Co-culture of ovine eggs with oviductal cells and trophoblastic vesicles

Three experiments were conducted to determine whether coculture of early sheep eggs with oviductal cells would improve the ability of eggs to survive in culture. Eggs recovered from superovulated ewes were cultured in Ham's F-10 medium supplemented with 10% fetal calf serum (F10FCS) at 37.5 degrees C in 95% air:5% CO(2). In Experiment 1, eggs with one to eight cells were either transferred into recipient ewes immediately after collection or were cultured for 24 h in 5 ml Ham's F10 medium supplemented with 10% fetal calf serum (F10FCS), 5 ml F10FCS on a confluent monolayer of oviductal cells; in 25 ml of fresh F10FCS; or in 25 ml of F10FCS removed cultures of oviductal cells, 25 mul of fresh F10FCS or 25 mul of F10FCS removed from cultures of oviductal cells. After 24 h, the cultured eggs were transferred to recipient ewes synchronous with donors and subsequently recovered at necropsy on Day 8 post estrus. Coculture of sheep eggs with oviductal cells improved (P < 0.05) the development of transferred eggs compared to culture in F10FCS alone. In Experiment 2, eggs recovered from superovulated ewes on Days 3 to 6 after estrus had undergone 1.8 cleavages by Day 3 and 4.1 cleavages by Day 6. In Experiment 3, single-cell eggs were cultured for 3 d in 5 ml F10FCS, cocultured with ovine trophoblastic vesicles or cultured on a confluent monolayer of oviductal cells. Coculture of eggs in F10FCS on a monolayer of oviductal cells supported in vitro egg cleavage to a greater degree than did F10FCS alone or F10FCS with trophoblastic vesicles (P < 0.05). In Experiment 4, single-cell eggs were cultured for 3 d then transferred to recipients. Eggs were cultured in 5 ml F10FCS on confluent monolayers of oviductal cells from luteal or estrous ewes or on cells that had been frozen after recovery from a culture of oviductal cells. After culture, the eggs were transferred to oviducts of recipients and recovered 3 d later at necropsy. Coculture of eggs for 72 h with oviductal cell monolayers did not increase the in vitro, or subsequent in vivo, cleavage rate regardless of the type of oviductal cells.     

Development of one-cell fertilized sheep ova following microinjection into pronuclei

Three experiments were conducted to identify, sources of loss of fertilized single-cell sheep eggs microinjected with DNA. In the first experiment, immediate transfer of eggs into synchronous recipients resulted in 86% of embryos developing (>32 cells) at Day 7. Incubating eggs in microdrops of Ham's F-10 medium + 10% fetal calf serum for 5 h at 37 degrees C in an atmosphere of 95% air: 5% CO(2) before transfer reduced development (65% >32 cells). Removing eggs from drops for 30 min of microscopic inspection, simulating manipulation during microinjection, caused no additional reduction in development (63% >32 cells). However, injection of eggs with buffer was detrimental to subsequent development (42% >32 cells). In Experiment 2, injection of buffer or injection of DNA in buffer into the pronuclei before transfer of eggs into recipient ewes resulted in 29 and 19%, respectively, of embryos developing to >32 cells at Day 7. In Experiment 3, more eggs developed when held in 5 ml of medium than in microdrops (P = 0.07). No difference in development was found between eggs held in bicarbonate-buffered BMOC or in phosphate-buffered saline with added fetal bovine serum. The development of sheep eggs appears to be greatly reduced after microinjection, but until alternate procedures are found, a high rate of loss of injected eggs may be an unavoidable cost of inserting foreign genes into sheep.     

