Effects of arginine-glycine-aspartic acid (RGD) containing snake venom peptides on parthenogenetic development and in vitro fertilization of bovine oocytes

The ability of synthetic arginine-glycine-aspartic acid (RGD)-containing peptides to induce intracellular calcium transients similar to those observed at fertilization by spermatozoa in the bovine has been reported (Campbell et al., 2000: Biol Reprod 62:1702-1709; Sessions et al., 2006. Mol Reprod Dev). These results also indicated the ability of synthetic RGD-containing peptides to induce activation and subsequent parthenogenetic development to the blastocyst stage, although, at numbers lower than observed with control in vitro fertilization (IVF). Evidence has been provided indicating the important effect of surrounding regions on the biological activity of the RGD sequence (Zhu and Evans, 2002; Sessions et al., 2006). The current experiments were designed to use natural RGD-containing sequences (disintegrins) to understand their effects. A total of three RGD-containing snake venom peptides (Kistrin (K), Elegantin (Ele), and Echistatin (Ech)) and one nonRGD-containing venom (Erabutoxin B (EB; control) were used at three concentrations (0.1, 1, and 10 micro g /ml) to induce parthenogenetic development to the blastocyst stage and in conjunction (1.0, 5.0, and 10 micro g/ml) with spermatozoa to evaluate competitive inhibition of fertilization and subsequent development. A (P < 0.01) higher number of bovine oocytes developed to the blastocyst stage after incubation with K, Ele and Ech at 1.0 micro g/ml, and was not different (P > 0.01) from IVF control. Fertilization was significantly reduced (P < 0.01) at all concentrations of K, Ele and Ech as compared to IVF control. No reduction (P > 0.05) was observed in EB (nonRGD) treated oocytes. These results support the involvement of a disintegrin-integrin interaction at fertilization in the bovine resulting in activation and subsequent development.     

Effect of sperm survival and CTC staining pattern on in vitro fertilization of porcine oocytes

Polyspermy in pig oocytes fertilized in vitro remains unacceptably high. In this study, we evaluated the effects of gamete coincubation time, and determined if the proportion of capacitated spermatozoa would be predictive of the fertilizing ability of frozen-thawed semen in vitro. Cumulus-oocyte complexes were collected from slaughterhouse prepubertal gilt ovaries and matured in vitro for 44 h in TCM199, with EGF, FSH, cysteamine and follicular fluid. Fertilization was induced with 2 x 10(5) frozen-thawed spermatozoa/ml in TBM. Penetration of oocytes as well as polyspermic fertilization occurred 2 h after insemination. A strong correlation between penetration and polyspermic fertilization rates has been demonstrated, but there was no correlation between the proportion of capacitated spermatozoa, as assessed by chlortetracycline staining, at the time of insemination and fertilization rates. We also compared the results of IVF in three IVF media: TBM, m199 and TALP. Penetration and polyspermy were very different in these three media: 71 +/- 19% and 25 +/- 13% in TBM, 37 +/- 11% and 6 +/- 2% in m199, 10 +/- 2% and 0% in TALP, respectively. Nevertheless, survival of spermatozoa or modifications of the capacitation status were not different in these media after 6 h incubation. We concluded that survival and capacitation characteristics of the semen used for IVF could not be predictive of the IVF results. It seems necessary to act at the oocyte level to control both variability between replicates and the incidence of polyspermy. Improving the spermatozoa penetration blocking system of the oocytes and reducing the number of sperm-binding sites on the zona pellucida (ZP) are our further objectives.     

小鼠体内外受精植入前胚胎全基因组甲基化模式比较

为考察体外受精、操作及培养环境对体外受精的小鼠植入前胚胎全基因组DNA甲基化模式的影响,本研究以体内受精的植入前胚胎作为对照,采用间接免疫荧光法检测小鼠体内外受精植入前胚胎基因组DNA甲基化模式.实验结果表明,体外受精各期植入前胚胎呈现出与之相应时期的体内受精植入前胚胎不同的DNA甲基化模式和水平,原核期甲基化水平较高,2-4-、8-细胞期明显降低,而桑葚胚和囊胚期又略有升高.各期体外受精植入前胚胎的基因组DNA甲基化水平都比同时期体内受精胚胎的甲基化水平低.本实验结果部分显示了体外受精、操作及培养环境可能对正常的DNA甲基化模式产生影响,造成体外受精植入前胚胎甲基化模式异常.     

