatment T1 (56.0%) and T3 (53.6%) were higher (P<0.05) compared to treatment T2 (26.2%) and T0 (control) (31.3%). Result of the other were showed that early bovine embryonic development on 4th day cultured which reached 9-16 cell on treatment T1 (26.2%) and T3 (27.4%) were higher (P<0.05) compared to that T2 (11.9%) and T0 (control) (10.8%). In conclusion, 1.00 mM GSH supplementation in medium was more effective in supporting normal pronucleus formation and early fertilization bovine embryonic development compared to in culture medium. ABSTRAK Penelitian ini bertujuan untuk mengetahui tingkat fertilisasi dan kompetensi perkembangan awal embrio sapi dengan penambahan glutathione (GSH) pada medium fertilisasi in vitro (IVF) dan kultur in vitro (IVC). Penelitian ini terdiri atas dua penelitian yang terdiri dari masing-masing empat perlakukan dan enam kali ulangan dengan rancangan acak lengkap (RAL) menggunakan 651 oosit. Penelitian I, sebanyak 317 oosit sapi dalam tissue culture medium (TCM) 199 dimatangkan pada inkubator 5% CO2 dan suhu 39°C selama 24 jam, kemudian difertilisasi dengan spermatozoa yang telah diseleksi menggunakan teknik swim up. Oosit dan spermatozoa diinkubasi pada medium fertilisasi dengan penambahan 0,25 mM, 0,50 mM, dan 1,00 mM GSH. Penelitian II, sebanyak 334 oosit sapi dimatangkan pada medium pematangan dan difertilisasi, kemudian dikultur pada medium modified synthetic oviduct fluid (mSOF), dengan perlakuan: penambahan GSH hanya pada medium fertilisasi (T1), penambahan GSH hanya pada medium kultur (T2), dan kombinasi penambahan GSH pada medium fertilisasi dan kultur (T3). Hasil penelitian I, menunjukkan bahwa penambahan 1,00 mM GSH pada medium fertilisasi dapat meningkatkan pembentukan pronukleus normal (2PN) yang lebih tinggi (86,9%) dibandingkan dengan perlakuan yang lain yaitu 0,50 mM (80,3%), 0,25 mM (73,8%), dan kontrol (58,9%) (P<0,05). Penelitian II menujukkan bahwa perkembangan awal embrio sapi pada hari ke-2 kultur yang mencapai pembelahan 5-8 sel pada perlakukan T1 (56,0%) dan T3 (53,6%) lebih tinggi (P<0,05) dibandingkan dengan perlakuan T2 (26,2%) dan T0 (kontrol) (31,3%). Hasil penelitian lain menunjukkan bahwa perkembangan awal embrio sapi pada hari ke-4 kultur yang mencapai pembelahan 9-16 sel pada perlakuan T1 (26,2%) dan T3 (27,4%) lebih tinggi dibandingkan dengan perlakukan T2 (11,9%) dan T0 (kontrol) (10,8%) (P<0,05). Dapat disimpulkan bahwa penambahan 1,00 mM GSH pada medium fertilisasi lebih efektif dalam mendukung pembentukan pronukleus normal dan perkembangan awal embrio sapi dibandingkan pada medium kultur.

Effect of anisomycin, a protein synthesis inhibitor, on thein vitrodevelopmental potential, ploidy and embryo quality of bovine ICSI embryos

Zygote ◽  
2016 ◽  
Vol 24 (5) ◽  
pp. 724-732 ◽  
Author(s):  
María Elena Arias ◽  
Raúl Sánchez ◽  
Ricardo Felmer
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Developmental Rate ◽  
Fertilization Rate ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Protein Synthesis Inhibitor ◽  
Oocyte Activation ◽  
Synthesis Inhibitor

