Selenium recovery from kiln powder of cement manufacturing by chemical leaching and bioreduction.

A novel process by using chemical leaching followed by bacterial reductive precipitation was proposed for selenium recovery from kiln powder as a byproduct of cement manufacturing. The kiln powder at a slurry concentration of 10 w/v% with 0.25 M Na2CO3 at 28°C produced wastewater containing about 30 mg-Se/L selenium. The wastewater was diluted four-fold and adjusted to pH 8.0 as preconditioning for bioreduction. A bacterial strain Pseudomonas stutzeri NT-I, capable of reducing selenate and selenite into insoluble elemental selenium, could recover about 90% selenium from the preconditioned wastewater containing selenium of 5 mg-Se/L when supplemented with lactate or glycerol. The selenium concentrations in the treated wastewater were low around the regulated effluent concentration of 0.1 mg-Se/L in Japan.

[Rapidly enlarging giant left ventricular pseudo-false aneurysm after myocardial infarction; report of a case].

A 71-year-old man was admitted to our hospital with acute myocardial infarction and cardiac tamponade. After pericardial drainage, his hemodynamics was improved. Because more than 3 days had been passed after the onset of myocardial infarction and he had severe renal dysfunction, emergent coronary angiography (CAG) was not performed. After improvement of his general status, coronary angiography and percutaneous catheter intervention was carried out, and his course was uneventful. But transthoracic echocardiography before discharge revealed a giant posterior psudoaneurysm. Patch closure and coronary artery bypass grafting was carried out under cardiopulmonary bypass, and postoperative course was uneventful. Postoperative left ventriculogram revealed disappearance of pseudoaneurysm, but relatively large akinetic area of posterior-inferior wall was left around a patch. Pseudo-false aneurysm was diagnosed by histological examination.

Effects of cooling and warming rates during vitrification on fertilization of in vitro-matured bovine oocytes.

The purpose of this study was to clarify the relationship of cooling rates (CR) and warming rates (WR) during vitrification with postwarming viability of in vitro-matured bovine oocytes. In Experiment 1, oocytes were vitrified in a solution containing 7.2 M ethylene glycol and 1.0 M sucrose by use of open-pulled glass capillaries with five different outer diameters and were warmed by placement of the capillaries into 0.25 M sucrose solution. The capillaries of 2000-, 1400-, 1000-, 630-, and 440-mm diameters provided CR of 2000, 3000, 5000, 8000, and 12,000 degrees C/min and WR of 5000, 8000, 17,000, 33,000, and 62,000 degrees C/min, respectively. In oocytes vitrified in capillaries of 1400-mm diameter (CR, 3000 degrees C/min; WR, 8000 degrees C/min), the morphological survival rate (86% of vitrified), penetration rate (79% of inseminated), and normal fertilization rate (69% of penetrated) were higher or tended to be higher than those in the other vitrification groups. In Experiment 2, oocytes cooled at 2000, 3000, or 12,000 degrees C/min were warmed at 8000 degrees C/min, and oocytes cooled at 3000 degrees C/min were warmed at 5000, 8000, or 33,000 degrees C/min. Among these CR-WR combinations, cooling of oocytes at 3000 degrees C/min regardless of the WR resulted in higher postwarming survival. These results indicate that survival of in vitro-matured bovine oocytes after vitrification and subsequent warming is improved by a slightly rapid cooling rate in open-pulled glass capillaries compared to that obtained in conventional straws.

Muscle high-energy metabolites and metabolic capacity in patients with heart failure.