Mouse sperm-egg interaction in vitro in the presence of neem oil

In vitro evidence is presented showing toxicity of neem oil on sperm-egg interaction in mouse. Cumulus oophorus-enclosed ova, inseminated with capacitated spermatozoa, were cultured in 1 ml of in vitro fertilization (IVF) medium and overlayered by 1 ml of different concentrations of neem oil (1, 5, 10, 25, 50 and 100%) for IVF duration of 4h. At the end of incubation, ova were allowed to grow in neem oil-free culture medium and assessed for fertilization, first cleavage (2-cell formation) and blastocyst formation in vitro at 4–14h, 24h and 108h post-insemination respectively. The study showed that the presence of neem oil at concentrations of 10, 25 and 50% caused inhibition of IVF in a dose-dependent manner. The toxic effect of exposure of 25 and 50% neem oil was further carried over to the first cleavage of the resulting fertilized ova and the toxic effect of 5, 10, 25, and 50% was carried over to the blastocyst formation from the resulting fertilized ova when grown in neem-oil free culture medium. A total of 94.1% inhibition of 2-cell formation and 100% inhibition of blastocyst formation from the inseminated ova was observed in 50 and 25% neem oil-treated groups respectively. Neem oil at 100% concentration caused 100% degeneration of ova at 1h of sperm-ova coculture. The study showed a direct toxic effect of neem oil on sperm-egg interaction in vitro and encourages research investigations of this herbal product as a pre-coital contraceptive.     

Effect of heparin and chondroitin sulfate on the acrosome reaction and fertility of bovine sperm in vitro

Glycosaminoglycans (GAGs) were reported to induce acrosome reactions (AR) in epididymal and ejaculated bovine sperm (4,5). The GAGs chondroitin sulfate A (CS-A) and heparin were tested on ejaculated bovine sperm for their ability to increase in vitro fertilization (IVF) frequencies. Regardless of treatment, a sperm-egg incubation time of 18 hr was sufficient to achieve maximal rates of fertilization. The IVF frequency of sperm incubated 6 hr with 10 mug/ml heparin (116 173 , 67%) was increased (P<0.05) above control levels (56 181 , 31%); however, 10 mug/ml CS-A (56 164 , 34%) was without effect (P>0.05). In contrast to previous reports, CS-A did not (P>0.05) induce AR in ejaculated (9.5-hr incubation) or epididymal sperm (22.5-hr incubation). Linear increases in fertilization frequency (40% to 81%; P=0.001) and AR (9% to 32%; P相似文献   

Observations on the fertilization and development of preimplantation bovine embryos in vitro in the presence of Tritrichomonas foetus

Tritrichomonas foetus, a world-wide distributed parasitic protozoan is a cause of infertility and abortion. There is no documented information on the susceptibility of bovine embryos to the parasite. To determine the effect of T. foetus on fertilization and embryonic development of preimplantation bovine embryos, we added approximately 10(4)/ml or 10(6)/ml T. foetus (Belfast strain) to sperm cells and oocytes prior to in vitro fertilization (IVF) or to presumptive zygotes 24 h post-fertilization. Light and scanning electron microscopy (SEM) revealed that exposure of oocytes or embryos at any stage of development to T. foetus caused rapid adhesion of the trichomonads to the embryonic intact zona pellucida (ZP) and to trophoblastic cells of hatched blastocysts. Treatment of contaminated embryos with 0.25% trypsin for 3 min did not render them free from T. foetus. Motile parasites were not observed after 18 h incubation in IVF medium, or after 72 h in synthetic oviductal fluid (SOF) embryo culture medium. The percentages of cleaved zygotes, blastocysts and hatched embryos resulting from culture of experimental and uninfected control groups of embryos were not different (P > 0.05). Tritrichomonas foetus was not detected in embryonic cells of ZP-intact or hatched embryos when examined by transmission electron microscopy (TEM). In conclusion, T. foetus has no detrimental effect on the fertilization and development of IVF embryos and the potential risk of transmission of trichomonosis is unlikely, due to the limited survival of the parasite in IVF culture conditions.     