SummaryIncreasing the efficiency of intracytoplasmic sperm injection (ICSI) in domestic animals has been attempted by many researchers, however embryonic development to the blastocyst stage remains low compared with that ofin vitrofertilization (IVF) embryos. One of the main problems observed in cattle is inadequate oocyte activation after ICSI. The present study compared the effect of cycloheximide (CHX), 6-dimethylaminopurine (DMAP), and anisomycin (ANY) on the fertilization rate, development, ploidy and quality of bovine embryos generated by ICSI. Although no differences were observed between treatments in terms of cleavage, higher blastocyst rates were observed for ANY (37.3%) compared with CHX (21.8%,P< 0.05) and DMAP (28.6%,P> 0.05) treatments. No differences were observed in the quality of embryos as assessed by the total number of cells, their distribution to the different embryo compartments [inner cell mass (ICM) and trophectoderm (TE)], the proportion of ICM cells to the total cell numbers and terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL)-positive cells. Similarly, no differences were observed in the normal ploidy of embryos (56, 67, and 55%) for ANY, CHX and DMAP, respectively. However, higher fertilization rates were observed for ANY (75%) and CHX (87%) treatments compared with DMAP (35%). In conclusion, ANY showed a superior developmental rate compared with CHX treatment. Although no significant differences were observed compared with an improved protocol of DMAP (2Io-DMAP), the lower fertilization rate recorded with DMAP strongly suggests that ANY could be a better alternative for oocyte activation than traditional chemical compounds used currently in ICSI.

scholarly journals Calreticulin from suboolemmal vesicles affects membrane regulation of polyspermy

Reproduction ◽  
2014 ◽  
Vol 147 (3) ◽  
pp. 369-378 ◽  
Author(s):  
María Dolores Saavedra ◽  
Irene Mondéjar ◽  
Pilar Coy ◽  
Miguel Betancourt ◽  
Humberto González-Márquez ◽  
...  
Keyword(s):  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Specific Binding ◽  
Calcium Ionophore ◽  
Ultrastructural Level ◽  
Oocyte Activation ◽  
Cortical Granules ◽  
Immunoreactive Band

This study was designed to determine whether calreticulin (CRT), a chaperone protein, is present in in vitro-matured (IVM) pig oocytes and to study its potential role in the block to polyspermy. Western blot analysis, using an anti-CRT antibody, of oocyte lysate showed an immunoreactive band of ∼60 kDa. Simultaneous labeling of IVM oocytes with anti-CRT antibody and peanut agglutinin lectin (PNA lectin, a porcine cortical granules (CG)-specific binding lectin) revealed localization of CRT in the subplasmalemmal region with a 27.7% colocalization with PNA staining. After IVF, PNA labeling was not observed and anti-CRT labeling decreased significantly in zygotes and disappeared in two-cell embryos. Western blot analysis of oocyte exudate obtained from zona pellucida (ZP)-free oocytes activated with calcium ionophore confirmed the presence of a band that reacted with an anti-CRT antibody. Anti-CRT antibody and PNA labeling were not observed in activated oocytes despite being detectable in non-activated oocytes. The presence of CRT in vesicles located under the oolemma was demonstrated using immunogold cytochemistry at the ultrastructural level. To study the role of CRT in fertilization, ZP-enclosed and ZP-free oocytes were incubated with exogenous CRT and then inseminated. Whereas ZP-free oocytes showed fewer penetrating sperm and lower polyspermy rates than untreated oocytes, the opposite effect was observed in ZP-enclosed oocytes. In conclusion, CRT is confined to subplasmalemmal vesicles partially overlapping with CG contents. Its exocytosis after the oocyte activation seems to participate in the membrane block to polyspermy in pigs but is not involved in the ZP block.

scholarly journals The role of nitric oxide in parthenogenetic activation of pig oocytes: A review

2008 ◽  
Vol 52 (No. 11) ◽  
pp. 363-377 ◽  
Author(s):  
J. Petr ◽  
E. Chmelíková ◽  
L. Tůmová ◽  
M. Ješeta
Keyword(s):  
Nitric Oxide ◽  
Calcium Ionophore ◽  
Signalling Pathways ◽  
Oocyte Activation ◽  
Parthenogenetic Activation ◽  
Complex Processes ◽  
Parthenogenetic Development ◽  
Adequate Stimulus ◽  
Signalling Cascade