UNLABELLED: OKITA, K., K. YONEZAWA, H. NISHIJIMA, A. HANADA, T. NAGAI, T. MURAKAMI, and A. KITABATAKE. Muscle high-energy metabolites and metabolic capacity in patients with heart failure. Med Sci. Sports. Exerc., Vol. 33, No. 3, 2001, pp. 442-448. BACKGROUND: Various abnormalities in skeletal muscle have been demonstrated by biopsy in patients with chronic heart failure (CHF). In mammalian muscles, high-energy metabolite composition at rest (HEMC) provides data on important metabolic characteristics; however, the significance of HEMC has not been clarified in patients with CHF. Therefore, we investigated HEMC in normal subjects and patients with CHF and examined its relation to muscle metabolic capacity and exercise tolerance. METHODS: High-energy metabolites (phosphocreatine (PCr), inorganic phosphate (Pi), and ATP) in resting calf muscle were measured by 31P-magnetic resonance spectroscopy (31P-MRS), and ratios of Pi to PCr, Pi to ATP, and PCr to ATP were calculated in 34 patients with CHF and 13 age- and size-matched normal subjects. Muscle metabolism was evaluated during local exercise of unilateral plantar flexion by 31P-MRS. Metabolic capacity was estimated by the rate of PCr breakdown in relation to the workload. Systemic exercise capacity was evaluated by a bicycle ergometer. RESULTS: The ratio of PCr to ATP was significantly increased in patients with CHF compared with controls (3.06 +/- 0.43 vs 2.72 +/- 0.36, P < 0.05) and was significantly correlated with metabolic capacity (r = -0.37, P < 0.01) and with peak oxygen uptake (r = -0.45, P < 0.01). There was a significant correlation between metabolic capacity and peak oxygen uptake (r = 0.53, P < 0.001). CONCLUSION: HEMC was altered in patients with CHF, and this change was related to metabolic capacity and exercise capacity. These findings provide new insight into the mechanism of impaired muscle metabolism in CHF.

Comparison of palpebral fissure obliquity in three different racial groups.

PURPOSE: To compare the obliquity of the palpebral fissures of three different racial populations. METHODS: Prospective observational study. Frontal views of the palpebral fissures of three different groups of subjects (Brazilian whites, Brazilian Japanese, and Brazilian Indians from the Upper Rio Negro Basin of the Amazonas State) were acquired with a photographic camera and transferred to a Macintosh computer. Using the National Institutes of Health Image software (NIH Image), the angle formed by the inner and the outer canthi was measured for all images. RESULTS: The mean fissure angle of the Japanese (9.39 degrees) was not statistically different from the mean angle of the Indians (9.64 degrees). On the other hand, both were significantly greater than the mean angle of the whites (4.60 degrees). CONCLUSIONS: Marked fissure obliquity is found more frequently among Asians than among whites.

Nuclear transfer in cattle : effect of linoleic acid-albumin on freezing sensitivity of enucleated oocytes.

The effect of linoleic acid-albumin (LAA) supplementation to the media for IVM, enucleation, and activation on the developmental potential of bovine embryos produced by nuclear transfer (NT) into frozen-thawed cytoplasts was investigated. Blastomeres derived from morulae was placed in the perivitelline space of frozen-thawed cytoplasts, which were then fused by a DC pulse. The proportion of fused embryos was similar between groups with and without LAA (87 vs. 90%). The proportion of development to blastocysts of NT embryos derived from the media with LAA (14%) was higher than that without LAA (4%), indicating that LAA treatment of bovine oocytes during IVM, enucleation and activation can improve the ability of such cytoplasts after freezing and thawing to develop into blastocysts after NT.

Dissociation between muscle metabolism and oxygen kinetics during recovery from exercise in patients with chronic heart failure.

OBJECTIVE: To estimate muscle metabolism and oxygen delivery to skeletal muscle in patients with chronic heart failure. METHODS: 13 patients with chronic heart failure and 15 controls performed calf plantar flexion for six minutes at a constant workload of 50% of one repetition maximum. During recovery from exercise, skeletal muscle content of oxygenated haemoglobin (oxy-Hb) and the level of phosphocreatine (PCr) were measured by near-infrared spectroscopy and (31)P-magnetic resonance spectroscopy, respectively. RESULTS: The mean (SD) time constants of PCr and oxy-Hb during recovery from exercise were significantly greater in patients with chronic heart failure than in normal subjects (tau PCr: 76.3 (30.2) s v 36.5 (5.8) s; tau oxy-Hb: 48.3 (7.3) s v 30.1 (7.7) s; p < 0.01). Both time constants were similar in normal subjects, while the tau PCr was significantly greater than the tau oxy-Hb in patients with chronic heart failure. CONCLUSIONS: The slower recovery of PCr compared with oxy-Hb in patients with chronic heart failure indicates that haemoglobin resaturation is not a major rate limiting factor of PCr resynthesis. It is suggested that muscle metabolic recovery may depend more on oxygen utilisation than on haemoglobin resaturation or oxygen delivery in patients with chronic heart failure.

Effects of timing of oocyte cryopreservation on in vitro development of nuclear-transferred bovine zygotes.