Bovine in vitro fertilization with frozen-thawed semen

A procedure to obtain high and repeatable fertilization frequencies for bovine in vitro fertilization (IVF) with frozen-thawed sperm was developed. IVF frequency of in vitro matured oocytes was increased by a swimup sperm separation procedure (P=0.01) or treatment of sperm with the glycosaminoglycan heparin (P=0.0001), but the two factors did not interact (P=0.23). Heparin was the most important factor in increasing IVF frequencies. The fertilization frequency was not affected by the batch of oocytes used (P=0.38), but bull effects were present (P<0.05). Within a bull, the IVF system was highly repeatable and varied between trials no more than +/- 12% in fertilization frequency with an overall fertilization frequency of 299 379 (79%) on four trials over four bulls. In vivo matured oocytes fertilized in vitro were transferred to ewe or heifer oviducts. Morulae or blastocysts were recovered from ewes after four to five days, while conceptuses were present in the bovine after 25 days (diagnosed by ultrasound). Embryonic development from the IVF system either pre- or postimplantation was normal.     

In vitro maturation, in vitro fertilization and embryonic development of canine oocytes

In this study we have investigated the efficiency of in vitro maturation (IVM) as a basic way to study the development of canine oocytes after in vitro fertilization (IVF). We decided, therefore, to perform two-part experiments. Firstly, experiment I compared the effects of TCM199 without fetal bovine serum (FBS) with TCM199 supplemented with 5% FBS on the in vitro nuclear maturation rate of canine oocytes. For the efficiency of meiotic development to the metaphase II (MII) stage, we found that 4.7% (4/64) of all oocytes grown in TCM199 without FBS developed to the MII stage compared with only 1.7% (1/59) of those grown in TCM199 with 5% FBS for 48 h. Therefore, FBS did not increase in vitro nuclear maturation. In experiment II, the cleavage rate of canine oocytes used for IVF was investigated following heparin treatment. Canine oocytes were fertilized in four groups: Fert-TALP medium without heparin (Fert I) or Fert-TALP medium supplemented with 10, 20 or 30 microg/ml heparin (Fert II, Fert III, Fert IV, respectively). Oocytes that were grown for 24 h in Fert I following fertilization showed the highest rate of all of the groups, 6.5% (5/77) and developed to the early morula stage. Markedly, the oocytes cultured in Fert I for 24 h following insemination had a higher rate of embryonic development than other groups. We can assert that, unlike findings in other mammals, heparin treatment in canine IVF does not increase the efficiency of the fertilization rate and is therefore not an important factor.     

Effect of exogenous glutathione on the in vitro fertilization of bovine oocytes

Increased amounts of reactive oxygen species (ROS) during in vitro culture may cause cytotoxic damage to gametes and embryos. The main purpose of this study was to investigate the effect of glutathione (GSH), a ROS scavenger, supplemented during IVF of bovine oocytes on embryo development using spermatozoa from different bulls. The following experiments were performed: 1) matured COCs were fertilized in the absence or presence of 1 mM GSH using semen from 4 bulls (Bulls A, B, C and D); 2) matured COCs were fertilized in the absence or presence of 1 mM GSH using semen from Bull C to examine sperm penetration, pronuclear formation and apposition; 3) COCs were fertilized with in the presence of either 0, 0.1, 1.0 or 10 mM GSH to examine the effect of GSH concentration using sperm from Bull C; 4) concentrations of GSH were measured both in the medium and in the oocytes during IVF. Glutathione at 1 mM in IVF medium affected the blastocyst formation, but not the cleavage rate. The effect on blastocyst formation was bull dependent: semen from Bull B and D had a negative, that from Bull C a positive and the one from Bull A no effect. The positive effect of Bull C semen increased the rate of blastocyst formation from 20.1 to 27.3% in control and GSH-treated samples, respectively. The increased rate was due to more zygotes reaching the 8-cell or greater stage by Day 4 after IVF. There was no change in the fertilization or cleavage rates. The GSH was still stable after 18 h incubation in IVF medium, and there was a dose-dependent increase in the GSH concentration in the oocytes. It is concluded that the effect of GSH during IVF on the proportion of blastocysts is dependent on both bull and GSH concentration.     