Parthenogenetic activation of mammalian oocytes with artificial stimuli is commonly applied in various reproductive biotechniques, e.g. cloning using nuclear transfer. For this reason, many studies focus on oocyte activation <I>in vitro</I>. Recently we have described the activation of pig oocytes using nitric oxide. This activating stimulus is very specific in many aspects. However, it does not provide an adequate stimulus for parthenogenetic development. It was shown that nitric oxide stimulated some signalling pathways which are inactive in conventional treatments for parthenogenetic activation, e.g. the cGMP-dependent signalling cascade. On the other hand, nitric oxide does not stimulate certain signalling pathways involved in oocyte activation after calcium ionophore, e.g. the PKC signalling cascade. The aim of this review is to characterize the complex processes induced in oocytes after treatment with nitric oxide. Perspectives for further research and the application of nitric oxide for parthenogenetic activation of oocytes are outlined.

245 ANALYSIS OF CORTICAL GRANULE EXUDATE OBTAINED BY CHEMICAL OOCYTE ACTIVATION IN PIGS

2011 ◽  
Vol 23 (1) ◽  
pp. 221
Author(s):  
R. Romar ◽  
M. J. Izquierdo-Rico ◽  
H. Funahashi
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Cortical Granule ◽  
Ionophore A23187 ◽  
Oocyte Activation ◽  
Cortical Granules ◽  
Isolation And Identification

Cortical granules (CG) are clue organelles in the mammalian oocyte because once released, their content modifies the zona pellucida (ZP) and oolema, thus preventing polyspermy. However, research on putative CG proteins has progressed slowly because of the picogram amount of proteins contained in CG. Isolation and identification of CG contents in porcine oocytes would help to elucidate the molecular mechanism involved in blocking polyspermic fertilization. Our objective was to study the contents of CG from in vitro-matured (IVM) porcine oocytes, and to achieve this objective, CG exudate was collected after its release from chemically activated oocytes. Oocytes were subjected to IVM in porcine oocyte medium supplemented with 50 μM β-mercaptoethanol for 44 h. After the IVM period, the ZP was removed by protease treatment (0.5% pronase in PBS), and the ZP-free oocytes were activated with calcium ionophore A23187 (6.5 μM, 2 min) in a medium consisting of 114.06 mM NaCl, 3.20 mM KCl, 0.50 mM MgCl2·6H2O, 10.00 mM sodium lactate, 0.35 mM NaH2PO4, 5.00 mM glucose, 25.07 mM NaHCO3, and 8.00 mM calcium lactate·5H2O. After activation, oocytes were transferred to fresh medium without calcium ionophore and kept for 30 min to allow release of the CG content. After this time, medium containing the CG exudate was collected, as well as the activated oocytes, and both samples were stored at –80°C until analysis. Samples were thawed and the CG proteins were concentrated by centrifugation in 10-kDa centrifugal devices (Microcon, Millipore, Billerica, MA) following the manufacturer’s instructions. The CG exudates from activated oocytes (n = 300) and activated oocytes (n = 125) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. In brief, 4% stacking and 12% separating gel was used and run using 25 mM Tris–0.2 M glycine buffer, pH 8.6, containing 0.1% SDS for 1.5 h at 150 V and room temperature. After electrophoresis, the gel was silver stained. Thirteen strong bands were identified in the CG exudate lane, with an approximate molecular mass from approximately 45 to 105 kDa. However, the lane for activated oocytes showed faint protein bands. The presence of well-defined bands in the CG exudate lane might correspond to different CG-derived proteins. These preliminary results show a new approach for studying CG content. Further proteomic analysis of the bands will help to describe specific proteins contained in these organelles, shedding light on the role of the cortical reaction in pigs. Supported by MEC and FEDER (AGL2009-12512-C02-01) and Okayama Universit R. R. was granted funding by JSPS (Ref. S-09210).