In vitro culture of sheep oocytes matured and fertilized in vitro

Sheep oocytes that matured and fertilized in vitro were cultured to evaluate their cleavage to the 8- to 16- cell stage and further development in five different media as follows: 1) CPMW (TCM199 + 20% ewe serum + 0.4% BSA), 2) Ham's F-10 + 10% ewe serum, 3) Brinster's pyruvate medium + 0.1% glucose (BPM-G), 4) co-culture with sheep oviduct epithelial cells in TCM199 + 10% fetal calf serum, and 5) co-culture with sheep granulosa cells in the same medium as 4. The culture duration was 4 or 7 d for 8- to 16-cell or further development. The proportions of 8- to 16-cell eggs were 1) 16% (8 49 ), 2) 25% (12 49 ), 3) 52% (58 112 ), 4) 63% (105 167 ) and 5) 45% (27 60 ). The co-culture with sheep oviduct cells resulted in a significantly (P < 0.05) higher rate of cleavage than the other media, except BPM-G. The proportion of noncompacted morula (35%, 24 68 ) was also significantly (P < 0.05) higher in the co-culture of sheep oviduct cells than the other media. The 8- to 16-cell eggs produced by BPM-G (n=38) and the co-culture with sheep oviduct cells (n=42) were transferred into the uterus of recipient ewes, but no elongated blastocysts were obtained 13 d later. On the other hand, 8 out of 55 one-cell eggs (15 to 18 h after in vitro insemination) transferred to the oviduct of recipient ewes were elongated blastocysts (24% of 34 recovered eggs). The data show that the co-culture of in vitro fertilized eggs with sheep oviduct epithelial cells could support development of 8- to 16-cell embryos or early morula, but their viability is still questionable.     

Fertilization in vitro of rabbit eggs with or without follicular cells by epididymal spermatozoa capacitated in a chemically defined medium

A portion of eggs from mature does treated with FSH and HCG to induce superovulation was freed from the follicular cells by treatment with hyaluronidase and repeated pipetting. Denuded and untreated eggs with follicular cells were placed in a defined medium with epididymal spermatozoa which had been exposed to the medium for 15 min and preincubated for 10–10.5 h in a CO₂ incubator. No significant difference was noted in the penetration rate of the eggs with (92%) and without (99%) follicular cells 5–5.5 h after insemination. Although there was no difference in the incidence of polyspermy, many more spermatozoa had penetrated into the perivitelline space of the denuded eggs than into those with follicular cells. Regardless of the presence of follicular cells, most of the penetrated eggs were undergoing cleavage or had developed to the 2- or 4-cell stage 24 h after insemination.     

Change in the Respiratory Rate of Sea Urchin Spermatozoa following Sperm-egg Interaction in the Absence of Mg2+

Spermatozoa of the sea urchin, Hemicentrotus pulcherrimus (10⁸ cells/ml), preincubated with unfertilized eggs deprived of jelly coats (more than l0⁵ cells/ml) at 20°C for 20min in Mg²⁺ free artificial sea water containing 1 mM Ca²⁺ (MFASW), exhibited very low respiration, which was enhanced by 2, 4 dinitrophenol (DNP). The fertilization rate in MFASW was usually less than 5% and was about 25% at most. Preincubation with fertilized eggs (with and without a fertilization membrane) in MFASW did not reduced the respiratory rate of spermatozoa. The rate of sperm respiration was lower in MFASW than in artificial sea water (ASW), but was higher than the respiratory rate of spermatozoa preincubated in MFASW with unfertilized eggs. Sperm respiration in MFASW or in ASW was not stimulated by 2, 4 dinitrophenol. Almost complete inhibition of sperm respiration was obtained with unfertilized eggs fixed with glutaraldehyde at concentrations of above 10⁵ cells/ml in MFASW and of about l0⁴ cells/ml in ASW. The respiratory rate of spermatozoa treated with fixed eggs was enhanced by DNP. It is concluded that the respiratory rate of the spermatozoa is reduced by their interaction with unfertilized eggs before their penetration into the eggs.     