The utility of cryopreserved bovine oocytes as recipient cytoplasts for nuclear transfer (NT) was examined. In vitro-matured (IVM), metaphase-II oocytes were enucleated by mechanical suction and activated parthenogenetically. The cytoplasts were fused with blastomeres of in vitro-produced day-5 morulae by a DC electropulse, and then cultured up to 8 days (non-frozen controls; group I). Oocytes were frozen-thawed in 1.5-M ethylene glycol and 0.1-M sucrose before enucleation (group II), after enucleation (group III), after enucleation and aging culture (group IV), or after activation (group V). In group I, 91% of IVM oocytes could be used for NT and 89% of them fused successfully. Finally, 36% of the fused zygotes developed into blastocysts. The proportions of morphologically normal oocytes after thawing in groups IV and V (70 and 69%, respectively) were higher than in group III (56%), and the proportion of IVM oocytes used for NT in group IV (56%) was higher than those in groups II, III, and V (33%, 35%, and 38%, respectively). Fusion rates of the NT zygotes in groups III, IV, and V (90%, 88%, and 88%, respectively) were higher than the rate in group II (75%). Rates of development into blastocysts of the fused zygotes in groups II, III, IV, and V were 0%, 3%, 2%, and 6%, respectively (P < 0.05, group II vs. groups III, IV, and V). Developmental kinetics and cell numbers of the blastocysts were similar among the groups. It was suggested that timing of oocyte cryopreservation is among the factors influencing efficiency of production of cloned embryos in cattle.

Effect of linoleic acid-albumin in the culture medium on freezing sensitivity of in vitro-produced bovine morulae.

The objective of this study was to improve the survival of in vitro-produced bovine morulae after cry opreservation. In Experiment 1, presumptive zygotes at 20 h post-insemination (hpi) were cultured in a mixture of modified synthetic oviduct fluid (m-SOF)/0.3% BSA and m-SOF/0.3% linoleic acid-albumin from bovine serum (LAA) at 39.0 degrees C in 5% O2, 5% CO2 and 90% N2 (final LAA concentration: 0, 0.01, 0.03, 0.1 or 0.3%). Morulae harvested at 138 hpi were frozen and thawed in m-PBS/0.3% BSA containing 1.5 M ethylene glycol and were cultured for 96 h in m-SOF/10% FBS to assess further development. The post-thaw survival of morulae derived from culture in 0.1% LAA (60%, P < 0.01) and in 0.03% LAA (55%, P < 0.05) was higher than that in 0% LAA (32%). Lowering the LAA concentration below 0.1% resulted in similar rates of morula development as in m-SOF/0.3% BSA. In Experiment 2, zygotes were cultured in m-SOF/0.1% LAA from 20 to 90 hpi and/or from 90 to 138 hpi. Post-thaw survival of morulae that had been exposed to LAA from 20 to 90 hpi (39%) or from 90 to 138 hpi (56%) was higher than that of morulae cultured without LAA from 20 to 138 hpi (12%, P < 0.02). These survival rates were lower than that of morulae cultured with LAA over a period of 20 to 138 hpi (76%, P < 0.001). The results indicate that cell-free culture of IVM/IVF bovine zygotes in m-SOF supplemented with LAA produces morula-stage embryos relatively tolerant to the process of freezing and thawing.

Skeletal muscle metabolism limits exercise capacity in patients with chronic heart failure.

BACKGROUND: Several studies have indicated that skeletal muscle is important in determining the exercise capacity of patients with chronic heart failure (CHF). However, this theory has been investigated only in experiments based on local exercise involving a small muscle mass. We investigated skeletal muscle metabolism during maximal systemic exercise to determine whether muscle metabolism limits exercise capacity in patients with CHF. We also studied the relationship between muscle metabolic abnormalities during local and systemic exercise. METHODS AND RESULTS: Skeletal muscle metabolism was measured during maximal systemic exercise on a bicycle ergometer by a combination of the metabolic freeze method and 31P magnetic resonance spectroscopy in 12 patients with CHF and 7 age- and size-matched normal subjects. We also evaluated skeletal muscle metabolism during local exercise while subjects performed unilateral plantar flexion. Muscle phosphocreatine (PCr) was nearly depleted during maximal systemic exercise in patients with CHF and normal subjects (12.5+/-0.04% and 12.3+/-0.07%, respectively, of initial level). PCr depletion occurred at a significantly lower peak oxygen uptake (peak VO2) in patients with CHF than in normal subjects (CHF, 20.2+/-3.0 versus normal, 31.8+/-3.7 mL . min-1 . kg-1, P<0. 0001). Muscle metabolic capacity, evaluated as the slope of PCr decrease in relation to increasing workload, was correlated with peak VO2 during maximal systemic exercise in patients with CHF (r=0.83, P<0.001). Muscle metabolic capacity during local exercise was impaired in patients with CHF and was correlated with capacity during systemic exercise (r=0.76, P<0.01) and with peak VO2 (r=0. 83, P<0.001). CONCLUSIONS: These results suggest that impaired muscle metabolism associated with early metabolic limitation determines exercise capacity during maximal systemic exercise in patients with CHF. There was a significant correlation between muscle metabolic capacity during systemic and local exercise in patients with CHF.