161 IN VITRO FERTILIZATION OF DROMEDARY CAMEL (CAMELUS DROMEDARIUS) OOCYTES WITH EPIDIDYMAL SPERMATOZOA

2012 ◽  
Vol 24 (1) ◽  
pp. 192 ◽  
Author(s):  
A. R. Moawad ◽  
G. M. Darwish ◽  
M. R. Badr ◽  
A. B. El-Wishy
Keyword(s):  
In Vitro Fertilization ◽  
Calcium Ionophore ◽  
Fertilization Rate ◽  
Dromedary Camel ◽  
Vitro Fertilization ◽  
Fertilization Rates ◽  
Significant Difference ◽  
Epididymal Spermatozoa ◽  
Normal Fertilization

Various techniques such as AI and ET have been reported to improve reproductive efficiency and genetic potential in camelids. In vitro fertilization and the development of IVP embryos are considered an alternative for genetic improvement in this species. This study investigated the effects of different sperm cell concentrations (1, 2, 3 and 4 × 106 sperm mL–1), different capacitating materials (5 mM caffeine, 10 μg mL–1 of heparin, 10 mg mL–1 of theophylline, 1 mM calcium ionophore A23178 and 10 μg of heparin + 5 mM caffeine), post-slaughter epididymal flushing time and fertilization media supplements (Fert-TALP + 6 mg mL–1 of BSA and Fert-TALP + 3 mg mL–1 of polyvinylpyrrolidone ) on fertilization rates and subsequent development of dromedary camel oocytes. Cumulus–oocyte complexes obtained at slaughter were matured in vitro in TCM-199 for 36 h at 39°C in a humidified atmosphere of 5% CO2. For IVF, spermatozoa were collected from epididymides of slaughtered male camels at 1 to 2 h post-slaughter or after 24 h of epididymal storage at 4°C. The spermatozoa were then prepared for IVF by the swim-up technique. Following sperm capacitation, oocytes and spermatozoa were co-incubated for 18 h. Oocytes were then stained using aceto-orcein for evaluation of fertilization events. Presumptive zygotes were cultured in vitro in TCM-199 medium supplemented with 5% FCS for 9 days at 39°C in a humidified atmosphere of 5% O2, 5% CO2 and 90% N2. At least 3 replicates were performed for each experimental group. Data were analysed by chi-square test. Fertilization rates were 55.5, 62.5, 62.7 and 47.2% in oocytes inseminated with 1, 2, 3, or 4 × 106 sperm mL–1, respectively. Normal fertilization rate (oocytes with 2 pronuclei) was higher (P =  0.06) in oocytes inseminated with 2 × 106 sperm mL–1 (29.7%) than in those inseminated by 4 × 106 sperm mL–1 (11.1%). Treatment of epididymal spermatozoa with 5 mM caffeine significantly increased (P ≤ 0.05) fertilization rate (61.9%) compared with calcium ionophore A23178 (32.4%). These values were not significantly different from other groups (38.5, 54.1 and 50.0% in heparin, theophylline and heparin + caffeine, respectively). Normal fertilization was highest (25.4%) in oocytes inseminated with caffeine-treated spermatozoa. Insemination of oocytes in Fert-TALP medium containing BSA resulted in a higher fertilization rate (21.4%) compared with oocytes in polyvinylpyrrolidone-supplemented medium (5.7%; P =  0.06). Storage of camel epididymides at 4°C for 24 h did not affect fertilization rates. Cleavage rate (48 h post-insemination) was higher in oocytes fertilized with caffeine-treated spermatozoa than in oocytes in the theophylline group (26.8 vs 10.5%; P =  0.08). No significant difference was observed in the frequency of blastocyst development (5 days post-insemination) between the 2 groups (5.4 vs 2.6%); based on the number of cleaved oocytes, the same proportions of blastocyst embryos were reported (20.0 and 25.0%). Taken together, these results suggest that dromedary camel oocytes can be matured, fertilized and subsequently developed in vitro with high developmental potential. Epididymal spermatozoa at a concentration of 2 × 106 sperm mL–1 prepared in a medium containing caffeine as a capacitating agent can be used effectively in IVF of camel oocytes.

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