Penetration of zona-free hamster eggs by liposome-treated sperm from the bull, ram, stallion, and boar

Spermatozoa from each of four rams, four stallions, and three boars (six semen samples) were treated with dilauroylphosphatidylcholine (PC12) liposomes and compared with control bull sperm to induce the acrosome reaction (AR) and study possible penetration of the sperm into zona-free hamster eggs. Diluted sperm were incubated with several concentrations of PC12 for 7 min at 39 degrees C prior to insemination of the hamster eggs in vitro. The sperm from the bull were diluted to 10(6) cells/ml, as previously studied. Sperm from the ram, stallion, and boar were diluted to 6 X 10(6) and 20 X 10(6) cells/ml. After addition to the eggs, the sperm concentration was reduced by 75 percent. Inseminated eggs were incubated with sperm for 3 h at 39 degrees C prior to being fixed, stained, and observed for sperm penetration. At an initial concentration of 6 X 10(6) cells/ml, bull sperm treated with 36.7 microM PC12 achieved an egg penetration rate of 92%, whereas under nearly identical conditions stallion spermatozoa achieved only 54% egg penetration. Under similar conditions, ram spermatozoa failed to penetrate eggs, but when the initial sperm concentration was increased to 20 X 10(6) cells/ml, sperm incubated with 51.1 microM PC12 achieved 52% egg penetration. Boar spermatozoa treated with PC12 at either sperm concentration failed to exhibit an AR or penetrate hamster eggs. In general, as PC12 concentration increased the percentage of sperm with an AR increased and sperm motility decreased. It is concluded that 1) PC12 liposomes are effective in inducing the AR in sperm from the bull, ram, and stallion, but under conditions tested are ineffective with boar sperm;(ABSTRACT TRUNCATED AT 250 WORDS)     

The sharing of male production among workers and queens in Scaptotrigona depilis (Moure, 1942) (Apidae, Meliponini)

The proportions of males produced by queens or workers of Scaptotrigona depilis, and the sex-ratio in the brood, were estimated. Thirteen young combs were collected; from one half of each comb the cells were opened and the number of eggs per cell was recorded. Later, upon maturation, from the other halves of the combs the individual inside each cell was classified according to sex. If from a cell containing initially two eggs a male would emerge (supposedly the son of a worker) the proportion of such cells in the comb would represent the maximum overall workers’ male production. Consequently, the difference with the number of males found in the second half of the comb would indicate the minimum contribution by the queen. In these 13 combs, on average, 7.3% of the cells contained 2 eggs, whilst in 30.9% of the cells a male developed. Males were found in 10 combs, in 2–79% of the cells. In 6 of the 13 combs 2–47% of the cells contained 2 eggs. In these 6 combs more than 40% of the cells had a male inside. In 5 of them, the frequency of males was significantly higher than the frequency of cells containing 2 eggs. Queens, therefore, produced the majority of the males. There was no correlation between worker and queen numerical investments in male production, indicating different response mechanisms of workers, compared to queens, to conditions favouring male production. There was also no correlation between the mean egg size of the queen and the percentage males in the combs.     

Wolbachia and the normal and incompatible eggs of Aedes polynesiensis (Diptera: Culicidae)

The development of eggs resulting from compatible and incompatible crosses of Aedes polynesiensis strains was studied. No difference was seen between newly laid compatible and incompatible eggs. Very little development occurs in most of the incompatible eggs; the eggs that develop mature but often produce abnormal embryos. Embryos developed in as many as 10% of the incompatible eggs and these developed into both males and females, which indicates that the surviving embryos were not parthenogenetic. In eggs infected with Wolbachia, the pole cells are infected at the time of formation and provide infection for the germ cells which develop later. Embryos that survive the incompatibility reaction and hatch from the egg develop normally.     