Comparison of mice born after intracytoplasmic sperm injection with in vitro fertilization and natural mating

The procedures of in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) are routinely used in modern medicine to overcome infertility and, in animal husbandry, to propagate lines with compromised fertility. However, there remains concern that manual selection and injection of whole sperm into oocytes could contribute to pre- and postnatal developmental defects. To address this, we have used gene expression profiling and immunophenotyping to characterize offspring generated by these procedures. We used gametes from glutathione peroxidase 1 knockout (Gpx1-/-) mice as a sensitized screen responsive to oxidative stress from artificial reproduction technologies (ART). There were no differences between IVF and ICSI derived offspring in gene expression patterns, and minor differences in hematopoietic parameters. Furthermore there were only minor differences between these IVF and ICSI pups and those derived from natural mating. These data demonstrate for the first time in that there is no significant phenotypic affects of ICSI when compared to IVF and we identified a relatively minor influence of the artificial fertilization methods on phenotype of offspring compared with natural mating. These observations would support the use of ICSI for derivation of mutant mouse lines and may be of some importance for the use of this technique in human ART.     

A protocol for rat in vitro fertilization during conventional laboratory working hours

In vitro fertilization (IVF) is a valuable technique for the propagation of experimental animals. IVF has typically been used in mice to rapidly expand breeding colonies and create large numbers of embryos. However, applications of IVF in rat breeding experiments have stalled due to the inconvenient laboratory work schedules imposed by current IVF protocols for this species. Here, we developed a new rat IVF protocol that consists of experimental steps performed during common laboratory working hours. Our protocol can be completed within 12 h by shortening the period of sperm capacitation from 5 to 1 h and the fertilization time from 10 to 8 h in human tubal fluid (HTF) medium. This new protocol generated an excellent birth rate and was applicable not only to closed colony rat strains, such as Wistar, Long-Evans, and Sprague–Dawley (SD), but also to the inbred Lewis strain. Moreover, Wistar and Long-Evans embryos prepared by this protocol were successfully frozen by vitrification and later successfully thawed and resuscitated. This protocol is practical and can be easily adopted by laboratory workers.     

In vitro deveropment of porcine oocytes fertilized in vitro with spermatozoa preincubated in two different media

This study was conducted to clarify the effects of sperm concentration and media during preincubation on fertilization and development of porcine oocytes fertilized in vitro (IVF). The effect of porcine oviduct epithelial cell aggregates (POECA) on in vitro development of IVF embryos was also examined. Oocytes matured in vitro for 48 to 50 h were inseminated with epididymal spermatozoa preincubated at 2 sperm concentrations (1 - 2 x 10(8)/ ml vs 4 - 5 x 10(8)/ ml) for 3 h in either Dulbecco's phosphate buffered saline (PBS) or Brackett and Oliphant medium (BO). For capacitation, spermatozoa were treated with heparin (100microg / ml) for 15 min at 38.5 degrees C under 5% CO(2) in air. Cleavage and development to the blastocyst stage were evaluated on Day 3 and Day 8 after culture with or without POECA. The effect of sperm concentration on preincubation did not affect the fertilization rate, but preincubation in PBS medium did result in a higher fertilization rate (P < 0.05) than did the BO medium. The proportion of embryos undergoing cleavage and development to the blastocyst stage was significantly higher (P < 0.05) in the POECA co-culture group than in the group without POECA co-culture. The present results indicate that PBS medium can be utilized as a simple preincubation medium for porcine spermatozoa and that the presence of POECA during in vitro culture improved the development of IVF oocytes to the blastocyst stage.     