Skeletal muscle metabolism in maximal bicycle and treadmill exercise distinguished by using in vivo metabolic freeze method and phosphorus-31 magnetic resonance spectroscopy in normal men.

This study indicates that skeletal muscle metabolism may affect the results of maximal bicycle and treadmill exercise differently, and that maximal bicycle exercise was limited by quadriceps muscle metabolism rather than by cardiopulmonary capacity. In contrast, maximal treadmill exercise was not limited, eliciting more cardiopulmonary reserve and attaining greater peak oxygen uptake than maximal bicycle exercise.

Effect of nuclear stages during IVM on the survival of vitrified-warmed bovine oocytes.

The effect of nuclear stages during IVM on the survival of vitrified-warmed bovine oocytes was investigated. Oocytes with compact cumulus cells were cultured for 0, 6, 12 and 24 h in TCM199 supplemented with 5% fetal bovine serum (FBS) in 3% CO2 in air. The oocytes were first exposed to 20% ethylene glycol solution and were subjected to vitrification in a solution containing 40% ethylene glycol, 18% Ficoll-70 and 0.3 M sucrose. After warming in 20 degrees C water, oocytes which had been vitrified at less than 24-h of IVM were again cultured to complete the 24-h of IVM period. Oocytes were then incubated with frozen-thawed spermatozoa in Brackett and Oliphant (BO) medium containing 60 micrograms/ml heparin and 0.25% BSA for 20 h. In vitro fertilization rates of oocytes vitrified-warmed at 0, 6, 12 and 24-h IVM were 75.2, 68.0, 82.0 and 72.4%, respectively, comparable to the rates for unvitrified control oocytes (80.6%). A higher incidence of polyspermic fertilization was observed in oocytes vitrified at 24-h IVM (44.9 vs 22.6% in the control group, P < 0.05). Vitrification of oocytes at 12-h IVM seemed to be better than that of other IVM groups, since the normal fertilization rate of all treated oocytes was the highest (36.0%) among the vitrification groups. Developmental competence of the oocytes following vitrification and in vitro fertilization (12-h IVM group) was examined by cell-free culture of presumptive zygotes up to 9 d in modified synthetic oviduct fluid (mSOF) in 5% CO2, 5% O2 and 90% N2. The cleavage rate of zygotes from vitrified oocytes 48 h after insemination was 29.8%, which was lower than that of the control group (57.0%, P < 0.05). Development to blastocysts from the vitrified oocytes (4.8%) was much lower than that of the control group (27.0%, P < 0.05). These results indicate that cryopreservation of bovine oocytes by vitrification may be affected by their maturation stage in vitro, and that developmental competence to blastocysts of cleaved oocytes following vitrification may be impaired compared with unvitrified control oocytes.

Metabolic abnormality of calf skeletal muscle is improved by localised muscle training without changes in blood flow in chronic heart failure.

OBJECTIVE: To investigate whether localised skeletal muscle training, which does not have a great influence on the heart, improves abnormalities of calf muscle metabolism in patients with chronic heart failure. METHODS: Seven cardiac patients in New York Heart Association class II and III undertook a random order crossover trial. Training consisted of unilateral calf plantar flexion exercise. Before and after training, the patients' metabolic responses were examined during the calf exercise test with phosphorus-31 nuclear magnetic resonance spectroscopy (31P-MRS) and calf blood flow with plethysmography. The new Borg scale was employed as a subjective fatigue scale. RESULTS: In a constant load exercise test (70% of maximum load achieved during the incremental exercise), standardised phosphocreatine and intracellular pH decreased less after training (p < 0.05, repeated measures analysis of variance). The new Borg scale improved significantly after training (p < 0.05). Blood flow did not change significantly in either test. CONCLUSIONS: In patients with chronic heart failure, localised calf skeletal muscle training improved oxidative capacity without changes in calf blood flow. This training also improved the subjective fatigue scale. This training method may therefore alleviate leg fatigue experienced in daily activities.