表达绿色荧光蛋白重组新城疫病毒 LaSota疫苗株的构建

新城疫病毒是理想的新型活病毒疫苗载体,具有巨大的优势和应用前景。采用生产实践中广泛应用、免疫效果良好的NDV LaSota弱毒疫苗株,建立了反向遗传操作系统。在此基础上,进一步构建了表达绿色荧光蛋白(GFP)的重组NDV基因组cDNA克隆,成功救获了重组病毒rLaSota-EGFP,病毒F1代尿囊病毒液按1×104EID50接种9~10日龄SPF鸡胚尿囊腔,接种后分别于24h、48h、72h及96h收获尿囊液,检测平均HA滴度分别为28、210.3、211.3和211,每mL尿囊液病毒量EID50分别为108.64、109.22、109.21和109.64,重组病毒与亲本株生长滴度在相近时间达到峰值,生长动力学特性与亲本株无明显差异。各代次重组病毒按1×106EID50病毒量接种9~10日龄SPF鸡胚,96h内完全不致死鸡胚。救获重组病毒保持了LaSota弱毒疫苗亲本毒株对鸡胚良好的高滴度生长适应和低致病特性,并且鸡胚连续传9代次仍保持GFP的稳定表达及生物学特性不变。重组病毒rLaSota-EGFP的成功救获为开展新城疫病毒活载体疫苗研制提供了可行的技术平台。     

不同品系萼花臂尾轮虫休眠卵的形态特征

对采自青岛和芜湖两地的萼花臂尾轮虫在3种温度(20 ℃、25 ℃和30 ℃)和2种藻类食物浓度(1.0×10⁶和5.0×10⁶ cells·ml^-1)下所产休眠卵的长径、短径和体积等形态特征进行了显微测量、计算和分析.结果表明,2种食物浓度下,培养温度以及培养温度和品系间的交互作用均对轮虫休眠卵的长径、短径和体积具有显著影响.当食物浓度分别为1.0×10⁶和5.0×10⁶ cells·ml^-1时,轮虫在20 ℃下所产休眠卵的长径、短径和体积均最大;在25 ℃和30 ℃下所产休眠卵的短径和体积均最小.品系对轮虫休眠卵长径、短径和体积的影响也取决于食物浓度.当食物浓度为1.0×10⁶ cells·ml^-1时,芜湖品系轮虫的休眠卵长径、短径和体积(156.00 μm、99.95 μm和12 269.11 μm³)均显著大于青岛品系轮虫的休眠卵(145.13 μm、91.97 μm和10 498.19 μm³);而当食物浓度为5.0×10⁶ cells·ml^-1时,芜湖品系轮虫的休眠卵长径、短径和体积(155.68 μm、100.85 μm和12 348.59 μm³)均与青岛品系轮虫的休眠卵(156.63 μm、98.04 μm和12 054.20 μm³)之间无显著差异.两品系中,仅芜湖品系轮虫休眠卵的长径、短径和体积分别与温度呈曲线相关.同一温度下,两品系轮虫的休眠卵体积均随着食物浓度升高而增大;但30 ℃下芜湖品系轮虫所产休眠卵体积却随着食物浓度的升高而减小.     

In vitro capacitation of stallion spermatozoa in calcium-free Tyrode's medium and penetration of zona-free hamster eggs