In vitro fertilization and cleavage of in vivo matured bovine oocytes

The effect of the interval between onset of estrus and oocyte collection on in vitro fertilization (IVF) rates has been investigated. The oocytes were surgically collected 6-18 h (Group I), 19-24 h (Group II), 25-29 h (Group III) and 30-36 h (Group IV) after the beginning of estrus. Recognizable stages of nuclear maturation were identified in 54.9% of the oocytes used for IVF (5.9% at germinal vesicle, 31.4% at metaphase I, 17.6% at metaphase II); the other 45.1% were degenerate. Considerable between- and within-cow variation in oocyte morphology, oocyte maturation and IVF results was observed. The cverall fertilization and cleavage rates (to four-cell stages) were 26.5 and 6.0%, respectively. The fertilization rate increased as the interval between onset of estrus and collection increased and was optimal 30-36 h after onset. Thus, onset of estrus proved an effective means of timing oocyte collection for IVF.     

Comparison of histone modifications in in vivo and in vitro fertilization mouse embryos

Histone modifications are thought to play important roles in various cellular functions. In this article, the distribution patterns of acetylation on histone H4, methylation on histone H3 lysine 9, and phosphorylation on histone H3 serine 10 were examined in in vivo and in vitro fertilization (IVF) preimplantation mouse embryos by using indirect immunofluorescence and scanning confocal microscopy. We desired to know whether the IVF, which has been widely used as a routine assisted reproductive technology in animal and human, was safe at the epigenetic level. As results, we found that there was no difference in these histone modification patterns in in vivo and IVF mouse embryos from zygote to blastocyst stage. Moreover, these histone modifications had different distributions at all examined stages, but they were consistent with the mouse embryo developmental stages.     

Brief coincubation of gametes in porcine in vitro fertilization: role of sperm:oocyte ratio and post-coincubation medium

In this study, a short coincubation time of 10 min was used to determine the effect of different sperm:oocyte ratios during in vitro fertilization (IVF), and different periods of post-coincubation in a medium that is not appropriate for IVF, on fertilization parameters. In the first experiment, a total of 1624 in vitro matured oocytes, from 4 replicates, were inseminated with frozen-thawed spermatozoa at different sperm:oocyte ratios (2000, 1500, 1000 and 500 sperm:oocyte) and coincubated for 10 min or 6 h. The oocytes from 10 min of coincubation were washed in IVF medium to remove spermatozoa not bound to the zona pellucida and transferred to another droplet of the same medium (containing no spermatozoa) for 6h. The oocytes from the other group remained with the spermatozoa for 6h. Oocytes from both groups were then cultured in embryo culture medium (IVC) for 12h to assess fertilization parameters. In the second experiment, 1872 in vitro matured oocytes, in 3 replicates were inseminated with frozen-thawed spermatozoa using the same sperm:oocyte ratios as in the first experiment. The oocytes were coincubated for 10 min and transferred directly to IVC medium for 18 h (group A), to IVF medium (containing no sperm) only for 2h and then to IVC medium for 16 h (group B), or to IVF medium (containing no sperm) for 6h and then to IVC medium for 12 h (group C or control). There was an effect of sperm:oocyte ratio on all fertilization parameters in experiment 1. The efficiency of IVF (number of monospermic oocytes/total number inseminated) was higher (P<0.05) for oocytes coincubated with spermatozoa for 10 min and inseminated with 1500 and 1000 sperm:oocyte (35.8+/-3 and 37.6+/-2.7%, respectively) and for those coincubated for 6h with 500 spermatozoa per oocyte (37.2+/-3.1%). In experiment 2, the penetration and efficiency rates obtained in group A were poor (between 3 and 15%) irrespective of the sperm:oocyte ratio. However, in group B the fertilization parameters were similar to the controls and were also affected by the sperm:oocyte ratio. These results demonstrate that coincubation time may be reduced to 10 min to increase the efficiency of fertilization depending on the sperm:oocyte ratio, and that the spermatozoa bound to the zona pellucida require a maximum of 2h in an appropriate medium to penetrate the oocytes.     