Incidence of chromosomal anomalies in early bovine embryos derived from in vitro fertilization.

The incidence of chromosomal anomalies in early bovine embryos derived from follicular oocytes fertilized in vitro using sperm separated by Percoll density gradient centrifugation was investigated. Overall, chromosomal anomalies were observed in 13.7% (138/1005) of embryos. There were 14 haploids (1.4%), 2 hypodiploids (0.2%), 6 hyperdiploids (0.6%), 101 triploids (10.0%), 12 tetraploids (1.2%), 2 diploid/triploid mosaics (0.2%), and 1 diploid/tetraploid mosaic (0.1%). The frequency of triploidy was caused mainly by polyspermy. There was a significant difference in the frequency of embryos with abnormal chromosomes between the two bulls used (P less than 0.005), but Percoll centrifugation did not affect the observed incidence of anomalies. The frequency of chromosomal anomalies in embryos at each stage increased with delay or arrest of development. These results suggest that the incidence of chromosomal anomalies depended on the conditions of in vitro fertilization and the arrest of development.

Sex ratio of early embryos fertilized in vitro with spermatozoa separated by percoll.

Early bovine embryos were obtained by in vitro fertilization and sexing carried out by chromosome analysis. Separation of bovine X- and Y-bearing spermatozoa was performed using Percoll density gradient centrifugation and the enrichment of X-sperm proportion was investigated. Through treatment with vinblastin sulfate and podophyllotoxin, 880 (48.6%) of 1812 embryos at two- to seven-cell stages at 48 to 53 h after sperm-egg incubation produced metaphase spreads, and 399 (45.3%) of these were successfully sexed; the sexable rate reaching 53.4% for four-cell embryos. Sexing rates for embryos from the original sperm of two bulls were 69.6% (32 46 ) in Bull A and 54.2% (58 107 ) in Bull B. Embryos fertilized in vitro with sperm sedimented at the bottom of sperm centrifuged under conditions (I) 50 to 85% of Percoll, 15 degrees C; (II) 30 to 80%, 10 degrees C; (III) 30 to 80% 20 degrees C; (IV) 30 to 90%, 20 degrees C, gave rise to male sex ratios of (I) 58.3% in Bull A and 53.5% in Bull B, (II) 65.9% in Bull A, (III) 49.3% in Bull B and (IV) 66.7% in Bull B. In conclusion, Percoll density gradient centrifugation under these four conditions was unsuccessful in separating X- and Y-bearing bull spermatozoa.

In-vitro fertilization of pig oocytes by frozen boar spermatozoa.

In Exp. 1 pig oocytes matured in vitro were used to evaluate the fertilizability in vitro of frozen epididymal (4 boars) and ejaculated (3 boars) spermatozoa that were preincubated in modified TCM-199 for 4 h at 37 degrees C. The percentages of penetrated oocytes with the frozen epididymal spermatozoa were 0-40%. In contrast, none of the oocytes were penetrated with the frozen ejaculated spermatozoa. In Exp. 2, oocytes matured in vivo were inseminated in vitro with the frozen epididymal spermatozoa that were known to penetrate oocytes matured in vitro. The penetration rate was 79% and the percentage of polyspermic oocytes was 57%. Culture for 30 h of oocytes matured in vivo and fertilized in vitro resulted in 51% (34/67) developing to the 2-cell stage. These embryos were transferred to 2 recipient gilts. One gilt became pregnant and a litter of 3 (1 live and 2 dead) was born. These results indicate that frozen epididymal spermatozoa can be used for in-vitro fertilization in the pig.

In vitro fertilization and cleavage capability of bovine follicular oocytes classified by cumulus cells and matured in vitro.