Capacitation of stallion spermatozoa in Tyrode's calcium-free (TCF) medium was assessed. Twelve gel-free ejaculates were collected. After removal from the seminal plasma, cells were washed three times with 0.85% saline containing 0.1% bovine serum albumin (BSA) and resuspended in TCF. Both washing and incubation media were adjusted to pH 8 and 300 to 310 mOsm. Final sperm concentration during incubation was 2 x 10(6) cells/ml. The diluted ejaculates were incubated for 2, 4, 6, 8 and 10 h at 37 degrees C in an atmosphere containing 5% CO(2). Acrosomes were stained with naphthol yellow and erythrocin B initially and after each incubation period and evaluated microscopically. Transmission electron microscopy was used to verify whether normal acrosome reaction was occurring or if cells were degenerating. Penetration of zona-free hamster oocytes was evaluated using 10(3) to 10(4) sperm/ml suspension and coincubating eggs for 3.5 to 4 h with sperm. Penetration tests were done for wash and incubation treatments and recorded positive when swollen sperm heads or male pronuclei were present. Incubation time affected acrosome integrity (P<0.001). Incubation for 8 to 10 h significantly improved acrosome reaction (P<0.001) and the percentage of reacted acrosomes increased sharply after 6 h of incubation (P<0.001). None of the washed sperm penetrated zona-free eggs at zero time, but sperm from all incubation treatments penetrated eggs. A peak penetration rate of 29.9% was observed at 8 h (P<0.001). Results indicate that under the conditions used, the requirement for Ca(++) in the medium for the process of capacitation and acrosome reaction can be substituted for by elevated pH.     

The block to sperm penetration in zonal-free mouse eggs

The rate of sperm penetration and the number of sperm penetrating zona-free mouse eggs were found to be dependent on sperm concentration. At the lowest sperm concentrations examined (10² cells/ml, sperm-egg ratios of approximately 1:1), most eggs were penetrated (75%), and polyspermy was low (19%) following 3 hr of incubation. The number of sperm penetrating the egg was logarithmically related to sperm concentration. All eggs showed a delay of at least 20 min between insemination and penetration, and penetration was complete in approximately 2 hr at 10⁴ sperm/ml; this penetration block was attributed to egg-related changes. The existence and timing of the egg plasma membrane block to polyspermy were evaluated by reinsemination experiments. In this approach, the block was triggered in zona-free eggs with a low concentration of capacitated epididymal sperm at time 0, and the eggs were subsequently challenged with high sperm concentrations. The presence or absence of a block was inferred from the degree of polyspermy observed in these eggs after 3 hr of incubation. Adjusting for sperm concentration-dependent delays between insemination and sperm penetration, a blocking time of approximately 40 min was obtained.     

日本血吸虫尾蚴经人工方法转变的童虫体外培养的研究

本文介绍了日本血吸虫体外培养系统。建立了较适于童虫生长发育的B41培养基。尾蚴经人工方法转变的童虫在体外可发育至雌雄合抱,雌雄生殖器官形成。雌虫可达产卵阶段,但未具备正常的产卵机能。培养的血吸虫在体外至少可存活110天。     

Production of cloned pigs from adult somatic cells by chemically assisted removal of maternal chromosomes

The present study demonstrated that brief treatment of in vitro-matured porcine oocytes with demecolcine results in a membrane protrusion that contains a condensed chromosome mass, which can be easily removed by aspiration. This simple, chemically assisted method for removing maternal chromosomes enabled the production of a large number of nuclear-transferred porcine eggs. The development of eggs whose chromosomes were removed by this procedure following transfer of somatic cell nuclei to the blastocyst stage was not significantly different among groups activated using different procedures (6% to 11%) and was also not different among donor cells of different origins (3% to 9%), except for cumulus cells (0.4%). After transfer of 180 to 341 nuclear-transferred eggs that received somatic cells to 6 recipients, 2 of the recipients produced 8 healthy cloned piglets from the heart cells of a female pig. The chemically assisted method for removing maternal chromosomes was also effective for bovine and rabbit eggs.     