Optimal conditions for successful in vitro fertilization and subsequent embryonic development in Sprague-Dawley rats

The present study was conducted to determine the optimal conditions for successful in vitro fertilization (IVF) in Sprague-Dawley (SD) rats. The IVF of oocytes from SD and Wistar rats was compared in different fertilization media (mR1ECM, IVF-20, and modified Krebs-Ringer bicarbonate solution [mKRB]), and IVF conditions were then optimized for oocytes of the SD strain. Results showed that in mR1ECM medium, fertilization rates were markedly lower in SD rats (15%) than in the Wistar strain (73%), although this response was significantly improved by increasing the NaCl concentration. In addition, fertilization rates in SD rats were higher in modified IVF-20 (73%) than in IVF-20 (18%) and mKRB (53%). In contrast, fertilization rates in Wistar rats were higher in IVF-20 and modified IVF-20 than in mKRB (78%, 74%, and 36%, respectively). Further investigation concerning the effects of the NaCl supplementation (10- 40 mM) in IVF-20 on the fertilization of oocytes in the SD strain indicated that significantly higher percentages of oocytes were fertilized in IVF-20 supplemented with 30 mM NaCl (66%) and developed to the blastocyst stage (47%) in vitro. After transfer, embryos derived from this IVF system developed to term at a percentage comparable to that of in vivo-fertilized controls. In conclusion, differences exist in optimal IVF conditions between rat strains, and a modified culture medium has been successfully developed for assessment of the developmental competence of oocytes in SD rats.     

Chromosome configuration during in vitro maturation of goat, sheep and buffalo oocytes

The competence of meiotic chromosome configuration at the time of co-culture of oocytes with spermatozoa is an essential prerequisite for successful in vitro fertilization (IVF). Although this technology has been used in several livestock species, various intrinsic and extrinsic factors affecting the high repeatablity of IVF have yet to be understood. The present study was conducted to determine the appropriate time for coculture of oocytes and spermatozoa in order to optimize the fertilization rate in sheep, goats and buffalo. Oocytes were collected from the ovaries of slaughtered animals. The oocytes were divided into 10 groups and cultured for maturation in TCM-199 supplemented with estrous cow serum for different durations at 38.5 x 0.5/C in a CO(2) incubator. Sheep and goat oocytes were removed from culture medium after 0,6,12,22,24,26,28,30,32 and 36 and buffalo oocytes after 0,6,12,16,20,22,24,26,28, and 36 h. The oocytes were treated with hypotonic solution (0.75 M KCl) and fixed in Carony's fixative on glass slides. The fixed oocytes were stained with Giemsa solution, and the meiotic chromosomes were evaluated under a compound microscope at x 1000 magnification. Observations were recorded on a total of 1328 oocytes (sheep, 409; goat, 727 and buffalo, 192). The sequential configurations of diffused chromatin, pachytene, diplotene (along with nucleoli), diakinesis and metaphase II (MII) were analyzed at different durations of culture. Control oocytes (fixed at 0 h without incubation) were mostly at the pachytene stage, and as the duration of culture increased the instances of diplotene, diakinesis and finally MII increased. Oocytes at the MII stage of meiosis are known to be at the optimal stage of development for co-culture with spermatozoa and successful in vitro fertilization. On the basis of sequential configuration of chromosomes, it was found that the optimal duration of in vitro maturation of oocytes is 32, 30 and 24 h for sheep, goats and buffalo, respectively.     

Ejaculate and type of freezing extender affect rates of fertilization of horse oocytes in vitro