Bovine oocytes were collected from ovaries obtained from an abattoir. They were classified according to the character of the cumulus cells using a stereomicroscope, and cultured in 25 mM Hepes buffered Tissue Culture Medium 199 supplemented with 10% fetal calf serum at 39 degrees C and inseminated by capacitated sperm. Maturation rates of Class A oocytes, with compact, dense cumulus cells; Class B, partially naked oocytes with thin cumulus layers or small remnants of cumulus cells and Class C, naked oocytes were 97.4% (38/39), 89.8% (106/118) and 52.9% (45/85), respectively. The fertilization rates for the three classes were 86.8%, 85.8% and 53.3%, respectively. The naked oocytes had a significantly lower fertilization rate than oocytes of the other two classes. Significantly more Class A oocytes cleaved (63.7%, 232/364) than those of Class B (29.5%, 36/122) and Class C (17.7%, 28/158).

Monoclonal antibodies to porcine zona pellucida antigens and their inhibitory effects on fertilization.

Five hybridomas which produce monoclonal antibodies (Mabs) to porcine zona pellucida (ZP) were established. The immunoglobulin classes were IgG2a from hybridoma B11C8 and IgM from the other four. Using immunofluorescent staining, the Mab from B11C8 stained only porcine oocytes, the Mab from G10G5 stained oocytes of pigs and hamsters but Mabs from C6H1, D3H4, G10F9 stained oocytes of pigs, humans, hamsters, rats and mice. Mabs from B11C8 and G10F9 strongly blocked boar sperm binding to porcine ZP but other Mabs produced only slight blocking. However, no Mabs could block sperm penetration during in vitro fertilization (IVF) of hamster oocytes. When a goat antibody (IgG) to mouse serum gamma-globulin was applied after each Mab as a second antibody, only Mab from G10G5 impaired IVF of hamster oocytes.

Cryoprotective effects of some amides on rabbit spermatozoa.

Semen was diluted 1:9 with egg yolk-citrate medium containing 0.31--3.1 M (final concentration) formamide, butyramide, acetamide, propionamide, dimethylformamide, lactamide, malomide, ethylene glycol, trimethylene glycol, dimethylsulphoxide (DMSO) or glycerol. After 30 min incubation at 20 degrees C, sperm motility was superior in hypertonic solutions of acetamide, lactamide, dimethylsulphoxide, trimethylene glycol and ethylene glycol. Some of these compounds were added to semen diluted 1:2 in an isotonic egg-yolk-glucose-lactose-raffinose solution and frozen by the pellet method. Relatively good survival of motility was obtained in 1.0 M-DMSO, -lactamide or -acetamide. Dimethylformamide (0.5 M), ethylene glycol (0.5--1.5 M), trimethylene glycol (1.5 M) and propionamide (0.75 M) also gave some protection. Insemination of does with semen frozen and thawed with 1.0 M-DMSO, -lactamide or acetamide gave fertilization rates of 68--88%, and 84% (38/45) of does gave birth to an average of 5.3 young.

Penetration of the zona-free or intact eggs by foreign spermatozoa and the fertilization of deer mouse eggs in vitro.

Zona-free eggs were introduced to fresh or preincubated sperm suspensions and the penetration of eggs by foreign spermatozoa was examined, as evidenced by enlargement of the sperm head and formation of the male pronucleus. It was found that zona-free hamster eggs can be penetrated by guinea-pig, deer mouse and rabbit spermatozoa but zona-free rat, mouse and rabbit eggs cannot be penetrated by guinea-pig spermatozoa. Furthermore, zona-free rat and mouse eggs cannot be penetrated by spermatozoa from two species of deer mice and the Mongolian gerbil. The zona pellucida of a few intact rat eggs can be penetrated by mouse (6%) and by P. leucopus spermatozoa (14%) but enlargement of the sperm head and formation of pronuclei were observed in the former but not in the latter. It seems that (1) sperm capacitation is required for the penetration of zona-free eggs, (2) the attachment of foreign spermatozoa to eggs may indicate their potential ability of penetration in some cases, (3) there is a certain affinity between the vitellus of one species and spermatozoa from another species, (4) the block to the entry of foreign spermatozoa is not only in the zona pellucida but also in the vitelline membrane, (5) zona-free hamster eggs can be penetrated by spermatozoa of six species, (6) mouse spermatozoa can penetrate zona-free eggs of three species, and (7) fertilization of intact P. maniculatus eggs can be achieved in vitro.