Maturation-associated changes in the rat zona pellucida

Rat follicular oocytes, arrested at prophase I, cannot be fertilized in vitro. This capacity is acquired following resumption of meiosis and a series of changes involving both the oocyte and the cumulus cells surrounding it. Oocytes exposed to sperm at different hours before ovulation show a gradual increase in the permeability of their zona pellucida (ZP). Our study examined whether the ZP, in response to the physiological stimulus for maturation and concomitant with the other oocyte--cumulus components, undergoes maturational changes. Two ZP characteristics were assessed, sensitivity to proteolysis and sperm binding. ZP surrounding oocytes and eggs were collected from five sources: 1) germinal vesicle (GV)-intact oocytes, 2) preovulatory eggs, 3) ovulated eggs isolated from oviducts of immature females, 4) fertilized eggs, 5) ovulated eggs isolated from oviducts of mature females. All ZP surrounding oocytes/eggs from groups 1-5 were dissolved by trypsin. When solubility by pronase and alpha-chymotrypsin was examined, a large variation between groups was found. All ZP from group 2 were dissolved by 0.001% pronase, compared to 0% solubility in group 4. Only 10% of the ZP surrounding GV-intact oocytes (group 1) were dissolved by this enzyme, compared to 82% in group 3. Solubility in 0.01% alpha-chymotrypsin showed a similar pattern. Capacitated sperm were incubated with eggs from groups 1 and 3. The number of sperm binding to ZP in group 3 was repeatedly higher than that in group 1. In both tests it was found that the ZP surrounding the mature eggs differ in their characteristics from ZP of GV-intact oocytes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Assessment of the consumption of walleye pollock eggs by nekton and jellyfish in the northern Sea of Okhotsk in the spring

In the northern Sea of Okhotsk, nekton and jellyfish consumed as many as 831 × 10⁹ walleye pollock eggs per day in 2011. The nekton exerted the highest pressure, viz., 98.3% of the overall predation on pollock egg by aquatic animals. Of the entire quantity of consumed eggs, 55.9% were eaten by herring, 35.9% by walleye pollock, 6.5% by Sakhalin sole, and 1.7% by jellyfish. Among jellyfish, scyphomedusae Cyanea capillata and Chrysaora melonaster, as well as the hydromedusa Tima sachalinensis consumed the largest quantities of eggs. The total consumption of pollock egg by aquatic animals in 2011 was estimated at 42.4 × 10¹², or 11.4% of the entire quantity of eggs that were spawned by walleye pollock in the waters of the northern part of the sea. The total amount of pollock eggs that were eaten by herring and pollock together for 51 days in 2011 amounted to 38.9 × 10¹², which was 5.7 times as much as that in 2002. Thus, a significant growth of predation on pollock eggs by their main consumers, viz., herring and walleye pollock, was observed in 2011. This was caused by an increase in the populations of both species during the recent years and also by a higher concentration of pollock eggs.     

Ovicidal effects of heat and disinfectants on Syphacia muris estimated by in vitro hatching

The ovicidal effects of heat and various chemical disinfectants on an oxyurid rat nematode Syphacia muris were investigated, using the hatching methods in artificial intestinal juice. The eggs were collected from the perianal skin of spontaneously infected rats by means of a piece of transparent adhesive tapes, and these eggs were treated with each disinfectant for two hours. It was found that 70% ethanol and 80 degrees C 30 min treatments killed almost all of the eggs. However, a small number of the eggs tested was killed by 0.02% chlorhexidine digluconate or 0.05% benzethonium chloride. Alcide, 3% saponated cresol solution, 50% isopropanol, 10 ppm sodium hypochlorite and 5 ppm iodophol had some effects against the eggs, but they didn't kill the eggs completely. A biological assay through infection of the eggs to rats might be necessary because the effects of 2% formalin on the eggs were not determined by the hatching methods.     