In vitro fertilization (IVF) was performed on in vitro-matured equine oocytes in three experiments. Frozen-thawed sperm were prepared using swim-up separation and heparin treatment. In Experiment 1, fertilization was achieved with sperm from only one frozen ejaculate of four obtained from the same stallion. Within this ejaculate, fertilization rates were higher with fresh media, as compared to media held for 6-8 days before use (39.6% versus 7.3%, respectively; P<0.001). The type of bovine serum albumin used affected fertilization rates (4% versus 39.6%; P<0.001). To determine if IVF rates were influenced by factors associated with the freezing process (Experiment 2), a single ejaculate from a second stallion was frozen using eight variations in timing of steps in the freezing protocol. There were no differences among treatments in fertilization rates (range, 0-3%). In Experiment 3, fertilization rates of semen frozen in an extender containing 21.5% egg yolk were lower than fertilization rates of semen from the same ejaculate but frozen with a 3% egg-yolk extender (0% versus 15%, respectively; P<0.01). We inferred that rates of equine IVF with frozen-thawed sperm were influenced by ejaculate, the composition and age of the media used, and freezing extender. To our knowledge, this is the first report of ejaculate or extender differences affecting in vitro fertilization in this species. These factors may help to explain the great variability in fertilization rates reported with equine IVF, both among and within laboratories.     

Effects of epigallocatechin-3-gallate (EGCG) on in vitro maturation and fertilization of porcine oocytes

The beneficial properties of green tea and especially of its principal active polyphenol, epigallocatechin-3-gallate (EGCG), have led to an increased demand for dietary supplements with highly enriched EGCG concentrations. In order to investigate the possible reproductive-related consequence of EGCG supplementation, the effects of this catechin on in vitro maturation (IVM) and fertilization (IVF) of oocyte, using the pig as experimental model, were examined. In the first series of experiments EGCG, at concentrations ranging from 0 to 25 microg/ml, was added during in vitro maturation of pig oocytes. EGCG had no effect on nuclear maturation of pig oocytes and on fertilization traits considered after IVF at any of the doses tested. By contrast, a significant (p<0.05) decrease in the number of embryos that developed to blastocysts following parthenogenetic activation was recorded when 25 microg/ml EGCG was added to IVM medium; in addition this catechin concentration significantly (p<0.05) inhibited progesterone production by cumulus cells after 48 h of culture. When induction of sperm capacitation was performed in presence of EGCG, a significantly lower percentage of spermatozoa showing a Hsp70-capacitated pattern and a significant reduction of sperm H(2)O(2) production were evident at a concentration of 25 microg/ml EGCG (p<0.05). During gamete coincubation EGCG reduced, in a dose response manner, the number of reacted spermatozoa suspended in fertilization medium and increased the number of sperm bound to ZP. Supplementation of 10 microg/ml EGCG during IVF significantly increased the fertilization rate while higher EGCG concentrations (25 microg/ml) decreased the percentage of fertilized oocytes (p<0.05). In conclusion, our data suggest that high EGCG concentrations could affect in vitro maturation and fertilization in pig; it cannot be totally excluded that excessive EGCG concentrations could induce reproductive-related consequences also in vivo.     

In vitro effect of malathion and diazinon on oocytes fertilization and embryo development in porcine

Diazinon and malathion are commonly used organophosphate insecticides in agriculture, industry, and in veterinary medicine as an ectoparasiticide. The importance to carry out in vitro reproductive toxicology assays lies on the need of knowing the alterations these insecticides may cause at cellular level, since they are endocrine disruptors that interfere with reproductive functions. The aim of this study was to evaluate in vitro oocyte viability, fertilization, and embryo development with different concentrations of diazinon and malathion. For in vitro fertilization (IVF), porcine oocytes and sperm were co-incubated for 7 h with increasing concentrations (50, 100, and 500 μM) of diazinon and malathion. For embryo development, fertilized oocytes were cultured in medium containing the same insecticide concentrations during 96 h for embryo development and 144 h for morulae formation. Diazinon did not affect oocyte viability and embryo divisions but decreased IVF (fertilization inhibition₅₀ = 502 μM) and morulae formation (morulae inhibition₅₀ = 344 μM). Malathion affected all the studied parameters: lethal concentration₅₀ = 1 mM, fertilization inhibition₅₀ = 443 μM, development inhibition₅₀ = 375 μM, and morulae inhibition₅₀ = 216 μM. The results of this study indicate that diazinon and malathion used in commercial formulation could be toxic, producing impairment in in vitro fertilization and embryo development. This is an approach for further investigations to find out cell damage mechanisms produced by these widely used insecticides.