Investigation of microvessel density after irradiation

It is believed that malignant cell populations need the development of microvessels to grow and metastasize. The aim of our investigation was to find out whether gamma irradiation can affect proliferation of endothelial cells and thus can affect microvessel density in vivo. We used fertilized chicken eggs. The vascularized part of the yolk sac membrane (area vasculosa) of the eggs received single doses of 2 to 10 Gy. Forty-eight hours after irradiation, the area vasculosa was photographed in vivo, and prints of known magnification were evaluated to determine the density of the blood vessels. Microvessel count is the well-established marker used to determine vascular density. In addition, the proliferative activity of endothelial cells in the yolk sac membrane was determined by estimating the expression of proliferating cell nuclear antigen (PCNA). PCNA immunostaining was assessed immunohistochemically. After a single dose of 10 Gy, a statistically significant increase in vascular density was found compared to values determined at 0, 2, 4 and 8 Gy (P < 0.05). Twenty-four hours after 10 Gy irradiation, 44.8% (mean) of the endothelial cells were PCNA-positive. This was significantly higher (P < 0.05) compared to the results 24 h after 4.0 Gy (22.7%) and compared to control (19.4%). Twenty-four hours later, i.e. 48 h after irradiation with 10 Gy, the endothelial cells also showed a significantly (P < 0.05) higher PCNA positivity with a mean of 34.1% compared to the nonirradiated area vasculosa (18.1%) and compared to the results after 4.0 Gy irradiation (12.0%). The prerequisite for blood vessel formation is the proliferation of endothelial cells. Thus a single-dose irradiation with 10 Gy induces endothelial cell proliferation and subsequent neovascularization in the area vasculosa of the fertilized egg.     

Fusion of hamster and pig zona-free eggs stimulated by boar and guinea pig sperm at fertilization in vitro

In the course of in vitro fertilization of zona-free hamster and pig eggs by boar and guinea-pig spermatozoa it was observed that homologous and heterologous eggs fused together, forming cell hybrids between two or more cells. The fusogenic activity was attributed to spermatozoa and this was the hypothesis tested. The fusogenic activity (coinciding with sperm penetration activity) was dependent on the duration of sperm preincubation, which may be regarded as capacitation in vitro. Fusion occurred only after 3 hr of sperm preincubation and a narrow optimum was detected at 4–4.5 hr. Fusion of eggs was also dependent on sperm concentration. A relatively high proportion of fusions was observed at a sperm concentration of 4.0 × 10⁴ per ml and an optimum was attained at a concentration of 5.0 × 10⁵ per ml. The first fusions were observed at 90 min after semination. After 3 hr more than a half of the eggs reacted, and by 20 hr of incubation 80% of ova were fused. The fusability of eggs was tested and found to occur at 14 hr after ovulation. The fusion process was also studied using transmission electron microscopy. It is supposed that the process of egg fusion may be caused either by a similar mechanism to sperm-egg fusion, or by products released during the sperm acrosome reaction.     

Pronuclear location before the first cell division determines ploidy of polyspermic pig embryos.

Polyspermy occurs frequently in the fertilization of mammalian eggs, but little is known about whether polyspermic eggs have developmental ability in vitro or in vivo. We previously reported that poly-pronuclear (PPN; 3 or more pronuclei) pig eggs developed normally to the blastocyst stage despite having fewer inner cell mass cell numbers as compared to blastocysts derived from two-pronuclear (2PN) eggs. Here it is shown that most PPN pig eggs have abnormal cleavage patterns (having 3 or more cells) in the first cell division and retarded development of pronuclei prior to syngamy as compared to 2PN eggs. Most blastocysts (14 of 18) that developed from PPN eggs showed abnormal ploidy (were haploid, triploid, and tetraploid) whereas 20 of 22 blastocysts derived from 2PN embryos were diploid. The size and morphology of most Day 40 fetuses that developed from PPN eggs appeared to be normal. Of 8 Day 40 fetuses analyzed, 1 was triploid (XXY) and another was a mosaic with both diploid (XX) and tetraploid cells (frequency of less than 10%, XXXX), and the others were diploid. Anomalies of chromosomal composition were not detected in these fetuses. Five live piglets and one dead piglet were born from two recipients of PPN eggs. It is proposed that not all pronuclei of PPN pig eggs participate in syngamy, resulting in diploid cells in the conceptus. Our data suggest that there are two types of pronuclei location in polyspermic pig eggs and that the resulting ploidy is determined at the zygote stage before the first cell division according to pronuclear location.